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Sensory transmission in peripheral neurons : effects of K+ channel blockers and autacoids Spigelman, Igor 1988

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SENSORY TRANSMISSION IN PERIPHERAL NEURONS: EFFECTS OF K -CHANNEL BLOCKERS AND AUTACOIDS +  By IGOR SPIGELMAN B. Sc. (Four Year), University of Toronto, 1983 M. Sc. The University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics. Faculty of Medicine  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA JULY 1988 © IGOR SPIGELMAN, 1988  In  presenting  this  degree at the  thesis in partial  fulfilment  of  the  requirements  for  an advanced  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  department  or  publication  thesis for by  his  or  scholarly purposes may be granted by the her  It  is  understood  that  my  copying  or  of this thesis for financial gain shall not be allowed without my written  permission.  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  representatives.  head of  2<T~J*4 _^  - ii ABSTRACT Sensory transmission was studied in trigeminal  root ganglia  guinea pigs, using intracellular recording techniques. examine blockers  in  detail  the  effects  of  on the membrane voltage  applications  (TRG) of  One approach was to  of  different  K -channel +  responses and outward currents of TRG  neurons, in order to better understand the fundamental processes that affect their excitabilities  and repetitive  spike discharge.  The second approach  was to examine several endogenous substances for their effects on the excitabil ities of TRG neurons. In addition, a strategy was developed for electrophysiological recording from neurons in human sympathetic  ganglia.  Successful  investigations  of  these neurons revealed properties similar to certain reported characteristics of sympathetic neurons in experimental animals, including high (~29 MoJ input resistances, channel  blocker  blockers - 10 mM).  pharmacological  sensitivity  tetrodotoxin  4-aminopyridine  The investigations  of  spikes  (TTX, 1 uM) and (4-AP,  1 mM)  to  to  and  the  specific  selective  Na +  K -channel +  tetraethylammonium (TEA,  demonstrated the potential  value of  these in  vitro preparations for studies of the human condition. The investigations in TRG neurons demonstrated that bath applications of TEA (0.1-10 mM) and 4-AP (0.05-5 mM) or Cs  +  applied internally from the  recording electrode, produced an increase in input resistance and a decrease threshold for spike generation in all neurons. increased subthreshold oscillations the repetitive pulses,  Also, applications of 4-AP  of the membrane potential  and enhanced  spike firing evoked by intracellular injections  or elicited  spontaneous  firing.  In contrast,  of current  TEA or Cs applica+  tions blocked the oscillations and the spike afterhyperpolarizations (AHPs) without exaggerating  repetitive  discharge.  These  investigations  suggested  - iii that  several  pharmacologically  control of excitability  distinct  in TRG neurons.  K -currents  contribute  to  the  Comparison of combined actions of  4-AP and TEA with those of C s , suggested that other ions in addition to +  K may contribute to postspike events. +  Single currents  electrode that  voltage-clamp  were evoked  at  the  commands from holding potentials  analyses  revealed  termination  of  near -40 mV.  transient  outward  hyperpolarizing voltage  The activation  was  rapid  (<5ms) and inactivation (T-19 ms) complete at potentials within the activation range (-40 to -75 mV). TEA  (10 mM), fast  During combined application of TTX (1 pM) and  activating,  sustained  currents  depolarizing commands from holding potentials  (>1 s)  were evoked by  near -70 mV.  These currents  were blocked completely by the additional applications of 4-AP (5 mM). Applications of TEA (0.1 mM to 10 mM) produced dose-dependent reductions of the transient currents.  outward currents.  However, administrations  Applications of Cs of 4-AP (0.05  to  also blocked the  +  5 mM) only  slightly  reduced these currents and high doses of muscarinic agonists had no effect. The high sensitivity  to TEA, and not to 4-AP, suggest a fundamental distinc-  tion from similar currents observed previously in other neurons of vertebrates  and invertebrates,  and hence this  transient  outward current  in TRG  neurons, is termed I(y)The  kinetics  Therefore,  of  blockade  re-activation  of  I^jj of  suggest  its  involvement  I ^ ^ by TEA may T  voltage-dependent  repetitive discharge ability.  interfere  Na -channels, +  in  the  spike  indirectly  leading  to  AHPs.  with  decreases  the in  The TEA-insensitive sustained outward current  presumably has an inhibiting influence on repetitive  discharge.  Conditions  that interfere with this current, such as blockade of K -channels by 4-AP +  without a significant  blockade of I / \ , T  strongly  favour the generation of  - iv repetitive discharge in TRG neurons. The investigations  using electrical  changes in the resting potential the perikarya,  or facilitate  stimulation of axons revealed that  could inhibit the invasion of spikes  the  generation  Applications of 4-AP (1 mM) facilitated  of ectopic  the perikaryal  spike  into  discharges.  invasion of  spikes  evoked by axonal stimulation, and also resulted in the appearance of fast (~10 ms) depolarizations applied  stimuli.  that  reached  These investigations  perikarya of sensory  spike  threshold  in the  absence of  provided direct evidence  neurons are capable of spike generation,  that  the  and suggest  that this behavior may occur in normal or pathophysiological conditions. The most notable  effects  of autacoids were those of  histamine, whereas bradykinin did not affect  substance  P and  neuronal membrane properties.  Applications of substance P in micromolar doses evoked large (up to 45 mV), reversible  depolarizations  in the majority of  neurons,  whereas  histamine  applications produced similar depolarizations only in a small portion of the TRG neurons. were evident  Increases in the during  substance  repetitive  discharge  abilities  P-induced depolarizations.  ionic mechanism of substance P action revealed that the resulted in activation of inward currents as well currents.  In addition,  i t was shown that Na  of  neurons  Studies  on the  peptide-applicati'ons  as blockade of outward  and Mg  were involved in  the mechanism of action. These findings represent the first demonstration of the profound actions of substance P on the perikaryal membranes of sensory The excitatory  actions  of this  endogenous peptide  neurons in mammals.  also give  rise  to  the  possibility of physiological actions of substance P at multiple sites in the trigeminal system.  - V -  TABLE OF CONTENTS CHAPTER 1  Page  INTRODUCTION  1  1.1  General introduction  1  1.2  Anatomical organization of the trigeminal root ganglion and other sensory ganglia  2  Embryology and morphology of neurons in sensory ganglia  3  1.3  1.4 Involvement of perikarya in sensory transmission 1.4.2 Role of satellite cells 1.4.2 Variability of neuronal populations in sensory ganglia  5 7 7  1.5 Sensory modalities transmitted by primary afferent fibers 1.5.1 Relationship of afferent fiber diameters to sensory modalities 1.5.2 Morphology of peripheral terminations of primary afferent fibers in relation to sensory modalities  8 9 9  1.6 Peripheral neural mechanisms of pain 1.6.1 Triple response 1.6.2 Axon reflex 1.6.3 Autonomic reflexes  10 11 11 12  1.7 Endogenous algogenic substances 1.7.1 Potassium 1.7.2 Changes in extracellular pH 1.7.3 Acetylcholine 1.7.4 5-Hydroxytryptamine 1.7.5 Histamine 1.7.6 Bradykim'n 1.7.7 Substance P  12 12 13 13 14 14 14 15  1.8  Substance P as neurotransmitter of primary afferent neurons  16  1.9  Membrane electrical properties of primary sensory neurons  18  1.9.1 2  Control of membrane excitability  METHODS  2.1 Animals 2.1.1 Source 2.1.2 Animal feed and housing 2.2 Surgical procedures for removal of the TRG 2.3 Procedure for obtaining human trigeminal root ganglia at autopsy  19 22 22 22 22 22 24  - vi -  2.4  Procedure for obtaining human sympathetic ganglia  24  2.5 Electrophysiological recordings 2.5.1 Ion substitutions 2.5.2 Drugs 2.5.3 Electrodes 2.5.4 Recording equipment  26 28 28 28 29  3  30  RESULTS  3.1  Dependence of resting membrane potential on extracellular cations , 3.1.1 Changes in extracellular [K J 3.1.2 Changes in extracellular [NaJ * 3.1.3 Changes in extracellular [Ca ] and Co^ -application  30 30 32 32  3.2 Membrane properties of TRG neurons 3.2.1 Membrane potential oscillations 3.2.2 Action potentials and afterhyperpolarizations  35 35 35  3.2.3  Effects of ionic channel blockade on spikes  37  Membrane properties of human sympathetic neurons  39  3.4 Effects of K -channel blockers 3.4.1 Current clamp: effects of tetraethylammonium on subthreshold potentials 3.4.2 Effects of TEA on spikes and repetitive discharge 3.4.2 Effects of 4-aminopyn'dine on subthreshold potentials of TRG neurons 3.4.4 Effects of 4-AP on spikes and repetitive discharge 3.4.5 Combined applications of 4-AP and TEA 3.4.6 Effects of intracellular Cs 3.5 Voltage-clamp analysis 3.5.1 Transient outward currents 3.5.2 Effects of ionic substitution 3.5.3 Ineffectiveness of muscarinic agents 3.5.4 Effects of 4-AP and TEA 3.5.5 Effects of internal Cs 3.5.6 Other outward currents 3.5.7 Combined applications of 4-AP and TEA  42  +  2  3.3  +  +  +  42 46 50 50 53 55 59 59 60 64 64 68 68 72  3.6 Studies on the perikaryal invasion of spikes in the TRG 3.6.1 Changes in resting membrane potential 3.6.2 Effects of 4-AP  76 76 79  3.7 Studies of autacoid effects in TRG neurons 3.7.1 Bradykinin 3.7.2 Histamine  83 83 83  - vii -  3.7.3 Substance P 3.7.3.1 Effects on subthreshold membrane properties 3.7.3.2 Effects of substance P on spikes and repetitive discharge  83 83 87  3.8 Studies on the ionic mechanism of substance P actions 3.8.1 Effects of changes in extracellular [Na ] 3.8.2 Effects of changes in extracellular [Mg ] 3.8.3 Effects of Ca -channel blockade 3.8.4 Voltage-clamp studies of substance P actions in the presence of K -channel blockers  87 87 91 91  4  97  +  2+  2+  94  +  4.1  DISCUSSION Membrane potential dependence on extracellular cations  100  4.2 Electrical membrane properties of TRG neurons 4.2.1 Subthreshold responses 4.2.2 Action potentials 4.2.3 Postspike afterhyperpolarizations  100 102 101 102  4.3  103  Membrane electrical responses of human sympathetic neurons  4.4  Differences in the actions of K -channel blockers on TRG neurons 4.4.1 Membrane potential oscillations 4.4.2 Repetitive spike firing +  4.5 Comparison of transient outward current [ I ( T ) J 4.5.1 Ionic species mediating I( ) 4.5.2 Membrane repolarization 4.5.3 Other outward currents 4.5.4 Significance 4.5.5 Bursts and ionic mechanisms 4.5.6 Significance T  105 106 108 w  l  t  n  *A  1  0  9  110 Ill 112 113 114 115  4.6  Spike initiation in the TRG  116  4.7  Membrane responses to autacoids  117  4.8 Ionic mechanism of substance P action 4.8.1 Implications for sensory transmission  118 121  4.9  123  Directions for future research  5  CONCLUSIONS  126  6  REFERENCES  130  - viii LIST OF TABLES TABLE Table I. Table II.  Page Summary of spike and afterhyperpolarization (AHP) characteristics  38  Effects of tetraethylammonium (TEA, 10 mM) on electrical properties  45  - ix LIST OF FIGURES FIGURE  Page  1.  Schematic diagram of the transport chamber  25  2.  Schematic representation of the recording chamber  27  3.  Dependence of membrane potential on extracellular [K ]  31  4.  Effects of Na -deficient solutions  34  5.  Two types of action potentials in TRG neurons  36  6.  Responses of human neurons  41  7.  Effects of combined TTX and TEA applications on spikes and subthreshold oscillations  44  Comparison of TEA and 4-AP effects on spikes and subthreshold oscillations  48  Effects of TEA and 4-AP on spikes in a human neuron  49 52  11.  K -channel blockade and burst activity 2+ Effects of Ca -channel blockade on spikes  54  12.  Effects of Cs on spikes  56  13.  Spike afterpotentials  58  14.  Transient outward currents in TRG neurons  62  15.  Transient outward currents in human neurons  63  16.  Effects of 4-AP and TEA  66  17.  Dose-response of K -channel blockers  67  18.  Effects of Cs on spikes and transient outward currents  69  19.  Voltage-clamp responses to depolarizing command steps  71  20.  Effects of K -channel blockers on other outward currents  74  21.  4-AP-sensitive outward current  75  22.  Comparison of responses to axonal and perikaryal stimulation...  78  8. 9. 10.  +  +  +  in TRG neurons  +  +  +  - X -  23.  Effects of changes in resting potential on spike invasion  81  24.  Effects of 4-AP on spike invasion  82  25.  Effects of histamine on a TRG neuron  84  26.  Depolarizing responses to substance P  86  27.  Facilitation of repetitve discharge  88  28.  Reduction of responses in Na -deficient perfusates  90  29.  Reduction of responses in Mg -deficient perfusates  93  30.  Effects of substance P during K -channel blockade  96  +  2+ +  - xi ACKNOWLEDGEMENTS I wish to express my gratitude to Professor Ernest Puil without whose support and encouragement this work would not have been possible. also  like  to  Therapeutics  thank for  other  their  members  patience  of  the  Department  with me these  last  of  I would  Pharmacology &  few years.  thanks are reserved for Drs. B. R. Sastry and D. M. J . Quastel  Special for their  helpful discussions of my work. The  financial  acknowledged.  support of the  Canadian Heart Foundation is  gratefully  - xii -  This thesis is dedicated to my parents - Tamara and George Spigelman who constantly struggle to improve my well-being.  "The real voyage of discovery consists not in seeking new landscapes, but in having new eyes." Marcel Proust  SPIGELMAN  1  1 INTRODUCTION 1.1  General introduction Physiological and pharmacological investigations  ties  of  sensory  neurons are  important to an understanding of peripheral  nervous system (PNS) function. peripheral membrane.  receptors Alterations  is  of the membrane proper-  Transmission of  mediated  in the  by  ionic  electrical  sensory  mechanisms  properties  of  signals  from the  in  the  neuronal  the  membrane can  profoundly influence impulse propagation along the primary afferent to the central nervous system (CNS).  fibers  Endogenous substances and therapeutic  agents may act through ionic mechanisms in the membrane, thereby influencing the neuronal excitabilities.  