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Calcium regulation in long-term changes of neuronal excitability in the hippocampal formation 1985

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CALCIUM REGULATION IN LONG-TERM CHANGES OF NEURONAL EXCITABILITY IN THE HIPPOCAMPAL FORMATION by ISTVAN MODY B.Sc.[Hon.], University of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1985 (cp Istvan Mody, 1985 2% 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 The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6(3/81) i i ABSTRACT The regulation of calcium (Ca^ +) was examined during long- term changes of neuronal e x c i t a b i l i t y in the mammalian CNS. The preparations under investigation included the kindling model of epilepsy, a genetic form of epilepsy and long-term potentiation (LTP) of neuronal a c t i v i t y . The study also includes a discussion of the possible roles of a neuron-specific calcium-binding protein (CaBP). The findings are summarized as follows: 1 ) The d i s t r i b u t i o n of CaBP was determined.in c o r t i c a l areas of the rat using a s p e c i f i c radioimmunoassay. The protein was found to have an unequal d i s t r i b u t i o n in various c o r t i c a l areas with preponderence in ventral structures. 2) Extending previous studies on the role of CaBP in kindling-induced epilepsy, i t s decline was correlated to the number of evoked afterdischarges (AD's) during the process of kindling. 3 ) Marked changes in CaBP leve l s were also found in the brains of the e p i l e p t i c strain of mice ( E l ) . The hippocampal formation and the dorsal o c c i p i t a l cortex contained s i g n i f i c a n t l y lower CaBP than the control (CF - 1 ) s t r a i n . The induction of seizures further decreased the leve l s of CaBP in the E l mice. These findings are indicative of a possible genetic impairment of neuronal C a 2 + homeostasis in the E l s t r a i n . 4) The l e v e l s of t o t a l hippocampal C a 2 + and Zn 2 + were measured by atomic absorption spectrophotometry in control and commissural-kindled animals. While no change was found in the t o t a l C a 2 + content of the region, hippocampal Z n 2 + of kindled preparations was found to be s i g n i f i c a n t l y elevated. 5) To measure Ca 2 +-homeostasis, the kinetic analysis of 4^Ca uptake curves was undertaken in the in v i t r o hippocampus. This technique was found to be a v a l i d method for assessment of C a 2 + - regulation in the CNS under both physiological and pathophysio- l o g i c a l conditions. The effect of various e x t r a c e l l u l a r C a 2 + concentrations, 2,3-dinitrophenol (DNP), c a l c i t o n i n , nifedipine and 3-isobutyl-1-methylxanthine (IBMX) on 4^Ca uptake curves was examined in order to i d e n t i f y the two exchangeable C a 2 + pools derived through kinetic analysis. 6) The k i n e t i c analysis of 4^Ca uptake curves revealed that C a 2 + - r e g u l a t i o n of the hippocampus i s impaired following amygdala- and commissural kindling. The changes r e f l e c t an enhancement of a C a 2 + pool that includes free c y t o s o l i c C a 2 + and a concomitant decrease in the amount of buffered calcium probably as a result in the decrease of hippocampal CaBP l e v e l s . 7) A novel form of long-term potentiation (LTP) of neuronal a c t i v i t y in the CA1 region of the hippocampus i s described. Perfusion of 100 uM of IBMX in the hippocampal s l i c e preparation induced a long l a s t i n g increase in the amplitude of the stratum radiatum evoked population spike and EPSP responses with changes in synaptic e f f i c a c y as indicated by the altered input/output relationships. I n t r a c e l l u l a r correlates of IBMX-induced LTP iv included lowering of synaptic threshold and enhancement of the rate of r i s e of the EPSP with no al t e r a t i o n s in the passive membrane c h a r a c t e r i s t i c s of CA1 pyramidal neurons. The fact that IBMX was able to exert i t s e f f e c t even in the presence of the calcium-blocker cation Co 2 +, taken together with the drug's action on hippocampal exchangeable Ca , raises the p o s s i b i l i t y that the C a 2 + necessary for induction of LTP may be derived from an intraneuronal storage s i t e . These studies indicate the significance of i n t r a c e l l u l a r Ca -regulatory mechanisms in long-term changes of neuronal e x c i t a b i l i t y which occur in experimental models of epilepsy and long-term potentiation. V TABLE OF CONTENTS Abstract i i Table of Contents v L i s t of Figures ix L i s t of Tables.. x i i i Acknowledgements xiv CHAPTER I. Introduction 1 1 . 1 . Calcium and the regulation of c e l l u l a r function.. 1 1.2. The role of calcium in the CNS 6 1.3. Calcium and long-term changes in neuronal e x c i t a b i l i t y ; The present study 14 CHAPTER I I . D i s t r i b u t i o n and a l t e r a t i o n s in calcium-binding protein (CaBP) in the CNS 18 2.1. General introduction 18 2.1.1. Calcium-binding proteins of the CNS 18 2.1.2. The calcium-binding protein CaBP 22 2.1.3. Possible roles of CaBP in the CNS 24 v i 2.2. C o r t i c a l d i s t r i b u t i o n of CaBP in rats 31 2.2.1. Introduction 31 2.2.2. Methods 32 2.2.2.1. Rat c o r t i c a l samples.. 32 2.2.2.2. CaBP radioimmunoassay (RIA) 34 2.2.3. Results 35 2.2.4. Discussion 44 2.3. Relationship of hippocampal afterdischarges to levels of CaBP during development of commissural kindling 46 2.3.1. Introduction 46 2.3.2. Methods 48 2.3.3. Results 50 2.3.4. Discussion 56 2.4. Di s t r i b u t i o n of CaBP in the c o r t i c a l areas of the ep i l e p t i c E l mouse.... 59 2.4.1. Introduction 59 2.4.2. Methods 61 2.4.3. Results 65 2.4.4. Discussion 73 CHAPTER I I I . Measurement of hippocampal C a 2 + homeostasis 77 3.1. Measurement of t o t a l hippocampal Ca* and Zn^ using atomic absorption spectrophotometry (AAS) 79 3.1.1. Introduction 79 3.1.2. Methods 81 3.1.3. Results 82 3.1.4. Discussion 84 v i i 3.2. Measurement of hippocampal exchangeable calcium using kinetic analysis of 4^Ca uptake curves 87 3.2.1. Introduction 87 3.2.2. Methods 89 3.2.2.1. Measurement of 4 5 C a uptake 89 3.2.2.2. Curve f i t t i n g 94 3.2.2.3. Compartmental analysis 99 3.2.3. Results. 103 3.2.3.1. " Eff e c t of e x t r a c e l l u l a r calcium ( [ C a 2 + ] 0 J 103 3.2.3.2. Effect of drugs that a l t e r C a 2 + metabolism 109 3.2.3.3. Theoretical manipulations of the S 3 pool. . 113 3.2.4. Discussion .116 3.3. Measurement of C a 2 + - r e g u l a t i o n during kindling using the kinetic analysis of 4^Ca uptake curves 123 3.3.1. Introduction 123 3.3.2. Methods 125 3.3.3. Results 126 3.3.4. Discussion 135 CHAPTER IV. Ef f e c t of IBMX on hippocampal e x c i t a b i l i t y 143 4.1. Introduction 146 4.2. Methods 149 4.2.1. E x t r a c e l l u l a r recording and analysis 149 4.2.2. I n t r a c e l l u l a r recording and data analysis 156 4.3. Results 159 v i i i 4.3.1. Effect of drugs on stratum radiatum evoked potentials 1 59 4.3.2. Effect of IBMX on paired-pulse i n h i b i t i o n 163 4.3.3. Effect of IBMX on input-output (I/O) curves 167 4.3.4. Calcium and IBMX-induced LTP 178 4.3.5. Effect of IBMX on bursting induced by low calcium 186 4.3.6. Effect of IBMX on passive membrane properties of hippocampal CA1 pyramidal c e l l s 187 4.3.7. Long-term e f f e c t of IBMX on the rate of ri s e of i n t r a c e l l u l a r l y recorded EPSPs 190 4.3.8. I n t r a c e l l u l a r input/output relationships following perfusion of IBMX 193 4.3.9. Effect of IBMX on in h i b i t o r y mechanisms of CA1 pyramidal neurons recorded i n t r a c e l l u l a r l y .196 4.3.9.1. Accommodation 196 4.3.9.2. Long-lasting hyperpolarization (LHP)....199 4.3.9.3. Inhibitory post-synaptic potentials (IPSPs) and paired-pulse i n h i b i t i o n 202 4.3.10. Long-term e f f e c t of IBMX on stimulus threshold...203 4.4. Discussion 206 CHAPTER V. Conclusions 216 REFERENCES 221 ix L I S T O F F I G U R E S Figure 2.1. CaBP content of the fron t a l region of rat cortex.. 36 Figure 2.2. Levels of CaBP in p a r i e t a l c o r t i c a l areas of the rat 38 Figure 2.3. CaBP levels in the caudal t h i r d of the rat cortex 40 Figure 2.4. Summary of CaBP d i s t r i b u t i o n in rat cortex 42 Figure 2.5. Development of afterdischarges during commissural kindling 51 Figure 2.6. Relationship between the number of evoked AD's and decline of hippocampal CaBP level s during development of commissural kindling 54 Figure 2.7. C o r t i c a l areas used for determination of CaBP lev e l s in mice 63 Figure 2.8. Levels of CaBP in c o r t i c a l areas of control and E l male mice 67 Figure 2.9. Levels of CaBP in c o r t i c a l areas of control and E l female mice 69 X Figure 3.1. Sequential flow-chart representation of the methods used for kinetic analyses of 4^Ca uptake curves.. 91 Figure 3.2. Graphical analysis and f i t t i n g of the 4^Ca uptake curve 96 Figure 3.3. Effect of alt e r a t i o n s in e x t r a c e l l u l a r C a 2 + concentrations on the hippocampal 4^Ca uptake curves and e l e c t r i c a l a c t i v i t y 105 Figure 3.4. Ef f e c t s of drugs and hormones on 4^Ca uptake and electrophysiological properties of hippocampal s l i c e s 110 Figure 3.5. Ef f e c t s of theoretical manipulations of the S 3 (buffered) C a 2 + pool 114 Figure 3.6. 4^Ca uptake curves in control, commissural- (HPC) and amygdala-kindled (AMY) hippocampal s l i c e s 129 Figure 3.7. Histograms showing exchangeable calcium pools in hippocampal s l i c e s obtained from control, commissural- (HPC) and amygdala-kindled animals 133 Figure 3.8. Schematic i l l u s t r a t i o n of a l t e r a t i o n s observed in calcium exchange kinetics of kindled hippocampi 136 Figure 4.1. Measurement of e x t r a c e l l u l a r potentials in the CA1 region of the hippocampal s l i c e preparation 152 Figure 4.2. Chemical formulae of the drugs used in the present study 154 xi Figure 4.3. Effe c t of various drugs on the amplitude of population spikes evoked in the CA1 region of the hippocampal formation 160 Figure 4.4. Effe c t of IBMX on paired-pulse i n h i b i t i o n - recorded e x t r a c e l l u l a r l y 165 Figure 4.5. Potentials recorded in the dendritic and somatic region of area CA1 during input/output curves .168 Figure 4.6. I/O curves having stimulus intensity (SI) on the abscissa 171 Figure 4.7. I/O curves having fiber volley amplitude (V) on the abscissa 174 Figure 4.8. I/O curves having the rate of r i s e of the EPSP (D) on the abscissa 176 Figure 4.9. Ef f e c t of C o 2 + on IBMX-induced LTP 179 Figure 4.10. Ef f e c t of a short duration (<30 min) perfusion of 0 mM Ca 2 +/9 mM Mg 2 + on the action of IBMX in the CA1 region of the hippocampus 182 Figure 4.11. Ef f e c t of a long duration (60 min) perfusion of 0 mM Ca 2 +/9 mM Mg 2 + on the action of IBMX in the CA1 region of the hippocampus 184 Figure 4.12. Ef f e c t of 100 uM IBMX on the passive membrane c h a r a c t e r i s t i c s of CA1 pyramidal neurons 188 Figure 4.13. Effect of IBMX on the rate of r i s e (dV/dt) of i n t r a c e l l u l a r l y recorded EPSPs 191 Figure 4.14. I n t r a c e l l u l a r I/O rel a t i o n s h i p following perfusion of IBMX in a CA1 pyramidal c e l l 194 Figure 4.15. Effect of IBMX on the accommodation of pyramidal c e l l discharge 197 Figure 4.16. Effect of IBMX on in h i b i t o r y events acting on CA1 pyramidal neurons 200 Figure 4.17. Effect of IBMX on stimulus- and f i r i n g threshold of CA1 pyramidal neurons 204 xi i i LIST OF TABLES Table 2.1. Effect of afterdischarges (AD's) on level s of hippocampal and cerebellar CaBP 53 Table 2.2. Levels of CaBP in various c o r t i c a l areas of control and e p i l e p t i c (El) mice 71 Table 2.3. Two-way analysis of variance (ANOVA) of the eff e c t s of s t r a i n , sex and seizures on the c o r t i c a l l e v e l s of CaBP in control and E l mice 72 Table 3.1. Calcium and zinc in the hippocampal formation of control and commissural-kindled rats as measured by atomic absorption spectrophotometry 83 Table 3.2. Eff e c t s of e x t r a c e l l u l a r calcium concentrations on hippocampal C a 2 + exchange 107 Table 3.3. Eff e c t s of drugs on hippocampal C a 2 + exchange 112 Table 3.4. Effect of kindling-induced epilepsy on C a 2 + exchange rates and compartment sizes of hippocampal s l i c e s 131 xiv ACKNOWLEDGEMENTS I would l i k e to express my gratitude to Dr. James J. M i l l e r for providing me the opportunity to pursue my s c i e n t i f i c goals in his laboratory. Many sincere thanks to Dr. Kenneth G. Baimbridge for his guidance regarding the CaBP radioimmunoassay and to Michael W. Oliver for his friendship and valuable collaboration in some of the experiments. The helpful discussions and comments of other fellow graduate students were also welcome. I appreciate the technical assistance of K. Henze, J . Nairn and R. Anderson. I would l i k e to thank the members of my advisory committee and Dr. G. G. Somjen, the external examiner, for reviewing the thesis. The receipt of the F. F. Wesbrook Fellowship from the University of B r i t i s h Columbia i s also acknowledged. F i n a l l y , I wish to dedicate t h i s thesis to my father who expressed so much interest in my work and to the memory of Kevin J. Pittman whose own thesis could never be completed. 1 C H A P T E R I . INTRODUCTION 1.1. Calcium and the regulation of c e l l u l a r function The discovery of calcium dates back to the f i r s t decade of the 19 t n century when H. Davy isolated the metal, together with other a l k a l i n e earths, and gave i t s name from the Latin word 'calx' meaning lime. Following i t s discovery, b i o l o g i s t s found that l i v i n g organisms exist in a milieu that contains Ca^ , but i t wasn't u n t i l 1882-83 when Sidney Ringer through a fortuitous discovery gave the f i r s t account of i t s functional s i g n i f i c a n c e . Ringer, examining the effects of various ions on the contractions of frog heart, f i r s t dismissed the role of C a 2 + , but later discovered that the solution he was using had been prepared not from d i s t i l l e d water but regular tap water, naturally containing high amounts of C a 2 + . Subsequent examinations have proven that the presence of C a 2 + was necessary for maintenance of normal cardiac contractions. Further evidence for the role of Ca^ in excitable tissue was obtained by Locke (1894) who showed that 2 nerve to nerve and nerve to muscle transmission both required e x t r a c e l l u l a r C a 2 + . Much of the 20*-̂  century research in biology, physiology and medicine has focused on the important functions of Ca in c e l l u l a r systems and has shown that the cation participates in numerous aspects of the physiology and pathophysiology of l i v i n g organisms. Calcium has major functions in both excitable and non-excitable c e l l s which include, to enumerate just a few, reproduction and development of c e l l s , c e l l movement, e l e c t r i c a l a c t i v i t y , mineralization of a bony or calcarous skeleton, exo- or endocytosis, photoreception, act i v a t i o n and inactivation of enzymes and proteins, and f i n a l l y , c e l l death. For a complete review of calcium's involvement in c e l l u l a r function the reader i s directed to the recent book by Campbell (1983). Only some of the recent developments w i l l be discussed below, focusing mainly on the regulation of i n t r a - c e l l u l a r calcium and i t s second messenger function. The t o t a l calcium content of l i v i n g c e l l s i s usually equal to the calcium concentration of the e x t r a c e l l u l a r f l u i d , but i t is d i f f e r e n t i a l l y d i s t r i b u t e d in a bound form into the various i n t r a c e l l u l a r organelles, membranes and proteins leaving only a minute cytoplasmic fracti o n (<0.1%) in the free ionic form (Borle, 1981a). Nevertheless, most of the c e l l u l a r calcium i s exchangeable, which means that i t may be replaced with time by i t s e x t r a c e l l u l a r counterpart. The organelles involved in the maintenance of i n t r a c e l l u l a r Ca 2 +-homeostasis are primarily the mitochondria and the endoplasmic reticulum. To complement the Ca 2 +-regulatory action of i n t r a c e l l u l a r mechanisms, c e l l s possess 3 various systems responsible for the extrusion of excess C a 2 + , which include the Na +-Ca 2 + exchange, and metabolic Ca2+-pumps. Because of the f a c i l i t y of the preparation, the mitochondrial calcium transport has been extensively studied (for reviews see Lehninger et a l . , 1977; Akerman and N i c h o l l s , 1983). In summary, accumulation of C a 2 + in the mitochondrial matrix is compensated by extrusion of H + which results from the respiratory chain and hydrolysis of ATP. To maintain the electrochemical potential difference across the mitochondrial membrane, two protons have to be exchanged for one C a 2 + . This however, in the case of a large Ca loading, would s i g n i f i c a n t l y a l t e r the pH of both the mitochondrial matrix and the cytoplasm. Therefore, a permeant anion, such as phosphate or acetate, must be present that w i l l transport H + back into the matrix, and following i t s dissoci a t i o n binds C a 2 + to form a C a 2 + - s a l t . Much controversy has surrounded the efflux pathway of C a 2 + from the mitochondrial matrix, which seems to be dependent on cytoplasmic inorganic phosphate (Akerman and Nicholls, 1983). In addition, since most of the experiments are carried out in isolated mitochondria, i t is d i f f i c u l t to speculate about the setpoints for release and uptake of calcium i_n s i t u . While there is no doubt that mitochondria contain a reasonable amount of the exchangeable c e l l u l a r Ca , their Ca^ - buffering role in the physiological range remains questionable (Somlyo et a l . , 1985). A more l i k e l y candidate for physiological regulation of i n t r a c e l l u l a r calcium and C a 2 + - b u f f e r i n g seems to be the endo- plasmic reticulum (ER), which has both a high a f f i n i t y and a high 4 capacity for calcium-binding. A recent study by Somlyo et a l . (1985) in which the d i s t r i b u t i o n of C a 2 + in l i v e r c e l l s iji vivo was examined, concluded that the endoplasmic fr a c t i o n of c e l l u l a r calcium may be as high as 23-27%, while mitochondria only contain about 5%, the amount necessary for the modulation of C a 2 + - sensitive mitochondrial enzymes. The functional significance of ER-calcium i s not only r e f l e c t e d in i t s calcium buffering capacity. Its muscle equivalent, the sarcoplasmic reticulum, i s involved in the release of Ca 2 + which is indispensable for activation of muscle contraction (Endo, 1977; Martonosi, 1984). The role of calcium-receptor and calcium-binding proteins w i l l be discussed in the context of the nervous system (see Section 2.1 . ). While the concentration of free calcium i s precisely regulated in the vast majority of c e l l s , transient increases in [Ca^ ] w h i c h may result from calcium influx or release of endogenous Ca would activate certain biochemical events that constitute the basis of the second messenger action of the cation (Kretsinger, 1981). The role of calcium as an i n t r a c e l l u l a r messenger is of major importance because C a 2 + translates the actions of substances (hormones, neurotransmitters, e t c . ) , which are unable to permeate the c e l l but have receptors on the outside of the plasma membrane, into s p e c i f i c c e l l u l a r biochemical reactions. The contribution of calcium as a second messenger to hormone or neurotransmitter action is not a simple and e a s i l y detectable process, since i t may be masked sometimes by activation of other Ca^ -dependent mechanisms. Al t e r n a t i v e l y , 5 one may argue about the s p e c i f i c i t y of f i r s t messenger action i f there was only one i n t r a c e l l u l a r second messenger. Nevertheless, in most cases Ca^ acts s y n e r g i s t i c a l l y with other messenger systems, such as c y c l i c nucleotides, or i t s i n t r a c e l l u l a r concentration is under the control of several concomitantly activated regulatory processes. The cooperatlvity between Ca and other second messenger systems has long been recognized (Rasmussen and Goodman, 1977). In a recent review, Rasmussen and Barret (1984) propose an integrated view of the calcium messenger system, in which c y c l i c nucleotides play an important r o l e . According to their hypothesis, the i n i t i a l response, which consists of a transient increase in [ C a 2 + ] ^ , together with a sustained response due to cAMP activation (e.g., protein phosphorylation) may produce an integrated response which constitutes the observed ef f e c t of hormone action. This mechanism would allow for a very sensitive gain control, since C a 2 + i t s e l f i s involved in several steps of the c y c l i c nucleotide metabolism, ranging from the ac t i v a t i o n or i n h i b i t i o n of the cyclase to the ac t i v a t i o n of phosphodiesterase. Cyclic nucleotides are not the only other second messenger system that has been involved in calcium's action. The hydro- l y s i s of a membrane l i p i d , phosphatidylinositol 4,5-biphosphate, into d i a c y l g l y c e r o l and i n o s i t o l triphosphate (InsPj) i s under the control of hormone or neurotransmitter action in several preparations (Berridge and Irvine, 1984). Although InsP 3 may have second messenger actions of i t s own, i t s p r i n c i p a l function appears to be the release of Ca from i n t r a c e l l u l a r storage 6 s i t e s , mainly the endoplasmic reticulum. This release mechanism could serve as an amplification of the c e l l u l a r signal, since i t has been suggested that for every molecule of hydrolyzed phosphatidylinositol 10 or more calcium ions could be liberated. F i n a l l y , considering the toxic effects of elevated [ C a 2 + ] ^ (Schanne et a l . , 1979) i t i s quite remarkable how c e l l s are able to maintain i t s concentration at a reasonably steady l e v e l and to use i t s regulatory properties as a messenger system. 1.2. The role of calcium in the CNS Several of the regulatory functions of C a 2 + mentioned in the previous section also apply to neurons. Moreover, in the case of the CNS, calcium plays a pi v o t a l role in the modulation of processes that stand at the very basis of neuronal e x c i t a b i l i t y (Erulkar and Fine, 1979). The variety of Ca 2 +-mediated phenomena is so large that in some cases i t is almost impossible to determine, following proof of the cation's involvement, which p a r t i c u l a r mechanism may be the most important in causing or maintaining the observed event. Careful examination i s required to d i s t i n g u i s h between i t s action on neurotransmitter release, regulation of membrane e x c i t a b i l i t y , activation of other ionic channels or even some biochemical events that could be triggered by C a 2 + . In many instances, p a r t i c u l a r l y in the mammalian CNS, 9 + . . d i r e c t measurements of Ca involvement are d i f f i c u l t to achieve, thus leaving the researcher to rely on indirect evidence, mainly 7 origi n a t i n g from invertebrate preparations. Despite a l l the technical obstacles in C a 2 + measurement (also see Chapter I I I ) , there i s now considerable evidence for some of the important functions that t h i s cation plays in the mammalian CNS. Calcium channels It i s now clear that there are several types of membrane 9 _|_ channels that regulate the influx of Ca into nerve c e l l s from the e x t r a c e l l u l a r milieu. Most of these channels are se n s i t i v e to the voltage across the plasma membrane, but there may also be channels that are regulated through the action of hormones and neurotransmitter substances (Hagiwara and Byerly, 1981; Reuter, 1983; Tsien, 1983a). The voltage-dependent Ca 2 + channels share some properties that are equally applicable to a l l the excitable tissues examined. In contrast to the Na + channels, they are i n s e n s i t i v e to tetrodotoxin (TTX) but are readily blocked by other polyvalent cations (Co 2 +, L a 3 + , Mn 2 +, C d 2 + and N i 2 + ) at concentrations approximately equivalent to ex t r a c e l l u l a r C a 2 + . In addition, organic blockers such as verapamil and D-600 (the methoxy derivative of verapamil), and in some cases dihydropyridines (e.g., nifedipine and nitrendipine) antagonize calcium currents. The channels are not absolutely s p e c i f i c for C a 2 + , thus barium 9 _ i _ and strontium ions are able to replace Ca* in permeating through the channel molecule. While inactivation of Na + channels i s dependent on membrane voltage and the presence of gating 9 4. p a r t i c l e s , the cause of Ca^ current reduction following 8 prolonged opening of the channel is less evident. The current hypothesis, derived from the study of molluscan nerve c e l l s ( T i l l o t s o n , 1979; Eckert and T i l l o t s o n , 1981), i s that C a 2 + ions accumulate on the inside of the plasma membrane and inactivation occurs when their concentration i s s u f f i c i e n t l y large to stop additional ions from entering the c e l l . This mechanism would allow for a longer channel opening time and for a more precise control of Ca^ entry into neurons, depending on intraneuronal ionic d i f f u s i o n and the presence of C a 2 + - b u f f e r i n g systems. However, voltage-dependent inactivation of C a 2 + channels has been shown to exist in several systems (Hagiwara and Byerly, 1981; Tsien, 1983a). The identity and functioning of neurotransmitter-dependent C a 2 + channels is characterized to a lesser degree (Reuter, 1983). Although some substances, that may well be neurotransmitter 9 + candidates, enhance Ca^ influx into neurons, i t i s not known whether their effect i s due to changes in voltage across the plasma membrane or to activation of specialized channels. A l t e r n a t i v e l y , these agents may simply modulate permeation of C a 2 + through the voltage-sensitive channels, as has been postulated for the mechanism of presynaptic i n h i b i t i o n in CNS neurons (Reuter, 1983). Neurotransmitter release 9 + Once Ca^ crosses the plasma membrane through the channel and enters the neuronal cytoplasm, i t is available for the act i v a t i o n of several regulatory mechanisms that are ultimately 9 underlying normal functioning of neurons. Induction of neuro- transmitter release, analogous to exocytosis in other systems, i s of primary importance in achieving chemical communication between elements of the CNS. Much of the evidence for calcium's involvement comes from the study of the neuromuscular junction, where i t was recognized 90 years ago that the presence of C a 2 + in the e x t r a c e l l u l a r medium is a neccessary requirement for transmission to occur (Locke, 1894). It took however more than seventy years for the 'calcium hypothesis' of neurotransmission to emerge (Katz and M i l e d i , 1965; 1967). Later, investigation of the squid giant synapse provided experimental proof for the depolarization-induced calcium entry into presynaptic terminals and the release of neurotransmitters (Llinas, 1977). The steps of a c t i v a t i o n from calcium entry to the f i n a l quantal release process have been subject to several reviews (e.g., Rahamimoff, 1976; Llinas and Heuser, 1977). Although direct evidence for the existence of mechanisms analogous to those found at the squid giant synapse is lacking in the mammalian CNS, i t i s reasonable to assume that calcium ions contribute in a similar manner to the central release of neurotransmitter molecules. It has been postulated (Llinas, 1977; Llinas and Heuser, 1977) that the delay of about 200 usee between depolarization of the synaptic terminal and actual release through exocytosis can be accounted for by accumulation of Ca at the inside of the plasma membrane (to le v e l s of about two orders of magnitude higher than resting [ Ca 2 +]^) and subsequent rapid ac t i v a t i o n of adjacent transmitter releasing s i t e s . 10 While the direct involvement of C a 2 + in the release of neurotransmitters seems to be well established, the exact mechanism of the calcium-dependent activation i s s t i l l somewhat obscure. Dodge and Rahamimoff (1967) examining the effect of various e x t r a c e l l u l a r Ca 2 + concentrations on the release process, proposed a cooperative action for several calcium ions (probably four) at a hypothetical Ca 2 +-binding s i t e . Equally plausible is the involvement of the i n t r a c e l l u l a r mediator protein calmodulin, as outlined in Section 2.1.1. Furthermore, the p o s s i b i l i t y that a Na -dependent release of Ca* from i n t r a - c e l l u l a r storage s i t e s may be responsible for the event has been suggested in l i g h t of the observation that transmitter release also occurs in the absence of [ C a 2 + ] Q (Rahamimoff et a l . 1980). Activation of other ionic conductances Entry of calcium through specialized channels i s not only important at the l e v e l of the presynaptic terminal, but also regulates permeability of the membrane to other ions, such as K +. Activation of such a K + conductance w i l l tend to hyperpolarize the neuronal membrane since the K +-equilibrium potential i s more negative than the resting membrane potential in most i f not a l l neurons. A Ca^ -activated K -conductance, or gK C a, discovered by inje c t i o n of C a 2 + into neurons (Krnjevic and Lisiewicz, 1972; Meech, 1972) has been shown to be present in many neuronal systems (for review see Schwartz and Passow, 1983). It i s also evident that activation of this conductance i s not a simple charge displacement, since B a 2 + readily crosses the membrane 11 through C a 2 + channels, but f a i l s to activate gK C a, and may even result in i t s i n h i b i t i o n (Gorman and Hermann, 1979). The importance of g^ca * n ^he control of neuronal e x c i t a b i l i t y i s outlined in Section 2.1.3. It i s now recognized that many neuro- modulators exert at least a part of t h e i r effects through a l t e r i n g t h i s Ca^ -activated conductance. Noradrenaline, histamine and c o r t i c o t r o p i n releasing factor have a l l been shown to regulate the e x c i t a b i l i t y of CNS neurons through i n h i b i t i o n of gK C a (Madison and N i c o l l , 1982; Aldenhoff et a l . , 1983; Haas and Konnerth, 1983), while dopamine may hyperpolarize c e l l s through i t s a c t i v a t i o n (Benardo and Prince, 1982). It has also been proposed that the gK^a system may be under the control of c y c l i c AMP, although the evidence i s highly controversial (Benardo and Prince, 1982; Madison and N i c o l l , 1982). As in the case of neurotransmitter release, the entry of C a 2 + through specialized channels does not seem to be an absolute requirement for a c t i v a t i o n of the 9 Kca* Endogenous calcium ions released from i n t r a c e l l u l a r storage s i t e s may be just as e f f e c t i v e in t r i g g e r i n g the K +-conductance (Kuba, 1980; Akaike et a l . , 1983), thus contributing to the o v e r a l l regulation of neuronal e x c i t a b i l i t y . In addition to the Ca^ -activated K -conductance, a chloride-conductance that seems to be dependent on C a 2 + has been recently observed in cultured spinal cord neurons (Owen et a l . , 1984). The presence of t h i s conductance, depending on the equilibrium potential for C l ~ in d i f f e r e n t neurons, may complement the inhibitory action of the gK C a system. 12 Regulatory functions of intraneuronal Ca* The intraneuronal actions of Ca are not limited to neuro- transmitter release or activation of other ionic fluxes. Several biochemical mechanisms are dependent on the presence of C a 2 + , a n d some of them may involve the special second messenger function of the cation as described for other systems (see Section 1.1.). Many of these actions are possibly mediated through intraneuronal o+ . . . . . Ca -receptor proteins, of which calmodulin is a prime example. The role of these proteins in the nervous system i s described in more d e t a i l in Chapter I I . Considering the variety of neuronal Ca 2 +-dependent phenomena and their many functional implications, i t is of no surprise that cyt o s o l i c free calcium i s under s t r i c t control in nerve c e l l s and is maintained at less than micromolar levels (e.g., Alvarez- Leefmans et a l . , 1981). The c e l l u l a r elements which achieve t h i s rigorous function are no d i f f e r e n t in neurons than in non- neuronal tissue. The endoplasmic reticulum, calcium-binding proteins and mitochondria together with a low resting permea- b i l i t y of the membrane to Ca^ , a l l contribute to the maintenance of low [ C a 2 + ] ^ . As in non-neuronal tissue, the share of mitochondria in the regulation of calcium levels within the physiological range i s questionable (Blaustein et a l . , 1978; 1980). In addition to the intraneuronal buffering and sequestering mechanisms, excess C a 2 + i s extruded from the cytosol with the aid of the Na +-Ca 2 + exchange and activation of C a 2 + 13 pumps, possibly linked to the Ca 2 -Mg2 ATPase (Erulkar and Fine, 1979). E x t r a c e l l u l a r C a 2 + Despite the many regulatory functions of intraneuronal C a 2 + , i t i s not necessary to postulate that a l l changes in nerve c e l l e x c i t a b i l i t y have to be a d i r e c t consequence of al t e r a t i o n s in [ C a 2 + ] j . E x t r a c e l l u l a r C a 2 + i s a very e f f e c t i v e modulator of membrane e l e c t r i c a l properties, as shown by the experiments of Frankenhaeuser and Hodgkin (1957) in the squid axon. The cation may neutralize the negative charges on the outer surface of the plasma membrane near the voltage sensitive channels, thus having a charge s t a b i l i z i n g e f f e c t . In central neurons, lowering of [Ca z ] Q induces spontaneous discharges (Richards and Sercombe, 1970; Jefferys and Haas, 1982), while iontophoresis of C a 2 + reduces their f i r i n g rate (Kato and Somjen, 1969; Wright, 1984). This i s probably why the calcium concentration of the cerebro- spinal f l u i d , in a manner similar to the regulation of cyto s o l i c C a 2 + , is maintained at a reasonably steady value of 1.5-1.8 mM (Campbell, 1983). 