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Studies on the asynchronous synaptic responses and endogenous potentiating substances of neurotransmission… Chirwa, Sanika Samuel 1988

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STUDIES ON THE ASYNCHRONOUS SYNAPTIC RESPONSES AND ENDOGENOUS POTENTIATING SUBSTANCES OF NEUROTRANSMISSION IN THE HIPPOCAMPUS By SANIKA SAMUEL CHIRWA B . S c , (Pharmacy), The University of British Columbia, 1981 M.Sc, (Pharmarmacology & Therapeutics.). The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics, Faculty of Medicine, The University of British Columbia)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA 1988 September ©Sanika Samuel Chirwa, 1988  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives.  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Pharmacology and Therapeutics  The University of British Columbia Vancouver, Canada  D a t e  DE-6 (2/88)  26 September 1988  CHIRWA  ii  ABSTRACT In the hippocampus, transient  tetanic  stimulations of inputs, or  simultaneous pairings of conditioning intracellular ations with activated presynaptic afferents  postsynaptic depolariz-  at low stimulation frequencies,  result in input specific long-term potentiation sion.  (LTP) of synaptic transmis-  LTP lasts for hours in vitro, or weeks in vivo, and it  be involved in memory and learning.  brief  is thought to  Experimental evidence in the literature  suggests that postsynaptic mechanisms mediate LTP induction, whereas presynaptic mechanisms are involved in its maintenance.  Since LTP is thought to  be generated by postsynaptic mechanisms and to be subsequently maintained by presynaptic processes, this suggests the presence of feedback during LTP development,  however, the experimental  actions is presently not available.  interactions  evidence for such inter-  Consequently, the present studies were  conducted to examine possible feedback interactions between postsynaptic and presynaptic  elements  in  the  hippocampus.  Furthermore,  the  tested the hypothesis that substances released during tetanic caused the release of endogenous substances that interacted afferents  resulting in alterations  experiments stimulations  with activated  in presynaptic functions and LTP produc-  tion. Experiments  were  hippocampal slices. and 0.5  mM Ca  chronous (minEPSPs)  Briefly,  (denoted  release in  conducted  of  CA^  During transient  Ba  ++  transversely  sectioned  guinea  physiological medium containing 3.5  as Ba  transmitters, neurons  b  using  after  applications,  medium)  was used to  observed stimulation  as  evoked  of  short bursts of  the  pig  mNi B a  ++  induce the  asyn-  miniature  EPSPs  stratum  radiatum.  evoked minEPSPs were  CHIKWA observed following  stimulations  of  depolarizing current injections  the  stratum  radiatum  into CA^ neurons.  or  iii  conditioning  Moreover, the frequen-  b  cies of minEPSPs were significantly increased immediately after simultaneous stimulations of the stratum radiatum and conditioning depolarizing current injections into CA^  neurons.  Significant increases in the frequencies of  evoked minEPSPs were also observed during LTP induced by tetanic tions.  The above  attributed, mitters  increases  in  the  frequencies  of  stimula-  evoked minEPSPs were  in part, to presynaptic changes resulting in increases in trans-  released.  However,  a thorough  quanta!  analysis  is  requirea  to  substantiate this conclusion. In order to determine whether  any substances released during  tetanic  stimulations were involved in the mooulation of presynaptic functions and induction of rabbit  LTP, samples were collected from guinea pig hippocampus and  neocortex.  It  was found that  samples that were collected during  tetanic stimulations of the guinea pig hippocampus in vivo or rabbit neocortex  in vivo produced LTP in the  guinea pig hippocampal  Applications of these samples after LTP.  heating and cooling failed  vitro.  to induce  Subsequent studies demonstrated that PC-12 cells incubated in growth  medium treated with samples collected during tetanic rabbit cell  slice in  neocortex developed extensive neurite cultures  incubated  in  (1)  heated  growths.  stimulations  of  the  In contrast, PC-12  and cooled samples,  (2)  samples  collected in the absence of tetanic stimulations of the rabbit neocortex, or (3)  plain growth medium, failed to develop neurite  growths.  In addition,  PC-12 cell cultures that were incubatea in growth medium containing samples collected during tetanic  stimulations plus saccharin (10 mM), a substance  known to inhibit N6F-dependent neurite growth, failed to develop neurites.  CHIRwA  iv  In separate experiments it was found that saccharin could block (1)  the  synaptic potentiating effects of the above collected and applied endogenous substances, and (2) LTP induced with tetanic stimulations, in the guinea pig hippocampus in vitro.  The concentrations of saccharin used in these studies  had insignificant effects on resting membrane potentials, input resistances, spontaneous neuronal  or  evoked  depolarizations  responses  of  CA^  neurons.  b  Furthermore,  induced by N-methyl-DL-aspartate  (NMDA)  CA^  or  with  tetanic stimulations of the stratum radiatum, were not altered by saccharin applications. pulse  In addition, saccharin had insignificant effects on paired-  facilitation,  CA^  neurons,  and  post-tetanic Schaffer  potentiations,  collaterals  minEPSP  terminal  frequencies  excitability.  These  results suggest that saccharin blocked LTP through mechanisms different either  non-specific alterations  activation. receptor  Perhaps  the  activation.  in CA^ cell  agent  That  properties  antagonized  LTP at  saccharin blocked  neocortical sample as well as by tetanic  a  in  from  or NMDA receptor step  beyond NMDA  LTP caused by the  applied  stimulation of the stratum radia-  tum, and that saccharin also blocked neurite growth in PC-12 cells induced by  the  neocortical  samples,  raises  the  prospect  substances are involved in LTP generation. was  found  that  the  potentiating  substances  were  not  antagonised  growth  of  atropine  the or  collected  CAj^  col laterals-CA^ synapses) s t i l l  population spike.  While  brief  dihydro-e-erythroidine.  maintained  applications  it  endogenous  Heated and then cooled solutions of glutamate (a putative transmitter Schaffer  related  In other control experiments,  effects by  that  of  their 2.5  actions  at the on the  yg/ml exogenous  NGF (from Vipera lebetina) during low frequency stimulations of the stratum  CHIKWA  v  radiatum did not consistently induce LTP, this peptide significantly f a c i l i tated the development of LTP when applied in association with tetanic stimulations  of  weak inputs  in  the  CA^ area.  These weak  inputs  could not  support LTP if tetanized in the absence of the exogenous NGF. The results of the studies in this thesis suggested that postsynaptic depolarizations modulated presynaptic functions in the hippocampus.  Tetanic  stimulations in hippocampus and neocortex caused the release of diffusible substances, which were probably growth related macromolecules, that acted  with  activated  elements resulting  presynaptic  afferents  in LTP development.  and/or  The precise  subsynaptic locus of  inter-  dendritic actions of  these agents awaits further investigations.  Research Supervisor  CHIRWA  vi  TABLE OF CONTENTS  Chapter  Title  Page No.  A.  TITLE PAGE  B.  ABSTRACT  ii  C.  TABLE OF CONTENTS  vi  D.  LIST OF TABLES  E.  LIST OF FIGURES  F.  ABBREVIATIONS  G.  ACKNOWLEDGEMENTS  H.  DEDICATION  I.  INTRODUCTION  1  2.  BASIC MORPHOLOGY OF THE HIPPOCAMPAL FORMATION  8  3.  i  xiv xv xviii xix xx  2.1  General  8  2.2  The hippocampal region  8  2.3  The hippocampus  9  2.4  The dentate gyrus  9  2.5  The hilus and CA^ region  11  2.6  The cornu ammonis field  11  CELLULAR PROPERTIES AND INTRINSIC CIRCUITRY  13  3.1  Dentate gyrus granule cells  13  3.2  Cornu ammonis pyramidal neurons  13  3.2.1  Subfield CA  3.2.2  Subfield CA  3.2.3  Subfield CA  X  14  3  15  0  17  CHIRWA Chapter  4.  5.  6.  Title 3.3  CA^ and Hi 1 us neurons  3.4  Interneurons  vii  Page No. 18 19  EXTRINSIC AFFERENTS TO THE HIPPOCAMPUS  21  4.1  Entorhinal-hippocampal inputs  21  4.2  Septo-hippocampal inputs  21  4.3  Miscellaneous inputs  22  ELECTROPHYSIOLOGY OF THE HIPPOCAMPUS  23  5.1  Electrical properties of neurons  23  5.2  Intrinsic ionic conductances  24  5.3  Bursting activity  25  5.4  Miniature postsynaptic potentials  26  5.5  Evoked field responses  26  5.6  Inhibitory postsynaptic potentials  27  5.7  Electrotonic couplings  29  5.8  Ephaptic interactions  30  SYNAPTIC PHARMACOLOGY OF THE HIPPOCAMPUS  31  6.1  GABA  31  6.2  GABA receptors  31  6.3  GABA receptors  32  6.4  Putative excitatory transmitters  33  6.5  Exogenous glutamate actions in hippocampus  34  6.6  NMDA and Quisqualate/Kainate receptors  34  6.7  Subsynaptic receptors  35  6.8  Other putative transmitters  36  6.9  Neuromodulators  37  A  B  CHIRWA Chapter 7.  Title  viii  Page No.  LONG-TERM POTENTIATION IN THE HIPPOCAMPUS  38  7.1  Introduction  38  7.2  Basic features of long-term potentiation  38  7.2.1  Long-term potentiation  38  7.2.2  Distribution of LTP  40  7.2.3  Homosynaptic and heterosynaptic LTP  40  7.2.4  LTP in single neurons  41  7.3  7.4  Production of LTP  41  7.3.1  Induction  41  7.3.2  Co-operative LTP  43  7.3.3  Associative LTP  43  7.3.4  Coupled LTP  44  LTP production with pharmacological methods  44  7.4.1  Raised extracellular K  44  7.4.2  Raised extracellular C a  7.4.3  Phorbol esters  46  7.4.4  Mast cell degranulating peptides  47  7.4.5  Glutamate  47  7.4.6  Miscellaneous  48  +  ++  45  7.5  Blockade of LTP induction  48  7.6  Maintenance of LTP  49  7.6.1  Biochemical and structural changes  49  7.6.2  Protein kinase C  51  7.b.3  Increased transmitter release  51  CHIRWA Chapter  Title 7.7  8.  Page No. 53  BARIUM AND SACCHARIN AS EXPERIMENTAL TOOLS  55  8.1  General  55  8.2  Barium  55  8.3  9.  Summary  ix  8.2.1  Chemistry  55  8.2.2  Transmitter release  56  8.2.3  K currents  57  +  Saccharin  58  8.3.1  Chemistry  58  8.3.2  Disposition  59  8.3.3  Tumor promoter  59  8.3.4  Neurite growth  61  8.3.5  Inhibition of enzymes  62  METHODS AND MATERIALS  64  9.1 Animals  64  9.1.1  Source  64  9.1.2  Animal feed and housing  64  9.2  Slice preparation  64  9.3  Slice selection  66  9.3.1  Slice chamber and perfusion method  66  9.3.2  Standard and test media  66  9.4  9.5  Endogenous sample collections  68  9.4.1  Guinea pig hippocampus  68  9.4.2  Rabbit neocortex  71  PC-12 Rat Pheochromocytoma cell line  71  CHIRWA Chapter  Title 9.6  9.7  10.  x  Page No.  Electrical instruments  73  9.6.1  Amplifiers  73  9.6.2  Stimulators  73  9.6.3  Oscilloscopes  73  9.6.4  Magnetic tape recorder  74  9.6.5  Paper plotter and chart recorder  74  9.6.6  Miscellaneous  74  Stimulating and recording electrodes  75  9.7.1  Stimulating electrodes  75  9.7.2  Recording electrodes  75  EXPERIMENTAL SCHEMES  76  10.1  Intracellular recordings  76  10.2  Extracellular recordings  78  10.3  Induction of long term potentiation  80  10.3.1 Tetanic stimulations  80  10.3.2  80  10.4  10.5  Paired depolarizations  Effects of B a  in hippocampus  80  and evoked responses  80  ++  10.4.1  Ba  10.4.2  Asynchronous release of transmitter  and LTP  81  Effects of released endogenous substances in the hippocampus  82  10.5.1 Collection of endogenous substances  82  10.5.2  10.5.3  Guinea pig hippocampal samples and LTP production  84  Rabbit neocortical samples and LTP production  85  CHIRWA Chapter  Title 10.6  10.7  11.  xi  Page No.  Effects of rabbit neocortical samples on cultured PC-12 cells  86  10.6.1 PC-12 cell growth  86  10.6.2  Preparation of PC-12 cells feeding media  87  10.6.3  Neurite induction  87  Studies on the possible mechanisms of action of saccharin  89  10.7.1 General  89  10.7.2  Dose-response curves  89  10.7.3  Saccharin and electrical  properties  of neurons  90  10.7.4  Saccharin and LTP  90  10.7.5  Saccharin and post-tetanic potentiation  91  10.7.6  Saccharin and presynaptic excitability  91  10.7.7  Saccharin and paired-pulse facilitation  92  10.7.8  Saccharin and NMDLA responses  93  10.8  Effects of exogenous NGF in the hippocampus  94  10.9  Data analysis  94  RESULTS 11.1  11.2  96  Recordings in CA^ field of the hippocampus  96  11.1.1  Features of intracellular  96  11.1.2  Miniature postsynaptic potentials  11.1.3  Recordings with C s electrodes  101  11.1.4  Features of extracellular  104  b  recordings  +  Saccharin dose-response curves  responses  98  106  CHIRWA Chapter  Title  xii  Page No.  11.3  Effects of barium in the hippocampus  109  11.4  Induction of long-term potentiation  111  11.4.1 Tetanic stimulations  111  11.4.2  114  Paired depolarizations  11.5  Asynchronous release of transmitter and LTP  116  11.6  Endogenous substances and synaptic potentiation  121  11.7  Effects of rabbit neocortical samples on cultured PC-12 cells  127  11.7.1  127  PC-12 cell growth and neurite induction  11.8  Effects of exogenous N6F in the hippocampus  11.9  Mechanisms of action of saccharin: Extracellular studies  129  134  11.9.1  Saccharin and LTP:  Dose-relationships  11.9.2  Saccharin and post-tetanic potentiation  136  11.9.3  Saccharin and paired-pulse facilitation  137  11.9.4  Saccharin and dendritic negative wave during tetanus  11.10 Mechanisms of action of saccharin:  134  140 Intracellular  studies  141  11.10.1 Effects of saccharin on spontaneous and evoked responses  141  11.10.2 Saccharin effects on presynaptic terminal excitability  143  11.10.3 Saccharin and NMDLA responses  145  CHIRWA Chapter 12.  Title  xi i i Page No.  DISCUSSION  148  12.1  General  148  12.2  LTP in C A  12.3  K efflux and LTP  12.4  Feedback interactions  -  lb  150 150  +  152 I  j  12.5  minEPSPs and depolarizations in Ba  153  12.b  minEPSPs and LTP  155  12.7  Quantal transmission in hippocampus  158  12.8  Endogenous substances and LTP  160  12.9  Endogenous substances and neurite growth  162  12.10 LTP and neurite growth  163  12.11 Possible mechanisms of saccharin  166  12.12 Implications  167  in LTP  13.  CONCLUSION  170  14.  REFERENCES  172  LIST OF TABLES  Table 9-1  • Title Composition of media (in mM) used for hippocampal slices  11-1  Small discrete potentials in CA^ neurons in the guinea pig hippocampus in vitro  11-2  Changes in frequencies of evoked minEPSPs in CAi neurons during simultaneous pairings of conditioning depolarizing current injections into CAi neurons and stimulation of the stratum radiatum in guinea pig hippocampus in vitro D  D  11-3  Effects of samples collected from guinea pig hippocampus in vivo on CA^b population spike in guinea pig hippocampus in vitro  11-4  Effects of saccharin on post-tetanic potentiation of the population spike in the CAjb area induced by stimulation of the stratum radiatum in the guinea pig hippocampus in vitro  CHIRWA  xv  LIST OF FIGURES  Figure 2- 1  Title General morphology of the hippocampal formation  Page No. 10  3-1  The major afferent systems in the hippocampus  16  5-1  Representative evoked field responses in the hippocampus  28  Recording chamber and perfusion method for the maintainance of transversely sectioned guinea pig hippocampal slices  67  Positioning of small cups onto rabbit neocortical surface for collection of samples in vivo  72  Determination of cell input resistances with intracellular injections of graded hyperpolarizing current pulses into CA^b neurons  77  A schematic illustration of the positioning of stimulating and recording electrodes in the guinea pig hippocampus in vitro  79  Preparation and composition of the different types of feeding media used for incubation of rat adrenal pheochromocytoma (PC-12) cell cultures  88  Characteristic features of evoked intracellular potentials in the CA^ neurons of the guinea pig hippocampus in vitro  97  Intrasomatic recordings of spontaneous small discrete potentials in CA^-, neurons in the guinea pig hippocampus in vitro  100  The occurence of miniature EPSPs potentials in CAit> neurons in guinea pig hippocampal slices incubated in tetrodotoxin  102  Characteristic features of intracellular potentials in a CAib neuron recorded with micropipettes f i l l e d with Cs  103  Characteristic features of evoked field potentials recorded in the CA15 area following stimulation of the stratum radiatum in the guinea pig hippocampus in vitro  105  9-1  9-2 10-1  10-2  10- 3  11- 1  11-2  11-3  11-4  11-5  Title Dose-response curves of saccharin obtained by the method of single application, randomised design Changes in amplitudes of population spikes in CAit> area evoked by stimulation of the stratum radiatum during applications of different doses of saccharin in guinea pig hippocampus in vitro Intracellular potentials in C A neurons in guinea pig hippocampal slices incubated in barium lb  Illustration of long-term potentiation induced by high frequency tetanic stimulations of the stratum radiatum in guinea pig hippocampus in vitro Representative recordings of intracellular and extracellular potentials in CA^b area evoked by stimulation of the stratum radiatum before and after induction of long-term potentiation Illustration of long-term potentiation induced by simultaneous pairings of conditioning depolarizing current injections into CA^b neurons and stimulation of the stratum radiatum in guinea pig hippocampus in vitro Comparisons of the frequency of minEPSPs in CA^ neurons following stimulation of the stratum radiatum before, and after development of long-te potentiation in^uinea pig hippocampal slices incubated in Ba Representative changes in evoked intracellular responses in a CAi neuron in guinea pig hippocampal slices incubated for 2 min in barium D  Illustration of long-term potentiation in C A ^ area of the guinea pig hippocampus in vitro induced with brief applications of samples collected during tetanic stimulations of the guinea pig hippocampus in vivo Effects of samples collected from rabbit neocortex in vivo on CA^b population spike in guinea pig hippocampus in vitro  Title Failure to induce long-term potentiation in in guinea pig hippocampus in vitro when samples collected during tetanic stimuI ation of the rabbit neocortex in vivo are applied in the presence of saccharin The blockade of tetanus-induced long-term potentiation by saccharin in the guinea pig hippocampus in vitro Effects of samples collected from rabbit neocortex in vivo on neurite growths in PC-12 cell cultures Effects of NGF (Vipera lebetina) on weak EPSP recorded in the CA15 dendritic region in guinea pig hippocampus in vitro Effects of different concentrations of saccharin on LTP production in the CA^b area of the guinea pig hippocampus in vitro Demonstration of lack of effects of saccharin on paired-pulse facilitation in the CA^ of the guinea pig hippocampus in vitro a r e a  Effects of saccharin on the extracellular negativ wave in CAi|~, apical dendrites induced during tetanic stimulations of the stratum radiatum in the guinea pig hippocampus in vitro Examination of the electrophysiological effects of saccharin in the guinea pig hippocampus in vivo Effects of saccharin on the intracellular depolarizations in CA15 neurons induced by NMDLA applications in the guinea pig hippocampus in vitro  CHIRwA  xvi i i  ABBREVIATIONS AP5 AP7 APV ACh AHP ATP CA cAMP cGMP DG DRG EMP EPP EPSP FANFT GABA GAD HTHS HTNS IPSP KA KC1 LTP MEPP MCD minEPSP minlPSP NAD NADP NMDA NMDLA NGF NMJ PC-12 PKC PP PS PTP PW RMP n Sch SKF10047 STP THS TNS UHS UNS R  D-2-amino-phosphonovalerate D-2-ami no-phosphonoheptanoate D-2-amino-phosphonovalerate Acetylcholine Afterhyperpolarization Adenosine triphosphate Cornu ammonis. Aaenosine 3':5'-cyclic phosphate Guanosine 3':5'-cyclic phosphate Dentate gyrus Dorsal root ganglion Embden-Meyerhof-Parnas pathway End-plate potential Excitatory postsynaptic potential N-[-4-(5-nitro-2-furyl)-2-thiazolyJ-formamide •y-aminobutyric acid Glutamic acid decarboxylase Heated-tetanised hippocampal sample Heatea-tetanised neocortical sample Inhibitory postsynaptic potential Potassium acetate electrode Potassium chloride electrode Long-term potentiation miniature end-plate potential Mast cell degranulating peptide miniature excitatory postsynaptic potential miniature inhibitory postsynaptic potential Nicotinamide-adenine dinucleotide Nicotinamide-adenine dinucleotide phosphate N-methyl-D-aspartate N-methyl-DL-aspartate nerve growth factor Neuro-muscular junction Rat adrenal pheochromocytoma cells Protein Kinase C Perforant pathway Population spike Post-tetanic potentiation Positive wave Resting membrane potential Input resistance Schaffer collaterals n-a1lynormetazoc i ne. Short-term potentiation Tetanised hippocampal sample Tetanised neocortical sample Untetanised hippocampal sample Untetanised neocortical sample  CHIRWA  xix  ACKNOWLEDGEMENTS I am d e e p l y i n d e b t e d t o my s u p e r v i s o r Dr. B h a g a v a t u l a R. S a s t r y f o r h i s encouragement, e m o t i o n a l s u p p o r t and academic  guidance.  My h e a r t f e l t  thanks  a r e due t o Mr. P a t r i c k May and Dr. Hermina M a r e t i c f o r t h e i r f r i e n d s h i p and c o l l a b o r a t i o n i n some o f t h e e x p e r i m e n t s  1 wish  in this thesis.  t o thank  d a n e l l e H a r r i s , E l a i n e Jan and M a r g a r e t Wong f o r t h e i r a s s i s t a n c e i n e d i t i n g and t y p e - s e t t i n g t h i s m a n u s c r i p t and L o u i s e Low f o r h e r h e l p i n t h e p r e p a r a tion of the Tables i n t h i s t h e s i s . These l a s t few y e a r s have been e x t r e m e l y r e w a r d i n g t o me, both c a l l y and s o c i a l l y . Pharmacology  I wish t o thank  ano T h e r a p e u t i c s ,  academi-  a l l t h e members o f t h e Department o f  particularly  D r s . David  Quastel,  Bernard  McLeod, E r n e s t P u i l , M o r l e y S u t t e r , and M i c h a e l Walker f o r t h e i r f r i e n d s h i p and c o n t r i b u t i o n s towards  t h e many l e a r n i n g e x p e r i e n c e s I have had h e r e .  My  e n d l e s s t h a n k s a r e r e s e r v e d f o r my f a m i l y ; l i i s e t s o , S a n i k a , and T h a b i s o ; my p a r e n t s ; b r o t h e r s and s i s t e r s : are second t o none.  Your s e l f l e s s  love, support  ano s a c r i f i c e s  I have never known a g r e a t e r p a i n t h a n I have had b e i n g  away from y o u a l l . S o r r y f o r my n o t b e i n g t h e r e , d u r i n g a l l y o u r t i m e s o f need. The f i n a n c i a l s u p p o r t s o f Graduate the Medical Research  Student  Research  A s s i s t a n t s h i p from  C o u n c i l (Canada) and World H e a l t h O r g a n i z a t i o n F e l l o w -  ship are g r e a t l y appreciated.  CHIRWA Dedicated to my f i r s t loves: Tiisetso and Sanika Jr. and Thabiso It would mean nothing without you  xx  CHIRWA 1.  1  INTRODUCTION An intriguing  aspect of  brain  function  is  how learning  operations are accomplished, at the cellular level.  and memory  Hebb (1949) postulated  changes in synaptic weightings, as a consequence of use-dependent activities in neural  networks.  It  was hypothesized that  simultaneous activities  in  pre- and postsynaptic neurons led to synaptic modifications for learning and memory (Hebb, 1949).  In recent years, the phenomenon of long-term potenti-  ation (LTP) that has been extensively studied in the mammalian hippocampus is taken to be consistent with the concept of the "Hebbian synapse" and, therefore,  provides a potential  and Wigstrom, 1988;  model for  learning and memory (Gustafsson  Kelso, Ganong and Brown, 1986).  LTP in the hippocampus  is described as an input specific increase in synaptic efficacy brief  tetanic  stimulations of the input  Bliss and Gardner-toedwin,  1973;  and in vitro:  Schwartzkroin and Wester, 1975). to  (in vivo:  The tetanic  following  Bliss and L*)mo, 1973;  Alger and Teyler, stimulation  induce LTP include those frequencies that occur  (Larson and Lynch, 1986; Rose and Dunwiddie, 1986).  1976;  frequencies used  in normal physiology The increase in synap-  tic efficacy lasts for several minutes to hours in vitro, or days to weeks in vivo  (Barnes,  1979;  Swanson,  Teyler  and  Thompson, 1983).  LTP  is  observed as decreases in onset latencies and/or increases in amplitudes of the evoked field responses. increased probability  With intracellular  in cell  EPSPs (Andersen, et a l . , 1980c;  recordings, LTP is seen as  discharges or enhancements of subthreshold Schwartzkroin and Wester, 1975).  Using transversely sectioned hippocampal slices, different experimental methods can be used to e l i c i t LTP. conditioning intracellular  For example, brief pairings of adequate  postsynaptic depolarizations with low frequency  CHIRWA activation of afferents induces LTP (Sastry, Goh and Auyeung, 1986). lasting synaptic potentiations the following:  also occur following transient  raised extracellular  1982), mast cell  Ca  (Turner,  2 Long-  exposures to  Baimbridge and Miller,  degranulating peptides from bee venom (Cherubini  et a l . ,  1987), phorbol analogs that activate protein kinase C (Madison, Malenka and Nicoll, 1986), and raised extracellular Ca  (May, Goh and Sastry, 1987).  ++  in  addition  to  increases  in  K  in the absence of  +  extracellular  During LTP several changes are observed  synaptic  efficacy.  These changes  release of newly synthesized proteins during tetanic  include  stimulations  (Duffy,  i  Teyler  and Shashoua, 1981),  1984;  Greenough, 1984;  synaptic  re-modeling  Desmond and Levy, 1981;  (Chang  and Greenough,  Fifkova and Van Harreveld,  1977), increased release of glutamate and aspartate, the putative ters  (Bliss,  et a l . ,  1986;  Skrede  and  decreased presynaptic terminal excitability whether  the above changes reflect  Malthe-Stf)renssen, (Sastry, 1982).  mechanisms mediating  transmit-  1981),  It  and  is not known  the expression of  LTP, or if they represent secondary changes associated with the phenomenon. Furthermore, proteins factor(s) LTP.  the  identities  and physiological  have not been established. control morphological  In  functions  addition,  alterations  of  it  of  is  the  not  released  clear what  synaptic structures  during  In fact, the exact mechanisms underlying the change(s) leading to this  synaptic  potentiation  still  remain  to  be  determined  (Gustafsson  and  Wigstrom, 1988). A popular hypothesis that has received much attention in recent years advocates the involvement of transmitter and voltage regulated NMDA receptor channels.  Briefly,  both  NMDA and non-NMDA receptors  are  thought  to be  present, for example, at the Schaffer collateral-commissural/CA^ pyramidal cell  synapses in the hippocampus (Collingridge,  Kehl and McLennan, 1983;  CHIRwA Harris, Ganong and Cotman, 1984; The  putative  transmitter,  non-NMDA receptors  3  Wigstrom and Gustafsson, 1984 and 1986).  glutamate,  is  an  agonist  (Mayer and Westbrook, 1987;  for  both  NMDA and  Watkins and Evans, 1981).  Presumably, non-NMDA receptor channels mediate the fast EPSPs, whereas NMDA receptor channels are blocked by Mg  and do not contribute  significantly  to low frequency synaptic transmission (Col 1ingridge, 1985; 1984;  Wigstrom and Gustafsson, 1985a).  stimulations  (or  blockade  NMDA receptor  them.  of It  has  adequate  been  However, it is thought that tetanic  postsynaptic channels,  postulated  Nowak, et a l • ,  depolarizations) resulting  that  these  in  Ca  remove ++  influx  postsynaptic  Ca  subsequently mediate secondary changes leading to long-lasting of  the  1988).  synaptic responses (Collingridge, The NMDA hypothesis  is  1985;  the Mg through influxes  potentiation  Gustafsson and Wigstrom,  based on experimental  data  showing that  amino-phosphonovalerate (APV) a presumed competitive antagonist at the NMDA receptors, blocks the induction but not the maintenance of LTP (Col 1ingridge, Kehl and McLennan, 1983; Gustafsson, 1984). extracellular  Ca  ++  Harris,  Ganong and Cotman, 1984;  Wigstrom and  Interestingly, APV also blocks LTP inducea by raised (Errington,  Lynch and Bliss,  1987), mast cell  degranu-  lating peptides from bee venom (Cherubini, et a l . , 1987), and paired presynaptic Nicoll,  ana  conditioning  1988).  postsynaptic  One intriguing  depolarizations  (Kauer,  Malenka  ana  feature concerning LTP expression is that  presynaptic mechanisms seem to be involved in the maintenance of this phenomenon (Sastry,  1982;  Teyler  and DiScenna, 1987).  Consistent with  this  notion are studies that have shown that LTP is associated with sustained increases  in the  et a l . , 1986;  release  of  glutamate,  the  putative  Skrede and Malthe-S^renssen, 1981).  transmitter  (Bliss,  How a process that is  generated via postsynaptic mechanisms is subsequently sustained by presynap-  CHIKWA  4  tic mechanisms has not been investigated. The studies reported tions.  If  in this thesis arose from the following predic-  LTP induction is postsynaptic but its maintenaince is presynaptic  (Bliss and Lynch, 1988), this suggests that during LTP production feedback interactions occur between postsynaptic and presynaptic regions (cf. 1983;  Sastry, Goh and Auyeung, 1986).  Eccles,  Consequently, studies presented in  this thesis have examined these interactions through the analysis of changes in  frequencies  of  evoked  miniature  excitatory  postsynaptic  (minEPSP) in the guinea pig hippocampus in vitro. medium containing low Ca transmitters  potentials  In these studies,  Ba  ++  was used to induce the asynchronous release of  (cf. Silinsky, 1978), and this was observed as increases in the  frequencies of evoked minEPSPs in CA^ neurons following stimulation of the stratum radiatum. method for  The assessment of evoked minEPSPs provided a  examining  presynaptic  functions.  In  this  regard,  potential  changes  in  evoked minEPSP frequencies were used to assess directly increases in transmitter release during LTP.  This was done for the following basic reason.  The data in the literature showing sustained increases in release of transmitters during LTP are based on biochemical assays of glutamate, the excitatory  transmitter  candidates  increased glutamate associated  with  release  metabolic  in  the  hippocampus.  could be due to processes  that  It  is  conceivable  enhanced glutamate  are  unrelated  to  that  turnover  transmitter  release per se. The major thrust of the studies, however, have been centered on determining some of the physiological effects of the substances that are released during tetanic stimulations.  These studies stemmed from the prediction that  proteins that are released during tetanic  stimulations  Shashoua, 1981), may be involved in LTP development.  (Duffy,  Teyler and  Consistent with this  CHIRWA  5  notion, for example, are reports in the literature showing that substances that  inhibit  protein  Sarvey, 1984).  synthesis  also  block  LTP production  (Stanton  and  The studies reported in this thesis involved the collection  of samples during tetanic stimulations of the guinea pig hippocampus in vivo and rabbit neocortex in vivo, and subsequently applying them onto guinea pig hippocampus in vitro.  Collections from the rabbit  used for the following reasons, made it  possible to  neocortex in vivo were  lhe bigger surfaces of the rabbit neocortex  use relatively  larger  collecting cups.  More impor-  tantly, LTP has been shown to occur in the mammalian neocortex (Lee, 1982). Related studies involved testing the effects of the samples collected during tetanic stimulations on neurite growth in cell cultures. performing  these  experiments  arose  from  the  The rationale for  following  observations.  Proteins that are known to be released during tetanus-induced LTP present with molecular weights ranging between 14-86 kD (Duffy, Teyler and Shashoua, 1981;  Hesse, Hofstein ano Shashoua, 1984), and this is strikingly similar  to the molecular weights of nerve growth related substances (Berg, 1984;  factors  Wagner, 1986).  (NGF)  and other  Interestingly,  growth  the hippo-  campus and neocortex have the highest levels of NGF messenger RNA in the brain (Korsching et a l . , 1985;  Whittemore, et a l • , 1986). . Furthermore, NGF  presumably is essential  for  occurs following  in the hippocampus (Crutcher  injury  Hendry et a l . , 1974; 1985). is  nerve cell  survival  and axon sprouting that  Nietro-Sampedro and Cotman, 1985;  and Collins,  Springer and Loy,  Since LTP is associated with structural alterations  conceivable that these structural  relatives of NGF.  If  the released proteins during tetanic macromolecules.  in synapses,  changes could be mediated  this is the case, then it stimulations  1986;  it  by close  raises the prospects that could be growth  related  Hence experiments were conducted to test the above ideas as  CHIRWA follows.  Briefly,  primary  (denoted as PC-12 cells)  tissue cultures  of  rat  6  pheochromocytoma cells  develop extensive neurite  networks when incubated  in growth medium containing NGF or related substances (Greene and Tischler, 1976).  Hence, PC-12 cells cultures were incubated in growth medium contain-  ing samples collected during tetanic saccharin was added to Saccharin is known to  the  growth  inhibit  stimulations.  ln  medium containing  NGF "receptor"  dorsal  root  ganglia  cell  cultures  the  above samples.  binding in a dose-dependent  fashion, and the drug decreases NGF-dependent neurite chick  some experiments,  (lshii,  growth  1982).  in embryonic Several  other  experiments examined, for example, the physiological effects of saccharin in the guinea pig hippocampus in vitro.  A few studies examined the effects of  applied NGF on synaptic transmission in the CA^ area. Taken together, the results of the studies in this thesis were found to be consistent with the following predictions: tions  modulate  apparent  presynaptic  increase  activities;  in transmitter  (2)  release;  (1)  Postsynaptic depolariza-  LTP was (3)  associated  with  Substances released  an  during  tetanic stimulations could e l i c i t LTP, thereby indicating that they could be involved in the production of this  phenomenon; and (4)  The above samples  contained neurite inducing factors, thereby indicating that these endogenous substances could be growth related macromolecules. that saccharin blocked both the neurite  induction of  growth in PC-12 cell cultures  Furthermore,  LTP in the  it seemed  hippocampus and  induced by samples collected during  tetanic stimulation of the rabbit neocortex via common mechanisms shared by these two processes.  The concentration of saccharin (10 mM) used in these  studies did not exert significant effects ties,  synaptic  transmission  or  on C A  NMDA receptor  lb  cell electrical  mediated  These results suggested that saccharin blocked the  proper-  depolarizations.  induction of LTP at a  CHIRWA step beyond NMDA receptor activation. this  thesis  raise  the  prospect  In summary, the results presented in  that  involved in the production of LTP.  7  growth  related  The above ideas,  substances may be and their  implications, are further developed in the discussion sections.  inherent  CHIRWA 2.  8  BASIC MORPHOLOGY OF THE HIPPOCAMPAL FORMATION  2.1  General A detailed knowledge of the neural networks involved in the generation  of responses is useful for any meaningful logical signals.  interpretation  of electrophysio-  For these reasons, comprehensive accounts of the hippo-  campal morphology and physiology will present  chapter  will  endeavour  to  features  of the hippocampus that will  presented in subsequent chapters.  be discussed illustrate  in this  the  basic  thesis.  The  morphological  have a bearing on the  discussions  The descriptive morphological terms used  here are consistent with the nomenclatures employed by Blackstad (1956), Cajal (1911) and Lorente De No (1934).  Wherever appropriate, sections have  been updated based on recent detailed anatomic studies (e.g. 1984; 2.2  Swanson, Wyss ana Cowan, 1978;  Schwerdtfeger,  White, 1959).  The hippocampal region During ontogenic development, the cortical  the allocortex and the isocortex. neocortex, and it cortical mantle.  mantle  is subdivided into  The isocortex is commonly known as the  is a homogenous unit that separates completely from the The allocortex consists of the archicortex and the palae-  cortex, and these are heterogenous units that remain attached to the c o r t i cal mantle (Filimonoff,  1947).  A separate region denoted as the periallo-  cortex is situated between the allocortex and the isocortex.  The periallo-  cortex gives rise to structures such as the peripalaeocortical entorhinal, cortices  presubicular,  (Blackstad, 1956;  Lorente De No, 1934;  retrosplenal  and  Brodmann, 1909;  Sanides, 1972;  White,  periarchicortical  claustral, cingulate  Chronister and White, 1959;  Vaz Ferreira,  1975; 1951).  The palaecortex gives rise to the olfactory bulb, accessory bulb, retrobul-  CHIRwA bal region, periamygdalar region, the olfactory  tubercle,  the septum, the  diagonal region and prepiriform region (Pribram and MacLean, 1953; 1972;  Schwerdtfeger, 1984).  9  Sanides,  The archicortex is comprised of the subiculum,  Ammon's horn, fascia dentata precommissural hippocampus and supracommissural hippocampus Cowan, 1978;  (Blackstad,  1956;  Lorente  De No,  Teyler and Discenna, 1984).  1934;  Swanson, Wyss and  Hence, the term "archicortex" is  synonymous with "hippocampus proper" or simply "hippocampus" (Schwerdtfeger, 1984; 2.3  Teyler and DiScenna, 1984). The hippocampus The hippocampus is seen as a curved elongated ridge that is  along the floor of the descending horn of each lateral ventricle.  situated A trans-  verse section of the hippocampus reveals two distinct interdigitating termed cornu ammonis and dentate gyrus (Figure 2-1). contain densely packed sheets of c e l l s . hippocampus are the  pyramidal  cells  Each of these fields  The predominant cell types of the  in the cornu ammonis field  granular cells in the dentate gyrus f i e l d . cells are distributed in both fields  fields  and the  However, several other types of  (Amaral, 1978;  Cajal, 1893;  Lorente  De No, ly34). 2.4  The dentate gyrus The dentate  The blade  (or  side) of the curvature that is adjacent to the subiculum (Figure 2-1)  is  termed as the suprapyramidal blade.  is  the  gyrus field  intraventricular  is curved into  localised  The infrapyramidal blade, therefore,  part of the curvature  Swanson, Wyss and Cowan, 1978). in a single  layer  a "V" shape.  (Chronister  and white,  1975;  The granular cells of the dentate gyrus are  termed the  stratum granulosum.  