In addition, certain pathophysiological condi-  tions in humans involve perturbations of neuronal membrane function. investigations  Hence,  of drug-induced alterations in membrane properties are prere-  quisite to an understanding of PNS function from a clinical standpoint. Although neurons of the dorsal root ganglion (DRG) have been subjected to extensive investigations  during the past century,  geminal root ganglion (TRG), especially been studied to the same extent. alized assumption that  neurons  supportive  are  role)  This oversight is due partly to the gener-  the membrane properties of TRG neurons are likely  believed in the  primary afferent axons.  tri-  their membrane properties, have not  similar to their counterparts in the DRG. these  neurons of the  to  In addition, the cell  participate  transmission  of  only  sensory  secondarily  bodies of  (i.e.,  in a  information along  their  The assumption does not have a firm basis because  the role of neuronal cell bodies in this transmission has not been thoroughly examined with electrophysiological recording techniques.  methods, particularly with intracellular  SPIGELMAN 2 The investigations membrane electrical  described  properties  in  this  thesis  focus  using these techniques  attention  on  the  and examine certain  mechanisms by which sensory impulse transmission can be modulated in the TRG neurons. ical  For a fuller comprehension of the experimental design, the anatom-  organization and involvement of  will be reviewed.  the  ganglia  in sensory  transmission  Secondly, it may be instructive to summarize, succinctly,  the peripheral mechanisms of sensation,  including endogenous agents that may  mediate nociception.  1.2  Anatomical organization of the trigeminal  root ganglion and other  sensory ganglia The dorsal root ganglia and cranial inal  root ganglion are collectively  spinal  or sensory  roots or cranial  ganglia. sensory  sensory ganglia such as the trigem-  referred to as craniospinal, cerebro-  Sensory ganglia  form swellings on the dorsal  nerves near their points of entry into the CNS.  Three exceptions to this arrangement are the primary sensory neurons of the visual and olfactory systems and the mesencephalic nucleus of the trigeminal nerve which lies within the brainstem.  Sensory ganglia contain the  cell  bodies (perikarya) of primary afferent neurons and the proximal portions of their axons.  The ganglia also contain the Schwann and satellite cells that  form sheaths which are associated with the axons and the perikarya, tively, of the neurons. pheral  respec-  In addition, the supportive elements of the peri-  nervous system also are present:  these include the connective and  vascular tissues. In mammals, the TRG is the largest sensory ganglion in the body and is composed of three divisions - - ophthalmic, maxillary and mandibular.  Each  ganglion is formed at the point of convergence of its three divisions on the  SPIGELMAN 3 floor of the cranial vault, with the largest (mandibular) branch in the most lateral position.  The bilateral branches provide innervation to the mucosal  lining of oral cavities, the skin, and the muscles of mastication (the latter are  innervated  nucleus  in  innervate  by neuronal  the  perikarya within  brainstem).  the cornea  Primary  the  afferents  (McNaughton, 1938)  as well  trigeminal  mesencephalic  in  the  trigeminal  as  blood vessels  nerve in  the  circle of Willis (Mayberg et a l . , 1981; Liu-Chen et a l . , 1984), one of the few  nociceptive  areas  vibrissae (whiskers) neurons.  within  the  cranium (Wolff,  1963).  The mystacial  possessed by most mammals are served by fibers of TRG  Also, sensory  innervation of the tooth pulp, surrounding gingiva  and periodontal membrane is supplied by the trigeminal nerve (Kelly, 1981). Sensory ganglia are surrounded by a multilayered perineuria! sheath and a fibrous outer epineurium that are very nerve trunks (Lieberman, 1976). perforate the inner meningeal cerebrospinal  fluid  similar to  those of peripheral  Since the cranial and sensory  nerve roots  layer of the dura mater, they are bathed in  (Gamble, 1976).  However,  the  ganglia  possess a rich  supply of blood vessels that do not share the protective mechanisms present in the brain; the contents of sensory ganglia are exposed to the ions and molecules  that  pass  across  the  capillary  walls.  Compared with  spinal  ganglia, the blood vessels of the TRG appear to be less permeable to the intravenous injection of protein tracers (Arvidsson, 1973).  This may account  for the relative immunity of the ganglion to many toxins and drugs that can affect peripheral nerves (Selby, 1984).  1.3  Embryology and morphology of neurons in sensory ganglia During the course of development, cells of the neural crest migrate into  clumps along the neural tube forming the primordia of  the  spinal  ganglia  SPIGELIAN (Horstadius, 1950; ganglia cranial  is  Weston, 1970;  more complicated,  Pannese, 1974).  because  unlike  4  Formation of the cranial  their  spinal  counterparts,  ganglia originate from the neural crest cells as well as from the  cells of the ectodermal placodes (Weston, 1970). of these two sources to the ganglion cells is ganglion.  The relative contribution specific  to each particular  Thus, the TRG receives contributions from the neural crest cells  and placodal ectoderm, whereas the petrossal and nodose ganglia are derived predominantly from the ectodermal placodes (Johnston and Hazelton, 1972). The undifferentiated ganglion cells send out several  small pseudopodia  but during further development these are reduced to two processes,  one at  each pole of  one  the  cell.  The processes become oriented  such that  is  directed towards the CNS and the other towards the periphery (Krieg, 1966). The peripheral process (Pannese,  1974).  is  usually  thicker  and grows  at  a  faster  pace  With continued growth the perikaryon begins to move away  from the aligned processes but remains connected by a stem process.  This  differentiation results in the formation of a T-junction at the point where the  stem process  (initial  segment or "connecting  piece"  of  Cajal,  1907)  bifurcates into the central and peripheral processes. In mammals, especially carnivores, the stem process becomes convoluted, sometimes with complete envelopment of its Scharf,  1958).  This  development  of  perikaryon (Svaetichin,  a glomerulus  during the first few postnatal weeks (Cajal, 1909).  takes place  only  1951; late  The exact significance  of the glomeruli is not known, but they may confer a delay in the invasion of the perikarya by impulses from the periphery (cf. Dun, 1955).  Sometimes,  at the point where the unmyelinated stem process emerges from its glomerular form, a myelin sheath may be acquired. thinner than that of the dorsal  This internodal myelin is usually  root fibers  (Rexed and Sourander, 1949).  SPIGELMAN 5 Myelinated stem processes bifurcate at the nodes of Ranvier and are normally constricted at these points,  whereas the T-junctions of unmyelinated pro-  cesses are usually enlarged (Ha, 1970). 1.4  Involvement of perikarya in sensory transmission The offstream position of the perikarya of sensory ganglia with respect  to their axonal processes that transmit information from the periphery to the CNS has led to the view that the perikarya are of limited in terms of an electrophysiological 1976).  