14 1.3. Calcium and long-term changes in neuronal e x c i t a b i l i t y ; The present study In view of a l l the possible s i t e s and mechanisms of Ca 2 + action i t is not d i f f i c u l t to envisage how an altered neuronal calcium metabolism may contribute to changes in the functional c h a r a c t e r i s t i c s of neurons. Some pathophysiological changes may indeed result from the altered a b i l i t y of nerve c e l l s to cope with a Ca challenge. For example, Heinemann et a l . , (1977) detected s i g n i f i c a n t declines in e x t r a c e l l u l a r C a 2 + a c t i v i t i e s during epileptiform discharges of c o r t i c a l neurons. Presumably the loss of [ C a 2 + ] 0 was p a r a l l e l e d by an increase in the i n t r a - neuronal concentration of the cation, indicating the involvement of calcium in epileptiform events. Similarly i t has been shown that regenerative C a 2 + spikes par t i c i p a t e in the bursting a c t i v i t y in the p e n i c i l l i n model of experimental epilepsy (Wong and Prince, 1978; Hotson and Prince, 1981) and neuronal calcium accumulation characterizes the status epilepticus induced by 1- a l l y l g l y c i n e ( G r i f f i t h s et a l . , 1982). A l l of these data indicate that the one possible mechanism underlying epilepsy may involve an impairment of the neuronal regulation of C a 2 + . One of the d i f f i c u l t i e s with the concept of calcium's involvement in epileptiform phenomena is the lack of substantial evidence for chronic a l t e r a t i o n s in calcium regulation. Acute changes in Ca homeostasis in various models of experimental epilepsy cannot possibly account for the sustained alterations in 15 e x c i t a b i l i t y which i s considered to be a general c h a r a c t e r i s t i c of e p i l e p t i c neurons (Ward, 1969). In order to result in a persistent e f f e c t on neuronal e x c i t a b i l i t y , calcium has to induce a 'permanent' biochemical change. A l t e r n a t i v e l y , due to some neurochemical mechanism(s), the regulation of calcium i t s e l f has to be offset as long as the neurons maintain their patho- physiological state. The recent findings that a neuron s p e c i f i c calcium-binding protein (CaBP) i s gradually l o s t in the granule c e l l s of the dentate gyrus during and following kindling-induced epilepsy are the f i r s t indications of a possible involvement of long-term al t e r a t i o n s in C a 2 + - r e g u l a t i o n (Miller and Baimbridge, 1983; Baimbridge and M i l l e r , 1984; Baimbridge et a l . , 1985). Since CaBP is also d i s t r i b u t e d in the cerebral cortex, in neurons prone to epileptiform a c t i v i t y , the purpose of the present study included measurement of c o r t i c a l CaBP d i s t r i b u t i o n with the aid of a s p e c i f i c radioimmunoassay to detect possible regional differences in i t s l e v e l s . Furthermore, extending previous studies on the kindling model of epilepsy, the present experiments address the question regarding the c o r r e l a t i o n between electrophysiological a l t e r a t i o n s during epileptogenesis (the development of afterdischarges) and the decline in CaBP. If neuronal regulation of calcium does indeed play a role in seizure mechanisms, then other models of experimental epilepsy should also be characterized by detectable changes in calcium regulation. To examine t h i s p o s s i b i l i t y , levels of CaBP were measured in the c o r t i c a l areas of the genetically e p i l e p t i c s t r a i n of mice ' E l ' . The results were compared to a control 16 st r a i n and the effect of seizures on CaBP levels was also studied within the E l s t r a i n i t s e l f . A l t e r n a t i v e l y , the decline in the levels of CaBP may not result in an altered calcium homeostasis and the changes in the protein content of brain tissue may be simply coincidental with the observed epileptiform a c t i v i t y . In order to determine whether CaBP plays a role in the regulation of neuronal C a 2 + the technique of kinetic analysis of 4 5 C a uptake curves was employed in hippocampal s l i c e s obtained from kindled animals. Since this technique has not been widely applied to neuronal tissue, the v a l i d i t y of the method for the CNS had to be determined. Once thi s was determined, the method proved to be a valuable tool in determination of brain calcium homeostasis. In addition to measurement of exchangeable C a 2 + using the radioactive tracer method, the amount of t o t a l hippocampal calcium was also determined by atomic absorption spectrophotometry (AAS). The zinc content of the hippocampus was determined alongside the calcium levels to determine changes that may have occurred during the process of kindling. In contrast to the more deleterious e f f e c t s of calcium in neurons, such as p a r t i c i p a t i o n in epileptogenesis, the cation is also involved in long-lasting changes in neuronal functions subserving physiological events. For example, p l a s t i c changes in synaptic function occur at molluscan synapses and the underlying process i s thought to be a persistent change in the regulation of C a 2 + channels, or some other calcium-mediated event, such as changes in K + conductance. The enhanced synaptic e f f i c a c y of the 1 7 system may d i r e c t l y determine the learning process (Klein et a l . , 1980; Kandel, 1981). In addition, calcium i s readily involved in yet another long-lasting change of neuronal e x c i t a b i l i t y , namely the phenomenon of long-term potentiation (Swanson et a l . , 1982; Turner et a l . , 1982; Eccles, 1983). Part of the present study was therefore undertaken to examine various aspects of Ca 2 +-regulation under experimental conditions where permanent or quasi-permanent alterations in CNS Ca'' -homeostasis are l i k e l y to occur. A novel form of long-term potentiation is described, caused by a methylxanthine derivative, that i s also caused by altered neuronal C a 2 + regulation. The p o s s i b i l i t y that release of calcium from i n t r a c e l l u l a r storage s i t e s , rather than calcium entry per se, may be responsible for the observed e l e c t r o p h y s i o l o g i c a l changes i s also examined with the aid of the ^ C a uptake method. In summary, the present study examines the regulation of C a 2 + in neuronal systems that present long-term alt e r a t i o n s in their function. The systems under investigation comprised the kindling model of experimental epilepsy, a genetic form of epilepsy and f i n a l l y , the phenomenon of long-term potentiation induced by a methylxanthine derivative. 18 CHAPTER I I . DISTRIBUTION AND ALTERATIONS IN CALCIUM-BINDING PROTEIN (CaBP) IN THE CNS 2.1. General introduction 2.1.1. Calcium-binding proteins of the CNS The regulation of i n t r a c e l l u l a r calcium is achieved by most eukaryotic c e l l s through a variety of factors. Apart from i n t r a c e l l u l a r organelles, such as mitochondria and the endo- plasmic reticulum, proteins that bind C a 2 + with a high a f f i n i t y (10~6-10 -^ M) p a r t i c i p a t e in the o v e r a l l physiological framework of calcium regulation (Borle, 1981a). Some of these proteins are involved in the modulation of enzymatic events thus being part of the second messenger role of C a 2 + (Kretsinger, 1981), while others subserve transport mechanisms, or constitute i n t r a c e l l u l a r C a 2 + - b u f f e r s . The important contribution of calcium-binding proteins to c e l l u l a r regulatory mechanisms i s probably best i l l u s t r a t e d by their presence in v i r t u a l l y a l l species and in 19 every tissue from e p i t h e l i a to brain. There are close to 100 'different' proteins that have been reported to bind calcium, but i t i s debatable whether they are a l l capable of exerting p h y s i o l o g i c a l l y s i g n i f i c a n t alterations upon binding of calcium ions (Kretsinger, 1976). Most of the calcium binding proteins of known structure share r e l a t i v e l y similar Ca 2 +-binding domains, c a l l e d the EF-hand regions (Kretsinger, 1976). These regions consist of the subunit 9 + where binding of Ca occurs flanked almost at right angles by two s t r u c t u r a l units consisting of alpha helices (helix-loop- helix conformation). The multiple EF-hand structures (usually four) of some calcium-binding proteins are thought to have resulted from gene duplication. Although several proteins have not retained t h i s quadruple arrangement, the large family of calcium binding proteins is considered to have evolved from a common ancestral protein (Demaille, 1982). The 'on' and ' o f f binding rate constants for the Ca 2 +-domains are remarkably fast, making these proteins ideal for rapid C a 2 + binding and release, while the conformational changes induced by calcium are of a r e l a t i v e l y slower time course (Robertson et a l . , 1981; Levine and Williams, 1982). The nature of the conformational changes upon C a 2 + binding has been investigated using nuclear magnetic resonance (NMR) studies (cf., Dalgarno et a l . , 1984). Binding of pairs of calcium ions is considered to be a cooperative event. The resultant conformational a l t e r a t i o n of the protein produces movement of the 'trigger zone' which in turn, in the case of regulatory proteins, 20 may be attached to other enzymes or macromolecules. Interference caused by drug binding to these proteins, for example, neuro- l e p t i c antagonism of calmodulin (Weiss and Levin, 1978), or modulation of the protein molecule i t s e l f through phosphorylation (Dalgrano et a l . , 1984) may hinder the effectiveness of their calcium-dependent regulatory action. Since the discussion of a l l calcium binding proteins is beyond the scope of the present study, only a r e l a t i v e l y few w i l l be b r i e f l y described below, p a r t i c u l a r l y those shown to exist within the central nervous system. Calmodulin (CaM) i s one of the most widely distributed calcium binding proteins found in plants as well as in a l l species of animals examined. It has been discovered as the C a 2 + - dependent regulator of brain c y c l i c nucleotide phosphodiesterase (Cheung, 1970; Kakiuchi and Yamazaki, 1970), and during the seventies many i n t r a c e l l u l a r messenger roles of C a 2 + have been linked to i t s action (Cheung, 1980; Means and Dedman, 1980; Klee et a l . , 1980). Other than i t s obvious function in regulating c y c l i c nucleotide metabolism in the brain, CaM has been found to be l o c a l i z e d in post-synaptic densities (Lin et a l . , 1980) as well as to participate through Ca 2 +adependent protein phosphorylation in the release of neurotransmitters from isolated synaptic v e s i c l e s (DeLorenzo et a l . , 1979). Therefore, the involvement of CaM in synaptic function seems quite well established (DeLorenzo, 1982). The phosphorylation of synaptic proteins (presumably tubulin) by CaM action may result in long term alt e r a t i o n s of neurotransmitter functions, which may in turn 21 subserve p l a s t i c changes in the CNS. One of these, the long-term potentiation phenomenon (LTP) in the hippocampus has indeed been shown to be blocked by CaM antagonist drugs (Finn et a l . , 1980; Mody et a l . , 1984). Recent studies on CaM suggest that i t may also regulate the calcium channels of nerve c e l l s or at least i t may be homologous to the calcium binding protein that i s involved in channel modulation ( c f . , Johnson et a l . , 1983). Another calcium binding protein, parvalbumin is found primarily in muscle and nerve tissue (Heizmann, 1984). It is a r e l a t i v e l y small protein (Mr of approx. 12,000), and was o r i g i n a l l y isolated from frog and carp muscles. The present working hypothesis concerning i t s mechanism of action, at least in muscle, states that i t serves as a 'shuttle' for C a 2 + between troponin-C (the C a 2 + binding subunit of troponin) and the sarcoplasmic reticulum. The l i b e r a t i o n of C a 2 + from troponin-C would then allow for the next contraction to occur. This may be the reason why parvalbumin is primarily l o c a l i z e d within the fastest contracting s k e l e t a l muscle f i b e r s (Celio and Heizmann, 1982). Parvalbumin's r o l e in the CNS i s less well defined, but immunohistochemical studies have shown i t s l o c a l i z a t i o n in small interneurons of the cortex, basket c e l l s of the hippocampus and cerebellum, Purkinje and s t e l l a t e c e l l s of the cerebellum and periglomerular c e l l s of the olfactory bulb (Celio and Heizmann, 1981). In general, there seems to be a good co r r e l a t i o n between parvalbumin d i s t r i b u t i o n and neurons that use GABA as a neurotransmitter, but the functional significance of these findings i s presently under investigation. 22 The S-100 protein i s also a s p e c i f i c calcium-binding protein, l o c a l i z e d mainly in astrocytes rather than neurons in the CNS. In astrocytes i t may activate their putative c o n t r a c t i l e apparatus. Although the protein i s equipped with the t y p i c a l EF- hand structures for C a 2 + binding, some of these domains are modified. A K -modulated Ca binding s i t e i s present, and another one may bind Zn^ with a higher than normal a f f i n i t y . Many of the early functional studies of the S-100 protein have implied a role in memory formation (e.g., Hyden and Lange, 1973), but according to more recent research t h i s may not be the case (Moore, 1982). 2.1.2. The calcium-binding protein CaBP Calcium-binding protein (CaBP) was f i r s t shown to be present in chick intestine and i t s synthesis to be dependent on vitamin-D (Wasserman and Taylor, 1966). Subsequently the protein has been isola t e d from the intestines of several species and shown to d i f f e r in size and the number of calcium-binding domains. For example, the avian protein has a Mr of 28,000 with four s i t e s for C a 2 + binding while the bovine and porcine proteins are of lower molecular weight (Mr=8,700-9,000) and have two and one s i t e respectively, a l l of which bind C a 2 + with an a f f i n i t y of approximately 10"^ M (Wasserman, 1980). The amino acid sequence and three-dimensional structure of these proteins has now been established (e.g., Szebenyi et a l . , 1981) and i t appears that the 23 calcium-binding domains generally show the EF-hand conformation proposed by Kretsinger (1976) for proteins of the same c l a s s . Immunologically similar, perhaps i d e n t i c a l , proteins to the avian gut CaBP have been found in several tissues that are involved in C a 2 + transport. Most of these proteins show a strong s i m i l a r i t y also with respect to their dependency on vitamin-D (Christakos and Norman, 1980). Because of the common calcium transporting function of the tissues where i t i s found, CaBP has been proposed to function as an i n t r a c e l l u l a r Ca c a r r i e r (Levine and Williams, 1982; Wasserman and Fullmer, 1983). In the gut for example, following the action of vitamin-D, there is a rapid uptake of calcium by the i n t e s t i n a l c e l l s , but calcium transport from one c e l l u l a r pole to the other does not occur unless CaBP i s present (Wasserman and Fullmer, 1983; Wasserman et a l . , 1983). The l o c a l i z a t i o n of a calcium-binding protein similar to the CaBP of the avian gut in the brain, a tissue where calcium i s d i r e c t l y involved in the control of neuronal e x c i t a b i l i t y , has opened new avenues of research for the functional role of this protein. F i r s t , i t has been demonstrated by radioimmunoassay procedures that avian, rat and even human brain samples contain s i g n i f i c a n t amounts of CaBP (Taylor, 1974;' Christakos et a l . , 1979; Baimbridge and Parkes, 1980; Baimbridge et a l . , 1980; 1982). Later, by using immunocytochemical techniques, i t has been shown that the protein i s not randomly dis t r i b u t e d throughout the CNS, but i t i s rather confined to par t i c u l a r neurons while being absent from others and from g l i a l c e l l s 24 (Jande et a l . , 1981; Roth et. a l . , 1981; Baimbridge and M i l l e r , 1982; Garcia-Segura et a l . , 1984). A l l of these studies have c l e a r l y i d e n t i f i e d the cerebellar Purkinje c e l l s and some neurons of the cerebral cortex and subcortical structures as containing large amounts of the protein. The conspicuous l o c a l i z a t i o n of CaBP in some neurons but not in others i s well i l l u s t r a t e d in the hippocampal formation, where dentate granule c e l l s and pyramidal c e l l s of the CA1 region show high degree of CaBP- immunoreactivity, while the pyramidal neurons of area CA3 do not (Baimbridge and M i l l e r , 1982). The question remains open whether CaBP of nerve tissue is sensitive to vitamin-D or not. The early study of Taylor (1974) showed some degree of vitamin-D dependency, but the discrepancy between the d i s t r i b u t i o n of 1,25(OH)2_D3 binding s i t e s (Stumpf et a l . , 1982) and CaBP l o c a l i z a t i o n in the brain tends to dismiss t h i s hypothesis. Furthermore, in rats, intracerebroventricular injections of 1,25(OH)2~D3 (the active metabolite of vitamin D) did not a l t e r brain CaBP leve l s (K.G. Baimbridge, unpublished observations), indicating that the regulation of CNS CaBP by vitamin-D i s unlikely. 2.1.3. Possible roles of CaBP in the central nervous system One of the f i r s t p o s s i b i l i t i e s that arises from the studies on i n t e s t i n a l c e l l s i s that CaBP may underlie a similar C a 2 + - transport function in neurons. However, transport of C a 2 + across the c e l l i s probably less important in brain than in other 25 tissues such as intestine, kidney and bone, where absorption and reabsorption of calcium i s a s i g n i f i c a n t part of the normal functioning of these structures. Nevertheless, CaBP may s t i l l be involved in intraneuronal transport of Ca z , and considering i t s high a f f i n i t y for Ca^ -binding, i t may serve as a temporary storage s i t e for the cation. This mechanism may operate especially under circumstances where the lower a f f i n i t y mito- chondria would be insensitive to changes in [ C a 2 + ] ^ . According to this hypothesis, following cessation of the C a 2 + signal, CaBP o 't-would 'migrate' to the mitochondria, or other more permanent Ca storage s i t e s within the neurons, and would release i t s bound calcium. Because of the di f f e r e n t Ca 2 + a f f i n i t i e s of the two systems, th i s mechanism has to involve an active process of Ca 2 + uptake by the mitochondria. Secondly, the presence of CaBP in certain nerve c e l l s and not in others would make the CaBP-containing neurons less sensitive to the ubiquitously d i s t r i b u t e d calmodulin (CaM). The r e l a t i v e l y similar Ca 2 +-binding constants of the two proteins would enable c e l l s that possess CaBP to bind the C a 2 + that otherwise could have activated CaM. The net result would be a dampening of CaM-regulated events and enzymatic reactions, including protein phosphorylation. Moreover, i f CaM i s indeed involved in neurotransmitter release (DeLorenzo, 1982), the presence of CaBP in the presynaptic terminals of some neurons would allow for a more precise and fine tuning of Ca 2 +-CaM regulated exocytosis of synaptic v e s i c l e s . In certain neurons where two or possibly more neurotransmitters may be co-localized 26 in the same synaptic terminal the presence or absence of CaBP may thus permit the selec t i v e release of one or the other neurotransmitter molecule, depending on the amount of C a 2 + entry. Although neurons tend to maintain their free ionic i n t r a - c e l l u l a r calcium at very low le v e l s (10~^-10 - 7 M), this may r i s e to 10-100 times i t s resting value during a c t i v i t y , neuro- transmitter action or certain pathological events. Therefore, bearing in mind the high a f f i n i t y of C a 2 + binding to CaBP, the protein may serve as an intraneuronal calcium buffering system, especially so when [Ca^ ]± i s raised to supranormal l e v e l s . This hypothesis appears reasonable since i t has been estimated that in cerebellar Purkinje c e l l s , which contain the highest CaBP levels in the CNS, the cytosolic concentration of CaBP may be as high as 0.1-0.2 mM (Baimbridge et a l . , 1982). This would allow for C a 2 + - buffering up to 0.4-0.8 mmol/litre of cytoplasm since 4 mol of C a 2 + are bound to each mol of CaBP. I n t r a c e l l u l a r free C a 2 + would probably never r i s e to such l e v e l s , indicating that the buffering capacity of CaBP would be able to cope with most, i f not a l l , of physiological and even pathophysiological changes of Ca* inside nerve c e l l s . The neuronal C a 2 + - b u f f e r function of CaBP seems to be an a t t r a c t i v e hypothesis, but raises the question as to how Ca i s buffered in the many neurons that lack the protein? In these c e l l s the only high s e n s i t i v i t y Ca 2 +-sequestering mechanism may be the endoplasmic reticulum (Duce and Keen, 1978), which would make them s e l e c t i v e l y vulnerable to a supranormal Ca^ - challenge. 27 The calcium-buffering hypothesis would also imply that CaBP could e f f e c t i v e l y regulate the magnitude of C a 2 + influx through calcium channels. As shown for molluscan nerve c e l l s calcium channels are not only sensitive to membrane voltage, but are 9 + inactivated due to accummulation of Ca^ on the inside of the plasma membrane ( T i l l o t s o n , 1979; Eckert and T i l l o t s o n , 1981). There i s reason to believe that similar events also occur in the mammalian CNS. If this proves to be the case, then C a 2 + binding to an i n t r a c e l l u l a r calcium-buffer, such as CaBP in the proximity of the calcium channel would remove free calcium ions which otherwise would hinder further entry of Ca 2 + through the channel as proposed by the kinetic model of Chad et a l . (1984). The diminished Ca 2 +-dependent C a 2 + inactivation would enable the 9 + CaBP-containing neurons to have larger Ca -spikes of longer duration. While regulating the amount of calcium entry into neurons, CaBP may also be involved, although somewhat i n d i r e c t l y , in the modulation of the C a 2 + - a c t i v a t e d K + conductance (gK^g) of nerve c e l l s . This hyperpolarizing conductance is present in v i r t u a l l y every nerve c e l l (Schwarz and Passow, 1983) and i t s a c t i v a t i o n in CNS neurons stops their bursting or rep e t i t i v e discharge (Alger and N i c o l l , 1980; Hotson and Prince, 1980). Furthermore, since signal encoding in the f i r i n g pattern of neurons i s an important feature of information transmission in the CNS (Stein, 1967), i t i s expected that sustained r e p e t i t i v e neuronal discharge is under rigorous c o n t r o l . The underlying regulatory mechanism must be the a c t i v a t i o n of an outward current (Jack et a l . , 1975). Both 28 the late K + current ( j A ^ a n d t n e C a 2 + - a c t i v a t e d K + current possess the time course and magnitude required to control the frequency of neuronal f i r i n g . In neurons of the mammalian CNS there i s evidence that adaptation (or accommodation) of spike discharge during a long depolarizing current pulse i s under the control of gK C a (Madison and N i c o l l , 1982; 1984). The suggestion has been made that calcium entry per se i s not the primary factor that controls the magnitude of the gK C a. Injection of the Ca^ - chelator EGTA into CA1 pyramidal c e l l s of the hippocampus, which f a c i l i t a t e s Ca^ -dependent potentials, abolishes 9&ca conductance re s u l t i n g in a prolonged and a more rapid (unaccommodated) f i r i n g of these neurons (Madison and N i c o l l , 1984). More di r e c t evidence 9 + + . . . . that the Ca^ neccessary for K conductance a c t i v a t i o n originates from the endoplasmic reticulum comes from the work of Kuba (1980) in the sympathetic ganglion or Akaike et a l . (1983) in s n a i l neurons. If the calcium neccessary for a c t i v a t i o n of gK^g i s derived from i n t r a c e l l u l a r storage s i t e s , how could CaBP be involved in the regulation of th i s conductance? The hypothesis regarding the release of intraneuronal Ca 2 + as activator of the K + conductance is based on the role of parvalbumin in rapidly contracting muscle fi b e r s (see Section 2.1.1.). The fi b e r s that contain parvalbumin are able to contract several hundred times a second due to the fast removal of Ca^ from troponin-C by binding to parvalbumin. If the neuronal gK^a i s activated by Ca 2 + released from i n t r a c e l l u l a r storage s i t e s , possibly the endoplasmic reticulum, then binding of C a 2 + to parvalbumin or CaBP would prevent C a 2 + from reaching 29 the K + channel and thus i t s a c t i v a t i o n . A l t e r n a t i v e l y , the calcium binding proteins, due to their high a f f i n i t y for the cation, may be able to remove the already bound Ca* from the K channel molecule resulting in the inactivation of the K + channel. A mechanism of inactivation somewhat similar to t h i s , although involving d i f f u s i o n of C a 2 + away from the channel, has been proposed by Gorman and Thomas (1980). Further evidence to support t h i s l a t t e r alternative comes from the work of Barish and Thomas (1983) in molluscan neurons where mitochondria do not seem to part i c i p a t e in removal of C a 2 + from the activated K + channel and therefore the presence of a high a f f i n i t y buffering system had to be proposed. Baldissera and Parmiggiani (1979) constructed a model of the spinal motoneurone, which includes t h i s form of gK C a i n a c t i v a t i o n . In their model, depending on the duration and magnitude of the inactivation process, the neuron i s capable of high rate rep e t i t i v e f i r i n g and changes of discharge frequencies. It has yet to be determined whether involvement in activation or inactivation of the gK^a proves to be the s i t e of action for calcium-binding proteins. In either case, Caz+- dependent K + channels would stay open for shorter times in neurons which possess parvalbumin and/or CaBP. This in turn would enable neurons that contain any of these proteins to maintain a longer duration r e p e t i t i v e discharge. As mentioned in Section 2.1.1., parvalbumin i s l o c a l i z e d mainly in interneurons, c e l l s that are capable of f i r i n g action potentials at extremely high frequencies for sustained i n t e r v a l s . The Purkinje c e l l s of 30 the cerebellum which show two types of f i r i n g patterns (Llinas and Sugimori, 1980a,b), both at r e l a t i v e l y high frequencies, contain both parvalbumin and CaBP in high concentrations. In general terms, there i s quite a good correlation between the a b i l i t y of neurons to sustain their frequency of discharge and the l o c a l i z a t i o n of CaBP within these c e l l s . The two notable exceptions are the granule c e l l s of the dentate gyrus and neurons of the i n f e r i o r olivary nucleus. In these c e l l s , r e p e t i t i v e discharge may be a d d i t i o n a l l y regulated by s p e c i f i c membrane ch a r a c t e r i s t i c s which also play a role in determining the f i r i n g pattern of neurons (Jack et a l . , 1975). However, both of these c e l l types show a depolarizing a f t e r - p o t e n t i a l (DAP) of a marked amplitude (Llinas and Yarom, 1981a,b; Fricke and Prince, 1984; M.W. Oliver, personal communication) that may be a r e f l e c t i o n of the lack of gKQa coupled with a concomitant long-lasting C a 2 + entry. The co r r e l a t i o n of CaBP l o c a l i z a t i o n in certain neurons together with their electrophysiological investigation w i l l result in the elucidation of the exact function of t h i s protein in the CNS. 31 2.2. C o r t i c a l d i s t r i b u t i o n of CaBP in rats 2.2.1. Introduction It has been established that nervous tissue contains a large amount of CaBP which i s unequally d i s t r i b u t e d in various areas of the CNS with l i t t l e species differences (Christakos et a l . , 1979; Baimbridge and Parkes, 1980; Baimbridge et a l . , 1980; 1982). Although the exact funtion of the protein was not determined, at the time i t appeared that i t p a r t i c i p a t e d in the regulation of neuronal calcium. Further immunohistochemical studies (Jande et a l . , 1981; Baimbridge and M i l l e r , 1982; Garcia-Segura et a l . , 1984) have established i t s confinement to some neurons but not others and demonstrated i t s presence in elements of the neocortex. Since there are profound changes in e x t r a c e l l u l a r calcium concentrations during c o r t i c a l epileptogenesis (Heinemann et a l . , 1977), i t i s possible that the presence of an intraneuronal C a 2 + - b u f f e r i n g protein, such as CaBP, may a l t e r the neurons' responsiveness to the calcium challenge. The present study was therefore undertaken to examine in d e t a i l the c o r t i c a l d i s t r i b u t i o n of CaBP considering the d i f f e r e n t i a l s e n s i t i v i t y of various n e o c o r t i c a l areas to aberrant forms of a c t i v i t y (Ward, 1969). With the use of a s p e c i f i c radioimmunoassay i t was also possible to determine the CaBP content of several c o r t i c a l areas to complement the av a i l a b l e immunohistochemical studies. 32 2 . 2 . 2 . Methods 2 . 2 . 2 . 1 . Rat c o r t i c a l samples Male r a t s of the W i s t a r s t r a i n (body w e i g h t s : 250-300 g) were s a c r i f i c e d through d e c a p i t a t i o n and t h e i r b r a i n s removed. V a r i o u s c o r t i c a l a r e a s were d i s s e c t e d f r e e on i c e , f o l l o w i n g removal of the h ippocampus , b r a i n stem and s u b c o r t i c a l s t r u c t u r e s i n c l u d i n g the b a s a l g a n g l i a . The c o r t i c a l r e g i o n s were d i s s e c t e d as f o l l o w s : f r o n t a l c o r t i c a l t i s s u e was o b t a i n e d by c u t t i n g the most r o s t r a l 2 . 0 - 2 . 5 mm of the two c e r e b r a l hemispheres; the p a r i e t a l r e g i o n c o n t a i n e d the t i s s u e between t h i s cut and the c e n t r a l s u l c u s t h a t was i d e n t i f i e d v i s u a l l y by the a r t e r y of the c e n t r a l s u l c u s r u n n i n g a l o n g s i d e ; o c c i p i t a l area i n c l u d e d the r e m a i n i n g c a u d a l p a r t s of the c e r e b r a l c o r t e x . Once these s e c t i o n s were o b t a i n e d in a f r o n t a l p l a n e , the p a r i e t a l and o c c i p i t a l p a r t s were f u r t h e r s u b d i v i d e d d o r s o - v e n t r a l l y i n t o four e q u a l p a r t s (on both s i d e s ) . These r e g i o n s were rough ly e q u i v a l e n t to the a r e a s d e f i n e d by K r i e g (1946): P a r i e t a l a r e a I : A . c i n g u l a r i s p o s t e r i o r v e n t r a l i s ; A . g i g a n t o p y r a m i d a l i s ; A . p o s t c e n t r a l i s o r a l i s . P a r i e t a l a r e a II : A . p o s t c e n t r a l i s c a u d a l i s . P a r i e t a l a r e a I I I : A . p o s t c e n t r a l i s c a u d a l i s ; A . i n s u l a r i s d o r s a l i s . 33 P a r i e t a l area IV : A. i n s u l a r i s v e n t r a l i s ; Cortex pyriformis anterior; A. amygdala anterior. O c c i p i t a l area I : A. r e t r o s p l e n i a l i s l a t e r a l i s ; A. o c c i p i t a l i s medialis. O c c i p i t a l area II: A. s t r i a t a ; A. o c c i p i t a l i s l a t e r a l i s . O c c i p i t a l a r e a l l l : A. a u d i t o r i a ; A. temporalis; A. i n s u l a r i s v e n t r a l i s . O c c i p i t a l area IV: A. i n s u l a r i s v e n t r a l i s ; A. e n t o r h i n a l i s ; Cortex pyriformis; A. amygdala posterior; C o r t i c a l amygdaloid nucleus. In addition to the aforementioned regions, the hippocampus was dissected free and divided.into two roughly equal parts: the dorsal and the ventral hippocampus. Two homologous c o r t i c a l samples o r i g i n a t i n g from one animal were pooled in a test-tube regardless of hemispheric o r i g i n ( l e f t or right) and were homogenized for 20 sec in 50 volumes of 20 mM T r i s - H C l , 5 mM NaCl, 1 mM EGTA (pH 7.4) at 4°C. The homogenates were then centrifuged for 30 min at 30,000 g (at 4°C) and the clear supernatant was used for CaBP radioimmunoassay (RIA) and t o t a l soluble protein (TSP) determinations. For CaBP RIA, 100 ul ali q o u t s of the supernatant were used in 6-10 f o l d d i l u t i o n s and assayed in t r i p l i c a t e s . The t o t a l 34 soluble protein content of the supernatant was determined in 0 t r i p l i c a t e s of 50 ul samples using the one step BioRad method and porcine gamma globulin as the protein standard. 2.2.2.2. CaBP radioimmunoassay (RIA) The RIA for CaBP used antibody raised in rabbits against the p u r i f i e d 28,000 dalton CaBP is o l a t e d from human cerebella (Baimbridge at a l . , 1982) at a f i n a l d i l u t i o n of 1:25,000. The RIA buffer contained 0.03 M Na-barbital, 6 g/L bovine serum albumin and 1 mM EDTA at pH 8.6. Standards for CaBP, containing 1.62-80 ug of p u r i f i e d protein were prepared in the buffer, using the technique of s e r i a l d i l u t i o n s . Each RIA tube contained 700 pi of buffer, 100 pi of antibody, or 100 ul of buffer (for non-specific binding tubes) and 100 pi standard or samples or buff er (for maximum binding tubes). Tubes were incubated for 6 h at 4°C and 1 2 5 I - l a b e l l e d CaBP was added in 100 ul RIA buffer (usually 9,000-10,000 cpm per tube) and the incubation was continued for another 20 h. Following the incubation, to p r e c i p i t a t e the antibody-bound f r a c t i o n of the la b e l l e d protein, 100 p i of 8 mg/ml of porcine gamma globulin and 700 pi of 25 w/v polyethylene g l y c o l (Carbowax PEG 6000, Fisher S c i e n t i f i c ) was added to each tube. After mechanical a g i t a t i o n of the tubes on a multi-vortexer, p r e c i p i t a t i o n was achieved by centrifugation for 30 min. The supernatant was discarded, the tubes were rinsed off with water and counted in a gamma-counter. The percentage of bound/total was calculated taking in account the corrections 35 for non-specific binding (usually <5%). A standard curve was constructed from the known CaBP concentrations and the CaBP content of unknown samples was determined using interpolation from t h i s curve between the range of 2.5-30 ng CaBP/tube. Tissue CaBP le v e l s were expressed as ng/mg t o t a l soluble protein (TSP). This RIA procedure had a s e n s i t i v i t y of 1-2 ng CaBP and has previously been shown to be s p e c i f i c for rat, mouse, bovine and human CaBP (Baimbridge et a l . , 1982). 2.2.3. Results The CaBP content of various c o r t i c a l areas of rats (n=l2) i s shown on the coronal sections of Figs 2.1.-2.3. The level s of CaBP ranged f rom about 600—1800 ng/mg TSP which are approximately 1/I0 t h the l e v e l of cerebellar CaBP content (e.g., Baimbridge et a l . , 1982). Both the pyriform and entorhinal c o r t i c e s contain the highest CaBP levels in the cerebral cortex of the rat, and represent about 14% of the t o t a l c o r t i c a l CaBP. The least amount of protein was found in the most dorsal o c c i p i t a l areas accounting only for about 4.5% of the c o r t i c a l CaBP content. From the coronal sections, as well as from the histogram representation in F i g . 