These cells  possess apical dendrites that project into the stratum moleculare layer that is situated between the stratum granulosum and the pial' surface within the  CHIRWA  10  Figure 2-1. General morphology of the hippocampal formation. The stratum py rami dales (str. pyr.) layer of the cornu ammonis fC~A~) field and the stratum granulosun (str. gran.) layer of the dentate gyrus (DG) field contain densely packed sheets of cells which are mostly neuronal bodies of pyramidal cells and granule cells, respectively. Several other types of neurons are found in these layers as well as the subiculum (SUB) and the hilus regions. Typically, the axonal and dendritic processes are distributed in the following layers: stratum oriens (str. o r . ) , stratum radiatum (str. rad.), stratum lacunosum (str. l a c ) , and stratum moleculares (str. mol.). The axons of CA^ pyramidal cells course through the alveus and exit out via the subiculum or the fimbria (FIM). [ f i s s . denotes fissure].  CHIRWA hippocampal fissure (Figure 2-1). found several  2.5  Inside the dentate gyrus curvatures are  layers of polymorphic cells that make up the  (Amaral, 1978;  11  hilar  region  Lorente De No, 1934).  The hilus and CA region f|  Within the dentate gyrus concavity and close to its apex is the hilus. The hilus is extremely variable malian brain, it  in appearance across species.  is least developed in rodents but increases in complexity  in the rabbit, monkey and man (Lorente De No, 1934). whether  the  (Blackstad,  In the mam-  hilus 1956;  constitutes Cajal,  1911;  the  third  layer  Opinions are split on of  the  Lorente De No, 1934).  caudal end of the cornu ammonis extends into the hilus.  dentate In  rodents,  Swanson, Wyss and Cowan, 1978).  Several  cell  types  Cajal, 1893;  Lorente De No, 1934).  (Amaral,  (e.g.  c e l l s , modified pyramidal cells) have been identified in the hilus, 1978;  the  Yet the boundaries  between the cornu ammonis and the hilus are not readily discernible 1978;  gyrus  basket (Amaral,  Some of the cell types in the  hilus are similar to those found in the stratum oriens layer of the cornu ammonis f i e l d . structural  In view of the above, the hilar  region is taken to be a  transition zone between the dentate gyrus and the cornu ammonis  (Lorente De No, 1934).  As proposed by Lorente De No (1934), the modified  pyramidal cells that stream out of the hilar region make up the CA region. 4  2.6  The cornu ammonis field The rostral end of the cornu ammonis (CA) merges with the subiculum.  The caudal part forms a continuum with the outer section of the CA^-hilar region described above (Lorente  De No, 1934;  cornu ammonis field has a curved cell In  addition,  the  Schwerdtfeger,  1984).  The  layer termed the stratum pyramidale  (Figure  2-1).  cornu ammonis field  has  the  layers.  The alveus, lies next to the epithelium of the lateral  following ventricle,  CHIRWA and this layer marks the outer boundary of this f i e l d . is  situated between the alveus and the  stratum pyramidale,  stratum  The stratum oriens  pyramidale.  but on the opposite side to the  12  Next to  the  stratum oriens,  are  found the stratum lucidum, stratum radiatum, stratum lacunosum and stratum moleculare, in that order (Figure 2-1). In vertebrates,  the extent of development of different  hippocampus is most varied in the cornu ammonis f i e l d . example, the  division between  somewhat a r t i f i c i a l .  stratum  radiatum  regions of the In  and stratum  (Lorente  for  lacunosum is  Consequently, in these animals, the stratum radiatum  and stratum lacunosum are often described as a single layer, radiatum  rodents,  De  No,  1934).  Similarly,  the  considered to be part of the stratum pyramidale layer.  stratum  i.e.  stratum  lucidum  is  CHIRWA 3.  13  CELLULAR PROPERTIES AND INTRINSIC CIRCUITRY  3.1  Dentate gyrus granule cells Granule cells are the predominant  layer. ovoid  These neurons are cell  bodies  Matthysse, 1983).  which  highly are  neurons in the  polar,  about  20  A short stem dendrite  and they by  stratum  possess  13 pm in  characteristic  size  emerges from the  granulosum  (Williams apical  pole of  each granule cell and extends into the stratum moleculare where it cates repeatedly 1934;  (Cajal, 1911;  Lindsay and Scheibel, 1976;  Williams and Matthysse, 1983).  with various types of spines.  The dendritic  and  bifur-  Lorente De No,  branches are covered  In general, spines are confined to segments  beyond the f i r s t branch of the stem dendrites. The granule cells possess thick axons, termed mossy fibers, which originate  from  the  basal  (Blackstad, et a l . ,  pole  1970).  and extend  transversely  Each mossy fiber  across  subfield CA^  has a distinct  axon hillock  situated at the basal tip of the soma (Williams and Matthysse, 1983).  Mossy  fibers are highly laminated and they form synaptic contacts with spines of proximal cells  dendrites  in the  cells.  infrapyramidal  across the  cells.  The mossy fibers  dentate gyrus blade  Granule cells in the  mossy fibers 1974;  of CA^ pyramidal  suprapyramidal  entire  CA^ field  Swanson, Wyss and Cowan, ly78).  1961)  CA  3c  granule pyramidal  dentate gyrus blade send out (Lorente  De No,  A small fraction  form synapses with some of the polymorphic cells (Blackstad and Kjaerheim,  innervate  of  and interneurons  1934;  Haug,  of mossy fibers  in the hilar/CA^ region in field  CA^ (Frotscher,  1985; Tombol, et a l . , 1979). 3.2  Cornu ammonis pyramidal neurons Pyramidal cells are the predominant neurons in the stratum pyramidale.  CHIRWA The somata of a pyramidal cell is pear-shaped, and it the long axis is vertical  to the alvear surface.  size is about 40 by 20 pm. body sizes.  The rostral  is oriented such that  On average the cell body  However, pyramidal cells exhibit a range of cell cornu ammonis has the smallest pyramidal  whereas the caudal cornu ammonis is endowed with the pyramidal  cells  structural  have  apical  differences  14  and  basal  largest  dendrites.  among pyramidal  cells  There  cells,  cells.  are  All  important  such as dendritic  profiles  and/or axonal ramifications.  Lorente De No (1934) used these morphological  differences  cells  various  among pyramidal  subfields,  Cajal, 1911; 3.2.1  namely  Golgi, 1886;  CA,  ,  delineate CA ,  The stratum  as a diffuse  region  of  the  CA~  9  Schaffer, 1892;  Subfield CA^.  ammonis starts  to  cornu and  ammonis  CA„  (see  also  in the rostral  cornu  Blackstad, 1956). pyramidales  mixed cells  before  it  densely packed single layer consisting primarily of pyramidal initial  part of  the  stratum  pyramidales  is  denoted  comprised of a mixed primary cell population (i.e to the subiculum: the  Schaffer  border  Lorente De No, 1934).  collaterals  between  CA,,  of  and  lb  c  The  CA,,  of  the  CA^ area,  except  This  and it  is  contains cells belonging  cells  contains  cease  much  at  the  smaller  but  lb  of the CA-^ subfield.  area is marked by the presence of  those  cells.  According to Lorente De No (1934),  la  similar pyramidal c e l l s , representative CA^  becomes a  as CA, , ia  CA^ and CA^ pyramidal  CA, .  into  that  small the  pyramidal  dendrites  The onset of  cells similar  to  of  CA^  pyramidal  cells  start  from  c  cells are smooth, with numerous side branches. In  general,  basal  dendrites  of  CA-^ pyramidal  the  soma in a bush-like fashion, with irregular branches that divide repeatedly in the stratum oriens.  The apical  dendrites  extend out  into the  radiatum for some distance before they begin to branch extensively.  stratum Both  CHIRWA  15  apical and basal dendritic branches are covered with various types of spines (Andersen, et a l . ,  1980a;  Scheibel and Scheibel, 1968).  The CA pyra1  midal cells have axons that arise from the basal side and branch out in the stratum oriens. ramify  in the  Some axonal  branches cross  the  stratum radiatum or distribute  stratum  pyramidale and  back to CA-^ and  pyra-  midal layers, where they presumably make synaptic contacts with interneurons (Lorente De No, 1934).  Most of the axonal collaterals descend down to the  alveus, where they form pathways in both directions and leave the hippocampus via the fimbria or the subiculum.  The CA^ axons project  hippocampus to other brain regions such as the prefrontal  cortex  (Swanson, 1981;  lateral  out of the  septal nuclei and  Swanson and Cowan, ly77).  None of the  axonal branches of CA^ pyramids make contact with the pyramidal cells in CA^ and CA^, or the dentate gyrus granule cells (Lorente De No, 1934). 3.2.2  Subfield  CA^.  The  CA-^  pyramidal  cells  possess  large  apical dendrites that cross the stratum radiatum and only begin to branch when they reach the stratum moleculare (Lorente De No, 1934).  In contrast,  the basal dendrites of CA^ pyramidal cells begin to branch early, traverse the stratum oriens.  Both the basal dendrites and initial  as they parts of  the apical dendrites are endowed with numerous thick spines. The pyramidal cells in CA subfields have thick axons that originate 3  from their  basal poles.  These axons cross the stratum oriens and travel  towards the fimbria and leave the hippocampus (Figure 3-1).  In addition,  these axons give off several collaterals which terminate within the stratum oriens or in the CA^ pyramidal cell neurons;  Lorente De No, 1934).  and travel 1934).  within the  stratum  layer  (presumably to innervate  inter-  Some collaterals cross the CA~ cell radiatum/lacunosum layers  (Lorente  layer  De No,  These collaterals are myelinated, and they constitute a horizontal  CHIRWA  16  Figure 3-1. The major afferent systems in the hippocampus. The granule cells in the dentate gyrus give out mossy fibers (mf) that innervate CA3 pyramidal c e l l s . The axons of CA3 pyramidal cells have thick axons, some of which branch and give rise to the Schaffer collaterals (Sch) that innervate CAj pyramidal c e l l s . The commissural input (com) represents afferents from the contralateral hippocampus. Note the perforant path (pp) which is a major extrinsic input into the hippocampus originating from the entorhinal cortex and innervating the granule cells of the dentate gyrus.  CHIRWA fiber  system termed Schaffer collaterals  which innervate  the  17  apical den-  drites of CA^ and CA^ c e l l s . C  Some CA  pyramidal  3  this  feature  subfield. erals. fibers  cells do not  is one criterion  that  give off  Schaffer  has been used to  collaterals ana  subaivide the CA^  CA^ pyramidal cells have axons that give out Schaffer collatFurthermore,  of  (Lorente  both  the  CA.  pyramidal  infrapyramidal  De No, 1934).  cells and the  In contrast,  CA  3b  are  innervated  suprapyramidal and CA  by the mossy granule  pyramidal  3fl  cells  cells  only innervated by the mossy fibers of the suprapyramidal granule The CA.^ area consists of mixed pyramidal  cells,  of  CA  do not have Schaffer  3a  collaterals  (Lorente  Instead, most of the CA.^ pyramidal cells have thick one or two myelinated collaterals.  cells.  ana presumably 50% of  these cells have Schaffer collaterals (Lorente De No, 1934). cells  are  The pyramidal De No,  1934).  axons that give off  These collaterals ascena to the stratum  radiatum where they form an associational pathway running within the stratum radiatum of the CA^ and CA^ subfields. b  3.2.3  Subfield CAg.  The CA subfield is rather 2  considered to be a transitional No', 1934;  small, and it  is  area, between CA^ ana CA.^ (Lorente De c  Schwerdtfeger, 1984).  The CA^ pyramidal cells are large  (i.e.,  similar to those of CA^), but their dendrites do not possess thick spines (Lorente  De No,  1934;  Haug,  1974;  Swanson,  Wyss and  Cowan,  1978).  Furthermore, the apical dendrites of CA^ pyramidal cells start to divide into branches within the stratum radiatum, shortly after body layer.  leaving the cell  The axons of CA^ pyramidal cells do not give Schaffer collat-  erals, but they possess thick collaterals that cross the cell body layer and enter  the  reaching  stratum radiatum where they constitute up to  the  CA  lh  region.  In  addition,  a strong axial the  CA  ?  pathway  pyramidal  cell  CHIRWA axons form horizontal  collaterals  which travel within the  18  stratum oriens,  towards the subiculum and entorhinal cortex. 3.3  CA^ and Hilus neurons The CA^ starts as a densely packed cell  layer which begins to scatter  towards the region bordering the dentate gyrus suprapyramidal cells  in this  latter  section appear  "unaligned",  ammonis layer had folded back on itself dendrites of CA^ pyramidal  as if  the  blade.  caudal cornu  (Lorente De No, 1934).  cells appose the  infrapyramidal  The  The basal  granule  cells.  Their apical dendrites, however, tend to branch within the region, or give branches that travel along the suprapyramidal blade to reach the molecular layer.  The CA^ pyramidal cell dendrites make synaptic contacts with mossy  fibers.  However, it  is unclear what other afferents make synaptic contacts  with the dendritic branches that extend into the molecular layer. of  CA^ pyramidal  cells  innervate  the  fields (bottlieb and Cowan, 1973). with CA^ cell layers.  dendrites  in the  stratum  oriens  These axons also have collaterals  sural projection to the contralateral CA^ cell  dendrites,  and contralateral  CA^  These fibers establish synaptic contacts and the  In addition, the axons of CA^ pyramidal  collaterals.  1981;  ipsilateral  The axons  mostly in the  stratum  radiatum  cells give out  Schaffer  that constitute  a commis-  CA^ subfield where they synapse with  stratum oriens  (Laurberg and Sorensen,  is  devoid  Schaffer, 1892). The  apex  zone of  the  hilus  essentially  consists mostly of mossy fiber bundles. polymorphic layer The rest of  the  is  comprised of  throughout the region (Amaral, 1978).  neurons  and  This small zone is taken to be the  of the dentate gyrus hilus  of  (Amaral,  1978;  diverse cells  Blackstad, 1956). that  are dispersed  These cells exhibit complex dendritic  profiles, much like those seen in CA..  The axons from some of the  hilar  CHIRWA  19  cells approach the granule cells and ramify within the stratum granulosum. Some axonal collaterals travel within the  inner third  along the suprapyramidal blade and terminate  of the dentate  gyrus molecular  layer.  Yet other  axonal collaterals form a commissural pathway that travels to the molecular layer  of  the  contralateral  dentate  gyrus f i e l d .  It  is  not  known with  certainty whether hilar projections terminate only on granule c e l l s , or  if  these inputs make synaptic contacts with interneurons in the stratum granulosum (Swanson, Sawchenko and Cowan, 1981). 3.4  Interneurons The pyramidal cells in the cornu ammonis and the granule cells in the  dentate  gyrus make up 96-98% of the neuropil  1984).  However,  a variety  regions (Cajal, 1911;  of  interneurons  in the hippocampus (Buzsaki, are  Lorente De No', 1934).  distributed  within  these  These interneurons have been  classified according to their morphological characteristics, e.g. pyramidal, horizontal, fusiform, inverted fusiform, multipolar and basket cells (Ribak and Seress, 1983;  Buzsaki, 1984).  Interneurons  provide the pathways for  inhibition (or modulation) of the principal cells in each hippocampal field (Andersen, Eccles and L i n i n g , 1963 and 1964; 1961;  Storm-Mathisen,  Ribak, 1984). the  Though it  overwhelming  inhibition  1977;  Vaugh and Saito,  evidence presently  implicates  Seress and excitatory,  as  mediating  Andersen, Eccles and L^yning, 1964;  Fox and  Finch, Nowlin and Babb, 1983;  interneurons  Turner and Schwartzkroin, 1980).  The basket cells are the best studied inhibitory hippocampus.  1978;  is likely that some interneurons are  (Andersen, 1975;  Ranck, 1981;  Ribak,  Kandel, Spencer and Brinley,  interneurons  These interneurons have spherical to triangular  measuring 50 by 50 nm on average (Andersen et a l . , 1969).  in the  cell bodies,  Each basket cell  gives out several dendrites that typically extend from the somata without  CHIRWA giving branches, but exhibit (Andersen, et a l . ,  1969).  spines (Amaral, 1978; and Saito, 1978).  "frequent  swellings like a string of pearls"  The dendrites of basket  Buzsaki, 1984;  20  cells have few or no  Ribak and Seress, 1983;  Ribak, Vaugh  In the cornu ammonis f i e l d , the somata of basket cells  are situated close to the pyramidal interneurons distribute  cell  bodies.  The dendrites of these  within the stratum oriens, or  stratum radiatum (Lorente De No, 1934).  ascend towaras  the  Ihe axons of basket cells are very  thin, but divide extensively, giving axonal terminals that form basket-like structures around the somata of pyramidal c e l l s .  Each basket cell  vates as many as 500 pyramidal cells (Anaersen, et a l . , 1969).  inner-  However, the  extent of basket cell axons distribution in the hippocampus is not known.  CHIRWA 4.  21  EXTRINSIC AFFERENTS TO THE HIPPOCAMPUS  4.1  Entorhinal-hippocampal inputs Afferents from the entorhinal cortices, termed perforant paths, consti-  tute the major  cortical  inputs  originates from layers 1-111 1974;  to  the  hippocampus.  of the entorhinal  Steward and Scoville, 1976).  cortex  The perforant (Segal  path  and Landis,  Cells from each of these regions send  out axons through the perforant path tnat distribute largely in the dentate gyrus molecular layers.  The projections of the perforant path run perpendi-  cular to the main axis of the hippocampus.  Hence, the lateral  entorhinal  cortex perforant path terminates in the outer one-third of the dentate gyrus molecular  layer  whereas  the  medial  entorhinal  cortical  perforant  paths  terminate in the middle one-third of this layer (Wyss, 1981). Axons dentate 1966;  in  the  perforant  gyrus granule cell  path  dendrites  Hjorth-Simonsen, 1973;  1976).  Since dentate  form synapses with the  gyrus  spines of  (Andersen, Holmquist and Voorhoeve,  Matthews, Cotman and Lynch, 1976; interneurons,  as well  as cells  in  Steward, CA^ and  hilus, have dendritic branches that extend into the molecular layer and Seress, 1983), it  However, detailed morphometric studies have not  yet confirmed such interactions.  In addition, the entorhinal cortex inner-  vates cells in CAp CA and subiculum fields 3  4.2  (Ribak  is likely that these dendritic projections are inner-  vated by perforant paths.  Steward, 1976;  the  (Gottlieb  and Cowan, 1972;  Witter, et a l . , 1988).  Septo-hippocampal inputs The septo-hippocampal pathways mainly originate from the nucleus of the  medial septum (Swanson, 1978).  However,  some septal-hippocampal pathways  start from the nuclei of the lateral septum.  Both pathways are topographi-  CHlkWA cally organized along the septo-temporal and Sieger, 1977).  22  axis of the hippocampus (Meibach  The medial septal pathway is thought to be non-cholin-  ergic (Amaral and Kurz, 1985;  Wainer, et a l . , 1985), and it  projects via  the medial aspect of the fornix body before reaching the hippocampus (Lewis and Shute, 1967). (Amaral  The lateral septal area is considered to be cholinergic  and Kurz, 1985;  Lewis and Shute,  Wainer, et a l . , 1985), and it  1967;  Nyakas, et a l . ,  projects through the lateral  1987;  portion of the  fornix column via the fimbria and then enters the ventral hippocampus (Lewis and Shute,  1967).  Studies  using  intra-axonal  markers  or  histochemical  methods have demonstrated that the septal-hippocampal pathways terminate the cornu ammonis and the dentate gyrus fields (Nyakas, et a l . , 1987). the  exact  target  cells  in  these hippocampal  fields  still  remain  in But  to be  identified. 4.3  Miscellaneous inputs Other  following.  inputs  that project  to  the  hippocampal  The locus ceruleus projects noradrenergic afferents  Nicoll,  1982;  Swanson and Hartman, 1975)  et a l . ,  1980;  Pasquier and Reinoso-Suarez, 1978).  receives  formation  serotonergic  afferents  from the  include  (Madison and  to the CA^ and subiculum (Loy,  raphe  The hippocampus also  nuclei,  and dopaminergic  afferents from the substantia nigra and the ventral tegmental area 1977;  Scatton, et al • , 1980;  Segal, 1980).  the  (Iversen,  Morphometric studies have also  revealed projections to the hippocampus that originate from the thalamus and the hypothalamus.  The inputs from the thalamus terminate  subiculum, whereas inputs from the  hypothalamus terminate  gyrus and subiculum (Schwerdtfeger, 1984).  in the CA^ and in the  dentate  CHIRWA b.  23  ELECTROPHYSIOLOGY OF THE HIPPOCAMPUS  5.1  Electrical properties of neurons Many of the experimental  campal slice preparation, found to Spencer, 1961). tials  be strikingly 1961;  data have come from studies using the hippo-  but these results similar  to  values  obtained obtained  Kandel, Spencer and Brinley,  in vitro in vivo  1961;  have been (Kandel  and  Spencer and Kandel,  Hippocampal pyramidal and granule cells have resting membrane potenof minus 50-70 mV, on average.  from the follows  slopes of (reported  and 35-70,  for  et a l . , 1983;  the  current-voltage  as ranges,  dentate  Their  in  gyrus  Turner, 1982;  Mn):  (brown,  input resistances, calculated  relationships, 26-45,  for  Fricke  typically  CA ; 1  and Perkel,  are  as  34-42,  for CA  1981;  Durand,  Turner and Schwartzkroin, 1980).  3  The membrane  time constants, which are the latencies from onset of the pulse to l - ( l / e ) of the peak voltage deflection, exhibit ranges, in msec); for  the following values (reported as  10-20 for the CA^ and dentate gyrus neurons, and 17-26  CA, neurons (Brown, Fricke and Perkel, 1981;  Turner, 1982;  Turner and Schwartzkroin, 1980).  Durana, et a l . ,  The large variability  resistances and time constants measurements probably reflect ences in the fields,  1983;  sampled neurons within and among the  real  in  differ-  different hippocampal  ln addition, hippocampal neurons have been modelled in order to  assess other electrical features such as electrotonic lengths, dendrite-tosoma conductance ratios, et a l . , 1983; Turner,  1984;  etc  Johnston, 1981;  (Brown,  Fricke  and Perkel,  Kawato and Tsukahara, 1984;  Turner and Schwartzkroin,  1984).  1981; Turner,  Durand, 1982;  The calculated estimates  for the electrotonic length and the dendrite-to-soma conductance ratios the  hippocampus are  0.8-1.0  and  1.5-3,  respectively  (Brown,  in  Fricke and  CHIRWA Perkel, 1981; and  Durand, et a l - , 1983;  Schwartzkroin,  1984).  Johnston, 1981;  These  values  Turner, 1982;  indicate  that  the  24 Turner  hippocampal  neurons are electrically compact. 5.2  Intrinsic ionic conductances Hippocampal neurons are  endowed with  a multitude  of  both chemical  and/or voltage gated membrane ionic channels (Colino and HalliwelT,  1987;  Lancaster and Nicoll, 1987; Llinas, 1984; Moore, et al • , 1988; Schwartzkroin and Slawsky, 1977). channels  The specific ionic conductances that occur when these  open contribute  propagated  potentials.  towards  the  Some of  the  genesis ionic  described in the hippocampus include the classic action potentials) vating Na High  +  dendrites,  inactivating  generate  local  conductances that  following.  and  have been  Sodium spikes  (i.e.  are generated via the Hodgkin-Huxley type inacti-  conductances (Llinas,  threshola  and outcome of  1984;  Ca  Schwartzkroin  conductances  Ca -dependent  action  ++  and Slawsky, 1977).  located  potentials  in  the  termed  soma and  Ca  ++  spikes  (Schwartzkroin and Slawsky, 1977; Wong and Prince, 1978). A second type of ++ Ca conductance does not inactivate, and this conductance is able to ++ ++ induce Ca spikes. This presumably somatic Ca conductance has a low threshold of activation and is involved in the induction of an outward K current  (Hotson  Presently, K  +  and  Prince,  1980;  Schwartzkroin  conductances constitute the largest  and  Slawsky,  number of  tances that have been described (Colino and Halliwell, Barker, 1984). Huxley,  1952)  is  1977).  ionic conduc-  1987;  Segal and  F i r s t , there is the classic Hodgkin-Huxley type (Hodgkin ana delayed  phase of the fast M-current,  +  rectifier  K  +  action potential.  a low  threshold  current  which  Another K  non-inactivating  +  generates  current, K  +  the  falling  denoted as the  conductance which  elicited by depolarizations and modulated by transmitters  is  (Adams, Brown and  CHIRWA halliwell, exhibit vated  Moore, et a l • ,  1988).  Furthermore,  hippocampal neurons  +  inactivating delayed rectifying K conductances which ++ ++ by Ca . During neuronal activations, Ca influxes  neurons dent  1981;  subsequently  K  effluxes  induce outward  K  cause intracellular  polarizations,  which diminish cell  Schwartzkroin  and  Stafstrom,  conductances.  +  membrane  2b  are  acti-  into  These Ca -depen++  shifts  termed  afterhyper-  discharges (hotson and Prince,  1980).  ln  addition,  CA^  fast  1980;  transient  conductances have been noted in the hippocampus (Gustafsson, et a l . ,  K  +  1982)  which presumably serve to prevent the rapid return of membrane potentials to baselines,  following  contributes  towards  hyperpolarizations. the  prevention  of  This  K  +  conductance  rebound excitations,  probably  as the  membrane potential returns to baseline following membrane potential  cell  pertur-  bations. 5.3  Bursting activity Hippocampal  neurons can generate  spontaneous bursts  of  2-10  potentials of decreasing amplitudes and increasing durations, i . e . , spikes  (Masukawa,  Bernado and Prince, 1982;  Prince and Basbaum, 1979). fire  bursts  Prince  but  CA^ cells  readily In  support  contrast,  bursting  1975;  activities  CA^ pyramidal  spikes but do not do so ordinarily  Bernado and Prince, 1982).  complex Wong,  Typically, dentate gyrus granule cells do not  and Basbaum, 1979).  bursts of  Schwartzkroin,  action  (Alger,  cells 1984b;  (Wong,  can give Masukawa,  The differences in bursting behaviour in differ-  ent cornu ammonis subfields or dentate gyrus, may be due to differences in distributions  and activities  of  recurrent/feed-forward  inhibitory  inter-  neurons (Alger, 1984a). Intracellular records have shown that complex-spikes are comprised of long  duration  action  potentials  and  depolarizing  after-potentials  CHIRWA (Schwartzkroin,  1975).  26  Wong (1982) postulated that sodium spikes initiated  by membrane  potential fluctuations in the hippocampal pyramidal neurons ++ ++ Ca conductances. Upon membrane repolarizations, the Ca  activated  conductances Ca  ++  decayed  spikes (Kandel  slowly,  resulting  and Spencer, 1961;  in  secondary  depolarizations  Schwartzkroin  and Slawsky,  and  1977;  Wong and Prince, 1978). 5.4  Miniature postsynaptic potentials Hippocampal neurons are relatively electrically compact.  This, and the  long membrane time constants, can make possible the intrasomatic  detections  of small and discrete, potentials that are generated at dendritic subsynaptic membranes. the quantal  These spontaneous small potentials are thought to be due to  release of transmitters  and Prince, 1979;  (Alger  and Nicoll, 1982a; Brown, Wong  Johnston and Brown, 1984; Voronin, 1983; Yamamoto, 1982)  and to be similar to the well characterized miniature  end-plate  at the neuromuscular junction (del Castillo and Katz, 1952; 1952;  Katz,  1962).  small potentials  On the basis of their  IPSPs are effectively 1984),  •"-dependent  Fatt and Katz,  pharmacological  profiles,  in the hippocampus have been termed miniature  and inhibitory synaptic responses (i.e.  Brown,  potentials  and  minEPSP and minlPSP).  blocked by picrotoxin these  inhibitory  pharmacological  or bicuculline agents  responses  mediated  CA^ pyramidal  cells  by  the  excitatory  The miniature (Johnston and  selectively  abolish  y-aminobutyric  acid  (Johnston, 1978). 5.5  Evoked field responses Stimulation  antidromic  of  the  responses in  the  CA^ subfield.  axons  in  following  alveus evokes  These responses are  population spikes with short onset latencies (Figure 5-1). responses are capable of  the  high tetanic  seen as  These antidromic  stimulation  frequencies  CHIRWA (Chirwa,  1985).  The evoked population  spikes are not  27  abolished by high  magnesium and/or manganese containing media ( i . e . , physiological medium with l i t t l e or no C a ) .  Stimulation of the afferents  ++  the  stratum  radiatum  (e.g.  commissural or  tively) cause a presynaptic potential  in the stratum oriens or  Schaffer  recorded negative deflection (arrow in Figure 5-1)  fibers  amplitude  activated.  responses  as  The  shown  in  respec-  in a strip-like region at the stimu-  lated level (Andersen, 1983; Andersen, et a l . , 1978).  volley and its  collaterals,  This extracellularly  is termed the presynaptic  is usually taken as an index of the number of presynaptic Figure  volley  5-1.  The  is  followed  by postsynaptic  negative-going  synaptic  field  responses generated by stimulation of commissural or Schaffer collaterals, have their maximum in the region where the activated fibers terminate and show reversal when recorded from distant positions along the dendritic axis (Andersen, et a l . , 1980c).  Depending on the magnitude of the orthodromic  excitatory potentials, action potentials are discharged in several pyramidal neurons.  The magnitude of the summated action potentials generated is maxi-  mum when recorded in the stratum pyramidale layer and shows polarity sal on both sides of the pyramidal layer.  Andersen, et a l .  selective activation of a small group of afferent fibers potentials proximal  in the CA^ neurons. and distal  f i e l d potentials.  These investigators  synapses in  LNB:  CA^ were largely  rever-  (1980a) caused  to e l i c i t  field  demonstrated that the equipotent  in evoking  The density of the excitatory synapses was the same  (see also Andersen, Storm and Wheal, 1987)j. 5.6  Inhibitory postsynaptic potentials The collaterals  These inhibitory  of  CA^ axons feedback onto  interneurons,  recurrent inhibition).  in turn,  innervate  inhibitory  interneurons.  the CA^ neurons  Hence, when a CA, pyramidal neuron  (i.e.,  CHIRWA  Antidromic  28  Orthodromic PW  PW  Somatic layer  1 mV  PS 10 ms Dendritic region  field EPSP  Figure 5-1. Representative evoked field responses in the hippocampus. The traces are from extracellular recordings in the somatic layer and the apical dendritic region of the CAj area. Stimulation of the CAj axons in the alveus evokes an antidromic population spike (PS) with short onset latencies (i.e. the time from start of stimulation artifact (arrow) to peak amplitude of population spike). Orthodromic stimulation of the stratum radiatum evokes the negative-going field EPSP in the dendritic region as well as the positive wave (PW) 1n the somatic region. The positive wave reflects mostly excitatory and inhibitory activities in CAj neurons. Increasing the orthodromic stimulation strength evokes a population spike in the somatic region (i.e. reflects the summation of synchronously discharging CAj neurons, see Andersen, Bliss and Skrede, 1971) that intoduces two peaks in the somatic positive wave (denoted as PWj and PW? in the illustration). In general, PWi mostly corresponds with excitatory Influences whereas PW* mostly corresponds with inhibitory influences in CAj cells (Andersen, Eccles and Loyning, 1964). In addition, the field EPSP recorded in the dendrites can be interrupted by a positive peak (*) and this is a reflection of the fields generated by the population spike in the somatic region. Note the presence of the presynaptic volley (PV) that 1s usually discernable in these dendritic recordings.  CHIRWA is  activated,  it  hyperpolarizing  subsequently drives responses in  the  an interneuron,  29  which then induces  same CA^ pyramidal  cell.  During  this  hyperpolarizing response in the CA^ pyramidal neuron, incoming excitatory responses are shunted (Andersen, Eccles and Loyning, 1964).  Some inhibitory  interneurons  in  are  innervated  directly  by other  afferents  stratum oriens and stratum radiatum (e.g. Frotscher, 1985).  the  alveus,  These inhibi-  tory interneurons then feed onto pyramidal neurons where they induce conductances that shunt excitatory responses (i.e.  feed-forward  inhibition).  The  presence of feed-forward inhibitions is supported by studies that have shown that stimulations  of the afferents  in the  radiatum  prominent  in  induce  (Buszaki, 1984).  It  IPSPs  stratum oriens or the  quiescent  CA^  pyramidal  stratum neurons  appears that these hyperpolarizing responses could not  have been caused by recurrent inhibitions, since these latter responses are dependent on prior discharges in CA-^ neurons. 5.7  Electrotonic couplings Nonsynaptic  interactions  have been demonstrated  in  the hippocampus.  MacVicar and Dudek (1981) reported the occurrence of electrotonic couplings, where activities  in one neuron are transmitted  directly  presumably via anatomically identifiable junctions.  to other neurons  The dye Lucifer Yellow,  which is very sparsely taken up from the extracellular  space and does not  cross chemical synaptic junctions, was used to reveal gap junctions (Dudek, et a l . ,  1983).  In these studies, however,  coupling being introduced by the electrode cells 1983).  has not been entirely This is particularly  ruled out  (cf.  the  possibility of mechanical  itself  partially  Alger,  impaling both  McCarren and Fisher,  important since pyramidal neurons are tightly  packed together (Lorente De No', 1934).  CHIRWA 5.8  30  Ephaptic interactions Ephaptic  interactions  are thought  to be the  caused by current flows via extracellular 1982;  Taylor and Dudek, 1982).  It  influences on a neuron  resistances  (Jefferys  and Haas,  was found that when hippocampal slices  in vitro were perfused for prolonged periods with a low C a  ++  to  development  block  rhythmic  synaptic bursts  Dudek, 1982).  transmission),  lasting  for  this  several  resulted  seconds  in  (Alger,  the  1984b;  Taylor and Dudek  differential  and adjacent  ated  (i.e.  Taylor  of and  The bursts occurred spontaneously, or they could be evoked  with direct or antidromic stimulations.  tials.  medium  recordings of intracellular  (1984) analysed  extracellular  poten-  These investigators found that during population spikes, the associextracellular  across  inactive  electrical  pyramidal  fields  cell  caused currents  membranes.  electrotonic or ephaptic interactions  It  has  to been  flow  passively  suggested that  could be involved in synchronization  of cell discharges in the hippocampus (Richardson, Turner and Miller, Yim, Krnjevic and Dalkara, 1988).  1984;  CHIRWA 6.  31  SYNAPTIC PHARMACOLOGY OF THE HIPPOCAMPUS  6.1  GABA Inhibitory synaptic influences, both recurrent  t-aminobutyric Mathisen,  acid  1977;  (GABA) as  Frotscher,  their  et a l . ,  principle 1984).  and feed-forward,  neurotransmitter  Glutamic  use  (Storm-  acid decarboxylase  (GAD) as well as GABA-ergic receptors are distributed in all layers of field CA^ (Storm-Mathisen,  1977;  Andersen, et a l . ,  1980b).  Evidence indicates  that inhibitory interneurons (at least the basket cell type; 1934)  release  dendrites,  GABA onto CA^ pyramidal  and  activate  conductances  (Andersen, Bie and Ganes, 1982; Interestingly,  the  cell that  bodies, shunt  axon hillock  excitatory  antidromic  shape of  activation  of  and/or  influences  Bowery, Hudson and Price, 1987). IPSPs  caused by antidromic  differs from those evoked during orthodromic stimulation. not  Lorente De No,  CA^ pyramidal  stimulation  Orthodromic, but  neurons e l i c i t s  larger  IPSPs  [NB:  ame size field potentials and associated IPSP measured concurrently;  Alger  and Ni col 1,  abolished exhibit  by  two  1982b].  GABA-ergic time-dependent  Furthermore,  antagonists.. components,  recurrent In i.e.,  IPSPs  contrast, early  (Alger and Nicoll, 1979; Hablitz and Langmoen, 1987).  are  completely  feed-forward and  IPSPs  late components  The early component  is abolished by picotroxin and bicuculline (GABA-ergic antagonists), whereas the late component is insensitive to these agents.  These differing pharma-  cological profiles for IPSPs have formed the basis for the conclusion that GABA actions are mediated by at least two types of receptors, termed GABA^ and GABA (Alger and Nicoll, 1979). B  6.2  GABA receptors A  GABA receptors are largely distributed on the soma, axon hillock and fl  CHIRWA proximal  parts of stem dendrites of pyramidal  receptors induce Cl" currents that cause  cells.  32  Activation of GABA^  hyperpolarizations.  The inhibi-  tory conductance induced by GABA appears to be due to Cl" ions since these fluxes  are  diminished  in  media  (Thalmann, Peck and Ayala, 1981).  containing  low  Cl"  Furthermore, the reversal  concentrations potential  for  these conductances range between minus 60-65 mV (Andersen, et a l . , 1980b). These values are thought to be consistent with those calculated using the Nernst equation, which take into account the  presumed chemical  between intracellular  While picrotoxin  block  and extracellular  the GABA^ receptor  prevent the interaction Olsen, 1982; 6.3  Cl".  coupled C l " channel, bicuculline  appears to  is thought  Peck, Schaffer and Clark, 1973).  GABAg receptors  polarizing contrast,  responses that applications  cells e l i c i t s  and Ganes, 1982).  depolarizing changes  in  are  layer e l i c i t s  blocked by picrotoxin  of GABA to the dendritic  or  bicuculline.  In  regions of CA^ pyramidal  This depolarizing  effectively  responses  shunts excitatory  induced  C l " gradients.  response to GABA is  by  GABA,  Moreover,  synaptic responses.  however,  the  ionic  presumably  are  not  sensitive  more negative than would be expected for C l " conductances. ionic conductances show reversal findings  led  to  subtype termed GABA . g  with GABA  R  the  potentials  implication  of  Hence GABA released  receptors  to  initiate  similar  to  that are  Instead these  to those of K .  The  +  a second GABA-ergic in the dendritic  inhibitory  The  conductances associated  with the depolarizing actions of GABA exhibit reversal potentials  above  hyper-  a depolarizing response, as recorded in the soma (Andersen,  inhibitory since it  acts  to  of GABA with the GABA^ receptors (Johnston, 1978;  Exogenous GABA applications to the CA^ pyramidal  Bie  gradients  receptor  sites  inter-  conductances that  are  CHIRWA probably mediated 1982b;  by K  fluxes  +  (Alger,  Andersen, et a l . , 1980b).  Ca  ++  1984a; Alger  Inoue, Matsuo and Ogata, 1985).  1979;  conductances have also been impli-  cated in the responses mediated by GABA^ activation 1985;  and Nicoll,  33  (Gahwiler  and Brown,  Recent pharmacological experiments  have shown that baclofen is a selective agonist at GABAg receptors  (e.g.  Bowery, Hudson and Price, 1987), and phaclofen is purported to be a specific antagonist at these receptors (Dudar and Nicoll, 1988). 6.4  Putative excitatory The evidence for  aspartate  an excitatory  transmitter  role  of  glutamate  in the hippocampal commissural and Schaffer collateral  based on biochemical uptake  transmitters  sites  and autoradiographic  (Storm-Mathisen  and  localizations  Iversen,  1979;  of  and/or  axons is  high  affinity  .Fonnum, et a l . ,  1979),  induction of changes in the endogenous levels of amino acids after selective lesions  (Fonnum and Walaas, 1978)  release  following  Wieraszko  K  +  and Lynch,  or  and the demonstration of C a  electrical  1979;  stimulation  Malthe-S^irenssen,  (Nadler,  Skrede  et a l . ,  putative  excitatory  Nadler, et a l . , 1976;  transmitters  (e.g.,  Koerner  1978;  and Fonnum, 1979).  