significance  role in the intact animal (Lieberman,  This view is supported by the evidence that the majority of neuronal  perikarya in the ganglia appear to be devoid of synaptic contacts.  However,  a very small number of synaptic bouton terminations have been demonstrated conclusively to impinge on perikarya in the cat DRG.  The axons that form  these contacts originate in the spinal cord (Kayahara et a l . , 1984). Spontaneous  electrical  activity  is  not  a feature  of  primary  sensory  neurons in the in vitro preparations, prior to passage of current through the recording electrode or on electrical stimulation of the axonal processes (cf.  Puil  et  al.,  1986;  Puil  and  Spigelman,  1988).  Furthermore,  Darian-Smith and colleagues (1965) showed that orthodromic impulses initiated in a large myelinated nerve fiber will invade the central terminations before excitation of the perikaryon is observed extracellularly in the TRG . Although some available evidence  indicates  that  synaptic  activity and  action potentials in TRG perikarya do not contribute significantly to ongoing electrical  activity  electrophysiological perikarya  in  compounds  necessary  processes.  along  the  afferent  observations  trigeminal for  Substances  afferent the  fibers,  certain  suggest an active transmission.  activities  of  their  biochemical  and  participation of  the  The  neurons  central  synthesize  and peripheral  synthesized within the perikarya travel by fast and  SPIGELMAN 6 slow axoplasmic transport to both (terminal) ends where presumably they are utilized for various functions (e.g.  transmitter release).  The synthesizing  or "factory" role has been localized to the perikarya by the biochemical and histochemical  demonstrations  of putative  transmitters  such as  S-glutamate  and substance P within primary afferent neurons, including those of the TRG (Hokfelt et a l . , 1975). Electrophysiological evidence from studies on DRG neurons also supports a more active role of perikarya in afferent transmission. Camino (1973) have found that  the  repeated  activation  Thus, Tagini and of  large diameter  neurons by electrical stimulation of the peripheral nerve evokes spikes that travel towards the ganglion where additional action potentials are generated in  the  vicinity of  potentials  the  perikaryon.  Curiously,  these  "anomalous" action  always seemed to propagate in the antidromic direction towards  the periphery, but not to the central terminations. Kirk  (1974) recorded spontaneous  impulses that periodically discharged  from the dorsal rootlets of ganglia that were isolated from the periphery by transection of the spinal nerve.  In contrast to the above observations of  Tagini and Camino (1973) these spontaneous impulses traveled only orthodromically  from the  sectioned  nerve.  ganglion  towards  Such ectopic  the  spinal  discharges  cord,  and not  towards  have been suggested to  be the  source of abnormal sensations which occur following amputation (Carlen al.,  the  et  1978) Recent  investigations  recording techniques,  of  TRG neurons  have revealed that  in  the  vivo,  using  perikaryal  intracellular  membranes of TRG  neurons may act as a f i l t e r of inputted signals (Puil et a l . , 1986, 1987). The ability of TRG neurons to either amplify or dampen signals, depending on the  input frequencies,  may provide an explanation  for  the  abilities  of  SPIGELMAN 7 sensory  neurons  to  generate  "new  action  potentials"  in  response  to  excitation of their axons. 1.4.1  Role  of  satellite  cells.  In  the  course  of  embryological  development each neuronal perikaryon becomes enveloped by a capsule formed by small flattened cells,  i.e.,  satellite cells  cells  of peripheral  nerve fibers,  crest  (cf.  1970).  Weston,  (Cajal, 1909). Like Schwann  these probably derive from the neural  Satellite  cells  are  closely  apposed  to  one  another and to the neuronal perikarya which they always envelop (Lieberman, 1976). spaces  Gap junctions even  more  between  (Pannese,  satellite  1969).  cells  However,  narrow large  the  intercellular  molecules  still  can  permeate freely to neuronal surfaces (Rosenbluth and Wissig, 1964). Although the cytology of satellite cells is well established, the functions of satellite unknown.  cells  and their interaction with neurons are virtually  Nevertheless, one role of satellite cells has been ascertained in  the glutamine-glutamate cycle in which satellite cells take up glutamate and transform i t to glutamine which is subsequently released (Schon and Kelly, 1974).  The glutamine may be taken up by neurons to provide a substrate for  synthesis of glutamate and its subsequent utilization in various ways. 1.4.2  Variability  of  neuronal  populations  in  sensory  ganglia.  The  perikaryal diameters of neurons in sensory ganglia differ considerably. For example, neuronal diameters have been described in the range of 15-110 Lim within the human sensory ganglia (Ohta et  al.,  1974).  We have observed  perikaryal  diameters of 100 um in trigeminal root ganglia obtained 4 hrs  postmortem  from  an  adult  human male  (Spigelman  and  Puil,  unpublished  observations). Histological studies of sensory neurons revealed separate populations of large light and small dark cells  (Andres,  1961;  Scharf,  1958).  In the  SPIGELMAN 8 trigeminal  and spinal ganglia of rodents these become apparent only post-  natally (Kalina and Wolman, 1970).  In many studies,  have been observed in ultrastructural features (Lieberman,  1976).  These differences  consistent  of the two neuronal classes  have yet  to be reconciled with the  possible functional differences of light and dark neurons. considered,  the  small  dark cells  differences  are more likely  If size alone is  associated  with small  diameter axons and therefore likely to transmit sensory information specific to these fibers (cf. Gobel, 1974;  Ohm'shi and Dyck, 1974).  1.5 Sensory modalities transmitted by primary afferent fibers The somatic sensory system of mammals is capable of conveying five basic qualities of sensations that can be evoked by stimulation of various tissues in the body.  Touch, warmth, cold and pain are best developed in the skin,  but also can be present in other body parts including visceral organs. modality is  served by a specific  alone each nerve fiber will However,  more complex  activity of different  set  of primary afferent  only evoke  sensations  are  a single  primary  produced within  sets of nerve fibers.  the position and movement of the joints  fibers.  The fifth  (kinesthesis)  Each Acting  sense quality.  the  CNS from  sensation  the  concerns  that depends on the  activity of primary afferent fibers which innervate ligaments and capsules of joints.  In addition, primary afferents  innervating skeletal  muscles may  also contribute to kinesthesis. Thus,  single  qualities  of sensation  can be identified  in humans when  stimulated appropriately, although most conscious sensations evidently arise from the central  synthesis of sensory  afferent  fibers.  Whereas only  elicited  by an appropriate stimulus,  input through the  one elementary changes  sensory in the  various sets of  experience  can be  strength or temporal  SPIGELMAN 9 characteristics modality.  of  the  stimulus  can derive  a variation of  the  sensory  For example, a submodality such as itch can be converted to pain  by increasing intensity, or touch can be converted to flutter by increasing the frequency of a stimulus (Bishop, 1946). 