2.4., i t i s evident that there is a clear dorso-ventral d i s t r i b u t i o n of CaBP in the cerebral cortex. Dorsal regions contain s i g n i f i c a n t l y less protein, while the highest l e v e l s in a given coronal plane are l o c a l i z e d to the most ventral areas. This observation also applies to phylogenetically older c o r t i c a l structures, such as the hippocampus, where the ventral 36 Figure 2.1. CaBP content of the fron t a l region of rat cortex. The section i s taken at 10,550 um from Konig and Klippel (1963) and corresponds to the A. f r o n t a l i s (Krieg, 1946). The number represents the average CaBP content expressed in ng/mg t o t a l soluble protein of 12 animals. 37 38 Figure 2.2. Levels of CaBP in p a r i e t a l c o r t i c a l areas of the rat. This section was taken from 7020 urn of Konig and Klippel (1963) or 1.2 mm anterior to bregma from Pellegrino et a l . (1979). The p a r i e t a l c o r t i c a l d i v i s i o n s as described in the text as 'Parietal I-IV going from dorsal to ventral correspond to areas based on Krieg (1946). The numbers represent average CaBP content in ng/mg TSP (n=12) . 39 40 Figure 2.3. CaBP l e v e l s in the caudal t h i r d of the rat cortex. The section corresponds to 2580 pm of Konig and Klippel (1963) or 3.2 mm posterior to bregma of Pellegrino et a l . (1979). The dorso-ventral c o r t i c a l d i v i s i o n s correspond to 'Occipital areas I.-IV.* indicated in the text. The dorsal and ventral regions of the hippocampal formation are also indicated. The numbers represent averaged CaBP content in ng/mg TSP of 12 animals.  42 Figure 2.4. Summary of CaBP d i s t r i b u t i o n in rat cortex. The various c o r t i c a l areas are indicated on the X-axis in terms of their dorso-ventral and rostro-caudal l o c a t i o n . 'HPC' stands for hippocampal formation. 43 44 hippocampal formation contains about twice the amount of CaBP found in the dorsal part. Immunohistochemical observations (unpublished) indicate that the protein i s mainly confined to the neurons of layers II-IV of the cortex while weaker staining is seen in layers I and V. The amounts of t o t a l soluble protein or tissue wet weight did not vary s i g n i f i c a n t l y from one c o r t i c a l area to another. Therefore, the marked differences in l e v e l s of CaBP in the various c o r t i c a l regions are not due to sample v a r i a b i l i t y of TSP or wet weight. 2.2.4. Discussion The present study extends the findings of Baimbridge et a l . (1982) who measured the l e v e l s of CaBP in various areas of the rat brain. CaBP content of the cerebral cortex was found to be comparable to the rest of the brain although with s i g n i f i c a n t regional varations. The most consistent finding was the dorsal to ventral d i s t r i b u t i o n of the protein. Some of these findings however, may be biased, at least in part, by preponderence of regions in the ventral sections that contain high l e v e l s of CaBP. The hippocampal formation i s a good example, since the ventral part has a s i g n i f i c a n t l y larger volume/volume contribution from the dentate gyrus, which alone contains about three times more CaBP than the CA1 or CA3 areas (Baimbridge et a l . , 1982). In other samples the regional differences may be due to the elevated CaBP content of the c e l l s themselves. For example, 45 only about 4% of the neurons of the entorhinal cortex have been shown to have immunoreactive CaBP (Garcia-Segura et a l . , 1984). Since CaBP in thi s c o r t i c a l region i s approximately 0.2% of the t o t a l soluble protein, the absolute amount of CaBP in the neurons that contain i t may be as high as 5% of TSP. This would be equivalent to a c y t o s o l i c CaBP concentration of about 30-70 uM, and considering 4 mols of C a 2 + bound per molecule of CaBP, the Ca* -buffering capacity would be 0.1-0.3 mmol/L. This value is about a t h i r d of that found in cerebellar Purkinje c e l l s (Baimbridge et a l . , 1982), but i s large enough to make the protein well suited for the possible functional role as an intraneuronal C a 2 + - b u f f e r in the cerebral cortex. It remains to be determined through the use of combined el e c t r o p h y s i o l o g i c a l and immunohistochemical methods, which one of the several l i k e l y functions of CaBP as a buffering/sequestering element applies in the case of c o r t i c a l neurons. 46 2.3. Relationship of hippocampal afterdischarges to l e v e l s of CaBP during development of commissural kindling 2.3.1. Introduction Kindling is one of the most widely used animal models of experimental epilepsy and resembles numerous c h a r a c t e r i s t i c s of the human disease (Racine, 1978; McNamara et a l . , 1980). In order to induce t h i s condition low i n t e n s i t y d a i l y e l e c t r i c a l stimulations are delivered to any part of the limbic system or other c o r t i c a l structures and this paradigm ultimately results in a permanent neurophysiological change leading to a convulsive state (Goddard, 1967; Goddard et a l . , 1969; Racine, 1978). With l i t t l e exception, the seizures and paroxysms r e s u l t i n g from kindling are usually not spontaneous, but show a t y p i c a l progression that involves several stages culminating in f u l l motor convulsions c l a s s i f i e d according to the schema of Racine (1972b) as being 'stage 5'. Although i t has been discovered in rodents, the phenomenon i s not s p e c i f i c to these animals since i t can be induced in a l l species studied thus far sampled from amphibians to mammals. While there has been a concerted e f f o r t to determine the physiological and biochemical c o r r e l a t e s of k i n d l i n g (Kalichman, 1983; McNamara, 1984), these have for the most part concentrated on a l t e r a t i o n s which occur in the period following i t s induction. Thus there i s l i t t l e evidence to suggest whether the observed 47 changes are due either to motor-seizures or to the process of kindling i t s e l f . One notable exception is the loss of hippocampal CaBP, shown e a r l i e r to be l o c a l i z e d to the granule c e l l s of the dentate gyrus ( M i l l e r and Baimbridge, 1983), that precedes the onset of motor seizures, and thus seems l i k e l y to be a true neurochemical correlate of kindling-induced epilepsy (Baimbridge and M i l l e r , 1984). This study however, did not examine the possible r e l a t i o n s h i p between changes in hippocampal CaBP levels and known ele c t r o p h y s i o l o g i c a l correlates that p a r a l l e l the development of kindling. Regardless of the limbic stimulation s i t e , the process of kindling i s generally characterized by electrographic events, termed afterdischarges (AD's), that show a t y p i c a l progression starting with the f i r s t stimulation t r i a l (Racine, 1972a; 1978). The form of the AD's (biphasic spike or spike and wave) i s rather simple at the early stages and t h e i r frequency i s quite low (lo2/sec). Following several stimulations the AD's become more complex and the frequency tends to double or t r i p l e . Although the occurrence of AD's precedes the development of c h a r a c t e r i s t i c motor a c t i v i t y in the stimulated animals, the changes in afterdischarge frequency and duration i s a good c o r r e l a t e of behavioral manifestations during the kindling process. The progressive lengthening of AD's duration i s i n d i c a t i v e of changes in neuronal functions, suggesting sudden recruitment of additional neuronal c i r c u i t r y , and occurs in steps p a r a l l e l i n g the behavioral changes (Burnham, 1975). Racine (1972b) has c l a s s i f i e d into fi v e stages these c h a r a c t e r i s t i c motor a c t i v i t i e s 48 which develop during kindling: 1 . - Mouth and f a c i a l movements; 2.- Head nodding; 3.- Forelimb clonus; 4.- Rearing; 5.- Rearing and f a l l i n g , i . e . , a f u l l motor seizure. Since no previous study has correlated the e l e c t r o - physiological a l t e r a t i o n s to possible neurochemical changes, the present experiments were undertaken to examine the r e l a t i o n s h i p of evoked AD's to l e v e l s of hippocampal CaBP in an attempt to e s t a b l i s h a causal l i n k between the change in Ca 2 + - r e g u l a t i o n and the development of kindling-induced AD's. 2.3.2. Methods Male rats of the Wistar s t r a i n (200-300g) were anesthetized with Nembutal (30 mg/kg i.p.) and c h r o n i c a l l y implanted with bipolar stainless s t e e l stimulating and recording electrodes (MS 303/2-Plastic Products Co.) positioned in the hippocampal commissure (AP from bregma: -1.8 mm; L: 0 mm; V: 4.2 mm below the surface of cortex) and h i l a r region of the dentate gyrus (AP from bregma: -3.3 mm; L: 1.8 mm; V: 3.7 mm below the surface of cortex) respectively. The diameter of the electrodes was 0.2 mm and the stimulating electrodes (placed in the commissure) had a t i p separation of 0.1-0.2 mm. The recording electrode of the dentate gyrus had one pole cut 0.5-1.0 mm shorter than the other to enhance the recording of hippocampal EEG a c t i v i t y . A l l implants were done with the a i d of a stereotaxic apparatus. A ground electrode c o n s i s t i n g of a s t a i n l e s s steel screw mounted in 49 the s k u l l and touching the surface of the cortex was also added before the electrode assembly was embedded in dental cement. Following one week of post-operative recovery, d a i l y kindling stimulation (100 uA, 60 Hz sine-wave, for 1s) was i n i t i a t e d . Each rat was brought d i r e c t l y from i t s home cage into a wooden stimulation/recording box, the respective leads were connected and the kindling stimulus was delivered following 0.5-1 min of baseline EEG recording. The e l e c t r i c a l a c t i v i t y of the hi l u s was amplified and led to an oscilloscope as well as to a Gould chart-recorder to obtain hard copies of the EEG a c t i v i t y . The number and duration of AD's were noted and six groups were defined on the basis of the number of AD's that had been evoked, i . e . , 0, 5, 10, 15, 20, and 'stage 5' motor-seizure. The control group was implanted but not stimulated. Twenty-four hours following the l a s t stimulation, the rats were s a c r i f i c e d and the hippocampi and cerebellum were removed. The electrode placements were v i s u a l l y i d e n t i f i e d in the hippocampi which were then either prepared for i n v i tro e l e c t r o p h y s i o l o g i c a l recordings (Oliver and M i l l e r , 1985) or CaBP radioimmunoassay (RIA) according to the procedures described e a r l i e r . In a l l elect r o p h y s i o l o g i c a l experiments only one • hippocampus was u t i l i z e d from one animal, the other was prepared for RIA. Cerebellar tissue was used as control/reference for the RIA. In the iji v i t r o preparation the e f f i c a c y of synaptic transmission and the con t r i b u t i o n of i n h i b i t o r y processes in the dentate gyrus were assessed using the paired-pulse paradigm 50 (Oliver and M i l l e r , 1985). The r e s u l t s of these experiments are however, not included in the present study. 2.3.3. Results The f i r s t kindling s t i m u l i , delivered to the hippocampal commissures, evoked a brief (approx. 10 s) afterdischarge in forty-one of f o r t y - f i v e animals. Subsequent t r i a l s resulted in the progressive lengthening of the AD's (up to 35-40 sec) with the occurrence of c h a r a c t e r i s t i c behavioral signs (e.g., grooming, rearing and wet-dog shakes; Racine, 1972b). A t y p i c a l record of the electrographic development of h i l a r AD's is shown in F i g . 2.5. (A-D) culminating in the generation of a kindling- induced seizure (Fig. 2.5.E), usually after 25-32 stimulation t r i a l s . Following the AD's, a period of low e l e c t r i c a l a c t i v i t y was observed which lasted for at least 3 min, and i t was usually during t h i s depressed portion of the EEG when wet-dog shakes appeared (Fig. 2.5.C and D). The duration of f u l l motor-seizures was in excess of 1 min in a l l cases observed. The l e v e l s of hippocampal CaBP, as measured by RIA, showed a decline c o r r e l a t e d to the number of recorded AD's (Table 2.1.). A s i g n i f i c a n t loss of CaBP (about 18%) was detected in animals following 10 AD's and the d e c l i n e continued up to 20 AD's when i t reached a maximal reduction of 32% (Fig. 2.6.). This change was not further a l t e r e d by motor-seizures, in fact a s l i g h t but not s i g n i f i c a n t increase was noted compared to the 20 AD's group. 51 Figure 2.5. Development of afterdischarges during commissural kindling. EEG a c t i v i t y recorded in the h i l u s of the dentate gyrus following a 1 sec kindling stimulus (large artefacts at the beginning of each record) delivered to the hippocampal commissures. A, electrographic response recorded from an animal with no AD's after 23 stimulation t r i a l s . The records in B, C, D show the progression of AD's development through the 5fc^, 10fc^ and 20*-̂  stimulation t r i a l s respectively. Note the lengthening of the primary AD's (immediately following the stimulus artefact) and the subsequent depression of e l e c t r i c a l a c t i v i t y . Wet-dog shakes usually occurred during the second, lower frequency AD's and are indicated by closed arrowheads. E, record of a f u l l motor seizure (during the interval between the open arrows) induced on the 3 0 t n stimulation t r i a l l a s t i n g t y p i c a l l y in excess of 1 min. Traces in B, C, D, and E are from the same animal. 29 T a b l e 2.1. Ef f e c t of afterdischarges (AD's) on l e v e l s of hippocampal and cerebellar CaBP. Afterdischarges recorded i n the h i l u s of the dentate gyrus were evoked through commissural stimulation as described i n the text. The l e v e l s of CaBP i n brain samples were,I determined by the use of a s p e c i f i c radioimmunoassay (RIA). Control 0 AD's 5 AD's 10 AD's 20 AD's Seizures HIPPOCAMPUS TSP (ug/mg wet wt) 34.1+.7 33.3+1.1 33.5+.7 34.6+.8^ 35.9+.8^ 36.2+1.3 CaBP (ng/mg TSP) 962+40 936+36 907+29 786+43* 635+31* 653+31* CEREBELLUM TSP (ug/mg wet wt) 30.8+1.6 31.9+1.1 30.6+1.3 32.2+1.8 31.9+.8 30.2+.9 CaBP (ng/mg TSP) 11852+603 11721+554 12547+406 12014+501 12537+540 12024+309 NUMBER OF ANIMALS 6 7 9 8 9 7_ NOTE: numbers represent mean + S.E.M. Denotes s i g n i f i c a n t d i f f e r e n c e from co n t r o l group (p<0.001) — Duncan's multiple range t e s t . 54 Figure 2.6. Relationship between the number of evoked AD's and decline of hippocampal CaBP levels during development of commissural kindling. "Control" refers to animals implanted with electrodes, but not stimulated, while the "0 afterdischarges" group consists of animals with no detectable afterdischarges after several (5-25) kindling stimuli probably due to their high afterdischarge thresholds. Error bars: S.E.M.; n for each point = 12-18. Number of Af terdischarges 56 Since in th i s study levels of CaBP were expressed as frac t i o n of t o t a l soluble proteins (TSP), i t i s important to note that TSP levels were unaltered by kindling. In addition, no s i g n i f i c a n t changes were found in either CaBP br TSP levels of the control/reference cerebellar tissue (Table 2.1.). 2.3.4. Discussion The present study confirms and extends the observations of Baimbridge and M i l l e r (1984) on the progressive decline of hippocampal CaBP during the process of kindling. The findings indicate that alterations in both biochemical and electro- physiological functions occur well before the onset of motor- seizures and further demonstrate that they are dependent on the number of hippocampal afterdischarges. Not only i s there a biochemical a l t e r a t i o n which precedes the onset of a f u l l motor- seizure there i s also an apparent c o r r e l a t i o n between these changes and the occurrence of AD's. It has been established that the loss of hippocampal CaBP is confined to the granule c e l l s of the dentate gyrus (Mil l e r and Baimbridge, 1983) and to be independent of the limbic stimulation s i t e (Baimbridge et a l . , 1985). This reduction in CaBP depends on the number of evoked hippocampal AD's with a half-maximal loss (16%) present by 10 AD's (cf., Baimbridge and M i l l e r , 1984). A s l i g h t decline in the levels of CaBP was already detected after 5 AD's, and although t h i s change was not s i g n i f i c a n t (6%), the absolute loss may be greater. It has to be considered that 57 measurement of CaBP in the whole hippocampus tends to underestimate the changes l o c a l i z e d to the dentate gyrus. While other c e l l types within the hippocampus, most notably the CA1 and CA2 pyramidal neurons, also contain CaBP, their CaBP content i s unaffected by kindling (Miller and Baimbridge, 1983). Thus the granule c e l l s p e c i f i c loss of CaBP even after 5 AD's i s probably larger than 12% when the volume to volume r a t i o of dentate gyrus/CA1+CA2 i s taken into account. The maximum loss (32% in whole hippocampus) was reached after 20 AD's confirming the previous studies of Baimbridge and M i l l e r (1984). Electrophysiological parameters other than afterdischarges are also affected in the process of kindling. It i s known for example, that in the dentate gyrus there is an enhancement of paired pulse i n h i b i t i o n following kindling-induced epilepsy (Tuff et a l . , 1983; Oliver and M i l l e r , 1985) probably r e f l e c t i n g the pronounced i n t e r i c t a l i n h i b i t i o n which is known to occur following kindling-induced seizures (Engel and Ackermann, 1982; F u j i t a et a l . , 1983). This increase in inhi b i t o r y events may serve as a protective mechanism which would retard the development and spread of kindling-induced seizure a c t i v i t y . It i s d i f f i c u l t to ascertain whether any of the changes observed in the present study ( i . e . , loss of CaBP and prolonged AD's) are causally related, and furthermore, whether the loss of CaBP may be responsible for the augmented inhib i t o r y processes of the dentate gyrus (Tuff et a l . , 1983, Oliver and M i l l e r , 1985). As described by the many possible functions of CaBP in the CNS (see Section 2.1.3.), a decline in a putative intraneuronal 58 C a 2 + b u f f e r might a l t e r multiple and perhaps competitive processes leading to both enhanced e x c i t a b i l i t y (AD's development) and increased i n h i b i t o r y events (paired-pulse). The loss of CaBP during kindling may result in an increased neuronal calcium influx analogous to c o r t i c a l structures where the bursting c h a r a c t e r i s t i c of epileptiform a c t i v i t y is p a r a l l e l l e d by a decrease of e x t r a c e l l u l a r C a 2 + (Heinemann et a l . , 1977) and seizures induced by 1 - a l l y l g l y c i n e , where the amount of i n t r a - neuronal C a 2 + becomes elevated ( G r i f f i t h s et a l . , 1982). Recent evidence suggests that t h i s may indeed be the case, at least when the kindling stimulus i s delivered d i r e c t l y to CA1 neurons (Wadman et a l . , 1985). Furthermore, the kindling model of epilepsy is also characterized by an altered calcium-homeostasis (see Section 3.3.), although t o t a l hippocampal C a 2 + remains constant (see Section 3.1). With the loss of CaBP, the elevated lev e l s of i n h i b i t i o n may be due to a sustained a c t i v a t i o n of the 9 KCa "hich would also promote a decrease in f i r i n g rate of the neurons to counteract the high frequency kindling stimulation. If the enhancement of inhibitory events of the dentate granule c e l l s during kindling proves to be Ca* -dependent, a link between the loss of a C a 2 +-buffer (CaBP) and the electrophysiological a l t e r a t i o n s may be revealed. A l t e r n a t i v e l y , the f a l l in CaBP and the change in Ca -regulation may be coincidental with the enhancement of paired-pulse i n h i b i t i o n . In this case the events could be part of global neurochemical and electrophysiological a l t e r a t i o n s c h a r a c t e r i s t i c of the kindling-induced epilepsy. 59 2.4. D i s t r i b u t i o n of CaBP in c o r t i c a l areas of the e p i l e p t i c E l mouse 2.4.1. Introduction It has been acknowledged that genetic factors, i f not solely responsible for epileptiform a c t i v i t y , may s i g n i f i c a n t l y contribute to the generation of seizures. P a r t i c u l a r l y in human epilepsy the importance of genetic determinants seems evident, as stressed in a review by Newmark and Penry (1980). In several species including the chicken, mouse, g e r b i l , rat, dog and baboon at least one genetic model of epilepsy i s available and numerous investigators have provided evidence for s p e c i f i c neurochemical changes associated with these models (cf., Jobe and Laird, 1981; Laird et a l . , 1984). Most of the animals exhibit seizures following presentation, in some cases repeated over several weeks, of one or more environmental factors such as postural, auditory or photic s t i m u l i . The genetic influences may be d i r e c t l y responsible for the enhanced s u s c e p t i b i l i t y for seizures. A l t e r n a t i v e l y , they may cause permanent changes in the CNS with resulting effects whereby neuronal tissue w i l l more e a s i l y contribute to epileptogenesis when adequately stimulated. In view of the role of C a 2 + in neuronal e x c i t a b i l i t y i t i s feasible that some of these permanent, genetically determined.alterations may involve some calcium-dependent mechanism(s). This seems to be the case with 60 the seizure prone stra i n of mice (DBA/2N) where a deficiency of the Ca 2 +-ATPase enzyme has been demonstrated (Rosenblatt et a l . , 1976). The e p i l e p t i c (El) stra i n of mice, as opposed to i t s more widely used audiogenic counterpart (the DBA/2J s t r a i n ) , is characterized by induction of seizures through vestibular stimulation. It has been developed in Japan and reported as the 'ep' mouse (Imaizumi et a l . , 1959), and later registered as the 'El s t r a i n ' (Imaizumi et a l . , 1964). The E l mouse has therefore been mainly studied by Japanese investigators who have shown that i t s induced seizures are true electrographic events (Suzuki, 1976; Suzuki and Nakamoto, 1977). The documented neurochemical findings include a lower than normal l e v e l of brain norepinephrine (Hiramatsu et a l . , 1976), and abnormally elevated acetylcholine, GABA and taurine concentrations (Naruse et a l . , 1960; Kurokawa et a l . , 1966; Iwata et a l . , 1979). In addition, a recent study using the 2-deoxyglucose technique, to detect metabolic a l t e r a t i o n s , has established the hippocampus as the presumed e p i l e p t i c focus (Suzuki et a l . , 1983). The involvement of the hippocampal formation in the generation of seizures, as i t i s in the kindling model of epilepsy (see Section 2.3.), could mean that s i m i l a r i t i e s may exist between the two models of experimental epilepsy regarding epileptiform phenomena. Some of these mechanisms may be related to altered regulation of Ca^ in certain CNS structures. The c o r t i c a l d i s t r i b u t i o n of CaBP was therefore examined in the e p i l e p t i c E l str a i n and in a control, non-seizure prone s t r a i n 61 (Swiss Albino CF-1) to detect possible genetic differences. Furthermore, sex differences in the levels of the protein were analyzed and the effect of seizures on c o r t i c a l CaBP content was determined using seizing and non-seizing E l mice. 2.4.2. Methods The e p i l e p t i c E l s t r a i n was obtained from Dr. J. Suzuki of the Psychiatric Research Institute of Tokyo and generations El-F-75 and El-F-76 were bred at the U.B.C. Department of Physiology's animal care f a c i l i t i e s . The animals were housed in p l a s t i c boxes covered with a mesh and were cleaned once a week to avoid excessive vestibular stimulation upon moving of the cages. Weight and age matched mice of the Swiss Albino s t r a i n (CF-1) were used as controls. The weights of a l l animals used in the study ranged from 20 to 38 g. The seizures of the E l strain may be induced by vestibular stimulation of various forms. Among the several stimulation paradigms including pendulum type swinging, repeated v e r t i c a l or horizontal movements, and tossing up of the animal, the most ef f e c t i v e method is a 15-20 cm throw in the a i r , whereafter the mice land on their paws on a soft surface (Kurokawa et a l . , 1966). In the present study the animals were stimulated mainly through t h i s l a t t e r procedure, although v e r t i c a l swinging was also used and found to be just as e f f e c t i v e . Each stimulation t r i a l consisted in careful removal of the animal from i t s cage and 30 consecutive throws or v e r t i c a l 62 movements. The f i r s t stimulation started when the animals reached the age of 3 weeks (Suzuki and Nakamoto, 1977) and continued every 4-5 days u n t i l the desired number of seizures was obtained. If the animal had a convulsion, no further stimuli were delivered during the session. Control E l mice did not undergo stimulation t r i a l s and their handling was minimal to avoid excessive vestibular input. Animals of both sexes were s a c r i f i c e d 2-4 days following the l a s t seizure together with non-stimulated counterparts and mice of the control s t r a i n . The brains were removed and dissected into the approximate c o r t i c a l areas of F i g . 2.7. following separation of the cerebellum, hippocampal formation and caudate nucleus. The landmark for distinguishing between p a r i e t a l and o c c i p i t a l areas was the artery of the central sulcus. The tissue was prepared for radioimmunoassay (RIA) as described in Section 2.1.2.2. with pooling of right and l e f t hemispheric samples. The procedure of RIA for CaBP was similar to that desribed for rat samples (Section 2.2.2.1.) and the CaBP content was expressed as ng/mg TSP ( t o t a l soluble protein). S t a t i s t i c a l analysis was done on the pooled data from several experiments and consisted of a two-factor ANOVA. One analysis involved the combined e f f e c t of strain and sex on levels of CaBP and compared the c o n t r o l st r a i n to non-seizing E l . The other comprized the s e i z i n g (animals exhibiting more than 7 f u l l motor seizures) and the non-seizing El group to find possible alterations due to the sex of animals and/or seizures. 6 3 Figure 2.7. C o r t i c a l areas used for determination of CaBP levels in mice. The drawings are diagramatic representations from Slotnick and Leonard (1975) with the numbers marked by an asterisk indicating mm from bregma. The abbreviations for the various c o r t i c a l areas as used in t h i s and subsequent figures (2.8 and 2.9.) are indicated on the lower part of the figure. HIP- Hippocampus DP - Dorsal Parietal Cortex VT - Ventral Temporal Cortex DO - Dorsal Occipital Cortex VO-Ventral Occipital Cortex 65 2.4.3. Results Following repeated vestibular stimulation, mice of the E l s t r a i n exhibited strong tonic or clonic convulsions mostly preceded by squeaks, as described by Kurokawa et a l . (1966). These seizures usually lasted for about 15-30 s and were t y p i c a l l y evoked between the and the 9fc^ stimulation sessions. Although not frequently, some of the previously stimulated animals exhibited spontaneous seizures following their removal from the common housing cage, probably due to the inadvertent mild vestibular stimulation. Histograms representing the c o r t i c a l d i s t r i b u t i o n of CaBP in control and E l mice are shown in Figs. 2.8. and 2.9., for males and females respectively. As has been shown for the c o r t i c a l d i s t r i b u t i o n of CaBP in rats (see Section 2.2.3.), there was a clear dorso-ventral d i s t r i b u t i o n of the protein with higher l e v e l s confined to ventral c o r t i c a l regions. The cumulative data and the s t a t i s t i c a l analysis are summarized in Tables 2.2. and 2.3 respectively. E f f e c t of s t r a i n Certain c o r t i c a l areas of the E l st r a i n were found to contain lower levels of CaBP than the control CF-1 s t r a i n . The hippocampi of male El mice have on the average 13% less CaBP than mice of the CF-1 st r a i n while female Els have 8% lower hippocampal CaBP levels than t h e i r CF-1 counterparts. The st r a i n 66 differences are more pronounced in the dorsal o c c i p i t a l cortex where the respective changes are 14% and 15%. In other c o r t i c a l areas the CaBP levels were quite comparable and no s i g n i f i c a n t differences could be detected with the two-way ANOVA. Eff e c t of sex S i g n i f i c a n t l y lower CaBP level s were detected in females of both str a i n s in the hippocampal formation (13% in CF-1 and 8% in El) and the ventral o c c i p i t a l c o r t i c a l areas (6% in CF-1 and 18% in E l ) . The CaBP content of the dorsal p a r i e t a l cortex in female mice was found to be elevated compared to males (19% in CF-1 and 17% in E l ) . Differences due to sex appeared to be s i g n i f i c a n t in the E l s t r a i n even after seizures have been induced. The seizing female E l mice retained the c h a r a c t e r i s t i c changes of the ventral o c c i p i t a l and dorsal p a r i e t a l c o r t i c e s , but hippocampal CaBP levels only showed the e f f e c t of seizures. E f f e c t of seizures in the E l s t r a i n The induction of seizures decreased the l e v e l s of CaBP in the E l s t r a i n , p a r t i c u l a r l y in the hippocampus and the ventral parietal/temporal c o r t i c e s . The largest change was observed in the ventral temporal cortex of male E l mice which contained 12% less CaBP than the corresponding c o r t i c a l region of their non- stimulated counterparts. A l l c o r t i c a l and cerebellar samples examined had a comparable TSP content and the r a t i o of TSP/tissue wet weight.was found to be constant, indicating that the observed al t e r a t i o n s in 67 Figure 2.8. Levels of CaBP in male mice. The c o r t i c a l regions 2.7. Error bars indicate S.E.M. represent the number of samples. c o r t i c a l areas of control and E l are abbreviated as shown in F i g . while the numbers in parentheses 68 00 f - £ c n c QQ O O 1300 n 1200 I 100 - 000 - 900 - 800 - 7 0 0 0 (3 (9) (14) (18) (19) 7/ (14) (26) T (ID (10)08) I 3 (ii) I (28) I (12) I (10) (18) I (18) II HIP DP VT DO V 0 (T) Control strain (Swiss) E L strain (no seizures) Seizing EL(>7seizures) 69 Figure 2.9. Levels of CaBP in c o r t i c a l areas of control and E l female mice. The c o r t i c a l regions are abbreviated as shown in Fig . 2.7. Error bars indicate S.E.M. while the numbers in parentheses represent the number of samples. 70 Q_ CO cn E CD o O 1300 1200 - 0 0 - 1000 - 9 0 0 - 8 0 0 - 7 0 0 - (17) I (9) I (12) ^(19) I 0 www (9) (9) lx (14) I (12) -^(20) (9) (ID (9) (9) 11 (10) (9!Z I HIP DP VT DO V0 ©Cont ro l strain (Swiss) E L strain (no seizures) Seizing EL(>7seizures) 71 Table 2.2. Levels of CaBP i n various c o r t i c a l areas of con t r o l and e p i l e p t i c (El) mice. CaBP was measured according to the RIA described i n the text. C o r t i c a l areas from mice of the control s t r a i n CF-1 (Swiss) and the e p i l e p t i c s t r a i n E l are- shown on F i g . 2.7. The cont r o l E l group ( E l n s ) received no stimulations and exhibited no seizures, whereas the se i z i n g E l ( E l g ) group had at least 7 f u l l motor seizures each. Number of samples = (n). C o r t i c a l Area CaBP content (ng/mg TSP) Swiss MALES E 1 n s E1 S Swiss FEMALES E i n s Els CEREBELLUM mean 12,506 13,233 13,036 13,322 12,361 12,660 S.D. 1,099 1,290 958 971 1,055 1,220 (n) (12) (10) (18) (17) (12) (20) HIPPOCAMPUS : mean 1,134 999 912 993 920 881 S.D. 184 119 83 81 120 110 :(n) (18) (14) (26) (17) (12) (19) DORSAL PARIETAL CTX. mean 859 802 843 1,025 939 910 S.D. 156 190 97 68 110 65 (n) (11) (10) (18) (9) <9) (9) VENTRAL TEMPORAL CTX. mean 1,141 1,157 1,018 1,292 1,101 1,035 S.D. 170 161 100 107 165 224 (n) (19) (14) (28) (14) (12) (20) DORSAL OCCIPITAL CTX. mean 918 792 772 917 787 730 S.D. 77 108 77 119 89 78 (n) (12) (10) (18) (11) (9) (9) VENTRAL OCCIPITAL CTX mean 1,139 1,225 1,160 1,082 1,010 1,032 S.D. 70 153 89 103 78 95 (n) ( I D (9) (18) (9) (9) (10) 72 Table 2.3. Two-way an a l y s i s of variance (ANOVA) of the effects of s t r a i n , sex and seizures on the c o r t i c a l l e v e l s of CaBP i n control and E l mice. The two-way ANOVA was done on the data presented i n Table 2.2. and involved two comparisons: 1.) between the control group (Swiss) and the non-seizing E l group ( E l n s ) to determine the e f f e c t s of sex and s t r a i n , and 2.) between the seizing ( E l g ) and non-seizing E l group to examine the e f f e c t s of gender and seizures on the c o r t i c a l CaBP l e v e l s . The e f f e c t was considered to be non- s i g n i f i c a n t (N.S.) i f p>0.01. The respective c o r t i c a l areas are represented i n F i g . 2.7. C o r t i c a l Area 1. Swiss vs. El. ns 2. E l n s vs. E l s Sex S t r a i n Interaction Sex Seizure Interaction s e x — s t r a i n sex-seizure CEREBELLUM HIPPOCAMPUS DORSAL PARIETAL CTX VENTRAL TEMPORAL CTX DORSAL OCCIPITAL CTX N.S. p<0.005 p<0.005 N.S. N.S. p<0.005 p<0.005 N.S. N.S. N.S. N.S. p<0.025 N.S. p<0.001 N.S. VENTRAL OCCIPITAL CTX p<0.001 N.S. p<0.001 N.S. N.S. N.S. N.S. p<0.025 N.S. p<0.025 N.S. N.S. N.S. p<0.025 N.S. N.S. N.S. N.S. p<0.001 N.S. N.S. 73 terms of CaBP levels were not due to abnormally elevated c e l l u l a r soluble proteins. In addition, the s l i g h t changes in cerebellar CaBP leve l s were found not to be s i g n i f i c a n t except for an interaction between sex and s t r a i n . 