While the abundant biochemical evidence implicates glutamate as  mediated  ++  ano  (or  aspartate)  Cotman,  1982;  White, Nadler and Cotman, 1979), it is far from clear  whether these substances constitute the actual endogenous neurotransmitters in the hippocampus.  The validity of any ligand binding technique is depend-  ent on the demonstration that the radioactive ligand selectively labels the physiological or pharmacological receptors under study. often  not f u l f i l l e d  Even though C a  ++  release studies, it  with glutamate  and voltage  or aspartate  This requirement  (Foster  is  and Fagg, 1984).  dependencies have been demonstrated  in  the  is not known for certain whether the released tritiated  transmitters come from the same intracellular compartments as the endogenous  CHIRWA transmitters themselves (Laduron, 1984).  34  In other systems, it is known that  tritiated ligands can be trapped in different intracellular  compartments of  intact cells (Maloteaux, et a l . , 1983). 6.5  Exogenous glutamate actions in hippocampus Glutamate application by iontophoresis near single hippocampal neurons,  causes a fast onset excitation followed by a rapid termination  of action.  This excitant action is thought to be mediated by a direct glutamate-induced depolarization of the hippocampal neurons (Mayer and Westbrook, 1987).  The  electrophysiological studies indicate that glutamate induces an inward movement  of  1987). cells,  cations,  mostly  Na  and/or  perhaps  Ca  (Mayer  There are "hot" spots along the dendritic  trees  at which the depolarizing actions of glutamate  (Dudar, 1974; ably reflect  Schwartzkroin and Andersen, 1975). receptor  sites  for  glutamate.  and Westbrook,  of CA^ pyramidal  are most prominent  These hot spots presum-  Hablitz  and Langmoen (1982)  reported that the reversal potentials for the glutamate-mediated tion in the hippocampus were comparable to those of the EPSPs.  depolarizaBoth shifted  in a negative direction in low Na medium. +  6.6  NMDA and Quisqualate/Kainate receptors Though many antagonists  of  excitatory  acidic  amino acids have been  discovered, their blocking actions have been against exogenous acidic amino acids. kainic 1987;  Many of the acidic amino acids, notably aspartate, quisqualate and acid,  exhibit  P u i l , 1981).  actions  similar  to  glutamate  (Mayer  and Wesbrook,  Their differences in potencies, 'antagonistic' profiles  and/or ionic conductances activated have led to the implication of different types of acidic amino acid receptors.  At least two types of acidic amino  acid receptors have been implicated;  the NMDA receptor  receptor(s)  (Dingledine, 1984;  Foster and Fagg, 1984;  and the non-NMDA  Mayer and Westbrook,  CHIRWA 1987;  McDonald and Wojtowicz, 1982; N-Methyl-D-aspartate  activated,  presumably  increase  Mg  NMDA receptor blockade near  ++  1987;  Watkins, 1984).  (NMDA) is selective for NMDA receptors which, when  (Cotman and Iversen, 1987; 1987).  35  a  cationic conductance  Mayer and Westbrook, 1987; Watkins and Olverman,  activation  the  voltage-dependent  resting  Mayer and Wesbrook, 1987;  is  highly  voltage-dependent,  membrane potential  (Cotman  due to a  and  Watkins and Olverman, 1987).  Iversen,  Experimental  evidence indicates that adequate depolarizations, however, remove the block by Mg  ions,  and Iversen,  leading 1987;  presumably to  Watkins  regenerative  and Olverman,  1987).  Ca  currents  (Cotman  Both quisqualate and  kainate exhibit preferences at non-NMDA receptors, whose activations Na  +  and possibly K  +  conductances.  Glutamate  is active  at  elicit  both NMDA and  non-NMDA receptors. A variety of substances have been shown to antagonise responses mediated by applied NMDA, quisqualate or kainate. mylaminomethylsulphate are  or  l-(p-chlorobenzoyl)-piperazine-2,3-dicarboxylate  non-specific antagonists of  However,  these  potencies.  quisqualate  NMDA responses, but  with  lower  Competitive NMDA receptor antagonists include (AP5;  APV),  D-2-amino-phosphonoheptanoate  3-3(2-carboxypiperazine-4-yl)propyl-l-phosphonate  Iversen, 1987;  Watkins and Olverman, 1987).  antagonists include n-allylnormetazocine Iversen, 1987; 6.7  and kainate responses.  Selective antagonists have only been discovered for responses  D-2-amino-5-phosphonovalerate and  both  substances also diminish  mediated by NMDA receptors.  (AP7)  Substances such as f-D-gluta-  Mayer and Wesbrook, 1987;  (Cotman  and  Non-competitive NMDA receptors  (SKF10047) and MK-801 (Cotman and Watkins and Olverman, 1987).  Subsynaptic receptors ln terms of excitatory  synaptic transmission in the hippocampus, the  CHIRWA antagonist profiles are somewhat incomplete.  36  Both NMDA and non-NMDA recep-  tor subtypes are thought to be distributed in the same subsynaptic regions of the hippocampus.  In addition,  (and possibly non-NMDA) receptors (Dingledine,  1983a).  non-NMDA receptors. antagonists  of  The fast  there is evidence indicating that NMDA are distributed  in  presynaptic regions  EPSPs are thought  to  be mediated  At the present time,  the  fast  synaptic  by the  specific (and indeed selective)  transmission  have  not  been  found.  Recently, it has been proposed that the slow depolarizing wave that develops during tetanic  stimulations of afferents  GABA-ergic inhibitors; receptor  activation.  responses. tic  (particularly  in the presence of  see Wigstrom and Gufstaffson, 1985b) was due to NMDA APV in  low doses has been found to  abolish these  However, in higher doses APV will also diminish the fast synap-  responses.  More studies are needed to  clarify  further  the synaptic  pharmacology of excitatory transmission in the hippocampus. 6.8  Other putative transmitters A variety of extrinsic modulatory pathways in the hippocampus have been  identified.  In  most cases,  the  exact  target  cells  inputs and/or .their origin are not fully known. seem to innervate  parts of CA^ include;  these  septum and diagonal  Lynch, Rose and Gall, 1978),  the noradrenergic outflow from the locus ceruleus (Lindvall 1974)  and the  serotonergic  projection  nuclei (Azmitia and Segal, 1979). these extrinsic  pathways is  extrinsic  Some of these inputs that  the medial  band cholinergic input (Storm-Mathisen, 1977:  for  from the  medial  and Bjorkland,  and dorsal  raphe  The probable modulatory role for some of  illustrated  by the  actions of  acetylcholine.  Acetylcholine is known to cause a reduction in the M-current which is active over the -70 mV to -40 mV membrane potential Kelly, 1981;  Bernado and Prince, 1982).  range (Dodd, Dingledine and  This antagonism of the M-current  CHIRWA causes  slow depolarizations  in the cells  and raises  input  37  resistances.  These actions probably improve electrical compactness of target c e l l s .  Such  an action would facilitate the invasion of small synaptic signals to the soma (Dingledine, inhibit  1984).  addition,  acetylcholine  the release of inhibitory and excitatory  CA^ hippocampal field It  In  6.9  Ben-Ari,  the cholinergic septal  axo-axonic contacts with inhibitory or excitatory  shown to  neurotransmitters  (Yamamoto and Kawai, 1967;  is not known, however, whether  has been  in the  et a l . , 1981).  inputs also form  afferents.  Neuromodulators Recent findings have revealed  a diverse distribution  substances in the hippocampus (Dingledine, 1984). lished whether  these neuroactive  co-exist with other  It  of  neuroactive  remains to be estab-  substances form separate pathways and/or  neurotransmitters.  It  can be speculated that neuro-  active substances might even reside and/or be released from dendritic spine structures and thereby influence synaptic interactions.  But the experimen-  tal evidence for these possibilities is presently lacking. The main neuroactive substances that have been characterized so far in the  CA^ field  cholecystokinin intestinal  include  enkephalin-1ike  and somatostatin  substances  (Greenwood,  (Gall,  et a l . ,  et a l . ,  1981),  1981),  vasoactive  polypeptides (Loren, et a l . , 1979), substance-F (Vincent, Kimura  and McGeer, 1981) and angiotensin-Il  (Haas, et a l . , 1980).  CHIRWA 7.  38  LONG-TERM POTENTIATION IN THE HIPPOCAMPUS  7.1  Introduction Long-term synaptic  model  for  cellular  potentiation  mechanisms  is  generally  involved  in  viewed  learning  as  and  use-dependent increase in synaptic efficacy invariably alters  a  potential  memory.  The  the  intricate  balances in synaptic weighting within and among neural networks.  Clearly,  signals that traverse potentiated synapses exert biased influences on their target cell(s).  The attractiveness  of  long-term  potentiation  (LTP)  as a  model for learning and memory is discernable in the following observations. LTP is readily inducible in the hippocampus, a structure that has long been considered to subserve learning and memory functions Thompson, 1983;  Teyler and DiScenna, 1984).  The basic requirements for LTP  induction are within physiological ranges (Byrne, 1986; Rose and Dunwiddie,  1986).  Even if  (Swanson, Teyler and  1987;  Larson and Lynch,  memory functions are ultimately  encoded as biochemical changes within selected neuronal groups,  preferential  mechanisms for the transfer or retrieval of stored information are probably present.  Enduring changes in synaptic efficacy in selected neural  could be one such method. priming is itself  circuits  Moreover, that synapses remain potentiated  an example of learning  (cf.  Eccles, 1977).  after  Whatever  is  the role of LTP in physiology, the understanding of this phenomena is bound to yield significant city.  It  insights into the diversity of nervous system plasti-  is for such reasons that the phenomenon of long-term  potentiation  continues to attract great interests among neurobiologists. 7.2  Basic features of long-term potentiation 7.2.1  Long-term potentiation.  co-workers gave the f i r s t  detailed  In the early seventies, Bliss and his account of long-term potentiation  (LTP)  CHIRWA in the hippocampus in vivo (Bliss and L^mo, 1973; 1973).  39  Bliss and Gardner-Medwin,  These investigators discovered that conditioning stimuli of 10-20 Hz  (for one or more seconds) given to a selected perforant path bundle, induced post-tetanic synaptic potentiations of the population spikes in the stratum granulosum and field  EPSPs in the molecular  layer  of  the  dentate  gyrus  evoked by the same inputs (pre- and post-tetanic test pulses were evoked at 0.5 Hz, same stimulation parameters; hours to several  days, and it  Bliss and L^mo, 1973).  was manifested  LTP lasted for  as decreases in  population  spike latency, and increases in amplitudes of population spikes and/or field EPSPs.  Often,  depressions of  evoked responses lasting  several minutes followed a low frequency tetanus observed.  from  seconds  to  (10-20 Hz) before LTP was  In contrast, the higher frequency conditioning trains  (i.e.,  100  Hz) induced LTP without prior post-tetanic depressions of evoked responses. But LTP elicited by high and low frequency tetanic trains exhibited the same characteristics (Bliss and L^mo, 1973;  Bliss and Gardner-Medwin, 1973).  LTP expression was confined to the only.  However, repeated tetanic  tetanized  perforant  paths bundle  stimulations of the same input caused an  augmentation of the established LTP until  an asymptote was reached.  Bliss  and co-workers found that LTP was not due to changes in stimulating electrode properties  after  simple upward shift 1973;  tetanus.  along the  Furthermore,  LTP was not  stimulus-response curves  Bliss and Gardner-Medwin, 1973).  ted bigger population spike relative  a result  (Bliss  of a  and L#mo,  The post-tetanus field EPSPs e l i c i to matched pre-tetanus  over a wide range of stimulation intensities.  field  EPSPs,  These studies also revealed  that the potentiation of the population spike was not always accompanied by the potentiation of the field EPSP. of LTP;  Hence there were two basic expressions  that which presented with the potentiation of the population spike  CHIRWA  40  alone, and that which presented with simultaneous increases to population spikes and field EPSPs.  These two forms of LTP expression were subsequently  classified as "E-S potentiation" and "synaptic potentiation",  respectively  (Andersen, et a l . , 1980c). 7.2.2  Distribution of LTP.  Recent studies have demonstrated that  many other afferent systems can be potentiated in the mammalian brain (see Byrne, 1987, for review). the  following  Wester, tion,  synapses:  1975),  Within the hippocampus, LTP has been produced at Schaffer  collaterals-CA^  and mossy fibers-CA^ (Alger  (Schwartzkroin  and Teyler,  1975).  and  In addi-  LTP occurs in the synapses of CA^ commissural projections and the  contralateral  CA-^ and CA^ neurons  Buzsaki, 1980).  (Bliss,  Lancaster  and Wheal,  1983;  LTP has also been induced in the excitatory projections to  feed-forward inhibitory interneurons (Buzsaki and Eidelberg, 1982;  Kairiss,  et a l . , 1987).  Outside the hippocampus, LTP has been found in the following  structures  vertebrates;  in  limbic  system  (Racine,  Milgram  and  Hafner,  1983), cerebral cortex (Lee, 1982), pyriform cortex (Stripling and Patneau, 1985), and the medial  geniculate  nucleus (Gerren  and Weinberger,  Long lasting synaptic potentiations have been studied in the  1983).  invertebrate  nervous systems as well (see Byrne, 1987, for review), but these will not be considered here. 7.2.3 like  the  Homosynaptic and heterosynaptic dentate  gyrus, only  LTP.  input-specific  In  the  CA^ subfield,  LTP has been demonstrated.  Hence, when the Schaffer collaterals are tetanized, only the Schaffer collateral-CA^ synapses become potentiated et a l . ,  198Uc).  (Andersen, et a l . ,  McNaughton (1983) described this  "homosynaptic LTP".  1977;  Andersen,  input-specific  LTP as  In the CA^ subfield, input specificity of LTP is not  always preserved (Yamamoto and Chujo, 1978)  since tetanic  stimulations of  CHIRWA inputs can result  in LTP across the  non-tetanized inputs (i.e. 1979;  synapses of  "heterosynaptic LTP";  Yamamoto and Chujo, 1978).  both the  41  tetanized and  Misgeld, Sarvey and Klee,  Significant differences between homosyn-  aptic LTP and heterosynaptic LTP have been reported.  Heterosynaptic LTP  development is apparently restricted to the polysynaptic components of the evoked response, and the expression of homosynaptic LTP is limited to the responses evoked by monosynaptic inputs in the CA^ subfield (Higashima and Yamamoto,  1985).  often e l i c i t latencies.  [Nb:  Orthodromic  stimulations  dual population spikes (or field  of  inputs  to  the CA^  EPSPs) with different onset  The early responses are due to the activation  of monosynaptic  inputs, whereas the late responses are caused by polysynaptic activations (Higashima and Yamamoto, 1985)]. 7.2.4  LTP in single neurons.  increases in probabilities  In single neurons, LTP is expressed as  for neuronal discharges and/or augmented ampli-  tudes of subthreshold intracellular EPSPs (Schwartzkroin and Wester, 1975). However, LTP is not accompanied by changes in input resistances or resting membrane potentials measured in the soma (Andersen, et a l . , 1980c). single-electrode voltage clamp methods, Barrionuevo, et a l . ,  Using  (1986) found  that the currents associated with the excitatory postsynaptic potentials are greatly increased after LTP development in the mossy fibers-CA^ synapses. In addition, the ability of intracellular EPSPs to propagate to the soma did not change during LTP. intracellular  Presumably, the changes that mediate increases in  EPSPs in CA^ neurons during LTP are localised to subsynaptic  and/or presynaptic elements (Barrionuevo, et a l . , 1986). 7.3  Production of LTP 7.3.1  Induction.  alone (Chirwa, 1985;  LTP can not be induced by antidromic  Schwartzkroin and Wester, 1975).  tetanizations  The classic LTP is  CHIRWA seen only with orthodromic tetanizations. frequencies are capable of  1978).  Though a whole range of tetanic  inducing LTP, higher  reliably induce synaptic potentiations  42  tetanic  (Chirwa, 1985;  frequencies most  Dunwiddie and Lynch,  Tetanic stimulations delivered during perfusions with physiological  medium without blockers,  (i.e.  Lynch, 1979; however,  Ca  or medium with raised concentrations  Mg  ++  and/or  ton ) ++  do  not  elicit  Wigstrom, Swann and Andersen, 1979).  is not reversed by subsequent transient  medium (Dunwiddie and Lynch, 1979).  LTP  of  Ca  entry  (Dunwiddie  and  Prior induction of LTP, exposures to  Ca -free ++  Hence, only the induction of LTP with  tetanic stimulations, but not its maintenance, is dependent on extracellular ++ Ca  levels. Conflicting reports have been presented on whether induction of LTP is  dependent on postsynaptic discharges.  Scharfmann and Sarvey (1985) reported  that LTP induction was blocked when bath applications of GABA were used to inhibit postsynaptic spiking during tetanic  stimulations of inputs.  results were interpreted  need for  during LTP production.  as reflecting In contrast,  the  Kelso,  These  postsynaptic spiking  Ganong and Brown (1986) used  intracellular  injections of QX-222, a quartenary anaesthetic agent, to block  intracellular  action potentials  in the  stratum radiatum  synapses.  induced LTP across  stimulation  Lynch, 1978).  stimulations  Schaffer collaterals-CA^  (Chirwa,  stimulation 1985;  Chirwa,  compared with higher, frequency et a l . ,  1983;  Dunwiddie  But the higher tetanic stimulation frequencies (i.e.  ated with minimal together,  the  Yet tetanic  Interestingly, postsynaptic discharges are much more pronounced  during lower frequency tetanic tetanic  in CA^ neurons.  postsynaptic spiking)  the available experimental  most reliably  induce LTP.  and  associTaken  evidence favors the notion that post-  synaptic spiking per se is not a necessary pre-requisite  for LTP develop-  CHIRWA ment.  43  Rather, postsynaptic membrane depolarization seem to be essential for  LTP induction in the hippocampus (Malinow and Miller, 1986). 7.3.2  Co-operative LTP.  (1973), it strengths further  was evident  In the studies of Bliss and Gardner-Medwin  that LTP induction was dependent on the  used during tetanus.  McNaughton,  Douglas  stimulus  and Goddard,  (1978)  examined the relationships of stimulus strength with LTP induction  in the dentate gyrus and found that low stimulus strengths mostly elicited brief  potentiations  develop).  that  lasted  for  3-5  minutes  (i.e.,  LTP failed  LTP production, however, was consistently evoked with high stimu-  lus strength tetanus, suggesting the presence of stimulus intensity hold"  for  LTP induction.  It  was inferred  that  LTP production  co-activation of a minimum number of afferents during tetanic This  to  effect  (McNaughton,  was  termed  "co-operative"  Douglas and Goddard,  also been demonstrated  in  the  interactions  1978).  Co-operative  CA^ (Yamamoto  "thresrequired  stimulations.  or  co-operativity  interactions  and Sawada,  1981)  have  and  the  CA subfields (Lee, 1983). 1  7.3.3  Associative LTP.  McNaughton, Douglas and Goddard, (1978) used  two separate weak inputs to the  same target  inputs supported LTP when tetanized  independently.  tetanizations of both inputs produced LTP. and Steward  neurons, and none of  (1979) found that conjoint  However,  these  simultaneous  In comparable experiments, Levy  tetanic  stimulations  input with a weak input, produced LTP in the weak input. input could support LTP, when tetanized independently.].  IHB:  of  a strong The strong  These co-operative  interactions between two separate inputs have now been classified as "associative interactions" or "associative LTP" (Johnston and Brown, 1984). was not determined how associative interactions  occurred between  It  separate  inputs to the same dendritic tree or those that impinged on the apical and  CHIRWA basal  dendrites.  McNaughton, Douglas and Goddard (1978)  postulated  postsynaptic neurons were, the conduit for these interactions. and Sastry  (1985) could induce transient  antidromic activation  of Schaffer  increases in the  collaterals,  44  following  that  However, Goh threshold for  tetanic  stimula-  tions of nearby but separate inputs to the same target cells in the CA^ subfield.  These studies  illustrated  that  interactions  among presynaptic  terminals were possible and could account for associative LTP. 7.3.4  Coupled LTP.  Sastry,  Goh and  Auyeung  (1986)  found  that  repeated pairings of test inputs that were activated at low frequencies  (0.2  Hz), with the simultaneous applications of adequate conditioning intracellular depolarizations of CA neurons, resulted in LTP that was localised to 1  the test inputs.  LTP induction was dependent on (1)  the intensity of the  depolarizing current injections, (2) concomitant activations of test inputs, and (3) the total number of pairings used.  Fewer pairing episodes elicited  transient short-term potentiations, lasting for 3-5 minutes. number  of  pairings  Auyeung, 1986).  It  (i.e.  more than  10)  produced LTP (Sastry,  if  picrotoxin  ist) was present in the physiological medium. pairing postsynaptic depolarizations  1986; 7.4  Goh and  was found that LTP production, using the pre- and post-  synaptic pairings, was facilitated  independently  Increasing the  reported  by other  (GABA^ receptor antagon-  That LTP could be induced by  with presynaptic activation  investigators  (Kelso,  was also  Ganong and Brown,  Wigstrom, et a l . , 1986). LTP production with pharmacological methods 7.4.1  Raised extracellular  K . +  May, Goh and Sastry  (1987) could  induce LTP in the CA^ subfield with the application of raised extracellular  K  +  (10-80 mM),  in  the  absence of  extracellular  presented an important finding showing that the  Ca . ++  This  report  induction of LTP was not  CHIRWA ++  absolutely dependent on extracellular  Rather,  adequate depolariza-  tions of pre- and postsynaptic regions were necessary.  In view of this, the  requirement tetanic  for  extracellular  stimulations  previously  Dunwiddie and Lynch, 1979; reflected  the  need for  evoked release  of  Ca  Ca  .  45  during  ++  reported  the by  induction other  of  LTP with  investigators  Wigstrom, Swann and Andersen, 1979)  Ca  ++  for  transmitters  evoked transmitter during  tetanic  release.  stimulations  (cf.  probably Hence the  subsequently  depolarized the postsynaptic regions, that were necesssary for LTP production.  Interestingly,  tetanic  stimulations  supports that  in  transmitter  Sr  release (cf.  whether  processes.  medium  release)  substituted  + +  transmitter known  Wigstrom and Swann  Sr  can  Even though  out the  Ca  in  ++  Sr  could e l i c i t (a  + +  also  mediating  substitute  extracellular  Ca  extracellular  Ca  for is  ++  LTP with  agonist  ++  Ca  .  It  that seemed  depolarization-coupled  Zengel and Magleby, 1980).  induce synaptic potentiations rule  containing  instead of  for  (1980)  Ca not  It  is presently not  in  other  always  biological  necessary to  (May, Goh and Sastry, 1987), this does not  possible involvement of  intracellular  Ca  in LTP.  Lynch,  et al • , (1983) reported that the induction of LTP in single neurons could be blocked  with  substance). 7.4.2  prior  intracellular  ejections  of  EGTA,  (a  Ca  ++  chelating  This intriguing study awaits replication by other investigators. Raised extracellular  Ca .  Turner,  ++  Baimbridge  and  Miller  (1982) caused long term increases in synaptic efficacy in the CA-^ subfield following transient  exposures to  perfused for 10 min;  elevated  control medium:  extracellular  2 mM C a ) . ++  Ca  ++  (4 mM C a  ++  Both the PS and the  field EPSPs evoked with stimulations of the Schaffer collaterals were potentiated for periods beyond 3 hrs.  There were no changes to the amplitudes of  the antidromic PS in the CA^ subfield evoked by stimulation of CA^ axons  CHIRWA in the alveus (Turner, was  associated with  Baimbridge and Miller, the  accumulation  of  1982).  The Ca -induced LTP ++  presumably  intracellular  However, it could not be determined whether the increased C a to presynaptic and/or postsynaptic regions. exposures to elevated  extracellular  Ca  46  ++  Ca  .  was limited  The induction of LTP with brief has also been reported by other  ++  investigators (e.g. Bliss, Dolphin and Feasey, 1984; Higashima and Yamamoto, 1985;  Sastry, et a l . , 1983;  Reymann, et a l . , 1986).  cance of these effects of Ca 7.4.3  is at present unclear.  Phorbol esters.  synaptic potentiations  However, the s i g n i f i -  Malenka,  Madison and Nicoll  in the CA^ subfield with transient  phorbol analogs known to activate protein kinase C (PKC).  (1986) induced applications of Both the popula-  tion spike and field EPSPs were potentiated for periods beyond 2 hrs postapplication. of  The probabilities for single cell discharges or the amplitudes  subthreshold intracellular  Nicoll,  1986).  EPSPs were increased  The characteristics  of  the  (Malenka,  potentiated  Madison and  responses induced  with phorbol esters were similar to those seen with tetanus-induced LTP (cf. Bliss and Lf)mo, 1973).  In a separate report,  that the synaptic potentiations with augmented transmitter  Nicoll and co-workers showed  induced with phorbol esters were associated  release, as evidenced by the increased frequen-  cies of minEPSPs ana minlPSPs (Malenka, Ayoub and Nicoll, 1987). tion, there was an increase in K  +  stimulated release of endogenous gluta-  mate (Malenka, Ayoub and Nicoll, 1987). were consistent  with  the  induced by phorbol esters.  In addi-  involvement However,  Taken together, of  presynaptic  the above studies mechanisms  in LTP  postsynaptic mechanisms also contri-  buted towards the expression of LTP since the phorbol esters blocked specif i c chloride conductances that were active (Madison, Malenka and Nicoll, 1986).  at resting membrane  Interestingly,  a report  potentials appeared in  CHIRWA the  literature  enzyme  that  protein  electrolytes  showed that  kinase  of  intracellular  C (PKC)  into  recording electrodes)  injections  of  CA^ neurons  (i.e.  resulted  synaptic  that were similar to LTP (Hu, et a l . , 1987)  in  47  the  active  PKC mixed  with  potentiations  However, PKC was continuously  present ( i . e . , in the recording micropipette) throughout the duration of the experiments (Hu, et a l . , 1987). 7.4.4  Mast cell degranulating peptides.  in the literature showing that brief  A report has also appeared  exposures to mast cell  degranulating  peptides (MCD) isolated from bee venom caused LTP development subfield (Cherubini, et a l . , increases (lasting  1987).  beyond 6 hr)  in the CA-^  LTP was expressed as post-application  in evoked EPSPs, following stimulation of  the Schaffer collaterals, without concomitant alterations in membrane resistance,  cellular  excitability  or  the  magnitude  of  afferent  volleys.  MCU  applications were associated with reversible depolarizations that could be blocked  if  TTX  or  Co  (a  ++  perfusion medium, indicating  Ca  ++  entry  blocker)  were  that these depolarizations  present  in  were synaptic  the in  origin.  The potentiating effects of MCD required synaptic activations since  TTX  Co  or  ++  blocked  the  induction  However, neither TTX nor C o Interestingly,  ++  of  LTP  (Cherubini,  et a l . ,  1987).  could reverse developed LTP induced by MCD.  prior treatment of the MCD with trypsin destroyed its poten-  tiating effects.  These authors suggested that MCD may be mimicking an endo-  genous peptide (Cherubini et a l . , 1987). 7.4.b  61utamate.  synaptic potentiation stimuli glutamate  to  Schaffer to  et a l . , 1987).  Recently,  it  was  reported  that  input-specific  could be induced with simultaneous pairings of collaterals  sensitive  spots  and brief along  the  iontophoretic CA^ apical  applications  dendrites  test of  (hvalby,  LTP was expressed as long lasting (beyond 1 hr) increases in  CHIRWA cell  discharge probabilities  and shorter  spike onset  latencies  48  following  stimulation of the Schaffer collaterals. 7.4.6  Miscellaneous.  GABA-ergic  blockade with  tates the induction of LTP (Douglas, 1978; It  picrotoxin  facili-  Wigstrom and Gustafsson, 1983).  is generally presumed that picrotoxin diminishes GABA mediated shunting  of excitatory  responses.  Reports have also appeared showing that  septal  input stimulation (which is partly cholinergic) enhances the E-S type of LTP (Robinson, 1986). itory  It is known that exogenous acetylcholine exerts disinhib-  influences on hippocampal neurons through  M-currents, Ropert,  and/or  1982).  (2) release of GABA ( i . e .  Some of the above effects  mimicked by septal serotonin  stimulations.  Depletion  diminishes the magnitude  Goddard and Riives, 1983;  the suppressions of  (1)  disinhibition;  Krnjevic and  of acetylcholine  are probably  of cortical  of LTP that  noradrenaline or  can be obtained  Hopkins and Johnston, 1984).  (Bliss,  Substances such as  noradrenaline are known to reduce neuronal accommodations in the hippocampus (Madison and Nicoll, 1986), depolarizations.  and these effects  Interestingly,  tances (e.g 4-aminopyridine)  probably prolong membrane  substances that block certain  K conduc+  facilitate the development of LTP induced by  tetanic stimulations (Chirwa, 1985;  Lee, Anwyl and Rowan, 1986).  Clearly,  pharmacological manipulations that enhance neuronal depolarizations generally facilitate LTP production, and vice-versa. 7.5  Blockade of LTP induction Several reports have shown that tetanic stimulations of the Schaffer  collaterals  in the presence of APV (in  doses that  did not  antagonise  non-NMDA receptors) blocked the induction of LTP across the Schaffer collaterals  and CA^ synapses (Col 1ingridge,  Ganong and Cotman, 1984;  Kehl  and McLennan, 1983;  Wigstrom and Gustafsson, 1984).  Harris,  Similarly, APV  CHIRWA has been found to block tetanus-induced LTP in the dentate gyrus et a l . , 1986). in  It  (Morris,  These results have led to the implication of NMDA receptors  the induction  1985a).  49  of  LTP (Collingridge,  1985;  Wigstrom  and Gustafsson,  has been suggested that the intense synaptic activation during  tetanic stimulations of afferents caused postsynaptic depolarizations that were sufficient  to remove the voltage-dependent  NMDA receptor-gated through  these  ionic  channels,  channels. and  the  This  blockade by Mg  led  intracellular  to  the  influx  of  accumulations  of  subsequently mediated the changes underlying LTP production 1985;  Wigstrom and Gustafsson, 1985a).  of the  ++  Ca  ++  Ca  (Collingridge,  That NMDA could be a common denom-  inator in the induction of LTP was reflected in recent findings showing that APV  also  blocked  Ca -induced  LTP (Errington,  Lynch and Bliss,  1987).  However, the anticipated universality of the NMDA hypothesis in LTP production was found to be limited.  Harris and Cotman (1986) found that APV did  not block tetanus-induced LTP at the mossy fiber-CA^ synapses. 7.6  Maintenance of LTP 7.6.1  Biochemical and structural changes.  Though LTP has been shown  to last for several weeks in vivo (Bliss and Gardner-Medwin, 1973), experimental  evidence  indicates  1979;  Swanson, Teyler and Thompson, 1983).  LTP is not clear. contribute  that  LTP gradually  decays over  A variety of changes have been described that probably  towards the maintenance of LTP, and some of the best studied Distinct ultrastructural  spines occur  exhibiting  in preparations  Van Harreveld and Fifkova, 1975).  enlargement  (Barnes,  However, the exact duration of  mechanisms are reviewed here.  1977;  time  of  spine  head  (Desmond  changes in dendritic  LTP (Fifkova  and Van Harreveld,  The observed changes included (1)  and Levy,  1981;  Fifkova  and Van  Harreveld, 1977), (2) widening and shortening of spine stalks (Fifkova and  CHIRWA Anderson,  1981,  1985), (4)  (3)  increased  length  of  synaptic  appositions  increased number of synapses (Greenough, 1984;  and Gorman, 1985;  Routtenberg, 1985), and (5)  50  (Fifkova,  Greenough, Hwang  increased number of sessile  spines and direct contact synapses (Chang and Greenough, 1984). ln view of the above structural  changes, it  has been speculated that  contractile proteins could be involved in LTP development Markham and Fifkova, 1986).  (Fifkova,  ln contrast, Lynch and co-workers have argued  for different types of dendritic morphological correlates investigators  reported  an  increase  in  the  binding sites, presumed to be excitatory induction  (Baudry  experiments,  1985;  and Lynch,  1979;  number  of  for  LTP.  specific  These  glutamate  synaptic receptors, following LTP  Baudry,  et a l . ,  Lynch and co-workers could not detect  1980).  In  significant  separate  changes in  spine area, spine number, spine neck diameter or length of the postsynaptic density  during  1984).  Baudry and Lynch (1980) subsequently proposed the  increased tetanic  LTP (Lee,  subsynaptic  stimulations  increased  et a l • ,  receptors caused C a  intra-dendritic  Ca  1980;  see  during  LTP.  influx  into  ++  ++  triggered  a  also  It  (calpain  I)  was activated  was  involvement  of  hypothesized  that  postsynaptic dendrites.  The  biochemical  involved phosphorylation of y-pyruvate dehydrogenase. proteinase  Chang and Greenough,  change  which  Then a membrane bound  which in turn caused proteolysis of  some membrane associated component(s)  (fodrin)  that  subsequently mediated  the uncovering of subsynaptic glutamate receptors previously not accessible in  synaptic  transmission  (Baudry  and  Lynch,  1980;  Eccles,  1983).  The  i n i t i a l promise that was inherent in this hypothesis has subsided, however, in  light  of  contradictory  reports  that have appeared  in the  literature.  Sastry and Goh (1984) replicated the glutamate binding studies used by Lynch and co-workers, but found that the increase in glutamate binding correlated  CHIRWA better with synaptic depressions rather than LTP.  51  Lynch, Feasey and Bliss  (1985) confirmed Sastry and Goh's finding that LTP is not associated with increases in glutamate binding sites. have de-emphasized their tors  and LTP.  Rather,  spectrin and other morphology of  In recent years, Lynch and co-workers  postulate correlating increased subsynaptic recepit  is now speculated that  cytoskeletal  synaptic contacts  proteins,  calpain degrades brain  leading  (Lynch, 1986;  to  cf.  alterations  in  the  Markham and Fifkova,  1986). 7.6.2 kinases  Protein  have  been  kinase  C.  implicated,  In for  membrane ionic channel functions.  recent  years,  example,  in  a  number  transmitter  of  protein  release  or  At least three types of protein kinases  have been identified, namely (1) cAMP or cGMP dependent kinases, (2) ++ ++ Ca -calmodulin dependent kinases, and (3) Ca -phospholipid dependent kinases  (also  activity  termed  "PKC")  (Nairn,  Hemmings and Greengard,  of PKC is dependent upon intracellular related  1985).  The  levels of diacylglycerol,  phosphatidylserine  (or  phospholipids)  (Nishizuka, 1984).  In addition, the actions of diacylglycerol are mimicked  by certain phorbol esters (Castagna, et a l . , 1982). soluble  forms  in  the  cytosol, or  they  are  and  intracellular  Ca  ++  PKC fractions exist in  bound to  membranes.  Akers,  et a l . , (1986) have recently reported that LTP is associated with the translocation of cytosol ic PKC to membranes.  The translocation of PKC was not  associated with any changes in the total PKC (i.e. It  soluble + bound form).  was concluded that the membrane bound PKC subsequently phosphorylated  "FI"  proteins, which are thought to be involved in the formation  of new  synapses (Routtenberg, 1985). 7.6.3  Increased transmitter  release.  Several studies have reported  that LTP is associated with increases in the release of the putative trans-  CHIRWA mitters  glutamate and aspartate.  radiolabeled  aspartate,  52  Using hippocampal slices pre-loaded with  Skrede  and Ma1 the-Sirenssen  (1981)  observed an  increase in the resting release of labelled aspartate following LTP induction.  Dolphin, Errington and Bliss (1982) subsequently reported long-last-  ing increases in the release of labelled glutamate that was newly synthesized from infused radiolabeled glutamine, during LTP in the dentate gyrus in vivo (see also Bliss, et a l . ,  1986).  At the present time,  interpreta-  tions of these release studies have been confounded by the fact that it  is  not known for certain i f glutamate is indeed the endogenous transmitter, and whether labelled ligands actually occupied the same sites as the endogenous neurotransmitter  (cf. Laduron, 1984).  Recently, different  experimental  approaches were utilized  in examining  probable increases in the release of endogenous transmitters during LTP.  ln  a combined electrophysiological and neurochemical study, Agoston and Kuhnt (1986)  observed a significant  relatively ing  increase  in  K -induced +  4  ^Ca  + +  uptake  in  pure synaptosomes prepared from minislices of area CA^ follow-  LTP  induction  across  the  Schaffer  collaterals-CA^  synapses.  Applegate, Kerr and Landfield (1987) observed that micrographs prepared from ultrathin (i.e.  sections of the CA^ subfield obtained from potentiated  LTP was i n i t i a l l y  established  in  CA^ of  rat  animals  hippocampus in vivo)  showed significant reductions in both local vesicle densities and distant vesicle  densities.  However,  these  investigators  observed  significant  increases in the number of vesicles attached to the presynaptic membranes of active zones (Applegate,  Kerr and Landfield, 1987).  found that the average area in the LTP tissue. the  notion  that  addition,  it was  and perimeter per spine significantly increased  Taken together,  LTP was,  In  in  part,  the above studies were consistent with due to  increases  in  Ca  dependent  CHIKWA  53  transmitter release. 7.7  Summary Much of the available evidence in the literature  indicates that LTP  production is dependent on the generation of sufficient depolarizations to presynaptic and postsynaptic regions.  Consequently, maneuvers that enhance  both presynaptic and postsynaptic depolarizations facilitate of LTP (Wigstrom and Gustafsson, 1983 and 1985a).  the induction  Conversely, manipulations  that diminish and/or interfere with the occurrence of these depolarizations antagonise LTP development (Malinow and Miller, 1986). predictions Wigstrom,  have been borne out 1988).  However,  with  there  experimental  are  many  To some extent these data  outstanding  (Gustafsson and features  questions) about LTP production that remain to be resolved.  