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Firing properties and Na⁺-dependent plateau potentials of neurons in nucleus principalis trigemini of… Sandler, Vladislav Michael 1996

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FIRING PROPERTIES AND Na -DEPENDENT PLATEAU POTENTIALS OF NEURONS IN NUCLEUS PRINCIPALIS TRIGEMINI OF THE GERBIL +  VLADISLAV MICHAEL SANDLER A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Neuroscience Program) We accept this thesis as conforming (\o  n  the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April  1996  © Vladislav Michael Sandler,  1996  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his or her representatives.  It  is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of Q^txplM^-^t  ^t^u^LLt^s.  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  lS^Q£ fif^t, J33£  11  ABSTRACT  We in  investigated the e l e c t r o p h y s i o l o g i c a l properties  the  nucleus  principalis  trigemini  (PrV),  using  whole-cell  recordings in in vitro slice preparations of brainstem. three groups mainly by their spontaneously doublets  active  or bursts;  firing  and able  properties:  to  discharge  We i d e n t i f i e d  type 1 neurons w e r e action  type 2 neurons, d e p o l a r i z e d  fired action potentials in a nonadapting (tonic)  of n e u r o n s  potentials  by current  pulses,  and the  less  c o m m o n l y encountered type 3 neurons also fired in s u c h patterns  but  with  and  biphasic  reconstruction between  types  dendritic  trees  pattern;  in  afterhyperpolarizations.  Neurobiotin  did not reveal s i g n i f i c a n t  morphological  1  and  2  neurons  distributed  mainly  which  were  staining  differences  multipolar,  along one axis.  with  Type 3 n e u r o n s  had more e x p a n s i v e a n d circular dendritic arborizations. Hyperpolarization potential  due to  beyond -75  current  pulse  mV or down to the  injection,  resulted  K  in  +  reversal  an  inward  rectification which w a s e x p r e s s e d a s a s a g in the voltage r e s p o n s e s of  types  1  and  type  2  neurons.  A  rebound  depolarization or spike burst w a s evident on t e r m i n a t i o n In type  1 neurons, the  application  of C s  +  subthreshold of a p u l s e .  (2 mM), a blocker  of  a  Ill  hyperpolarization-activated voltage  cation  current  (l ), H  eliminated  the  sag and the dependence of the rebound spike-latency  on  membrane voltage, but did not alter the main features of the rebound response.  We attribute the inward rectification  an l -like  current.  H  Depolarization by current pulse injection hyperpolarized "plateau  with  potentials".  DC to  prevent  This feature,  neurons, consisted of an initial that  decreased in amplitude,  firing,  2 +  oscillatory and then  free media, with  burst of 3 or 4 s p i k e s  plateaued  (TEA)  2+  for  a variable  We always observed pulse injection  or without  antagonists, C o o r C d , and during external 2 +  evoked  not observed in types 2 or 3  these voltage shapes on depolarizing current Ca  of  into type 1 neurons, occasionally  duration, followed by an abrupt repolarization.  perfusion with  to the activation  during  the C a - c h a n n e l 2+  tetraethylammonium  application. An analysis of the depolarizing voltage  responses evoked by  current pulses in type 1 neurons during blockade of persistent transient  and  N a conductances with TTX (600 nM) and K conductances +  +  with T E A (10 mM) and 4-aminopyridine presence of inward rectification.  (4-AP; 0.5 mM), revealed the  This had a peak activation  near  iv  the  plateau  itself  and was  completely  blocked  by N i  T h e s e o b s e r v a t i o n s are consistent with the activation Ca -conductance.  Hence, we  2+  conductance  mechanism  propose  controls  the  that  a  2 +  (600  uM).  of a t r a n s i e n t  Ca -dependent 2+  generation  of  the  K  +  plateau  potential. T h e application of T T X , as low a s 0.6 nM, i n c r e a s e d the to onset a n d d e c r e a s e d the duration of the plateau potential, greatly  affecting  manner,  action  potentials.  T T X enhanced the  descended  towards  concentrations potential  an  negative  abrupt  terminal  [Na ]-perfusion,  plateau potentials  and fast  of  the  plateau,  +  plateau  genesis.  Low  reduced the a m p l i t u d e s  Evidently,  N a conductance can produce  Higher  a b o l i s h e d the  blocking action potential  spikes.  as i t  repolarization.  6 min)  however, s i m u l t a n e o u s l y  +  without  concentration-dependent  slope  of T T X (e.g., 60 nM for  before c o m p l e t e l y  persistent  In a  latency  small  marked  of  c h a n g e s in a  c h a n g e s in  firing  current  pulses  behavior of type 1 neurons. A producing  long-lasting the  suprathreshold  hyperpolarization  plateau  potential.  depolarization  also evoked a h y p e r p o l a r i z a t i o n  in C a  followed Indeed,  2 +  free  subthreshold  A C S F with  at the offset  Co  2 +  of the current  (1  or mM) pulse.  This hyperpolarization varied with  was blocked by TTX (5 nM and 300 nM) and  changes in the duration  semiquantitative  analysis  hyperpolarization  depended on the  conclude,  therefore,  that  revealed  Na  +  entry  of the plateau that  the  potential.  magnitude  of  A the  neuronal  depolarization.  We  during  a depolarization  can  increase a K conductance in type 1 neurons. +  From represent  our the  studies,  we  conclude  contributions  conductances, high threshold  of  +  persistent  plateau and  potentials  transient  Ca -dependent rectification, 2+  as C a - and Na -dependent K 2 +  that  +  conductances.  The ability  neurons of primary sensory nuclei.  In nucleus principalis  burst responses to mechanical stimuli neurons  that  messenger  likely  regulation.  are  subject  +  as w e l l to  bursts as part of Na -dependent plateaus is an unusual property +  Na  fire in  trigemini,  represent a normal output of to  intra-  and  extracellular  -  vi  TABLE OF CONTENTS ABSTRACT  i i  TABLE O F CONTENTS  v i  LIST O F F I G U R E S  ix  ACKNOWLEDGEMENTS  x i  1.  INTRODUCTION  1  2. M E T H O D S  ....4  3. R E S U L T S  7  3.1  7  IDENTIFICATION O F N U C L E U S PRINCIPALIS TRIGEMINI ( P R V ) N E U R O N S  3 . 2 PHYSIOLOGICAL PROPERTIES OF P R V NEURONS  3.2.1  10  Inward rectification in type 1 neurons  12  3.2.2 Contribution of inward rectification to spike generation in type 1 neurons  12  3.2.3  Firing patterns of type 2 neurons  14  3.2.4  Firing patterns of type 3 neurons  16  3 . 3 P L A T E A U POTENTIALS IN T Y P E 1 N E U R O N S  18  3 . 4 IONIC MECHANISM O F PLATEAU POTENTIAL GENERATION IN T Y P E 1 N E U R O N S  20  Vll  3.4.1  Relationship of plateau potential firing to extracellular Ca 20  3.4.2  Involvement of a persistent Na  3.4.3  Sensitivity of plateau potentials to TTX.  25  3.4.4  Extracellular replacement of Na  27  3.4.5  Involvement of Ca -dependent rectification  3.4.6  Involvement of Ca -activated K  2+  conductance  +  +  with choline 29  2+  2+  +  conductance  3 . 5 T H E P O S T - P U L S E HYPERPOLARIZATION ( P P H )  4.  23  DISCUSSION  4.1 DEPOLARIZING R E S P O N S E S IN T Y P E S 2 AND 3 NEURONS  31 3 3  3 7 37  4 . 2 HYPERPOLARIZING C U R R E N T PULSE INJECTIONS INTO P R V NEURONS  3 8  4 . 3 CLASSIFICATION O F T Y P E S 1 A N D 2 N E U R O N S  3 9  4 . 4 B U R S T FIRING IN T Y P E 1 NEURONS  3 9  4 . 5 IS T H E BURST FIRING PATTERN O F T Y P E 1 NEURONS REPRESENTATIVE O F P R V NEURONS  IN V I V O ?  40  4 . 6 T H E PLATEAU POTENTIALS O F T Y P E 1 NEURONS  41  4.6.1  Influence of cation currents  4.6.2  Repolarization of plateau potential  4 . 7 P O S T - P U L S E HYPERPOLARIZATION ( P P H )  4 . 8 FUNCTIONAL CONSIDERATIONS IN T Y P E 1 NEURONS  42 43 44  4 6  Vlll  5. R E F E R E N C E S  5 0  6.  5 8  APPENDIX  1  6.1 D A T A ACQUISITION A N D P R O C E S S I N G P R O G R A M V M S  5 8  ix  LIST OF FIGURES  FIGURE 1 C A M E R A LUCIDA DRAWINGS OF NEUROBIOTIN-STAINED  NEURONS  8  FIGURE 2 CHARACTERISTIC FIRING BEHAVIOR OF T Y P E 1 NEURON  11  FIGURE 3 INWARD RECTIFICATION, EVOKED BY HYPERPOLARIZATION, A N D REBOUND FIRING IN  T Y P E 1 NEURON  1 3  FIGURE 4 CHARACTERISTIC TONIC FIRING BEHAVIOR O F T Y P E 2 NEURON  15  FIGURE 5 CHARACTERISTIC TONIC FIRING BEHAVIOR O F T Y P E 3 NEURON  17  FIGURE 6 T Y P E 1 N E U R O N S GENERATE PLATEAU POTENTIALS DURING C A - F R E E PERFUSION 1 9 2 +  FIGURE 7  T I M E COURSE FOR DEVELOPMENT OF PLATEAU POTENTIAL  UNDER  CA -FREE 2 +  CONDITIONS WIRH 1 M M C O I N T H E A C S F  22  2 +  FIGURE 8  B L O C K A D E OF PERSISTENT N A  +  CONDUCTANCE IN A T Y P E 1  NEURON ELIMINATES  PLATEAU POTENTIAL A N D P O S T - P U L S E HYPERPOLARIZATION ( P P H )  FIGURE 9  REDUCTION IN SLOPE OF PLATEAU DUE TO T T X  2 4  BLOCKADE OF PERSISTENT N A  C O N D U C T A N C E IN A T Y P E 1 NEURON UNDER C A - F R E E CONDITIONS WITH 1 M M 2 +  FIGURE 1 0  EFFECTS OF REDUCED EXTRACELLULAR [ N A ] +  DURING PERFUSION WITHOUT [ C A ] AND WITH C O 2 +  CO  2 +  +  26  ON PLATEAU POTENTIALS EVOKED  2 8  2 +  F I G U R E 1 1 HIGH-THRESHOLD C A - A C T I V A T E D K C O N D U C T A N C E IN A T Y P E 1 NEURON.. 3 0 2+  FIGURE 1 2  B L O C K A D E OF K  +  +  CONDUCTANCE BY T E A APPLICATION (1  M M ) TRANSFORMS  SPIKE BURST FIRING OF A T Y P E 1 NEURON INTO PLATEAU POTENTIAL GENERATION  3 2  FIGURE 1 3  NA -ACTIVATED K +  +  CONDUCTANCE IN A T Y P E 1  WITH C A - F R E E A C S F A N D C O 2 +  2 +  (1 M M )  FIGURE A 1 MAIN PANEL  NEURON DURING PERFUSION  3 6  5 9  FIGURE A 2  SUBPANEL "CONTINUOUS D A Q "  60  FIGURE A 3  "BUILD P R O T O C O L " VI  61  FIGURE A  4  "BUILD  PROTOCOL" VI  GENERATION  FIGURE A  5  IN THE MODE OF THE C H I R P - T Y P E STIMULUS  62  "BUILD PROTOCOL" VI  GENERATION  IN THE MODES OF THE S I N E W A V E - T Y P E STIMULUS  63  FIGURE A 6  " C H A R T R E C O R D E R " VI  6 4  FIGURE A 7  " P R O T O C O L D A Q " VI  6 5  FIGURE A 8  " P L A Y BACK" VI  FIGURE A 9  " D S P " VI  67  "FIT" VI  6 9  FIGURE A  10  FIGURE A 11  " D X / D T " VI  6 6  70  xi  ACKNOWLEDGEMENTS I would  like  to express my gratitude  to Dr. Dietrich  W. F.  