1.5.1 In  the  Relationship of afferent course  of  fiber diameters to sensory  investigations  on  peripheral  sensory  modalities.  mechanisms  a  correlation was observed between the various modalities of sensation and the diameters of primary afferent fibers that mediate these sensations.  In the  current classification  (Mountcastle, 1980) the large myelinated fibers  6-12 um in  35-75 m/s  diameter,  conduction  sensations that  are grouped together  touch-pressure,  flutter-vibration  myelinated fibers  (A6:  1-5 m,  velocity)  serve  as mechanoreceptive.  and  position  5-30 m/s)  a  (AB:  number of  These include  sensibility.  The  small  and the unmyelinated fibers (C:  0.2-1.5 um, 0.2-2 m/s) that innervate the furry skin of many mammals, such as rodents, provide the crude sense of contact. in  the  skin of  innervation  the  primate  hand is  by larger myelinated  almost  fibers  However, mechanoreception entirely  (Perl,  dependent  1968).  Pain  pinpricks and cold sensation also are mediated by fibers  on  the  induced by  in the A 6 range,  whereas sensations of burning pain and warmth are served by the C-fibers (Zotterman, 1933;  Bishop and Heinbecker, 1935;  Clark et a l . , 1935;  Bessou  and Perl, 1969). 1.5.2  Morphology of peripheral terminations of primary afferent  in relation to sensory modalities.  The many varieties  of sensory  fibers endings  found in the skin and visceral tissues of mammals can be classified broad groups: (1) free (bare) nerve endings,  into  (2) endings with expanded tips  and (3) encapsulated endings (Miller et a l . , 1960).  The free nerve endings  are densely distributed throughout all layers of the skin.  Both the A6 and  SPIGELMAN C-fibers  10  terminate in free endings which are appropriate for fibers  mediate the more "primitive" sensations such as pain.  that  Endings with expanded  tips (Merkel's tactile disks) are those associated with specialized epidermal cells.  As in the case of free nerve endings, a single nerve fiber supplies  many tactile disks (Merzenich and Harrington, 1969). Encapsulated nerve endings myelinated nerve fibers.  are formed around the  large  The different types of end organs seem to prede-  termine the dynamic sensitivities example,  terminals of  Meissner afferents  are  of mechanoreceptive nerve endings. velocity  detectors  that  discharge  For brief  bursts of impulses which decline rapidly (adapt) following a step indentation of the skin, and recur briefly on removal of the stimulus.  Such endings are  sensitive selectively to low-frequency stimuli in the 30-40 Hz range and are thought to serve the mechanoreceptive submodality of flutter.  In contrast,  Pacinian corpuscles which provide the submodality of vibration may function optimally at stimulus frequencies of 250-300 Hz (Merzenich and Harrington, 1969).  1.6 Peripheral neural mechanisms of pain It is known from animal experimentation as well as from stimulation of human subjects  that  propagated  the  polymodal  via  because  impulses  initiated by cutaneous  A6 and C-fibers. they  are capable of  Both  fiber  painful  groups  responding to  stimuli  are  are considered  different  forms of  stimuli to  impulses  destructive energy. It takes  is  not known how the transduction of painful  place  possibility  at is  the  peripheral  that free  terminals  of  nociceptive  afferents.  One  nerve endings are activated directly by noxious  stimuli in a manner analogous to that observed in encapsulated endings of  SPIGELMAN mechanoreceptive afferents.  11  Another suggestion involves the release of pain  producing substances (algogens) either from damaged tissue or from the nerve terminals themselves.  These algogens in turn act as agonists on receptors  located on the free nerve endings, leading to excitation of primary afferent fibers. 1.6.1  Triple response.  This phenomenon is i n i t i a l l y apparent as sudden  and intense pain subsequent to a damaging stimulus to the skin.  An unpleas-  ant  follows.  state  of  low  intensity,  pronounced vasodilation  poorly  in the  localized  pain  usually  injured area leads to  the  A  formation of a  wheal which is soon surrounded by a wider area of less intense vasodilation of the skin.  Local reddening, wheal formation and flushing surround of the  skin comprise the sequence described by Lewis (1942) as the triple response. The reduction in threshold for pain (hyperalgesia) in  part of  the  surrounding flare  may persist  in the injured area and  for  days.  The secondary  hyperalgesia in a wide region outside the flare may last for several after  the  initiating  stimulus.  The two hyperalgesias  may have  hours  different  causal mechanisms (Mountcastle, 1980). 1.6.2  Axon reflex.  The flare  formation  injured site can be produced only when the Sectioning  the  degeneration  of  central  portion  peripheral  (Chapman et a l . , 1961).  of  axons,  the  flare  These observations  cutaneous  vasodilation  antidromic activity  surrounds  locally  peripheral axons are  intact.  roots  flare  does  formation  not is  lead  to  preserved  In contrast, degeneration of peripheral axons does  vasodilation and wheal  lost.  the  dorsal  and the  not deter local is  that  which  is  formation, whereas the surrounding  have led to apparent  as  the a  conclusion  flare  is  in the peripheral branches of nociceptive  a  that  the  result  of  afferents.  SPIGELMAN Vasodilation  occurs concomitantly  with  plasma extravasation  and the  12 two  processes have been termed neurogenic inflammation (Jancso et a l , 1967). 1.6.3  Autonomic reflexes.  mediated by the aggressive  autonomic  response  of  the  Noxious stimuli can evoke  nervous  system  as  part  organism (Mountcastle,  of  reflex the  1980).  reactions  defensive Some of  or  these  reactions arise as a result of synaptic activation of neurons in the CNS, or the  antidromic activation of  collaterals  of  peripheral  nociceptive  sensory  fibers  afferents. on  Impingements  postganglionic  neurons have been observed; these can be excited  by afferent  of  the  sympathetic stimulation  (cf. Jessell, 1983).  1.7  Endogenous algogenic  substances  Extracts from damaged tissue can cause intense pain when injected into skin  (Mountcastle,  1980).  This  observation  led  to  nearly  60 years  of  research on the endogenous algogen that may be released from the tissue by a damaging stimulus, leading to activation of nociceptive afferents. substances  Several  that may be present in extracts of damaged tissue are known to  cause pain and each has been proposed as the chemical mediator of pain. 1.7.1  Potassium.  K  +  administered as a salt  solution  by intradermal,  intramuscular, intraarterial or intravenous injections produces pain (Keele and Armstrong, 1964).  In contrast to other pain-producing substances  that  may require a specific membrane receptor, an increase in extracellular + + [K J will cause neuronal depolarization simply through a change in [K J gradient between the extracellular and intracellular compartments.  A rise  in extracellular [K ] e l i c i t s pain that has a more rapid onset and briefer +  duration than pain produced by extracts of damaged tissue, such as blister  SPIGELMAN fluid  (Keele and Armstrong,  1964).  