It  for  that  certain  described  whether  in  the  the  various  types  hippocampus (e.g.  of  Ca  potentiations  (and  is not known have been  induced LTP, homosynaptic LTP,  heterosynaptic LTP, co-operative LTP, etc)  represent the same phenomenon.  Furthermore, it has not been directly tested whether inhibitory synapses can support LTP.  This may very well be likely, in view of the finding showing  that transient  exposures to  phorbol esters  spontaneous minlPSPs and minEPSPs Nicoll, 1986). then  the  induction  CA^ neurons  frequencies of  (Malenka,  Madison and  Presuming that LTP across inhibitory synapses is possible,  requirements of  in  increased the  for  LTP would  be  pre- and  postsynaptic depolarizations  unattainable  at  these  inhibitory  in  the  synapses.  Clearly, there are insufficient data on the exact role of depolarizations during LTP production. Studies  that  have utilized  agents  such as APV or  picrotoxin, have  tended to disregard potential presynaptic interactions of these drugs. explanations given for the facilitatory  effects  of  The  picrotoxin during the  CHIRWA induction of LTP tend to ignore the powerful by activated rather  "dendritic"  GABAg receptors.  inhibitory Dendritic  insensitive to picrotoxin or bicucculine.  influences  54 exerted  GABAg receptors  Then there is the more  basic question of the identity of the endogenous excitatory transmitters the hippocampus.  The failure  are  of many exogenous antagonists  of  in  glutamate  (and its analogs) to selectively block synaptic transmissions in the hippocampus raises the real prospect that the endogenous transmitters may not be glutamate or aspartate.  Moreover, the failure of APV to block the induction  of LTP in the mossy fiber-CA^ system is puzzling since glutamate putative  transmitter  fiber-CA^  in  synapses are  mossy fibers.  It  is  not  endowed with a class of  known  if  receptors  is the  the  that  mossy  function  like NMDA receptors, though insensitive to APV. The physiological significance of the observed synaptic morphological changes during LTP will dendritic  spines is  only be established once the  elucidated.  It  has been proposed that  serve to attenuate synaptic signals (Chang, 1952) of signals from different afferents 1970). tic  role  of  spines might  or permit the  'weighting'  impinging on the same dendrite  (Rail,  Spines could be a structural mechanism for the separation of synap-  apparatus  studies  functional  that  and have  thereby used  delimit  structural  synaptic  cross-talk.  dimensions  obtained  Yet from  simulation hippocampal  histological studies indicated that subsynaptic signal transients were only attenuated by less than 2 1984; Turner, 1984).  across the  spine neck  (Kawato and Tsukahara,  Presuming that these simulations accurately  synaptic  transmission  inherent  in increased spine sizes during LTP, for example, become somewhat  less important.  It  in  the  hippocampus,  then  the  apparent  reflected advantages  is evident from the above discussions that more studies  will be needed to clarify the mechanisms underlying LTP.  CHIRWA 8.  55  BARIUM AND SACCHARIN AS EXPERIMENTAL TOOLS  8.1 General ++  ba  and  substances  saccharin  useful  have  experimental  coupled asynchronous release Silinsky,  certain  1978).  Silinsky  characteristics  tools.  of  Ba  that  supports  transmitters  (Quastel  (1978) found that,  in  make  these  depolarization-  and Saint,  the  1988;  presence of  Ba , ++  stimulation of the motor axons caused a burst of miniature end-plate potentials  (MEPP) at the neuromuscular junction  (NMJ).  These MEPPs were caused  by the asynchronous release of transmitter.  Changes in MEPP frequencies at  the NMJ reflect  Castillo and Katz, 1954).  presynaptic mechanisms (del  view of the above, the effects of B a  on depolarization-coupled  ++  In  transmit-  ter release provide a method for examining presynaptic functions in neural circuitries  (Quastel, et a l . , 1988).  Tn the case of saccharin, this agent  interferes with the binding of nerve growth factor (NGF) to its "receptors", and it  also decreases NGF-dependent neurite growth in embryonic dorsal root  ganglia  cell  saccharin  (DRG)  on  NGF-dependent  substances that Ba  cultures  (Ishii, cell  Therefore,  differentiation  have neurite-inducing  and saccharin were used  ++  1982).  in  can  activities.  the  the  be  effects  used to  screen  The above features  experiments  in  this  of  thesis.  of The  characteristics of these substances are briefly reviewed in this chapter. 8.2  Barium 8.2.1  Chemistry.  Ba  number, valence and chemical radius Ca , + +  logical  of Mg  Ba  and  ++  Ca  ++  ++  is Sr  smaller + +  channels,  is  closely  properties than  (Stokes, and B a  that 1964).  ++  related  to  Ca  (Rosseinsky, 1965). of  the Ba  currents  ++  are  other  alkali  readily usually  in  atomic  The hydrated earth  permeates larger  metals physio-  than  cur-  rents  carried  by  Blaustein, 1982; physiological  Ca  (Augustine  and  Eckert,  Potreau and Raymond, 1980).  effects  of  Ba  that  have  1984;  CHIRWA  56  Nachshen  and  In this regard, most of the  been  examined  pertain  to  its  actions on membrane ionic currents and transmitter release. 8.2.2  Transmitter  release.  Transmitter  release  mission is thought to be c r i t i c a l l y dependent on Ca  during neurotrans-  entry into presynap-  tic terminals via specific voltage sensitive channels which open in response to  membrane  (Augustine,  depolarizations Charlton  induced  and Smith,  by  1987;  Baker,  Ridgway, 1971;  Dodge and Rahamimoff, 1967;  Miledi,  Quastel  1973;  et a l . ,  mediates the synchronous quantal  presynaptic  1988;  1972;  action Baker,  Katz, 1969;  Rubin,  1970).  potentials Hodgkin and  Krnjevic, Unce  release of transmitter.  1974;  inside,  Ca  ++  At the NMJ, the  vesicles in nerve terminals are taken to be the morphological correlates of acetylcholine (ACh) quanta (Ceccarelli and Hurlbut, 1980, Whittaker, 1959). Each MEPP recorded at the  postsynaptic junction  reflects  the  all-or-none  discharge of the ACh content of one synaptic vesicle (ael Castillo and Katz, 1954;  Fatt and Katz, 1950;  Fatt and Katz, 1952).  The end-plate  potential  (EPP), therefore, consists of the "synchronous" release of two or more ACh quanta  (del  Castillo and Katz, 1954).  According to stochastic models of  transmitter release, the mean number of quanta released by an action potent i a l (i.e. quantal content, m) is equal to the product of the number (n) of quanta capable of  responding and the  respond (del Castillo and Katz, 1954;  average  Katz, 1969;  probability  (p)  that  McLachlan, 1978);  they  thus  m = np  Under conditions where "p" is very small (e.g. in the absence of extracellu-  CHIRwA Tar  Ca ),  the  ++  Poisson's (e.g.  distribution  statistics.  of  "m" in  time  When conditions  in the presence of extracellular  binomial statistics.  is  adequately  increase Ca ),  "p",  feasible  "m" is best described with  ++  In general, the above models have adequately described  (Augustine,  Greenfield Jr, 1986; The entry of Ca causes  described by  and presumably "n"  quanta! events at various chemical synapses wherever been  1987;  Aurbach,  Johnston and Brown, 1984;  increases in  such recordings have  1971;  into presynaptic terminals,  Hackett,  Cochran and  Katz, 1969;  Kuno, 1971).  in response to an impulse,  "p" and "n" and results  in multiquantal  transmitters, which is necessary to support the EPP or EPSP. permeates these Ca  Quastel the  Ba  ++  readily  the "asynchronous" quantal discharge of  in response to a single nerve impulse (Quastel and Saint, 1988;  et a l . ,  1988;  multiquantal  However,  release of  channels in response to a single nerve impulse during  depolarizations but only initiates transmitter  57  EPP or  Silinsky  presence of  Silinsky, EPSP  1978).  in  response to  (1978) found that  Ba  produced " . . . a  Therefore, a  repetitive  slowly  Ba  cannot  ++  single  nerve  developing  nerve  stimulation  avalanche  of  support impulse. in  the  MEPPs",  which was associated with an underlying slow depolarization of the postsynaptic  membrane  (Silinsky,  1985).  Interestingly,  the  slow  produced by repetitive high frequency nerve stimulation Ba  ++  was  linearly  concluded that  8.2.3 have  been  ++  to  frequencies  of  correlate  of  ACh release  in the presense of  MEPPs.  increases in MEPP frequencies at  electrophysiological bathed in B a  related  depolarization  Silinsky  (1978)  the NMJ represented  by stimulated  the  preparations  solutions (cf. Douglas, Lywood and Straub, 1961). K*  currents.  demonstrated  in  The  effects  various  of  Ba  preparations  ++  on  such as  K  +  conductances  spinal  neurons  (Ribera and Spitzer, 1987), mammalian hippocampus (Hotson and Prince, 1981),  CHIRWA dorsal  root  ganglion  acinar cells apparently  cells  and Matsuda,  (Iwatsuki and Petersen,  all  the  K  preparation,  Ba  1985).  conductances are  +  and voltage-activated  vated K  (Yoshida  K  pancreatic  accounted for  substitute  for  Ca  in  conductance (Iwatsuki and Petersen,  +  and  acinar  by the  inducing  1985).  cells,  same  the  Ca  In  ++  this  Ca  acti-  Presumably B a  ++  Ca  pancreatic  channels (Iwatsuki and Petersen, 1985).  +  can  In  1980),  58  ++  and  +  possess  similar  efficacies  in  subsequently  inhibiting  K  channels,  ++  from both sides of the membrane. for  inhibition  more  potent  1985).  of at  ++  the  Ca  activating  However, Ba +  activated these  K  K  has a much greater  channels, whereas  channels  +  (Iwatsuki  potency  ++  Ca  is much  and  Petersen,  In the hippocampus, Hotson ano Prince (1981) found that bath appli++  cations  of  +  ++  Ba  augmented  Ca  potentials  hyperpolarizations.  lhese effects  of B a  the  through  channels  influx  reduction  of of  demonstrated  Ba K  +  Ca  conductances.  a Ba -mediated  In  but  attenuated  were presumed to be caused by  ++  followed  presynaptic  increase  K -dependent  in the  by the  terminals,  presynaptic  Ba  -mediated  Sastry  (1979)  terminal  action  potential refractory period, presumed to be a reflection of a widened action potential  caused by the  Hence the effects membranes Ba  ++  8.3  that  of B a are  blockade ++  on K  endowed  +  of  the  delayed  K  +  rectifier  currents seem to be present  with  Ca  ++  and  K  in all  conductances.  +  current. cell  Whether  interferes with all types of K currents in cells is unclear. +  Saccharin 8.3.1  Chemistry.  Saccharin  (chemical  name:  zisosulfonazole), discovered by Fahlberg in 1879,  2,3-dihydro-3-oxoben-  is a potent non-caloric,  synthetic, non-sucrose sweetening agent (Arnold, Krewski and Munro, 1983 for review). but  its  Saccharin has a low solubility sodium salt  is  very  soluble  (1  (1 g dissolves in 290 ml water), g dissolves in 1.5  ml water).  CHIRWA Furthermore,  saccharin is very stable,  particularly  3.3-8.0, and only decomposes at temperatures Krewski and Munro, 1983; 8.3.2  at  59  pH ranges between  in excess of 230° C (Arnold,  Swinyard and Lowenthal, 1980).  Disposition.  The use of saccharin as an a r t i f i c i a l  sweetener  was considered to be safe, since it was thought to be an inert substance in humans.  Saccharin is not metabolized in the body (Byard and Golberg, 1973;  Lethco and Wallace, 1975;  Sweatman and Renwick, 1979), and it  is  largely  excreted via the kidneys (mostly through tubular secretions without reabsorption)  (Colburn,  ly79).  The substance crosses the placenta in pregnancy (Ball, Renwick and  Williams, 1971;  1977;  Bekersky  and Blumenthal,  Matthews,  Fields  West, 1979), and it  Krewski  and Munro,  blood-brain barrier  Renwick  et a l . ,  is excreted into milk during lactation  (Arnold,  It  is  Fishbein,  unclear  1973;  and Sweatman,  Pitkin,  1983).  and  1981;  whether  saccharin crosses  in any significant amounts (Pitkin,  et a l . , 1971).  the An  extensive search of the literature only produced a few studies demonstrating direct physiological effects of saccharin, with the exception of controversial reports related to its  presumed involvement  in certain tumors of the  urinary bladder  (Arnold, Krewski and Munro, 1983 for review).  than any other  single factor,  saccharin as an a r t i f i c i a l 8.3.3  has led to the  This, more  almost complete disuse of  sweetener.  Tumor promoter.  On reviewing the literature it was interest-  ing to note that the causal relationship between saccharin and carcinogenesis remains unresolved, due to the equivocal nature of the toxicological studies (Arnold, Krewski and Munro, 1983 for review).  Some of the  early  studies on saccharin failed to control for the mutagenic and/or carcinogenic properties of impurities that were present in the preparations. in most animal  Moreover,  studies, excessively large doses of saccharin (relative to  CHIRWA  60  the maximum saccharin concentrations used in food and beverages) were used to  demonstrate  purported  saccharin-induced tumors  Munro, 1983 for review).  (Arnold,  Krewski  and  In recent years, saccharin has been suggested to  be a tumor promoter in'carcinogenesis (Bryan, Erturk and Yoshida, 1970). The process of  carcinogenesis  biochemical phases, namely (1) and Boutwell, 1978).  changes.  initiation,  to  and (2)  involve  two  distinct  promotion (Scaga, Sivak and it  is thought to be  In contrast, promotion is the result of epigene-  Substances that induce cancer presumably act through one or  both of these phases. initiation  thought  Initiation is irreversible,  due to a mutagenic event. tic  is  requires  the  Initiation alone does not result action  of  a promoting  agent  in cancer, rather to  produce cancer.  Batzinger, Ou and Bueding (1977) examined directly the effects of several saccharin preparations on base pair substitution and frameshift mutations in his" Salmonella typhimurium tester  strains TA10Q and TAy_8.  gators could not detect  any mutagenic  preparations  Ou and Bueding, 1977).  (Batzinger,  reported by several other investigators Trosko,  et a l . ,  1980;  Mondal,  activities  with purified saccharin Similar results have been  (Bryan, Erturk  Brankow  and  These investi-  and Yoshida, 1970;  Heidelberger,  1978),  using  different direct assay methods (e.g. Ames salmonella assay with or without rat In  liver  microsomal enzymes, host-mediated assay, dominant  contrast,  the  tumor-promoting  studies such as the following. "subthreshold"  doses of  of  saccharin  is  test).  suggested  in  Cohen, et a l . , (1979) fed Fischer rats with  a tumor  furyl)-2-thiazolyj-formamide  actions  lethal  initiating  (FANFT), for  six  substance, N-L-4-(5-nitro-2consecutive weeks at  time the animals were separated into  several  were subsequently fed as follows:  control rat chow, (2) rat chow mixed  with 2% or 5% saccharin, (3)  (1)  groups.  which  The various groups  a period of six weeks post-FANFT was allowed  CHIRWA before  instituting  rat  chow mixed with  2% or  controls, some rats were maintained on (a) with FANFT, or  (c)  rat  chow with 2% or  5% saccharin.  In  separate  control rat  chow, (b)  rat chow  5% saccharin.  After  animals were sacrificed for histological examinations. only rats  fed with FANFT followed  presented with urinary  bladder  by saccharin  tumors  results were consistent with the  61  (Cohen,  notion that  It  (groups  et a l . ,  two years,  was found that 2 and 3 above)  1979).  The above  saccharin might  be a tumor  promoter. 8.3.4 altering  Neurite the  growth.  process of  Tumor  cellular  mechanisms (Cohen et a l . , 1977; 1978;  lshii  et a l . ,  et a l . , 1977).  1978;  lshii  (DRG)  cell  act  by  Diamond, O'Brien and Rovera, 1977;  lshii,  O'Brien  through  to  unknown  development  cultures.  thought  as yet  Rovera,  neurite  are  differentiation  and Diamond, 1977;  (1982) tested the effects  growth factor-dependent ganglion  promoters  Yamasaki  of saccharin on the nerve  in embryonic chick dorsal root  The DRG cell  cultures  were  incubated  in  growth media containing the e-subunit of the mouse submaxillary gland nerve growth factor  (pNGF).  In addition, possible interactions between saccharin  and the binding of radiolabeled eNGF to intact cells obtained from dissociated GDR were examinea ( l s h i i , 1982).  It  was found that 48.8 mM saccharin  reversibly inhibited neurite outgrowth  in DRG cell  cultures  (lshii,  1982).  In contrast, sucrose, glucose or sodium chloride (50 mM used in each case) did not antagonise eNGF-dependent neurite development in DRG cell cultures. These findings illustrated the specificity of the saccharin mediated inhibition  of  eNGF-dependent neurite  changes in osmolality,  lshii  development,  and that  it  was not  due to  (1982) noted that developing neurites dia not  retract when subsequently exposed to saccharin, rather their rates of growth were greatly  diminished by saccharin,  ln the binding studies, 10-100 mM  CHIRwA saccharin significantly bound to cells (48.8 1982).  diminished the  amount  of  labelled  62  eNGF that was  mM saccharin reduced 3NGF binding by 60-65%;  The relationship between the saccharin-mediated  inhibitions  lshii, of  (1)  BNGF binding, and (2) eNGF-dependent neurite growth, was not established. 8.3.5  Inhibition of enzymes.  At about the same time that saccharin  was being reported to be a potential  tumor promotor, other studies began to  show that this agent inhibited the activities mid-seventies, Lygre (1974, 1976)  of specific enzymes.  In the  discovered that saccharin caused a reduc-  tion of close to 50% in the enzymatic activities of beef and rat glucose-6phosphatases.  Later, Vesely and Levey (1978) showed that saccharin s i g n i f i -  cantly inhibited guanylate cyclase in different tissues, including that from the urinary bladder.  In recent years, the effects of enzyme inhibition by  saccharin have clearly  been established in micro-organisms.  For example,  Streptococcus mutans is considered to be the major etiological agent responsible  for  dental  caries  (Hamada  and Slade,  1980).  Many reports  literature demonstrated that saccharin inhibited  the  growth and acid production of S. mutans (Linke,  1977;  and Chang, 1976;  Tanzer and Slee, 1983).  in  the  carbohydrate-dependent Linke, 1980;  Linke  Linke and Kohn (1984) assayed the  specific activities of glycolytic enzymes in cell-free extracts of S. mutans NCTC 10449 and found that saccharin caused significant reductions in the order of 37-58%; mg/ml, respectively) phate  dehydrogenase,  (maximum  for saccharin concentrations of 0.02-20  in the activities phosphoglycerate  inhibition of specific activities  reductions  of hexokinase, glyceraldehyde-3-phosmutase  and  pyruvate  kinase.  The  of glycolytic enzymes was proposed to be  the mechanism by which saccharin inhibited  the  growth of micro-organisms.  Micro-organisms such as S. mutans generate their ATP via the Embden-MeyerhofParnas (EMP) pathway  (Brown and wittenberger, 1971).  ln view of this,  it  CHIRWA was suggested that the diminution of the specific activities the EMP pathway resulted  in the reduction  63  of enzymes in  of both vegetative  growth and  fermentative acid production in these micro-organisms (Linke and Kohn, 1984). A recent study has extended the number of enzymes that are inhibited by saccharin.  Brown and Best (1986) reported that saccharin also diminished  the activities of the following enzymes; phosphate  dehydrogenase,  lactate dehydrogenase, sorbitol-b-  mannitol-l-phosphate  hyde-3-phosphate dehydrogenase, glutamate  dehydrogenase,  glyceralde-  dehydrogenase, glucose6-phosphate  dehydrogenase and 6-phosphogluconate dehydrogenase.  More importantly, Brown  and Best (1986) found that saccharin competitively  antagonised the binding  of reduced coenzyme, NAD, to lactate dehydrogenase (saccharin increased K values for  NAD from 0.033 to  0.250 mM;  K  i  for  saccharin was 6.2  m  mM).  Interestingly, all the enzymes inhibited by saccharin were those that bound to substrates or coenzymes which contained  adenine  and/or  pyridine  ATP, NAD(H), NADP(H)) (Brown and Best, 1986, cf. Linke and Kohn, 1984). was noted that saccharin shared spatial  and structural  similarities  (i.e. It with  adenine and pyridine, the functional groups of NAD, and it was surmised that these similarities  accounted for the observed competitive antagonism (Brown  and Best, 1986). It  is  intriguing  to  note  that  saccharin  activities as well as inhibits enzyme activities. ses are related  is not clear.  antagonises  NGF-dependent  Whether these two proces-  Brown and Best (1986) suggested that the  negative interactions of saccharin with the EMP pathways, for example, could result in the accumulation of intracellular  carbon.  This carbon could then  be channelled into other metabolic pathways in the c e l l . ling  of  carbon  differentiation  subsequently  modulates  in general is not clear.  NGF-dependent  How this channelgrowth  or  cellular  CHIRWA 9. 9.1  64  METHODS AND MATERIALS Animals 9.1.1  rabbits  Source.  (either  sex)  Male Duncan Hartley guinea-pigs and New Zealand White were obtained  from the  Animal  University of British Columbia, Vancouver (Canada). used standard animals.  animal  Their  care  procedures  for  the  guinea-pigs were weaned after  were fed  rabbit  maintenance 14 days,  indoor and outdoor  chow supplemented  with  Centre  of  The  The Animal Care Centre  guinea pig chow that was supplemented with vitamin C. in a communal pen comprising of  Care  laboratory  and were fed on  The rabbits were kept areas.  cabbage.  of  Both  These animals  guinea  pigs and  rabbits had access to water ad libidum. 9.1.2 8-12  Animal feed and housing.  male guinea-pigs  from the animal  Once a week, typically on Mondays,  (200-250 g, approximately  unit and used for  28 day old) were received  studies in that same week.  Rabbits of  either sex (2-3 kg, approximately 42-56 days old) were usually obtained in sets of two, 2-3 times in a week. of  the  Department  British Columbia.  of  Animals were housed in the animal rooms  Pharmacology and Therapeutics  the  University  of  About 4-6 guinea pigs were placed in each wire cage (58 x  35 x 53 cm, in size) in the animal room. to food (guinea pig chow) and water. cage (58 x 35 x 53 cm, in size; pigs).  at  These guinea pigs had free access  Each rabbit  was kept in a separate  different room from that used for guinea  The animal rooms had controlled temperatures  (22-23° C) and humidity  (50-55 ) with set 12-hourly day and night periods. 9.2  Slice preparation Hippocampal  slices  were prepared  from male  guinea  pigs  as follows.  Animals were i n i t i a l l y cooled (30-40 min) on an ice pack in a dessicator to  CHIRWA  65  a rectal temperature of 28-30° C and maintained on a mixture of 1.5-2% halothane  in  carbogen  brated with this  (95% 0  a n 2  d  5%C0 ).  The  2  halothane-carbogen  mixture  dessicator  (in  was  pre-equili-  concentrations  for general anaesthesia) before introducing the animal.  sufficient  To obtain  slices,  the skin on the head was cut and an insertion made under the base of the skull. sagittal  A pair of small scissors was used to cut through the skull along the suture line and the sides pulled apart to expose the brain.  brain was carefully removed and placed on dissecting paper. pus was dissected free  and quickly  transferred  medium that was continuously being oxygenated.  to  The  Each hippocam-  cooled physiological  Subsequently, each hippocam-  pus was then sectioned transversely to the septotemporal axis at a thickness of 450 um on a Mcllwain tissue chopper.  Serial sections were separated with  fine stainless steel spatulae in a plate containing previously cooled (5° C) physiological  medium and equilibrated  surgery  to  slice  cooling  of  the  preparation  animal  was  with carbogen.  completed  significantly  the  slice  chamber.  Slices  were  within  increased  slices obtained from each hippocampus.  120; 2.0  KC1,  the  proportion  Initial of  viable  positioned  between  two  nylon  nets  NaHC0 , 3  (see  26;  NaH P0 ,  1.3;  Table  9-1,  list  2  4  for  to to  The chambers were perfused  mL/min with the standard medium containing in mM:  and glucose, 10.0  used). 7.4)  3.1;  3 minutes.  from  Finally, slices were transferred  minimise movement as well as permit submersion. at a rate of 1.5-2  The procedure  CaCl , 2  of  2.0;  physiological  NaCl, MgCl , 2  media  The standard medium was pre-gassed with carbogen (pH of medium, ca.  and maintained at 32 ± 0.5° C.  In addition, the carbogen flowed over  the top of the slices in the slice chamber.  Slices were allowed to equili-  brate with the standard medium in the slice chamber for minutes prior to recording.  a minimum of 60  CHIRWA 9.3  66  Slice selection About 8-10 slices were selected from the middle portion of each hippo-  campus in vitro. following c r i t e r i a .  The selection of  slices to  be used was based on the  Each slice had to be intact  unmashed borders, i . e . ,  slice edges.  with well  defined and  Only slices with a clearly discernable  cornu ammonis and dentate gyrus cellular  layers were chosen.  Furthermore,  the selected slices had to have clean and even (smooth) surfaces, i . e . , 'mashy' or 'flaky'.  Such slices could support physiological responses for  periods up to 12 hours. tion.  not  Each experiment was typically of 2-4  hours dura-  Hence 2-3 of the selected slices from a given animal could have been  used sequentially.  But to minimise variability between slices from the same  animal (and those of other animals)  caused by different  exposure times to  the standard medium, only one slice per animal was usually used.  Conse-  quently, the total times of exposures to bathing medium and experimentation for  different  slices were essentially  comparable  during  each series  of  experiments. 9.3.1  Slice chamber and perfusion method.  the present experiments Department Columbia.  of  (Figure 9-1)  The slice chamber used in  was manufactured by Mr. C. Caritey,  Pharmacology and Therapeutics,  The  University  of  British  The full descriptions of the slice chamber used were reported in  a publication from this  laboratory  (Pandanaboina and Sastry,  basic components of the slice chamber were as follows;  (a)  1984).  The  a raised stage  constructed of plexiglass with (b) a circular chamber of diameter 7.5 cm and depth  0.7 cm bored into the top surface, and (c)  regulating (Figure  aluminum plate  that was attached  a special  beneath  the  temperature-  circular chamber  9-1).  9.3.2  Standard and test  media.  The standard and test  contained in separate 50-mL polyethylene barrels.  media were  Carbogen lines for media  CHIRWA  Figure 9-1. Recording chamber and perfusion method for the maintainance of transversely sectioned guinea pig hippocampal slices, lhe slice chamber consisted ot a raised stage made from plexiglass that had a circular incubating chamber (*) bored into the top surface and, a temperature-regulating bar was fixed within the stage. The standard physiological medium was added into the reservoir (feed-tank) with a flow-line leading into one of the media barrels. The test physiological media were added into the remaining media barrels. Any one of these madia barrels could be used to perfuse the incubation chamber through flow-lines (continuous lines with arrows) that travelled via the common manifold then made turns within the temperatureregulating aluminum bar before entering the incubation chamber. The oxygenation flow-lines are indicated by the dotted lines.  CHIRWA oxygenation terminated  in each of  these  barrels.  The barrel  68  containing  standard medium was in turn connected to an elevated feeding tank (volume, ca.  2 L)  feed-tank barrel  which was the  source Of  was continuously oxygenated  was connected to  medium  as well.  a common manifold.  opposite end of the manifold polyethylene tube.  standard  led to the  (Figure  A tube A single  9-1).  The  from each 50-mL outlet  from  the  slice chamber via a connecting  Control and/or test solutions were introduced into the  slice chamber via an inlet at the bottom of the slice chamber.  Continuous  drainage was via a suction outlet created for that purpose (Figure 9-1).  A  balanced inflow and outflow of solutions ensured the maintenance of constant solution levels medium at all  in the slice chamber.  times during the experiments.  whole perfusion set-up permitted; test  The slices were submerged in  solutions; (b)  spaces within the  adequate  (a)  properties,  the  the rapid exchange of standard and  oxygenation  system; and (d)  Among other  the  of  solutions;  the regulation  (c)  minimum dead  of solution  temperature  with a maximum of 0.5° C fluctuation. The standard and test media used in the experiments are summarized in Table 9-1.  Most of these media were freshly prepared to their final consti-  tuent concentrations on the day of the experiment.  However, stock solutions  of  sodium saccharin, were  substances  such  as  prepared once a week.  N-methyl-DL-aspartate  or  These stock solutions were refrigerated  when not  being used. 9.4  Endogenous sample collections 9.4.1  Guinea pig hippocampus.  Guinea pigs were i n i t i a l l y  tized with 1.5 g/kg urethane, given intraperitoneally.  anaesthe-  When each guinea pig  was adequately anaesthetized, it was transferred and then positioned into a  Table  9-1.  Medium  2  Control  Raised Ca  + +  Low  Ca  free Ba  Moderate High  + +  Ba  Ba + +  + +  Composition  NaCl  2 KC1  2  of media  NaHC0  2 3  (in mM)  NaH P0„ 2 4 o  used  2 d-Glucose  for  1  hippocampal  CaCl  2 2  MgCl  slices  3 2  BaCl  120  3.1  26  1. 8  10  2.0  2.0  -  120  5.0  26  -  10  4.0  4.0  -  120  3.1  26  1.8  10  -  3.5  120  5.0  26  -  10  3.5  4.0  0.5  120  5.0  26  -.  10  2.0  4.0  2.0  120  5.0  26  -  10  0.5  4.0  3.5  4 2  MnCl  0.5  Source: (1) F i s h e r S c i e n t i f i c C o . ; (2) BDH C h e m i c a l s L t d . ; (3) S i g m a C h e m i c a l s C o . ; ( 4 ) J.T. B a k e r C h e m i c a l s Co.  2  CHIRWA stereotaxic  holder.  For continued anaesthesia,  each ginuea pig  maintenance doses of urethane whenever necessary (typically during the collection experiments.  Once in  following  skin  surgery  retracted. sagittal  was  done.  The  the  above  Two spherical holes (4 mm in diameter; suture line) were carefully  drilled  received  every 1-3  stereotaxic the  70  skull  holder, was  cut  hr) the and  one on each side of the  into the exposed skull.  The  centre of these holes was around the following co-ordinates (with the bregma as the  reference  point);  posterior,  8 mm, and  lateral  5 mm.  It  was  important to prevent bleeding by ensuring that the dura was not punctured when the holes were made in the skull.  Small spherical cups (10 mm long,  outside diameter approximately 4 mm) constructed from drinking straws, were positioned into each hole as follows. fine scissors and forceps.  The dura was removed carefully with  Then one end of the straw-cup was pushed deeper  into the brain tissue down to a depth of ~ 7 mm from the surface of the skull.  Dental wax was applied between the straw and the skull, and around  much of the exposed skull. Suction was used to clear the cortical tissue within the cup, but never beyond the  depth  of  the  inserted  cups.  Once the  unwanted  tissue  was  cleared, the cups were rinsed with oxygenated physiological medium until the fluid was clear. experiments.  At this time, the animals were ready for the collection  Using micromanipulators,  introduced into each cup.  a bipolar stimulating  The stimulating electrode was inserted down to a  distance of about 7.25 mm from the surface of the skull (i.e. the CA-^ area of collect 10.5.1).  fluids  the  electrode was  hippocampus).  from each cup at  Modified the  suction  appropriate  lines  intervals  to penetrate were used to (see  section  When all the collections were done, the animals were sacrificed,  and their brains removed.  The hippocampi were dissected out and examined.  CHIRWA I t was  c l e a r f r o m t h e markings  on t h e hippocampal  surfaces that they  been p e n e t r a t e d by t h e s t i m u l a t i n g e l e c t r o d e down t o t h e s t r a t u m regions.  71 had  radiatum  In some c a s e s , t h e bottom end o f t h e cup had c u t t h r o u g h i n t o t h e  a l v e u s ; samples from t h e s e a n i m a l s were d i s c a r d e d . 9.4.2  Rabbit  maintained  on  neocortex.  halothane  Rabbits  (1.5-2%)  (2-3  and  kg)  were a n a e s t h e t i z e d ( 9 5 % Gv,  carbogen  m i x t u r e s and were r e s t r a i n e d i n a s t e r e o t a x i c a p p a r a t u s . made i n t h e s k u l l , two cups ( d i a m e t e r : o f t h e n e o c o r t e x t h a t was  exposed  was  suction  in contact with line  was  lowered  the with  f i x e d to the  i n n e r end  In a d d i t i o n , a  micromanipulators fluid  C0 ) 2  bore-holes  o f t h e d u r a ( F i g u r e 9-2).  neocortex.  s u c t i o n l i n e s were used t o c o l l e c t  Through  5%  were p o s i t i o n e d on t h e s u r f a c e  by removal  r i n g - s h a p e d s t i m u l a t i o n e l e c t r o d e was that  8 mm)  and  and  i n each  into  o f each  A cup  single  on-off  cup.  These  each  cup, whenever n e c e s s a r y .  The s u c t i o n l i n e s l e d t o a common c o n t a i n e r t h a t was kept on d r y i c e . 9.5  PC-12 Frozen  Rat Pheochromocytoma c e l l  c u l t u r e s o f r a t pheochromocytoma PC-12  F-5876) were o b t a i n e d from American were m a i n t a i n e d 4x10^  PC-12  line  Type C u l t u r e s , USA.  i n s e a l e d ampules.  cells.  These  cell  Each  ampule  ampules were packaged  lines  (Batch  No.  These f r o z e n c e l l s  contained  approximately  on d r y i c e d u r i n g  shipment  and d e l i v e r y was e f f e c t e d w i t h i n t h r e e days ( i . e . between R o c k v i l l e (U.S.A.) and  Vancouver  (Canada)).  Upon r e c e i p t , each  a t t a c h e d t o a s t r i n g and suspended The  j a r with  i t s c o n t e n t s was  ampule w i t h PC-12  in a j a r f i l l e d with  placed  in a walk-in  cells  was  liquid nitrogen.  refrigerator.  Every  a l t e r n a t e day, l i q u i d n i t r o g e n was added t o t h e j a r , t o r e p l a c e amounts l o s t through evaporation.  CHIRWA  Figure 9-2. Positioning of small cups onto rabbit neocortical surface for collection of samples in vivo, ihrough bore-holes made in the skull, two cups (diameter: 8 mm) were positioned on top of the neocortex that was exposed by removal of the dura.  CHIRWA 9.6  73  Electrical instruments 9.6.1  Amplifiers.  Extracellular responses were amplified with the  Western  Precision  Instruments  (WPI)  differential  pre-amplifier  DAM-5A.  This pre-amplifier had a maximum gain- of 1000X.  model  During recordings,  the low frequency (10 Hz) and high frequency (10 KHz) f i l t e r s were set at 0.1 Hz and Wide-Band, respectively.  The amplified physiological potentials  were then led to the Data Precision 6000 waveform analyser 9.6.3).  Intracellular  responses were fed into the Dagan single electrode  system, model 8100-1. pre-amplifier current clamp.  This unit had three operational modes namely:  only (bridge-current  stimulator  (see section  (switched  clamp),  current  (b)  clamp),  pre-amplifier and  (c)  (a)  and switched  switched  voltage  The current clamp or the voltage clamp mode, had sample and hold  times of 10 microseconds, and an adjustable switching frequency of 500 Hz-25 KHz.  The probe had an input  current of 1 pA.  resistance of  10^  M  with an input bias  Some other operational features of this unit included an  adjustable DC offset (range ± 1000 mV) and adjustable capacitance compensations (0-15  pF).  Most of the intracellular  recordings were done with the  unit set in the switched current clamp mode (filters at wide-band), and 10X gain. 9.6.2  Stimulators.  Square wave pulses were delivered  through an  isolation unit type DS2 regulated by the digitimer programmer D4030. natively,  the  Grass  S88 stimulator  was used to  drive  the  Alter-  photoelectric  constant current units. 9.6.3 the  Data  Oscilloscopes. Precision 6000  The amplified field potentials were fed into Universal  Waveform  Analyser  model  611.  This  programmable unit had capabilities for digitizing and storing analog signals (sampling speeds;  100 KHz;  digitizing resolution up to 14 bits;  memory:  CHIRWA 48K bytes). An outlet  74  The processed signals were displayed on the cathode ray tube. from the Data Precesion 6000 unit fed into a plotter.  Outputs  from the Dagan amplifying system were fed into the Tektronix 5113 Dual Beam Storage Oscilloscope. Tektronix  This unit  5A14N four-channel  amplifier,  amplifier,  (b)  (DC-offset,  and high  plug-in modules;  Tektronix  and (c) Tektronix 5B12N dual time base.  outputs from the Dagan amplifier module  had the following  5A22N  (a)  differential  Intracellular  recoraing  were fed into the differential  amplifier  frequency  filters  at  0.1-1  KHz).  signals were fea into one or more of the four-channels amplifier.  Current Any of  these signals could be viewed on the oscilloscope. 9.6.4  Magnetic tape recorder.  taped in their 3968A  instrument  amplifier  entirety  (on-line).  on magnetic tape  recorder.  were fed  into  Typically,  Most intracellular  Hewlett Packard  Outputs from the Tektronix one of  the  recorder was set at 9.52  using the  the  recording  channels of (and  cm/sec (FM Data,  experiments were  the  play-back)  5A22N  differential  HP tape speed  (HP)  of  band-width 1250 Hz).  recorder the  HP  Selected  segments of these tapings could be charted out on the HP-7404A plotter  or  analysed on the oscilloscopes, as necessary. 9.6.5  Paper plotter  and chart  recorder.  Permanent records of  all  observed signals could be plotted on the HP 7404A recorder and the HP 7470A plotter.  This last plotter was driven and controlled by the Data Precesion  6000 programmable acquisition unit.  In addition, signals displayed on the  oscilloscope type 5113 could be photographed directly. 9.6.6 able to  Miscellaneous.  During intracellular  pick up characteristic  recordings, it was desir-  sounds associated with  activities, as the recording electrode approached c e l l s .  specific neuronal From these sounds,  it was possible to determine the "position" of the electrode tip within the  CHIRWA tissue.  For this purpose, an output from the Dagan amplifier  the Grass AM-8 Audio Monitor  (low  filter,  100 Hz;  was fed into  and high f i l t e r ,  KHz).  To facilitate the penetration of c e l l s , the intracellular  trode  assembly was attached  model 607W.  With this unit,  to  75  0.3  micro-elec-  a David Kopf Instrument Microdrive  unit,  the microelectrode could be lowered into  the  recording region in pm steps (100-200 steps/sec). 9.7  Stimulating and recording electrodes 9.7.1  Stimulating  electrodes,  model  SNEX-100 with shaft  Instruments) were used. Each stimulating  electrodes.  