Schwarz without whose support and encouragement this work would not have been possible. I would like to thank Dr. Ernest Puil for his invaluable advice and his endless time on direction preparation of this thesis.  during process of experiments and  I  1. INTRODUCTION  The nucleus principalis station  trigemini  (PrV) is the primary  in the lemniscal pathway mediating somatosensory s i g n a l s  from facial regions to the cortex.  In mammals, the spinal trigeminal  nucleus, consisting of subnuclei oralis, constitutes cortex  central  interpolaris,  and c a u d a l i s ,  a second neuron system in the sensory pathway to the  (Olszewski 1950).  defined and distinct  In the gerbil,  the  PrV is large,  from the spinal nucleus, situated  the brainstem and just rostrally to the bifurcation  well-  laterally  in  of the t r i g e m i n a l  root (Ramon y Cajal 1910). The PrV ascending information  receives  fibers  of  mostly  ventroposteromedial  the by  the  primary  fifth way  nerve of  the  nucleus of the  Torvik 1957; Williams et al. 1994).  sensory  input  from  short  and, as  an output,  sends  medial  thalamus  lemniscus  to  the  (VPM; Jones 1 9 8 5 ;  The PrV also receives  afferent  fibers from sensorimotor regions of the cerebral cortex (cat: Brodal et al. 1956), red nucleus (cat: Edwards 1972), periaqueductal and dorsal  raphe nuclei  (rat:  Li et  al. 1993).  gray  Hence, neuronal  2  operation and sensory transduction  in the PrV are likely  subject  to  conveys  a  various modes of control. The  peripheral  input,  predominance of tactile  although  multimodal,  sensory afferents  the dorsal column nuclei.  to the PrV, analogous to  In the PrV of rodents which have w e l l -  developed whiskers, barrelets occupy a large portion of the neuropil, representing patterns  afferents convey  from  individual  information  vibrissae.  about  topographically  in a point-to-point  V P M , en route  to the cortical  vibrissal  projection,  barrel  Their  field  discharge deflection,  to the barreloids (Belford  in  and K i l l a k e y  1979a; Ma and Woolsey 1984; Van der Loos 1976; Woolsey 1970). Developmentally,  such maps are present first  in PrV at birth,  and  later in V P M and cortex (Belford and Killakey 1979b; Erzurumlu and Killakey 1983; Killakey and Belford 1979). Peripheral stimulation neurons that electrical  involve  stimulation  produces single unit responses in P r V  a variety  of  the phasic and tonic  Smith 1960).  properties.  of the skin, the patterns  rapidly or slowly adapting afferent for  firing  Following  of impulses  from  fibers, however, cannot account  response modes of PrV neurons (Darian-  The input-output  transformations, therefore, infer  the  3 e x i s t e n c e of different  cell c l a s s e s .  nonmonotonic s t i m u l u s - r e s p o n s e certain  neurons, showing  beyond the strength the  spike  rate.  that  Darian-Smith  an i n c r e a s e  an inhibition  ('surround'  in receptive  projection  neurons and interneurons  hand,  repertoire  of  stimulus  fields).  membrane  that  reduction and s p a c e  a p r e s e n c e of  (Mountcastle  patterns  properties,  of  types  the of  above, the  neurons.  morphology,  connectivity,  PrV,  is  there  a  On t h e  reflect  according  to  various  a  P r V should  contain  neuron's  knowledge  development and spike firing  properties  absence of its  of  functionally  information  neurons.  are not  stations.  conductance  For e x a m p l e , an interaction with  Ca  2 +  and  K  +  of  conductances  in the  work,  we  primary  properties sensory  a persistent is  the  about  In this  known to occur in neurons of other  of  patterns  report on three distinct cell types with certain m e m b r a n e  relay  distinct  1984).  can  Despite a detailed  surprising  electrophysiological  that  reduced  this  adaptation)  This implies  response  probability,  is  (phasic  intensity  of voltage- a n d ligand-gated c o n d u c t a n c e s .  In view different  over time  different  contributions  in  interpretation  represents  other  described  curves for the p h a s i c r e s p o n s e s o f  needed for maximum firing  A classical  (1960)  Na  +  particularly  interesting  because it  endows the  capability limited by a long-lasting  PrV neuron  with  a  bursting  inhibition.  2. M E T H O D S  Using isoflurane, (Meriones  unguiculatus)  we deeply anesthetized  Mongolian  gerbils  aged between 11 and 17 days (P11 to P17).  After decapitation, the brain was removed and immersed in i c e - c o l d artificial cerebrospinal fluid (ACSF) for 1-2 min. into two with a quasi-sagittal  The brain was cut  (i.e., a 20 to 30° horizontal  deviation  from the ideal sagittal plane) incision and further trimmed to form a block containing brainstem and caudal cerebellum.  For making slices  (with a Vibratome), oriented rostro-laterally to ventro-caudally,  we  used the  the  tissue  block  brainstem.  The cut  thickness.  Slices,  trigeminal  nucleus  temperature  that  contained  slices  were  containing (PrV),  the  between  a visually  were  submerged  larger  portion  300  and 350  identifiable into  u,m  in  principal  A C S F at  (23 °C) and allowed to recover for at least  A C S F contained  of  room  1 h.  The  (in mM): NaCI, 125; KCI, 2.5; CaCI , 2; MgCI , 1; 2  2  glucose, 25; NaHC0 , 25; N a H P 0 , 1.25 and was saturated with 95% 3  0  2  4  and 5% C 0 which maintained the pH near 7.4. Low [Na ] s o l u t i o n s +  2  2  were made by partially substituting 'Na-deficient'  NaCI with choline chloride.  perfusion, the [NaCI] in the 1/4,  1/8,  For  and 1/16  NaCI  solutions were, respectively, 57.5, 42.1, and 34.1 mM. For C a f r e e 2 +  A C S F , 2 mM CaCI was omitted  or substituted  2  with 1 mM CoCI , or 1 2  mM CdCI . 2  For whole-cell recording (Blanton et al. 1989; Strohmann et a l . 1994),  we  used  patch  electrodes,  borosilicate glass with filament  pulled  from  (WP Instruments)  thin-walled  and filled  with a  solution containing (in mM): K-gluconate, 115; ethylene glycol-bis(paminoethylether)  A/,A/,A/',A/'-tetraacetic  acid  hydroxyethylpiperazine-A/'-2-ethanesulfonic  (EGTA), acid  10;  A/-2-  (HEPES),  10;  MgATP, 4; NaGTP, 0.3; and KCI, 20. The pH was adjusted to 7.25 w i t h KOH. The signals, recorded in the current-clamp 2B, Axon Instruments), kHz with Instruments  were filtered  a data acquisition  custom-made data acquisition developed  with  Labview  Instruments) (Appendix 1).  at 3 kHz and sampled at  board (16  Corp.) in a Macintosh  mode ( A x o c l a m p -  bit  resolution,  Quadra 950  computer  National running  and processing programs which instrumentation  software  5-10  were  (National  The electrode resistances ranged from 7  6  to 9 MQ and access resistances were <100 MQ.  All  experiments  were conducted at room temperature (22-24 °C). In eleven experiments, pipette solution (5 mg/rhl). was  withdrawn  from  the  neurobiotin  was added to the  At the end of the recording, the pipette neuron  and  the  slice  was  immediately, with 4% paraformaldehyde in phosphate buffer (pH = 7.2) containing  20% sucrose.  The fixed  neurobiotin  were  visualized  using  an avidin  1988).  cells  under  observations  microscope using a camera lucida attachment (Zeiss).  kit,  tetrachloride  (Sigma) as a chromogen (Horikawa and Armstrong from  were  biotinylated A B C Elite  Vector Laboratories) and 0.05% 3,3'-diaminobenzidine  reconstructed  solution  Neurons f i l l e d  horseradish peroxidase (HRP) complex (Vectastatine  were  fixed,  brain slices  frozen and resectioned at a thickness of 75-90 urn. with  patch  Stained a  light  7  3. Results 3.1  Identification of nucleus principalis  In  quasi-sagittal  principalis light  as  trigemini an  dorsoventral cerebellar  was rostral spinal  opaque,  pale  direction.  Its  the  brainstem,  ovoid location  Anatomical  structure,  to the  that  and ventral  to the PrV, as well  nucleus  which  caudally  the PrV (Figure 1, A-C).  the  inferior its  nerve w h i c h  as the cigar shaped, descended  in  the  these brain  (n = 6 ) , to confirm  identity of the PrV and surrounding structures. of recorded neurons provided confirmation  in  helped in  We also studied frozen sections, cut from  slices and Nissl-stained with cresyl violet  nucleus  or r e f l e c t e d  elongated  was ventral landmarks  neurons  the  were the sensory root of the trigeminal  trigeminal  brainstem.  of  (PrV) appeared under translucent  peduncule.  identification  sections  trigemini (PrV)  the  Neurobiotin s t a i n i n g  of their  location  within  Figure 1. Camera lucida drawings of neurobiotin-stained neurons in thenucleus principalis trigemini. A: type 1 neuron. B: type 2 neuron. C: type 3 neuron. Scale, 100 u..  9  We recorded from  62 neurons in the  three types on the basis of neurobiotin  PrV and distinguished  staining  and firing  modes.  The initial membrane potentials were similar,  falling  range (-52  of the neurons w e r e  to -58  mV).  More than one half  spontaneously active, frequently (type 1; Figures 1A, 2; n = 33).  firing  action  into a narrow  potentials  in bursts  Neurons of another group fired  repetitive action potentials only in response to depolarizing pulses (type 2; Figures 1B, 4A; n = 23). types  1  and  2  neurons  afterhyperpolarizations  (AHPs).  large,  long-lasting  morphological features.  