2.3.4. Discussion Genetic models of epilepsy have s i g n i f i c a n t l y contributed to our current understanding of neurochemical and neurotransmitter a l t e r a t i o n s in seizure disorders (Jobe and Laird, 1981; Laird et a l . , 1984). The present study confirms the results of numerous Japanese investigators who have demonstrated that the E l st r a i n of mice i s a r e l i a b l e model of experimental epilespy in which seizures may be triggered through vestibular stimulation in a highly reproducible fashion. Since repeated stimuli (4-9) are necessary to evoke f u l l motor seizures, this model may be considered as being related to kindling-induced epilepsy where successive e l e c t r i c a l stimuli are applied in order to induce convulsions (Goddard et a l . , 1969). Therefore, analogous to kindling, some l a s t i n g alterations must take place during the time of stimulation that ultimately result in the abnormal functioning of nerve c e l l s . In contrast to kindling which may be induced in any s t r a i n of a given species, the E l mice should have some additional neuronal determinants that make i t prone to abberrant neuronal a c t i v i t y . It has been reported that whole brain acetylcholine and GABA l e v e l s of the E l mice are s i g n i f i c a n t l y higher than those of 74 control strains while excitatory amino acids (glutamate and aspartate) are reduced (Kurokawa et a l . , 1966). This may r e f l e c t a genetic factor that i s part of a global defence mechanism against, rather than a direct cause of the seizure disorders. This hypothesis i s further supported by the elevated l e v e l s of taurine in E l mice and the fact that taurine injections raised their convulsion threshold (Iwata et a l . , 1979). However, some la s t i n g neurochemical a l t e r a t i o n i s probably reponsible for the propensity for seizures in the El s t r a i n since these mice have a s i g n i f i c a n t l y lowered metrazol- and e l e c t r i c a l stimulation- induced seizure threshold (Kurokawa et a l . , 1966). Albeit no detailed electrophysiological investigation is available concerning the E l s t r a i n , i t may very well be that inh i b i t o r y events are enhanced in various c o r t i c a l areas due to larger than normal GABA l e v e l s . By analogy, supranormal inhib i t o r y events, although not neccessarily GABA-mediated, have been described in the dentate gyrus following kindling-induced epilepsy (Tuff et a l . , 1983; Oliver and M i l l e r , 1985) which may be due to the selective loss of CaBP from the granule c e l l s of t h i s structure (Miller and Baimbridge, 1983). The decreased levels of CaBP, as measured by RIA, in certain c o r t i c a l structures of the E l mice may thus relate to the variety of neurochemical findings in this s t r a i n (Naruse et a l . , 1960; Kurokawa et a l . , 1966; Hiramatsu and Mori, 1977; Iwata et a l . , 1979) or a l t e r n a t i v e l y , may r e f l e c t an a l t e r a t i o n in c o r t i c a l C a 2 + - r e g u l a t i o n of the E l s t r a i n . Since no detectable morphological a l t e r a t i o n s are noted in neurons of the E l mice 75 under l i g h t - or electron microscopic investigation (Kurokawa et a l . , 1966), the loss of CaBP i s not l i k e l y to be a result of neuronal degeneration processes. If mechanisms underlying C a 2 + homeostasis in certain c o r t i c a l structures of the E l mice are disturbed, i t i s expected that s i g n i f i c a n t changes in neuronal e x c i t a b i l i t y would p a r a l l e l these a l t e r a t i o n s . The hippocampal formation i s the only c o r t i c a l area where s i g n i f i c a n t st r a i n and seizure related differences were found in terms of CaBP l e v e l s . It is this stucture that presumably constitutes the focus of paroxysmal discharges in the e p i l e p t i c st r a i n as measured by l o c a l glucose u t i l i z a t i o n (Suzuki et a l . , 1983). In addition to the hippocampus, the ventral parietal/temporal cortex showed a s i g n i f i c a n t decline in l e v e l s of CaBP following seizures. This area has previously been associated with the electrographic focus of aberrant discharge patterns (Suzuki, 1976; Suzuki et a l . , 1977). It is not known what the s i g n i f i c a n t s t r a i n related a l t e r a t i o n in CaBP content of dorsal o c c i p i t a l c o r t i c a l areas may be a r e f l e c t i o n of. The sex related differences in both control and E l strains are also unclear. No study i s available with regard to the effect of sex hormones on brain CaBP l e v e l s . However, administration of e s t r a d i o l markedly lowers seizure threshold in male or ovarectomized female rats (Millichap, 1969), indicating that i t has a profound e f f e c t on seizure generation in neurons. Whether the s i g n i f i c a n t l y lower levels of CaBP in the hippocampi and ventral o c c i p i t a l c o r tices 76 of female mice represent the effects of sex hormones remains to be determined. Further immunohistochemical studies are necessary to ascertain the precise anatomical l o c a l i z a t i o n and confinement of the CaBP loss observed in the E l mice both with or without seizures. In addition, electrophysiological investigations should determine the nature of e x c i t a b i l i t y changes that occur in th i s valuable model of genetic epilepsy. 77 C H A P T E R I I I . MEASUREMENT OF HIPPOCAMPAL Ca2+-HOMEOSTASIS There are a considerable number of neuropathological conditions where abnormalities in regulation of i n t r a c e l l u l a r Ca could explain many of the altered functions of nerve c e l l s . Several methods are available for detection of Ca 2 + a c t i v i t i e s in various tissues (Borle, 1981a; Blinks et a l . , 1982; Campbell, 1983; TSien, 1983b). These methods include measurement of t o t a l Ca^ content usually by atomic absorption spectrophotometry (AAS), chelating agents or colorimetric t i t r a t i o n ; measurement of free i n t r a c e l l u l a r C a 2 + by the use of metallochromic or o p t i c a l indicators; or determination of ionic compartmentalization by kinetic analysis of radioactive tracer fluxes; and more recently, measurement of C a 2 + ionic a c t i v i t y (either extra- or i n t r a - c e l l u l a r l y ) with the aid of ion-sensitive microelectrodes (ISMs). Although the large arsenal of methods for detection and measurement of C a 2 + would suggest that determination of i t s ionic a c t i v i t i e s i s a r e l a t i v e l y easy task, in practice this is rarely the case. P a r t i c u l a r l y in the mammalian CNS, where one has to deal with . aggregates of small, sometimes not e a s i l y accessible 78 nerve c e l l s , there are only a few applicable procedures for assessement of Ca 2 +-homeostasis. So far AAS and p r e c i p i t a t i v e techniques (e.g., oxalate-pyroantimonate) have been proven successful for determination of t o t a l Ca 2 + while ionic flux measurements have been d i r e c t l y estimated using 4^Ca or by p + monitoring Ca* -dependent e l e c t r i c a l events. The use of ISMs has generally been r e s t r i c t e d to the mere estimation of changes in i n t r a c e l l u l a r C a 2 + alterations by monitoring e x t r a c e l l u l a r C a 2 + a c t i v i t i e s . The present study examines calcium regulation in the hippocampal formation under normal and various experimental and neuropathological conditions using two dir e c t methods of measurement: the AAS procedure for t o t a l C a 2 + determination, and the kinetic analysis of 4 5 C a uptake curves for investigation of i n t r a c e l l u l a r Ca* d i s t r i b u t i o n and compartmentalization. 79 3.1. Measurement of t o t a l hippocampal Caz and Znz using atomic absorption spectrophotometry (AAS) 3.1.1. Introduction Atomic absorption spectrophotometry (AAS) i s one of the most sensitive and s p e c i f i c methods available for t o t a l c e l l or tissue 2 + Ca^ estimates. The technique depends on the excitation of electrons in free atoms that are obtained by vaporization of compounds in a high temperature flame. The atoms in the flame are excited by the absorption of l i g h t at a certain wavelength (e.g., for C a 2 + at 422.7 nm) after the sample has been atomized in an air-acetylene flame (2570 K) or in nitrous oxide-acetylene flame (3230'K) ( W i l l i s , 1963). The presence of Ca 2 +-binding anions, such as phosphate reduces atomization and therefore decreases the s e n s i t i v i t y of the method for calcium. However, the interference caused by these calcium ligands can be eliminated by the addition of La3"1" which produces a maximum s e n s i t i v i t y for assay of C a 2 + (Pybus et a l . , 1970). Since the preparation of tissues for AAS measurements i s usually similar . regardless of the nature of the ion involved, tissue content of a variety of other ions may be determined in conjuction with t o t a l C a 2 + . Of p a r t i c u l a r relevance to the present study was the determination of hippocampal Z n 2 + l e v e l s . The presence of zinc in the hippocampal formation and in p a r t i c u l a r the dentate granule c e l l — mossy fiber system has 80 been demonstrated in a number of histochemical studies (Timm, 1958; Crawford and Connor, 1972; Haug, 1974; Danscher, 198.1; Stengaard-Pedersen et a l . , 1983), but the role of this t r a n s i t i o n metal in the CNS remains poorly understood (Crawford, 1983). Electrophysiological investigations have shown some alterations in the function of the mossy fiber input to CA3 pyramidal c e l l s using H2S chelating techniques (von Euler, 1962), while l i t t l e change was detected with the Zn 2 +-chelator diethyldithiocarbamate (Danscher et a l . , 1975). More recent studies have suggested that high lev e l s of zinc may be responsible for convulsive behavior in cert a i n experimental models of epilepsy (Chung and Johnson, 1983; Pei et a l . , 1983), and have found that iontophoresis of Zn 2 + enhances the f i r i n g rate of c o r t i c a l neurons (Wright, 1984). In view of these findings and the fact that s i g n i f i c a n t biochemical alterations take place in the dentate granule c e l l -- mossy fiber system during kindling-induced epilepsy (Miller and Baimbridge, 1983; Baimbridge and M i l l e r , 1984; Baimbridge et a l . , 1985; also see Section 2.2.), i . e . , the s p e c i f i c loss of a calcium-binding protein (CaBP), the present study was undertaken 9 j . 9 j _ to investigate possible changes in hippocampal Zn* and Ca l e v e l s following kindling. Alterations in the t o t a l tissue content of these metals could be responsible for some of the aberrant discharge properties of dentate gyrus granule c e l l s during and following kindling-induced epilepsy (see Section 2 • 2 • 3 • ) • 81 3.1.2. Methods Adult male Wistar rats were kindled through the hippocampal commissures according to the procedures described in Section 2.3.2., with the exception that no recording electrodes were implanted to monitor afterdischarge a c t i v i t y . Following recovery from surgery the animals received d a i l y kindling stimuli (100 uA, 60 Hz for 1 s) or sham stimulation (controls). Animals were divided into three experimental groups: a) controls (n=4); b) p a r t i a l l y kindled , i . e . , 20 stimulation t r i a l s but no evidence of motor seizures (n=4); c) f u l l y kindled, i . e . , 5-10 motor seizures evoked through 30-40 stimulation t r i a l s (n=6). The day following the last stimulation the animals were decapitated, the brains were removed using p l a s t i c instruments washed in HC1 to avoid ionic contamination and then placed in HCl-rinsed polystyrene v i a l s containing 4% formaldehyde solution (Fjerdingstad et a l . , 1974; Kemp and Dansher, 1979). Analysis has shown that the formaldehyde solution was devoid (<0.01 part per mi l l i o n (ppm)) of Z n 2 + and C a 2 + contamination both before and after storage of the brain samples (see also Fjerdingstad et a l . , 1974). Following a 3-day storage period at 4°C the hippocampi were dissected free and dried overnight at 110°C. The samples were then weighed and dissolved in 1 ml of concentrated HNO3 (Baker Analyzed Reagent) upon gradual heating. Further d i l u t i o n s were made using b i d i s t i l l e d water which contained 10 mmol/1 LaCl3 and 50 mmol/1 HCl (Baker Analyzed Reagent) for samples prepared 82 for Ca 2 + analysis (Pybus et a l . , 1970). Standard solutions containing appropriate concentrations of zinc and calcium (Aldrich) were prepared in HNO3 diluted to the same extent as the tissue samples, with 10 mM LaCl3 and 50 mM HC1 added to the C a 2 + standards. For detection of the metals a Jarrel-Ash 280 atomic absorption spectrophotometer (with air-acetylene flame) was used connected to a Sargent recorder. Readings were done at 422.7 and 213.9 nm, the p r i n c i p a l absorption l i n e s for C a 2 + and Z n 2 + respectively. The standard curves were linear in the range of 0.01-0.3 ppm for Z n 2 + and 0.1-10.0 ppm for C a 2 + . Each sample was measured three times and the metal concentration was interpolated using linear regression from the standard curve. Although measurements were made on parts per million/dry weight basis, the values were extrapolated to wet weight, considering dry weight to be 22% of t o t a l tissue weight (Chung and Johnson, 1983). 3.1.3. Results Calcium and zinc content in whole hippocampi of control and commissural-kindled rats is presented in Table 3.1. The dry weight of tissue samples was considerably constant in a l l three experimental groups and was not d i f f e r e n t from values published in previous reports (e.g., Frederickson et a l . , 1982). The amount of calcium in control hippocampi (1.81 mmol/kg wet weight extrapolated) i s somewhat lower than found by other investigators 83 Table 3.1. Calc ium and z i n c i n the hippocampal formation of c o n t r o l and commissural- k i n d l e d ra t s as measured by atomic absorpt ion spectrophotometry. Commissural-kindled Sham st imulated No se izures 5-10 Seizures (n=8) (n=8) (n=12) Whole hippocampus d r y weight (mg) 12 .15 + 1.0 12.85 + 1 .3 12.46 + 1.3 Hippocampal Ca , ppm/dry weight 329 .80 + 39.6 357.70 + 32 .4 356.70 + 72.1 mmol/kg wet weight 1 .81 + 0.22 1.96 + 0 .17 1.96 + 0.39 Hippocampal Zn, * * ppm/dry weight 88 .30 + 4.6 92.90 + 5 .9 101.60 + 7.1 umol/kg wet weight 297 .00 + 15.5 312.70 + 19 .9 342.00 + 23.9** NOTE: Numbers represent means + S . D . , n = number of hippocampi. E x t r a p o l a t e d from parts per m i l l i o n dry weight using wet weight dry weight = 100/22 (Chung and Johnson, 1983) and the atomic weights: Ca=40.08, Zn=65.37. S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (p<0.001) and p a r t i a l l y k ind led group (p<0.05) — Duncan's m u l t i p l e range t e s t . 84 (cf., Borle, 1981a) but is similar to that obtained by Kemp and Danscher (1979) using X-ray emission spectroscopy in tissue fixed with formaldehyde. Commissural kindling tended to elevate t o t a l C a 2 + of the hippocampus, but th i s 8% change was found not to be s i g n i f i c a n t l y d i f f e r e n t from controls. The values for basal Zn 2 + content of the hippocampal formation are in good agreement with other studies using various methods of measurement (Kemp and Danscher, 1979; Frederickson et a l . , 1982; Baraldi et a l . , 1983; Chung and Johnson, 1983; Sato et a l . , 1984). In contrast with C a 2 + , zinc l e v e l s in the hippocampi of commissural-kindled rats with more than five motor seizures showed a marked 15.1% increase over basal Z n 2 + concentrations and a 9.4% enhancement compared with p a r t i a l l y kindled animals. In view of the fact that hippocampal zinc is mainly confined to the dentate granule c e l l -- mossy fiber system, which only accounts for a fraction of t o t a l hippocampal dry weight, the magnitude of the changes in the p r i n c i p a l Zn 2 +-containing elements may be as large as 40-50%. 3.1.4. Discussion Several studies have summarized neurochemical and neuro- transmitter function a l t e r a t i o n s produced by kindling-induced epilepsy (McNamara et a l . , 1980; Kalichman, 1982; Peterson and Albertson, 1982; McNamara, 1984). The finding that a neuron- s p e c i f i c CaBP i s s e l e c t i v e l y decreased in the granule c e l l s of the dentate gyrus (Miller and Baimbridge, 1983) points toward 85 s p e c i f i c changes regarding calcium regulation in these c e l l s . However, measurements of t o t a l hippocampal calcium did not reveal any major changes in i t s tissue content, suggesting that i f C a 2 + - homeostasis is altered at a l l following kindling-induced epilepsy, this change has to occur via a d i s t i n c t mechanism involving r e d i s t r i b u t i o n of the ion rather than an absolute change in i t s concentration. The increase in zinc content may or may not be linked to an altered hippocampal calcium regulation. Enhanced levels of zinc may be epileptogenic (Chung and Johnson, 1983; Pei et a l . , 1983) but there i s no indication about the exact mechanism(s) whereby Zn* may trigger these pathophysiological changes. Recent studies have shown that granule c e l l s of the dentate gyrus take up zinc and release the cation upon stimulation (Assaf and Chung, 1984; Howell et a l . , 1984). Therefore, i t is reasonable to assume that most of the changes presented in this study are confined to the granule c e l l s and their mossy fi b e r s . Because zinc participates in the regulation of a wide variety of enzymes (Prasad, 1979; Ebadi et a l . , 1981; Wolf and Schmidt, 1982), i t i s d i f f i c u l t to speculate how an elevated zinc concentration may influence biochemical events and ultimately synaptic transmission in the hippocampus. For example, chronic zinc deficiency a l t e r s the function of the mossy fibers while CA3 pyramidal c e l l s are unaffected (Hesse, 1979). Furthermore, a decrease in brain zinc during experimental hepatic encephalopathy is p a r a l l e l e d by a decrease in GABA-receptors (Baraldi et a l . , 1983). Whether the converse is true for an increase in zinc 86 concentration remains to be determined. The synthesis of CaBP i t s e l f may in turn be impaired by larger than normal amounts of Zn 2 +, at least this seems to be the case in the ga s t r o - i n t e s t i n a l tract where Z n 2 + has been shown to i n h i b i t the synthesis of vitamin D-dependent CaBP (Corradino and Fullmer, 1980). The c o l o c a l i z a t i o n of zinc with peptide neurotransmitters in the hippocampal formation (Stengaard-Pedersen et a l . , 1983) may indicate a similar function of the cation to that shown in pancreatic c e l l s , i . e . , the presence of Z n 2 + protects i n s u l i n from proteolytic cleavage (Emdin et a l . , 1980). The progressive increase in hippocampal' zinc content during commissural kindling, probably due to an enhanced uptake of the cation from the CSF, i s a novel correlate of kindling-induced epilepsy. If zinc i s released from the mossy fibers following stimulation (Assaf and Chung, 1984), then i t s elevated levels during kindling may cause abnormal discharges of CA3 pyramidal c e l l s , analogous to the effects seen in c o r t i c a l neurons (Wright, 1984). On the other hand, out of the several possible actions of th i s t r a n s i t i o n metal, the manner in which i t may play a role a l t e r i n g the electrophysiology (Tuff et a l . , 1983; Oliver and M i l l e r , 1985) or the neurochemical parameters (Mil l e r and Baimbridge, 1983) of dentate granule c e l l s during and following kindling, remains yet to be determined. 87 3.2. Measurement of hippocampal exchangeable calcium using kinetic analysis of ^ C a uptake curves 3.2.1. Introduction In the majority of physiological systems the l e v e l of i n t r a c e l l u l a r calcium i s maintained within s t r i c t l i m i t s by an energy dependent membrane pump linked to the Ca 2 +/Mg 2 +-ATPase, the Na +/Ca 2 + exchange system and a variety of i n t r a c e l l u l a r Ca 2 + chelating and buffering mechanisms consisting primarily of mitochondria, endoplasmic reticulum and calcium-binding proteins (Borle, 1981a). Alterations in any one of these processes w i l l have s i g n i f i c a n t effects on the l e v e l of i n t r a c e l l u l a r Ca which in turn may lead to changes in the physiological functioning of the c e l l s . The measurement of i n t r a c e l l u l a r Ca 2 + concentrations has therefore been an important objective of many investigators. Although there are a number of well-documented methods of measurement, the l i m i t a t i o n s of their a p p l i c a b i l i t y to aggregates of small nerve c e l l s , as i s the case in the mammalian nervous system, are obvious (cf. Blinks et a l . , 1982). Calcium-sensitive microelectrodes that impale molluscan neurons with . ease have rarely been used to measure intraneuronal Ca* concentrations of mammalian CNS neurons (Morris et a l . , 1983). In addition, the amount of Ca 2 + d i s t r i b u t e d into the several sequestering and buffering systems of these nerve c e l l s cannot be determined using t h i s approach. 88 A r e l a t i v e l y simple method that provides some insight into the compartmentalization of C a 2 + and i t s buffering i s the kinetic analysis of 4^Ca uptake or efflux curves. Provided the system i s at a steady state, the tracer is introduced into the extra- c e l l u l a r compartment and i t s d i s t r i b u t i o n is monitored during a period of time in the c e l l u l a r network under investigation. The uptake curve i s f i t t e d by a mathematical function and according to the p r i n c i p l e s of compartmental analysis (Robertson, 1957; Sheppard, 1962; Jacquez, 1972) the exchange rates of the cation and i t s i n t r a c e l l u l a r d i s t r i b u t i o n may then be determined. Kinetic analysis of the 4^Ca uptake curves has been widely used in several non-neuronal preparations, such as kidney, l i v e r and myocardial tissues. (Borle, 1969; 1970; 1975a,b; 1981a; Claret-Berthon et a l . , 1977; B a r r i t t et a l . , 1981; Uchikawa and Borle, 1981; Wakabayashi and Goshima, 1981). Its counterpart, the k i n e t i c analysis of 4^Ca efflux patterns, has been successfully applied to similar preparations (Uchikawa and Borle, 1978a,b) and in some cases to heterogeneous tissues, such as s l i c e s of the anterior p i t u i t a r y (Moriarty, 1980) or brain (Rubiales de B a r i o g l i o and Orrego, 1982). Measurements of calcium using this approach have usually agreed with the results obtained through other techniques and methods (Borle, 1981a; Blinks et a l . , 1982). The aim of the present study was therefore to apply the kinetic analysis of 4^Ca uptake curves to the hippocampal s l i c e preparation in view of the variety of calcium-mediated phenomena that have been shown to exist in this CNS structure. The objectives were two-fold: 1) to determine the uptake function for 89 compartmental analysis and 2) to i d e n t i f y the nature and factors that influence the d i f f e r e n t C a 2 + pools and fluxes in t h i s system. 3.2.2. Methods A flow-chart of the methods used i s presented in Fig. 3.1. Following incubation of the s l i c e s and c a l c u l a t i o n of calcium uptake, graphical and computerized methods of curve f i t t i n g were used to determine the exchange rates and calcium compartments of the tissue. The procedures underlying each step in the kinetic analysis of ^ C a uptake curves are described below. 3.2.2.1. Measurement of 4 5 C a uptake Adult male Wistar rats were s a c r i f i c e d , t h e i r brains quickly removed and the hippocampus dissected free. S l i c e s , 450 urn in thickness, were prepared using a Sorvall tissue chopper. Twenty- four to t h i r t y s l i c e s were routinely obtained from both hippocampi and then were randomly d i s t r i b u t e d into six porous v i a l s and later transferred into the incubation chamber (a Petri dish of 500 ml capacity) containing 300 ml of a r t i f i c i a l cerebro- spinal f l u i d (CSF). The chamber was kept in a water bath at a constant temperature of 35 + 0.5°C, while the medium inside the chamber was continuously oxygenated with a 95% C>2 / 5% CO2 gas mixture. The a r t i f i c i a l CSF contained in mM : NaCl 124; KCl 3.75; 9 0 KH 2P0 4 1.25; CaCl 2 and MgS04 1.5; NaHC03 24 and D-glucose 10; at a pH o f 7.4 . Before addition of the tracer to the incubation medium, the s l i c e s were allowed at least one hour of e q u i l i b r a t i o n . At time zero 1 uCi of 4^Ca added and individual holding v i a l s (containing 4-5 s l i c e s each) were removed at 2.5, 5, 10, 30, 60 and 90 min following introduction of the tracer. In some experiments the uptake was carried out up to 120 min post tracer addition. The uptake was terminated by placing the s l i c e s within the porous holding v i a l s into ice-cold LaCl3 buffer (160 mM T r i s HC1 + 10 mM L a C l 3 , adjusted to pH 7.4 with NaOH). The s l i c e s were then removed from the holding v i a l s , placed in test tubes containing 3.0 ml of LaCl3 buffer and washed f i v e times (for 10 min each). Previous studies have demonstrated that t h i s wash procedure e f f e c t i v e l y removes excess e x t r a c e l l u l a r calcium while leaving i n t r a c e l l u l a r calcium pools unaffected (Baimbridge and M i l l e r , 1981; Hellman, 1978; Hellman et a l . , 1975; Van Breemen and McNaughton, 1970). Following the La-wash the s l i c e s were in d i v i d u a l l y weighed on a precision balance and transferred into separate counting v i a l s containing 500 ul of Protosol [NEN] to allow for digestion of the tissue. Omnifluor [NEN] - 5 ml/vial - was used as a s c i n t i l l a t i o n f l u i d and r a d i o a c t i v i t y was measured the following day on a Beckman LS 9800 l i q u i d s c i n t i l l a t i o n counter. Total counts were obtained from 100 ul samples of the incubation medium removed at various time intervals following addition of the tracer. 91 Figure 3 .1 . Sequential flow-chart representation of the methods used for kinetic analyses of 4^Ca uptake curves. 92 INCUBATION OF S L I C E S FOR 1 HR. (CA ) q - 1 . 5 MM: 02/C02:95/5Z: T°-35±.2° C ADDI T I O N OF 1 pCi ^ C A AT T q - 0 MIN REMOVAL OF S L I C E S AT 2.5. 5. 10. 30. 60. AND 90 MIN. 10 MIN WASH IN 160 nfi T R I S . H C L + 10 MM LACI .3 (PH 7.2) AT T ° - 1 ° C ^ » 5X WEIGHING AND COUNTING U P T A K E T I S S U E CPM/MG WET WEIGHT NMOL CA CPU/100 pi MEDIUM MG WET WEIGHT 150 NMOL CA/100 JJL MEDIUM G R A P H I C A L A N A L Y S I S UJ I * Q- X < T I M E it slope, = -A, 5 B - i ^ slope2=-A2 T I M E COMPUTER A I D E D NON-LI HEAR L E A S T SQUARE FOR B E S T F I T : A[1-EXP(- AJT ) ] • B [ I - E X P ( - A 2 T ) J DERIVATION OF POOLS AilD FLUXES ACCORDING TO AN OPEN SERIES SYSTEM MODEL C F . U C I U K A W A A N D B O R L C . 1 9 8 1 93 Uptake was calculated according to the formula: Tissue CPM / mg wet weight [Ca](nmol) UPTAKE = = medium CPM / [Ca] in medium mg wet weight Where CPM represents counts/min. In experiments where the calcium concentration of the a r t i f i c i a l CSF was varied or addition of drugs was necessary, s l i c e s were allowed to eq u i l i b r a t e for at least 1 hour to the novel conditions. Drug concentrations used were 100 uM for 3- isobutyl-1-methylxanthine (IBMX) and 2,4-dinitrophenol (DNP) and 1 uM for nifedipine respectively. A l l drugs were obtained from Sigma Chemical Company. Salmon c a l c i t o n i n was used in a concentration of 4 U/300 ml (Armour Pharmaceutical Co.). 4 ^ C a C l 2 (2 mCi/ml) containing 176 ug Ca/ml was purchased from Amersham Corporation. To determine the v i a b i l i t y of the incubated tissue, some s l i c e s were removed after approximately 2 hours and transferred to a super fusion-type electrophysiological recording chamber. Evoked f i e l d potentials were recorded in medium similar to the incubation solution. If the c h a r a c t e r i s t i c synaptic responses could not be evoked from d i f f e r e n t c e l l types of the hippocampal formation, s l i c e s for the 4^Ca uptake experiments were discarded. This procedure also allowed for assessment of drug effects on the electrophysiological properties of neurons in the incubated hippocampi. In order to establish e q u i l i b r a t i o n of the s l i c e s with regard to t o t a l calcium concentration during the f i r s t hour of 94 incubation, t o t a l s l i c e calcium was measured by atomic absorption spectrophotometry (AAS) according to the method described e a r l i e r , except for the formaldehyde fixa t i o n (Section 3.1.2.). 3.2.2.2. Curve F i t t i n g To perform the kinetic analysis of 4^Ca uptake curves, i t is essential to determine the theoretical function that f i t s the experimental data points with maximum accuracy. Since previous studies using other preparations have shown that the uptake of lab e l l e d calcium i s best described by a double exponential equation (Borle, 1969; Borle, 1970; Claret-Berthon et a l . , 1977; B a r r i t t et. a l . , 1981) the f i r s t approach was to test an uptake function that i s the sum of two exponentials, of the form: f( t ) = A * [ 1 - e x p ( - * t)] + B-[1-exp(-A 2't)] (1) where t i s time; A and B are two exponential constants and X'i , X 2 a r e t n e reciprocals of the two time constants respectively. Each uptake curve consisted of 3-5 data points (individual s l i c e calcium uptake expressed per mg of s l i c e wet weight) at every time i n t e r v a l (2.5, 5, 10, 30, 60 and 90 min). The maximum uptake (MAX) was calculated from the mean uptake value at 90 min + 5% and was used for the i n i t i a l graphical analysis of the curve. The c a l c u l a t i o n of the maximum uptake using t h i s formula assumes that 95.5% of the asymptote has been reached at 90 min although t h i s may not have been the case in every experiment 95 especially those having a slow f i r s t exponential. However, the use of multiple iterations and the Simplex method (see below) as the f i n a l curve f i t t i n g algorithm have overridden any errors due to graphical analysis. To derive the f i r s t estimates for the constants of the double exponential equation "MAX - UPTAKE" (at each time) was plotted on a semilogarithmic scale (Fig.3.2.A.). Since the semilogarithmic plot could not be f i t t e d by a single straight l i n e (indicating a double exponential function), standard exponential peeling techniques (Riggs, 1963; Jacques, 1972) were applied to y i e l d the intercepts (A and B) and the slopes ( A 1 and A 2 ) of the two linear regression l i n e s . Each individual data point was assigned equal weight for contribution to the regression l i n e . The seemingly larger standard error bars at 60 and 90 min of Fig.3.2.A. are due to the logarithmic scale of the Y-axis. The graphical analysis of the uptake curve only provided the i n i t i a l estimates of the exponential constants. The constants obtained through the graphical method were further adjusted to y i e l d the "best f i t " to the experimental data points through the use of an i t e r a t i v e computer program or by the application of the Simplex method for curve f i t t i n g . The i t e r a t i v e method consisted of adjusting the four individual parameters of the double exponential uptake function (A, B, A1 and A2) u n t i l the sum of squares of the differences between the theoreti c a l values and the observed experimental values was minimal, i . e . least squares (Berman et a l . , 1962b). This rather lengthy method is prone to errors depending on the precision of the i n i t i a l estimates that 96 Figure 3.2. Graphical analysis and f i t t i n g of the 4^Ca uptake curve. A. Representative uptake curve obtained for a control preparation ( l e f t ) and i t s semilogarithmic transform ( r i g h t ) . On the semilogarithmic plot the MAX value was determined as being 105% of the uptake at 90 min. Linear regression and standard exponential peeling techniques were used to derive the two intercepts (A and B) and the two slopes CS^ a n d ^ 2 ) from lines Y1 and Y 2 respectively. B. Following i t e r a t i v e or Simplex non- linear least squares curve f i t t i n g methods a double exponential equation of the indicated form was adjusted to the experimental data points. From the parameters of t h i s uptake function the exchange rates and compartment sizes for C a 2 + could be calculated. The inset shows the t h e o r e t i c a l model of hippocampal calcium regulation in a system with a s e r i a l arrangement. The f i r s t term of the double exponential represents exchange between ex t r a c e l l u l a r Ca z and the S 2 pool (sum of membrane-bound and free ionic i n t r a c e l l u l a r calcium). The second exponential term characterizes the exchange between the S 2 compartment and the sum of a l l i n t r a c e l l u l a r buffered/sequestered C a 2 + (S3). Ca2+ Uptake (nmoles/mg wet weight) Z3 98 were derived through graphical analysis. However, i f s u f f i c i e n t time was allowed (usually 10,000 i t e r a t i o n s ) , the program converged at parameter values that represented the best f i t (error l e v e l < 1 0 - 4 ) . Use of the Simplex algorithm provides an al t e r n a t i v e method to overcome d i f f i c u l t i e s and l i m i t a t i o n s of the i t e r a t i v e procedure. The "simplexes" ( i . e . , matrices formed by the parameters to be f i t t e d and the sum of residuals squared) expand and contract during each cycle and automatically converge towards the best f i t , i . e . , the minimal sum of residuals (Nedler and Mead, 1965). Using a modified computer program of the Simplex algorithm (Caceci and Cacheris, 1984), usually less than 200 cycles were required to obtain the best f i t to the experimental data points with an error level less than 10~ 4. The simplex method provided the further advantage that equations other than a double exponential could e a s i l y be tested for the p o s s i b i l i t y of better f i t s . In the present experiments single- or t r i p l e exponential functions and Michaelis-Menten type kinetics did not y i e l d better approximations to the observed data points. Once the exponential constants were derived through the non-linear least squares analysis the kinetic parameters of the 4^Ca uptake could be calculated. 99 3.2.2.3. Compartmental Analysis An uptake equation that consists of two exponential terms means that two exchangeable calcium compartments are present in the system under investigation (Berman et a l . , 1962a; Berman, 1965). The t h i r d compartment i s the e x t r a c e l l u l a r [Ca 2 +] in which the tracer i s introduced at time zero. The mathematical derivation of pools, fluxes and rate constants is beyond the scope of the present study and can be found in several reviews (Robertson, 1957; Sheppard, 1962; Berman, 1965; Jacquez, 1972). Since the experiments yielded a three compartment system (with two c e l l u l a r pools of exchangeable calcium and the calcium of the e x t r a c e l l u l a r medium), the analyses, of Robertson et a l . (1957) and of Uchikawa and Borle (1981) were adopted to determine the exchange rates in hippocampal s l i c e s . A three compartment system may be schematically represented by the following equation according to the notations of Robertson et a l . (1957): k l 2 k23 > > Si $12 S 2 #23 S 3 (2) < < k21 k32 Depending whether the investigator has access to the end compartment (S 1) or the middle compartment (S2) to introduce the tracer, the system i s considered to be "in series" (catenary) or" i n p a r a l l e l " (mammillary) respectively. 