Concentric lengths  of  bipolar  stimulating  50 mm (Rhodes Medical  These electrodes had resistances of around 1.0 Mft.  electrode  was replaced whenever  its  resistance  signifi-  cantly increased to greater than 5 Mfi (occurred after 5-7 weeks of continuous use), i f  this resistance could not be lowered by basic techniques of  cleaning the electrode. 9.7.2  Recording  glass micropipettes  electrodes.  Standard  (internal diameter,  WPI) were used to prepare extracellular  fiber  1.02 mm;  filled  borosilicate  outside diameter,  recording electrodes.  1.5 mm:  These micro-  pipettes were pulled to fine tips (tip diameter, 1-3 ym) on Narishige Scientific  Instruments'  NaCl  (resistances;  vertical electrode puller type PA-2, and f i l l e d with 4 M 0.5-1.5 Mn.  fiber f i l l e d micropipettes  Intracellular  (internal diameter,  1.0 mm:  WPI) pulled to fine tips  resolved  under  Scientific  microscope set  Instruments'  at  vertical  electrodes 0.76  (submicrons tip  mm;  outside  diameters,  400X magnification) electrode  were made from  puller  could not be  on the  type  diameter,  Narishige  PA-81.  pipettes were f i l l e d with either 3 M potassium chloride (resistances; Mo,), 2 M potassium acetate 50-90 Mfl).  These 50-90  (50-90 Mn) or 3 M cesium chloride (resistances;  CHIRWA 10.  76  EXPERIMENTAL SCHEMES  10.1 Intracellular recordings Standard intracellular Briefly,  microelectrode  "null-bridge" method.  techniques were used in the present  studies.  impedances were determined with the "Z-test" and  The Z-test was done by using the Dagan 8100-1 to pass  1 nA (10 ms, at 100 hz) through the microelectrodes, and the resulting voltage  outputs  were proportional  in amplitude  ances.  The null-bridge method used external  to microelectrode  trigger  imped-  sources to generate  currents (0.2-1 nA, for 200 msec) that were passed (via the Dagan 8100-1) through the microelectrodes.  The amplifier bridge was subsequently adjusted  to provide a zero stimulus voltage  at the output.  The magnitude of the  adjustment needed to balance the bridge, corresponded linearly to the resistance of each microelectrode (in Mfi). Microelectrode impedances were determined at three different  times (null-bridge method):  trations; (2) upon impalements; and (3) after retrieval the experiments were completed. similar  in each experiment.  (1) before cell penefrom the c e l l s , when  The values obtained for (1) and (3) were  The values for (2) were higher  contributions of membrane resistances in these measurements. resistances could be estimated by subtracting values those of (2).  due to the  Hence membrane  in (1) or (3) from  In some experiments, the membrane input resistance (R ) was n  continuously monitored with constant hyperpolarizing intracellular (0.5-1 nA, 200-300 msec at 1 Hz).  Whenever  appropriate,  determined with graded hyperpolarizing intracellular nA,  100-200 msec, at 1 Hz).  The amplitudes  current  R  n  currents was also  pulses (0.5-1  of the resulting  membrane  potential shifts at plateau were plotted as a function of the currents used (Figure 10-1).  The calculated slopes of these curves corresponded to the  input resistances in Mn.  200 ms  B  0-5 nA 200 ms INWARD CURRENT (nA)  0-7  0-5 »  i  0-3 •  0-1  t i l l  X •< •v  rO  m  73  s  r>  70  Control  1  0  z  3  03  70  in 79 m M  2  z 3 <  Figure 10-1. Determination of cell input resistances with intracellular injections of graded hyperpolarizing current pulses into lb l .' ™ ® are shown the membrane potential shifts from rest in a CA^ neuron induced by graded intracellular current pulses (BJ applied in random order. Note that action potentials could be induced with a depolarizing current pulse thereby confirming that this cell was a neuron. [NB: Low resolution and truncation of action potentials is due to photographic reproduction of the traces from the oscilloscope.] In determining the cell input resistances, the amplitudes of the membrane shifts at steady-state (*) were plotted as a function of the hyperpolarizing current pulses as illustrated in (C_). The slopes obtained from these I-V curves yielded the cell input KC1 [CA neuron; RMP, -60 mV; R , ~27 M resistances in Mn. electrode.] L A  n e u r 0 f  s  lb  n  CHIRWA The values of directly  the  resting  membrane  from the oscilloscope.  potentials  Typically, the  (RMP)  cell  were continuously monitored during the experiments.  78  were obtained  membrane Whenever  potentials  appropriate,  the cell membrane potential could be clamped to desired voltage levels, with passage of appropriate steady DC currents generated by the Dagan 8100-1 but this was not necessary for most cells studied.  Only cells that presented  with stable  or more negative  (i.e.  non-fluctuating)  RMP of  -60  greater than 25 Mft, were used for data collection. in  CA^  or  b  (0.1-7  nA,  radiatum  neurons  were evoked  50-400 msec; 0.2-0.02  electrodes  10-2),  and  where  neurons.  positioned Schaffer  These  in CA^ in  the  direct  current  stimulations  of  injections the  stratum  In some experiments,  neurons were evoked with stimula-  CA^ apical  collaterals  antidromic  n  Intracellular responses  (10-150 uA, 0.01-0.3 msec, at 0.01-0.2 Hz).  antidromic action potentials ting  Hz)  with  and R  make  action  dendritic  synaptic  potentials  region  contacts  presented  (Figure  with  with  CA^  distinct  thresholds of activation and could be evoked in Ca -free medium. ++  10.2 Extracellular recordings Square wave pulses (10-150 uA, 0.02-0.8 msec, at 0.2 Hz) were used to stimulate the stratum radiatum (Figure 10-2). dendritic  recorded  Ca  medium.  -free  presynaptic parameters  volleys  potentials This  were  latter  and antidromic  in each experiment  examined procedure  initial  population  in  standard  permitted  responses.  were those that  amplitudes of 1.0-1.5 mV or dendritic these  Components of the somatic and  The  medium and  visualization  selected  elicited  responses were obtained,  their  of  stimulation  population  EPSP amplitudes of 0.5-1  in  mV.  spike Once  amplitudes were  constantly monitored for stability (at 0.2 Hz) for a minimum of 30 min.  CHIRWA  Figure 10-2. A schematic illustration of the extracellular ositioning of stimulating and recording electrodes in the guinea pig ippocampus in vitro. Orthodromic extracellular and" intracellular responses in the CA^ area were evoked with stimulating electrodes positioned in the, stratum radiatum (2_). Antidromic action potentials in individual CA3 neurons were evoked with stimulating electrodes positioned in the Schaffer col laterals-CA^ pyramidal cell synaptic regions (5_). Orthodromic field EPSPs were recorded with recording electrodes positioned in the CAi dendritic region (4_) and, population spikes were recorded with recording electrodes positioned in the CA^ pyramidal layer (3_). Intracellular potentials in CAi neurons were recorded with intracellular electrodes positioned in the CA]_ pyramidal layer (3) and, these electrodes could also be used for intracellular stimulations. Similarly, antidromic intracellular action potentials in CA3 neurons were recorded with stimulatingrecording intracellular electrodes positioned in the CA3 pyramidal layer  CHIRWA These responses constituted the control responses.  80  Subsequent experiments  were conducted only if these control responses remained stable. 10.3 Induction of long term potentiation 10.3.1  Tetanic  stimulations.  were evoked with stimulations 0.02 or 0.2 Hz).  Postsynaptic responses in CA^ area  of the  stratum  radiatum  (test  frequencies,  The following train frequencies were used to induce synap-  tic potentiations of the population spike, field EPSP or intracellular EPSP; (1) 50 Hz, 5-250 pulses, (2)  100 Hz, 100, and (3)  400 Hz, 200 pulses.  each case, the same stimulus intensity was used throughout the  In  experiment.  In some experiments, intracellular responses were recorded using micropipettes f i l l e d  with 3 M CsCl.  induced  even  applied  internally.  10.3.2  if  most  This was done to  postsynaptic  K  Paired depolarizations.  with picrotoxin  (50  uM)  added to  test whether  currents  +  were  blocked  control  medium to  induction by this method (cf. Sastry, Goh and Auyeung, 1986).  (3-7  by  Cs  +  The following experiments were done  the  an impaled CA^ neuron was directly  LTP could be  facilitate LTP In each case,  depolarised with current  injections  nA, 300-400 msec) while the inputs in the stratum radiatum were being  activated at the onset of the intracellular  depolarization.  stimulations, termed pairings, were evoked at 0.2  Hz.  These conjoint  Typically, 5 to 15  consecutive pairings were given at any one time. 10.4 Effects of Ba  was  10.4.1  Ba  used  these  in  minEPSPs in  in hippocampus and evoked experiments  responses. to  C a ^ neurons following  A Ba  facilitate  the  stimulation  of  medium with occurrence the  stratum  low Ca of  evoked  radiatum.  It was anticipated that changes in the frequency of evoked minEPSPs could be used in assessing presynaptic functions in the hippocampus (cf,  Silinsky,  1978).  In order  to  select  following media were tested +4"  low Ba  medium, 3.5  Ba  (in  (1)  mM):  0.5  Ba  Ba  ++  81  and C a ,  the  + +  and 3.5  ++  Ca ;  and 2 Ca  4*4*  ;  termed  4*4"  Ca  termed  ++  4*4"  2 Ba  and 0.5  of chapter 9).  combination of  ,| 11  (2)  4*4"  and (3)  an ideal  CHIRWA  moderate  Ba  medium,  +4*  ; termed  high Ba  medium (see  Table  Each impaled cell was exposed to one type of Ba  perfused for 2-10 min.  9-1  medium  Whenever appropriate, picrotoxin (50 pM) or tetrodo-  toxin (1 uM) was added to these solutions to block GABA-ergic inhibition or inhibit  Na  dependent  +  membrane potential both  in  control  spontaneous  action  levels and R medium  and evoked  experiments, B a  ++  vals of 30 min.  control  and  p  during  Ba  Changes  applications.  ++  responses were  applications were repeated  in  In  addition,  recorded.  In  in the same c e l l s ,  at  some inter-  The results obtained in the above experiments were used to medium to be utilized in subsequent studies.  ++  Asynchronous release  and  respectively.  were determined for each impaled neuron,  intracellular  select an appropriate B a 10.4.2  potentials,  the  selected  Ba  of  LTP.  Both  the The  CA-^ neurons examined were impaled with recording electrodes f i l l e d  with  either 3 M KC1 (KC1 electrode) concentric  stimulating  electrode  contained  and  50 pM picrotoxin.  ++  media  transmitter  or 2 M CH C00K (KA electrode). was positioned  within 50 pm distance from the CA^ pyramidal  in  the  layer,  stratum  During 2-5 min Ba  tions,  were  following;  frequencies (1)  evoked  minEPSPs  determined  (2)  direct  intracellular  depolarizing current  mostly  applicaimmediately  single subthreshold or suprathreshold stimulations  stratum radiatum, (3-7  of  radiatum  to stimulate  proximal synapses (Andersen, et a l . , 1980a). the  A bipolar  3  of  the  injections  nA, 200-300 msec), and (3) after pairings of subthreshold stimulations  of the stratum radiatum with direct current injections into a CA^ neuron. In  studies  with  tetanic  stimulations,  slices  had  their  CA ~CA 2  4  CHIRWA pyramidal cell layers removed.  This was done to minimise the occurrence of  minEPSPs due to action potentials during picrotoxin and B a  ++  82  generated  applications.  in these fields,  particularly  The present experiments aimed at  determining whether evoked minEPSPs in the CA^ neurons in the presence of Ba  ++  were increased during  assessing 1978). Ca  increases  in  LTP.  This provided one method for  released  transmitter  Each slice was exposed to  (0.5  mM) for 2 min whenever  during  LTP  medium containing B a necessary.  (cf.  During Ba  Silinsky,  (3.5  ++  directly  mM) and  perfusion, the  number of minEPSPs following the EPSP evoked with stimulation of the stratum radiatum was determined.  Slices were then reexposed to normal medium for 15  min and the stratum radiatum was tetanized  (400 Hz, 200 pulses; stimulation  strength adjusted to evoke subthreshold EPSP only) to induce LTP. potentiation  was  detected  as  previously subthreshold EPSPs, then reexposed to  Ba  LTP.  second Ba  During this  following  the  determined.  ++  addition,  the  same  Ba  ++  LTP-inducing tetanus.  medium  increases  sometimes reaching threshold.  of  Slices were  the  induction of  application, the number of evoked minEPSPs  EPSP evoked with In  post-tetanus  containing medium 15 min after  applications were monitored. to  long-lasting  Long-term  the  stimulation  of  presynaptic  the  stratum  volleys  radiatum was  during  these  Ba  ++  In separate experiments, slices were exposed twice  with  During these B a  a ++  30  min  interval  without  the  applications, the frequencies of  minEPSPs after stimulation of the stratum radiatum were determined. 10.5 Effects of released endogenous substances in the hippocampus 10.5.1  Collection of endogenous substances.  It has been reported in  the literature that proteins are released during the induction of LTP with tetanic  stimulations  (Duffy,  Teyler  and Shashoua, 1981).  Whether  proteins and other substances that are released during tetanic  these  stimulations  exert  any effects  tested.  The  on synaptic  present  transmission,  experiments,  for  therefore,  substances released during tetanic  example,  were  hippocampal  slices)  has not  been  designed  to  collect  The methods used to collect samples  from guinea pig hippocampus in vivo were as follows. oxygenated medium (i.e  83  stimulations with the view of examining  their effects on synaptic transmission.  of  CHIRWA  control  Every 5 min, 0.05 ml  medium used for  was added into  each cup.  incubating  An extra  guinea pig  oxygen  line was  positioned in each cup, on top of the f l u i d , to ensure that the added medium remained adequately oxygenated.  A stimulating electrode was placed 250 um  inside the hippocampal tissue through the cup.  At the end of each 5 min  incubation period, each hippocampus was tetanized  (bipolar  pulses of  0.5  msec duration at 100 Hz, 100 pulses, 15 V, given^every 5 sec, 6 trains), and the suction lines were opened during the fifth second to collect the fluids in  the  cups.  These samples were denoted  as the  "tetanized  hippocampal  samples" (THS), and they were collected in a common container that was kept on dry ice.  The above procedures were repeated until the desired volume of  THS (4 ml) was collected. samples", i.e.  Control samples (2 ml of "untetanized hippocampal  UHS) were collected into a separate container using the same  techniques as described but without tetanization  of the hippocampus. [NB:  From each guinea pig 2 ml of UHS were collected before collecting 4 ml of THS.] Similar methods as described above were used to collect substances from the rabbit neocortex in vivo.  Briefly, every 5 min, 0.1  medium was added into each cup (the described in chapter 9).  100  pulses,  30 V),  placing the cups was  At the end of each 5 min incubation period, the  neocortical surface was tetanized Hz,  procedures for  ml of oxygenated  and the  (bipolar pulses of 0.5 msec duration at 50 samples were collected  (i.e.  tetanized  CHIRWA neocortical samples; TNS; 4 ml).  84  Two ml samples of "untetanized neocortical  samples" (UNS) were collected prior to any TNS collections. The samples (i.e. stored  in  separate  UNS, UHS, TNS, or THS) from a particular animal were  collection  indicate the following;  tubes  that  (1) animal type,  (3) type of sample, i . e .  UNS.  were adequately  identified  to  (2) date of sample collection, and  At the end of each collection experiment, the  samples were stored at around -60° C until used in subsequent experiments. 10.5.2  Guinea  pig  hippocampal  samples  and  LTP  production.  To  examine the effects of the collected samples on synaptic transmission in the hippocampus in vitro,  the following procedures were followed.  The samples  (UHS and THS) were quickly thawed by placing the sample tubes in  luke-warm  water, and the THS samples were split into two portions of 2 ml each.  One  portion of the THS was then heated by suspending the capped tube containing the sample in boiling water for 30 min and then cooled. termed  the  heated-tetanized  hippocampal  sample  This sample was  (HTHS).  This  heating  procedure was done to inactivate heat sensitive macromolecules that could be present in the THS (cf. Duffy, Teyler and Shashoua, 1981). Typically, samples collected from one animal single experimental  series in vitro.  sample was transferred oxygenated  for  at  least  into  in vivo were used in a  Prior to application, the  one of  the  5 minutes.  perfusion  To allow  for  barrels  appropriate  where  longer  contact  it  was times  between the applied samples and the hippocampal slices, the flow rates were adjusted to 1 ml/min.  The samples were applied blind to the guinea pig  hippocampal slices, as each slice was exposed to one sample only (i.e. THS, HTHS or UNS). recorded radiatum),  in  After .obtaining  CA^ pyramidal the  stable  layer  responses (i.e.  evoked  sample was perfused  by stimulation  (about  2 min)  onto  population of the  the  spikes stratum  hippocampal  CHIRWA slice.  The population spikes were evoked at 0.2  85  Hz and monitored during  these experiments. 10.5.3  Rabbit neocortical  samples and LTP production.  Effects of  the samples collected from rabbit cerebral cortex in vivo on synaptic transmission in the  hippocampus in  vitro,  were examined  in  the  same way as  described in section 10.5.2, with the following modifications.  Briefly, the  rabbit neocortical samples were identified as follows; (1) UNS, for untetanised neocortical samples, (2) (3)  THS, for tetanised neocortical samples, and  HTNS, for heated-tetanised  neocortical samples,  ln these experiments,  the order of application was UNS, HTNS and TNS and these applications were given during low frequency stimulations  (0.2  Hz) of the stratum radiatum.  Each application was followed by a 60 minute wash with standard medium. separate  slices,  TNS was applied  in the  absence of  stimulations  of  In the  stratum radiatum, and at least 5 min after stopping any such stimulation. Stimulations  of  applications.  the In  hippocampus in the  stratum  another  radiatum  series of  presence of  were  reinstituted  experiments,  10 mM saccharin.  5 min post-TNS  TNS was applied to Saccharin is  known to  inhibit binding of nerve growth factor to its receptors ( l s h i i , 1982). was anticipated substances if  that  saccharin would antagonise  they were present in TNS.  effects  TNS with  NGF-like  saccharin perfusion, TNS was  This was followed by washing with standard medium for 60 minutes.  At this time a second application of TNS was repeated saccharin.  of  It  The saccharin was applied for 10  minutes and during the 7-8th minute of this applied.  the  the  in the absence of  In other experiments, the above procedures were used to apply (1)  atropine  (100 pM),  or  (2)  dihydro-e-erythroidine  (100 pM).  These experiments were done to check for possible effects of any acetylcholine that could be present in the TNS.  In addition, the effects of 2 min  CHIRWA applications glutamate  of exogenous glutamate  on  the  stratum  (100 yM),  radiatum-induced  and, pre-heated  CA^  population  86  and cooled spikes were  examined. 10.6 Effects of rabbit neocortical samples on cultured PC-12 cells 10.6.1 adrenal  PC-12 cell  growth.  cells  are  clonal  pheochromocytoma cells which develop neurites  nerve-growth Therefore,  factor  or  PC-12 cell  related  cultures  with NGF-like activities. that  PC-12  samples  collected  substances.  It  to  structures  synaptic  compounds provide  (Greene  and  a method for  tetanic  of  rat  when incubated with Tischler,  1976).  screening substances  In the present experiments during  lines  it  stimulations  was hypothesized  contained  NGF-like  is known that LTP is associated with morphological changes (Teyler  and DiScenna, 1987,  for  conceivable that substances released during tetanic  review).  stimulations  It  is  could be  growth related macromolecules that mediate the above structural changes.  In  view of this, the present experiments were done to determine whether samples collected during tetanic cell  cultures.  The  stimulations could induce neurite  growth  medium  used  for  cell  growth in PC-12  cultures  contained  Dulbecco's modified Eagles medium, 5% fetal calf serum and 10% heat-inactivated horse serum (ingredients obtained from Gibco Laboratories).  Suffici-  ent Gentamicin was added to make a final antibiotic concentration of 0.005%. The frozen PC-12 cells were activated  as follows.  PC-12 cells  warm water  beaker.  was quickly  submerged in  The ampule containing (temp:  38-40° C)  in a  With agitation, the frozen contents in the ampule melted within a  minute, at which time the ampule was quickly immersed in 70% ethanol at room temperature (25-26° C). The neck of the ampule was carefully broken and its contents  (i.e.  PC-12  cell  suspensions)  emptied  into  a centrifuge  tube  CHIRWA containing 6 ml of growth medium. suspensions were transferred  Using a pipette,  0.2  87  ml of PC-12 cell  into each culture dish (35 x 10 mm size),  to  which 1.3 ml of the growth medium was added (final volume of 1.5 ml in each dish).  A total of 30 culture dishes were plated in this way.  dishes were placed in one large tray that was transferred tor.  After twenty-four  were randomly  10.6.2  30 culture dishes.  assigned to  culture dishes.  into the incuba-  hours of incubation, each culture dish was examined  for cell growth using a phase-contrast microscope. to be growing in all  These culture  five  PC-12 cells were found  At this time, the culture dishes  groups.  Each group,  therefore,  had six  Colored dots were used to identify the five groups. Preparation of PC-12 cells feeding media.  The various rabbit  neocortical samples were thawed and mixed with freshly prepared growth media as illustrated  in Figure 10-3.  Briefly, 5 ml of double-concentrated growth  medium was added to each one of four separate centrifuge  tubes.  Then the  following neocortical samples were added blind to the above tubes (5 ml in each case): (1) (Figure 10-3).  TNS, (2)  HTNS, (3)  UNS, and (4)  The contents of each tube were f i l t e r - s t e r i l i z e d ,  in color coded tubes to maintain blinding. feed  the  PC-12 cell  neocortical  TNS with 10 mM saccharin  cultures.  sample, similar  [NB:  collections  The above media were used to  To obtain (i.e.  and stored  5 ml  of  each type  of  TNS) from two rabbits were  pooled together.] 10.6.3 after plating), dishes.  Neurite  induction.  On day two of  incubation  (i.e  the growth medium was carefully decanted from the  24 hours culture  Culture dishes from the same color-coded group were replenished  CHIRWA  G r o w t h medium ( s i n g l e - cone)  G r o w t h medium ( d o u b l e - cone)  1 Neocortical samples  U  T S  H  Feed m e d i a  P C - 1 2 cell cultures  i  i  i  i  1  i  i  i  Figure 10-3. Preparation and composition of the different types of feeding media used for incubation of rat adrenal pheochromocytoma (PC-12) cell cultures. The control growth medium (single-conc.) contained Uulbeccos' modified Eagle's medium, 5% fetal calf serum, 10% heat-inactivated horse serum and 0.005% gentamicin. The growth medium (double-conc.) contained twice the concentration of the control growth medium (single-conc). To make feed media, aliquots of growth medium (double-conc.) were mixed with equal volumes of (1) untetanised neocortical samples, (U), (2) tetanised neocortical samples, (T), (3) heated-tetanised neocortical samples, (H), and (4) tetanised neocortical samples containing 10 mM saccharin, (TS). The feed media, therefore, contained normal concentrations of Dulbeccos' modifield Eagles' medium and 0.005% gentamicin. The above feed-media were used to maintain the various PC-12 cell cultures as illustrated. One group of PC-12 cell cultures was continued on control growth media (singleconc).  CHIRWA with 1.5  ml of the  feed media described in  section 10.6.2  89  above.  The  different types of feed media comprised of equal mixtures of growth medium and: (1) TNS, (2) UNS, (3) HTNS, (4) TNS and saccharin (Figure 10-3). growth medium was maintained in one group of PC-12 cell  cultures  Plain (Figure  10-3).  Saccharin, a substance that inhibits NGF-dependent neurite  growth  (lshii,  1982), was added to the feeding medium of another group.  In  the  present studies, it was wondered whether NGF-related compounds were present in TNS.  It was anticipated that if such factors were present in TNS, their  effects  on PC-12 cell  growth  could be antagonised by saccharin.  adding the feed media, the culture dishes were returned to the Thereafter,  After  incubator.  the cultured PC-12 cells were examined under a phase-contrast  microscope each day, starting with the second day and ending on the eighth day of incubation. 10.7 Studies on the possible mechanisms of action of saccharin 10.7.1  General.  Experiments were done to  determine  if  could prevent the effects of samples collected during tetanic  stimulations  when applied to the hippocampus, or block tetanus-induced LTP. tate analysis of results obtained from experiments used,  it  was necessary to  determine  the  saccharin  To f a c i l i -  in which saccharin was  electrophysiological  effects  of  spike  in  saccharin in the hippocampus. 10.7.2 ^lb  a  r  e  a  w a s  Dose-response curves. e w 0  ^  e c i  w  ^  t n  stimulation  Briefly,  the  population  of the stratum radiatum at 0.2 Hz.  In a given s l i c e , dose-response curves to saccharin were obtained using the single  application,  randomised  examined were (in mM); 2.5. was perfused for 10 minutes.  design.  The  5, 10, 20, 40 and 80.  saccharin  concentrations  Each drug concentration  This was generally followed by a wash period  of 15 minutes, which was found to be a sufficient  interval  for the popula-  CHIRWA tion spike to return to pre-drug levels.  90  The only time the wash time was  extended was after applications of 80 mM saccharin, when 20-30 min of wash were required to bring the population spikes back to pre-drug levels. 10.7.3  Saccharin and electrical  properties  of  neurons.  Possible  effects of saccharin on electrical properties of neurons in the hippocampus were examined as follows.  In these experiments, CA^ neurons were impaled  with microelectrodes f i l l e d with either 3 M KC1 or 2 M potassium acetate. In addition, a stimulating electrode was positioned in the stratum radiatum within 50-100 microns distance to the CA^ pyramidal layer. in order to stimulate  mostly proximal  This was done  synapses on CA^ apical  dendrites.  Under these conditions, the effects of saccharin on the following responses were examined: (1)  RMP and R ;  (2)  n  spontaneous minEPSPs and minlPSPs; (3)  evoked EPSPs, IPSPs, action potentials saccharin  was  perfused  for  2-10  and AHPs.  minutes.  In these studies, 10 mM  In  some experiments,  these  saccharin applications were repeated at 30 min intervals. 10.7.4 determining  Saccharin and LTP. the minimum dose of  The present  experiments  saccharin that could interfere  development of LTP following tetanic stimulations. the CA^ area was evoked by stimulation Hz.  were aimed  of  the  at  with the  The population spike in stratum radiatum  at  0.2  After obtaining control population spikes, 2.5 mM saccharin was applied  for 10 minutes, and the responses were monitored. saccharin applications, the pulses).  stratum  radiatum was tetanized  (400  Hz,  200  This was followed by re-institution of the standard medium to wash  out the saccharin. tum was tetanized  After a washing period of 30 minutes, the stratum radia(400 Hz, 200 pulses, same stimulus strength and durations  as used in the f i r s t tetanus). minutes.  During the last minute of  In different  The responses were monitored for another 30  slices, the above procedures were repeated with the  CHIRWA following saccharin conncentrations (in mM); 5,  7.5  91  and 10 saccharin.  ln  some control experiments, tetanic stimulations were applied twice, separated by 30 min intervals,  in standard medium only  (i.e.  the  slices were not  exposed to saccharin). 10.7.5  Saccharin  potentiation Eccles  post-tetanic  potentiation.  Post-tetanic  (PTP) is thought to be mediated by presynaptic mechanisms (e.g  and  Therefore,  and  Krnjevic,  1959;  changes in the  McNaughton,  magnitudes  of  Douglas  and  PTP during  Goddard,  different  provide one method for assessing presynaptic functions.  1978).  treatments  In view of this,  the present experiments examined the effects of saccharin on PTP size, these experiments, the control medium used contained reduced Ca to  Mg  ++  concentrations  (low  Ca  medium:  ++  1 mM C a  ++  and 3  ln  relative mM Mg ). ++  Secondly, the stimulation parameters were adjusted to evoke small population spike of  about  0.3-0.5  stimulations  of  post-tetanic  potentiation  cell  layer  (cf.  the  mV amplitudes.  stratum radiatum of the  Under (400  population  Hz,  these 200  spike  conditions, pulses)  in  Dunwiddie, Madison and Lynch, 1978).  the  tetanic  only induced  CA^ pyramidal  In each s l i c e , PTPs  were evoked (1) in control medium, (2) during 10 mM saccharin applied for 10 min, and (3) after drug applications. 10.7.6  Saccharin and presynaptic excitability.  Another method for  assessing presynaptic functions involves determination of the threshold for antidromic activations of presynaptic terminal regions.  Changes in values  of antidromic  indicators  thresholds during treatments  conditions in the presynaptic regions.  are  useful  of  For example, hyperpolarizations  the in  presynaptic regions are associated with increases in antidromic thresholds (e.g. Wall, 1958; Saint, Quastel and Chirwa, 1986; Sastry, 1982).  Hence the  methods of presynaptic excitability testing were used to assess further the  CHIRWA effects  of  saccharin on presynaptic  physiological  medium  used  did  not  regions. contain  ^+  Ca  ++  and 0.5  mM  Mn  3b  neurons.  c  experiments,  (i.e.  2  mM C a  ; see Table 9-1  in order to abolish synaptic transmissions.  were obtained from C A _  these  Intracellular recordings  The selection of cells for inclusion impale-  b  the  was  in chapter  in the experiments was the same as that described for CA^ neuron ments.  ++  the  4"4"  substituted with 1.5 mM Mg 9),  In  92  In these experiments, the stimulating electrode was positioned in  Schaffer  antidromic neurons.  col laterals-CA^  action  b  potentials  apical  dendritic  in Schaffer  region  collaterals  and used to evoke that  invaded  CA _  Stimulation test pulses at each fixed duration (given at 0.2  3b  c  Hz)  were adjusted to threshold for antidromic invasion on 50% of 6-8 consecutive trials.  The threshold values were determined for a range of stimulus dura-  tions (i.e.  0.1-5 msec stimulus pulse durations).  The rheobase was estima-  ted from the strength-duration curves of each recording, and these stimulus durations were used to determine threshold values in (1) control medium, (2) during 10 mM saccharin applied for 10 min, and (3) cations.  15 min after drug appli-  In saccharin, the threshold values were obtained in the last min  of drug application. 10.7.7  Saccharin and paired-pulse f a c i l i t a t i o n .  Paired-pulse f a c i l -  itation is well characterised in the hippocampus and it  is thought to be due  to an increase in transmitter released with the second pulse in the stimulation  pair  effects  (e.g.  (e.g.  Ca  McNaughton, ++  influx  1980). into  Presumably,  presynaptic  "residual"  terminals)  presynaptic  associated with  depolarizations induced by the f i r s t pulse add up with those of the second pulse and augment the effects of the latter pulse on transmitter In principle, alterations the  first  release.  in the ratio of the second response relative to  response during  paired-pulse  stimulations  reflect  changes  to  CHIRWA presynaptic mechanisms.  93  In view of the above, the effects of saccharin on  paired-pulse facilitations were examinea.  briefly, stimulus pairs that were  separated by an interval of 30 msec were applied to the stratum radiatum to evoke a pair pulse  interval  of  population  used was sufficient  response (hence facilitated) pair.  spikes in the  relative  to  CA^ field  respectively.  cause an increase  to the f i r s t  in  The  the second  response in each evoked  These responses were obtained during control medium perfusions.  was then followed by the application of 10 mM saccharin for 10 min.  This During  saccharin applications, responses to paired pulse stimulation of the stratum radiatum were recorded during the 1,  3,  5 and 9 min intervals.  Fifteen  minutes after saccharin applications, the responses to paired pulse stimulations of the stratum radiatum were also determined. 10.7.8  Saccharin and NMDLA responses.  N-methyl-DL-aspartate  (NMDLA)  responses  in  The effects of saccharin on CA^  neurons  since NMDA receptors are thought to be involved in the (Collingridge,  Kehl  and McLennan, 1983).  It  were  examined  induction of  was necessary to  test  LIP if  saccharin antagonised NMDLA responses since at this stage in the studies it was clear that saccharin blocked the induction of LTP with tetanic stimulations.  Intracellular  responses  previously described elsewhere.  were  obtained  from  In these experiments,  CA^  neurons  as  the racemate NMDLA  was used since it was available in the laboratory when these studies were planned.  Moreover, it  is known that the effects of NMDLA are similar  those of N-methyl-D-aspartate 1984).  to  (NMDA), the active enantiomer (e.g. Dingledine,  After successful impalements, NMDLA media were applied for 1 or 1.5  min and then washed out.  One or two different  concentrations of NMDLA  (in uM; 25, 50, 75 and 100) were tested during each intracellular recording. Typically, NMDLA applications were repeated at different  times in the same  CHIRWA c e l l , usually at intervals of 8-10 min. perfused for effects  10 min.  During the  94  Subsequently, 10 mM saccharin was  7-9th min of  of NMDLA concentrations were tested.  saccharin perfusion,  After  returning  the  to control  medium (i.e. post-saccharin), NMDLA applications were repeated. 10.8 Effects of exogenous NGF in the hippocampus Again  these  next  series  of  experiments  stemmed  from  the  results  obtained from experiments described in sections 10.5-10.6, which showed that substances released during tetanic stimulations induced LTP when applied in the hippocampus in vitro,  and these same substances could initiate .neurite  growth in cultured PC-12 c e l l s .  This raised the possibility that growth  related substances were present in the collected samples. of exogenous nerve growth factor examined in some experiments. was available  in the  (2.5  Hence the effects  ug/ml NGF from Vipera lebetina) were  This NGF was used in these studies since it  laboratory when these experiments were planned.  In  these studies, NGF was applied for 5-10 min with or without stimulation of the  stratum  layer.  radiatum  to  evoke  In separate experiments,  population  spikes  the effects  of  in  the  CA^ pyramidal  NGF were also tested as  follows.  Stimulation of the stratum radiatum was adjusted to evoke a weak  dendritic  EPSP in the C A  Hz,  100  pulses)  applied  lb  to  field. the  Repeated tetanic stratum  McNaughton, Douglas and Goddard, 1978). for 5-10 min.  radiatum  stimulations  only  (50-100  induced PTP  (cf.  Slices were then perfused with NGF  Then a similar tetanus to that described above was applied to  the stratum radiatum, during the last minute of NGF application.  In other  experiments, this tetanus was applied during perfusions of NGF and saccharin. 10.9 Data analysis Standard control procedures, with blinding wherever applicable, formed the protocol of all  experiments.  The following  statistical  methods were  CHIRWA used to analyse the data.  Briefly, o was set at 0.05,  were employed unless the  research hypothesis specified the  which a difference would occur. used.  and two-tailed tests direction  t-test  was used for comparisons  For this purpose, a variate (e.g. amplitude of  population spike) before treatment was compared with its counterpart treatment.  in  In this latter case, one-tailed tests were  Furthermore, the paired Students'  between two related samples.  95  after  However, for comparisons among a series of Means for which there  was one criterion  of  classification  (e.g.  Mean amplitude  of  population  spike), one-way ANOVA was used and, i f statistical differences were indicated, Duncans' multiple comparisons method was the a posteriori test used to determine which pairs of Means were statistically different.  CHIRWA 11.  96  RESULTS  11.1 Recordings in CAj^ field of the hippocampus 11.1.1  Features of intracellular  recordings.  penetrations was i n i t i a l l y examined in 20 CA^ c e l l s .  The quality  of  cell  In these and subse-  quent results, each cell represents a complete recording in one hippocampal slice prepared from a different pipettes f i l l e d electrode).  with either  Cells were impaled with micro-  3 M KC1 (KC1 electrode)  or 2 M CH C00K (KA 3  Successful impalements presented with stable RMP  and -80 mV and R  n  cells).  guinea pig.  between -55  values between 7 and 57 Mn (null-briage method; n = 20  Seventeen out of 20 cells could support action potentials when  challenged with depolarizing current injections (0.2-3 nA, 200-400 msec, 0.2 Hz).  These same cells presented with fast EPSPs and synaptically activated  action potentials following stimulations of the stratum radiatum (10-150 pA, 0.1-0.3 msec, 0.2 Hz; 17 of 17 c e l l s ) .  Action potentials had amplitudes of  80 ± 20 mV and widths, at half-maximum height, of ~1 msec. cells  were taken  to  be neurons, and their  The above 17  characteristics were  further  examined. Most potentials  of  the  above  CA^ neurons  did  give  spontaneous  action  (11 of 17 neurons), but some neurons presented with occasional  spontaneous  single  action  potentials  (< 6  However, sporadic small discrete potentials tered  not  (see section 11.1.2).  evoked intracellular  Figure 11-1  per  min;  6  of  17 neurons).  (< 5 mV) were commonly encounillustrates  responses in CA^ neurons.  the features  of the  The EPSPs and/or action  potentials evoked with direct depolarizing current steps or synaptic activations,  were  reflected 1987).  