Both  somata and dendritic  trees  extending mainly along one axis (cf. Figure 1A,B). fired  of  It was not possible to d i s t i n g u i s h  types 1 and 2 neurons had multipolar  "tonically"  current  The action potentials  included  type 1 from type 2 neurons by their  neurons  single  action  potentials  Another group of  with  fast  AHPs  in  response to depolarizing current pulses (type 3; Figures 1C, 5A; n = 6).  Type  arborizations  3  neurons  had  more  expansive,  than types 1 or 2 neurons.  dendritic  In each type, the soma  usually was elongated, sometimes triangular about 10-15 (im (long axis).  radial  in shape, measuring  10  3.2  Physiological  properties  Firing patterns  of PrV  neurons  of type 1 neurons.  In response to depolarizing  current injection type 1 neurons started firing  with a burst  consisting of doublets, i.e., a superposition of two action on a slower  hump  (n  =  33).  Because  spontaneously active (Figure 2B), we injected hold cells at potential  (V ) of -60  investigation  injections.  Figure 2 shows that depolarization  pulses elicited  responses  to  to  mV for  current  a  pulse  to threshold  with  one or two bursts of action potentials.  increase in the depolarizing current initial burst,  were  a constant current  systematic  current  their  potentials  neurons  mV, -65 mV, or -70  h  of  these  pattern,  An  decreased the latency to the  as well as the duration of the hump and the amplitude  of the AHP. The primary  spike burst remained steadfast  secondary doublets eventually disappeared and the firing  while  the  of s i n g l e  action potentials  increased (Figure 2A), with an increase in current  pulse magnitude.  Eleven type 1 neurons fired a burst of three s p i k e s  ("triplet")  at the onset of a depolarizing current pulse, followed  a doublet and/or single spike firing.  by  Only one neuron fired a burst of  four action potentials. For all type 1 neurons, the burst duration w a s <21 ms and the intraburst frequency was between 100 and 250 Hz.  Figure 2. Characteristic firing behavior of type 1 neuron(holding potential, V = -60 mV). h  A: voltage responses evoked by 500 ms current pulses of increasing amplitude (right). B: spontaneous activity of a type 1 neuron at its "resting" membrane potential ( V = 0). Note that the neuron fired mostly doublet and triplet action potentials h  (not shown) and that variations in amplitude reflect low sampling rate (500 Hz).  12  3.2.1  Inward rectification in type 1 neurons The  injection  duration)  into  exhibited  a large  depolarizing  of  type  1  hyperpolarizing neurons  depolarizing  current  such a s l  H  evoked  (Figure  eliminated  the voltage  current-voltage  3C),  c o m p l e t e l y b l o c k e d the pronounced r e c t i f i c a t i o n -70 m V , a n d developed fully mV.  K rectifier +  1994) s e e m e d unlikely in our e x p e r i m e n t s reversal  potential  w a s -98 mV.  C s - b l o c k a d e , w e c o n c l u d e d that +  produced the inward  H  rectification  and - 1 0 0 m V in type 1 neurons.  H  this  of C s (2 mM) +  A s evident Cs  in t h e  application  +  that a c t i v a t e d  IR  near  ( V ) of - 1 0 0 m  ( l ; Travagli  and G i l l i s  b e c a u s e the c a l c u l a t e d K  From the a c t i v a t i o n an l  a  (McCormick and  by a membrane potential  A contribution of an inward  of  We investigated  1 neuron.  (Figure  that  an a c t i v a t i o n  the a p p l i c a t i o n  s a g in a type  relationships  3A).  (500 m s  responses  blocker of l  +  Figure 3 B s h o w s that  pulses  voltage  s a g , implying  possibility by applying C s , a s e l e c t i v e P a p e 1990).  current  w a s the major  in a voltage  +  voltage a n d current  that  range between - 7 0  Figure 3. Inward rectification, evoked by hyperpolarization, and rebound firing in type 1 neuron (V = -60 mV). A,B: voltage responses evoked by hyperpolarizing h  current pulses of increasing amplitude under control conditions (A) and during C s application (B) show blockade of inward rectification and increased latency to rebound firing. C: current-voltage relationships for early and late responses (measured as indicated in A,B) to current pulse injections show blockade of inward rectification by +  Cs . +  D: dependence of latency to first action potential of rebound on amplitude of  current pulse amplitude under control conditions and during C s application. +  Os abolished the voltage-dependence of the latency to rebound firing. +  Note that  14  3.2.2  Contribution of inward rectification to spike generation in type 1  neurons  A  brief,  hyperpolarizng  depolarizing current  hump  pulses.  remained  at  This "rebound  the  offset  of  response" led to a  burst of action potentials and often a second burst or a single a c t i o n potential  (Figure 3). The latency to the rebound response became  progressively  shorter  with  current  pulse injections  of increasing  amplitude (Figure 3D). Extracellular C s application (2 mM) did not +  significantly rebound  change the amplitude  (Figure  3B).  However,  or the number of spikes in the Cs  +  eliminated  the  voltage-  dependence of the latency along with the sag (Figure 3C). Evidently, an l -like tail current did not greatly contribute  to the amplitude of  H  the depolarizing rebound response but was an important  contributor  to the latency of firing emerging from hyperpolarization.  3.2.3  Firing patterns of type 2 neurons  Figure 4 shows the characteristic depolarizing potentials  current  pulses, generating  with slow AHPs.  firing  of a type 2 neuron t o  a tonic  pattern  of a c t i o n  As in the case of the bursting type 1  neuron, an increase in the current pulse injected into a type 2 neuron  15 A  B  20  0 -  0  >  If  >  E  -20  E  >  E  -40 -60  20-  Amm  > ^  55 pA  -20-40-60-80-100-  18 8  i—H 0.0  0.2  1  r  0.4  0.6  T  T  r  0.8  1.0  0.0  0.2  t (s)  T  T  T  T  0.4  0.6  0.8  1.0  t (s)  Figure 4. Characteristic tonic firing behavior of type 2 neuron (V = -58 mV) h  evoked by current pulses of increasing amplitude (right). A: depolarizing voltage responses. B: hyperpolarizing voltage responses show evidence of inward rectification.  16  produced a decrease in the latency  to the first  action  potential,  interspike interval and amplitude of the A H P (Figure 4A). Similarly, hyperpolarizing depolarizing membrane  current voltage  pulse  displacement  sag, resulting potential  hyperpolarizing  4B).  We did not  produced  a  - 1 5 mV activated  a  In all type  produced  larger  consisting of a single action potential hump.  of  that  in a new steady-state  (Figure  pulses  injections  investigate  level  2 neurons,  rebound  of the larger  responses,  on top of a slow depolarizing  the  ionic  mechanism  of the  depolarizing sag or rebound responses in type 2 neurons. 3.2.4  Firing patterns of type 3 neurons  Figure 5 shows the characteristic neuron  depolarized  distinguishing  by current  of fast  firing  injection  feature of the action potential  was a biphasic A H P , consisting Figure 5C).  pulse  tonic  of a type 3  (n  = 6).  A  in this type of neuron  and slow components (cf.  Type 3 neurons exhibited little, if any, adaptation  during  the tonic firing to depolarizing current pulses of 0.5 s duration and no voltage sag in the hyperpolarized  range (e.g., to V = -100 mV). m  We did not observe rebound responses at the offset of current in type 3 neurons (Figure 5B).  pulses  t (s) 0.0  C  0.2  0.4  0.6 t (s)"  0.8  1.0  -35-,  0.28  0.32 . 0.36 t (s)  0:40  Figure 5. Characteristic tonic firing behavior, biphasic afterhyperpolarization, and hyperpolarizing voltage responses of type 3 neuron (V = -58 mV) evoked h  by current pulses of increasing amplitude (right). A: depolarizing responses. B: hyperpolarizing responses. C: faster time scale shows afterhyperpolarization with fast and slow components, from an action potential marked by the asterisk in A.  18  3.3  Plateau potentials in type 1 neurons During  the  course  of  these  (n=3) found that  a depolarizing  of three or four  action  until  the  towards  potential  potentials  plateaued  with  for  r e - a p p e a r e d , abruptly  6B).  W e also  we  occasionally  pulse produced an o s c i l l a t o r y  observed  burst  d e c r e a s i n g peak a m p l i t u d e s  a variable  the end of the plateau, a s i m i l a r  amplitude (Figure  investigations,  period  (-100  ms);  oscillation  of i n c r e a s i n g  ending in c o m p l e t e  repolarization  a  replacement  of  initial  burst  r e s p o n s e s to injected p u l s e s by s u c h "plateau potentials" a n d then, a resumption  of the regular  type  1 firing  behavior.  In contrast,  the  plateau potentials  were not apparent in the types 2 and 3 n e u r o n s .  W e a s s u m e d that  the  ability  of type  plateau  1 neurons, with  potentials  may  relate  to  bursting  a s p e c i a l role in P r V function,  p r o c e e d e d to investigate their ionic m e c h a n i s m .  and  1  20 -  Control  0 >  E  -20-  E  >  -40-60-  \ A  —AA w I F J I  0.08  i  i  0.12  0.16  1  t  B  1  V  i  1  0.20  i  0.24  (S)  200 -  >  -20-  £  >  -40-60-  9 i  0.0  1  1  1  1  0.2  0.4  0.6  0.8  pA r~  1.0  (s)  t  120 CO  l  0.0  1  1  0.2  0.4  1——I  0.6 t  (s)  0.8  r~  1.0  E  100H o c CD  H—*  co  80H 60' 800  400 t  (s)  2+  Figure 6. Type 1 neurons generate plateau potentials during C a -free perfusion. A: time course for plateau potential development (V = -63 mV). The records to h  the same current pulse (27 pA) were obtained at the indicated times, before and 2+  after starting C a -free perfusion. B: current pulse-evoked plateau potential response in a different neuron under normal ionic conditions (inset is on faster time scale). C a reduction in latency to the first spike in the burst accompanies 2+  the development of the plateau potential during time of C a -free perfusion (as in A). The plot shows the latency as a function of time after initiation of perfusion without C a .  