100 If scheme (2) is applied to the experimental conditions, the notations are as follows: Sj- is the amount of calcium in compartment / (in nmol/mg s l i c e wet weight). ij i s the flux (or rate of exchange) at steady state in either direction between compartments / and j (in nmol/mg s l i c e wet weight/min). kj-y i s the rate constant of exchange from compartment i to j , i . e . , the fraction of Ŝ- transported to S- in unit time (min~ 1). Taking into account the assumptions of Uchikawa and Borle (1981) with regard to open or closed systems, the hippocampal s l i c e s in the present experiments may be regarded as being part of an "open" system. This requires the e x t r a c e l l u l a r compartment to be i n f i n i t e l y larger than the sum of the i n t r a c e l l u l a r exchangeable calcium compartments. Although this i s c l e a r l y not the case, since the s l i c e s were incubated in a f i n i t e amount (300 ml) of a r t i f i c i a l CSF, the r a d i o a c t i v i t y of the extra- c e l l u l a r environment during the course of experiments was constant, indicating that the Ca^ of the e x t r a c e l l u l a r medium was not s i g n i f i c a n t l y affected by uptake into the incubated s l i c e s . Therefore, the error introduced by the open- system assumption is in the range of 0.01% (Uchikawa and Borle, 1981). A t y p i c a l uptake curve f i t t e d by the double exponential equation i s shown on Fig.3.2.B. The insert depicts the s e r i a l model system which i s considered to be a more accurate r e f l e c t i o n 1 0 1 of c e l l u l a r C a 2 + d i s t r i b u t i o n in the CNS. In this model, the fast component of the exchange i s associated with the rapidly exchangeable C a 2 + pool (the t o t a l of calcium bound to the inside of the plasma membrane and free cytoplasmic C a 2 + ) , i . e . , unbuffered pool. The slow component stands for exchange between the unbuffered and the sequestered/buffered pools of calcium. In the p a r a l l e l model no communication is allowed between the two c e l l u l a r calcium pools, i . e . , both the unbuffered and buffered pools of calcium are in d i r e c t connection only with the e x t r a c e l l u l a r environment. The calculations required to obtain the kinetics of the calcium exchange are presented below. The exponential parameters from equation ( 1 ) may be used to derive the calcium pools, fluxes and rate constants for an open system from the time d i f f e r e n t i a l of the uptake (Uchikawa and Borle, 1 9 8 1 ) : dU/dt = a-exp(-^ 1-t) + b*exp(-A 2't) (3) where U i s the uptake, t i s time and a = k'X] and b = B ' A 2 > From t h i s d i f f e r e n t i a l equation the solutions for pools, fluxes and rate constants of the calcium exchange may be calculated for both models as follows: 1 02 In series (catenary) system [S^ = ext r a c e l l u l a r ] a> 2 l = a + b (4) S 2 + S 3 = a /A, + b/A2 (5) (a + b ) 2 5 2 = (6) a'-A, + b'X 2 a-b- (A, - A 2 ) 2 5 3 = (S 2 + S 3) " S 2 = (7) • %2 ' (a 'A, + b*A2') A 1 -A 2 -s 2 -s 3 $23 = " (8) a + b k12 = ^ 2 l / s 1 = 0 (since S, = » ) (9) k 2 l = ^ 2 l / S 2 (10) k23 = <t>23/S2 ( 1 1 > k32 = 4>23/S3 (12) In p a r a l l e l (mammillary) system [S 2 = ex t r a c e l l u l a r ] = a /A, (13) S 3 = b/A 2 (14) <|>1 2 = a (15) $ 2 3 = b (16) k21 = e $ 2 \ / s 2 = 0 (since S 2 = oo) (17) k12 = $ 2 l / S 1 = (18) k23 = < $ 2 3 / s 2 = 0 (since S 2 =00) (19) k32 = $>23/s3 = ^2 ( 2 0 ) 1 03 3.2.3. Results The uptake of l a b e l l e d calcium into hippocampal s l i c e s could be best f i t t e d by a double exponential function in every experimental condition examined. This finding indicates that the s l i c e s as a whole behave in terms of calcium compartmentalization as having two i n t r a c e l l u l a r exchangeable C a 2 + pools (Berman et a l . , 1962b; Uchikawa and Borle, 1981). The uptake curves presented in the figures of this section have been generated by computer from the average values of the exponential constants derived from individual experiments, rather than f i t t i n g a single curve to the averages of the i n d i v i d u a l data points obtained in d i f f e r e n t experiments. This representation is considered to be mathematically more accurate, since the exponential f i t t i n g the averages may not be a true r e f l e c t i o n of the average values of the exponential terms (Riggs, 1963). 3.2.3.1. Effect of e x t r a c e l l u l a r calcium U C a 2 + ] 0 ) In t h i s series of experiments hippocampal s l i c e s were prepared in a r t i f i c i a l CSF containing the standard 1.5 mM Ca , but were incubated in media with calcium concentrations of 0.1, 2.0 and 4.0 mM. Control s l i c e s were always incubated in 1.5 mM [ C a 2 + ] c . The e x t r a c e l l u l a r Mg 2 + concentration was kept constant at 1.5 mM except for the 4.0 mM C a 2 + containing medium where i t 1 04 was lowered to 1.0 mM, for t h i s p a r t i c u l a r ionic composition has been shown to result in long-term alt e r a t i o n s in the e x c i t a b i l i t y of hippocampal neurons (Turner et a l . , 1982; Mody et a l . , 1984). Alterations in e x t r a c e l l u l a r calcium concentrations had marked e f f e c t s on the shape of the 4^Ca uptake curves (Fig. 3.3.A.). Increasing [ C a 2 + ] Q caused a s i g n i f i c a n t l y faster i n i t i a l uptake rate than i t would be expected from changes in the ionic gradient across the plasma membrane. The electrophysiological properties of the s l i c e s incubated at various calcium concentrations were also affected. The evoked responses of a control s l i c e taken from the 4^Ca incubation chamber with the c h a r a c t e r i s t i c population spikes recorded in the CA1 and dentate regions of the hippocampal formation are shown in Fig.3.3.B. The feed-back i n h i b i t i o n , as measured by activation of inh i b i t o r y interneurons by an antidromic conditioning stimulus, was found to be normal. When s l i c e s were incubated in medium containing 0.1 mM C a 2 + subsequent recordings showed the c h a r a c t e r i s t i c evoked multiple discharges and later the spontaneous, regular bursting in the CA1 area (Fig 3.3.C.). The C a 2 + pools, fluxes and rate constants in hippocampal s l i c e s at d i f f e r e n t [ C a 2 + ] Q are summarized in Table 3.2. The data have been obtained through kinetic analysis of the uptake curves according to equations (4)-(20) presented in Section 3.2.2.3. The two time constants of the uptake function are the reciprocals of the rate constants ( k l 2 and k 3 2 ) of the p a r a l l e l model. Regardless whether the s e r i a l or the p a r a l l e l model i s used for interpretation, the size of the rapidly 1 05 Figure 3.3. Effect of alterations in ext r a c e l l u l a r C a 2 + concentrations on the hippocampal 4^Ca uptake curves and e l e c t r i c a l a c t i v i t y . A. Computer-fitted 4^Ca uptake curves at four d i f f e r e n t e x t r a c e l l u l a r C a 2 + concentrations. The numbers on the curves represent [ C a 2 + ] Q and [ M g 2 + ] Q in mM respectively. B. Electrophysiological properties of s l i c e s transferred from the uptake chamber into a recording chamber. Recordings were taken approx. 5 min following the transfer procedure. Extracellular population spikes (top traces) were recorded from the pyramidal c e l l s of CA1 region (•) evoked by stratum radiatum stimulation ( l e f t ) and the granule c e l l s of the dentate gyrus (O) subsequent to a c t i v a t i o n of the perforant path ( r i g h t ) . The bottom traces represent i n h i b i t i o n of the population discharge when orthodromic stimulation i s preceded by an antidromic stimulus that activates feed-back inhi b i t o r y interneurons, indicating good v i a b i l i t y of the s l i c e s in the uptake chamber (Calibration: 2mV/l0ms). C. Lef t : Synaptically evoked multiple population discharge recorded in the CA1 region during incubation with 0.1 mM [ C a 2 + ] c ( C a l i b r a t i o n : 2mV/20ms). Right: Spontaneous, regularly occurring f i e l d bursts in the absence of synaptic responses following e q u i l i b r a t i o n with the low calcium. 4^Ca was added to the incubation chamber when the s l i c e s have presumably reached this stage ( C a l i b r a t i o n : 2mV/l0s). In this and subsequent figures, in a l l records of e l e c t r i c a l a c t i v i t y positive i s upwards. 1 06 107 Table 3.2. E f f e c t s of e x t r a c e l l u l a r calcium concentrations on hippocampal Ca* + exchange. The pools, fluxes and rate constants f o r both the s e r i a l model system ( e x t r a c e l l u l a r compartment: S-̂ ) and the p a r a l l e l model system ( e x t r a c e l l u l a r compartment: of hippocampal c e l l u l a r C a 2 + exchange have been calculated according to equations (4)-(20) from the text. Data represented as mean + S.D. derived from the k i n e t i c a n a l y s i s of 4-8 uptake curves each (no. of s l i c e s = 20-27 for every uptake curve). [ C a 2 + ] Q i s indic a t e d i n mmoles/L (mM), while e x t r a c e l l u l a r Mg 2 + was 1.5 mM unless shown otherwise. Control .,, ,[Ca2+]o [Ca2+]o, [Ca2]o 4 mM [Ca2]o 1.5 mM 0.1 mM 2 mM [Mg2]o 1 mM IN SERIES POOL S 2 POOL S3 TOTAL FLUX S1-S2 FLUX S 2-S 3 k 2 3 [xlO 3 ] k32 2218 1052 3270 489 36 .224 16.5 .034 198 151 50 49 9 .04 5.3 .006 3 4 7 * -160* + 507* + * — 25 + * — 3^ + .072* + 10.0* + 2.3 .021* +.001 22 3 4 4 7 * ± 1 7 1 17 1 6 0 3 * . 1 1 2 8 6 5 0 5 0 * 1 4 8 3 1 1 4 4 * 1 4 5 .5 19* + 3 .01 .332* + .01 5.6* + 1.1 .012* +.001 6165^ + 120 1495* + 30 7660* + 150 2593* + 728 3 0 * - 4 .418* + .11 4.8* + .61 .020* +.002 IN PARALLEL POOL S± POOL S3 TOTAL FLUX S2-^i FLUX S 2 - S 3 k12 k32 1883 + 1387 + 3270 + 493 + 43 + .266 + .030 +, 227 190 50 142 10 .09 005 236^ 271* 507* 20" 1 5( .086] .018* 14 8 6 3 .4 .02 + .001 3330^ + 186 1720* + 142 5050* + 48 1124* + 47 20* + 3 .338 .01 + .012* +.001 6 0 1 0 , 1650* 7660* 2561* 32* .424* .019* + 140 + 10 + 150 + 723 1 4 + .11 + .003 UNITS: pmoles'mg - 1 s l i c e wet weight for pools, pmoles'mg - 1 s l i c e wet weight •min--'- for fluxes, min-1 for rate constants. Denotes s i g n i f i c a n t d i f f e r e n c e from c o n t r o l (p<0.01 one-way ANOVA), 108 exchangeable pool (S 2 of the s e r i a l model or of the p a r a l l e l model) increases l i n e a r l y 18-25-fold with a 40-fold increase in e x t r a c e l l u l a r C a 2 + concentration (from 0.1 to 4.0 mM). The calcium flux between th i s compartment and the e x t r a c e l l u l a r environment shows a comparable linear enhancement. In contrast to the fast component of the uptake, the slowly exchangeable calcium pool of hippocampal s l i c e s seems to be saturated at a [ C a 2 + ] D of 2.0 mM while the exchange rate between the slow and fast compartments i s maximal at the physiological range of [Ca 2 +]„ of 1.5 mM. Similar data have • been obtained by Borle (1970) in isolated kidney c e l l s that have a single i n t r a c e l l u l a r compartment and by B a r r i t t et a l . (1981) in l i v e r c e l l s with two i n t r a c e l l u l a r exchangeable calcium compartments. The t o t a l exchangeable C a 2 + of control preparations, as measured 'by the kinetic analysis, was found to be 3.27 nmole/mg wet weight. This value i s comparable to results of previous studies in s l i c e s of brain or other tissues (Stahl and Swanson, 1971; 1972; B a r r i t t et a l . , 1981; Borle, 1981a). Measured by atomic absorption spectrophotometry (AAS) s l i c e s incubated in control conditions have a t o t a l calcium of 4.52 +0.12 nmole/mg wet weight indicating that about 72% of t o t a l s l i c e calcium is exchangeable. Following the preparation of the s l i c e s , their e q u i l i b r a t i o n with regard to t o t a l calcium was remarkably rapid. The f i n a l value of t o t a l calcium was attained in less than 5 min of incubation. This finding indicates that during the 60 min of preincubation before addition of the tracer the s l i c e s have 109 surely attained the ionic steady state required for kinetic studies. 3.2.3.2. Effect of drugs that a l t e r C a 2 + metabolism This series of experiments involved testing the ef f e c t of drugs that may a l t e r the C a 2 + homeostasis of hippocampal s l i c e s . The dihydropyridine calcium channel antagonist nifedipine in a concentration of 10~ 6 M was used to block C a 2 + entry but had no marked eff e c t on the shape of the 4^Ca uptake curves (Fig. 3.4.A.). However, kinetic analysis revealed that the drug enhanced the slowly exchangeable pool of C a 2 + resulting in a larger than normal exchange between i n t r a - and e x t r a c e l l u l a r compartments (Table 3.3.). In contrast, c a l c i t o n i n (4U/300 ml) enhanced the fast component of the uptake while depressing the slow phase (Fig. 3.4.A.). The net effect of the hormone was to reduce t o t a l hippocampal exchangeable C a 2 + by mobilizing C a 2 + from the slowly exchangeable S 3 pool (Table 3.3.). The mitochondrial i n h i b i t o r 2,4,-dinitrophenol (DNP) in a concentration of 10~ 4 M had an ef f e c t similar to that observed by Borle (1981b) in the kidney. The fast component of the uptake became more rapid while the slow exchange phase was reduced (Fig. 3.4.B.). Kinetic analysis of ^^Ca uptake in the presence of DNP indicates an enhancement (about 38%) of the rapidly exchangeable compartment with a concomitant increase in calcium flux and a s i g n i f i c a n t reduction (about 57%) of the sequestered/buffered pool of C a 2 + (Table 3.3.). These al t e r a t i o n s were reflected in 110 Figure 3.4. Effects of drugs and hormones on 4^Ca uptake and ele c t r o p h y s i o l o g i c a l properties of hippocampal s l i c e s . A. 4^Ca uptake curves in the presence of 10~ 6 M nifedipine and 4U/300ml of salmon c a l c i t o n i n . B. Effects of 10~4 M 2,4-dinitrophenol (DNP) and 3-isobutyl-1-methylxanthine (IBMX) on the shape of the 4^Ca uptake curves. C. L e f t : Control CA1 population spike in a s l i c e removed from the uptake chamber. Right: Enhancement of the evoked response with induction of a second population spike (arrow) following incubation with 10~ 4 M DNP. (Calibration: 4mV/l0ms). D: Le f t : Population spike recorded in the pyramidal c e l l layer of the CA1 region under control conditions. Right: Potentiation of the evoked response in the presence of 10~4 M IBMX. (Cali b r a t i o n : 2mV/l0ms). A. 5 a> $ cn .E o E c + CN CD u CALCITONIN NIFEDIPINE 30 60" TIME ( min ) - . — , — | 90 CONTROL c . A CONTROL CONTROL IBMX DNP 1 r 1 i r- 30 60 TIME ( min ) IBMX 1 1 2 Table 3.3. E f f e c t s of drugs on hippocampal Ca2+^;exchange. Data represented as mean + S.D. derived from the k i n e t i c analysis of 4-8 uptake curves each (number of s l i c e s = 19-29 for each uptake curve). For explanation of pools, fluxes, rate constants, and the i r respective units see text and the the legend for Table 3.2. Drug concentrations are indicated i n moles/L (M) except for c a l c i t o n i n ( i n t e r n a t i o n a l units/300 ml). ==================================================̂ =========================== Control i Nifedipine ., Ca l c i t o n i n ;DNPa, IBMXb [ 1 0 - 6 M] ; \ [4U/300ml] [ 1 0 - 4 M] [ 1 0 - 4 ,;M] IN SERIES POOL S 2 2218 + 198 2022 ir + 86 2272^ + 24 3 0 5 6 * + 78 2837^ + 60 POOL S 3 1052 + 151 1513 + 109 703* + 59 476* + 115 658* + 15 TOTAL 3270 + 50 3 3 0 5 * + 35 2975* + 35 3532* + 106 3495* + 75 FLUX S 1-S 2 489 + 49 827* + 20 948* + 18 878* + 137 735* + 21 FLUX S 2-S 3 36 + 9 3 2 * + 8 6* + 1 7* + 2 15* + .4 k21 r .224 + . 04 .409* + , .08 .417* * + . 01 .287 .04 .259 + .01 k 2 3 [xlO 6 k 3 2 [xlO" 3 ], 16.5 ]. 34 + + 5 6 15.5 21* + + 5 7 2.8 * 9 + . + 6 1 2.3* * 16 +, + .9 7 5.2 * 23 + + .02 .1 IN PARALLEL POOL S 2 1883 + 227 1865 w + 125 2 2 4 0 * + 30 3003* + 100 2710* + 50 POOL S 3 1387 + 190 1670* + 70 735* + 65 528* + 117 785* + 25 TOTAL 3270 + 50 3305^ + 35 2975* + 35 3532* + 106 3495* + 75 FLUX S^S-L 493 + 142 793* + 29 941* + 19 870* + 137 718* + 22 FLUX S 2-S 3 43 + 10 3 4 « + 0 7* + 1 8 + 3 17* + .6 k12 , .266 + . 09 .426* +. .01 .420* .01 + , .04 +. .01 k 3 2 [xlO J ] 30 + 5 21* + 7 9* + 1 • 15* + 6 22* + .1 a DNP — 2,4-dinitrophenol b IBMX — 3-isobutyl-l-methylxanthine Denotes s i g n i f i c a n t d i f f e r e n c e from co n t r o l (p<0.01 one-way ANOVA). 113 the abnormal electrophysiological properties of hippocampal neurons consisting in an increase of the amplitude of ortho- dromically evoked responses and occurrence of multiple spike discharges (Fig. 3.4.C.) analogous to the results of Godfraind et a l . (1972) obtained iin vivo. The e f f e c t of IBMX (3-isobutyl-1-methylxanthine) on the 4 5 C a uptake curves was much l i k e that of DNP but of a lesser magnitude (Fig. 3.4.B. and Table 3.3.). The drug i s a potent i n h i b i t o r of the c y c l i c nucleotide phosphodiesterase (Chasin and Harris, 1972) and i t has been shown to cause an increase in the levels of cAMP in brain s l i c e s (Smellie et a l . , 1979). This data i s consistent with the results of Borle and Uchikawa (1979) who showed that cAMP and i t s dibutyryl derivative enhanced calcium uptake in c e l l cultures. The evoked potentials of the CA1 region were also affected by the drug showing a marked potentiation of both the population spikes (Fig. 3.4.D.) and the e x t r a c e l l u l a r l y recorded EPSPs. 3.2.3.3. Theoretical manipulations of the S3 pool The derivation of C a 2 + pools, fluxes and exchange rates through a double exponential equation allows for experimental modelling of the system under investigation. Each individual term of the exponential equation may be changed as desired to y i e l d compartments of d i f f e r e n t sizes and to af f e c t the exchange between various compartments of the model. F i g . 3.5.A. shows the s e r i a l model system in which the size of the buffered pool may be 1 1 4 Figure 3.5. Effects of theoretical manipulations of the S3 (buffered) C a 2 + pool. A. Schematic representation of the s e r i a l model of hippocampal i n t r a c e l l u l a r calcium regulation. The S3 compartment consists of C a 2 + sequestered/buffered by the mitochondria and endoplasmic reticulum as well as Ca 2 + bound to i n t r a c e l l u l a r proteins. B. By changing the exponential terms of the f i t t e d uptake equation the size of the S3 compartment may ea s i l y be manipulated. The t h e o r e t i c a l uptake curves are shown as they would result from a 50% reduction or a l t e r n a t i v e l y , from a 50% enhancement of the S3 pool. The control curve was generated from the experimental data obtained from hippocampal s l i c e s incubated under normal conditions. 1 1 5 s, Co 2+ [Co 2 + ] fast slovv / SP<^=>S^ o B. membrane x Ca 2 t -Proteins C O N T R O L S 3 - 5 0 % S 3 + 5 0 % 1 1 1 1 1 1 1 1 1 j 3 0 6 0 9 0 TIME ( min ) 1 1 6 altered by changing the value of the second exponential term of the uptake equation. The computer-generated curves following a 50% enhancement of the buffered S3 pool or i t s reduction to half of the control value are shown in F i g . 3.5.B. These curves are only of a the o r e t i c a l significance since no experimental condition w i l l result in a pure and isolated e f f e c t on the S3 pool alone. Examination of their shape is however useful for comparisons to various experimental situ a t i o n s . 3.2.4. Discussion Kinetic analysis of 4^Ca uptake curves revealed the existence of two separate, k i n e t i c a l l y d i s t i n c t pools of exchangeable i n t r a c e l l u l a r calcium in the hippocampal s l i c e preparation. In a model of th i s system, based on the p r i n c i p l e s of compartmental analysis (Robertson, 1957; Sheppard, 1962; Jacques, 1972), these two pools may be considered to have either a s e r i a l (catenary) or a p a r a l l e l (mammillary) arrangement (Robertson et a l . , 1957; Uchikawa and Borle, 1981). The present results do not show any s i g n i f i c a n t differences between these two; however, the s e r i a l model more clos e l y resembles the physiological schema of i n t r a c e l l u l a r calcium homeostasis (Brinley, 1978; Blaustein et a l . , 1980; McGraw et a l . , 1982). Notations applicable to t h i s model are therefore used in subsequent references to exchangeable Ca* pools. 1 1 7 Several essential c r i t e r i a must be met before application of kinetic analysis may be considered v a l i d for physiological systems. Most importantly the preparation has to be viable, and in the present study t h i s was demonstrated by the routine electrophysiological recordings in s l i c e s removed from the uptake chamber. Also of importance is the assumption regarding the system's steady state for both the t o t a l ionic concentration of C a 2 + and flux of the tracer (Sheppard, 1962; Borle, 1975; Uchikawa and Borle, 1981). This was ensured by a long pre- incubation period of the s l i c e s in the presence of drugs or the altered e x t r a c e l l u l a r ionic environment as well as the fact that s l i c e s equilibrated quite rapidly in terms of their t o t a l calcium content. Furthermore, during the tracer experiments, the uptake of calcium reached an asymptotic value without a subsequent decline indicating that the system was at isotopic equilibrium (Borle, 1981b). These factors together with the s i m i l a r i t y with other studies (Borle, 1981a) in terms of the levels of hippocampal exchangeable calcium (3.33 nmoles/mg wet weight) and the exchangeable fraction of t o t a l Ca 2 + (about 72%) provide strong evidence for the a p p l i c a b i l i t y of 4^Ca uptake kinetics to the ir\ v i t r o hippocampus. One d i f f i c u l t y however with using t h i s • p a r t i c u l a r preparation i s the fact that the hippocampal formation consists of a large variety of heterogeneous neurons and g l i a l s c e l l s , a l l of which may participate in C a 2 + regulation (Brostrom et a l . , 1982; MacVicar, 1984). While the calcium compartments derived through kinetic analysis cannot be attributed to any one of 118 these populations in p a r t i c u l a r , they are most l i k e l y l o c a l i z e d within the c e l l s because the bulk of e x t r a c e l l u l a r C a 2 + has been washed off by LaCl3 following termination of the uptake (Van Breemen and McNaughton, 1970; Hellman et a l . , 1976; Hellman, 1978; Baimbridge and M i l l e r , 1981). In view of these r e s t r i c t i o n s , the present experiments do not provide a way of distinguishing between the di f f e r e n t elements of the s l i c e preparation but rather characterize the system as a whole with regard to i t s calcium homeostasis. The two exponential terms of hippocampal 4^Ca uptake r e f l e c t two d i s t i n c t components of C a 2 + regulation in t h i s system. The fast component represents exchange between e x t r a c e l l u l a r Ca 2 + and that located within the plasma membrane and/or in i t s immediate v i c i n i t y . The rapidly exchangeable compartment (S 2) may thus consist of calcium t i g h t l y bound to the glycocalyx (inaccessible to LaCl3), Ca 2 + bound to the inside of the membrane and free ionic i n t r a c e l l u l a r C a 2 + . I d e n t i f i c a t i o n of S 2 as the sum of these C a 2 + pools i s supported by the findings that changes in ex t r a c e l l u l a r calcium concentrations have a s i g n i f i c a n t effect on the magnitude of the fluxes between S 2 and the a r t i f i c i a l CSF that bathes the s l i c e s . The driving force resulting from an increase in the C a 2 + gradient across the c e l l membrane results in an enhancement of the rapid phase of the exchange with a concomitant increase in the size of the S 2 compartment. In an attempt to further characterize the fast component of hippocampal calcium uptake, the effect of the putative calcium channel blocker nifedipine was tested on the kinetics of 4^Ca 1 1 9 exchange. This drug f a i l e d to produce the expected antagonism, which i s not surprising in l i g h t of previous observations that have shown i t s ineffectiveness on depolarization-induced calcium uptake into synaptosomes (Daniell et a l . , 1983). The enhancement of the S3 pool produced by nifedipine may r e f l e c t a novel action of t h i s drug in the CNS. The hormone c a l c i t o n i n is a potent regulator of Ca homeo- st a s i s in several systems (Borle, 1975b; 1981a) and c a l c i t o n i n receptors are widely d i s t r i b u t e d in the rat CNS (Rizzo and Goltzman, 1981; Henke et a l . , 1983). In the present study c a l c i t o n i n reduced the l e v e l of t o t a l exchangeable C a 2 + by enhancing the efflux rate of Ca^ from the elements of the hippocampal s l i c e . Since the size of the unbuffered compartment (S 2) was not s i g n i f i c a n t l y affected, i t might be suggested that the Caz+ normally present in the buffered compartment (S3) was mobilized by c a l c i t o n i n . This finding does not resemble c a l c i t o n i n ' s action in isolated kidney c e l l s (Borle, 1975b), but i t is possible that CNS receptors mediate a d i f f e r e n t e f f e c t than that observed in the periphery. While the fast calcium compartment is readily altered by changes in e x t r a c e l l u l a r calcium concentrations, the identity of the slowly exchangeable compartment (S3) i s less obvious. The application of the mitochondrial i n h i b i t o r DNP (10~ 4 M) resulted in a marked reduction of the slowly exchangeable Ca compartment indicating that mitochondrial calcium i s a s i g n i f i c a n t part of the S3 pool. These data are similar to those observed by Borle (1981b) in isolated kidney c e l l s . 1 20 In addition to mitochondria, neuronal tissue i s known to have several C a 2 + buffering/sequestering systems, p a r t i c u l a r l y the endoplasmic reticulum and various calcium-binding proteins (Brinley, 1978; Duce and Keen, 1978; Blaustein et a l . , 1980; McGraw et a l . , 1982). The contribution of these calcium- regulatory mechanisms to the slowly exchangeable Ca compartment i s d i f f i c u l t to assess for the lack of s p e c i f i c a l l y acting drugs and blocking agents. Alternative factors regulating the slow compartment may involve c y c l i c AMP as has been shown in kidney c e l l s (Borle and Uchikawa, 1979). The methylxanthine derivative IBMX is a potent phosphodiesterase i n h i b i t o r (Chasin and Harris, 1976) and also releases C a 2 + from i n t r a c e l l u l a r storage s i t e s such as the sarcoplasmic reticulum in the case of muscle fibers (Miller and Thieleczek, 1977). Its effect on hippocampal C a 2 + - uptake kinetics i s similar to that of DNP in reducing the slowly exchangeable pool of calcium and simultaneously increasing the fast compartment ( S 2 ) . This would suggest that in the hippocampus c y c l i c nucleotides p a r t i c i p a t e in the regulation of the slow compartment. A l t e r n a t i v e l y , i t would appear that the endoplasmic reticulum of neuronal elements may play a s i g n i f i c a n t role in hippocampal C a 2 + - b u f f e r i n g . At t h i s point no conclusions may be reached with regard to the contribution of various i n t r a c e l l u l a r proteins to the slowly exchangeable S 3 compartment. However, i t should be emphasized that one of these proteins, calcium-binding protein (CaBP), has been shown to be confined to nerve c e l l s of the hippocampal formation and has been postulated to buffer intraneuronal calcium 121 (Jande et a l . , 1981; Baimbridge and M i l l e r , 1982; Garc. ia-Segura et a l . , 1984). The s p e c i f i c loss of th i s protein from the granule c e l l s of the dentate gyrus during and following kindling-induced epilepsy ( M i l l e r and Baimbridge, 1983; Baimbridge and M i l l e r , 1984) may provide an approach to study i t s role in hippocampal calcium homeostasis (also see Section 3.3.). Since C a 2 + p a r t i c i p a t e s in many events that involve neuronal e x c i t a b i l i t y , changes in i t s regulation should be readily r e f l e c t e d in a l t e r e d electrophysiological c h a r a c t e r i s t i c s . When a s h i f t of Ca^ from the buffered into the unbuffered pool was produced with l i t t l e change in t o t a l exchangeable calcium (the ef f e c t s of DNP and IBMX), s i g n i f i c a n t a l t e r a t i o n s were observed in the e l e c t r o p h y s i o l o g i c a l properties of hippocampal neurons. Both drugs had a marked potentiating effect on the evoked responses and in the presence of DNP aberrant discharge patterns were induced. S i m i l a r l y , when the buffering capacity of the system i s severely challenged by the high e x t r a c e l l u l a r calcium concentration (4.0 mM Ca 2 +/1.0 mM Mg 2 +), long-lasting a l t e r a t i o n s in neuronal function may occur (Turner et a l . , 1982; Mody et a l . , 1984). The reduction of hippocampal exchangeable Ca* during incubation of the s l i c e s with low [ C a 2 + ] 0 i s reflected in the spontaneous bursting of hippocampal CA1 pyramidal c e l l s (Taylor and Dudek, 1982; Yaari et a l . , 1983; Haas and J e f f e r y s , 1984). In addition, the steady state C a 2 + influx into the c e l l s under control conditions may be the underlying mechanism for the slow, persistent inward Ca 2 +-current of hippocampal pyramidal c e l l s (Brown and G r i f f i t h , 1983). 122 The c o r r e l a t i o n between these a l t e r a t i o n s in neuronal a c t i v i t y and calcium regulation i s not l i k e l y to be coinc i d e n t a l , indicating that the 4^Ca uptake measurements provide a v a l i d assessement of neuronal Ca* homeostasis. The method would appear to be valuable for studying the action of hormones and various pharmacological agents on calcium related phenomena. ' Further characterization of the system should involve separation of the neuronal from g l i a l elements and determination of C a 2 + regulatory c h a r a c t e r i s t i c s of certain neuronal populations possibly through the use of cultured c e l l s . 123 3.3. Measurement of Ca1 -regulation during kindling using the kinetic analysis of 4^Ca uptake curves 3.3.1. Introduction Epileptogenic phenomena may r e s u l t , in part, from long-term changes in the e x c i t a b i l i t y of nerve c e l l s . Of the several ions within the milieu of the CNS, special interest i s attached to calcium because of i t s important role in neuronal e x c i t a b i l i t y (see Sections 1.2 and 1.3). In the hippocampal formation t h i s cation has been suggested to be involved in somatic and dendritic spike generation (Wong and Prince, 1978; Traub and L l i n a s , 1979; Brown and G r i f f i t h , 1983b), activation of hyperpolarizing conductances (Alger and N i c o l l , 1980; Hotson and Prince, 1980; Schwartzkroin and Stafstrom, 1980; Brown and G r i f f i t h , 1983a) and p a r t i c i p a t i o n in aberrant forms of pyramidal c e l l discharge (Wong and Prince, 1978; Traub and L l i n a s , 1979; Hotson and Prince, 1981). Furthermore, calcium regulates long-lasting changes in CNS a c t i v i t y as i s the case for potentiation phenomena c h a r a c t e r i s t i c of the hippocampal formation (Dunwiddie and Lynch, 1979; Baimbridge and M i l l e r , 1981; Turner et a l . , 1981; Eccles, 1983; Mody et a l . , 1984; also see Section 1.3.). Recents studies have also suggested the involvement of C a 2 + in the predisposition of neuronal tissue to epileptiform a c t i v i t y . Using the experimental model of kindling-induced epilepsy, M i l l e r and Baimbridge (1983) demonstrated a selective 124 loss of calcium-binding protein (CaBP) from the granule c e l l s of the dentate gyrus. The protein, which binds C a 2 + with high a f f i n i t y , i s normally l o c a l i z e d in particular neuron types of the mammalian CNS (Jande et a l . , 1981; Baimbridge and M i l l e r , 1982; Garcia-Segura et a l . , 1984) and i s considered to function as an important buffer for intraneuronal C a 2 + (Baimbridge et a l . , 1982; M i l l e r and Baimbridge, 1983). Inasmuch as the decrease in hippocampal CaBP i s not correlated with seizure a c t i v i t y per se but rather to the progression of kindling (Baimbridge and M i l l e r , 1984), i t has been suggested that this change represents a functional a l t e r a t i o n of calcium homeostasis in neurons of this region predisposing them to aberrant epileptiform a c t i v i t y ( M i l l e r et a l . , 1985). If calcium regulation i s altered following kindling-induced epilepsy, then i t i s important to determine what changes have occurred in the concentration of i n t r a c e l l u l a r C a 2 + as well as in i t s compartmentalization and the extent of i n t r a c e l l u l a r buffering. The kinetic analysis of the uptake of radioactive Ca is a widely used method for the determination of Ca 2 +-compartmentalization and buffering in several non-neuronal preparations (Borle, 1975; 1981a,b; Uchikawa and Borle, 1981). In view of the successful application of 4^Ca uptake kinetics to determine calcium homeostasis in the hippocampal formation (see Section 3.2.), the technique was used to investigate possible a l t e r a t i o n s in C a 2 + - r e g u l a t i o n of amygdala- and commissural- kindled preparations. 125 3.3.2. Methods Adult male Wistar rats (200 - 300 g) were divided into three groups: a) controls (n=8); b) commissural-kindled (n=6) and c) amygdala-kindled (n=6). The kindling group and two of the control animals were anesthetized with Nembutal (70 mg/kg i.p.) and were ste r e o t a x i c a l l y implanted with bipolar stimulating electrodes positioned either in the midline commissural pathway (AP: -1.8 mm from bregma; L: 0; V: 4.2 mm below surface of cortex) or the right amygdala (AP: -2.5 mm; L: 3.6 mm; V: 7.8 mm). Following recovery from surgery the animals, except for controls, were stimulated once a day (150 uA, 60 Hz, for 1 sec) u n t i l a minimum of five consecutive motor seizures were induced (stage 5 - according to Racine, 1972b). Hippocampal s l i c e s , 450 urn in thickness, were prepared on the day following the l a s t seizure according ' to the methods described previously (see Section 3.2.2.1.). The placements of stimulating electrodes in the hippocampal commissures and the amygdala were v e r i f i e d macroscopically by v i s u a l examination during d i s s e c t i o n . Usually 24-30 s l i c e s were obtained from both hippocampi of one animal and were randomly d i s t r i b u t e d into six porous uptake v i a l s (4-5 s l i c e s / v i a l ) . This procedure was necessary to obtain an adequate number of data points at each time in t e r v a l for the uptake curve f i t t i n g routine. Experiments involved determining the 4^Ca uptake in s l i c e s obtained from one animal. Double-blind experiments were also performed in which 126 control and kindled s l i c e s were incubated in the same uptake chamber although in separate holding v i a l s . To test neuronal v i a b i l i t y , some of the s l i c e s were routinely removed from the incubation chamber 1-2 hrs following incubation, and transferred to a superfusion type recording chamber for electrophysiological examination. The procedures for determination of 4^Ca uptake, curve f i t t i n g and compartmental kinetic analysis were similar to those described e a r l i e r (see Section 3.2.2.). In twenty s l i c e s obtained from control and kindled animals e x t r a c e l l u l a r space was also determined using the [^H]inulin method ( K l e i z e l l e r et a l . , 1964). 