Therefore K  is  +  unlikely to  be  13 the  sole or main agent acting peripherally to evoke painful sensations following injury to tissue. 1.7.2  Changes in extracellular pH.  cause pain on application to normally associated cut.  Low pH or alkaline solutions  a blister  base,  but  such changes  with the injuries produced by a pinprick  also  are  not  or a razor  Also, analgesia follows the initial painful effects of applications of  acidic solutions.  Changes in extracellular pH can e l i c i t  perikarya of sensory neurons (Gruol et a l . , 1980;  responses in the  Krishtal and Pidoplichko,  1981).  For example, a rapid decrease in the extracellular pH can evoke an  inward  current  Pidoplichko,  that  1981).  is  carried  by  Na  and  K  ions  (Krishtal  and  These authors observed that a greater number of TRG,  than DRG, neurons were capable of  responding to pH changes,  significance of this finding remains obscure.  although the  However, the sensory endings  of these neurons also may be endowed with receptors for protons and serve as sensors of [H ] in various regions of the body (Krishtal +  and Pidoplichko,  1981). 1.7.3  Acetylcholine.  Subcutaneous  or  intravascular  injections  of  acetylcholine (ACh) as well as its applications to blister base produce pain (Keele and Armstrong, 1964).  The onset of pain is fast and a refractoriness  that develops to subsequent applications of ACh is very pronounced.  Nico-  tinic receptors may be involved in the production of pain by ACh, because applications of muscarinic agonists such as methacholine or pilocarpine are not painful,  whereas  d-tubocurarine or hexamethonium antagonize  the pain  induced by ACh. The sensory afferent fibers that are excited by Ach belong both to the myelinated and unmyelinated categories respond to  pressure  (Fjallbrandt and  Iggo,  and include fibers that  1961).  The relatively  high  SPIGELMAN concentrations of ACh that are needed to e l i c i t pain on subcutaneous  14  injec-  tion are not likely to be released by tissue damage even in the presence of anticholinesterases  (Hurley and Koelle, 1958). Hence, i t  is  unlikely that  ACh is a prime candidate for the pain-producing substance. 1.7.4  5-hydroxytryptarnine.  The pain produced  by 5-hydroxytryptamine  (5-HT) on application to the blister base has a delayed onset and much longer duration than the 1964).  pain caused  Close arterial injections  skin produce a long-lasting fibers  by ACh application  is  discharge of  impulses  along cutaneous  van Gelder, 1962).  liberated during clotting  causes vasoconstriction  and Armstrong,  of 5-HT into an innervated area of  (Fjallbrandt and Iggo, 1961;  serum platelets  (Keele  (Mills et a l . , 1968).  the  sensory  This component of  (Janeway et  al.,  1918)  and  Mast cells in the trigeminal  ganglia of rats and guinea pigs contain 5-HT (Lehtosalo,  1984), but their  exact physiological role in the ganglia has not been identified. 1.7.5  Histamine.  The role of this particular autacoid in the vascular  responses to various noxious  stimuli  early work of Lewis and Grant  has been established  largely by the  (1924) and Lewis (1927).  The response  histamine has been associated closely with the name triple response. (1942) suggested that histamine produced itch but not pain. investigators  have  demonstrated  the  algogenic  actions  of  application to abraided skin or by intradermal injections  to  Lewis  However, other histamine (Rosenthal  on and  Minard, 1939). 1.7.6 Bradykinin.  This nonapeptide was among the f i r s t substances shown  to be present in exudates from painful tissue (Chapman et a l . , 1961; and Armstrong,  1964).  endogenous algogen, First,  appearance of  Although bradykinin  several this  observations autacoid  is  are at  in blister  a good candidate  Keele  for  the  odds with this proposal. fluid  collections  is  only  SPIGELMAN detected after contact with glass, which activates (Argent et  al.,  fibers  associated  is  1954).  the production of ki ni ns  Second, a complete destruction of afferent with  a  loss  of  the  15  nerve  pain-producing substance  in  subcutaneous perfusate collected from damaged areas (Chapman et a l . , 1961). Furthermore, bradykinin is highly potent for activating peripheral afferent fibers but does not have a selective action on nociceptive afferents A-beta mechanoreceptive  afferents  activated  Mense,  (Hiss  and  as well  1976).  as nociceptive Bradykinin  because  fibers  also  applications  are  produce  excitatory effects in the majority of TRG neurons in culture (Baccaglini and Hogan,  1983).  On  the  autoradiography that  other  receptors  hand,  it  was  recently  shown  using  for bradykinin are only associated  with a  subset of small diameter neurons in the TRG of guinea pigs (Steranka et a l . , 1988). Tachyphylaxis repeated  or  refractoriness  applications  of  are  bradykinin,  not  whereas  observed both  in  events  responses  to  are prominent  features of responses to applications of ACh, histamine and 5-HT (Keele and Armstrong,  1964;  sites  involved  are  Hiss and Menze, in  the  1976).  activation  compounds (Hiss and Menze, 1976).  of  Therefore,  different  nociceptive  afferents  by these  The fast adaptation of peripheral nerve  fibers to application of the above agents is consistent that these algogens may be responsible for the i n i t i a l a damaging stimulus.  receptor  with the proposal  fast pain felt  after  It is likely that the above mentioned substances may  all contribute something to the final painful response (Perl, 1976). 1.7.7  Substance  P.  The observations  of  (1961) revealed that the presence  of afferent  damaged tissue  for the  substance.  is  This  a prerequisite implied  a  release  of  Chapman and his fibers  in the  appearance of the  substance  colleagues vicinity of  a pain-producing from  the  fibers.  SPIGELMAN Substance P, a good candidate for the role of the endogenous already  known  established This  to  be  a  potent  vasodilator  in the peripheral nerve fibers  led Lembeck (1953) to  vasodilation  in  the  propose  triple  and  (Pernow,  substance  response  to  its  P as  tissue  algogen was  presence  1953;  16  has  been  Gaddum, 1960).  the  mediator of  injury.  In  the  addition,  substance P was reported to be a potent algogen in studies of the blister base  (Armstrong et  intra-arterial  al.,  1954)  and in observations  of  effects  following  infusions of substance P preparations in mammals (Potter et  a l . , 1962). Later,  it  was realised  that  impure preparations of  contaminated with bradykinin or related kinins (Stewart,  substance 1970).  P were  Following  the separation and sequencing of substance P (Chang and Leeman, 1970;  Chang  et a l . , 1971), the earlier experiments of Armstrong and her colleagues on the human blister base were repeated using synthetic substance P (Stewart et al.,  1976).  These investigations  revealed that substance P did not possess  algogenic properties, even at doses up to 1 mg/ml.  Substance P application  to cat tooth pulp does not produce excitation in the afferent nerve fibers (Gaselius et a l . , 1977).  Thus, the evidence for a role of substance P as  the mediator of neurogenic inflammation is very convincing.  