9  20  3.4 Ionic mechanism  of plateau potential generation in type 1  neurons  3.4.1  Relationship of plateau potential firing to extracellular C a  On perfusion of Ca -free A C S F to reduce C a 2+  2 +  2+  influx (n = 4), w e  observed a dramatic change in the firing pattern of the type 1 neuron evoked by depolarizing current pulses. [Ca ], the neuron fired 2+  potential  (Figure  a doublet  6A, Control).  During perfusion with 2 mM  followed  During  by a single  the C a - f r e e 2+  action  perfusion,  however, we observed a progressive reduction in the latency of the initial  action potential,  potential  (Figure 6A,C).  enhanced bursting, After  and onset of a plateau  ~2 min. of perfusion with the C a 2 +  free A C S F , the neuron fired triplets  followed  by a doublet  6A, 130 s). By 200 s, the neuron fired an initial  burst comprised of  four spikes and AHPs of decreasing amplitude followed burst and an action potential neuron transformed  (Figure 6A). The initial  into a plateau at V  (Figure 6A, 370 s), although  m  (Figure  by another burst of the  = ~-20 mV for - 1 0 0 ms  the plateau duration  varied  greatly  between experiments. We performed  similar  experiments  using blockade of  conductance by perfusion of Ca -free ACSF containing C o 2+  2+  Ca  2 +  (1 mM; n  21  = 16).  Figure 7 shows the effects  changes in firing  of this blockade which produced  behavior of a type 1 neuron, similar  to those of  C a - f r e e perfusion but with a much more rapid transition  from  2+  normal firing min.,  mode to the plateau potential.  we observed a well-developed  - 2 0 0 ms at V  m  the  Thus, in less than 3  plateau potential,  lasting  for  = - - 2 0 mV (Figure 7, 160 s), and in less than 4 min.,  its duration increased to nearly 500 ms (Figure 7, 200 s). neurons, application  of  Co  2 +  (1  mM) in C a - f r e e A C S F for 2+  minutes  resulted  in plateau potentials  duration  of the depolarizing current  conducted a set of experiments [Ca ] (0 mM) in the A C S F with C d 2+  (n = 6; not shown).  In s e v e r a l  that were longer than  pulse (not  using partial 2 +  shown). substitution  (1 mM) with very similar  6-8 the  We a l s o of  the  results  2+  Figure 7. Time course for development of plateau potential under Ca -free and 1 mM C o conditions (V = -60). Note prominent oscillations on plateau during perfusion for 200 s. 2+  h  23  3.4.2  Involvement of a persistent N a  +  conductance  Based on the data obtained during C a - f r e e perfusion  without  2+  and with  Co  persistent  or C d , we hypothesized that  2 +  2 +  Na  motoneurons  +  conductance,  (Llinas  such  and Sugimori  as  Purkinje  1980a,b;  1980), produced the plateau potentials. potentials  in  of a  cells  Schwindt  and  and C r i 11  As a test, we evoked plateau  by applying C o i n C a - f r e e A C S F and then, tetrodotoxin 2 +  2+  (TTX) to block the persistent N a conductance.  After  +  duration, plateau potential  in  <1  min.  evoking a l o n g -  (Figure 8A, Control), the application  TTX (600 nM) annihilated the plateau potential potentials  the activation  (not  shown).  and the fast  We also  of  action  applied  lower  concentrations of TTX, bearing in mind that lower concentrations the hydrophilic toxin would take longer to saturate the tissue.  of The  application of 60 nM TTX caused, after 40 s, a division of the plateau potential  into two  shorter  depolarizations  (Figure 8A).  subsequent 3 min., there was a progressive reduction and duration,  and after  6-7  min., an elimination  in amplitude  of  the  potential, leaving behind a burst of two action potentials reduced amplitude (Figure 8A).  Over the  plateau  of s l i g h t l y  With this concentration, then, it w a s  possible to annihilate plateau potentials without markedly  affecting  24  ->—r 300  19 pA 0.0  0.2  0.4  0.6  0.8  1.0  t (s)  Figure 8. Blockade of persistent Na+ conductance in a type 1 neuron eliminates plateau potential and post-pulse hyperpolarization (PPH). A: time course of T T X application 2+ 2+ (60 nM) under C a -free and 1 mM Co conditions shows splitting of plateau potential into two (40 s), a shortening of its duration (160-240 s) and complete blockade (340 s). After 420 s, TTX application blocked the burst of 3 action potentials (incompletely resolved) present at 340 s. B: changes in temporal sum of P P H ( W ^ , see Eq. 1 and text) measured in same neuron as a function of time of T T X application.  25  the neuron's ability  to fire  action potentials.  slope of the rising phase of the fast  action potential  after a total blockade of the plateau potential potentials  disappeared  5-6  A reduction  min. after  was apparent  with TTX. The action  TTX application.  findings show that small changes in the persistent due to TTX application  in the  can produce dramatic  These  N a conductance +  changes in the f i r i n g  behavior of the neuron. 3.4.3  Sensitivity of plateau potentials to T T X .  We investigated TTX blockade  the sensitivity  by performing  of the plateau potentials  concentration-response  (Figure 9). First, we changed the firing perfusion of Ca -free solutions with C o 2+  potentials then  with depolarizing current  applied  TTX at  different  experiments  mode of type 1 neurons by 2 +  (1 mM) and evoked plateau  pulses (500 ms, duration). concentrations  for  long-lasting  effects.  criterion  For quantification  behavior, we used a ratio TTX and control conditions.  We  6-10 min.  Application of TTX for 10 min. allowed observations of full which we considered an important  to  recovery  for assessment of i t s of  effects  on f i r i n g  of slopes of the plateau potential  under  We obtained the slope from a linear f i t  of the voltage points between the local minimum  after  the second  26  t (s)  Figure 9. Reduction in slope of plateau due to T T X blockade of persistent N a 2+  +  2+  c o n d u c t a n c e in a type 1 neuron under C a -free and 1 m M C o conditions. A : superposition of plateau potentials s h o w s the more rapid descent of the plateau, o b s e r v e d at - 1 0 min of T T X application in 0.6, 1.2, and 1.8 n M concentrations. Solid lines are a linear fit to voltage response between the local minimum after the s e c o n d spike at the start of the plateau and the local minimum before the plateau terminated in a rapid repolarization. B: concentration-response relationship for T T X effects on the s l o p e of the plateau (as in A). E a c h point (± S E ) represents 8 to 11 m e a s u r e m e n t s taken from e a c h neuron (n = 5) and solid line represents a Langmuir m o d e l fit.  27 action potential  in the initial  oscillation,  and the local  minimum  just before termination of the plateau potential. Figure 9A shows that TTX application increased the latency to the  first  spike  and decreased spike  magnitude and duration dependent manner.  amplitude  of the plateau itself,  as well  However, the slope of the plateau decay provided of TTX action.  shows that the concentration-response  relationship  a Langmuir  the  in a c o n c e n t r a t i o n -  a more sensitive, reproducible indicator  with  as  model, representing  the  binding  Figure 9B  is of  consistent TTX to N a  +  channels.  3.4.4  E x t r a c e l l u l a r replacement of N a  As additional  confirmation  N a conductance, we investigated +  replacement  with  choline  +  with choline  for an involvement the effects  during blockade of C a mM C o . 2+  2 +  In contrast  the  plateau  (Figure  10).  2+  to  TTX, the  +  As  pulses to induce the plateaus  effects  of  extracellular  1 Na  +  Thus, a sequential reduction of the N a  +  caused corresponding reductions in the amplitude potentials  Na  currents with C a - f r e e A C S F containing  deficiency were unspecific. concentration  persistent  of extracellular  on plateau potentials  before, we used depolarizing current  of  as well  as  action  potentials.  of  These  28 200 -  >  Control  -20-  E  >  -40-60-  [Ca ] = 0 mM, 2+  [Co ] = 1 mM 2+  Choline CI Replacement 1/4 [NaCI]  24 pA l 0.0  1  1  0.2  0.4  1  1  T  0.6  0.8  1.0  -  t (s)  Figure 10. Effects of reduced extracellular [Na ] on plateau potentials e v o k e d 2+ 2+ during perfusion without [ C a ] and with 1 m M C o . Choline CI w a s used to replace NaCI in the A C S F . All measurements were m a d e at - 1 0 min after exchanging the perfusion. Note the systematic reduction in plateau (and spike) amplitude, in contrast to the reduction in duration o b s e r v e d under T T X (cf. Figure 8). +  29 observations are in good agreement with the expectations based on the calculated change in the N a reversal potential due to the change +  in extracellular [Na ]. +  At low N a concentrations, the duration of the +  plateau was determined by the injected current pulse (cf. Figure 1 0 , 1/8 and 1/16  [NaCI]). Therefore, the terminal  plateau may require a critical  extracellular  repolarization [Na ]. +  In summary, the  marked reductions in the amplitude of plateau potentials deficiency in the extracellular TTX  provide  strong  [Na ] and their +  evidence  for  a Na  due to a  complete blockade by involvement,  +  of the  likely  a  persistent N a conductance. +  3.4.5  Involvement of Ca -dependent rectification 2+  From our results  it  seemed likely  that  activation  of  Ca  2 +  currents in type 1 neurons did not cause and, indeed, prevented the formation  of plateau potentials.  why a blockade of C a  2 +  holding the neuron at V  m  influx  We investigated supported their  possible reasons  generation.  While  near -60 mV, we applied TTX (600 nM), 4 -  aminopyridine (4-AP; 0.5 mM) and tetraethylammonium (TEA; 10 mM) in order to unmask this possible C a in Figure 11 A,  depolarizing  2 +  current  conductance (n = 5). As shown pulses evoked voltage  during the initial 200-300 ms of the responses.  