3.3.3. Results Animals kindled by stimulation of the hippocampal commissures required substantially more d a i l y stimulations (range: 26-31) than their amygdala-kindled counterparts (range: 13-16). No attempt was made to record afterdischarges during the process of kindling, but the t y p i c a l behavioral signs described by Racine (1972b) were observed in both groups. Hippocampal s l i c e s prepared from control and kindled animals exhibited normal v i a b i l i t y during incubation in the 4^Ca uptake chamber. Elec t r o p h y s i o l o g i c a l recordings in these s l i c e s indicated that the c h a r a c t e r i s t i c synaptic a c t i v i t y could be evoked from d i f f e r e n t c e l l types of the hippocampal formation. Although a detailed e l e c t r o p h y s i o l o g i c a l analysis was beyond the 127 scope of the present study, changes in i n h i b i t o r y mechanisms were noted in kindled s l i c e s , similar to those described by Oliver and M i l l e r (1985). In both control and kindled preparations the uptake of radioactive calcium could be best f i t t e d by a double exponential equation of the form: A • [ 1 -exp( - T\ 1 * t,) ] + B-[ 1-exp(- X 2*t) ] ; where A and B are two exponential constants, A-j and A 2 are the reciprocals of the time constants, and t i s time. The asymptote of t h i s function at time t = co represents t o t a l exchangeable calcium of hippocampal s l i c e s and the two exponential terms indicate the presence of two k i n e t i c a l l y d i s t i n c t i n t r a c e l l u l a r C a 2 + pools (see Section 3.2.4.). The computer f i t t e d uptake curves for control and kindled hippocampal s l i c e s are shown in F i g . 3.6. Each uptake curve was drawn using the average values of the exponential parameters obtained in individual experiments. This representation i s j u s t i f i e d since the average of the exponentials i s a more accurate mathematical representation than the exponential equation that would f i t the cumulated data points (Riggs, 1963). The r e s u l t s obtained through kinetic analysis of the 4^Ca uptake curves for two models of i n t r a c e l l u l a r Ca compartmen- t a l i z a t i o n are summarized in Table 3.4.. The s e r i a l (catenary) organization assumes that transfer between the e x t r a c e l l u l a r pool ( S ^ and the slowly exchangeable i n t r a c e l l u l a r compartment (S3) has to occur v i a a rapidly displaceable pool ( S 2 ) . In the 1 28 p a r a l l e l model e x t r a c e l l u l a r C a 2 + (S 2) i s allowed to exchange simultaneously with the two i n t r a c e l l u l a r compartments (S, and S3) while no communication between S, and S3 i s assumed. In either preparation (control or kindled) s i g n i f i c a n t differences regarding Ca* fluxes were not present between these two models, although the p a r a l l e l model consistently indicated a larger slowly exchangeable C a 2 + pool (S3 ) . Both kindled preparations showed marked alterations in the shape of the uptake curves (Fig. 3.6.) r e f l e c t e d by the pool sizes and exchange rates of C a 2 + (Table 3.4.). Si g n i f i c a n t changes in C a 2 + compartment sizes were only detected in s l i c e s obtained from commissural-kindled animals. A 38% enhancement of the S 2 and a marked 55% reduction of the S3 pool was observed. The amygdala-kindled preparation exhibited a s l i g h t but not s i g n i f i c a n t decrease of the S3 compartment. In both kindled preparations the t o t a l of exchangeable i n t r a c e l l u l a r calcium remained f a i r l y constant and was not s i g n i f i c a n t l y d i f f e r e n t from control s l i c e s (Fig. 3.7.). The 3.33 + .12 (mean + S.D.) nmol/mg wet weight of t o t a l exchangeable C a 2 + found in control hippocampal s l i c e s i s in good agreement with previous studies on s l i c e s of brain and other tissues (Stahl and Swanson, 1971; 1972; B a r r i t t et a l . , 1981; Borle, 1981; also see Section 3.2.3. and Table 3.2. ) . The changes in compartment sizes observed in commissural- kindled preparations were p a r a l l e l e d by concomitant alt e r a t i o n s in C a 2 + fluxes and rate constants. Due to the s i g n i f i c a n t decrease of the S3 pool, the exchange rate (FLUX S 2 <--> S3) 1 29 Figure 3.6. 4^Ca uptake curves in control, commissural- (HPC) and amygdala-kindled (AMY) hippocampal s l i c e s . The double exponential curves have been generated by computer from the average values for the kinetic parameters presented in Table 3.4. (n=6-8). Time zero represents addition of 4^Ca and i t i s at least 1 hr following preincubation. Note the s i g n i f i c a n t change in the shape of the kindled uptake curves, p a r t i c u l a r y that of the fast exponential components. JZ 'CD CD CD JE " o E C LU < + CN CD (J o 30 60 TIME ( min ) 131 Table 3.4. E f f e c t of k ind l ing - induced ep i l epsy on C a 2 + exchange rates and compartment s izes of hippocampal s l i c e s . The poo l s , f luxes and ra te constants for both the s e r i a l model system ( e x t r a c e l l u l a r compartment: S^) and the p a r a l l e l model system ( e x t r a c e l l u l a r compartment: S 2 ) of hippocampal c e l l u l a r C a 2 + exchange have been ca l cu la ted according to Sect ion 3 . 2 . 2 . 3 . POOLS ( S l f S 2 , S 3 ) represent the amount of exchangeable C a 2 + , FLUX i s the ra te of C a 2 + exchange at steady state between any compartment, and k i s the ra te constant for exchange between the re spec t ive compartments. Data expressed as mean + S .D. der ived from the k i n e t i c ana lys i s of 6-8 uptake curves each (number of s l i c e s = 24-27 for each uptake curve) , un i t s are as i n d i c a t e d i n Table 3 .2 . Contro l ;Commissural-Kindled Amygdala-Kindled IN SERIES POOL S 2 POOL S, TOTAL [S 2+S. 3] FLUX S-L-S2 FLUX S 2 - S 3 l21 k 2 3 [xlO 3 ] '32 2148 1179 3327 585 41 .280 19.0 .037 204 227 115 171 15 .1 6.6 .017 2954^ + 222 530* + 189 3484^ + 216 868* + 101 19* + 7 .294^ + .03 6.3* + 2.4 .039 .015 2403 + 539 1097 +504 3500^ + 175 947* + 82 91* + 50 .421* + .12 44.1 + 29.7 .056 + .028 IN PARALLEL POOL S x POOL S TOTAL ?S 1 +S 3 ] FLUX S2~S1 FLUX S 2 - S 3 k 12 k 32 1758 1569 3327 532 53 .303 .033 188 194 115 185 25 .1 .01 2792^ 692* 3484* 842" 25* .301 .038 + 214 + 204 + 216 + 107 + 13 + .03 + .01 1956 1544^ 3500* 834* 113* .474^ .069* + 669 + 644 75 90 62 .15 .02 Denotes s i g n i f i c a n t d i f f e r e n c e from contro l (p<0.01 one-way ANOVA). 132 between the respective compartments of the s e r i a l model was diminished, as was the f r a c t i o n of S 2 transported to S 3 per unit time ( 1 ^ 2 3 ) . In the p a r a l l e l model the rate constants were unaffected but the flux between the e x t r a c e l l u l a r compartment and the slowly exchangeable i n t r a c e l l u l a r C a 2 + pool ( S 3 ) was reduced about 50%. Amygdala-kindled s l i c e s , although not characterized by marked alterations in pool sizes, showed a s i g n i f i c a n t f a c i l i - t a t i o n of C a 2 + transport and exchange between a l l compartments. No discrepancies were found between the experiments in which separate incubations of control and kindled hippocampi were made and those in which the two preparations were simultaneously incubated. Results obtained using both procedures are combined and included in Table 3.4. Moreover, the two animals that had implanted stimulating electrodes but received no kindling stimuli exhibited similar hippocampal Ca compartmentalization and exchange rates to that of controls. The differences in 4^Ca uptake ki n e t i c s of kindled hippocampi did not seem to be correlated to the number of f u l l motor seizures. No tendency for further a l t e r a t i o n s was noted in animals with 10 motor seizures when compared with those exhi b i t i n g only 5 convulsions. Therefore the data has been pooled for a l l the kindled preparations regardless of the number of evoked stage 5 seizures. The e x t r a c e l l u l a r space fracti o n derived through [ 3 H ] i n u l i n measurements in control (n=l0) and kindled (n=10) hippocampal s l i c e s yielded similar r e s u l t s , in the range of .29-.31. In 1 33 Figure 3.7. Histograms showing exchangeable calcium pools in hippocampal s l i c e s obtained from control, commissural- (HPC) and amygdala-kindled animals. Note that the t o t a l exchangeable C a 2 + (S 2 + S3) i s similar in a l l three preparations.(* - denotes: s i g n i f i c a n t l y d i f f e r e n t from 'CONTROL'; p<0.0! - one-way ANOVA). 2 + Exchangeable Ca (p moles/mg wet weight) 3 ? o_ CO C>J Control ;HPC-Kindledg-HO • Amygdala - Kindled; 00 Exchangeable Ca (pmoles/mg wet weight) 135 addition the wet weight of hippocampal s l i c e s was found to be highly consistant: control (n=116) = 2.69 + .06 mg; commissural- kindled (n=111) = 2.65 + .06 mg; and amygdala-kindled (n=l09) = 2.68 + .06 mg. 3.3.4. Discussion Hippocampal s l i c e s maintained at a steady state may be characterized in terms of calcium compartmentalization as having two k i n e t i c a l l y d i s t i n c t C a 2 + pools (See Section 3.2.3.1.). The rapidly exchangeble pool has been shown to be l i n e a r l y related to e x t r a c e l l u l a r [ C a 2 + ] and represents the amount bound to the plasma membrane and in a free ionic state. The more slowly exchangeable compartment i s readily modified by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) and by the alkylxanthine derivative 3-isobutyl-1-methylxanthine (IBMX) and i s therefore considered to r e f l e c t Ca 2 + bound by i n t r a c e l l u l a r sequestering and buffering systems (See Section 3.2.3.2.). S l i c e s obtained from the hippocampi of commissural- and amygdala-kindled animals showed marked a l t e r a t i o n s in the kinetics of 4^Ca uptake indicating an abnormal c e l l u l a r C a 2 + regulation. These changes are diagramatically summarized in F i g . 3.8. The size of the buffered C a 2 + compartment is dramatically reduced in commissural kindling and i t i s p a r a l l e l l e d by a s i g n i f i c a n t increase in the rapid calcium flux as r e f l e c t e d by the faster i n i t i a l component of the uptake curve. 136 Figure 3.8. Schematic i l l u s t r a t i o n of a l t e r a t i o n s observed in calcium exchange kinetics of kindled hippocampi. In a l l three cases the diagrams refer to the s e r i a l arrangement of the exchangeable C a 2 + pools. The r e l a t i v e size of the l e t t e r s (representing calcium pools) and arrows (indicating C a 2 + fluxes) r e f l e c t proportional changes in these parameters of c e l l u l a r calcium regulation. E x t r a c e l l u l a r calcium (S-| ) was constant in a l l three conditions. Note the s i g n i f i c a n t enhancement of pool S 2 and reduction of compartment S3 in commissural-kindled s l i c e s and the large calcium fluxes in amygdala-kindled preparations. 1 37 CONTROL c fast [Ca2+] -Proteins COMMISSURAL-KINDLED AMYGDALA-KINDLED Membrane 138 It appears that during commissural kindling, C a 2 + i s transferred from the buffered S3 pool into the unbuffered S 2 compartment. The lack of changes in C a 2 + compartment sizes during amygdala- kindling is more d i f f i c u l t to interpret. The results show a large degree of v a r i a b i l i t y that i s probably due to pooling of i p s i - and c o n t r a l a t e r a l hippocampal s l i c e s with regard to the s i t e of stimulation. This however, was necessary to obtain four or more data points for every uptake curve at each time interval to s a t i s f y the s t a t i s t i c a l accuracy of the curve f i t t i n g 9 + algorithm. Nevertheless, Ca homeostasis i s l i k e l y to be altered in t h i s preparation as well, since there i s a s i g n i f i c a n t enhancement of C a 2 + fluxes and their rate constants. The data does not distinguish which of the variety of factors involved in the control and regulation of i n t r a c e l l u l a r Ca 2 + are altered during kindling. The shapes of the 4^Ca uptake curves presented in F i g . 3.6. resemble those produced by the application of pharmacological agents that interfere with mitochondrial calcium regulation (Borle, 1981b; also see Section 3.2.3.2.). On the basis of t h i s observation, i t may be suggested that mitochondrial C a 2 + uptake i s impaired in kindled hippocampi. Although changes in mitochondrial calcium transport are known to p a r a l l e l neuronal p l a s t i c i t y in the hippocampus (Baudry et a l . , 1983), there i s no evidence as yet to indicate that alterations in mitochondrial function occur during kindling-induced epilepto- genesis (McNamara et a l . , 1980; Kalichman, 1982; Peterson and Albertson, 1982;. McNamara, 1984). The endoplasmic reticulum may also play an important role in C a 2 + sequestration and long-term 1 39 changes in i t s function may e f f e c t i v e l y a l t e r neuronal C a 2 + homeostasis (Duce and Keen, 1978). One recently i d e n t i f i e d biochemical factor involved in i n t r a c e l l u l a r Ca regulation which i s correlated with kindling i s the neuron-specific calcium- binding protein (CaBP) (M i l l e r and Baimbridge, 1983). The fact that there i s a selective decrease in the hippocampal levels of this protein during the process of kindling raises the p o s s i b i l i t y that the a l t e r a t i o n s seen in the 4^Ca uptake kinetics of kindled hippocampal s l i c e s may be due to the loss of this putative intraneuronal Ca^ -buffer (Miller and Baimbridge, 1983; Baimbridge and M i l l e r , 1984). If C a 2 + bound to t h i s protein i s indeed part of the buffered (S3) Ca pool then the marked reduction of th i s compartment following commissural kindling may be explained by the depletion of hippocampal CaBP. Assuming this to be the case, the small change in the size of the S3 pool observed in amygdala-kindled s l i c e s could be a t t r i b u t e d to the r e l a t i v e l y small decrease in hippocampal CaBP produced by amygdala-kindling (Baimbridge et a l . , 1985). Further evidence in favor of the relationship between the S3 compartment and CaBP was obtained from experiments using microdissections of hippocampal s l i c e s . These studies, although preliminary, indicate that the change in 4^Ca uptake k i n e t i c s during commissural kindling i s mainly l o c a l i z e d to the dentate gyrus, where the decline of CaBP is observed, with l i t t l e a l t e r a t i o n in the CA2/CA1 regions of the hippocampal formation (M i l l e r and Baimbridge, 1983). The fact that the number of evoked f u l l motor seizures was not correlated to the magnitude of alterations in C a 2 + homeo- 140 st a s i s indicates that convulsions, themselves are not the primary cause of these changes. Preliminary findings (n=2) in p a r t i a l l y kindled animals with no motor seizures show alterations similar to those of the f u l l y kindled preparations after 20 commissural stimulations. These data again suggest a correlation between the altered ^^Ca kinetics and the loss of hippocampal CaBP since i t has been shown that seizures do not produce a further drop in the le v e l s of the protein that reach a half-maximal decline after only 10 commissural stimulations (Baimbridge and M i l l e r , 1984). It should be noted that t o t a l exchangeable Ca 2 + (the sum of the two c e l l u l a r exchangeable pools) was not altered in either of the kindled preparations. This result is in good agreement with other findings in which measurements of t o t a l hippocampal calcium by atomic absorption spectrophotometry show no s i g n i f i c a n t differences between control and kindled hippocampi (see Section 3.1.3.). Therefore, i t i s concluded that kindling results in a r e d i s t r i b u t i o n rather than an absolute change of the t o t a l hippocampal i n t r a c e l l u l a r C a 2 + . This process involves the mobilization of Ca^ from a pool that appears to be the sum of sequestered/buffered c e l l u l a r C a 2 + . Although many i n t r a c e l l u l a r organelles and systems may be part of th i s C a 2 + compartment (Blaustein et a l . , 1978; 1980; McGraw et a l . , 1982), u n t i l evidence i s available to the contrary, the most l i k e l y p o s s i b i l i t y for the cause of C a 2 + r e d i s t r i b u t i o n during kindling i s the loss of hippocampal CaBP. The observations of th i s study are v a l i d regardless of whether the two exchangeable c e l l u l a r compartments are assumed 141 to be connected in series or in p a r a l l e l with the ex t r a c e l l u l a r C a 2 + . The reason for the s i m i l a r i t y of the two models i s the small f r a c t i o n (0.019) of the unbuffered pool that is normally exchanged per unit time with the Ca* of the buffered compartment in the s e r i a l model. The p a r a l l e l model does not assume such exchange, i . e . , both compartments are only connected to the ex t r a c e l l u l a r space without any reciprocal communication. Based on the method of compartmental analysis developed in th i s study, i t i s evident that kindling results in s i g n i f i c a n t changes in calcium homeostasis in the hippocampal formation. Although this method does not dis t i n g u i s h which c e l l u l a r elements of t h i s heterogeneous structure are involved, i t i s reasonable to suggest that i f the a l t e r a t i o n i s associated with neurons, then their discharge properties would also be altered. In this regard, i t i s notable that following kindling, the granule c e l l s of the dentate gyrus exhibit s i g n i f i c a n t l y modified passive membrane properties (Oliver et a l . , 1983) as well as an enhancement of long-duration i n h i b i t o r y processes (Tuff et a l . , 1983; Oliver and M i l l e r , 1985). Recently Wadman et a l . (1985) have also been able to demonstrate an increase in the stimulus- or amino acid-induced loss in [ C a 2 + K in the dendritic region of CA1 neurons following kindling, indicating a chronic a l t e r a t i o n in C a 2 + - r e g u l a t i o n . The manner in which the change in C a 2 + homeostasis described in the present study may account for the observed electrophysiological changes, or a l t e r n a t i v e l y for some other modifications in biochemical and neurotransmitter functions (McNamara et a l . , 1980; Kalichman, 1982; Peterson and Albertson, 1 42 1982; McNamara, 1984) remains to be determined. Further studies are needed to establish the precise timing of the altered C a 2 + regulation during development of kindling and to establish a direct link to the loss of hippocampal CaBP and the number of recorded afterdischarges. 143 CHAPTER IV. EFFECT OF IBMX ON HIPPOCAMPAL EXCITABILITY In contrast to some damaging long-term a l t e r a t i o n s in nerve c e l l a c t i v i t y , as is the case during epileptogenesis, other p l a s t i c neuronal changes may subserve more v i t a l functions, for example learning and memory. Although the exact molecular mechanisms subserving these important functions have not yet been elucidated, neurophysiological evidence indicates that the hippocampal formation i s a major s i t e of p l a s t i c a l t e r a t i o n s . The f i r s t accounts for the involvement of the hippocampus in learning and memory in humans come from the studies of B. Milner, W. Penfield and W.B. S c o v i l l e on a patient ("H.M.") with b i l a t e r a l hippocampal lesions (Milner and Penfield, 1955; for review see Milner et a l . , 1968). The syndromes of H.M. and those of several other similar patients could be replicated only later on animals with b i l a t e r a l lesions of the hippocampus and the amygdala (Mishkin, 1978). Meanwhile B l i s s and colleagues (Bliss and Lomo, 1973; B l i s s and Gardner-Medwin, 1973) described the phenomenon of long-term potentiaton (LTP) in the hippocampal formation of the rabbit, an event that may represent the 144 underlying p l a s t i c changes necessary for memory formation and learning. It consists of an enhancement of the input/output function of the system that may l a s t for several weeks, or even months in an ir\ vivo preparation following short periods of tetanic stimulation of the perforant pathway. This form of LTP was readily reproducible in rats (Douglas and Goddard, 1975) and i t was subsequently shown that the phenomenon may also be induced in the isolated hippocampal s l i c e preparation (Schwartzkroin and Wester, 1975; Alger and Teyler, 1976; Andersen et a l . , 1977). Naturally, due to the l i m i t a t i o n s of the _iri v i t r o preparation, the LTP of the hippocampal s l i c e i s not as long- l a s t i n g as in chronic experiments but the isolated hippocampus provides the advantage of more dire c t experimental manipulations (Teyler et a l . , 1977). It was however with the hippocampal s l i c e that investigators were able to demonstrate that LTP i s not r e s t r i c t e d to the dentate gyrus but occurs at e s s e n t i a l l y every synaptic relay within the hippocampal formation. Although LTP has since been shown to exist in several other areas of the mammalian CNS such as the superior c e r v i c a l ganglion (Brown and McAfee, 1982; Briggs et a l . , 1985), the medial geniculate nucleus (Gerren and Weinberger, 1983), c o r t i c a l (Lee, 1982) and limbic forebrain structures (Racine et a l . , 1983), the hippocampal formation remains the most extensively studied region with regard to this p l a s t i c a l t e r a t i o n in neuronal function. In spite of several comprehensive review a r t i c l e s , a l l dealing with possible mechanisms underlying LTP ( B l i s s , 1977; B l i s s and Dolphin, 1982; Swanson et a l . , 1982; Eccles, 1983; Voronin, 1983; 1 45 Lynch and Baudry, 1984; Teyler and Discenna, 1984), the exact events responsible for the phenomenon are far from being well understood. The enhanced effic a c y of hippocampal synapses following LTP may be achieved in a multitude of manners. A presynaptic change may lead to a long-term a l t e r a t i o n in neurotransmitter release (Skrede and Malthe-Sorenssen, 1981; Dolphin et a l . , 1982) or al t e r n a t i v e l y , there could be a change in the s e n s i t i v i t y or absolute number of active postsynaptic receptors (Baudry et a l . , 1980; Lynch and Baudry, 1984). A further complexity is introduced by norepinephrine, which under certain circumstances may modulate the induction of LTP (B l i s s et a l . , 1983; Neuman and Harley, 1983; Hopkins and Johnston, 1984). The picture is not much clearer when one examines the i n t r a c e l l u l a r correlates of LTP in the CA1 region (Andersen et a l . , 1977; 1980; Lynch et a l . , 1983) or in the CA3 region of the hippocampus (Yamamoto and Chujo, 1978; Yamamoto et a l . , 1980). The major finding of these studies is the enhancement of the i n t r a c e l l u l a r excitatory postsynaptic potential (EPSP) during LTP while other c e l l u l a r parameters do not seem to be altered. 146 4.1. Introduction Regardless of the exact mechanism(s) involved, the induction of LTP is s t r i c t l y dependent upon the presence of C a 2 + , indicating an important role for the cation in th i s form of neuronal p l a s t i c i t y (for review see Eccles, 1983). Although strontium may support LTP in the hippocampus (Wigstrom and Swann, 1980), e x t r a c e l l u l a r Ca^ appears to be an e s s e n t i a l requirement (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). It has also been established that intraneuronal C a 2 + p a r t i c i p a t e s in the process of LTP since injection of EGTA into hippocampal pyramidal c e l l s blocks the occurrence of the phenomenon (Lynch et a l . , 1983). Furthermore, LTP was found to be associated with a s i g n i f i c a n t uptake and a transient retention of calcium (Baimbridge and M i l l e r , 1981) and could be produced either in v i t r o (Turner et a l . , 1981; Mody et a l . , 1984) or iji vivo ( B l i s s et a l . , 1984) by a' brief elevation of the e x t r a c e l l u l a r Ca 2 + concentration ('calcium-induced LTP'). Several lines of evidence suggest however, that calcium i s not d i r e c t l y involved in the mechanism of LTP but rather through an i n t r a c e l l u l a r messenger system that t r i g g e r s some long-lasting biochemical alterations (for reviews see Eccles, 1983; Lynch and Baudry, 1984). One of the prime candidates as the mediator of C a 2 + - e f f e c t s is the i n t r a c e l l u l a r calcium receptor protein c a l - modulin (Cheung, 1980). Its involvement in LTP i s l i k e l y , since calmodulin blocking agents such as neuroleptic drugs were shown 147 to i n h i b i t t etanic- and calcium-induced LTP in the hippocampus (Finn et a l . , 1980; B l i s s et a l . , 1984; Mody et a l . , 1984). Calmodulin may part i c i p a t e at v i r t u a l l y every l e v e l of control of neuronal e x c i t a b i l i t y (Cheung, 1980; Means and Dedman, 1980), but in view of the role of c y c l i c nucleotides in the CNS (Rasmussen and Goodman, 1977; Rasmussen and Barrett, 1984), calmodulin's influence on c y c l i c nucleotide metabolisms seems of primary importance. Fa'cilitation of LTP induction by norepinephrine ( B l i s s et a l . , 1983; Neuman and Harley, 1983; Hopkins and Johnston, 1984) may occur via the cAMP system which is known to enhance the e x c i t a b i l i t y of hippocampal neurons (Segal, 1981; Madison and N i c o l l , 1982). Therefore, a c t i v a t i o n of calmodulin by C a 2 + i s probably only the f i r s t step in a chain of biochemical events involving c y c l i c nucleotide metabolism and/or protein phosphorylation that ultimately lead to LTP. 9 + The source of Ca for ac t i v a t i o n of LTP i s not known. Although enhanced uptake and retention of the cation have been shown to be correlates of LTP (Baimbridge and M i l l e r , 1981), the release of C a 2 + from intraneuronal storage s i t e s cannot be excluded from the process. The aim of the present study has therefore been to elucidate some of the steps and mechanisms subserving LTP by the use of drugs that interfere with i n t r a c e l l u l a r calcium homeostasis and the catabolism of c y c l i c nucleotides. While a large variety of compounds are available that block calcium entry into nerve c e l l s , agents that interfere with the regulation of intraneuronal calcium are less common. For the present experiments, methylxanthine derivatives were the 1 48 drugs of choice, since they are believed to have two properties that may interfere at two d i f f e r e n t steps in the process of LTP induction. F i r s t l y , these compounds have been shown to release C a 2 + , at least in the case of sk e l e t a l muscle, from i n t r a c e l l u l a r storage s i t e s (Endo, 1977; M i l l e r and Thieleczek, 1977; Martonosi, 1984). Secondly, they are e f f e c t i v e i n h i b i t o r s of the enzyme phosphodiesterase (PDE) responsible for the breakdown of c y c l i c nucleotides (Chasin and Harris, 1976). Hence i f the Ca z - calmodulin-cyclic nucleotide step-by-step activation subserves the mechanism of LTP, i t would be expected that methylxanthines could act as long-lasting potentiators of neuronal a c t i v i t y . The augmentation of neuronal e x c i t a b i l i t y could be quite large, since the compounds act at two possible synergistic steps of LTP a c t i v a t i o n : the increase in c y t o s o l i c calcium and the elevation of c y c l i c nucleotide levels by i n h i b i t i o n of the enzyme responsible for their breakdown. The present study was c a r r i e d out using the methylxanthine compounds theophylline (1,3-dimethylxanthine) and 3-isobutyl-1- methylxanthine (IBMX) primarily because of their potent i n h i b i t o r y e f f e c t s on the enzyme phosphodiesterase (Chasin and Harris, 1976; Smellie et a l . , 1979). In addition, IBMX has been shown to release C a 2 + from i n t r a c e l l u l a r storage s i t e s , in the sarcoplasmic reticulum of skeletal muscle (Miller and Thieleczek, 1979) and the hippocampal s l i c e preparation (see Section 3.2.3.2). Papaverine, a non-methylxanthine compound which has some r e l a t i v e l y s p e c i f i c PDE-inhibitory action, was also used in 1 49 order to distinguish between possible Ca 2 + and c y c l i c nucleotide effects on LTP. 4.2. Methods 4.2.1. Ex t r a c e l l u l a r recordings and analysis Sl i c e s (450 urn in thickness) were prepared from the hippocampi of male Wistar rats (200-250 g) using a Sorvall tissue chopper. They were transferred with the aid of a pipette to a chamber that allowed long-term storage as well as experimental manipulation and electrophysiological recordings to be under- taken. The a r t i f i c i a l cerebrospinal f l u i d (CSF) was perfused at a rate of 2 ml/min in the recording chamber, 0.5 ml/min in the storage chamber and contained 124 mM NaCl, 3.75 mM KC1, 1.25 mM KH 2P0 4, 24 mM NaHC03, 1.5 mM CaCl 2, 1.5 mM MgS04 and 10 mM glucose dissolved in b i - d i s t i l l e d water. The 'Ca 2 +-free' solution was prepared by omitting C a C l 2 . When 3 mM CoCl 2 was added, MgSO^ and KH 2P0 4 were replaced by equimolar amounts of MgCl 2 and KC1 respectively. The medium was continuously warmed (35 + 0.5 °C) and oxygenated with a 95% 0 2, 5% C0 2 gas mixture. In addition to the oxygenation of the medium, warmed and humidified gas mixture was allowed to flow on the top of the s l i c e s . The a r t i f i c i a l CSF used during preparation of the tissue also contained .003% H 20 2 to maintain a better oxygen uptake (Llinas et a l . , 1981). 150 Elec t r o p h y s i o l o g i c a l recordings were started following one hour of e q u i l i b r a t i o n of the s l i c e s at a constant temperature of 35 + 0.5 °C. Recording electrodes (resistances 4-10 Mohms) were pulled (using a Narashige or a Frederick Haer microelectrode puller) from glass c a p i l l a r i e s (outer diameter of 1.5 mm with Omega Dot, Frederick Haer) and were b a c k f i l l e d with 2 M NaCl solution using a 31 gauge hypodermic needle. The electrodes, mounted on micro-manipulators, were positioned under v i s u a l guidance with the aid of a 4x dissecting microscope in the stratum pyramidale and stratum radiatum to record the e x t r a c e l l u l a r population spikes and EPSPs respectively. Stimulating electrodes, also on micromanipulators, consisted of twin nichrome wires (62 um in diameter) and were used to deliver 0.1 ms square wave pulses of 0.5 - 50 V intensity from i s o l a t i o n units (Medical Systems. Corp., Model DS2) with a baseline frequency of 0.133 Hz. These electrodes were positioned in the Schaffer collateral/commissural fiber system of the stratum radiatum for orthodromic activation of the CA1 pyramidal c e l l s . In some experiments an additional stimulating electrode was placed in the alveus to evoke antidromic responses. To test the degree of recurrent i n h i b i t i o n on the pyramidal neurons, an antidromic (conditioning - C) stimulus was delivered followed by orthodromic (test - T) stimulation (C-T i n t e r v a l : 20 ms). Input/output curves for the EPSP and population spike responses were obtained in each experiment by setting the orthodromic stimulus in t e n s i t y to evoke an i n i t i a l EPSP response of 1 mV 151 amplitude in the stratum radiatum and subsequently incrementing the stimulation intensity in 15-20 equal steps. Potentials, referenced to a s i l v e r - s i l v e r chloride bath ground, were recorded with a precision electrometer (M-707 WP Instruments) and displayed on a storage oscilloscope (Tektronix) afte r being f i l t e r e d at 0.1-10 KHz. The amplified signals were led to a PDP 11/23 computer for on-line averaging, data storage and analysis. Usually four consecutive sweeps were averaged over a period of one minute. The amplitude of the c h a r a c t e r i s t i c positive-negative-positive CA1 population spike was measured peak to peak from the f i r s t p o s i t i v i t y ( P ^ to peak negativity (N-j ) (Fig. 4.1.A.). The amplitude of the antidromic population spike recorded in the stratum pyramidale and that of the fiber volley and EPSP recorded in the stratum radiatum was measured as peak negativity compared to baseline (Fig. 4.1.B.). The rate of r i s e of the EPSP response was taken as the peak of the f i r s t order voltage d i f f e r e n t i a l : dV/dt (Fig. 4.1.C.). Drugs were dissolved in the a r t i f i c i a l CSF to y i e l d the f i n a l concentration (usually 100 uM) and were applied by perfusion. A three-way valve system allowed switching between perfusates. The design of the recording/incubation chamber made i t possible to have the experimental s l i c e s exposed only once to a drug and then another s l i c e from the storage part of the chamber could be transferred into the recording compartment. Addition of the drugs to the medium did not change i t s pH, which remained constant at 7.4 + 0.03. Theophylline, papaverine- hydrochloride and 3-isobutyl-1-methylxanthine (IBMX) were 1 52 Figure 4.1, Measurement of e x t r a c e l l u l a r potentials in the CA1 region of the hippocampal s l i c e preparation. A. Stratum radiatum evoked population spike recorded at the l e v e l of pyramidal c e l l s . Its amplitude was measured as the voltage difference between the f i r s t p o s i t i v i t y (P^) and the negative defl e c t i o n (N,). B. E x t r a c e l l u l a r EPSP response recorded in the stratum radiatum at the l e v e l of the apical dendrites. Stimulation was delivered to the Schaffer collateral/commissural fiber system. The amplitude of the response was measured as peak negativity (marked with a cross) calculated from baseline (BL). The negative def l e c t i o n which precedes the EPSP marked 'fv' represents the a c t i v a t i o n of presynaptic fibers and is termed fiber v o l ley. C. F i r s t order voltage d i f f e r e n t i a l (dv/dt) of the EPSP p o t e n t i a l . Its peak amplitude (as measured from baseline) corresponds to the fastest rate of r i s e of the EPSP ( l e f t arrow) while i t s value becomes zero during the peak of the EPSP (right arrow). C a l i b r a t i o n bars: horizontal: 5 ms; v e r t i c a l : 2 mV (for A. and B.) and 5 mV/ms (for C.) 153 154 Figure 4,2. Chemical formulae of the drugs used in the present study. Note the s i m i l a r i t y in structure between IBMX and theophylline (1,3-dimethylxanthine). Papaverine is an iso- quinoline derivative and a potent phosphodiesterase i n h i b i t o r . 1 5 5 3 - Isobutyl - l-mefhylxanfhine (IBMX) o H,C II H S W N > , - N 1ST I C H 2 H X - C H - C H - Theophylline H 3 C ^ H 1 N' •N CH-, Papaverine 1 56 obtained from Sigma Chemical Company. Their respective chemical formulae are presented in Fi g . 4.2. 