followed  IPSPs  and/or  by  transient  membrane  afterhyperpolarizations  hyperpolarizations, (AHP;  cf.  which  Schwartzkroin,  Typically, intracellular recordings could be maintained for periods  CHIRWA  K A electrode  KCI electrode  a  b  | 20 200 m s  200  ms  Figure 11-1. Characteristic features of evoked intracellular potentials in the CAih neurons of the guinea pig hippocampus m vitro" lntrace I lular potentials Tn CA^ neurons were evoked by stimulation of the stratum radiatum or by direct depolarizing current injections into these neurons. The evoked intracellular potentials were recorded with micropipettes f i l l e d with either 2 M potassium acetate (KA electrode) or 3 M potassium chloride (KCI electrode). The undershoots following the evoked EPSP (la) or the synaptically activated action potential (Ha) probably reflected IPSPs and/or afterhyperpolarizations (AHP). Leakage of Cl~ from KCI electrodes caused reversal of IPSPs and, this probably accounts for the apparent increase in duration of the "EPSP" (lb) or the "hump" following the synaptically activated action potential (lib) when compared to similar responses in column (a). The occurrence of "reversed IPSPs" tended to mask the underlying AfTPs in recordings using KCI electrodes. However, AHPs could clearly be observed after direct depolarizing current injections into CAi^ neurons in recordings using both KA electrodes and KCI electrodes ( i l l ) . [NB: Traces in column (a) were recorded on a strip-chart recorder and, traces in column (b) were photographed directly from the oscilloscope. Some traces have been re-touched to compensate for loss of clarity during photographic reproduction.  97  mV  CHIRWA of 60-150 min with no significant resistances  (RMP,  start:  31 * 1.5 Mn, ena: p > 0.05 results  changes to membrane potentials  -63 ± 2  mV,  end:  mV;  32 * 3.1 Mn, values are Mean ± S.E.M.;  by two-tailed paired Student's t-test formed  -62 * 2  the  basis  for  establishing  (2)  R  n  greater  than 25 Mn ;  synaptically activated action potentials;  R, n  (3)  and (4)  input start:  n = 17 neurons;  criteria  neuronal impalements for data collections, namely: (1) or more negative;  or  in both cases).  the  98  The above  used to  select  stable RMP of -60 mV clear  direct  fast  EPSPs and  action  potentials  induced with depolarizing current injections.  -ve  11.1.2  Miniature  membrane  potentials  electrodes CA^  were detected  (n = 7 neurons).  neurons  transient  postsynaptic potentials.  impaled  Only small  with  KC1  in +  frequencies Table 11-1).  (1-20  CA^ neurons  ve  potentials  electrodes  postsynaptic responses exhibited per sec) among different  Small  (n = 10  varied  discrete impaled  amplitudes  and  with KA  were detected neurons).  CA^ neurons  +ve  in  These  (< 5 mV) and  (n = 17 neurons;  ln recordings with KA, 10 uM picrotoxin reduced the frequen-  cies of small -ve potentials by at least 50% and 50 uM picrotoxin abolished all  -ve potentials.  Recordings with KC1 electrode  revealed  decreases of  about 40-60% in frequencies of ve potentials in 10 uM picrotoxin, and these +  decreases were as much as 90% in 50 uM picrotoxin. frequencies of small potentials  The changes  in  the  in the presence of picrotoxin were s i g n i f i -  cantly different from controls, as determined by one-way ANOVA with Duncan's multiple comparison tests (n = 17 neurons; quantitative data in Table 11-1). Typically, the effects of picrotoxin were reversible within 15-20 min after returning to control medium.  The above small potentials  were taken to be  simultaneous recordings of "miniature" EPSPs and "miniature" IPSPs (minEPSPs  CHIRWA  Table 11-1. Small discrete potentials pig hippocampus in vitro  in CA^ neurons in the guinea  KA electrodes -ve Control 10  +ve  KC1 electrodes +  ve  9*3  3 * 1  12 * 5  4 ± 2*  3 * 1  8 ± 2*  3*1  2*1*  3 * 1  14 * 2  uM  picrotoxin 50 uM picrotoxin 15 min post-drug  8*2  Values are Mean * S.E.M. per sec (numbers rounded up to nearest integer other than 0; n = 7 neurons for recordings with KA electrodes and, n = 10 neurons for recordings with KC1 electrodes. In these experiments, each hippocampal slice was exposed to 10 uM picrotoxin (applied for 10 min) followed by 50 uM picrotoxin (applied for 10 min). Subsequently, control medium was reperfused. Astericks indicate significant differences as determined by one-way ANOVA with Duncans' multiple comparisons tests.  control  50pM picrotoxin  100 m s Figure 11-2. Intrasomatlc recordings of spontaneous small d i s c r e t e p o t e n t i a l s 1n C A i neurons In the guinea p i g hippocampus in vitro, both -ve and •'•ye spontaneous small p o t e n t i a l s were p r e s e n t In r e c o r d i n g s w i t h m i c r o p i p e t t e s f i l l e d w i t h p o t a s s i u m a c e t a t e (2 M CH3COOK). However, the -ve spontaneous small potentials were a b o l i s h e d In 50 pM p i c r o t o x i n . By c o m p a r i s o n , o n l y *ve spontaneous small p o t e n t i a l s were p r e s e n t In r e c o r d i n g s w i t h m i c r o p i p e t t e s f i l l e d w i t h potassium c h l o r i d e (3 M KC1) b u t , as much as 90% o f t h e s e v e spontaneous small p o t e n t i a l s were a b o l i s h e d i n 50 pM p i c r o t o x i n . The spontaneous small p o t e n t i a l s recorded In the presence of 50 pM p i c r o t o x i n ( e . g . a r r o w s ) , w i t h both types o f , r e c o r d i n g m i c r o p i p e t t e s , were taken t o be " m i n i a t u r e " EPSPs i n CA^, neurons. The t r a c e s were recorded on a s t r i p - c h a r t r e c o r d e r a n d , the t h r e e sweeps i n each s e t were taken i n immediate s u c c e s s i o n . h  +  0  2 g 5 9  >_ g  CHIRWA and minlPSPs; Figure 11-2).  It  was inferred that picrotoxin diminished or  abolished minIPSPs that were probably due to the quantal (cf.  101  release of GABA  Alger and Nicoll, 1980; Brown, Wong and Prince, 1979;  Turner, 1988).  Recordings with KCI electrodes revealed only small +ve potentials because of the presence of minEPSPs and reversed minlPSPs, the latter being a result of equilibrium shifts in Cl~ conductances due to  leakage of  Cl~ from micro-  electrodes. In  separate  experiments  conducted  in  50 yh  picrotoxin  (different  neurons from above), minEPSPs were detected even though tetrodotoxin was present  in  the  medium (n = 4 neurons; Figure  demonstrated that the observed minEPSPs were not action potentials.  11-3). all  (1 pM)  These results  due to presynaptic  However, tetrodotoxin was not used in subsequent experi-  ments since it was necessary to have activatable afferents recorded minEPSPs had amplitudes of < 2 mV (Figure 11-2  (see below).  The  and Figure 11-3).  But the frequency of spontaneous minEPSPs was found to be low (< 10 per sec) and extremely  variable  among CA^ neurons  (1-20  per min).  Occasionally,  however, stimulation of the stratum radiatum evoked EPSPs or synaptically activated  action potentials  minEPSPs.  that were immediately  followed by a burst of  These were denoted as evoked minEPSPs.  This discovery motivated  the search for experimental methods that could consistently produce evoked ++  minEPSPs.  For  release  transmitters  Saint,  of 1988;  this  reason, in  Ba  was  used to  some experiments  Silinsky, 1978;  [NB:  induce  (Chirwa,  The use of B a  suggested by Dr. Quastel, who has been using B a  ++  ++  the asynchronous 1985;  Quastel  and  in these studies was to examine presynaptic  functions at the neuromuscular junction]. 11.1.3  Recordings with  typical intracellular CsCl.  Cs* electrodes.  Figure  11-4  illustrates  responses recorded with micropipettes f i l l e d with 3 M  After cell impalements, membrane potentials gradually shifted to  B Control  9 min  In T T X  15 min p o s t - a p p l i c a t i o n  2mV 20 ms  Figure 11-3. The occurence of miniature EPSPs potentials in CAjb neurons in guinea pig hippocampal slices incubated in tetrodoIbxin^ Spontaneous miniature tPbPs were detected Tn UAi neurons both in control medium containing 50 uM picrotoxin (A) or test medium containing 1 uM tetrodotoxin (TTX) and 50 uM picrotoxin (B_). Note the decrease in the frequency of spontaneous miniature EPSPs recorded at 9 min after starting TTX applications (i.e. TTX was applied for 10 min in this experiment). The frequency of spontaneous miniature EPSPs returned to pre-TTX levels after about 15 minute washing in control medium (CJ. The sweeps in each set are of continuous recordings taken from a strip-chart recorder, respectively. b  o zc  t—•  CHIRWA  12  9  S  103  min  B  A  (-51)  (-40)  (-43)  J  20 mV  50 ms  F i g u r e 11-4. C h a r a c t e r i s t i c f e a t u r e s o f i n t r a c e l l u l a r p o t e n t i a l s i n a C A i h neuron "recorded w i t h m i c r o p i p e t t e s f i l l e d w i t h C s ' . Tn I A J i s shown a s y n a p t i c a l l y a c t i v a t e d a c t i o n p o t e n t i a l Tn a C A j u neuron f o l l o w i n g s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m , b min a f t e r c e l l impalement w i t h a m i c r o p i p e t t e f i l l e d w i t h 3 M C s C l . Four min l a t e r ( i . e a t 9 m i n ) t h e same s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m evoked i n t r a c e l l u l a r p o t e n t i a l s w i t h two peaks t h a t a r e , denoted as " I " and " I I " i n t h e i l l u s t r a t i o n . The f i r s t peak, I j was taken t o be a "widened" sodium s p i k e due t o b l o c k a d e o f some K c u r r e n t s b y i n t e r n a l Cs . The second peak, I I , was p r o b a b l y a Ca** s p i k e . S u b s e q u e n t l y , s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m ( e . g a t 12 min a f t e r c e l l impalement) evoked a s i n g l e , v e r y l o n g - l a s t i n g peak (C_). [NB: The c e l l membrane p o t e n t i a l in mV a t time o f each r e c o r d i n g a r e g i v e n i n b r a c k e t s . ] +  CHIRWA  104  depolarized levels (cell  depolarizations of 10-50 mV, from i n i t i a l  resting  values; n = 10 neurons).  In the early stages, membrane depolarizations were  associated with increased frequencies of spontaneous action potentials which subsequently stopped, probably due to Na the prolonged membrane depolarizations.  channel inactivations  +  caused by  Prolonged subthreshold EPSPs could  s t i l l be evoked, however, with stimulations of the stratum radiatum (Figure 11-4).  In addition, Ca  spikes could be evoked with suprathreshold EPSPs  or adequate intracellular depolarizing current injections (0.5-2 nA, 100-200 msec; Figure 11-4). 11.1.4  Features of extracellular  responses.  Figure 11-5  illustrates  features of evoked responses recorded in the CA^ pyramidal cell layer and apical  dendritic  The evoked  regions, following  field  stimulus-dependent negative-going slices).  responses in  the  biphasic positive  stimulation  strengths  between  the  CA^ pyramidal waves  that  peaks, which were population  Stimulus  of  could  pA  radiatum.  layer  exhibited  cell  spikes  50-150  stratum  be  (Figure  (0.1-0.8  bisected with 11-5;  n = 10  msec; 0.2  Hz)  applied to the stratum radiatum elicited population spikes with amplitudes of 1.0-1.5 mV and onset latencies ( i . e . , time from onset of artifact to peak negativity of the population spike; Figure 11-5) Evoked field  responses recorded  in  the  CA^  b  ranging between 6-10 msec. apical  dendrites  presented  with negative-going waves which were caused by sinks associated with dendritic  field  EPSPs.  Positive-going  peaks  could  be  superimposed on these  dendritic field EPSPs ( i . e . , reflected sinks of population spike in the soma region), by increasing stimulus strengths applied to the stratum radiatum. Both the population spike and the field EPSP could be abolished by transient 4-4*  perfusions with Mg -Mn  ++  medium (Figure  11-5).  During these recordings  in Ca -free medium, however, the presynaptic volleys (especially with  b  c  CHIRWA  10 ms b Control  Mn* -Mg +  c 2 +  5 min FUcovory  II  10 ms  Figure 11-5. Characteristic features of evoked field potentials recorded in the CAih area following stimulation of the stratum radiatum in tne guinea pig hippocampus m vUro~ A" "weak positive wave" (PW) recorded in the somatic layer (Ia) and a "weak EPSP" recorded in the dendritic region (Id) could be evoked with low stimulation strengths applied to the sTratum radiatum (i.e. taken to be stimulation of "weak inputs"). However, higher stimulus strengths evoked population spikes in the pyramidal layer (Ib-c) and dendritic f i e l d EPSPs (Ie-f). The onset latencies (horizontal arrows) and amplitudes (vertical arrows) of evoked responses were measured as illustrated in (Jk) for the population spike and in (_Ie) for the dendritic field EPSP. The electrical fields generated by the population spike in the somatic layer sometimes appeared as a small peak (*) that contaminated the dendritic field EPSP as shown in (I_f_). In (II) are presented recordings taken from a different s l i c e . Both the somatic population spike (Ila) and the dendritic field EPSP (Hd), evoked by stimulation of the stratum radiatum in control medium, were abolished during a 10 min application of Ca -free medium (lib and H e ; responses taken at 5 min after starting Mn -Mg medium) but, these evoked responses recovered within 5 min after returning to control medium (ljk and I If). The presynaptic volley (PV) is readily discernable during blockade of synaptic transmission. t  105  CHIRWA  106  dendritic recordings) were clearly discernible (Figure 11-5). 11.2 Saccharin dose-response curves Saccharin (10-100 mM) competitively antagonises the binding of NGF to its  "receptors" and inhibits  NGF-dependent neurite  growth  (lshii,  1982).  Saccharin, therefore, provided a method for screening NGF-dependent activities.  This feature was utilized in certain experiments in this thesis (see  later sections).  In this regard, it was necessary to establish the dose-  response characteristics of saccharin in the hippocampus and this is i l l u strated in Figure 11-6. CA^  In these experiments, the population spike in the  area was evoked by stimulation  of  the  stratum radiatum.  exhibited  steep dose-response relationships,  suggestive of  mechanism  of  2  actions.  During  (saccharin was applied for  the  initial  10 min each time),  min  of  there  some specific  drug were  Saccharin  application increases  population spikes in the CA^ area evoked by stimulations of the  in  stratum  radiatum, at all drug concentrations tested (amplitudes of population spikes as % of  controls:  105-117;  n = 6 slices)  but  these  were  insignificant  changes as determined by one-way ANOVA (quantitative data in Figure 11-7). During the last 5-7 min of drug applications, saccharin concentrations > 5 mM but < 20 mM were associated with insignificant decreases of the population spikes in the CA^ area evoked by stimulation of the stratum radiatum (amplitudes of population spikes as a% of controls: p > 0.05,  one-way  ANOVA;  saccharin concentrations  quantitative  data  in  87-92; n= 6 slices;  Figure  > 20 mM induced significant  11-7).  However,  depressions of  population spike (amplitudes of population spikes as a % of controls: n = 6 slices;  p < 0.05,  the 0-35;  one-way ANOVA with Duncan's multiple comparisons  tests; quantitative data in Figure 11-7).  ln the case of 80 mM saccharin,  CHIRWA  150.0  Figure 11-6. Dose-response curves of saccharin obtained by the method of single application, randomised design. Plotted are amplitudes of evoked population spikes in CA^ area recorded during the 9th minute of a 10 min application of saccharin, expressed as a % inhibition of evoked population spikes obtained in control medium (values are Mean * S.E.M., n = 6 slices). Saccharin presented with a steep dose-response relationship which was distributed within two logarithmic units as illustrated.  CHIRWA  108  150  c o o o UJ  125  2.5 NaS n= 6  5 NaS n=6  10 NaS n=6  13 5 9  13 5 9  13 5 9  20 NaS n=6  40 N a S .n = 6  80 N a S n=6  100-  75-  QL CO  50  5  ID CL  25 -  o  Q_  0  Time  13 5 9  13 5 9  13 5 9  (min)  Figure 11-7. Changes in amplitudes of population spikes in CAih area evoked by stimulation of the stratum radiatum during applications ot different doses ot saccharin in guinea pig hippocampus in vitro. The graph shows the amplitudes of evoked population spikes recorded during a 10 min application of saccharin, expressed as a of evoked population spikes recorded in control medium. The time intervals along the X-axis illustrate the time when the evoked population spikes were recorded for each concentration of saccharin tested (values are Mean ± S.E.M.). £NB. These are the same experiments from which the dose-response relationships for saccharin were obtained (see Figure 11-6).] The asterisks indicate significant differences between population spikes evoked during saccharin application and population spikes evoked in control medium (n = 6 slices; p < 0.05; one-way ANOVA with Duncan's multiple comparisons test).  CHIRWA the evoked population spike was rapidly abolished (Figure 11-7).  109  From these  dose-response curves, 10 mM saccharin was selected and used in subsequent experiments since this drug concentration did not induce significant depressions of the population spike during applications. 11.3 Effects of barium in the hippocampus The B a Ca  ),  media tested were classified as low  ++  moderate  mM Ba  and 0.5  ents).  Within  (2  mM Ba  Ca 1-3  and 2  )(see  Table  minutes  Ca  9-1  of  Ba  mM), of  all  in high Ba  applications,  initial (e.g.  1-3  Ba  min of  EPSPs  and  Chapter  increased  R  by  n  applications).  ++  action  increased.  for  potentials)  However,  in  evoked  delayed  and  media  (3.5  media constitu-  CA^  at  least  neurons  became  Typically,  changed  as  media.  75% (checked the  In  in  the  evoked responses  follows.  In  low Ba  EPSP and associated IPSP were  moderate  and  high  i n i t i a l increases in amplitudes of evoked intracellular IPSP rapidly diminished.  and 3.5  ++  The onset of these depolarizations  medium, the amplitudes of evoked intracellular generally  9,  Ba  medium than in low or moderate Ba  ++  Ba  mM B a  and high  perfusions,  ++  depolarized by 3-20 mV (n = 30 neurons). was quicker  (0.5  Ba  media,  ++  the  EPSP and associated  Subsequently, stimulations of the stratum radiatum  staggered  synchronous  synaptic  responses  that  were  followed by bursts of miniature postsynaptic potentials  as illustrated  Figure 11-8.  (0.2-1 nA, 50-200  msec)  into CA^ neurons caused large  miniature min)  Injections of depolarizing current  postsynaptic  triggered  (similar  to  potentials.  spontaneous  epileptogenic  depolarizing  Continued  membrane discharges)  pulses  Ba  ++  depolarizing as  shifts  with bursts of  perfusions shifts  illustrated  in  in  with  (beyond  spikings  Figure  Subsequently, synchronous synaptic responses were abolished, except  4  11-8.  CHIRWA A  B  C  I  _JlO 200  mV  ms  Figure 11-8. Intracellular potentials in CA^ neurons in guinea pig hippocampal slices incubated in barium. The Ba media tested contained 3T5 mM Ba" arfd O Ca . Typically, an intracellular EPSP (IA) or a synaptically activated action potential (IB), and their associated afterhyperpolarizations (i.e picrotoxin not adHed to media), were evoked in a CA15 neuron by stimulation of the statum radiatum. In adition, action action potentials were evoked by injection of depolarising current pulses (0.4 nA, 200 msec) into a CA^t, neuron.. In Ba medium (applied for 10 min), stimulation of the stratum radiatum subsequently evoked delayed and staggered synchronous synaptic responses that were followed by bursts of miniature postsynaptic potentials {U_» number next to each trace in column A indicates time in min when record taken). Injections of depolarizing current pulses (0.4 nA, 200 msec) into a CA^ neuron evoked several action potentials, followed by^a bursts of miniature postsynaptic potentials (HC). Continued Ba perfusions triggered spontaneous membrane depolarizing shifts with sp_ikings as shown by the continuous trace in (III). The effects of Ba applications were essentially reversible within 15 min of re-perfusing with control medium (JJT, see also Figure 11-13). [NB: Action potentials truncated by strip-chart recorder. Some traces have been re-touched to compensate for loss of clarity during photographic reproduction.] +4  44  CHIRWA in low B a  medium.  ++  However, bursting discharges could s t i l l  with stimulations of the stratum radiatum. it  difficult  to visualise miniature  111  be triggered  These bursting discharges made  postsynaptic responses that might be  present. ++  The effects  of  Ba  applications were essentially  reversible  15 min of re-perfusing with control medium (Figure 11-8). experiments, high Ba since this Ba ions.  within  From the above  medium was selected and used in subsequent studies  medium exerted its effects relatively  More importantly, high B a  early during perfus-  medium was found to be most efficacious  ++  in evoking the asynchronous release of transmitters  following  stimulations  of the stratum radiatum. 11.4 Induction of long-term potentiation Tetanic stimulations.  11.4.1  tions of the stratum radiatum the  population  amplitudes: slices).  200 pulses) induced synaptic LTP of  (400Hz,  spike and field  population  Single high frequency tetanic stimula-  spikes,  EPSPs  in  250-600;  the  CA^ area  field  EPSPs,  (% increases  in  10 of 10  150-200;  Typically, LTP development was preceded by post-tetanic  potentia-  tions (PTP) that rapidly decayed in 3-5 min, revealing the underlying longlasting synaptic potentiation  as illustrated  in Figure 11-9.  The potenti-  ated responses were associated with reductions in onset latencies, and LTP showed l i t t l e or no decay even after 60 min as illustrated With  intracellular  recordings,  LTP  in  9  out  of  10  in Figure 11-10. CA^ neurons was  observed as increases in the amplitudes of subthreshold intracellular EPSPs (intracellular 167 * 7.9; paired  EPSPs as a % of  control  values are Mean ± S.E.M.;  Student's  t-test).  In  most  15 min after  induction  n = 10 neurons; p < 0.05; experiments,  previously  of LTP:  one-tailed subthreshold  EPSPs reached threshold after LTP development as illustrated in Figure 11-10  112  CHIRWA  600 H  c o u o  500 PS  400  \I  CO Ul  CO  o  Q_ CO UJ Cd  300 field EPSP  200  •i  o UJ  o  h  — i  =1=  100  400 Hz, 200 pulses  0 0  10  20  30  40  50  (n=10) 60  70  80  TIME (min)  F i g u r e 11-9. I l l u s t r a t i o n o f l o n g - t e r m p o t e n t i a t i o n i n d u c e d b y h i g h frequency t e t a n i c s t i m u l a t i o n s o f the stratum radiatum i n guinea p i g hippocampus i n v i t r e i T h e graph shows both t h e p o p u l a t i o n s p i k e (PS) r e c o r d e d i n t h e C A i p y r a m i d a l l a y e r and t h e f i e l d EPSP ( f i e l d EPSP) r e c o r d e d i n t h e d e n d r i t i c r e g i o n evoked by s t i m u l a t i o n o f t h e stratum radiatum i n these experiments. Tetanic stimulation of the s t r a t u m r a d i a t u m s u b s e q u e n t l y i n d u c e d p o s t - t e t a n i c p o t e n t i a t i o n s (*) t h a t decayed w i t h i n 5 min t o r e v e a l the u n d e r l y i n g long-term p o t e n t i a t i o n o f b o t h t h e p o p u l a t i o n s p i k e and t h e f i e l d EPSP. Note t h a t LTP was p r e s e n t t h r o u g h o u t t h e one-hour 1 o b s e r v a t i o n p e r i o d ( v a l u e s a r e Mean * S.E.M.). D  90  CHIRWA  113  Figure 11-10. Representative recordings of intracellular and extracellular potentials in CAi area evoked by stimulation of the stratum raalatum oerore ano after inouction or long-term potentiation. Tn (T) are shown pre- and post-tetanus intracellular potentials in representative CAi neurons recorded with micropipettes f i l l e d with 3 M potassium chloride (2a) or 3 M cesium chloride {lb). Note that in both cases, LTP is seen as increases in previously subthreshold intracellular EPSPs sufficient to ^voke a synaptically activated Na spike (la) or a presumed Ca spike (lb). In (II) are shown pre- and post-tetanus population spikes (ITa) recorded in the CAjb pyramidal layer and field EPSPs (lib) recoTded in the C A i dendritic region. The reduction in onset latencies of the potentiated synaptic responses is discernable in the field responses (II). n  D  +  -  b  CHIRWA (6 of 9 neurons). resistances before: after:  In these neurons, resting membrane potentials and input  recorded  -63.4 ± 1.3, 32.2 * 1.9;  in  the  after:  values  soma remained  unchanged  -63 ± 2.2;  in  R  n  are Mean ± S.E..M.;  Mn:  CA  with those  reported  by Andersen et a l .  neurons recorded with micropipettes  1b  during  LTP (RMP,  before:  32.5 * 2.5,  n = 9 neurons; p > 0.05  determined by two-tailed paired Student's t-test). agreement  114  filled  These results (1980c). with  Cs  +  In  are  as in  separate  (same neurons  described in section 14.1.3), tetanus-induced LTP was expressed as increases in subthreshold EPSPs in 7 of 10 neurons tested a% of  control  15  Mean * S.E.M.;  min  after  n = 10  induction  neurons;  of  strated still  in Figure 11-10.  be induced even if  LTP: 150.8 * 5.7;  p < 0.05;  t-test), even reaching threshold for Ca  (intracellular  one-tailed  EPSPs as values  paired  are  Student's  spikes (4 of 7 neurons) as i l l u -  The above results demonstrated that LTP could K  +  effluxes  in a neuron were diminished by Cs  +  applied internally. 11.4.2  Paired depolarizations.  In  these experiments,  slices were  incubated in physiological medium containing 50 uM picrotoxin, to facilitate LTP development  (Sastry,  Repeated pairings  Goh and Auyeung, 1986;  (10-15 pairings)  tions of a CA^ neuron (3-7 b  of  the  stratum  radiatum  at 0.2  Hz of  Wigstrom, et a l . , intracellular  198b).  depolariza-  nA, 200 msec) with subthreshold stimulations  (30-100  uA, 0.1-0.5  adjusted to 50-60% of orthodromic threshold)  msec;  stimulus  strength  resulted in subsequent long-  lasting increases in amplitudes of intracellular EPSP evoked by stimulation of the stratum radiatum (intracellular simultaneous Mean ± S.E.M.;  pre- and postsynaptic n = 6;  p < 0.05;  EPSPs as a % of control 15 min after  activations:  146.6 ± 8.3;  ANOVA with Duncans' multiple  tests; more quantitative data in Figure 11-11).  values  are  comparisons  This post-pairing LTP of  CHIRWA  115  200 c o o o  175 150  T  1  125 QL  CO Q_  Ld  (Z  UJ O <  cr  100 pre— and postsynaptic pairings  75 50 25 0  (values are Mean t S . E . M . , n = 6) 10  20  30  40  50  TIME (min)  Figure 11-11. Illustration of long-term potentiation induced by simultaneous pairings of conditioning depolarizing current injections into CAib neurons and stimulation of the stratum radiatum in guinea pig hippocampus in vitro, ihe graph shows the amplitudes ot intracellular EPSP recorded in CA^ neurons evoked by stimulation of the stratum radiatum. Consecutive 10-15 pairings of conditioning intracellular current injections into CA^ neurons with activations of the stratum radiatum at the beginning of each intracellular depolarization, subsequently induced short-term potentiations (*) that decayed within 3-6 min to reveal the underlying long-term potentiation of the intracellular EPSPs (i.e evoked by single stimulations at 0.2 Hz of the stratum radiatum). The potentiated intracellular EPSPs were maintained throughout the 30 min observation period. Picrotoxin (50 pM) was present in the medium.  60  CHIRWA evoked responses to  stimulation  of  the  116  stratum radiatum was present  for  periods beyond 30 minutes (Figure 11-11). 11.5 Asynchronous release of transmitter and LTP During minEPSPs  2-4  min applications  (minlPSPs  observed following  and  of  high  medium,  IPSPs were blocked with  single stimulations  of  single conditioning depolarizing current cases) into CA^ neurons (n = 26 neurons; b  data).  Ba  the  50  bursts of  pM picrotoxin)  stratum  injections  short  radiatum  or  were after  (0.01-0.2 Hz in both  see Table 11-2 for  However, the frequencies of evoked minEPSPs in B a  quantitative  were increased  ++  by at least 50%following the pairings of conditioning depolarizing current injections  into  CA^ neurons with concurrent  radiatum (p < 0.05;  at  of  the  stratum  n = 26 neurons; one-tailed Student's t-test; see quanti-  tative data in Table 11-2). initiated  stimulation  [NB:  Stimulation of the stratum radiatum was  the beginning of the  direct  intracellular  depolarization.]  These transient bursts of minEPSPs were greatly exaggerated with increasing pairings of conditioning depolarizing current  injections  and stimulations of the stratum radiatum, such that it  into CA^ neurons b  was not possible to  quantify them accurately. Increases in evoked minEPSPs frequencies were also observed during LTP produced CA2-CA2  by cell  tetanic  stimulations.  body layer  removed)  If  hippocampal  were exposed to  slices  high  Ba  (with  the  medium for  2 min, stimulation of stratum radiatum at 0.02-0.2 Hz resulted in a burst of evoked minEPSPs that  followed  activated action potential  the  synchronous EPSP or  (24 of 31 neurons).  the  synaptically  Slices were then re-exposed  to control medium for 15 min and the stratum radiatum was tetanized (400 Hz, 200 pulses; stimulation strength adjusted to evoke subthreshold EPSP only) to induce LTP (18 of the above 24 neurons).  LTP was detected as long-last-  CHIRWA  Table 11-2. Changes in frequencies of evoked minEPSPs in CAih neurons during simultaneous pairings ot conditioning depolarizing current injections into CAih neurons and stimulation of the stratum radiatum in guinea pig hippocampus in vitro  minEPSP amplitudes in mV 0.25 - 0.5  0.5 - 1.0  Unpaired  11 * 2  7 * 2  Paired  17 ± 3*  11 ± 3*  1.0 - 1.5  1.5 - 2.0  1*1 4*2*  1*1  Values are Mean * S.E.M. per 5 sec (n = 26 neurons). Astericks indicate significant differences as determined by one-way ANOVA with Duncans' multiple comparisons tests. In these experiments, 50 pM picrotoxin was present in the medium. [NB: Numbers rounded up to the nearest integer other than O.j  CHIRWA ing post-tetanus EPSPs as a%of activations:  increases of previously subthreshold EPSPs control  15  min  161.0 * 10.8;  after  values  simultaneous  are  118  (intracellular  pre- and postsynaptic  Mean * S.E.M.;  n = 18;  p < 0.05;  one-tailed paired Student's t-test), sometimes sufficient to reach threshold and e l i c i t  orthodromic  stratum radiatum.  action  potentials  following  stimulation  When slices were re-exposed to high B a  of  the  medium 15 min  ++  after the induction of LTP, the number of minEPSPs following stimulation of the  stratum  radiatum  before LTP: 6 * 3  was  at  least  minEPSP frequencies,  two-tailed paired Students' t-test; Figure  The presynaptic volleys during the second Ba  not different from those during the f i r s t  applications were  applications, indicating that the  increases in minEPSPs were not due to the axons.  (evoked  per 5 sec, after LTP: 22 * 7 per 5 sec; values are Mean *  S.E.M; n = 18 neurons; p < 0.05, 11-12).  doubled  activation  of more presynaptic  Furthermore, if slices were exposed to high B a  a 30 min  interval  without  minEPSPs were not  the  significantly  LTP-inducing tetanus, increased  (evoked minEPSP frequencies; during f i r s t second B a  ++  application:  neurons; p > 0.05,  6*2  ++  per  during Ba  the  the  frequencies  second  application:  ++  sec; values  two-tailea paired Students'  medium twice with  are  application  7*2;  Mean * S.E.M.;  t-test).  of  The input  during n= 6 resis-  2+ tances determined during the last min of Ba applications increased by at least  75% relative  to  "widened" as illustrated  controls,  and the  in Figure 11-13.  EPSPs and/or  action  potentials  But input resistances checked in  2+  Ba  at fixed intervals following the EPSP evoked with stimulation  stratum radiatum remained unaltered in Mn determined radiatum in Ba  at  before  100 msec from start  , before LTP: 56.3 * 2.9;  and after of  of the  LTP development  stimulation  15 min after  of  the  (R^  stratum  induction of LTP:  58.2 * 3.6; n = 6; p> 0.05; two-tailed paired Students' t-test).  A  During 2 min B a  B + +  application  Control  During 2 miri B a  + +  application  15 m i n p o s t 4 0 0 H z , 0-5s  j^w^-X  NuvjV_ J2 mV 500 m s  Figure 11-12. Comparisons of the frequency of minEPSPs 1n CAJK neurons following stimulation of the stratum radiatum before, ana after development of long-term potentiation in 9uipea pig hippocampal s l i c e s incubated in Ba . During a 2 min Ba application tfie synaptically activated action potential (truncated) was followed by a burst of minEPSPs (A). [NB: In these experiments spontaneous. minEPSPs were usually not present in the control medium or in Ba medium in the absence .of stimulation of the stratum radiatum.] After 15 min of washing out Ba the stratum radiatum was tetanized (400 Hz, 200 pulses; stimulation strength adjusted to evoke a subthreshold EPSP) to induce LTP. Then 15 min after the induction of LTP ( i . e . detected as a long-lasting post-tetanic Increase 1n amplitude of a previously subthreshold EPSP), the s l i c e was re-exposed to a second £ min Ba application ( i . e . an interval of 30 min between Ba applications). The number of minEPSPs Immediately following the synaptically activated action potential in Ba was more than doubled during LTP [B) ( i . e . same stimulation parameters as 1n (A). The traces 1n (A) and (Bj were recorded on a s t r i p - c h a r t recorder during the last 30" s of Ba application. In (A) and in (IB) the sweeps were taken 1n immediate succession. Picrotoxin (50 pM) was present in the medium. +  S g ^  +  ^ VO  CHIRWA  Control  2 min in B a  + +  10 min p o s t - B a  120  + +  4^—• llll  120 mV 10-5 nA 200 ms  Figure 11-13. Representative changes in evoked intracellular responses in a CAi slices neuron in guinea^ pig hippocampal incubated for 2 min in B a . EPSP (first intracellular [first row) and The " u 1la synaptically driven orthodromic action potential (second row) were evoked following subthreshold and suprathreshold stimulation of the stratum radiatum, respectively. In addition, action potentials in this neuron were also elicited with intracellular depolarizing current pulses (third row; current traces shown in fourth row). The undershoots associated with responses in rows 2 and 3 probably were a mixture of both IPSPs and afterhyperpolarizations (AHP^ [NB: Picrotoxin was not added to the media.] During a 2 min Ba application, the durations of the EPSP, the synaptically activated action potent i a l , the action potential due to current injections, and all their associated undershoots (IPSPs and/or AHPs) were* increased (middle columns in rows 1-3). Furthermore, during Ba application, note the increase in input resistance which is reflected as an increase in the magnitude of the membrane depolarizing response to intracellular current injection (middle trace versus i n i t i a l or last traces of row 3). The ^served changes during B a ^ application were presumably due to B a mediated blockade of some K effluxes from presynaptic and postsynaptic regions. However, the changes in evoked responses during B a ^ were reversible as early as 10 min post-application (last column in row 1-3). n  TT  CHIRWA  121  11.6 Endogenous substances and synaptic potentiation Applications of samples collected during tetanic  stimulations of the  guinea pig hippocampus in vivo (denoted as "THS") for 2 min onto hippocampal slices  during  stimulations  of  stratum  radiatum  at  0.2  Hz subsequently  produced synaptic LTP (population spike in CA^ area as a % of control, 15 b  min after  exposure: 130.5 * 4.8,  p < 0.05,  one-way ANOVA with Duncans' multiple  11-3).  The potentiated  values are Mean ± S.E.M.;  synaptic  (decreases of 10-30 % relative  slices,  comparisons tests;  responses had  reduced  onset  Table  latencies  to controls), and LTP was present beyona 60  min (quantitative data in Table 11-3; Figure 11-14). tions of HTHS (i.e.  n = 8  ln contrast, applica-  heated THS; see methods in chapter 10) (n = 6 slices) or  of samples collected in the absence of tetanic  stimulations of the guinea  pig hippocampus in vivo (denoted as "UHS") had insignificant effects on the population spikes in CA-^ area evoked by stimulation of the stratum radiatum (one-way ANOVA, quantitative data in Table 11-3). Similarly,  applications of  samples collected during tetanic  stimula-  tions of the rabbit neocortex in vivo (denoted as "TNS") caused LTP of the population spike in the CA^ area induced by stimulation  of  radiatum  exposure:  * 0.22,  (population values  are  spike expressed in mV 15 min after Mean ± S.E.M.,  n = 16,  multiple comparison tests, quantitative  p > 0.05,  the  stratum 2.64  ANOVA with Duncan's  data in Figure 11-15), with associ-  ated reductions in onset latencies (onset latencies of population spike as a % of control  15 after  induction of  S.E.M., n = 16, p < 0.05;  LTP:  86.3 ± 4.8;  are  one-tailed unpaired Student's t-test).  cal samples collected in the absence of tetanic slices)  values  and those TNS fractions  stimulation  that were pre-heated  n = 10 slices) did not produce significant potentiations  Mean ±  Neocorti-  (UNS;  n = 10  and cooled (HTNS; (quantita-  CHIRWA  Table 11-3. Effects of samples collected from guinea pig hippocampus in vivo on_CA_2b population spike in guinea pig hippocampus in vitro Control  Post-application  30  10  15  30  60 min  THS:  Mean S.E.M. n  100.6 1.5 8  136.6* 3.3 8  130.5* 4.8 8  138.7* 3.2 8  138.0* 3.1 8  UHS:  Mean S.E.M. n  100.0 2.7 6  97.0 3.3 6  102.0 3.2 6  99.3 1.5 6  103.0 2.0 6  HTHS:  Mean S.E.M. n  99.5 2.8 6  102.0 1.7 6  98.0 1.8 6  101.3 2.3 6  98.0 2.7 6  Reported values are amplitudes of population spikes expressed as of control. Astericks indicate significant differences (relative to control responses) as determined by one-way AN0VA with Duncans' multiple comparisons tests. Abbreviations: THS, tetanized neocortical sample; UHS, untetanized neocortical sample; and HTHS, heated-tetanized neocortical sample.  122  CHIRWA  Control  Post-application  10ms  15  123  15 min  Figure 11-14. 11 lustration of long-term potentiation in CA|h area of the guinea pig hippocampus in vitro induced with brief applications of samples collected during tetanic stimulations of the guinea pig hippocampus in vivoJ The effects of samples collected from guinea pig hippocampus in vivo on population spike in CA]k area evoked by stimulation of the stratum radiatum in representative experiments. Each sample was applied in a different s l i c e . Application of 2ml for 2 min of THS (I_) but not UHS {II) or HTHS {III) caused LTP (postapplication records taken 15 min after return to control medium).  CHIRWA tive data in Figure 11-15). TNS or THS. of  the  Presynaptic volleys were not altered by either  In addition, TNS applications without concomitant  stratum  radiatum  caused insignificant  changes in  responses (population spikes expressed in mV:  stimulations  evoked synaptic  controls, 1.03 ± 0.20;  min after application of TNS without stimulation of the stratum 0.99 * 0.17; Student's  values are Mean ± S.E.M.,  t-test).  124  n = 6;  p > 0.05,  radiatum:  two-tailed  When slices were exposed to TNS in the  and 15  last  paired 2-3 min  during saccharin (10 mM, applied for 10 min) LTP of the population spike in the  CA^  controls,  area  was  not  1.31 * 0.14;  1.22 ± 0.16;  values  and  are  ANOVA, Figure 11-16).  induced 15  Mean ±  min  (population after  S.E.M.,  spikes  application  n = 6  (population spikes expressed in mV:  p < 0.05,  TNS:  2.53 ± 0.35;  ANOVA with  mV:  saccharin/TNS:  p > 0.05,  one-way  values  are  Duncan's multiple  population  spike was observed  controls, 1.34 * 0.30;  and 15 min after  Mean ± S.E.M.,  n = 6  comparisons tests).  