humps  The current-voltage  2+ ' + Figure 11. High-threshold C a -activated K c o n d u c t a n c e in t y p e l neuron. A : voltage r e s p o n s e s evoked by 500 ms depolarizing current pulses of increasing amplitude (36, 4 1 , 54, 5 8 , 6 3 , 68 and 72 pA) during N a and K c o n d u c t a n c e b l o c k a d e by application of T T X (0.6 m M ) , T E A (10 mM) a n d 4 - A P (0.5 m M ) . B: current-voltage relationships for peak and steady voltage r e s p o n s e s (as in A ) . 2+ C : a s in A , but during application of Ni (0.6 mM) which completely blocked inward rectification. +  +  31  relationships for the peak and steady voltage responses (Figure 11B) show that this application  rectification  of N i  2 +  activated  at - 4 0 mV. E x t r a c e l l u l a r  (600 mM) completely  blocked the r e c t i f i c a t i o n  (Figure 11C). These data are consistent transient,  high-threshold  Ca  contribute  to the initiation  2 +  with  conductance.  the activation  of a  This rectification  may  of the plateau potential  and have some  bearing on its limitation. 3.4.6  Involvement of Ca -activated K 2+  +  conductance  We considered the possibility that a C a the influx necessary for activation 'controlling  conductance provided  2 +  of a K conductance and hence, a +  A H P ' . For. example, a plausible  plateau potential  is that the blockade of C a  activation of a K conductance that normally +  explanation  2 +  for the  entry prevented the  repolarized the neuron.  As a check, we applied TEA (1 mM) to see if a K -channel blocker +  also produced a propensity caused by a blockade of C a course of effects mM).  The first  for plateau generation, similar 2 +  influx.  Figure 12 illustrates  produced by extracellular  application  sign of TEA action on the firing  neuron depolarized with  a current  to t h a t the t i m e  of T E A (1  behavior  of the  pulse was a conversion  of the  initial burst of two spikes into a broader hump crowned by fast  32 20H  38 pA 0.0  0.2  0.4  0.6  0.8  1.0  t (s)  Figure 12. B l o c k a d e of K conductance by T E A application (1 mM) transforms s p i k e burst firing of type 1 neuron into plateau potential generation ( V = -57 m V ) . T h e s a m e pulse amplitude w a s used for the recordings before and after the indicated times after initiation of T E A - a p p l i c a t i o n . +  h  33 spikes  (Figure  repolarization, potentials. the  120  s).  of  12,  repolarization,  spikes  resulted  from  a blockade  160  s).  By  as reflected  transformed  action  this  exaggerating time,  the  initial  TEA application  burst delayed  in a 20-30% increase in the duration of the burst (not shown).  into plateau potentials  the bursts (Figure 12, 260 s).  of  Such bursts  whereas doublets  later, bursts and plateaus, replaced the single action potentials followed  of  depolarizing hump became broader and  increased,  the second action potential slowly  This  also evident from the reduced AHPs of single  Later, the initial  number  (Figure  12,  These effects  and that  of TEA are  comparable to the changes in the firing pattern of the same types of neurons deprived of external C a the concept that a C a potential  3.5  activated  (cf. Figures 6 and 12) and support K conductance regulates +  plateau  expression.  The post-pulse  An  2 +  2 +  abrupt  hyperpolarization  repolarization  (PPH)  terminated  the  plateau potentials in type 1 neurons (Figures 7, 8). for the sudden repolarization remains unclear. initial resting potential  Na -dependent, +  The exact reason  The undershoot of the  resulted in a long-lasting  hyperpolarization  34  after termination the  of the stimulus  PPH occurred  blockade  of  activation  K  during  blockade  conductances  +  pulse (PPH; Figure 8A). of  Ca  influx  2 +  by TEA, we  but  Because  not  hypothesized  during  that  the  of a Na -dependent K conductance (cf. Bader et al. 1 9 8 5 ; +  +  Schwindt et al. 1989) produced the PPH. In order to estimate  the  effect of the N a conductance blockade on the P P H size, we measured +  the temporal sum of the P P H amplitude ( W  PPH  ) which is (1)  where V is an initial  value and V (t) is membrane potential  h  given time. The integral  was taken from the point where  repolarized to V (t = 0), to the end of the recording («>). h  reduction  of  Na  +  influx  due to  TTX blockade  corresponding reduction in the P P H (n = 4). course of the reduction roughly  to  the  at a  m  in  the  V  m  Indeed, a  nM) caused a  Figure 8B shows the time  of the normalized W  decrease  (60  the  plateau  PPH  which corresponded  potential  during  TTX  application. A long-lasting  hyperpolarization  dependent manner, following absence of a plateau hyperpolarization  a depolarizing  potential.  at the offset  also occurred, in a v o l t a g e current  We used Eq. 1 to of depolarizing  pulse  in  quantify  pulses that  the the  evoked  35  sub- and suprathreshold responses under conditions of blockade of Ca  2 +  influx, before and after TTX application (n = 4).  For this s e r i e s  of experiments, we only partially blocked N a conductances with l o w +  concentrations  (e.g., 5 nM) of  TTX.  At  15-20  min. of  the TTX  application, the neuron did not generate a plateau potential  but s t i l l  was able to discharge one or more action potentials. Figure 13 shows the results of such an experiment in a type 1 neuron where V = ~-70 mV. In this neuron, a substantial h  of the PPH was apparent during TTX application pulse amplitude exceeded 25 pA (Figure 13A.B). TTX had little  or no effect  subthreshold voltage effects  on the  of TTX were  pronounced with  when the  mV (Figure  larger  current  evoked plateaus, bursts, and single action potentials. also observed these effects  current  The application  hyperpolarization  responses below -60  reduction  of  following 13C).  The  pulses t h a t Note that  when the voltage responses were  we still  under the threshold for a plateau potential (—48 mV) in this neuron. These observations  provide strong support for a N a activated  conductance mechanism in the P P H .  +  K  +  36  0.7  0.8  1.0  0.9  1.1  1.2  t (s)  -70  -65 V  m  -60 (mV)  Figure13. N a - a c t i v a t e d K conductance in a type 1 neuron during perfusion 2+ 2+ with C a -free A C S F and C o (1 m M ) . A : temporal s u m of post-pulse hyperpolarization (Wp^, s e e Eq.1 and text) a s a function of current pulse amplitude during control conditions and T T X application (5 nM). B: e x a m p l e s of hyperpolarizations s h o w n at high gain in Control (1 in A) and T T X (2 in A) conditions. C : d e p e n d e n c e of Wp-,. on the level of subthreshold depolarization. +  +  37  4. DISCUSSION  In  this  principalis  first  study  trigemini  of  the  electrophysiology  neurons, we distinguished  neurons (types 1, 2, and 3), according to their current-pulse repetitive  injections.  (tonic)  classes  firing  in response to depolarizing strength.  of  This  currents,  in  receptive  fields  (cf.  of at  stimulus-response of  which, classically, is the basis for quantifying neurons  of  responses to DC and  may represent a simple rate code for the intensity  sensory stimuli responses  three  nucleus  In all neurons, we observed a pattern  rates that depended on current relationship  of  the  thalamo-cortical  levels, Mountcastle 1984).  4.1  Depolarizing  responses  in types 2 and 3 neurons  The simplest transformation count /  intensity  depolarization pattern.  of an input current into a s p i k e -  code was evident  with  a ramplike  in type  3 neurons.  slope preceded  Because this slope increased with  a regular  current  orderly inverse relationship resulted between stimulus latency.  A  slow firing  magnitude,  an  strength  and  The responses of type 2 neurons to depolarizing  currents  38  were similar,  except that they exhibited slow AHPs and, therefore,  somewhat lower firing rates.  In type 3 neurons, the biphasic AHPs  led to complex depolarizing slopes that preceded repetitive  firing of  single  rate  action  potentials  and seemed to  control  the  and  regularity of firing (e.g., Figure 5).  4.2 Hyperpolarizing  current pulse injections into PrV neurons  Types 1 and 2 neurons exhibited a rectifying, in their type  responses to hyperpolarizing  current  depolarizing sag  pulses.  3 neurons responded to such pulse injections  changes that appeared passive, possibly influenced dendritic neurons  tree.  After  a hyperpolarizing  responded with  potentials  a rebound  occurred at shorter  In c o n t r a s t , with  by a radiating  pulse, both type  depolarizing  voltage  hump.  1 and 2 Action  latencies on top of the hump a f t e r  larger hyperpolarizing pulses. The voltage sags and rebounds w e r e reminiscent  of an I -Iike rectification H  that would tend to limit  (McCormick and Pape 1990)  long hyperpolarizations.  In type 1 neurons,  we found that Cs application, a blocker of l , eliminated the sag a s +  H  well  as the voltage  dependence of the latency  for the rebound  39  response.  It is likely,  then, that l  contributes  H  to  post-inhibitory  rebound responses in type 1 neurons.  4.3  Classification  of types 1 and 2 neurons  In many aspects, types 1 and 2 neurons were similar.  However,  type 1 neurons had other outstanding features that invited a detailed examination of their electrical behavior: burst  firing  of two  potentials;  and,  hyperpolarization. classification +  As  spikes a  in conjunction  long-lasting,  discussed  below,  with  these  1" relate to the activation  functional  indistinguishable states  plateau  criteria  for  of a p e r s i s t e n t  neuronal  of one class.  (2)  post-excitatory  If it were possible to regulate this  morphologically  represent  four  (3)  as "type  N a conductance. these  to  (1) spontaneous firing;  conductance, groups  Our results,  could  however,  suggest the separate classification of types 1 and 2 neurons.  