4.2.2. I n t r a c e l l u l a r recording and data analysis The method for preparation and maintenance of hippocampal s l i c e s was the same as described in the previous section. I n t r a c e l l u l a r recording electrodes were pulled from glass c a p i l l a r y tubing (outer diameter of 1.50 mm with Omega Dot, Frederick Haer) and were f i l l e d with 2 M potassium acetate (MCB) or in some cases with 2 M potassium methylsulphate (ICN Pharma- c e u t i c a l s ) . The e l e c t r o l y t e solutions were c a r e f u l l y f i l t e r e d through mil l i p o r e f i l t e r s to eliminate p a r t i c l e s and impurities. Electrode impedances ranged from 30 to 75 Mohms as measured with the aid of an electrometer in the medium of the recording chamber. The. electrodes were mounted on a variable speed p i e z o e l e c t r i c advance manipulator (Burleigh Inchworm PZ 550) and were lowered into the stratum radiatum under visual guidance with the a i d of a dissecting microscope. The ch a r a c t e r i s t i c s of the recording electrodes were examined before penetration of the c e l l s was attempted. While in the a r t i f i c i a l CSF, the electrodes were balanced through a Wheatstone bridge c i r c u i t and were considered adequate for i n t r a c e l l u l a r analysis i f passing . 75 - 1.0 nA of inward or outward current produced a minimal voltage defl e c t i o n (<2 mV). Impalements of hippocampal CA1 pyramidal c e l l s were obtained by a brief activation of capacitive feedback through the 157 electrode. Following penetration of the c e l l the voltage response dropped -40 to -60 mV and action potentials caused by membrane-injury were also noted. A variable DC source was used for hyperpolarizing current injections (~ 1.0 nA) into the c e l l s immediately following their penetration as well as for variable amplitude command pulses. Potentials, referenced to the bath Ag-AgCl2 ground, were recorded with a precision electrometer (M 707, WP Instruments). The signals were led to a storage oscilloscope, f i l t e r e d 10 kHz (DC), displayed for photography and further led to a PDP 11/23 computer for on-line averaging, data storage and analysis. The passive membrane c h a r a c t e r i s t i c s of hippocampal pyramidal c e l l s were determined as follows: (i) resting membrane potential (RMP) was taken as the potential difference between i n t r a - and ex t r a c e l l u l a r environment following removal of the electrode from the c e l l . ( i i ) Input resistance ( R n^ w a s calculated by the computer as the slope of the regression l i n e between current injected through the microelectrode and membrane voltage at 6-9 di f f e r e n t values of injected current up to 1.0 nA. The current pulses were of 100-150 ms duration and the resulting membrane voltage perturbation was measured after i t has reached a steady value (usually after 40-50 ms). When injection of multiple current pulses was not undertaken, R n was estimated from the voltage deflection caused by a single inward current inj e c t i o n of 0.5 or 1.0 nA. ( i i i ) The membrane time constant (T c) was also derived by computer from the slope of the regression l i n e drawn to the plot of time vs. the natural logarithm of the changes in 1 58 membrane charging p r o f i l e . The changes in membrane voltage were sampled every 32 us ensuring high accuracy of the measurements. The f i t t i n g of the regression l i n e was repeated at d i f f e r e n t levels of hyperpolarizing current injections and an average time constant was calculated from several determinations. To study the accommodation properties of hippocampal pyramidal c e l l s during a long-duration (600 ms) depolarizing current pulse, the signals were led to the PDP 11/23 computer through a peak detector. This ensured accurate counting of the evoked spikes as well as their continuous raster-type display. Since there is a reasonable v a r i a b i l i t y in the current-evoked discharge patterns of pyramidal c e l l s , trends of discharge and minor changes with time are d i f f i c u l t to observe through vis u a l examination of oscilloscope photographs or chart-recordings. The continuous raster procedure however, allowed for complete elimination of bias in the estimation of the number of spikes evoked during a given current pulse. In addition, post-stimulus time histograms (PSTs) were also constructed by the computer to check for the significance in changes of pyramidal c e l l f i r i n g patterns. 1 59 4.3. Results 4.3.1. Effect of drugs on stratum radiatum evoked potentials Within 5 min of i t s perfusion 100 pM IBMX caused a marked increase in the size of the population spike responses recorded in the pyramidal c e l l layer. This e x t r a c e l l u l a r potential represents the synchronous discharge of pyramidal neurons after their synaptic a c t i v a t i o n (Andersen et a l . , 1971). The potentiation was further enhanced during the perfusion and reached a maximum of 300-900% of the i n i t i a l response at the end of the 10 min administration period. Following return to the normal a r t i f i c i a l CSF the population spike f a i l e d to decline or showed only a s l i g h t recovery after 2-3 hours of wash (Fig. 4.3.). This effect was maximal with IBMX concentrations of 100 pM. At lower drug concentrations (e.g., 50 uM) this e f f e c t was not as dramatic but was as long-lasting (n=3). At a concentration of 10 pM only a s l i g h t potentiation of the stratum radiatum evoked response was observed which following the wash period returned to pre-drug lev e l s (n=5). Invariably, no effect of IBMX was recorded on the antidromically evoked synchronous discharges of pyramidal neurons, suggesting that a change in pyramidal c e l l e x c i t a b i l i t y did not occur in the presence of the drug (Fig. 4.4.). The effect of IBMX on the e x t r a c e l l u l a r EPSP response recorded at the l e v e l of the api c a l dendrites was similar to that 1 60 Figure 4.3. Effect of various drugs on the amplitude of population spikes evoked in the CA1 region of the hippocampal formation. Each response represents the average of four consecutive sweeps as displayed by the computer. Left column: control responses taken prior to the drug perfusions. Middle column: changes in the amplitude of the responses at the end of a 10 min perfusion period. Right column: population spikes recorded at indicated times following washout of the drugs. Note that IBMX and theophylline both caused a marked potentiation of the evoked responses, but only the effect of IBMX was long- l a s t i n g . Papaverine produced a non-significant potentiation during i t s perfusion and caused a s l i g h t depression of the population spikes during the washout period. Control 10 min. Drug Wash I B M X ,50 min 100 /JM Theophylline 15 m j n 100 >uM 5 mV 5 ms Papaverine 1 0 0 /JM 10 min 162 observed for the population spike, but of lesser magnitude (50-400 % ) . Concurrent with the increase in EPSP amplitude there was a c h a r a c t e r i s t i c reduction in the latency to peak amplitude (Andersen et a l . , 1980). Following stimulation of afferent fibers of the stratum radiatum a small negative f i e l d potential may be recorded in the region of the api c a l dendrites of CA1 pyramidal c e l l s . This potential precedes the EPSP and has been termed 'fiber volley' since i t i s thought to represent impulse propagation through presynaptic fibers (Andersen et a l . , 1978). The amplitude and latency of the fiber volley was unchanged by perfusion of IBMX and no long-lasting e f f e c t s were detected. As shown in Fig . 4.3., the effect of IBMX on .the CA1 population spike responses was to induce a long-lasting potentiation. This action was observed in more than 40 s l i c e s and in some cases, where long and stable recordings could be obtained, the evoked responses f a i l e d to return to control levels even after 3-3.5 hours of washout. Therefore, i t may be stated that analogous to the tetanic stimulation- or calcium-induced LTP, IBMX has caused long-lasting enhancement of the stratum radiatum evoked responses in the CA1 region of the hippocampal formation. The potent LTP-inducing effect of IBMX was not mimicked by the other methylxanthine derivative theophylline. Theophylline (at 100 uM), also an e f f e c t i v e adenosine antagonist (Dunwiddie and Hoffer, 1980; Dunwiddie et a l . , 1981) and a c y c l i c nucleotide phosphodiesterase i n h i b i t o r (Chasin and Harris, 1976), caused a reversible increase in the size of the population spikes and 1 63 EPSPs. In a l l s l i c e s examined (n=8) the potentiation induced by theophylline was approximately half of that caused by IBMX at a similar concentration and the recorded potentials returned to pre-drug l e v e l s within 10-15 min of the wash period (Fig. 4.3.). These results are similar to data reported by Dunwiddie and Hoffer (1980) who attributed the changes to blockade of adenosine receptors. Perfusion of papaverine in seven s l i c e s (100 pM) caused an i n i t i a l potentiation then a s l i g h t i n h i b i t i o n of stratum radiatum evoked responses (Fig. 4.3.). Since papaverine has been shown to be a potent inh i b i t o r of [ ] a d e n o s i n e uptake into c o r t i c a l synaptosomes (Bender et a l . , 1980) i t s effect may be related to enhancement of adenosine responses in addition to i n h i b i t i o n of phosphodiesterase. The unique LTP-inducing effect of IBMX was not shared by compounds that have presumably similar actions on adenosine receptors and the enzyme phosphodiesterase, indicating that some other property of IBMX may be responsible for the LTP. To further examine the LTP induced by IBMX, the drug's ef f e c t was tested on some parameters of neuronal e x c i t a b i l i t y known to be altered by other forms of LTP. 4.3.2. Effect of IBMX on paired-pulse i n h i b i t i o n Haas and Rose (1982) concluded that changes in inhibitory events are not responsible for LTP in the CA1 area of the hippocampus. However, when i n h i b i t i o n i s blocked in the presence 1 64 of GABA antagonists, the induction of LTP i s f a c i l i t a t e d (Wigstrom and Gustafsson, 1 983). If the LTP-induc.ing e f f e c t of IBMX i s p a r t i a l l y due to a decrease of GABA-mediated responses, then the drug should reduce paired-pulse i n h i b i t i o n of hippocampal pyramidal c e l l s . The paired-pulse paradigm consists of de l i v e r i n g two st i m u l i , usually separated by 20-40 ms, to hippocampal fiber systems that w i l l activate the pyramidal neurons. A conditioning ortho- or antidromic stimulation of CA1 pyramidal c e l l s results in subsequent activation of basket c e l l s which in turn have in h i b i t o r y actions on pyramidal c e l l s . If i n h i b i t i o n i s present, the next orthodromic test stimulation w i l l evoke a population spike of smaller magnitude (Fig. 4.4.A.). The duration of the i n h i b i t i o n following the conditioning pulse is dependent upon the stimulation paradigm, but i t has been shown to la s t in excess of 40 ms (Haas and Rose, 1982). Perfusion of IBMX did not a l t e r synaptic events responsible for paired-pulse i n h i b i t i o n of CA1 pyaramidal neurons (n=5). In spite of the c h a r a c t e r i s t i c potentiation of the population spike responses in the presence of IBMX, the test (T) stimulus delivered 20 ms following antidromic conditioning (C) invariably evoked an inhi b i t e d population spike (Fig. 4.4.B.). When the intensity of orthodromic stimulation was reduced to evoke a response comparable to pre-drug values the potency of i n h i b i t i o n was found to be unaltered (Fig. 4.4.C.). 165 Figure 4.4. Effect of IBMX on paired-pulse i n h i b i t i o n recorded e x t r a c e l l u l a r l y . The traces were photographed from a storage oscilloscope and represent population spikes (top) and EPSPs (bottom) following stimulation of the Schaffer c o l l a t e r a l - commissural afferent fibers ( • ) or antidromic activation (•) of the pyramidal c e l l s . A. Control population spike and EPSP (right) and their paired-pulse i n h i b i t i o n when an antidromic conditoning stimulus (C) precedes the orthodromic a c t i v a t i o n (T) of pyramidal c e l l s (C-T i n t e r v a l = 20 ms). B. Same as in A. but following IBMX-induced LTP. Note the large potentiation of the orthodromic population spike (~400%) with no s i g n i f i c a n t change in the antidromic response. Inhibition was s t i l l present as indicated by the smaller amplitude of the test response. Stimulus i n t e n s i t i e s for ortho- and antidromic a c t i v a t i o n were the same as in A. C. To test whether the potency of paired-pulse i n h i b i t i o n was the same as under control conditions, the orthodromic stimulation intensity was reduced (about 50%) while keeping the antidromic stimulation constant. No s i g n i f i c a n t change in the magnitude of the i n h i b i t i o n can be detected following IBMX-induced LTP. 166 167 4.3.3. Effect of IBMX on input/output (I/O) curves LTP i s characterized by sp e c i f i c a l t e r a t i o n s in the relationship of input/output curves (Schwartzkroin and Wester, 1975; Alger and Teyler, 1976; Andersen et a l . , 1977; Andersen et a l . , 1980). By monitoring individual parameters such as stimulus intensity, fiber volley amplitude, rate of r i s e of the EPSP or the amplitude and latency of EPSP and population spike i t i s possible to treat each variable as 'independent' and to express the changes in their relationships accordingly. To use the short notations somewhat similar to those of Andersen et a l . (1980) the curves w i l l be referred to as SI-S, SI-V, V-E, V-D, D-E and D-S relat i o n s , where SI = stimulus intensity, S = population spike amplitude, V = fiber volley amplitude, E = EPSP amplitude and D = rate of r i s e of the EPSP (dV/dt). As pointed out by Andersen et a l . (1980) some of these relations may be indicative of pre- synaptic changes (e.g., SI-V, V-E and V-D), while others may r e f l e c t postsynaptic events (e.g., D-E and D-S) that occur during LTP. The I/O curves shown on Figs. 4.6.-4.8. represent plots as measured from the potentials shown in F i g . 4.5. Input/output relationships using 15-20 di f f e r e n t stimulation i n t e n s i t i e s were examined in 15 s l i c e s . Twenty averaged and superimposed EPSPs (Fig. 4.5.A.) and population spikes (Fig. 4.5.B.) are displayed, as part of a t y p i c a l I/O experiment. The net ef f e c t of IBMX was to reduce the threshold for evoking a population spike. 1 68 Figure 4.5. Potentials recorded in the dendritic and somatic region of area CA1 during input/output curves. Each trace i s an average of four sweeps superimposed by the computer and represents the EPSPs (A.) and populations spikes (B.) evoked following 20 successive stimulations of the Schaffer c o l l a t e r a l - commissural f i b e r s (from 2.0 to 5.8 V in increments of 0.2 V). Control responses are shown on the l e f t panel while the responses on the right have been obtained after 40 min of washout following a 10 min IBMX perfusion of 100 uM concentration. Note the increase in the amplitude of the evoked responses at every stimulation i n t e n s i t y and the lack of change in the f i b e r volley potential which precedes the EPSP. 169 1 70 Therefore, at every stimulus in t e n s i t y , larger population spikes were recorded in the pyramidal c e l l layer after exposure to the drug. The SI-S relationship shown in F i g . 4.6.A. indicates that during IBMX-induced LTP, maximum potentiation (800-900%) occurs at intermediate stimulus i n t e n s i t i e s of about 1.5x threshold for evoking a population spike. The change in threshold after IBMX- induced LTP represents a s h i f t to the l e f t of the SI-S curve, similar to that obtained by Andersen et a l . (1980) following LTP produced by tetanic stimulation. A given stimulus intensity (Si) evoked comparable fiber volley (V) potentials both before and after perfusion of IBMX (Fig. 4.6.B.) indicated by the fact that no s i g n i f i c a n t s h i f t was observed in the SI-V re l a t i o n s h i p . Since the fiber volley represents impulse propagation through presynaptic fibers (Andersen et a l . , 1978), there was probably no change in the number of presynaptic f i b e r s activated by a given intensity of stimulation following perfusion of IBMX. This finding, observed in a l l preparations examined, i s analogous to the SI-V r e l a t i o n s h i p following tetanus-induced LTP (Andersen et a l . , 1980), which also does not seem to change presynaptic e x c i t a b i l i t y . The I/O curves most sensitive to synaptic changes are the V-E and V-D relationships. The amplitude (E) and the rate of r i s e or dV/dt (D) of the e x t r a c e l l u l a r EPSP represent current flow during synaptic a c t i v a t i o n of pyramidal neurons. When these parameters are plotted against the amplitude of the fiber volley (V), they usually show a li n e a r r e l a t i o n s h i p . As shown on F i g . 171 Figure 4.6. I/O curves having stimulus intensity (SI) on the abscissa. A. Plot of stimulus intensity (SI) vs. population spike amplitude (S), c a l l e d the 'SI-S' curve. Note the change in threshold stimulus intensity for evoking a population spike following perfusion of IBMX. The maximal potentiation was observed between stimulation i n t e n s i t i e s of 4-4.5 V, which were approximately 1.5x the o r i g i n a l threshold value. B. Plot of stimulus intensity (SI) vs. the amplitude of the fiber volley response (V), i . e . , the 'SI-V curve. Both regression l i n e s were f i t t e d by computer and no s i g n i f i c a n t changes were detected in either correlation c o e f f i c i e n t or slope of the relationship after LTP induced by IBMX. In the present and the following I/O curves 'IBMX' refers to potentials recorded after 40 min of washout, i . e . , following LTP induced by a 10 min perfusion of 100 pM IBMX. 172 173 4.7.A and B.), the l i n e a r i t y was not changed during LTP induced by IBMX but the slopes of the regression l i n e s were s i g n i f i c a n t l y enhanced (40-60%). This increase in slope occurred concomitant with a s h i f t of the l i n e s to the l e f t , i . e . , a comparable f i b e r volley response (V) invariably evoked a larger amplitude EPSP (E) and a greater dV/dt (D) after IBMX-induced LTP. The change in slope and the s h i f t to the l e f t of the V-E and V-D relationships, found in a l l s l i c e s examined (15/15), indicates that a similar presynaptic activation (V) results in a s i g n i f i c a n t l y larger synaptic drive and e f f i c a c y (characterized by E and D) following perfusion of IBMX. As shown by the overlapping data points of F i g . 4.8.A. and B. taken before and after IBMX perfusion, no s i g n i f i c a n t differences were found between the curves representing D-E and D-S relationships. Following IBMX-induced LTP both D-E and D-S curves showed some overlap with their control counterparts appeared as extensions of the control curves. This however was only due to the greater dV/dt (D) of the EPSPs evoked after LTP. While the D-E curves did not appear shifted in every preparation examined, the D-S curves exhibited a l t e r a t i o n s in 6/15 s l i c e s . In these remaining s l i c e s the D-S curves were shifted to the right (4/15) or to the l e f t (2/15), but these s h i f t s were found not to be s t a t i s t i c a l l y s i g n i f i c a n t . The lack of s i g n i f i c a n t changes in the D-E and D-S curves indicate that similar synaptic activation (reflected by D) evokes a comparable amplitude EPSP (E) or population spike (S) even after LTP has been induced by IBMX. 174 Figure 4.7. I/O curves having fiber volley amplitude (V) on the abscissa. A. The V-E relationship, or the plot of fiber volley amplitude (V) vs. the the amplitude of the e x t r a c e l l u l a r l y recorded EPSP (E). B. Plot of the fiber volley amplitude (V) vs. the rate of r i s e of the EPSP (dV/dt or D), i . e . , the V-D rela t i o n s h i p . In both graphs the regression l i n e s were f i t t e d by computer. Note the s h i f t to the l e f t of both l i n e s after IBMX- induced LTP as well as the s i g n i f i c a n t difference in slopes (p<0.1 for A. and p<0.005 for B. as measured by two t a i l e d t- test) . 175 0-50 0.75 | FIBER VOLLEY AMPLITUDE ( mV ) 1 7 6 Figure 4.8. I/O curves with the rate of r i s e of the EPSP (D) on the abscissa. A. Plot of the rate of ri s e of the EPSP (D) vs. i t s own amplitude (E), or the D-E curve. B. The D-S relationship, i . e . , the EPSP's dV/dt vs. the amplitude of the population spike before and after IBMX-induced LTP. In either plot no s i g n i f i c a n t differences were detected in the region of overlap of the 'control' and 'IBMX' curves. This i s to show that aft e r LTP induced by IBMX a comparable size dV/dt evokes a similar EPSP or population spike response. The reason for the apparent extension of the 'IBMX' curves beyond the 'controls' is the s i g n i f i c a n t enhancement of the EPSP's rate of r i s e following IBMX-induced LTP.  178 4.3.4. Calcium and IBMX-induced LTP The presence of e x t r a c e l l u l a r C a 2 + i s required for tetanus- induced LTP in the hippocampal s l i c e preparation (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979) and s l i c e s retain s i g n i f i c a n t l y larger amounts of calcium when challenged with a high frequency stimulus (Baimbridge and M i l l e r , 1981). In addition, Turner et a l . (1982) have shown that the increase in t o t a l i n t r a c e l l u l a r Ca* resulting from brief exposure to a higher than normal e x t r a c e l l u l a r calcium concentration may by i t s e l f cause LTP. To examine the contribution of calcium entry to the LTP induced by IBMX, the. e f f e c t of the Ca 2 + -channel blocker C o 2 + was tested. Within 30-35 min, perfusion of 3 mM CoCl 2 in 6 hippocampal s l i c e s gradually decreased the amplitude of the CA1 evoked responses, indicating that synaptic transmission was impaired (Fig. 4.9.A. and B.). When 100 pM IBMX was perfused for 10 min in the presence of Co 2 +, the responses became potentiated to levels 200-300% larger than pre-cobalt values. Upon return to normal CSF both the population spike (6/6) and EPSP (3/6) remained potentiated, i . e . , LTP had occurred (Fig. 4.9.C.) indicating that Ca^ entry per se was not a prerequisite of IBMX- induced LTP. In order to determine whether synaptic events were required to produce the IBMX-induced LTP, synaptic potentials were reduced or abolished by perfusing 'Ca 2 +-free' medium for a variable time. 179 Figure 4.9. Effect of Co 2 + on IBMX-induced LTP. The potentials are photographs of oscilloscope traces following stimulation of the Schaffer collateral/commissural fiber system and represent population spikes recorded at the le v e l of CA1 pyramidal c e l l s (top panel) or EPSPs recorded near the apical dendrites (bottom panel). A. Control evoked potentials. B. Reduction of the 9 + evoked responses following a 30 min perfusion of 3 mM Co - containing a r t i f i c i a l CSF. C. In spite of the presence of the calcium entry bocking agent Co 2 +, IBMX caused a potentiation of the evoked responses , p a r t i c u l a r l y evident for the population spikes, since the EPSP response contains an i n t e r f e r i n g r e f l e c t i o n of the large population spike. These potentials were recorded at 20 min of washout following a 10 min IBMX perfusion.  181 (Note: omission of CaCl 2 from the a r t i f i c i a l CSF does not ensure t o t a l loss of the cation since the glassware used for preparation of solutions i s a considerable source for C a 2 + . It is therefore reasonable to assume that the 'Ca 2 +-free' medium contains at least 10-50 uM C a 2 + , as measured by Yaari et a l . , 1983). The concentration of Mg 2 + was raised in this medium (from 1.5 mM to 9 mM) to reduce synaptic transmission and to avoid spontaneous bursting (Richards and Sercombe, 1970; Haas and Jefferys, 1982). In the presence of the above perfusate, synaptic responses were greatly diminished and decreased to non-detectable levels within 35-45 min (n=6). When IBMX was perfused prior to the complete loss of synaptic transmission at a point when a reasonable size (>1 mV) EPSP (but no population spike) could s t i l l be evoked, the drug caused the c h a r a c t e r i s t i c potentiation (Fig. 4.10.A. and B.). However, the potentiation lasted only as long as IBMX was present in the medium. Following return to normal perfusate the responses returned to control values showing no LTP. When s u f f i c i e n t time (~ 60 min) was allowed for the preparation to reach the state of zero synaptic transmission in the absence of e x t r a c e l l u l a r C a 2 + (Fig. 4.11.A. and B.), IBMX- induced potentiation was not observed . The antidromic population spike and the fiber volley were only s l i g h t l y affected during the 30-60 min of '0 mM C a 2 + ' perfusion (Figs. 4.10.C. and 4.11.C). It was only during the perfusion of IBMX (Figs. 4.10.C. and 4.11.C.) under these conditions that the amplitude of the antidromic population spike reversibly decreased. This finding i s somewhat suprising since 182 Figure 4.10. Effect of a short duration (<30 min) perfusion of 0 mM Ca 2 +/9 mM Mg 2 + solution on the action of IBMX in the CA1 region of the hippocampus. When potentials were s t i l l evokable (after about an 18 min perfusion of the low Ca z medium) 100 uM IBMX caused only a reversible potentiation of the EPSP (A.) and population spike (B.) responses. The amplitude of the fiber volley (C. X) declined s l i g h t l y during the perfusion of the '0 mM C a 2 + ' medium but returned to control levels during IBMX perfusion. The changes in the amplitude of the antidromically evoked population spike (C. A ) were not s i g n i f i c a n t in the lack of e x t r a c e l l u l a r Ca . However, perfusion of IBMX during the low C a 2 + medium produced a transient reduction of the antidromic spike. The bars above panel A. indicate the respective perfusion durations and refer to a l l of the panels. 1 ro O" m 3 Z3 u O " o AMPLITUDE ( mV ) 1 3 O POPULATION SPIKE AMPLITUDE ( mV ) CD EPSP AMPLITUDE ( mV ) O _ • ' • I . I .1 * oi o> %4 CD -l i l u * O l C k • J — I — I — . I— CO _l o R CO CD 1 X co O J O l o O ' > 184 Figure 4.11. Effe c t of a long duration perfusion (60 min) of 0 mM Ca 2 +/9 mM Mg 2 + solution on the action of IBMX in the CA1 region of the hippocampus. When the duration of the perfusion of low Ca medium was prolonged, within approximately 40 min a si g n i f i c a n t impairment of synaptic transmission was observed as indicated by the very small amplitude EPSP (A.) and no detectable population spike (B.) . Perfusion of IBMX under these circumstances produced only non-significant changes in the amplitude of the evoked reponses. No long-lasting change was detected, since the evoked potentials returned to control values following perfusion of normal a r t i f i c i a l CSF. The fiber volley response (C. X ) declined steadily during perfusion of the low C a 2 + and was l i t t l e affected by IBMX. In contrast, the antidromically evoked population spike (C. A ) exhibited l i t t l e change in the absence of extr a c e l l u l a r Ca^ , but perfusion of 100 pM IBMX caused i t s transient decline. The bars above panel A. indicate the respective perfusion durations and refer to a l l of the panels. AMPLITUDE (mV ) POPULATION SPIKE AMPLITUDE { mV ) (JJ EPSP AMPLITUDE ( mV ) 186 IBMX did not influence the antidromic responses in control conditions. Input/output curves (n=4) were also examined during perfusion of '0 mM Ca 2 +'. Again, i f the responses were allowed to reach maximal depression in the 'Ca -free' medium, IBMX did not result in potentiation. 4.3.5. Effect of IBMX on bursting induced by low calcium The r a t i o of Ca 2 +/Mg 2 + in the ex t r a c e l l u l a r f l u i d i s an ef f e c t i v e regulator of neuronal e x c i t a b i l i t y (Frankenhaeuser and Hodgkin, 1957; Richards and Sercombe, 1970). The hippocampus is p a r t i c u l a r l y prone to periodic bursting in the absence of synaptic transmission induced by lower than normal Ca concentrations (Jefferys and Haas, 1982; Taylor and Dudek, 1982; Yaari et a l . , 1983). The postsynaptic effect of drugs may be examined in this preparation since no synaptic a c t i v i t y i s present (Haas et a l . , 1984). Bursting of hippocampal CA1 neurons was induced in 9 s l i c e s by perfusion of CSF containing '0 mM' C a 2 + and 1.5 mM (normal) Mg 2 +. Following exposure to thi s medium regular bursts consisting of negative DC s h i f t s and superimposed spiking were recorded in the CA1 pyramidal c e l l layer. The bursts, which occurred with a c h a r a c t e r i s t i c frequency of about 0.05 Hz, were not changed by addition of 100 pM IBMX to the perfusate in 5/9 s l i c e s examined. In the remaining cases IBMX tended to accelerate the frequency of the bursts while having no marked effects on 187 their magnitude or duration. Following return to normal C a 2 + the bursts were abolished and the evoked responses returned to normal. No long-lasting e f f e c t of IBMX was recorded under these conditions. 4.3.6. Effect of IBMX on passive membrane c h a r a c t e r i s t i c s of hippocampal pyramidal c e l l s Perfusion of 100 pM IBMX did not cause s i g n i f i c a n t changes in the resting membrane potential (RMP) of CA1 pyramidal neurons, which i s similar to the finding of Segal (1981). A s l i g h t (2- 3 mV) depolarization was sometimes noted, but th i s e f f e c t was not consistent. The average (+ S.D.) RMP of 41 CA1 pyramidal c e l l s was -61.3 + 2.9 mV and their input resistances (R n) ranged from 25 to 37 Mohms (note however, that only c e l l s with input resistances higher than 25 Mohms were included in the study). The membrane time constant (T c) of pyramidal neurons was found to be in the range of 11-14.7 ms. The two passive membrane parameters (R n and T c) were unaltered by IBMX (Fig. 4.12.A. and B.) and therefore changes in their values are unlikely to be responsible for the long-lasting e f f e c t s of the drug. In four c e l l s , spontaneously occurring fast prepotentials (FPPs) of 2-5 mV amplitude were recorded but their frequency or magni-tude was not affected by IBMX treatment. 188 Figure 4.12. Effe c t of 100 uM IBMX on the passive membrane c h a r a c t e r i s t i c s of CA1 pyramidal neurons. Panels A. and B. represent current-voltage plots of a t y p i c a l pyramidal c e l l before and during IBMX perfusion respectively. The membrane input resistances are indicated as derived through the f i t t i n g of a regression l i n e to the lin e a r part of the I-V relationship. Insets show the change in membrane voltage upon injec t i o n of one of the the 7 steps of current pulses. The v e r t i c a l l i n e of the inset denotes the membrane potential at which measurements were taken. Panels C. and D. represent the ca l c u l a t i o n of the membrane time constant (T) for the same c e l l through the f i t t i n g of a regression l i n e to the natural logarithm of the voltage changes ( AV). The slope, of the relationship taken during the linear portion (indicated by v e r t i c a l lines) r e f l e c t s the time constant of the membrane and i t s value i s shown both before (C.) and during (D.) a 100 uM IBMX perfusion. No s i g n i f i c a n t e f f e c t s of IBMX were noted on either RMP, input resistance or time constant of CA1 pyramidal neurons. Control A. • - 2 0 - Rj a 33.7Mii 1 \ - • 1 * s 100- 1 1 1 1 — 1 — — —"1 ... 1 IBMX ( 1 0 0 / J M ) B. Ri = 34.9Mn •1.5 0 Current (nA) + 5 - 7 C. 0 ^ -5-J D. r-13.7 ms OA T-13.9 ms 25 —I i — 100 25 Time(ms) 100 190 4.3.7. Long-term e f f e c t of IBMX on the rate of r i s e of i n t r a c e l l u l a r l y recorded EPSPs The f i r s t order voltage d i f f e r e n t i a l ( d V / d t ) of the i n t r a - c e l l u l a r l y recorded EPSP following stimulation of the stratum radiatum represents current flow across the synaptic membrane of CA1 pyramidal c e l l s i f membrane capacitance remains constant. At just below threshold stimulation i t s value t y p i c a l l y ranges between 3 and 6 mV'ms-1. In the presence of IBMX (Fig. 4.13.) the rate of ri s e of the EPSP gradually increased and f a i l e d to return to control values even after 1 hour of washout period. The rapid rate of r i s e and the enhanced amplitude of the EPSP during and following IBMX resulted in spike-activation of pyramidal neurons as shown in insets b. and c. of Fig . 4.13. This long-lasting e f f e c t of IBMX was observed in a l l 14 c e l l s where stable recordings could be maintained in excess of 45 min of post-drug washout. These findings also agree with the potentiation of e x t r a c e l l u l a r l y recorded population spikes since neurons brought to f i r i n g threshold by the action of IBMX w i l l add to the size of the negative f i e l d p o t e n t i a l . Therefore, analogous to tetanic stimulation (Andersen et a l . , 1980; Lynch et a l . , 1983), i t is reasonable to suggest that IBMX produced LTP at the single c e l l l e v e l in the hippocampal CA1 region. 191 Figure 4.13. Effe c t of IBMX on the rate of rise.(dV/dt) of i n t r a c e l l u l a r l y recorded EPSPs. Perfusion of 100 uM IBMX (between arrows) caused a long-lasting enhancement of the rate of ris e of the i n t r a c e l l u l a r l y recorded EPSP in a representative CA1 pyramidal neuron. The inset shows the synaptic potentials recorded following stratum radiatum stimulation corresponding to the times marked by small l e t t e r s . Note that an i n i t i a l l y subthreshold EPSP (a) became suprathreshold during (b) and following (c) perfusion of IBMX. 192 1 93 4.3.8. I intracellular input/output relationships following IBMX perfusion Analogous to e x t r a c e l l u l a r potentials, single c e l l LTP should a l t e r the input/output (I/O) relationship of individual pyramidal c e l l s . To test t h i s hypothesis varying i n t e n s i t i e s of stimulation were delivered to the stratum radiatum during i n t r a c e l l u l a r injection of a 70 ms hyperpolarizing current pulse. This allowed for simultaneous measurement of membrane properties, such as input resistance and time constant, and c h a r a c t e r i s t i c s of the EPSP. In addition, several stimulus i n t e n s i t i e s could be applied before the c e l l reached spike threshold (Fig. 4.14.B.). The I/O curves were obtained by p l o t t i n g the stimulus intensity vs. the rate of r i s e (dV/dt) of the EPSP (Fig. 4.14.A.) before and at least 40 min after perfusion of IBMX. In a l l c e l l s examined (n=5) the slope of the I/O relat i o n s h i p was s i g n i f i c a n t l y enhanced following the drug but i t s l i n e a r i t y was unaltered. The rate of r i s e and the amplitude of the EPSP were potentiated at every stimulus i n t e n s i t y . In the absence of changes in time-constant and input resistance of the c e l l s , t h i s finding indicates that a larger synaptic current flow has been induced by stimuli of equal magnitude. In addition, EPSPs that were i n i t i a l l y subthreshold for f i r i n g the pyramidal c e l l s became suprathreshold after perfusion of IBMX. The stimulus i n t e n s i t i e s that were most e f f e c t i v e in potentiating the responses were of intermediate strength (Fig. 4.14.A.), a finding similar to that 1 94 Figure 4.14. I n t r a c e l l u l a r I/O relationship following perfusion of IBMX in a CA1 pyramidal c e l l . A. The rate of r i s e (dV/dt) of the i n t r a c e l l u l a r l y recorded EPSP plotted against the intensity of stimulation. Values were obtained from the traces in B. and C. and represent dV/dts at 9 di f f e r e n t stimulus i n t e n s i t i e s (range: 3.5-7.5 V in 0.5 V increments) delivered to the Schaffer collateral/commissural fiber system before ('CONTROL') and following 45 min of washout of 100 uM IBMX. The regression lines were f i t t e d by computer and their slopes are s i g n i f i c a n t l y d i f f e r e n t from each other (p<0.0l: two t a i l e d t - t e s t ) . B. and C. represent the computer-averaged EPSPs superimposed on a 0.25 nA hyperpolarizing current pulse. Note that membrane resistance as i t can be deducted from the voltage deflection before the stimulus artefact did not change during IBMX-induced LTP. Also note that while none of the control EPSPs produced c e l l discharge, the two potentials evoked at the l a s t two steps of stimulation i n t e n s i t i e s (7 and 7.5 V) both brought the neuron to f i r i n g threshold after LTP has been induced. 