controls, 2 min applications of glutamate depressions of  in  When TNS was subsequently applied without concurrent b  to  of  slices;  applications of saccharin, ,LTP of the CA^ population  exposure  expressed  (100  In  If  glutamate  separate  pM) caused post-application  spikes in CA-^ area by 30-70% that  10-35 min (n = 5 slices).  slices,  lasted  for  samples were pre-heated and cooled  (as in the case of HTHS or HTNS) before being applied on hippocampal slices the actions of glutamate were not altered % of controls 15 min after:  exogenous glutamate,  heated then cooled exogenous glutamate, Mean * S.E.M.; p > 0.05;  (population  71 * 8.2;  spikes expressed as  73.6 * 10;  and following  n = 5 slices; values are  two-tailed Student's t-test),  suggesting that the  LTP inducing substances in TNS could not have been endogenous glutamate. other controls, atropine (100 uM; n = 4) or dihydro-8-erythroidine  In  (100 pM;  n = 4) did not block LTP when TNS was applied in the last 2-3 min of 10 min  CHIRWA  125  Post-TNS • n=16 3-  I Control  Post-UNS  Post-HTNS  n=1 6  n=10  n=10  I I  I I I  0  10 2 0 3 0  I  5 10 15 3 0 60  Time  5 101530  60  5 10 15 3 0 6 0  (min)  Figure 11-15. Effects of samples collected from rabbit neocortex in vivo on CAih population spike in guinea pig hippocampus in vitro. Ihe errects of samples collected from rabbit neocortex in vivo on the population spike in CAib area evoked by stimulation of the stratum in representative slices are shown. The graph shows Mean ± S.E.M. of the population spike amplitudes (in mV). Note that TNS but not UNS or HTNS caused LTP (asterisks indicate significant differences at p < 0.05; one-way ANOVA with Duncan's multiple comparisons test).  CHIRWA  10 mM  Control  126  Post-application  Saccharin TNS  10  20  0-5  SO  J  S  10  20  30  40  «0  20  30  40  40  TNS  20 mt 30  40  10 OS  j 5  to  Figure 12-16. Failure to induce long-term potentiation in guinea pig hippocampus in vitro when samples collected during tetanic stimulation of the rabbit neocortex In vivo are applied in the presence of saccharin. The population spike in CA15 area were evoked with stirnulation of the stratum radiatum. The top row shows evoked population spikes obtained in control medium, followed by population spikes evoked during a 10 min application of 10 mM saccharin and TNS (i.e. 2 ml of TNS was applied during the last 2 min of saccharin applications), and population spikes recorded after these drug applications. The last three population spikes in the f i r s t row (i.e. at 30, 40 and 60 min responses) are shown again at the beginning of the second row. These responses are followed by subsequent population spikes obtained during application of TNS without saccharin, and population spike obtained after the TNS application. Each population spike is an average of 8 consecutive records (0.2 Hz). The time (in min) at which the averaging of the 8 records was initiated are shown underneath each response. Note that TNS applied alone, but not during saccharin, caused LTP.  CHIRWA perfusions  of  atropine  or  dihydro-p-erythroidine  (population  127 spikes  expressed as % of control 30 min after exposure of TNS in the presence of (1)  atropine:  203.5 ± 35.0;  or  (2)  dihydro-g-erythroidine;  values are Mean ± S.E.M., n = 4 in each case; Students' that  t-test).  might  have  These results been  present  p < 0.05,  226 ± 46.3;  one-tailed paired  suggested that endogenous acetylcholine in  TNS,  potentiating  effects of THS or TNS.  substances,  probably macromolecules, that  was  not  responsible  for  the  However, TNS contained heat-sensitive were  involved  in  potentiating  synaptic transmissions. Interestingly, ment of tetanus pulses)  induced LTP, if  were delivered  saccharin  to  applications  I. 11 ± 0.33; 0.47;  10 mM saccharin applied for 10 min blocked the develop-  the  high frequency trains  stratum  (population  and 30 min after  Figure 11-17).  the  radiatum  spikes  tetanic  during  expressed  stimulations  But a subsequent tetanus  (400  the  in  in  Hz, 200  last  mV:  min of  controls,  saccharin:  given in the  1.13 ±  absence of  saccharin produced LTP in the same slices (population spike in mV, 30 min post-tetanus:  2.84 ± 0.44;  values  are  Mean ± S.E.M.;  n= 9  slices;  p < 0.05; ANOVA with Duncan's multiple comparisons test; Figure 11-17). II. 7 Effects of rabbit neocortical samples on cultured PC-12 cells 11.7.1 these  studies  PC-12 cell was  to  growth and neurite  examine  whether  the  induction. collected  The objective of rabbit  neocortical  samples (see methods in chapter 10) contained neurite-inducing factors. quantification,  PC-12 cells were considered to have developed neurites  they  with one or  presented  diameter of the cell bodies.  more extensions  that  were  longer  than  For if the  On day 2 after plating PC-12 cells in various  stages of growth were in small evenly dispersed clusters that were anchored to the floor of each culture dish.  None of these cultures showed any  CHIRWA  >  £  400 Hz, 200 pulses v  400 Hz, 200 pulses y 10 mM Saccharin  4-  3  U  Q_ CO  Z  o h-  128  A  2  < _J ZD  Q_ O CL  T o J-  T o-  I  I  1  x  (values are Mean ±S.E.M., n = 9) 0  0  20  40  60  80  100  120  140  TIME (min)  Figure 11-17. The blockade of tetanus-induced long-term potentiation by saccharin in the guinea pig hippocampus in vitro. The graph shows the population spike in CAit> area evoked by stimulation of the stratum radiatum. Tetanic stimulation of the stratum radiatum during a 10 min application of 10 mM saccharin did not cause LTP. However, the same tetanus produced LTP when delivered to the stratum radiatum 60 min after application of saccharin.  160  CHIRWA significant  neurite  growth.  When PC-12  cell  cultures  129  were subsequently  incubated in different feeding media, the following results were obtained. PC-12 cell cultures incubated with TNS (n = 6) showed neurite growth on day 3, and these neurites continued to grow throughout the eight-day observation period.  However, PC-12 cells did not develop any significant neurites when  incubated n = 6),  in  (2)  feeding  HTNS (n = 6),  were incubated exhibited  media  in plain  containing  (1)  TNS with  or (3)  UNS (n = 6).  growth  medium throughout  insignificant neurite  extensions.  growth media containing UNS or  saccharin  (10 mM;  Similarly, PC-12 cells that these  experiments  also  Some PC-12 cells incubated in  HTNS presented with small  protrusions  (a  fraction of the PC-12 cell diameter in length) that seemed to have failed to develop into neurites. PC-12 cell cultures.  Figure 11-18  illustrates  the results obtained with  These results suggested that substances with NGF-like  activities were present in the samples collected during tetanic  stimulations  of the rabbit neocortex. 11.8 Effects of exogenous NGF in the hippocampus The results collected  during  in section 11.7 tetanic  above raised the prospects that samples  stimulation  contained NGF-like substances.  of  the  rabbit  neocortex  in vivo  Since these neocortical samples could induce  LTP when applied in the hippocampus (see results in section 11.6)  it  was  wondered whether similar effects could be mimicked by application of exogenous NGF.  When exogenous NGF (2.5  ug/ml, from Vipera lebetina) was applied  for 10 min during stimulation of the stratum radiatum (0.2  Hz), the popula-  tion spike in the CA^ area evoked by stimulation of the stratum was not potentiated during the subsequent one hour of recording spike expressed in mV:  controls, 1.36 * 0.2;  NGF with stimulation, 1.34 ± 0.3;  radiatum  (population  and 30 min after exposure to  values are Means ± S.E.M., n = 9 slices;  p > 0.05, two-tailed paired Student's t-test).  Similarly, if the stimula-  CHIRWA  Figure 11-18. Effects of samples collected from rabbit neocortex in vivo on neurite growths in PC-12 cell cultures. PC-12 cells were incubated with l.b ml ot growth medium containing 0.75 ml each of UNS (A), TNS (B_), HTNS (£) and TNS with 10 mM saccharin. PC-12 cells incubated in the presence of TNS (i.e. (BJ) and not the other samples developed extensive neurites, thereby indicating the presence of NGF-like substances in TNS. LNB: PC-12 cells were considered to have developed neurites if they presented with one or more extensions that were longer than the diameter of the cell bodies.] The micrographs were taken on the fourth day of incubation. The bar represents 50 pM; n = 6 culture plates for each sample.  130  CHIRWA tion of the stratum radiatum was interrupted peptide, there was no potentiation expressed in mV:  without stimulation, 1.29 * 0.45;  during the application of the  in any of the slices  controls, 1.38 ± 0.5;  132  (population spike  and 30 min after  exposure to NGF  values are Mean ± S.E.M.,  n = 4 slices;  p > 0.05, two-tailed paired Student's t-test). However, post-tetanic  a tetanic potentiation  induced LTP if illustrated  stimulation  of  stratum  of the weak CA^ field  radiatum  which  EPSP (i.e.  elicited  "weak input")  the tetanus was given in the presence of exogenous NGF as  in Figure 11-19  (fiela EPSP expressed in mV:  controls, 0.37 *  0.02; and 30 min after exposure to NGF and tetanus, 0.59 ± 0.01; values are Means ± S.E.M., n = 6 slices; p < 0.05, one-tailed paired Student's t-test). The above potentiating actions of exogenous NGF were blocked if the peptides were given in the last 10 min of 10 mM saccharin (saccharin was applied for a total of 12 min) (field EPSP expressed in mV: 30 min values  post-tetanus are  to  weak  Mean ± S.E.M.,  student's t-test).  input  n = 6  during  slices;  control, 0.45 ± 0.02; and  saccharin/NGF: p > 0.05,  That exogenous NGF facilitated  0.42 ± 0.0.05;  two-tailed  unpaired  the development of LTP  following stimulation of a weak input was fascinating since it  is known in  the literature that a weak input can develop LTP when tetanized in conjunction with a strong input (Levy and Steward, 1979). tion of a strong input  in the release of  chemical signals that  subsequently interact with a co-activated weak input.  In this regard, it is  conceivable  that  results  Perhaps tetanic stimula-  exogenous  NGF in  the  present  studies  exerted  effects  similar to those produced by a strong input that is concurrently tetanised with a weak input.  OS  Time p o s t - t e t a n u s (min)  Time in control m e d i u m (min)  1  OS  3 5  Yi  40  10 20 30  1  3 S  " I I I !  i  I  50  L  LL  Iii!  2-5 ug/ml N G F Time p o s t - t e t a n u s (min)  0-5 1  3 S  7 10 20  'I  0-5 mV 20 ms  T i m e in NGF (min)  F i g u r e 11-19. E f f e c t s o f , NGF ( V i p e r a l e b e t f n a ) o n weak EPSP recorded i n the C A i d e n d r i t i c r e g i o n i n g u i n e a p i g hippocampus in vitrol I he s t i m u l u s s t r e n g t h s used t o s t i m u l a t e t h e s t r a t u m r a d i a tum i n t h e s e experiments were those t h a t evoked a weak f i e l d EPSP ( i . e . , "0.4-0.5 mV) i n t h e d e n d r i t i c r e g i o n s a s i l l u s t r a t e d . T h e arrow denotes a t e t a n i c s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m (50 Hz, 10 p u l s e s ) . The numbers on top o f t h e r e c o r d s g i v e t h e p o s t - t e t a n i c times ( i n min) when responses were r e c o r d e d . Tetanic stimulation o f the s t r a t u m r a d i a t u m induced o n l y a s h o r t - t e r m p o t e n t i a t i o n i n c o n t r o l medium ( t o p row). However, t h e same t e t a u s d e l i v e r e d d u r i n g a 10 min a p p l i c a t i o n o f exogenous NGF (2.5 ug/ml a p p l i e d f o r 10 min) p r o d u c e d LTP (bottom row). Each r e c o r d r e p r e s e n t s an a v e r a g e o f 8 c o n s e c u t i v e sweeps. ;  n  CHIRWA 11.9 Mechanisms of action of saccharin: 11.9.1  Saccharin and LTP:  134  Extracellular studies  Dose-relationships.  The following series  of experiments were done to determine whether saccharin possessed non-specif i c electrophysiological effects that could account for LTP development in the hippocampus.  its  antagonism of  Figure 11-20 illustrates the dependency  of LTP blockade on saccharin concentrations perfused during tetanic stimulation of the stratum radiatum.  Saccharin concentrations of 5 mM (applied for  10 min), failed to block LTP development when the tetanic  stimulation was  delivered during the last min of drug application (population spike expressed as % of  control  saccharin:  201 ± 40;  15 min after values  are  tetanus  to  the  Mean ± S.E.M.;  ANOVA with Duncans' multiple comparisons tests).  stratum n = 6;  radiatum  p < 0.05,  during one-way  A second tetanic stimula-  tion given after 30 min of washing with control medium was not significantly different data  from LTP obtained during applications of saccharin (quantitative  in Figure 11-20).  These results  indicated that the  initial  tetanic  stimulations applied during 5 mM saccharin had induced near maximal LTP and, therefore, could not be potentiated any further with a second tetanus given during wash (cf.  Bliss and L^mo, 1973; Bliss ano Gardner-Medwin, 1973).  In  similar experiments, tetanic stimulations to the stratum radiatum during 7.5 mM saccharin applications resulted in relatively (population stratum n = 6;  reduced magnitudes of LTP  spike expressed as % of control 15 min after  radiatum p < 0.05,  Figure 11-20).  during  saccharin:  126 •* 11;  values  one-way ANOVA with Duncans' multiple  are  tetanus  to  the  Mean ± S.E.M.;  comparisons tests;  Subsequent tetanic stimulations of the stratum radiatum in  control medium (i.e.  30 min post-drug) resulted in a significantly  LTP in all cases (quantitative data in Figure 11-20).  greater  Tetanic stimulations  of the stratum radiatum during 10 mM saccharin application consistently  CHIRWA  500  c o u  Control n= 6  *  10 NaS n= 6  7.5 N a S n= 6  5 NaS n= 6  400  300 UJ Q_ CO  z g  135  200  I  100-  s \ s  Q_ O Q_  0  <cu  CD  CN CD  CD  IN  /  s s s  /  s \ \ \ \ s  / /  1 / / /  / / /  _/_  CN  CM  CD  CD  I—  Figure 11-20. Effects of different concentrations of saccharin on LTP production in the CAi area of the guinea pig hippocampus in vitro. Ihe grapn shows trie population spike in ik^ area evoked by stimulation of the stratum radiatum expressed as a of control. Each slice was exposed to a single concentration of saccharin that was applied for 10 min, respectively. ln saccharin, the f i r s t tetanic stimulation (Tet 1) was applied to the stratum radiatum during the 9th min of the drug application. This was followed by the second tetanus (Tet 2) that was applied 30 min after each saccharin application. In slices not exposed to saccharin (0 NaS), there was a 30 min interval between the f i r s t tetanus (Tet 1) and the second tetanus (Tet 2). Note that 10 mM saccharin blocked LTP development following tetanic stimulation of the stratum radiatum However, the same tetanus produced LTP when delivered to the stratum radiatum 30 min after 10 mM saccharin. [NB: 400 Hz, 200 pulses used in all experiments; asterisks indicate significant differences relative to control responses; p < 0.05; ANOVA with Duncan's multiple comparisons test.j n  /  CHIRWA blocked LTP induction (population  spike expressed as % of control  after tetanus to the stratum radiatum during saccharin: are  Mean ± S.E.M.;  n = 6;  p > 0.05,  one-way  ANOVA),  136 15 min  118 * 18; values and  yet  tetanic  stimulations of the stratum radiatum after 30 min wash elicited LTP in the same experiments tetanus  to  the  (population spike expressed as % of control 15 min after stratum  radiatum  during  wash:  290 ± 50;  values  are  Mean * S.E.M.; n = 6; p < 0.05, one-way ANOVA with Duncans' multiple comparisons tests; relatively  quantitative  data  in Figure 11-20).  By comparison, LTP of  greater magnitude coulo be obtained with tetanic  stimulation of  the stratum radiatum in slices not exposed to saccharin (population spike expressed as % of control 15 min after tetanus in control medium: values  are  multiple  Mean ± S.E.M.;  comparisons  n = 6;  tests;  p < 0.05,  Figure  one-way  11-20).  290 * 50;  ANOVA with Duncans'  Taken  together,  the  above  results illustrated that the minimum concentrations of saccharin that could consistently block LTP induction were between 7.5 and 10 mM. 11.9.2  Saccharin and post-tetanic  medium (i.e. 1.5  2 mM C a  mM Mg ;  ++  see table  ++  potentiation.  reduced  in control medium replaced with 0.5 9-1  in  chapter  9),  stratum radiatum (at 10 - 15 min intervals) potentiation  ln  tetanic  mM C a  stimulation  Ca  ++  ++  and  of  the  repeatedly induced post-tetanic  (PTP) of the population spike lasting for 3 - 6  min.  Interest-  ingly, the same tetanic stimulations delivered in the last min of saccharin perfusions (10 mM saccharin applied for 10 min) significantly  different  from that obtained  induced PTP that was not  in control  spike expressed as % of control 3 min after tetanus in: 190 * 10;  and  (2)  saccharin  204 * 23;  n= 6  medium  (population  (1) control medium,  slices;  values  Mean * S.E.M.; p < 0.05, two-tailed paired Student's t-test; Table 11-4).  are  CHIRWA Post-tetanic potentiation the  peripheral  mechanisms  in the hippocampus, like  nervous system, is thought  (McNaughton,  1980).  It  137  its counterpart  in  to be mediated by presynaptic  has been  suggested that  PTP in  the  peripheral nervous system (where it was f i r s t described) is due to transient hyperpolarizations that  cause  in presynaptic terminals  increases  in  amplitudes  invading the presynaptic terminals  following tetanic  and durations  (cf.  of  action  potentials  Eccles and Krnjevic, 1959).  changes presumably result in the enhancement of transmitter synaptic transmission.  stimulations  These  released during  Presuming that similar mechanisms underlie PTP in  the hippocampus, then results of the present studies indicate saccharin did not interfere with the release of transmitter  that 10 mM  during synaptic  transmission in the hippocampus. 11.9.3  Saccharin  and  paired-pulse facilitation  paired-pulse  facilitation.  was used to examine further  saccharin on presynaptic functions.  The  method  of  possible effects  of  Paired-pulse facilitation  be due to increases in released transmitter the stimulation pair (McNaughton, 1980).  is thought to  induced by the secona pulse in  Presumably, "residual" effects on  transmitter release associated with the f i r s t pulse ado. up with those of the second pulse  in  the  stimulation  pair.  In  this  characteristics of the evoked responses reflect  regard,  population  illustrated larger  than  spike  denoted  in Figure 11-21. the  first  as  "P^"  and  of 30 msec evoked a  "P ", 2  The second response, P ,  response,  2  P^  (ratio  n = 6 experiments; values are Mean ± S.E.M.).  the  Paired-pulse stimulation  of the stratum radiatum at a fixed inter-pulse interval of  in  presynaptic conditions and,  this feature was utilized in the present studies.  set  changes  of  ^2 1 :P  respectively,  as  was consistently w a s  3  * * 3  ±  1*25;  Interestingly, during 10 mM  CHIRWA  Table 11-4. Effects of saccharin on post-tetanic potentiation of the population spike in the CAih area induced by stimulation of the stratum radiatum in the guinea pig hippocampus in vitro.  Time after tetanus (min)  Control medium  10 mM Saccahrin  3  1.90 ± 0.18  2.04 ± 0.23  5  1.50 ± 0.20  1.84 ± 0.21  Values are Mean ± S.E.M. in mV (n = 5 experiments). The stimulation strength was adjusted to evoke pre-tetanus population spikes with amplitudes of 1 mV. Tetanic stimulation of the stratum radiatum (400 Hz, 200 pulses) during saccharin (10 mM saccharin applied for 10 min; tetanus given at 9 min of drug application) induced PTP that was not s i g n i f i cantly different from PTP obtained in control medium (p > 0.05; twotailed Student's t-test). LNB: Medium used contained reduced C a levels (see text).] ++  CHIRWA  Figure 11-21. Demonstration of lack of effects of saccharin on paired-pulse facilitation in the C A I K area of the guinea pig hippocampus in vitro. TwTfi puise stimulation of the stratum radiatum evoked a set of two population spikes (PI and P2) in the C A ] h area as illustrated. The interval between the two pulses in the stimulation pair was 30 msec and, this interval was found to be adequate in causing facilitation of the second response (P2) relative to the f i r s t response (PI). Plotted are the calculated ratio of P2 : PI for each set of evoked responses, respectively. The ratio for sets of responses obtained during 10 mM saccharin that was applied for 10 min were not significantly different from those obtained in control medium before or after application of saccharin.  139  CHIRWA saccharin cantly  (applied  for  different  10 min)  from that  the ratio  obtained  in control medium, 3.31 ± 1.25; 3.07 * 1.18;  in  of  Pg to P^ was not  control  medium (ratio  t-test;  signifiof  ^2 1 :P  :  and in saccharin at 9 min of application,  values are Mean * S.E.M.; n = 6 slices;  paired Student's  140  Figure 12-21).  p > 0.05;  These results  two-tailed  further  indicated  that 10 mM saccharin applied for 10 min did not significantly alter  trans-  mitter release during synaptic transmission. 11.9.4  Saccharin and dendritic  negative  wave during tetanus.  At  this stage in the studies it was becoming evident that 10 mM saccharin had insignificant frequency  effects  on synaptic responses in CA^ area produced by low  stimulation  of  the  stratum  radiatum.  However,  there  still  remained the possibility that saccharin might interfere with the development of extracellular 1988,  for  fields that occur during tetanus (Gustafsson and Wigstrom,  review).  It  is thought  mediate  low frequency excitatory  CA^  the  in  receptor  hippocampus  (see  that  non-NMDA receptors  6).  Under  blocked by Mg  conditions, NMDA  and, therefore,  ++  responses.  However, tetanic stimulations induce adequate dendritic depolarare sufficient  channels making it open channels.  generation  to remove the Mg  possible for cations (e.g.  of  excitatory  do not  significantly  that  the  these  contribute  izations  towards  of  synaptic responses in areas such as the  chapter  channels are apparently  activation  block of  ++  Ca ) ++  synaptic  NMDA receptor  to flow through these  A component of the dendritic negative wave that is abolished  by D-2-amino-5-phosphonovalerate (presumed to be a competitive antagonist at the NMDA receptor)  is thought to be the electrophysiological correlate of  NMDA receptor activation during tetanus. rin on the development of the dendritic examined.  Hence the effects of 10 mM sacchanegative wave during tetanus was  CHIRWA  141  When the slices were exposed to 10 mM saccharin for 10 min, the extracellular  negative  generated  during  wave  control,  0.98*0.4; values  apical  dendritic  stimulation  of  in Figure 11-22.  subsequent  1.05 * 0.25;  the  a tetanic  present as illustrated potentiated  in  to  this  are  ANOVA; Figure 11-22).  stratum  The field  tetanus  ano 15 min after  area  (field  tetanic  Mean * S.E.M.;  n= 5  of  CA^ neurons,  radiatum,  was  still  EPSP, however, was not EPSP  expressed  stimulation slices;  in  mV:  in saccharin,  p > 0.05,  one-way  A second tetanus given in the absence of saccharin  also resulted in a negative field that was not different during saccharin applications (Figure 11-22).  from that observed  However, the population EPSP  was potentiated (field EPSP expressed in mV:  control, 0.98 ± 0.4,  controls as above; and 15 min after tetanic stimulation  i.e. same  in control medium,  I. 55 * 0.63; values are Mean ± S.E.M.; n = 5 slices; p < 0.05,  one-way ANOVA  with  This  Duncans'  showing that  multiple  comparisons  saccharin did not  test;  affect  Figure  the  11-22).  development  of  the  finding dendritic  negative wave during tetanus was important for the following reason. receptors are thought to be involved in the Lynch,  1988;  reviews).  Collingridge,  It,  1985;  NMDA  induction of LTP (Bliss and  Gustafsson  and  Wigstrom,  1988,  for  therefore, appeared that 10 mM saccharin was not blocking LTP  by interfering with the negative wave in the dendritic region thought to be generated by the activation of NMDA receptors during tetanic stimulations. II. 10  Mechanisms of action of saccharin: 11.10.1  Effects  of  Intracellular  studies  saccharin on spontaneous and evoked responses.  Applications of 10 mM saccharin for 10 min did not induce any significant changes  in  resting  neurons tested  membrane  potentials  or  input  (RMP in mV, controls: -61 * 1.9;  resistances  during  the  last  saccharin application: -58.5 * 2.0; and R in Mfi determined from I-V  of  CA^  min of  CHIRWA  50 Hz, 5 pulses  20 min p o s t - t e t a n u s  A  10 mM Na Saccharin  Figure 11-22. Effects of saccharin on the extracellular negative waveinCAjb apical dendrites induced during tetanic stimulations of tne straTDm raaiatum in the guinea pig hippocampus in vitro. nTe field EPSP recorded in the CA]k dendritic region was evoked by stimulation of the stratum radiatum. In these experiments, 50 yM picrotoxin was added to the physiological medium. Five stimulation pulses, given at 50 Hz, revealed a dendritic negative wave that was superimposed with field EPSPs (A; record taken at 8 min during a 10 min application of 10 mM saccharin). In [b] is shown a dendritic field EPSP evoked by low frequency stimulation (0.2 Hz) of the stratum radiatum in control medium, 20 min after the tetanus of (A). Subsequently, a similar tetanus (50 Hz, 5 pulses), applied "in control medium, also induced a dendritic negative wave that was superimposed with dendritic field EPSPs (C). A dendritic field EPSP evoked by a low frequency stimulation (072 Hz) of the stratum radiatum was found to be potentiated (D), when checked 20 min after the tetanus of ( £ ) . In (EJ, note that super imposition of traces (A) and (£) gave a perfect f i t thereby showing that the responses induced by the tetanus during saccharin applications was of the same magnitude as similar responses obtained in control medium.  CHIRWA relationships, application:  controls:  31.5 * 1.54;  31.9 * 1.64;  p > 0.05;  two-tailed  frequency  of  values  unpaired  spontaneous  during  are  and minlPSPs, since  picrotoxin  affected  by the  of  (number  t-test;  postsynaptic  minEPSPs  drug  last  not  spontaneous  unpaired  Student's  t-test).  saccharin  n = 10  neurons;  Figure  11-23).  potentials  (i.e.  added to small  media)  intracellular  mixed not  control:  10 ± 1 per sec in  n = 10 neurons; p > 0.05;  Typical  The  were  potentials,  12 ± 2, and during last 2 min of saccharin application: both cases; values are Mean * S.E.M;  min of  Mean * S.E.M.;  Student's  miniature  the  143  two-tailed  responses  in  CA^  neurons obtained in control medium and during application of 10 mM saccharin are illustrated  in Figure 11-23.  Saccharin applications induced i n s i g n i f i -  cant changes in the magnitudes of EPSPs, IPSPs, action potentials (Figure 11-23). was not  or AHPs  The above results supported the notion that 10 mM saccharin  exerting  non-specific effects  on electrical  properties  of CA^  b  neurons that could account for the drugs' antagonism of LTP production. 11.10.2  Saccharin  Presynaptic terminal  effects  excitability  assessing some of the electrical (1958)  suggested  that  on  presynaptic  testing  terminal  excitability.  provides an indirect  method for  properties of presynaptic regions.  hyperpolarizations  of  presynaptic  terminals,  example, resulted in a decrease in presynaptic terminal excitability.  Wall for These  methods can also be used to identify possible mechanisms mediating increases or decreases in presynaptic terminal  excitability  1973; Saint, Quastel and Chirwa, 1986). excitability electrical  (e.g.  Cooke and Quastel,  Hence, the methods of presynaptic  testing were used to assess possible effects of saccharin on  properties of the presynaptic regions in the CA^ area of the  hippocampus.  b  Stimulus rheobasic values extrapolated  from  strength-duration  curves in each experiment, were typically around 3 msec (n = 5 neurons).  CHIRWA  Figure 11-23. Examination of the electrophysiological effects of saccharin in the guinea pig hippocampus in vivo. The intracellular tPi>P (JJ and synapticaIly activated action potential (I_I_) were evoked following subthreshold and suprathreshold stimulation of the stratum radiatum, respectively. The action potentials in this neuron were also elicited with intracellular depolarizing current pulses ( I l i a, current traces shown in II lb). The undershoots associated with responses in (II) and (Ilia) probably were a mixture of both IPSPs and afterhyperpolarizations TA"RP). In addition, input resistances were checked by recording membrane potential shifts (IVa) evoked with graded hyperpolarizing current injections (IVb). Saccharin (10 mM applied for 10 min, shown records taken during the 6-9th min interval of drug application) exerted insignificant effects on evoked responses as illustrated in (I-1V, middle responses). Furthermore, 10 mM saccharin did not cause changes in input resistance, as can be seen from the nearly identical membrane shifts, both in saccharin (IVa, middle responses) and in control media (1Va, f i r s t and last responses), induced by the same graded hyperpolarizing current injections (IVb).  144  CHIRWA Activations  of  the  Schaffer  antidromic action potentials had constant onset Ca  -free medium.  collaterals  at  in CA^ neurons.  latencies  of  5-6  values  Student's t-test).  evoked  These all-or-none responses  Applications of 10 mM saccharin (for  are  thresholds  msec, and they could be evoked  change the rheobasic thresholds (controls in 10.9 * 0.2;  rheobasic  145  Mean ± S.E.M.;  W  10 min)  A: 11.3 ± 0.4,  n = 5;  p >0.05;  in  did not  in saccharin:  two-tailed  paired  Ihese results suggested that 10 mM saccharin had insign-  ificant effects on the electrical properties of Schaffer collaterals  termi-  nals as determined by the methods described above. 11.10.3  Saccharin and NMDLA responses.  The  following  experiments  examined the effects of saccharin on depolarizations induced by NMDLA, the acidic amino acid that is thought to be a selective agonist for NMDA receptors.  Applications of NMDLA (25-100 uM) for 1 and/or 1.5 min were associ-  ated with  long-lasting  Baudry and Lynch, 1983).  "desensitizations"  of  the  responses  (cf.  Fagni,  These desensitizations were seen as diminutions in  the amplitudes of the drug-induced depolarizations during applications.  If  each cell was re-exposed to the same NMDLA concentration within 5 min after the f i r s t  application, the resulting depolarizations  application) were decreased by as much as 15-40%.  (to  the second NMDLA  However, repeated appli-  cations of the same NMDLA concentration elicited similar depolarizations the drug was given at intervals of at least 10 min.  if  ln view of the above  results, repeated NMDLA applications were given at intervals of at least 10 min.  It  was found that NMDLA (25 or 50 uM; applied for 1 min produced  depolarizations of 6 to 33 mV in C A tions  in  CA  lb  neurons  in  mV:  25  lb  M  U  neurons (intracellular NMDLA, 9 * 1 ;  and  50  depolarizauM NMDLA,  23.5 * 5; values are Mean * S.E.M.; n = 5 neurons in each case) and, these  CHIRWA  146  Control  50 p M  NMDLA  10 m M  Saccharin  II  50 p M N M D L A  !20mV  Figure 11-24. Effects of saccharin on the.intracellular depolarizations in CAih neurons induced by NMDLA applications in the guinea pig mppocampus in vitro, me top row shows intraceIIular recordings of the cell membrane potential and input resistances (as measured with 1 nA hyperpolarizing pulses of 100 msec duration given at 1 Hz) in control medium (A) and during 1 min bath application of 50 pM NMDLA (B). The bottom row shows similar responses in the same cell (i.e. cell membrane potential and input resistances) during a 10 min application of 10 mM saccharin (£) and when 50 pM NMDLA is applied for 1 min towards the end of saccharin application.  CHIRWA intracellular  depolarizations were not decreased even if the amino acid was  applied in the presence of 10 mM saccharin (i.e. zations in mV: saccharin,  147  intracellular  depolari-  25 uM NMDLA/10 mM saccharin, 8.5 * l ; and 50 uM NMDLA/10 mM  24 * 3;  values  are  Mean * S.E.M.;  two-tailed paired Student's t-test).  n = 5  neurons;  p > 0.05;  Furthermore the changes in R  n  during  NMDLA applications were not altered in the presence of saccharin as i l l u s trated in Figure 11-24. Taken together,  the results from studies with saccharin indicated that  this agent blocked LTP development through mechanisms different from either non-specific  alterations  receptor activation.  It  in  CA^  k  neurons  electrical  properties  seems logical to think that saccharin antagonized  the induction of LTP at a step beyond NMDA receptor activation. regard,  or NMDA  saccharin may turn out to be a useful  substance in  In  this  elucidating  mechanisms involved in the production of LTP, subsequent to the postulated NMDA receptor involvement step.  CHIRWA 12.  148  DISCUSSION  12.1 General Most of the electrophysiological studies were conducted in the CA-^ area CA  since homosynaptic LTP is well region itself  lb  characterized  in  this  region.  The  is comprised of cell distributions that are taken to be  representative of the whole CA^ subfields (Lorente De No, 1934).  Further-  more, the Schaffer collaterals from CA^ area constituted a readily accessible  input  discernable accurate Prince  to  the  CA^ neurons.  in  the  transverse  positioning of (1982),  subfields.  hippocampal  electrodes.  CA^ pyramidal  bursting activities,  Moreover,  the slice,  According to  neurons exhibit  unlike pyramidal  CA^ area and  was  readily  this  facilitated  Masukawa,  Bernado and  little  or  neurons of the other  no spontaneous cornu ammonis  In the present study, this feature was particularly desirable in  experiments examining minEPSPs since bursting activities would have interfered with the recording of small potentials. Samples collectea from the neocortex were used since several reports have described the development of  LTP in this  neocortex, LTP can be induced with tetanic  brain  structure.  stimulations of inputs  In  the  (Artola  and Singer, 1987; Komatsu, et a l . , 1988; Lee, 1983) or pairing of conditioning postsynaptic intracellular tic afferents  depolarizations with activation of presynap-  (Bindman, Meyer and Pockett, 1987).  Furthermore, the induc-  tion of LTP in the neocortex can be blocked by D-2-amino-5-phosphonovalerate and, this suggests that NMDA receptors are involved in the production of LTP in this structure. ly  similar  1988).  to  The above features of LTP in the neocortex are striking-  those observed in the  hippocampus (cf.  Bliss  and Lynch,  In terms of the studies presented in this thesis, the larger surface  CHIRwA of the rabbit  neocortex  and its  149  position in the brain was conducive for  collection of samples in reasonably adequate quantities. ln general, recorded intracellular  and extracellular  potentials  in the  hippocampus exhibited the same features, as have been described for similar responses in the  literature  Eccles and L i n i n g , 1963  (Andersen, Bliss  and 1964;  and Skrede,  1971; Andersen,  Johnston and Brown, 1984;  Kandel and  Spencer, 1961; Kandel, Spencer and Brinley, 1961; Spencer and Kandel, 1961; Schwartzkroin, 1987).  Furthermore,  small discrete potentials  were readily  detected in CA^ neurons and minlPSPs, but not minEPSPs, could be blocked b  with picrotoxin  (an  antagonist  at  the  GABA  receptors).  A  Several  investigators have also reported the occurrence of small discrete potentials 1988).  in  the  CA.^ region  (Malenka,  Ayoub and Nicoll,  other  excitatory  1987;  Turner,  Presuming that minEPSPs in the hippocampus are analogous to minia-  ture end-plate potentials minEPSPs  in  CA^  (MEPP) at the neuro-muscular junction  neurons  reflected  spontaneous  quantal  del Castillo and Katz, 1952).  (NMJ), then  (or  vesicular)  release of transmitters  (cf.  That vesicular  release of transmitters  occurs in the hippocampus is supported by recent  morphometric studies that have identified vesicles in presynaptic boutons in the  stratum  radiatum,  where  excitatory  synaptic  contacts  are  (Agoston and Kuhnt, 1986; Applegate, Kerr and Landfield, 1987). clear  why minEPSPs in  CA  lb  cells  had a mixed  size  numerous  It was not  distribution  (e.g.  Figure 11-2).  Since these minEPSPs recorded in the soma were coming from  synapses with  different  spatial  account for the variability  distributions  on dendrites,  in minEPSPs amplitudes.  It  this  could  is also conceivable  that minEPSP amplitudes reflected activation of different receptor subtypes (i.e.  NMDA, Quisqualate/ Kainate receptors).  resolved in future experiments.  Moreover,  These issues will need to be the  size of these minEPSPs is  CHIRWA  150  around 1 mV and, therefore, accurate analysis of size distribution is d i f f i cult.  In view of this, changes in rise times or amplitudes of spontaneous  minEPSPs could not be used as indices for changes occurring at synapse (see also section 12.7).  This, and the low and extremely  frequencies of spontaneous minEPSPs among CA^ neurons k  precluded the use of classic quantal (cf. del Castillo and Katz, 1954). Ba , + +  a potent  stimulation  a single  analytical  (e.g.  variable  Table  methods in the CA^ area b  Instead, it was found appropriate to use  agonist for the asynchronous release of transmitter  of  afferents  (Chirwa,  11-1)  1985; Quastel, et a l . ,  1988;  during  Silinsky,  1978 and 1985; Zengel and Magleby, 1980), to facilitate the occurrence of evoked minEPSPs.  This provided a potential method for assessing changes in  the release of transmitter, thereby indicating presynaptic mechanisms  (cf.  Silinsky, 1978). 12.2 LTP in C A  lb  Long-term potentiation  (LTP) in the CA^ area was readily b  (1) tetanic stimulations of the stratum radiatum,  (2)  induced by  simultaneous pairings  of conditioning postsynaptic depolarizations in a CA-^ neuron and activab  tion of the stratum radiatum, or (3) brief applications of samples collected during tetanic stimulation of the guinea pig hippocampus in vivo and rabbit neocortex  in vivo.  The synaptic potentiations  that were observed in  the  present studies exhibited features as described in the literature, namely, decreased onset latencies, unaltered presynaptic volleys, unchanged RMP or R  n  as recorded in  the  soma (Alger  and Teyler,  1976;  Andersen, et a l . ,  1980c; Bliss, and Gardner-Medwin, 1973; Bliss and Lfrio, 1973;  Schwartzkroin  and Wester, 1975; Swanson, Teyler and Thompson, 1983). 12.3 K efflux and LTP +  It was interesting to find that LTP could s t i l l be elicited even though  CHIRWA K  +  channels  That  K  were  currents  blocked  by Cs  +  leaking  from  were diminished by Cs  membrane depolarizations  that  inactivations of the Na  recording  was clearly  developed in  CA  micropipettes.  