4.4  Burst firing in type 1 neurons  In these slice preparations, type 1 neurons fired spike bursts spontaneously.  In response to  neuron characteristically  started  depolarizing with  current  a brief  burst  injection,  a  discharge,  40  followed  by a tonic pattern of single action potentials.  The burst  discharge consisted of 2-4 action potentials superimposed on a s l o w depolarizing  hump.  The application  depolarization and eliminated  of  TTX blocked  the action potentials.  the  slow  A slow,  Ca 2 +  dependent depolarization was apparent, during blockade of both N a and K c u r r e n t s (cf. Figure 11).  Under these conditions,  +  of N i  2 +  application  annihilated the slow depolarizing response to current  injection variations  which  had a threshold  of —40  mV.  This may  pulse reflect  in a Ca -channel protein combination that binds to N i 2+  (cf. Zamponi et al. 1995) or a distribution  +  2 +  of Ca -channels in the 2+  dendrites, which are located remotely from a likely somatic site of recording. While such a transient  Ca  2 +  current  may provide a minor  contribution, the spike burst is largely attributable to the activation of persistent and transient N a conductances. +  4.5  Is the burst firing pattern of type 1 neurons representative of  PrV neurons in vivo?  In the early extracellular Smith  (1960)  observed that  investigations  of the PrV, D a r i a n -  some neurons always  started  response to a stimulus with a spike burst but dismissed this  their pattern  41  as evidence for  possible cell damage.  increase in the intensity  In the in vivo studies,  of electrical  cutaneous (lip)  stimulation  did not change the interval between the two action potentials initial  burst.  burst  to  Much stronger  appear at  shorter  occurred repetitively similar  to  the  depolarizing further  current  we  representative  latencies;  responsiveness  of  single rate.  typel  action  the  suggest  in  that  vivo the  and  neurons  in  vitro  striking  evidence for the firing  the  potentials  This behavior is very injected  pulses of increasing amplitudes. of  in the  caused these spikes after  at a greater firing  comparison  problematic,  stimuli  an  Although data  may  similarities  patterns  with a be  provide  of PrV neurons  in  vivo.  4.6  The plateau potentials of type 1 neurons  A distinguishing  feature  of type 1 neurons was an ability  generate a plateau potential.  In many respects, this  similarities  plateau  (Llinas  to  the  somatic  and Sugimori  latency on stimulus from the stimulus  1980a),  amplitude; amplitude;  potential  including  (1)  of  potential Purkinje  has cells  a dependence of  (2) an independence of its (3) plateau oscillations,  to  its  duration  usually  of  42  inactivating  action potentials;  a membrane potential amplitudes. cell  of  and, (4) a relatively  ~-20 mV, despite the various  Also, TTX-application  plateau potentials.  fixed plateau a t stimulus  blocked both PrV and P u r k i n j e  The Ca -dependent plateau potentials 2+  in  cerebellar and spinal neurons (Hounsgaard and Kiehn 1989; Llinas and Sugimori  1980b),  and the Na -dependent  plateau  +  potentials  in  striatal and hippocampal neurons (Hoehn et al. 1993) are i n s e n s i t i v e to high TTX-concentrations. without  significant  concentrations  The elimination  alterations  provides firm  to action  of plateau potentials  potentials  by low TTX  evidence that their generation in P r V  neurons requires the activation of a persistent N a current. +  4.6.1  Influence of cation  currents  Occasionally, depolarizing stimuli in  neurons  minimized  under Ca  2 +  Ca -activated  currents +  1984),  extracellular  with  K currents  2+  Sedlmeir  normal  elicited  plateau  conditions.  potentials When w e  Ca-free, Co -application, or blocked 2+  with  such stimuli  T E A application always  (cf. Galvan and  evoked plateau  potentials.  During the presumed, progressive reduction of these currents, was a gradual prolongation  there  of the plateau (cf. Figures 7 and 12).  Therefore, Ca -dependent currents may contribute to the generation 2+  43  and limit  the duration of the plateau.  conditions, an interaction Na  +  currents  2 +  2+  +  would produce a depolarizing  of a hyperpolarizing input. currents  extracellular  of C a , Ca -activated K , and p e r s i s t e n t  spike burst in response to an excitatory  interactive  Under normal  hump, causing a b r i e f  stimulus or, on t e r m i n a t i o n  In view of possible modulation of these  (cf. Schwindt  et al. 1992),  modulate various aspects of plateau potentials  mechanisms t h a t seem likely  under  physiological conditions (cf. Zheng and Gallagher 1995). In their  investigations  of Purkinje cells,  Llinas and Sugimori  (1980a) observed an increased plateau magnitude after conductances  with  Ba  replacement  2+  of  blocking K  extracellular  Ca  +  or  2 +  intracellular application of high TEA-concentrations.  We observed a  reduced plateau  conditions  magnitude  reduced extracellular by TTX. These findings  in PrV neurons  [Na ] or partial  of  blockade of N a conductances  +  directly  under +  support the possibility  (Llinas and  Sugimori 1980a) that the plateau may represent a balance of N a and +  K  +  currents.  4.6.2 Repolarization of plateau potential  What terminates  the plateau in such an abrupt  have observed that a reduction of C a  2 +  influx  manner?  We  causes a prolongation  44  of the plateau (Figures 6, 7). The application of TEA also gradually prolonged the total  plateau time.  Therefore,  one explanation  termination of the plateau involves activation of a K internal  Ca . 2 +  activates  +  conductance by  Another is that the depolarization-initiated  a similar,  repolarizing  K conductance. +  for  Na influx +  Neither a c r i t i c a l  intracellular [Ca ] nor [Na ], alone, can explain the decreased plateau 2+  duration  when  +  Na  influx  +  was  gradually  conditions where there is very little  Ca  reduced influx  2 +  by TTX, under  (cf. Figure 8).  Oi  reduction of the "persistent" N a influx, the K currents that balance +  +  with a persistent Na current to limit +  mV, are sufficient justify  the plateau potential  to repolarize the neuron.  a hypothesis that a combination  current  with  These considerations  of a voltage-dependent  a Ca -dependent, or a Na -dependent, 2+  to ~ - 2 0  +  K  +  K  +  current  repolarizes the plateau.  4.7  Post-pulse  Whatever  hyperpolarization  its  exact  (PPH)  mechanism,  the  repolarization  plateau potential presumably relates to the hyperpolarization occurred on termination  of a depolarizing current  of  the  which  pulse (post-pulse  hyperpolarization or PPH). The PPH was not likely a consequence of  45  the sole activation first  of a voltage-dependent  K conductance.  In the  +  place, the TTX application, blocked the PPH in a voltage  expected for the activation  of the persistent  N a current.  voltage  range where  the  TTX-sensitive  Indeed,  +  blockade of the PPH required only low concentrations  range  of TTX. The  PPH activated  includes  membrane depolarizations that were subthreshold for the generation of plateau potentials.  We observed that the temporal sum (or area)  of the P P H correlated roughly to the magnitude of the stimulus  pulse  or depolarization. On the other hand, we did not observe PPHs during TEA-application activation  which is consistent  the hypothesis that  of a K conductance and not, electrogenic +  produces the PPH in these neurons.  potential  the  N a pumping, +  In axons, the equivalent of the  P P H (hyperpolarizing afterpotential) reversal  with  is affected by changes in the K  and extracellular  TEA application,  +  in a manner  that is predictable for an activated K conductance, but not a f f e c t e d +  by inhibitors  of electrogenic N a pumping (Poulter et al. 1995). +  We  conclude that a "persistent" increase in internal [Na ] (rather than in +  voltage) during the depolarization  triggered  the activation  conductance, producing the P P H in type 1 neurons.  of a K  +  46  4.8  Functional  considerations  in type 1 neurons  The early burst of a doublet during a depolarizing input in type 1 neurons implies that the onset of a natural stimulus would receive emphasis.  The spike firing  within  over a wide range of input current rate of firing  the burst usually was constant amplitudes.  Hence, the  in the burst cannot provide coding for the  amplitude, unlike the subsequent firing  of single action  It  would  seems probable  that  spike  bursts  occur  responses to natural excitatory (and post-inhibitory) the tonic firing  pattern  the nature (or modality) brief excitatory from  rapidly  bursts. receptors  fiber  inputs.  postsynaptic potentials  adapting  threshold  inputs whereas duration  of  of slowly  sufficient  If rapidly adapting afferents  and  On the one hand, activity  may evoke only  On the other, the extended activities  tonic firing.  potentials.  (EPSPs) mediating  mechano-receptors  could cause depolarizations  stimulus  as  would depend on the stimulus of afferent  initial  spike  adapting  duration  were to provide input  for to  type 1 neurons, the latency to the spike burst may represent the only available  code  for  the  stimulus  intensity,  consideration in somatosensory physiology.  an  uncommon  A more likely  is that type 1 neurons have specialized response patterns dynamic aspects of mechanical stimuli.  The larger  scenario for  the  EPSPs a r i s i n g  47  from the faster the burst  vibrissal  latency  and automatically  frequency for vibratory follicle) strict  afferents relationship  deflections,  inputs.  