195: 196 observed with potentiation of ex t r a c e l l u l a r responses (see Sections 4.3.1. and 4.3.3.). 4.3.9. Effect of IBMX on inhibitory mechanisms of CA1 pyramidal c e l l s recorded i n t r a c e l l u l a r l y Potentiation of i n t r a c e l l u l a r potentials may result i f the pyramidal c e l l s were to be released from the influence of an i n t r i n s i c or ex t r i n s i c inhibitory process. To account for this p o s s i b i l i t y the effect of IBMX was tested on some inhibitory events of CA1 neurons. (i) Ac c ommoda t i on When challenged with a long duration (600-700 ms) depolarizing current pulse, hippocampal pyramidal c e l l s f i r e several action potentials but the i n i t i a l high frequency discharge declines s i g n i f i c a n t l y towards the end of the current pulse. The underlying mechanism is the activation of a C a 2 + - dependent K + conductance (gK^ a) with a contribution from the M- current (Madison and N i c o l l , 1984). In addition, Madison and N i c o l l (1980) have shown that c y c l i c nucleotides and their derivatives are e f f e c t i v e regulators of accommodation. If elevated in the tissue, these compounds reduce the 9 Kca thus diminishing the accommodation properties of CA1 pyramidal c e l l s . The e f f e c t of 100 pM IBMX was tested on the accommodation properties of 8 hippocampal pyramidal neurons. Depolarizing 1 97 Figure 4.15. Effe c t of IBMX on the accommodation of pyramidal c e l l discharge. Raster display of the number of spikes during 600 ms depolarizing current injections (between open arrows on the abscissa) delivered every 30 sec. Note the s l i g h t decrease in accommodation produced during and following a 10 min IBMX perfusion as refl e c t e d by the two insets. These represent oscilloscope traces at the times indicated by closed arrowheads- The continuous raster display i t s e l f , which i s considered to be a more accurate measurement of accommodation, shows a sl i g h t decrease in accommodation during and following perfusion of IBMX. 90 J oo . 9 SO- 'c i c CD _̂ 3 30- d OJ r 0 X c "E QQ o. « « < t t \ • • • • • • : : • 9 t • V \ • • « • i i • « u •: \< • « • « t \ • • • 200 —1 — - i 400 . 6 0 0 800 milliseconds i oo 60mV3nA 300ms T 1 1000 1200 199 current pulses (600 ms in duration) were delivered every 30 s and the number of evoked spikes was continuously monitored as described in Section 4.2.2. A t y p i c a l example of a raster display i s shown on F i g . 4.15. A s l i g h t enhancement of the accommodation was noted in 5/8 c e l l s , the remainder showing no a l t e r a t i o n . This finding i s rather inconsistant with the in h i b i t o r y action of IBMX on the enzyme phosphodiesterase, since elevation of c y c l i c nucleotide lev e l s should have decreased the accommodation. ( i i ) Long-lasting hyperpolarization (LHP) Following their synaptic a c t i v a t i o n , hippocampal pyramidal neurons usually display a hyperpolarizing potential l a s t i n g in excess of 200 ms, that i s termed LHP (Alger, 1984; Lancaster and Wheal, 1984). Lancaster and Wheal (1984) using i n t r a c e l l u l a r injections of the C a 2 + chelator EGTA provided evidence that gK C a a c t i v a t i o n may have l i t t l e contribution to the LHP. According to the recent study of Alger (1984) the underlying mechanism appears to be a synaptically activated K + conductance. As shown on F i g . 4.16.A. the amplitude and duration of the LHP was in most cases s l i g h t l y but not s i g n i f i c a n t l y increased by IBMX. 200 Figure 4.16. Effect of IBMX on inh i b i t o r y events acting on CA1 pyramidal neurons. A. Slight enhancement of the long l a s t i n g afterhyperpolarization (AHP) that follows synaptic a c t i v a t i o n of CA1 pyramidal c e l l s in the presence of 100 uM IBMX. B. No s i g n i f i c a n t change was produced by IBMX in a t y p i c a l IPSP evoked following orthodromic stimulation of pyramidal neurons. C. In t r a c e l l u l a r paired-pulse i n h i b i t i o n was also unaltered by IBMX as re f l e c t e d by the f a i l u r e of the second orthodromic pulse to evoke c e l l discharge. The dotted l i n e s indicate the resting membrane potentials (RMP) of the respective c e l l s . RMPs were not changed by addition of IBMX to the perfusate. 201 Control A. AHP i X B. IPSP -4 C. Paired Pulse JlOmV 50 ms JlOmV 20 ms J20mV 5 ms 202 ( i i i ) Inhibitory post-synaptic potentials (IPSPs) and i n t r a - c e l l u l a r paired-pulse i n h i b i t i o n A decrease in the feed-back i n h i b i t i o n of hippocampal pyramidal c e l l s mediated through GABA-releasing basket c e l l s i s not of primary importance for the induction of LTP, but changes in the level s of i n h i b i t i o n may modulate the degree of potentiation (Haas and Rose, 1982). IPSPs of pyramidal neurons can be triggered by their ortho- or antidromic a c t i v a t i o n . F i g . 4.16.B. depicts the lack of eff e c t of 100 pM IBMX on an IPSP evoked through orthodromic stimulation of the Schaffer collateral-commissural f i b e r s ; similar results were obtained for the antidromic stimulation-evoked IPSPs (not shown). To estimate the effectiveness of the i n h i b i t i o n , a supra- treshold conditioning stimulus was delivered to the afferent fi b e r system followed, 20 ms l a t e r , by a test stimulus of equal i n t e n s i t y (Fig. 4.16.C). As a result of the activated i n h i b i t i o n , the second stimulus evoked a smaller EPSP that f a i l e d to trigger an action p o t e n t i a l . The paired-pulse paradigm was then repeated in the presence of 100 uM IBMX but no changes were detected in the potency of i n h i b i t i o n confirming the results obtained under similar circumstances in the study of extra- c e l l u l a r p o t e n t i a l s . 203 4.3.10. Long-term effect of IBMX on stimulus threshold By d e f i n i t i o n , when the stimulation intensity of afferent fibers is adjusted to 'threshold' only 50% of the stimuli w i l l evoke an action potential in the neurons under study. In F i g . 4.17.A. an experiment i s shown where the intensity of stimulation delivered to the Schaffer collateral/commissural fibers was adjusted to this threshold value. Before perfusion of the drug 6 out of 10 successive stimuli evoked an action potential in the CA1 pyramidal neurons. In the presence of IBMX (Fig. 4.17.B.) a l l stimuli delivered (10/10) caused a spike discharge. A reduction of stimulus intensity to approximately half of i t s o r i g i n a l value (Fig. 4.17.C.) could now evoke spikes with 6 out of 10 pulses thus indicating that a new threshold for synaptic activation was established following IBMX-induced LTP. A c h a r a c t e r i s t i c 22-50% reduction in threshold stimulation intensity was observed in a l l c e l l s examined (n = 7) and persisted after washout as long as steady recordings could be obtained. To c l a r i f y whether the lowering of stimulation threshold was due to a general somatic a l t e r a t i o n of f i r i n g l e v e l , a short (20 ms) depolarizing current pulse was used to evoke spiking of pyramidal c e l l s (Fig. 4.17.E.). The f i r i n g l e v e l and the amount of current needed to evoke spiking were not affected by IBMX (Fig. 4.17.F.), indicating that the CA1 c e l l soma i s an unlikely s i t e of drug action. 204 Figure 4.17. Effect of IBMX on stimulus- and f i r i n g threshold of CA1 pyramidal neurons. Panels A.-D. represent a t y p i c a l experiment in which the change in synaptic stimulus threshold (T) was determined. On each trace, 10 successive synaptic activations of a pyramidal c e l l are shown, which have been manually shifted for c l a r i t y by 20 ms on the oscilloscope. The threshold stimulus intensity evoked f i r i n g of the c e l l in 6/10 cases (A.) while the same stimulus intensity produced discharge of the c e l l in 10/10 cases during perfusion of 100 uM IBMX (B.). When stimulus intensity was reduced to approximately half of i t s o r i g i n a l value (1/2 T) in the presence of IBMX ( C ) , the i n i t i a l 6/10 spikes could be evoked, indicating a lowering of the synaptic threshold. This phenomenon persisted for a long time following washout of the drug (D.), indicating that LTP was produced. Panels E. and F. represent the f i r i n g l e v e l of a pyramidal neuron as measured by a short duration (20 ms) depolarizing current i n j e c t i o n . Since the resting membrane potential was not changed by IBMX, i t is reasonable to say that voltage at which spiking of the pyramidal c e l l was observed ( f i r i n g l e v e l ) , indicated by the two arrowheads, was not altered by the drug. Therefore, the observed changes in synaptic threshold are not due to a decreased f i r i n g l e v e l caused by IBMX. 205 STIMULUS THRESHOLD ( T ) A. B. i i i i i i A !A[n |A(r. AiV | A p A A A A A MA A D. "̂MMj-j j*-' A ,A | jnjA j 1 J 2 0 m V 2 0 ms Spike/Current Threshold E . Control J 5 m V / 5 n A IBMX 2 0 ms 206 4.4. Discussion Despite considerable research the exact mechanism of the long-lasting potentiation (LTP) in the hippocampal formation has not yet been elucidated. Several pharmacological agents have been successfully employed in blocking the long-term enhancement of synaptic responses and thus have provided useful information about the possible molecular events involved in the process (Finn et a l . f 1980; Mody et a l . , 1984; Stringer et a l . , 1984). While certain drugs are e f f e c t i v e in hindering the induction of LTP, only a limited number of agents other than tetanic stimulation and a brief elevation in e x t r a c e l l u l a r C a 2 + were shown to trigger i t s occurrence. Neuman and Harley (1983) have found that' iontophoretic application of norepinephrine in the dentate gyrus results in a long-lasting enhancement of the e x t r a c e l l u l a r f i e l d responses evoked by perforant path stimulation. The present findings indicate that synaptic p l a s t i c i t y in the CA1 region of the in v i t r o hippocampus may also be induced by the methyl- xanthine derivative IBMX. Several mechanisms of action may subserve t h i s LTP-inducing effect of IBMX. The drug may block adenosine receptors or i n h i b i t the enzyme phosphodiesterase and f i n a l l y , could cause release of Ca from intraneuronal storage s i t e s . Methylxanthine compounds are known for their double antagonism of adenosine responses in the central nervous system by i n h i b i t i n g purine release and by blocking postsynaptic 207 adenosine receptors (Stone, 1981). Previous studies on these substances in the hippocampus have focused on their properties to antagonize adenosine receptors (Schubert and Mitzdorf, 1979; Dunwiddie and Hoffer, 1980; Okada and Ozawa, 1980; Dunwiddie et a l . , 1981). These investigators have demonstrated that adenosine is an e f f e c t i v e i n h i b i t o r of hippocampal evoked responses and suggested the p o s s i b i l i t y for i t s tonic release since applications of adenosine receptor antagonists lead to enhanced synaptic a c t i v i t y . . Dunwiddie and Hoffer (1980) reported that evoked potentials were potentiated only as long as theophylline was present in the bathing medium. These investigators also noted that although i t antagonized adenosine responses in a concentration of 400 pM, IBMX f a i l e d to have any effect on the EPSP responses. In another region of the hippocampus, the CA3, Okada and Ozawa (1980) observed epileptiform a c t i v i t y upon exposure of the s l i c e s to high (500 pM) levels of IBMX. In the present study drug concentrations were considerably lower, res u l t i n g in comparable findings regarding the effect of theophylline but s i g n i f i c a n t differences with regard to IBMX's action. The reason for the discrepancy i s not known since potentiation of the Schaffer collateral/commissural evoked responses recorded at the pyramidal c e l l layer as well as at the le v e l of a p i c a l dendrites was observed in a l l of the s l i c e s examined (n=48). In addition, in 44/48 preparations the effect of IBMX was long-lasting, i . e . the responses f a i l e d to return to pre-drug lev e l s u n t i l the experiment was discontinued (usually in excess of 2 hours). Only one other methylxanthine derivative 208 (Saphenyl-theophylline) has been reported to cause somewhat analogous long-term enhancing e f f e c t s in the rat hippocampus when the stimulation frequency was 2 Hz (Corradetti et a l . , 1984). However, such a frequency of stimulation may by i t s e l f cause potentiation of the evoked responses and therefore a v a l i d conclusion regarding the drug's action cannot be reached. If the actions of IBMX on adenosine receptors are considered as being responsible for i t s LTP-inducing e f f e c t , then a long- term blocking action has to be postulated, or a l t e r n a t i v e l y a very slow washout time of the drug has to be present. These alternatives seem unlikely, however, in l i g h t of the short retention times and fast recoveries for IBMX as measured by Snyder et a l . (1981) using radioactively l a b e l l e d methylxanthine compounds. An ef f e c t of IBMX on adenosine receptors alone i s also not probable in view of i t s d i f f e r e n t i a l e f f e c t from that of theophylline. Theophylline has a two-fold lower IC50 for adenosine receptors than IBMX (Snyder et a l . , 1981) and yet the magnitude of potentiation produced during perfusion of IBMX was invariably larger than that caused by theophylline. Furthermore, while the washout times of the two drugs are similar (Snyder et a l . , 1981), theophylline f a i l e d to produce any long-lasting enhancement of the hippocampal evoked responses. The deviant properties of IBMX which d i s t i n g u i s h t h i s drug from a variety of other methylxanthines were also noted in several behavioural studies (e.g., Snyder et a l . , 1981; Yarbrough and McGuffin- Clineschmidt, 1981). In view of these observations and the results of the present study i t i s concluded that antagonism of 209 adenosine receptors may be p a r t i a l l y responsible for the potentiation of the evoked responses during IBMX perfusion, but cannot play a role in the LTP-producing effect of IBMX. As a second possible mechanism of action, i n h i b i t i o n of the enzyme phosphodiesterase (PDE) would result in elevated i n t r a - c e l l u l a r concentrations of c y c l i c nucleotides. According to Chasin and Harris (1976) the r e l a t i v e potencie of theophylline on PDE i n h i b i t i o n i s considered to be 1, then IBMX's and papaverine's potencies are 15 and 10-1,000 respectively (see also Smellie et a l . , 1979). In addition to i t s inh i b i t o r y action on PDE, papaverine has been shown to activate the adenylate cyclase of the cerebral cortex thus causing added elevations of c y c l i c AMP levels (Iwangoff and Enz, 1971). The results of the present study show that papaverine had an opposite effect to that of IBMX ( s l i g h t l y depressed the population spike and EPSP responses) suggesting that elevations in c y c l i c nucleotide levels are not responsible for the action of IBMX. Bursting induced by low Ca2+ is also modulated by drugs that interfere with c y c l i c nucleotides (Haas et a l . , 1984) whereas IBMX had no major effect on this type of epileptiform a c t i v i t y . The lack of effect on c y c l i c nucleotide metabolism i s further underscored by the sl i g h t enhancement of pyramidal c e l l discharge accommodation caused by IBMX. Drugs that elevate cAMP of hippocampal s l i c e s and c y c l i c nucleotides themselves have an opposite effect on the accomodation of CA1 pyramidal neurons (Madison and N i c o l l , 1980). Since the contributions of adenosine receptor antagonism and phosphodiesterase i n h i b i t i o n to the IBMX-induced LTP have been 210 ruled out, a t h i r d possible mechanism, namely the effect of the drug on i n t r a c e l l u l a r C a 2 + - r e g u l a t i o n has to be considered. The observations that IBMX retained i t s LTP inducing potency even in the presence of the C a 2 + channel antagonist C o 2 + and caused potentiation of f i e l d responses while e x t r a c e l l u l a r C a 2 + was s i g n i f i c a n t l y lowered indicate that the action of IBMX does not 9 + occur through augmentation of Ca entry into neurons. An enhanced C a 2 + influx would have also manifested i t s e l f during depolarizing current pulses in the form of a long-duration depolarizing potential as is c l e a r l y v i s i b l e in CA1 pyramidal c e l l s when the C a 2 + gradient i s increased by i n t r a c e l l u l a r a pplication of the C a 2 + chelator EGTA (see F i g . 2. of Madison and N i c o l l , 1984). No such effect was observed in t h i s study during IBMX perfusion. The fact that the dependency of IBMX-induced LTP on extra- 9 + c e l l u l a r Ca was not c r i t i c a l correlates well with the results of 4^Ca uptake experiments presented in Section 3.2.3.2. As shown by the kinetic analysis of 4^Ca uptake curves in the presence of IBMX, the drug was able to mobilize C a 2 + from an i n t r a c e l l u l a r buffered/sequestered pool. Total exchangeable 9 + intraneuronal Ca^ was not affected by IBMX suggesting that calcium entry per se did not play a s i g n i f i c a n t r o l e . Therefore, i t i s l i k e l y that the end result of drug action consists of a re d i s t r i b u t i o n of i n t r a c e l l u l a r C a 2 + from a slowly exchangeable (buffered/sequestered) compartment into a more rapidly 9 + exchangeable pool that includes free cytoplasmic Ca^ . Similar s h i f t s in calcium homeostasis have been observed in the kindling 21 1 model of epilepsy possibly due to the loss of an intraneuronal C a 2 + - b u f f e r , i . e . , the calcium-binding protein, CaBP (Section 3.3.3.). In the case of the IBMX-induced LTP there i s no evidence for a decrease in the levels of an intraneuronal calcium binding- and buffering system but rather an activation of release from intraneuronal storage s i t e s . The neuronal equivalent of sarcoplasmic reticulum, i s the endoplasmic reticulum. One of the several functions associated with t h i s c e l l u l a r element has been shown to be a manor Ca - buffering system in nerve c e l l s whereas the mitochondria seem to play a less s i g n i f i c a n t role (Duce and Keen, 1978; Blaustein et a l . , 1980; Brinley, 1980; McGraw et a l . , 1982). A calcium- dependent C a 2 + release from the endoplasmic reticulum of neurons is therefore feasible, similar to the process described for the sarcoplasmic reticulum of skeletal muscle (for reviews see Endo, 1977; Martonosi, 1984). Thus the C a 2 + sequestering pool shown to be affected by IBMX in the analysis of ^^Ca uptake kinetics may be the neuronal endoplasmic reticulum rather than the lower a f f i n i t y mitochondrial compartment. If t h i s i s the case, IBMX may reset the s e n s i t i v i t y of the endoplasmic reticulum for Ca , analogous to i t s effect in skeletal muscle (Miller and Thieleczek, 1978) or to that of caffeine in b u l l f r o g sympathetic neurons (Akaike et a l . , 1983), res u l t i n g in a larger Ca 2 +-induced C a 2 + release. A l t e r n a t i v e l y , IBMX may elevate the levels of i n o s i t o l triphosphate (InsP3) and enhance the release of endoplasmic r e t i c u l a r C a 2 + through the pathway described by Berridge and Irvine (1984). Although there is no evidence yet 212 whether IBMX has any eff e c t on phosphatidylinosotol metabolism, t h i s alternate route of drug action seems plausible since tetanus-induced LTP has recently been shown to enhance the levels of InsP3 ^ n t n e hippocampal formation (Bar et a l . , 1984). If release of i n t r a c e l l u l a r calcium i s indeed responsible for the neuronal p l a s t i c i t y caused by IBMX, several molecular mechanisms, at either pre- or postsynaptic s i t e s , may in turn be activated by the excess of available Ca^ . The synaptic l o c a l i z a t i o n of the changes following perfusion of IBMX i s demonstrated by the a l t e r a t i o n s of the I/O curves observed in the present study. The e f f i c a c y of the synapses became enhanced without any considerable increase in c e l l u l a r e x c i t a b i l i t y as shown by the l i ' t t l e change detected in the relationship between the rate of rise- of the EPSP and population spike amplitude (D-S curves). Consequently, when ppst-synaptic current was the same (as r e f l e c t e d by the rate of ri s e of the EPSP) i t produced the synchronous f i r i n g of approximately the same number of CA1 pyramidal neurons (since a comparable size population spike was evoked) both before and after administration of the drug. However, synaptic transmission had to be enhanced since under equivalent input conditions (stimulus intensity or fiber volley amplitude) a larger output function (synaptic current flow or EPSP amplitude) was generated following IBMX-induced LTP. The synaptic changes which occur during any form of LTP may be l o c a l i z e d at the le v e l s of the postsynaptic c e l l or could be caused by a l t e r a t i o n s in the presynaptic terminal (Bliss and Dolphin, 1982; Swanson et a l . , 1982; Eccles, 1983; Voronin, 213 1983). There may be a selective postsynaptic change responsible for the mechanism of LTP (Dunwiddie et a l . , 1978; Baudry et a l . , 1980). The release of C a 2 + from intraneuronal storage s i t e s as a result of IBMX in the post-synaptic c e l l may increase the number of postsynaptic glutamate receptors as proposed by Lynch and Baudry (1984) and thus cause a l a s t i n g change in synaptic e f f i c a c y . The increase in the size of the i n t r a c e l l u l a r EPSP and i t s rate of r i s e would be explained by this phenomenon. As shown for a computer-derived model of a motoneuron when synaptic input is held constant, the voltage change during an EPSP i s most sensitive to the density of active synapses on the postsynaptic membrane (Lev-Tov et a l . , 1983). In this model, changes in passive membrane c h a r a c t e r i s t i c s of the c e l l seem to be parameters of lesser s i g n i f i c a n c e . In the present experiments however, there i s no reason to assume that the synaptic input was not altered by IBMX, although the number of activated presynaptic f i b e r s , as shown by the constancy of the fiber volley p o t e n t i a l , i s probably unaffected. As in the model of Lev-Tov et a l . (1983) no changes were detected in tha passive membrane c h a r a c t e r i s t i c s (RMP, R n and T c) of hippocampal pyramidal c e l l s during perfusion of IBMX. But somatic recordings cannot r e f l e c t changes in the el e c t r o p h y s i o l o g i c a l properties of distant dendrites and dendritic spines. If these spines are assumed to have active channels, then small changes in their diameter which either increase or decrease spine shaft resistance produce considerable amplifications of the EPSP (Miller et a l . , 1985; Perkel and Perkel, 1985). If IBMX causes a long-lasting a l t e r a t i o n in the 214 morphology of the spine apparatus, the f i n a l result may well be a persistent enhancement of the synaptic responses. As an alternate mechanism, the excess free calcium in the presynaptic terminals may trigger the biochemical substrate for a long-lasting increase in neurotransmitter release. Such an enhanced release has been shown to be a correlate of tetanus- induced LTP in the hippocampus (Skrede and Malthe-Sorenssen, 1981; Dolphin et a l . , 1982) and presynaptic mechanisms have thus been implicated (Sastry, 1982; Dolphin, 1983). The long-term alterations r e f l e c t e d by the i n t r a c e l l u l a r input/output curves may also be accounted for by a presynaptic action of IBMX i f induction of a larger neurotransmitter release i s assumed. The excess Ca^ in the presynaptic terminal, p a r t i c u l a r l y originating from intraneuronal storage s i t e s , may produce an enhanced resting neurotransmitter release as demonstrated for the neuromuscular junction (Rahamimoff, 1976; Rahamimoff et a l . , 1980). This phenomenon would only l a s t while the ' drug is present in the perfusate and responses should return to control values following washout. This however, i s c l e a r l y not the case since reduction of synaptic threshold and enhancement of evoked i n t r a c e l l u l a r EPSPs were observed as long as recordings were maintained from the CA1 pyramidal c e l l s . Since the retention of C a 2 + after tetanic stimulation i s only transient (Baimbridge and M i l l e r , 1981), a long-lasting biochemical change of the presynaptic terminal would have to be postulated for the chronic increase in transmitter release. 215 The resulting enhancement of EPSP amplitude and i t s rate of r i s e , recorded inside the pyramidal c e l l s , was sustained for a longer period of time than in the case of LTP induced by conventional high frequency stimulation techniques thus stressing the potency of IBMX. However, as found by other investigators for the LTP induced by tetanic stimulation (Yamamoto and Chujo, 1978; Andersen et a l . , 1980; Yamamoto et a l . , 1980; Lynch et a l . , 1983) the event took place without any observable long-term changes in the passive membrane properties (RMP, R n and T c) of CA1 pyramidal neurons suggesting that the major factors responsible for the phenomenon are l o c a l i z e d to the synaptic junction. The present study does not distinguish between the two possible s i t e s of the LTP-inducing effect of IBMX. However, the results show a novel form of LTP in the mammalian CNS whereby release of intraneuronal Ca , possibly from the endoplasmic reticulum, i s responsible for the sustained potentiation of synaptic responses. This hypothesis awaits further testing e s p e c i a l l y since i t may be applicable to the tetanus- and calcium-induced forms of LTP. 216 C H A P T E R V . CONCLUSIONS The important role of calcium in the control of nerve c e l l function i s well established. E x t r a c e l l u l a r calcium exerts profound actions on the e x c i t a b i l i t y of neuronal membranes while cytosolic C a 2 + can by i t s e l f , or through the act i v a t i o n of second messengers, regulate the a c t i v i t y of neurons. It is therefore of no surprise that i t s concentration both i n t r a - and e x t r a c e l l u l a r l y i s under rigorous control. Inside the c e l l , several mechanisms, including buffering by i n t r a c e l l u l a r organelles and proteins, participate in the maintenance of a steady Ca^ concentration at an optimal l e v e l . Changes in the capacity of neurons to cope with a C a 2 + challenge may result in their altered discharge patterns which, i f they occur on a large enough scale, may influence the a c t i v i t y of the whole CNS. Therefore, any durable change in neuronal Ca 2 +-regulatory mechanisms could result in severe consequences with regard to the normal functioning of the nervous system. The present study examined some aspects of the intraneuronal Ca 2 +-homeostasis during long-term alterations in neuronal e x c i t a b i l i t y . 217 Kindling-induced epilepsy, whereby status epilepticus i s induced by d a i l y e l e c t r i c a l stimulation, i s a prime example of such a gradual but persistent change in nerve c e l l e x c i t a b i l i t y . It i s characterized by the early occurrence of r e p e t i t i v e , synchronous f i r i n g s of groups of neurons (afterdischarges or AD's), which culminate with time in f u l l t o nic-clonic seizures, analogous to the human disease. As presented in Section 2.3., the development of AD's was correlated to the loss of an intraneuronal calcium-binding protein (CaBP) in the hippocampal formation. Although the role of th i s protein i s not f u l l y established, one of i t s main functions may be the buffering of excess intraneuronal C a 2 + . The exact causal link between CaBP and AD's could not be established, but changes in the levels of the protein p a r a l l e l l e d the progressive lengthening of the AD's in the hippocampus. An impaired calcium regulation, as ref l e c t e d by diminished lev e l s of CaBP in certain c o r t i c a l areas, was found in yet another model of experimental epilepsy. The genetically e p i l e p t i c s t r a i n of mice ( E l ) , in which seizures are induced by successive vestibular stimulations, had s i g n i f i c a n t l y lower c o r t i c a l l e v e l s of- the protein than a control s t r a i n . This finding i s relevant, since one of the CaBP-deficient c o r t i c a l areas, the hippocampus, is also thought to be the focus of the paroxysms. It i s therefore possible that the enhanced s u s c e p t i b i l i t y to seizures in this s t r a i n i s due to a genetically Un-altered intraneuronal Ca^ regulation. 218 Measurement of intraneuronal C a 2 + i s a d i f f i c u l t task and can only be achieved with high accuracy in the large nerve c e l l s of molluscs. With the presently available techniques i t i s only possible to estimate calcium a c t i v i t i e s in elements of the mammalian CNS. In the more accessible non-neuronal preparations, the kinetic analysis of 4^Ca uptake curves has long been used to r e l i a b l y determine exchangeable C a 2 + l e v e l s . In the present study this technique was successfully applied to the in v i t r o hippocampus. The v a l i d i t y of the method was assured by comparing the hippocampal C a 2 + measurements to the data of other studies that used the same procedure in non-neuronal preparations as well as to available C a 2 + determinations in the mammalian CNS. Although a d i s t i n c t i o n could not be made between g l i a l and neuronal elements using the current technique, due to the heterogeneity of the hippocampal s l i c e preparation, i t was established that exchangeable C a 2 + is distributed, as in other systems, into two k i n e t i c a l l y separate pools. The f i r s t rapidly exchangeable compartment includes among other e n t i t i e s free ionic i n t r a c e l l u l a r C a 2 + , while the second more slowly exchangeable compartment consists of the sum of buffered c e l l u l a r Ca* . Once the r e l i a b i l i t y of the 4 5 C a uptake kinetics was established, the method was used to detect possible a l t e r a t i o n s . in Ca*- homeostasis following the chronic predisposition of neuronal tissue to epileptiform a c t i v i t y . S i g n i f i c a n t alterations in exchangeable Ca z were observed in hippocampal s l i c e s obtained from amygdala and commissural kindled animals. Although there was no change in the absolute 219 lev e l s of i n t r a c e l l u l a r calcium, there was a s i g n i f i c a n t r e d i s t r i b u t i o n of calcium from a buffered compartment into an unsequestered pool. If the calcium-binding protein CaBP i s considered to pe part of the intraneuronal C a 2 + buffering system, i t is evident that following i t s loss during kindling-induced epilepsy Ca^ would be transferred to other i n t r a c e l l u l a r exchangeable compartments. The finding that t o t a l exchangeable C a 2 + of kindled hippocampi was unaltered, an observation confirmed by atomic absorption spectrophotometry measurements, adds important conceptual consequences to our present understanding of neuronal Ca'' regulation. It indicates that persistent changes in neuronal e x c i t a b i l i t y do not neccessarily depend upon the amount of t o t a l C a 2 + in the system but rather on the quantity of the cation f r e e l y available for regulatory processes. This finding stresses the importance of intraneuronal Ca^ stores which may release or take up Ca according to the physiological (or pathophysiological) needs and s t i m u l i . It i s t h i s calcium release mechanism that most l i k e l y underlies the action of 3-isobutyl-1-methylxanthine (IBMX) in the hippocampal s l i c e preparation, although the exact s i t e of release could not be determined by 4^Ca k i n e t i c s . Analogous to the e f f e c t s of tetanic stimulation and brief exposure to elevated e x t r a c e l l u l a r C a 2 + , IBMX was shown in the present study to produce long-term potentiation (LTP) of the stratum radiatum evoked f i e l d p o t e n t i a l s in the CA1 region of the hippocampal s l i c e . Regardless whether the C a 2 + release occurs at a pre- or postsynaptic s i t e or both, the IBMX-induced LTP i s the f i r s t 220 available evidence indicating that an agent which translocates intraneuronal C a 2 + causes a l a s t i n g change in neuronal e x c i t a b i l i t y . The present study has only dealt with some aspects of the contribution of intraneuronal Ca 2 +-regulatory systems to long- duration changes in neuronal function. Naturally, many alternate pathways of research w i l l be available in the future to establish the precise roles of the several mechanisms that p a r t i c i p a t e in the control of C a 2 + within nerve c e l l s . In th i s context, p a r t i c u l a r emphasis should be placed on the various neuronal calcium-binding proteins and the endoplasmic reticulum which has received l i t t l e attention so fa r . F i n a l l y , the method of kinetic analysis of 4^Ca uptake curves, extended to the CNS by the present study, should prove to be a valuable tool in determining differences in calcium homeostasis among homogeneous, possibly cultured, populations of neurons. 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