reflected  neurons,  lb  1983; Johnston, Hablitz and Wilson, 1980).  transient  the  and subsequent  At f i r s t the above results were  puzzling since Haas and Rose (1984) reported that intracellular LTP induction  in  spike generating mechanisms (Brown and Johnston,  +  bited  151  in  four  CA^ neurons  accumulations of extracellular  K  tested.  This  Cs  inhi-  +  suggested that  that occur during depolariza-  +  tions (e.g. Benninger, Kadis and Prince, 1980; Chirwa, 1985), were necessary for LTP development.  On reviewing the literature, however, recent studies  of other investigators appeared to be inconsistent with the findings of Haas and Rose. LTP  For example, when Barrionuevo, et a l .  was  associated  fiber-CA^  synapses,  containing Cs  +  with  increased  these  Barrionuevo, et a l . , 1986). agents  that  diminish  K  currents  used  experiments  (e.g.  at  the  recording Figure 6,  mossy  electrodes page 545 of  In addition, it has now been demonstrated that currents  +  excitatory  investigators  in some of their  (1986) demonstrated that  during  depolarizations  facilitate  induction of LTP (e.g. Chirwa, 1985; Lee, Anywl and Kowan, 1986).  the  Moreover,  adequate conjunctive depolarizations of presynaptic and postsynaptic regions are thought to be necessary for LTP production (Bliss and Lynch, 1988; Goh, 1986; Gustafsson and Wigstrom, 1988; Kelso, Ganong and Brown, 1986; Malinow and Miller, 1986).  1986;  May,  Goh and Sastry,  At the single cell  resulting  from  production.  blockade  But it  1987;  level, therefore, of  K  +  currents  is feasible that K  +  Sastry,  Goh and Auyeung,  intracellular  could  depolarizations  predictably  enhance LTP  released from nearby neurons or  glia during a tetanic stimulation of afferents could be involved by assisting  release  of  substances  that  depend  on  depolarizations  and  these  CHIRwA  152  substances might further trigger events leading to LTP (see section 12.4). 12.4 Feedback interactions Reports in the literature indicate that postsynaptic mechanisms mediate LTP production and that presynaptic mechanisms are involved in its maintenance.  However,  it  is not known how the induction of LTP in postsynaptic  regions subsequently "triggers"  presynaptic mechanisms that are thought to  be involved in the maintenance of LTP. that  feedback  interactions  occur  regions during LTP production. that K act  effluxes  +  (1988)  large,  would be self-limiting  also  this  postsynaptic  is valid,  suggested  the following  Smith  (see  between  then  and it  presynaptic  seems unlikely  subsequent to subsynaptic membrane depolarizations  as feedback signals for  excitability  If  In this regard, it can be speculated  that  raised in  would progress to Somjen,  1979).  facilitate recruitment  reasons.  extracellular  synaptic  K,  increases  of  Na  in  +  review,  particularly  +  potentiations  inactivations  Even if  ln a recent  would  since  spikes  in  extracellular  if  increased afferents K  +  could  of inactive synapses, the inductive phase of LTP at  these synapses would s t i l l have to occur in postsynaptic regions, in keeping with the current ideas implicating postsynaptic mechanisms in the induction of LTP (Bliss and Lynch, 1988; Collingridge and Bliss, 1987; Gustafsson and Wigstrom, therefore,  1988;  Teyler  and  that once LTP is  DiScenna,  1987;  for  reviews).  induced postsynaptically  in  It  follows,  these  recruited  synapses, a "linking" mechanism with the presynaptic regions should subsequently occur to facilitate the development of the  postulated  presynaptic  changes involved in the maintenance of LTP (cf. Bliss and Lynch, 1988). Alternatively,  let  us  suppose  that  increased  extracellular  depolarizes nearby c e l l s , which might include glia cells (cf. May and Chirwa, 1988).  If  K  +  Sastry, Goh,  these cells mediate the changes associated with  CHIRWA LTP development,  it  still  has to be wondered how these c e l l s ,  in  153 turn,  "communicate" with activated synapses to cause increases in synaptic transmission efficacy.  In view of the above, it seems logical to think that  if  LTP production involves feedback interactions, then these are likely to be mediated by voltage-sensitive signals whose major effect to depolarize neuronal elements per se. such feedback signals, if initiate  secondary  facilitations. tetanic  in vitro  processes  leading  to  long-lasting  synaptic  Hence, it was exciting to find that samples collected during of  the  guinea  pig  hippocampus  in vivo  or  rabbit  in vivo induced LTP when applied in the guinea pig hippocampus (see section 12.7).  could be likely tions.  Rather, it can be hypothesized that  present, would act as "primary" messengers that  cellular  stimulations  neocortex  is not necessarily  It  became conceivable that these substances  candidates that mediate  the  postulated  feedback  interac-  These ideas are further developed in later sections.  12.5 minEPSPs and depolarizations in Ba The experimental in section 12.4  evidence for possible feedback interactions discussed  above are apparent  used successfully in facilitating neurons following stimulation  in the  following  1978).  increases  in  It  frequencies  of  ++  was b  of the  is exciting  Ba  the occurence of evoked minEPSPs in CA^ stratum radiatum,  reflected the asynchronous release of transmitters Silinsky,  results.  to  and this  probably  (Quastel, et a l . ,  note that transient  evoked minEPSPs occur  but  immediately  1988;  significant following  simultaneous pairings of postsynaptic depolarizations with activated presynaptic  afferents  in  the  CA^ area.  Ba -containing medium partly ++  campus, then the postsynaptic  simplest  reflect  Presuming that  presynaptic functions in the hippo-  interpretation  depolarizations  were  evoked minEPSPs in  of  these results would be that  modulating  presynaptic  activities,  CHIRWA resulting in the facilitation of evoked minEPSPs. on the following knowledge:  This conclusion is based  At the neuromuscular junction  (1978) observed an elevation of miniature  154  end-plate  (NMJ), Silinsky  frequencies  (MEPP) to  5-20 times the control level, if the motor nerve was stimulated at 1 Hz for a brief  period  (< 1 min).  However,  Silinsky  (1978)  found that  during  tetanic stimulations at frequencies > 1 Hz, there were massive increases in MEPP frequencies which were associated with a steady depolarization of the postsynaptic membranes.  In  view of the  guinea pig hippocampal slices  incubated  the asynchronous release of transmitter  above, it in Ba  ,  seems likely  that  evoked minEPSPs  following stimulation of  in  reflect  afferents.  Therefore, increases in the frequencies of evoked minEPSPs during conjunctive depolarizations of activated presynaptic afferents  with  intracellular  depolarizations of CA^ neurons may be correlated with increases in transb  mitter release.  If  this conclusion is valid, then the above results demon-  strate that postsynaptic depolarizations modulate the functions of activated inputs in the hippocampus. Indeed,  it  is possible that evoked minEPSPs in the presence of  Ba  ++  could reflect hitherto unknown events that are unrelated to quantal release of transmitter  (see section 12.6).  This possibility should be examined in  future studies before firm conclusions can be made.  However, it  is  inter-  esting to note that Sastry, Goh and Auyeung (1986) previously found that simultaneous pairings of activated afferents currents  into  CA^ neurons  induced  lasted for 3-5 min and/or LTP.  and injections of depolarizing  short-term  potentiations  (STP)  This STP, as well as LTP whenever present,  was associated with corresponding decreases in antidromic excitability Schaffer co-workers  collaterals  (Sastry,  (1986) noted the  that  Goh  striking  and  Auyeung,  similarity  1986).  between  Sastry,  of and  STP produced by  CHIRWA concurrent pairings of activated currents  into  afferents  a CA^ neuron in the  with injection  155  of depolarizing  hippocampus and post-tetanic  potenti-  ation (PTP) that has been described in the peripheral  nervous sytem (Feng,  1937; Guttmann, Horton and Wilber, 1937; Hughes, 1958)  and is thought to be  mediated by presynaptic mechanisms such as transient polarizations 1949;  Wall  of presynaptic terminals  and Johnson, 1958).  post-tetanic  (Eccles and Krnjevic,  In this  regard,  Sastry,  (1986) suggested that STP in the hippocampus, like  hyper-  1959;  Lloyd,  Goh and Auyeung  PTP in the  peripheral  nervous system, could also be mediated by presynaptic mechanisms.  However,  these authors also suggested that STP might be due to other unknown postsynaptic mechanisms in the hippocampus. and LTP induced by the injection  Auyeung, 1986)  is intriguing to note that STP  simultaneous pairing  of depolarizing currents  decreases in Schaffer  But it  collaterals  into  of  activated  afferents  with  a CA^ neuron was associated with  terminal  excitability  (Sastry,  Goh and  since this supports the possible involvement of presynaptic  regions in the above processes (cf. Wall, 1958). The results minEPSPs  during  in  the  present  conjuctive  thesis  showing  depolarizations  of  increases  in  presynaptic  bursts inputs  of and  postsynaptic neurons provides further evidence for thinking that postsynaptic depolarizations can modulate presynaptic functions in the hippocampus. How these interactions are achieved and/or their role in LTP production will need to be investigated in future experiments (see section 12.8). it  would be interesting  to examine whether  prevention of  However,  the speculated  feedback interactions might also block the induction of STP and/or LTP. 12.6 minEPSPs and LTP Another major  outcome from the  demonstration of significant  studies with evoked minEPSPs is  the  increases in evoked minEPSPs in CA,, neurons  CHIRWA  156  during LTP induced by tetanic stimulations of the stratum radiatum in slices incubated in Ba  .  There was no evidence to suggest that the increases in  evoked minEPSP frequencies during LTP were caused by changes in presynaptic volleys and altered  input resistances or resting membrane potentials,  as  recorded in the soma.  As previously discussed in section 12.5  above, the  simplest interpretation  for increases in frequencies of evoked minEPSPs is  that they reflect increases in transmitter release during LTP (cf. Silinsky, 1978 and 1985).  This is further discussed below.  Sustained  increases  in  the  release  of  synaptic  frequently cited as being the presynaptic change that  transmitter  is  maintenance of LTP (Bliss and Lynch, 1988 for review).  involved in the These conclusions  are based on biochemical studies that have shown significant between increased release of glutamate, in the hippocampus, and presence of et a l . ,  1986;  Dolphin,  Malthe-S^renssen,  1981).  Errington However,  correlations  the putative excitatory LTP (Bliss  the  and Lynch,  and  Bliss,  full  impact  1982; of  transmitter 1988;  Bliss,  Skrede  transmitter  glutamate  is  in  in the hippocampus.  fact  the  endogenous excitatory  For example,  it  and  these biochemical  results in relation to maintenance of LTP will only be realised once it confirmed that  is  may very well  indirect measurements of glutamate release during LTP reflects  is  synaptic be that  increases in  metabolic turnover of this excitatory amino acid that is unrelated to transmitter release per se. cal  Clearly, it is necessary to complement the biochemi-  studies on glutamate  release  with  electrophysiological  might demonstrate directly increases in transmitter transmission.  methods that  release during synaptic  The studies on evoked minEPSPs represent one such method for  directly assessing possible changes in transmitter release, before and after LTP development.  CHIRWA  157  Indeed, it is tempting to conclude that the observed increase in evoked minEPSPs seen during LTP was due to  an increase  in transmitter  release.  Again, this conclusion follows from what is known about the behaviour of MEPPs at the NMJ, where presynaptic mechanisms control the frequencies of MEPPs and their end-plates  amplitudes are directly  (Fatt  and Katz,  between activities  1952;  related  to the conditions of the  1962).  These direct comparisons  Katz,  at NMJ and intrasomatic  recordings in the hippocampus,  however, need to be made with caution for the following reasons.  At the  NMJ, the recording of MEPPs is at the end-plate where these discrete signals are generated (Fatt and Katz, 1952).  In contrast,  in the present studies  intrasomatic electrodes were used to pick up minEPSPs that were generated at dendritic sites. with  These dendrites branch extensively, and they are inundated  a multitude  minEPSPs.  of  synapses.  Moreover, it  these  synapses could  give  is feasible that the recruitment of inactive synap-  ses or latent excitatory and Wong, 1987)  Each one of  pathways (cf.  Chirwa, Goh and Sastry, 198b; Miles  could add to the number of observed minEPSPs during LTP  rather than just an increase in the release process per se.  It  is  likely  that the increase in the number of minEPSP could also be due to the formation of new synapses.  These possibilities will need to be tested in future  experiments. Alternatively,  changes  in  during LTP that could facilitate generated  at  However,  the  distal studies  dendritic  properties  may occur  the propagation of small discrete signals  synapses that of  membrane  previously failed  Barrionuevo,  et a l . ,  (1986)  to  reach the soma.  indicated  that  the  ability of the EPSP to propagate to the soma does not change during LTP and that any change that contributed to the increase in the intracellular EPSP in the  CA, neurons during  LTP was due to  an increase  in  the  synaptic  CHIRWA current.  158  But they could not distinguish between presynaptic and subsynaptic  contributions  to  et a l . , 1986).  the  enhancement  in  the  synaptic  A similar situation may exist  this regard, the possibility that the  current  (Barrionuevo,  in CA^ neurons as well.  increase in the EPSP is due to an  increase in the number of subsynaptic receptors appears unlikely. have shown that  LTP is  subsynaptic receptors  not  necessarily  (Goh, 1986;  In  associated with  an  Goh, Ho-Asjoe and Sastry,  Studies  increase 1986;  in  Lynch,  Errington and Bliss, 1985; Sastry and Goh, 1984). Interestingly, in vitro  the  potentiated  application  of  phorbol  esters  in  the hippocampus  synaptic responses that were similar to LTP, and this  potentiation was associated with increased minEPSP frequency (Malenka, Ayoub and Nicoll, 1987; Malenka, Madison and Nicoll, 1986).  Quantal analysis of  synaptic transmission between mossy fibers  and CA^ neurons in the hippo-  campus in vitro  by phorbol esters was accompan-  indicated that potentiation  ied by increases in quantal content  (Yamamoto,  and 1988).  In  it  increase  the  in  view of  the  above,  frequencies  of  Higashima and Sawada, 1987  is tentatively concluded that  evoked minEPSPs observed  in  the  studies support the idea that there is an increase in transmitter  the  present released  during LTP. 12.7 Quantal transmission in hippocampus Perhaps it will be useful to discuss some aspects of quantal transmission in the hippocampus that complicate the interpretation Both  inhibitory  voltage-clamp  and  excitatory  quantal  conditions, have been resolved  et a l . , 1988; Johnston and Brown, 1984). exist  events,  in the CA^ region (e.g.  has been described in earlier  in  under the  of these events. both  CA  3  current- and  region  (Brown,  A similar situation is thought to  Johnston and Brown, 1984,  for  review).  As  sections (12.1, 12.5 and 12.6), these quantal  CHIRWA events are taken to reflect of presynaptic vesicles.  all-or-none discharges of transmitter  159  contents  At the present time, however, unequivocal quanti-  tative extraction of classical quantal parameters, as has been done at the vertebrate  neuromuscular  junction  (cf,  del  Castillo  and  Katz,  1954;  McLachlan, 1978) is confounded by factors such as the following. Many reports have indicated that the dendritic membranes of hippocampal neurons have active properties (Andersen and L^mo, 1966; Andersen, Storm and Wheal, 1987: Cragg and Hamlyn, 1955; Fujita and Sakata, 1962; Miyakawa and Kato, 1986; Schwartzkroin and Slawsky, 1977; Spencer and Kandel, 1961; Wong, Prince and Bausbaum, 1979).  The extent to which these active  properties can modify quantal amplitude not clear how the expansive spatial  (q)  is not known.  dendritic  Moreover, it  is  distribution of synapses on dendrites  (Lorente De No, 1934) affects the stochastic distribution of quantal content (m).  In this regard, "success" or "failure" to observe a quantal event with  intrasomatic  recordings may be a function of  synapses on dendrites, capable of  rather  responding or the  the  location of  than changes in the average probability  number of (p)  Therefore, postsynaptic mechanisms could contribute distributive patterns of "m".  individual quanta  (n)  that they respond.  towards changes in the  Another troubling feature of quantal studies  in the hippocampus pertains to the fact that the role of spine structures in synaptic mechanisms is not known. example, are sites for currents.  It  is feasible  the all-or-none  Assuming that this  situation  spine structures would be expected  to  transfer exists directly  of in  that  spine necks, for  transient the  subsynaptic  hippocampus, then  influence  the  stochastic  behavior of "m" under different conditions of synaptic transmission.  Taken  together, the above factors raise the real prospect that the relationship, m = np  CHIRWA  160  may not be directly applicable in the hippocampus, unless modifications are incorporated in this equation that take into account factors such as synapse location, spine neck and active dendritic properties. 12.8 Endogenous substances and LTP Perhaps the most important finding of the present studies is the demonstration  for  the f i r s t  time that brief  during tetanic stimulations  applications of samples collected  of the guinea pig hippocampus in vivo or the  rabbit neocortex in vivo, induce LTP of the population spike in CA^ area b  evoked by stimulation of the stratum radiatum when applied onto guinea pig hippocampal  slices.  As previously  indicated,  samples presented with reduced onset  the  latencies  of  LTP induced  by these  evoked responses and,  these synaptic potentiations were not associated with changes in the size of presynaptic volleys.  These features  are strikingly  with LTP induced by tetanic stimulations  similar  of afferents  to those seen  in the hippocampus  (cf. Bliss and Gardner-Medwin, 1973; Bliss and L^>mo, 1973; Bliss and Lynch, 1988).  This suggests that the synaptic potentiation  induced by application  of the above samples has properties that are similar to tetanus-induced LTP. An intriguing  finding  in these  studies  is the  observation  that  the  above samples only induced LTP if applied during low frequency activation of the  stratum radiatum.  In  section 12.12  it  will  be speculated as to how  synaptic activations may have been necessary in order to these samples.  induce LTP with  Because only samples collected during tetanic  stimulations  of the guinea pig hippocampus in vivo or rabbit neocortex in vivo induced LTP, this raises the prospects that endogenous substances in these samples could mediate feedback interactions during depolarizations, as postulated in section 12.4 substance(s)  above.  In  terms  responsible for  of  possible identities  inducing LTP, it  of  the endogenous  is fascinating to note the  CHIRWA following.  Neither  atropine  nor  dihydro-e-erythroidine,  which  161  are  both  cholinergic antagonists (e.g. Gilman et a l . , 1985), blocked the induction of LTP by the above samples.  These results indicate that the observed synaptic  potentiations were not mediated by endogenous acetylcholine that could be present in the collected samples. the above samples. ous glutamate  The activity is therefore unlikely to be due to endogen-  since prior heating of exogenous glutamate does not abolish  the effects of this excitatory in vitro. results  In contrast, it in  Prior heating abolishes the effects of  loss  Lehninger, 1982).  of  amino acid when applied in the hippocampus  is known that heat denatures proteins ano this  biological  activities  In view of this,  endogenous substances in  the  it  collected  of  these  substances  seems likely that the  (e.g.  potentiating  samples could be macromolecules,  possibly proteins. On reviewing the  literature  it  was interesting  to note that  protein  synthesis inhibitors (Stanton and Sarvey, 1984) and monoclonal antibodies to proteins  (Lewis and Teyler,  1986;  the induction of LTP with tetanic campus.  and Shashoua, 1981;  logical  stimulations of afferents  Endogenous peptides ranging from 14 to  released into the extracellular Teyler  Stanton, Sarvey and Moskal, 1987)  to postulate  that,  86  Hess,  Hofstein  in the present  pig hippocampus in vivo and rabbit  treatment with trypsin  known to be  studies, similar  neocortex  (MCD;  see  inactivated  the  It  is  proteins were  stimulations of guinea  in vivo and these endogenous In this regard, a report  has appeared in which LTP was induced through brief peptides  kD are  and Shashoua, 1984).  proteins may be responsible for the observed LTP.  degranulating  in the hippo-  fluid during LTP in the hippocampus (Duffy,  released into the samples collected during tetanic  cell  block  Cherubini,  applications of mast et a l . ,  potentiating  1987).  effects  of  Prior MCD and  CHIRWA  162  Cherubini et a l . , suggested that MCD may have been mimicking the actions of an endogenous peptide. that  proteins  hippocampus.  could  Slearly, the above reports give credence to the idea be  involved  Future experiments  in  the  production  should examine whether  of  LTP  in  the  prior treatment of  the above samples with trypsin would abolish their potentiating actions. 12.9 Endogenous substances and neurite growth The possible identity samples discussed results. (NGF)  for  some of  in section 12.8  the  endogenous substances in  above is  illustrated  in the  the  following  The growth of neurites in PC-12 cells requires nerve growth factor  or  related  cultures  incubated  substances in  growth  (Greene media  and  Tischler,  containing  1976).  samples  PC-12  collected  cell during  tetanic stimulations of the rabbit neocortex in vivo, presentee with extensive neurite  growths.  These results indicat that NGF-like substances were  present in the above neocortical samples and were probably responsible for initiating  neurite  growth in PC-12 cell  cultures.  In  this  regard,  it  is  intriguing to note that the molecular weights of NGF and related compounds range between 14 to 90 kD (Berg, 1984; Wagner, 1986).  Coincidentally, these  values happen to be similar to the molecular weights of proteins that are known to be released during the induction of LTP with tetanic in the hippocampus (cf.  Hess, Hofstein and Shashoua, 1984).  that there may be similarities  in biological  activities  proteins that are released during tetanic stimulations.  It  stimulations is possible  between  NGF and  In fact, this idea  was one of the reasons for testing the effects of exogenous NGF (from Vipera lebetina) on synaptic transmission in the hippocampus. At f i r s t ,  it  was disappointing to note that application of exogenous  NGF during low frequency stimulation of the stratum radiatum did not induce LTP  in  the  hippocampus.  This  contradicted  the  notion  that  NGF-like  CHIRWA substances could be involved in LTP development!  But then it was discovered  that exogenous NGF could consistently induce LTP if with tetanus of a weak input.  163  applied in association  One possible interpretation  of these findings  is that exogenous NGF substituted for the endogenous substances that would have otherwise been released by an associative strong tetanus to a strong input  (cf.  Barrionuevo  reasons for the failure alone without tetanus  and Brown,  1983;  It  are unclear.  The  Perhaps this reflects species-specific or potencies of the applied exogen-  is also possible that tetanic  NGF-like substances but  1979).  to produce LTP with applications of exogenous NGF  differences in the intrinsic activities ous NGF.  Levy and Steward,  other  "co-factors",  stimulation as well,  releases not only that  would not be  present in a "purified" sample such as commercially available NGF. The possible involvement of NGF-like substances in LTP holos promise in view of the following reports in the literature.  LTP development is associ-  ated with synaptic morphological changes (Desmond and Levy, 1981; 1983; 1986; Fifkova and van Harreveld, S^renssen, 1981;  1977;  Voronin, 1983;  Lee, et a l . ,  1980;  Skrede and Malthe-  Lynch and Baudry, 1984).  that mediate these synaptic differentiations  are not fully  The mechanisms understood.  It  may be that these post-tetanic morphological changes are regulated by growth related  substances, which  tetanic  stimulation  Shashoua, 1984).  could be the  same substances released  (Duffy, Teyler and Shashoua, 1981;  Hess,  during  Hofstein and  Future experiments should further examine these possibili-  ties. 12.10 LTP and neurite growth It  is  clear  that  samples  collected  contained endogenous substances that caused neurite  during  induced LTP in  growth in PC-12 cell cultures.  tetanic the  stimulations  hippocampus and  Whether the same substances  CHIRWA in the samples mediated all However, other experiments  the above changes was not determined  164  directly.  in this thesis provid indirect evidence for the  involvement of the same substance(s)  in both LTP and neurite growth.  This  evidence will now be discussed. Both LTP development and neurite expression exhibit parallel changes to the  same treatments that  alter  the  effects  of  the  above  samples.  For  instance, prior heating abolishes the activities of the collected samples in causing synaptic potentiating  or neurite  growth.  Saccharin, a substance  that is known to antagonise the NGF-dependent neurite growth ( l s h i i , 1982), inhibits both the effects of the above samples in inducing neurite in  PC-12  cell  cultures  or  synaptic  potentiations  in  the  growths  hippocampus.  Interestingly, saccharin (in concentrations that did not significantly alter synaptic transmission in the hippocampus) antagonises the induction of LTP with tetanic stimulations as well as LTP produced by tetanus of a weak input in the presence of exogenous NGF. The actions of saccharin were not due to non-specific alterations the electrical view  of  the  spread over feature of  properties of CA^ neurons or NMDA receptor b  following two  results.  logarithmic  drugs exhibiting  Rand, 1980).  activation,  The dose-response curve to  scale  units,  selective  and this  pharmacologic  is  a  actions  in in  saccharin is characteristic (Bowman and  More importantly, the concentrations of saccharin (10 mM) used  do not exert significant effects on evoked synaptic responses in the hippocampus.  This is clearly illustrated  of saccharin on paired-pulse Paired-pulse facilitation  in the results showing lack of effects  facilitations  is well characterised  Kuhnt and Voronin, 1987; MacNaughton, 1980) increases  in transmitter  or  that  post-tetanic  potentiations.  in the hippocampus (Hess,  and it  is thought to be due to  is released with the  second pulse  in  the  CHIRWA stimulation pair (e.g. Hess, Kuhnt and Voronin, 1987). presynaptic depolarizations  (or their  165  Presumably residual  associated effects,  i.e.  Ca  influx  ++  into terminals) caused by the f i r s t pulse add up with depolarizations of the second pulse, thereby augmenting the effects stimulation  pair  (cf.  del  Castillo  of this  and Katz,  latter  1954).  pulse in the  Similarly,  PTP is  thought to be mediated by presynaptic mechanisms which are responsible for transient  increases in transmitter  (McNaughton, 1980). by saccharin.  release  following  Both paired-pulse facilitation  tetanic  and PTP were unaffected  Moreover, the occurrences of minEPSPs and minlPSPs during  saccharin applications were the same as in control medium. thresholds  stimulations  for  antidromic  altered by saccharin.  activations  of  Schaffer  In addition, the  collaterals  were not  All these results point to the fact that saccharin in  doses used in these studies lacked electrophysiological effects  that can  account for its blockade of the induction of LTP. Moreover, it alter  is particularly  important to note that saccharin did not  NMDLA-induced depolarization  of  the  CA-j^  neurons.  Furthermore,  saccharin did not antagonise the development of the dendritic negative field that occurs during tetanic  stimulations, and is thought to reflect  tions of NMDA receptors (Wigstrom and Gustafsson, 1984; findings were particularly area  is  thought  (Collingridge,  to  Kehl  important  be mediated and  via  MacLennan,  1986).  activa-  The above  since tetanus-induced LTP in the CA^ the  activations  1983).  These  of  results  NMDA receptors suggest  that  saccharin does not antagonize the development of depolarizations that are thought to be necessary for LTP induction (Gustafsson and Wigstrom, 1988). Taken together, the results with saccharin indicate that the blockade of LTP induction by this agent is probably occurring at a step beyond the activation of NMDA receptors.  CHIRWA  166  12.11 Possible mechanisms of actions of saccharin A prominent  feature of  the  abundant  literature on saccharin is  the  absence of studies that have examined the electrophysiological effects this  drug.  The studies on saccharin reported  provide some of  this  information.  However,  in this the  describes some mechanisms that could help to saccharin on LTP production or neurite  thesis,  literature  account for  growth.  of  therefore,  on saccharin  the  effects  of  These mechanisms will be  briefly reviewed here (see chapter 8). Firstly,  saccharin antagonises NGF binding  (lshii,  1982).  If  it  is  tentatively accepted that the endogenous substances in the samples collected during tetanic  stimulations  were close relatives  of  NGF, then  saccharin  could have been antagonising the binding of these substances to NGF receptors in the experiments reported in this thesis. against  the  substances  above is  possibility  known to  be  in  is  that  the  pM ranges.  However, a major argument  binding  of  Yet  blockade  the  NGF and  saccharin requires drug concentrations greater than 7.5 mM. to explain  the  need for  antagonising the  mM concentrations  binding of  of  tion is that the agent acts at intracellular ted into the c e l l .  saccharin in  substances with activities  plausible explanation for requiring a relatively  It  is  however,  difficult  "selectively" A  high saccharin concentra-  site(s) and is poorly transpor-  Hence, to achieve significant intracellular  Once internalized,  LTP by  in pM rangesl  it is necessary to have mM concentrations of saccharin in the space.  of  related  drug levels extracellular  saccharin may then antagonise some of  the NGF-dependent reactions within the c e l l s . Secondly, saccharin inhibits the activities of specific phosphorylating enzymes (Best and Brown, 1987; Brown and Best, 1986; Linke and Kohn, 1984; Lygre,  1974;  1976).  In  this  regard,  it  is  interesting  to  note  that  CHIRWA  167  NGF-dependent phosphorylating enzymes have been described in the literature (Halegoua and Patrick, inhibited  by  1980).  saccharin  It  is conceivable that some of the enzymes  incluaed  NGF-dependent  phosphorylating  enzymes.  Perhaps high saccharin concentrations are necessary to diminish the enzyme activities  significantly  mechanisms of  (cf.  Best  saccharin actions,  and  Brown,  these will  1987).  need to  Whatever,  the  be investigated  in  future experiments. 12.12 Implications in LTP The studies in this thesis show for the f i r s t time that substances that are released during tetanic  stimulations are capable of inducing LTP when  applied in the hippocampus and neurite growth in PC-12 cell cultures. exact mechanisms that trigger  the release of these substances or how they,  in turn, exert changes that lead to LTP or neurite remain to be determined. insights LTP.  into certain  F i r s t l y , it  The  growth in PC-12 cells  However, the present results begin to provide some  intriguing  features  is known that tetanic  cause the release of proteins  pertaining  to  the  phenomena of  stimulations that induce LTP also  (Duffy, Teyler and Shashoua, 1981), but the  effects of these released proteins on synaptic transmission are not known. Secondly, LTP development is associated with morphological changes to synaptic  structures  (e.g.  Fifkova,  1986).  The  factors  that  initiate  morphological changes or how they relate to LTP are not clear. since  the  induction  mechanisms, it "relayed"  to  of  LTP is  thought  is not known how this presynaptic  regions  to  be mediated  these  Thirdly,  by postsynaptic  postsynaptic change is subsequently  in  order  to  incorporate  presynaptic  mechanisms which are implicated in the maintenance of LTP (e.g. Bliss and Lynch, 1988). The studies  in  this  thesis  raise  the  following  prospects.  Since  CHIRWA tetanic  stimulations cause the release of  168  NGF-like substances capable of  inducing neurite growth in PC-12 cell cultures, it is conceivable that these same substances mediate the synaptic re-structuring that occur during LTP. It might be that depolarizations (through NMDA receptor activations) induced by tetanic that  act  stimulations trigger  the release of  as "feedback" messengers that  growth relatea substances  interact  with presynaptic  subsynaptic membranes and orchestrate changes that  produce LTP.  regard, the proteins that are released during tetanic Teyler and Shashoua, 1981)  could be close relatives  stimulations of NGF.  and/or In  this  (Duffy,  In a global  sense, if the release of NGF-like substances by intense neuronal activity is a common feature of various areas in the nervous system, it  becomes impor-  tant to determine if these substances are ubiquitously involved in improving the quality of synaptic transmission and, therefore, functions in general.  the quality of brain  In terms of LTP, a speculative case can be made for  the involvement of NGF-like substances in its production. NGF receptors are present on presynaptic terminals and the peptide is known to be taken into the terminals (Hendry et a l . , 1974; Springer and Loy, 1985).  Hence released peptides  with NGF receptors (and/or  subsynaptic  on active  (but  dendritic  could facilitate  enhancement of  and  presynaptic  thereby  terminals  initiate  changes  changes in dendritic morphology, synaptic  transmitter  release).  of the peptide(s).  these substances with their  peptide(s)-receptor it  quiescent)  interact  Presynaptic  activity  the interaction of NGF-like substances with receptors that  trigger the internalization of  not  membranes)  leading to LTP development (i.e. rearrangement,  from postsynaptic regions could  Therefore, the  receptors or the  events  interaction may be voltage dependent.  is interesting to note that electrical  activity  that  interaction follow  the  In this regard,  in neurons shapes the  CHIRWA  169  patterns of synaptic connections and that neurite growth on cells in culture is modified by the polarity of the electrical  field  (Frank,  1987; McCaig,  1987; 1988). In  summary, results  in the present study raise the possibility that  substances released by tetanic  stimulations contain NGF-like proteins that  may be involved in synaptic potentiation.  These agents could act as one of  the messengers in the chain of events leading to LTP production. bility  The possi-  that NGF-like substances are universally involved in LTP i s , there-  fore, worth investigating.  CHIRWA  170  13. CONCLUSION The major findings of the studies in this thesis can be summarized as foilows: 1.  In  hippocampal  slices  incubated  in  Ba  ,  evoked minEPSPs  following  stimulation of an input probably reflect the asynchronous release of synaptic transmitter. 2.  During depolarizations, postsynaptic modulation of presynaptic functions  occur in the hippocampus and, this is illustrated  in the observed transient  increases in frequencies of evoked minEPSPs immediately conditioning intracellular  after  pairing of  depolarizations of a CA^ neuron and activation  of the stratum radiatum. 3.  The increase in evoked minEPSPs during synaptic potentiation are consis-  tent with previous evidence that  LTP may partly  be due to  an apparent  increase in released synaptic transmitter. 4.  The  tetanic  induction  of  stimulations  LTP with  application  of  indicates  that  samples  these  samples  collected  during  contained endogenous  substances, possibly proteins, that may be involved in the production of synaptic potentiations. 5.  The  induction  of  neurites  in  PC-12  cell  presence of samples collected during tetanic  cultures  incubated  in  the  stimulations of the neocortex  illustrates that these samples also contained NGF-like substances. 6.  The finding that LTP can be produced by tetanic  stimulation of a weak  input in conjunction with application of exogenous NGF further  supports the  notion that close relatives of NGF may be involved in LTP development. 7.  Saccharin (a substance known to antagonise NGF binding to its receptors  and to inhibit  NGF-dependent neurite  growth) blocks both the induction of  CHIRWA  171  LTP in the hippocampus and neurite growth in PC-12 cell cultures induced by samples collected during tetanic stimulations of the rabbit neocortex; this supports the idea that common mechanisms may be involved in LTP production and neurite growth. 8.  Saccharin also blocks both the induction of LTP by tetanic  stimulation  of (a) a strong input, or (2) a weak input in conjunction with application of exogenous NGF; this  further  suggests that  NGF-like substances may be  involved in LTP development. 9.  The concentrations of saccharin used in these studies had insignificant  effects on CA^ cell electrical b  properties,  synaptic transmission or NMDA  receptor activation; this suggests that saccharin may be blocking induction of LTP at a step beyond NMDA receptor activation. 10. Taken  together,  the  studies  in  this  thesis  indicate  that  intense  depolarizations cause the release of diffusable substances, probably growth related  proteins,  that  subsequently  interact  with  activated  afferents and/or subsynaptic dendritic elements resulting in LTP.  presynaptic  CHIRWA 13.  172  REFERENCES  ADAMS, P. R., D. A. BROWN, J . V. HALLIWELL. Cholinergic regulation of M-current in hippocampal pyramidal c e l l s . J . Physiol. 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