for example, would decrease tune system  Since the vibrissal  are often directionally  for  the  higher  (and other  sensitive, the  hair  relatively  between the magnitude of hyperpolarization  the latency to a rebound burst  and  response (Figure 3) would tend  to  emphasize certain aspects of stimulus dynamics. It  had been shown  (Schultz,  Galbraith  et  al.  1976)  that  deflection of the sinus hair of the second type (St11) in the opposite to the optimal direction reduced background activity of both p r i m a r y afferent  and cortical  neurons to a zero-level. When the hair  released a rebound response was evoked so that peri-stimulus-time-histogram own estimation  (PSTH) was 3-5  its  amplitude  times  bigger  was in (our  based on the Fig. 1C, Fig. 4A and Fig. 4B in the  (Schultz, Galbraith et al. 1976) than background level. Although an effect  of either change of the deflection  speed of vibrissae  angle or the change of the  release to the neutral  position  on the  rebound  response magnitude had not been studied systematically in this work one can get some insight about it analyzing the data presented in the paper (Schultz, Galbraith et al. 1976). It is visible (see Fig. 1C, Fig. 4  48  A', Fig. 4 B \ Fig. 4 C ) in the (Schultz, Galbraith et al. 1976) that f a s t vibrissae  return  optimal deflection  to the neutral  position  after  higher  angle non-  evoked responses that were bigger in amplitude  (impulses/bin in PSTH) and more sharply tuned in time. The fact  that  the same dependence can be observed at the two extreme levels of the sensory pathway leads us to assume that similar  responses can  be recorded in the secondary sensory neurons such as those located in the brainstem  trigeminal  assumption is correct  complex,  (unfortunately,  work where this or similar  including  the  PrV. If  we could not find  hyperpolarisation  suddenly appears to be an important  relation  sensory  to  temporary  the  information  background firing  innervating vibrissae follicle can result  in a reduction  a single  question was addressed) the ability  the Type I PrV neurons to generate rebound response after  processing.  this  of  transient feature  in  For  example,  suppression in the primary  afferent  caused by non-optimal  hair d e f l e c t i o n  of a background excitatory  transmitter  release and thus in hyperpolarization of the postsynaptic site w h i c h in its  turn can contribute  to the whole  neuron hyperpolarization.  Then, transient increase of the primary afferent activity a neuron depolarization  from the more hyperpolarized  can lead to level of  its  49  membrane potential. Such type of depolarization can evoke rebound response  in  the  characteristics  neuron  so that  would reflect  its  certain  probability parameters  and  temporal  of the  incoming  signal such as duration and depth of background activity reduction as well  as the rate of its change. These parameters in their  directly  connected to the real-world  turn are  mechanical stimulation  of the  vibrissae. Based  on the  data  obtained  in  the  presented  work  one  can  hypothesize that the activation of an l -like current, which markedly H  affects the latency-amplitude relationship (Figure 3), may represent an adaptation stimuli.  for  transmission  An empirical  of dynamic change in mechanical  examination  of these considerations  would  allow determination of the types of mechanoreceptive afferents that produce depolarization  and hyperpolarization  the nucleus principalis trigemini.  in type 1 neurons of  50  5. R E F E R E N C E S  BADER,  C. R. A N D  BERHEIM,  L. AND  BERTRAND,  D. Sodium-activated  potassiun  current in cultured avian neurones. Nature Lond. 317: 540-542, 1985.  BELFORD,  G.  R.  A N D KILLA'CKEY,  subcortical trigeminal  H.  Vibrissae  P.  representation  in  centers of the neonatal rat. J. Comp. Neurol.  183: 305-321, 1979a.  BELFORD,  G.  R.  representation  AND KILLACKEY,  H.  The development  P.  188: 63-74, 1979b.  B L A N T O N , M . G . , L O T U R C O , J . J . A N D KRIEGSTEIN,  neurons  Neurosci.  BRODAL,  in  Methods  A.,  trigeminal  vibrissae  in subcortical trigeminal centers of the neonatal rat.  J. Comp. Neurol.  from  of  slices  of  reptilian  A. R. Whole cell  and mammalian  recording cortex.  J.  30: 203-210, 1989.  SZABO,  T.,  nuclei  and nucleus of solitary  TORVIK,  A.  study in the cat. J. Comp. Neurol.  Corticofugal tract.  fibers  to  sensory  An experimental  106: 527-556, 1956.  51  DARIAN-SMITH,  I.  Neurone activity in the cat's trigeminal main sensory  nucleus elicited by graded afferent stinulation. J. Physiol. 52-73,  Lond. 153:  1960.  EDWARDS, S . B .  The ascending and descending projections  of the red  nucleus in the cat: an experimental study using an autoradiographic tracing method. Brain Res. 48: 45-63, 1972.  ERZURUMLU,  R.  S . AND KILLACKEY, H .  P. Development of order in the  trigeminal system. J. Comp. Neurol.  G A L V A N , M . A N D SEDLMEIR, C .  rat  213: 365-380, 1983.  Outward currents  in voltage-clamped  rat  sympathetic neurones. J. Physiol. Lond. 356: 115-133, 1984.  HOEHN, K . , W A T S O N ,  T.  W . AND M A C V I C A R ,  B. A .  sensitive, slow sodium current in striatal Neuron  10: 543-552, 1993.  A  novel  tetrodotoxin-  and hippocampal neurons.  52 HORIKAWA, K. A N D ARMSTRONG, W . E .  labeling:  injection  of  conjugates. J. Neurosci.  biocytin  and  its  means of detection  Serotonin-induced  with  bistability  caused by a nifedipine-sensitive  potential. J. Physiol.  intracellular avidin  Methods 2 5 : 1 - 1 1 , 1 9 8 8 .  H O U N S G A A R D , J . A N D KIEHN, O .  motoneurones  versatile  A  Lond.  414: 265-282,  turtle  calcium  plateau  1989.  JONES, E. G .  The Thalamus. New York, Plenum,  KILLACKEY, H .  P. A N D B E L F O R D ,  G . R.  of  p.  1985,  325-360.  The formation of afferent  patterns  in  the somatosensory cortex of the neonatal rat. J. Comp. Neurol. 1 8 3 : 285-303,  Li,  Y.  1979.  Q.,  TAKADA,  Identification neurons  of  M.,  MATSUZAKI,  S.,  periaqueductal  projecting  to  both  the  gray  Res.  623: 267-277,  Y.  and dorsal  trigeminal  forebrain structures: a fluorescent in the rat. Brain  SHINONAGA,  sensory  A N D MIZUNO,  raphe  nucleus  complex  retrograde double-labeling 1993.  N.  and study  53 LLINAS,  R.  Purkinje  Electrophysiological  A N D SUGIMORI, M .  cell  somata in mammalian  properties  cerebellar  slices.  of in v i t r o J.  Physiol.  Lond. 305: 171-195, 1980a.  LLINAS,  R.  Purkinje  Electrophysiological  A N D SUGIMORI, M . '  cell dendrites  properties  of in v i t r o  in mammalian cerebellar slices. J.  Physiol.  Lond. 305: 197-213, 1980b.  MA,  P.  M . A N D WOOLSEY, T .  vibrissae  A.  Cytoarchitectonic  in the medullary trigeminal  correlates  of  complex of the mouse.  the Brain  Res. 306: 374-379, 1984.  MCCORMICK, D.  activated  A.  cation  AND P A P E ,  current  H. C.  Properties  and its  role  of a h y p e r p o l a r i z a t i o n -  in rhythmic  oscillation  in  thalamic relay neurones. J. Physiol. Lond. 431: 291-318, 1990.  MOUNTCASTLE,  V. B. Central nervous mechanisms in mechanoreceptive  sensibility.  In: Handbook of Physiology. Sect. I, Vol. Ill,  Physiological Society, Bethesda, M D , 1984, p.789-878.  American  54  OLSZEWSKI,  J . On the anatomical  and functional  trigeminal nucleus. J. Comp. Neurol.  POULTER,  M.O.,  dependent  T.,  HASHIGUCHI,  potassium  Neuroscience  68: 487-495,  RAMON Y C A J A L , S .  92: 401-413,  AND PADJEN,  conductance  organization  A.  L.  in  of  the  1950.  Evidence for a s o d i u m frog  myelinated  axon.  1995.  Histologie du Systeme Nerveux de I'Homme et des  Vertebres. Madrid, Consejo Superior de Investigaciones  Cientificas,  1910.  S C H U L T Z , W.,  GALBRAITH,  G. C ,  GOTTSCHLADT,  comparison of primary afferent  K.  and cortical  M.,  CREUTZFELDT,  neurone activity  sinus hair movements in the cat." Exp. Brain Res.  SCHWINDT,  P. C.  AND CRILL,  W. E.  Properties  O.  24: 365-381,  of a persistent  D.  coding 1976  inward  current in normal and TEA-injected motoneurons. J. Neurophysiol. 1700-1724,  1980.  "A  43:  55  SCHWINDT, P . C , S P A I N , W . J . A N D C R I L L , W .  excitability  by  a  sodium-dependent  neocortical neurons. J. Neurophysiol.  E. Long-lasting potassium  current  of  in  cat  61: 233-244, 1989.  SCHWINDT,  P . C , S P A I N , W . J . AND CRILL, W .  calcium  chelation  currents  in cat  E. Effects  on voltage-dependent neocortical  reduction  and  of  intracellular  calcium-dependent  neurons. Neuroscience  47:  571-578,  1992.  STROHMANN,  B.,  rectifying  properties  SCHWARZ,  D. of  W.  F.  A N D PUIL,  nucleus  ovoidalis  auditory thalamus. J. Neurophysiol.  TORVIK,  A.  The ascending fibers  nucleus: An experimental  E.  Mode of neurons  firing  in  the  and avian  71: 1351-1360, 1994.  from the main trigeminal  sensory  study in the cat. Am. J. Anat. 100:  1-16,  1957.  TRAVAGLI, R.  A.  AND GILLIS, R.  A. Hyperpolarization-activated currents,  l  H  and IK , in rat dorsal motor nucleus of the vagus neurons in vitro. J. IR  Neurophysiol.  71: 1308-1317, 1994.  56  VAN  DER L O O S ,  Neurosci.  Barreloids  in  mouse somatosensoty  thalamus.  Lett. 2 : 1 - 6 , 1 9 7 6 ,  N.,  WILLIAMS, M .  synaptic  ZAHM,  Differential  D. S . AND JACQUIN, M . F.  organization  projections 453,  H.  of  the  principal  and  spinal  foci  trigeminal  to the thalamus in the rat. Europ. J. Neurosci.  T. A. A N D  V A N DER  Loos, H. The structural  organization of l a y e r  I V in the somatosensory region ( S I ) of the mouse cerebral Brain Res.  17: 205-242,  Z A M P O N I , G . W . , BOURINET,  distinct  F.  E., D U B E L , S . J .  effects  A N D GALLAGHER,  dicarboxylic  cortex.  1970.  on  neuronal  ANDSNUTCH,  calcium  inhibition of activation-gating. Soc. Neurosci.  ZHENG,  6: 4 2 9 -  1994.  WOOLSEY,  two  and  J . P.  acid -induced  (1S,  burst  3R)-1  firing  T. P. Nickel modulates channels:  block  and  Abstr. 2 1 : 1 7 5 3 , 1 9 9 5 .  -aminocyclopentane-1,3-  is  mediated  by a native  57 pertussis toxin-sensitive  metabotropic  septal nucleus neurons. Neuroscience  receptor at rat 68: 423-434, 1995.  dorsolateral  58  6. APPENDIX  6.1  1.  Data acquisition  and processing  program  VMS  In order to perform the experiments presented above, it necessary to design and write a new data acquisitionprogram  for  Instruments boards.  the  Macintosh  data  LabView  Quadra 950  acquisition  instrumentation  and a n a l y s i s  computer  and DMA (direct development  was  and  National  memory  access)  environment  was  chosen for this purpose. LabVIEW includes  is  libraries  specifically programs  for are  a  general-purpose  of functions  and developmental  data acquisition called  and instrument  tools  that  designed  control. LabVIEW their  appearance on a computer monitor and their operation imitate  real-  amplifier).  Instruments  system  because  world instruments  Virtual  programming  (Vis)  (e.g. oscilloscope, tape-recorder,  The main advantage of using the virtual  chart  recorder,  instrumentation  approach, compared with conventional  programming  C, Pascal), is that every VI is a fully  functional  unit, similar  stand-alone  instrument,  be  separately  combination  with  other  which Vl's,  can  supplied  used  languages (e.g.  commercially  or  to a or  in  written  59  independently. Thus, one can assemble an entire new instrumentation complex  by  simply  connecting  different  laboratory. In addition, one can, in future, already working instruments  without  Vl's  in  an  imaginary  add new Vl's to a set of  modification  of existing V l ' s ,  or change parameters or functionality of the Vl's that are already at work. The VMS data acquisition  and processing program used in the  present work consists of seven main Vl's. These are: 1. A hardware setup; 2. The build protocol; 3. A play back module; 4. The chart recorder;  5. The protocol  data  acquisition  module  (DAQ); 6. A  waveform generator; 7. A digital signal processing unit (DSP). A user can access each of these Vl's by clicking on the corresponding button in the main panel (Fig. A1) appearing on a computer monitor when the VMS program is loaded. The "Stop" button terminates the V M S .  execution of  60  The VI "Hardware Setup" is used to change data a c q u i s i t i o n board settings board, and the  such as the device number of the A-to-D number of input  configuration  used  channels  voltage  for  in  the  and output  present  and current  project  conversion  channels. The t y p i c a l includes  recordings  two  expandable  input to  maximum of 8 and one output channel (maximum 2) for current  a or  voltage command generation. The subpanel "External Gains" (Fig. A2) is used to feed information  about external  amplifier  parameters  (voltage gain, current gain and headstage gain) into the V M S program. The subpanel "Continuous  DAQ" (Fig. 2A) defines parameters  chart recorder VI (explained later).  Figure A 2 Subpanel "Continuous DAQ"  of a  The VI "Build Protocol" is used to create a voltage or current command protocol.  Every protocol  can be a stimulus  stimuli repeated in time. Three types of stimuli A  pulse-type  modulated example  stimulus;  sinewave); of  the  "Build  2.  A  chirp-type  3. Sinewave stimuli. Protocol"  VI  for  or a set  of  are implemented: 1. stimulus  (frequency  Figure A3 shows a pulse-type  an  stimulus  protocol generation. In the given example the protocol consists of the 10 voltage pulses (Fig. A3., right-hand slide dial) increasing in amplitude by 5 mV (Fig A3., right-hand  column of controls).  Each stimulus  episodes (E-1, E-2, E-3, E-4, E-5; Fig. A3).  Figure A 3 "Build Protocol" VI  consists  of 5  62  Stimuli with  can be created visually  a mouse and simply  by marking parts of the  dragging and placing the  waveform  corresponding  cursor in a specific position on the display (e.g. Fig A3, E-1 cursor i s in the position x = 0.1 s and y= 0 mV). X- and Y-scaling on the display can be adjusted  automatically  or manually.  The total  number  episodes available for a user is 7 in the visual mode of construction protocol  and unlimited  can be saved in  in  an alternative  a file  text  and loaded  stimulus  mode.  later  for  of  Every use  or  modification. Figures A4 and A5 show the "Build Protocol" VI in the modes of  the  chirp-type  and sinewave-type  stimulus  protocols  respectively.  $WM i  $$:m -i  liiiii  Figure A 4 "Build Protocol" VI in the mode of the chirp-type stimulus generation  i m  63  Figure A 5 "Build Protocol" VI in the modes of the sinewave-type stimulus generation  The VI "Chart Recorder" is designed to substitute  a standard  chart recorder (Fig. A6). It is capable of continuously logging data to the disk without  interruption  of the data acquisition.  In order  to  start data logging a user has to chose a file name for a record of the acquired information. The VI automatically minute, adding the current  time to the file  creates a new file  every  name specified by user  (e.g. cell1_5-37  PM). The sampling rate for the data acquisition  displayed time  period are specified in the VI "Hardware Setup"  the "Continuos DAQ." panel  and in  64  Figure A 6  "Chart recorder" VI  The front panel of the "Protocol DAQ" VI is shown in the Figure A7. This VI is used to conduct single-cell recording in discontinuous mode using pulse protocols (created with the "Build Protocol" VI) or arbitrary waveforms (created with the "Waveform generator" VI) or imported from a text file.  65 l i g f i i l  input limits  f  5  hlph limn | M  !  low llfmt  1 bean  rat?  output limits  m\  509 0  UKHJEFOBM  IsSTRUMtM  PROTOCDl  <*CQUI&iTIOS  Figure A 7 "Protocol DAQ" VI  A protocol "Protocol"  or arbitrary  waveform  or "Waveform"  can be loaded by pressing the  buttons.  Stimuli  can be  delivered  manually (by pressing button "Next", Fig A 7 ) or automatically with a delay that is specified on the slide dial "Delay" (Fig. A 7 ) . Dials in the  panels  amplification respectively.  "Input for  limit" signal  and "Output acquisition  limit"  and signal  Acquired data are saved in a file  The file name is shown in the window  control  on-board generation  defined by the user.  "Path to save". The buttons  "Record" and "Approach" permit changing the voltage scale on the screen to a predefined value. In addition,  both voltage  and current  66  windows  can be transferred  into  an auto-scale  mode so that,  independently of the input signal magnitude or duration, scaling i s maintained at an optimum. Figure A8 shows the front panel of the "Play back" VI. This VI is designed to provide  easy access to  the  data obtained  during  experiment. A user can load a data file, like a video-tape into a video player, so that every experimental  trial  (frame)  can be accessed  sequentially in both forward and backward playback. To play back a l l frames from a file the user should press the "Total" button.  , J,  A  A —•+•  \  IBRD  EJOCKli'HilQ  F i g u r e A 8 "Play back" VI  lOMulflJi!!  67 The button "Print" screens with file media  (hard  permits  a transfer  of both voltage and current  name and location information  drive)  to  the  printer,  creating  from  the storage  a hardcopy  of  the  displayed data. The main VI for data analysis is called " D S P " (fig. A9).  F i g u r e A 9 "DSP"  VI  All data processing operations in this VI are performed on the data from  either  position  voltage  or current  trace  between  two  of these cursors can be changed and fixed  cursors. at  The  selected  positions with a mouse. As a segment of a trace is chosen, the user  68 can perform function.  following  Only  implemented "Max/Min"):  operations: 1. fit  linear  to  and  single  date; 2. define  data (button  exponential  maximum  cursors are positioned  "Fit")  to a  have  been  fits  and minimum  automatically  to the  (button maximum  and minimum of the chosen trace segment; 3. calculate the average over the  trace  segment  (button  (button "dX/dt") using an arbitrary Filter  data  (button  "lnt(mV*ms)").  "Filter");  "Mean"); 4. take  derivative  time window (dial "Window");  6. take  the  An example of a linear fit  shown, with the front panel of the "Fit" A11 shows front panel of the "dX/dt" temporal derivative  the  time  integral  5.  (button  to a plateau potential  is  VI, in Figure A10. Figure  VI with  an example of the  taken from the voltage trace segment between  the cursor markings in the Figure A9. A user can transfer current  trace  directly  (National Instrument  a chosen data segment of the voltage  to the HiQ numerical  computation  software) for on- or off-line  or  package  analysis (fig. A 9 ;  button "HiQ"). The data captured into a text file can be imported into any standard button  spread-sheet  "Capture",  option  or data processing "ASCII",  not  shown).  program  (fig  A9;  Finally  any  data  segment can be captured and stored in binary format  as an a r b i t r a r y  69  waveform  to  experiment  be used as current(fig.  A9;  button  or  voltage-command  "Capture",  options  during  "Command",  "Voltage" or "Current" (not shown). The V M S program is a new flexible tool designed for whole c e l l recording  and  data  analysis.  programs is, that new functions introducing  errors  in  already  The  advantage  can be added without existing  modules.  experiment control and data analysis, therefore, expansion in any direction neurophysiology.  Figure  A  10  "Fit"  VI  over  of potential  The  alternative the risk  of  design  of  is ideally suited to  development of the  modern  Figure  A  11 " d X / d t "  VI  


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