However, this  peptide does not seem likely to be by itself the pain-producing substance in the periphery. In the  present  investigations,  the  actions  of  several  mentioned autacoids were examined for their ability to affect electrical properties of TRG neurons.  of  the  above  the membrane  These studies were undertaken because  the pharmacology of autacoids in sensory ganglia is poorly understood and the mechanisms of actions of such substances afferent neurons are virtually unknown.  on the membranes of primary  SPIGELMAN 1.8  17  Substance P as neurotransmitter of primary afferent neurons A physiological  role for substance P has been of  when the peptide was first several  isolated  interest since  by von Euler and Gaddum.  1931  There are  reasons for identifying substance P as a neurotransmitter, particu-  larly in sensory systems.  Substance P is generated from a larger precursor  peptide in small diameter perikarya of sensory ganglionic neurons (Harmar et al.,  1981)  and transported  (Holton, 1959;  along  their  Brimijoin et a l . , 1980).  peripheral  and central  processes  Immunoreactivity for substance P  is localized in synaptic vesicles (Cuello et a l . , 1977), axonal terminations without clearly defined synaptic structures and neuronal contacts with blood vessels (Barber et a l . , 1979). (Otsuka and Konishi, 1976) electrical  Peripheral (Olgart et a l . , 1977), and central  release of substance P has been demonstrated on  stimulation of the peripheral nerve fibers.  Motoneurons in the  spinal cord and neurons of the cuneate nucleus in the brainstem are depolarized on application of 1977).  substance P (Konishi and Otsuka, 1974;  Krnjevic,  Furthermore, an association of the excitatory actions of substance P  with nociceptive  synaptic inputs has been observed in second order sensory  neurons of the spinal trigeminal tract (Andersen et a l . , 1977; the excitabilities  of terminals of primary afferent  1978).  Also,  fibers were shown to be  greatly affected by substance P applications (Randic et a l . , 1982).  However,  sensory neurons themselves do not respond to substance P applications in in vitro preparations  (Krishtal and Pidoplichko, 1981;  Nowak and Macdonald,  1982). A mediator role for substance P is likely at synapses of sensory fibers with neurons of inferior mesenteric its  application  Konishi  et  (Dun and Karczmar,  al.,  1979);  the  1979;  afferent  ganglia that are depolarized by Krier  non-cholinergic  and Szurszewski, excitatory  1979;  postsynaptic  SPIGELMAN potentials  are  evidently  generated  by  the  postganglionic  18  actions  of  substance P released by dorsal root stimulation (Konishi et a l . , 1980).  1.9  Membrane electrical properties of primary sensory neurons The membrane electrical properties T>f DRG neurons were among the  to be studied with intracellular recording techniques  (Svaetichin,  first 1951).  Several features of DRG neurons have made them readily accessible to electrophysiological analyses.  The presence of only a single stem process which  is attached to a spherical or ovoid perikaryon facilitates electrophysiological employed.  the analysis of  membrane properties when intracellular recordings are  This relatively simple geometry obviates some of the  in interpretation of the voltage  responses to  difficulties  intracellular injections  current that are used to assess membrane electrical  properties.  contacts or inputs are virtually absent and this further simplifies  of  Synaptic analysis  and interpretation of results. The membrane properties of TRG neurons have not been subjected to the same scrutiny as their DRG counterparts because of the notion that perikaryal membrane properties of TRG neurons are similar to those in the DRG, and also due to the relative anatomical inaccessibility (e.g.  rabbit).  of the TRG in many species  However, several features of the TRG neurons make it likely  that their membrane properties may differ from those of First,  neurons  in the TRG derive  the DRG neurons.  from two embryologically  separate  cell  groups of the neural crest and the ectodermal placodes, whereas DRG neurons derive exclusively from the neural crest. logical origin could result in functional In humans,  and perhaps  in  other  These differences  in the embryo-  differences.  mammals,  trigeminal  neuralgia  (tic  douloureux) is a disorder characterized by lightning-like attacks of pain of  SPIGELMAN  19  extreme intensity that can be precipitated by light stimuli from the skin, face  or gums,  in an apparent absence  of  neurological  deficit.  considered by some investigators to have a central pathogenesis 1984), more convincing evidence  Although (cf.  Selby,  points to peripheral origins of the neur-  algia, with a more likely site of pathology within the TRG itself  or in its  sensory  which has  root  (Kerr,  1979).  In the  glossopharyngeal  ganglion,  embryological origins similar to the TRG, neuralgias occur very infrequently, and almost never in other sensory ganglia such as the DRG (Selby, 1984). The possibility  that  impulses  can be  neurons in pathophysiological conditions  initiated  is  inferred from the  of some types of epilepsy and trigeminal neuralgia. of baclofen and particularly of the antiepileptic phenytoin in the 1983;  treatment  of  trigeminal  in perikarya  of TRG  similarities  The clinical  efficacies  agents carbamazepine and  neuralgia  (Fromm and Terrence,  Rail and Schleifer, 1985) suggest that these drugs may have inhibitory  actions  on putative epileptiform discharges  in the  TRG.  Sensory neurons  that have sustained some injury to their peripheral processes also would be expected  to  discharge  However, investigations have been applied  spike  bursts  (cf.  Burchiel  and  Russell,  of the chronic effects of epileptogenic  1985).  agents that  directly to the TRG in cats have not revealed any evidence  of abnormal spontaneous activity or physiologically evoked bursts of spikes in TRG extracellular recordings (Burchiel et a l . , 1978). convulsants  produce their gross  effects  Nevertheless, many  on motor and sensory  function by  actions on synapses, which are absent in the TRG (Lieberman, 1976). extracellular  recordings  as  compared to  intracellular techniques  provide a sensitive reflection of changes in membrane properties. more,  spontaneous  discharges  certain conditions (cf. section  in sensory 1.4).  neurons  Also, do not  Further-  have been recorded under  SPIGELMAN 20 1.9.1  Control  of membrane excitability.  patterns that travel  The transmission  along the primary afferent  fibers  is  of  impulse  dependent  on a  strict control of membrane excitability at the sites of impulse generation and  propagation.  The  control  of  neuronal  excitability  is  largely  attributable to currents mediated by potassium channels (Hille, 1984). of  these currents are activated  Most  with membrane depolarization and can be  partially resolved using voltage clamp techniques because of differences their  kinetics  of  activation  and  inactivation.  These  currents  in  can be  separated by changing the ionic species in the extracellular and intracellular  environments as well  as with selective K -channel blockers.  commonly used K -channel blockers,  tetraethylammonium  +  dine  (4-AP)  and  K -channels  Cs ,  (Adams  +  pharmacological  have  +  The most  +  and  blockade  different  Galvan, is  the  affinities  1986).  (TEA), 4-aminopyri-  for  An example  transient  outward  various of  types  specificity  K -current  U )  +  A  of in in  molluscan neurons (Hagiwara et a l . , 1961), which has been implicated in the control  of  1971).  In this case, as well as in neurons of vertebrates (except bullfrog,  cf.  repetitive  spike discharge  Adams and Galvan,  with  4-aminoyridine  applications Other  of  1986),  (4-AP)  another  K -currents with +  I  A  and  is not  (Connor and Stevens,  usually very  K -channel that  are  susceptible  sensitive  blocker,  +  kinetics  very  1971;  to  Neher,  to blockade  similar  external  tetraethylammonium  suggestive  of  I  (TEA).  have  A  been  investigated with patch-clamp techniques in dorsal root and nodose ganglion neurons where this  pharmacological characteristic,  was demonstrated (Kasai et a l . , 1986; contrast  to  the  patch-clamp  i.e.,  Bosu et a l . , 1985;  results,  the  blockade by 4-AP, Oyama, 1987).  putative  intracellular^ in rat nodose neurons is not much affected of 4-AP (Stansfeld et a l . , 1986).  I  A  In  recorded  by applications  SPIGELMAN  21  Voltage-clamped sensory axons do not exhibit currents with features of I  A  (Chiu and Ritchie,  1985). spike  1980;  Grafe  et  al.,  1985;  Brismar and Schwarz,  However, applications of 4-AP to myelinated axons produce repetitive discharges  (Kocsis  et  al.,  1987)  actions on currents other than I . A  that  are  suggestive  of blocking  Administrations of TEA do not  elicit  spike discharge, but enhance 4-AP-initiated repetitive firing and block the afterhyperpolarizations  (AHPs)  that  usually  follow  the  repetitive  spike  discharges. In our previous abilities  investigations  to discharge  spikes  on TRG neurons  repetitively  in  a decrease  response  to  in  their  current pulse  injections was observed with TEA applications which did not block the AHPs following the bursts (Puil and Spigelman, 1988; ting certain differences  from other sensory neurons and axons (cf.  Therefore, one of the main objectives examine  in  detail  the  Puil et a l . , 1988), sugges-  effects  of  in the present investigations applications  of  blockers (4-AP, TEA and Cs ) on the membrane voltage +  currents processes  of  TRG neurons,  that  affect  in order to  their  above).  different  K -channel +  responses and outward  understand better  excitabilities,  was to  the fundamental  including repetitive  firing.  The other objective was to examine the effects of applications of several endogenous substances (autacoids) on the excitabilities of TRG neurons.  SPIGELMAN  22  2 METHODS 2.1  Animals 2.1.1  Source.  from the Animal  Duncan Hartley guinea-pigs Care Centre of the  (either  University of  sex)  British  were obtained Columbia.  The  Animal Care Centre used standard animal care procedures for the maintenance of  laboratory animals.  days.  At the  Centre, guinea-pigs  were weaned after  14  These animals were fed on vitamin C-supplemented guinea pig chow and  had access to water ad 1ibitum. 2.1.2 Animal feed and housing. guinea-pigs  (200-300 g,  Once a week, typically on Mondays, 4-6  approximately 28 day old)  animal unit and used for studies in that same week.  were received  from the  Once the animals were  acquired, they were placed in a wire cage (58 x 35 x 53 cm, in size) in the animal care room of the Department of Pharmacology and Therapeutics. guinea pigs had free access to food (guinea pig chow) and water.  These  The animal  care rooms had controlled temperatures (22-23° C) and humidity (50-55%) with set 12-hourly day and night periods.  2.2  Surgical procedures for removal of the TRG Anesthesia  chamber.  was  induced with  4% halothane  administered  in  The trachea was cannulated and the animal's skull  with scissors.  The animal then was placed in a stereotaxic  a  2-liter  hair removed  head holder.  A  craniotomy was performed while anesthesia was maintained endotracheally with 2% halothane.  After midcollicular decerebration with a scalpel  blade, the  encephalon was aspirated to reveal the underlying posterior fossae.  At this  stage, the administered halothane concentration was reduced to 0.5%. Heart rate was monitored via electrodes inserted subdermally in the thorax region  SPIGELMAN throughout the surgical procedure in some experiments.  The left trigeminal  root ganglion was carefully isolated from the surrounding connective and  cartilagenous  instruments  bone with the aid of  (e.g.  No. 5 jeweler's  a dissecting  forceps  23  microscope  and miniature  tissue  and fine  scalpel  blade).  Care was taken not to disturb blood vessels in the vicinity of the ganglion for two reasons:  (1) minimization of the duration of hypoxia was considered  desirable,  and  (2)  dissection  of  the  profuse  bleeding  ganglion.  interfered  The central  with  visualization  and peripheral  stumps  and  of  the  ganglion were severed such that the total length of the excised tissue was 1 to 1.5 cm.  Usually, the central end was cut as close as possible  entry into the brainstem.  Following total  to  removal from a posterior  the ganglion was immersed quickly into cold a r t i f i c i a l cerebrospinal (ACSF) which was oxygenated with a 95/5% gaseous mixture of O2/CO2. remaining connective tissue, rounding the  ganglion,  arachnoidal and dura!  and its  central  (capsular)  and peripheral  its  fossa, fluid The  sheaths sur-  root stumps, were  removed from the ganglion with the aid of a dissecting microscope (Leitz). The ganglion was placed on a Teflon stage of a mechanical  tissue chopper  which was covered with ACSF-saturated porous tape and then cut parasagittal^ into thin  (250-450 um) slices;  this  procedure preserved some of the axon  bundles in the stumps of both central and peripheral branches. Slices were transferred immediately into a beaker (50 ml) with a nylon mesh immersed in continuously oxygenated ACSF at room temperature until of  the  required for recording.  The procedures for dissection,  second ganglion were identical  to  (19-24°C)  slicing  etc.  those described above and were  carried out within 30 min after preparation of slices of the f i r s t ganglion.  SPIGELMAN 2.3  24  Procedure for obtaining human trigeminal root ganglia at autopsy Trigeminal root ganglia were obtained 4 hrs postmortem from a male donor  (age,  81 years)  at the autopsy f a c i l i t i e s  of the Acute Care Hospital, The  University of British Columbia.  Following removal of the skullcap and the  brain,  were  trigeminal  root  ganglia  (procedure carried out by Dr. E. Puil). connective  excised  from  the  posterior  fossae  The ganglia, including surrounding  tissue, were immersed into cold (4°C) ACSF that was oxygenated  with 95% Og, 5% C 2 mixture 15 min prior to removal of the ganglia. n  remaining dissection  The  and slicing procedures were similar to those employed  for trigeminal root ganglia from guinea pigs and took place in the electrophysiological laboratory.  2.4  Procedure for obtaining human sympathetic ganglia Sympathetic  ganglia  were  harvested  from 6 human donors  (ages,  9-54  years) of certain organs for the Pacific Organ Retrieval for Transplantation programme.  The brain-dead individuals had no recent  history of  cardiovascular dysfunction or medication and were a r t i f i c i a l l y for  the  tive^, sion.  surgical  procedures.  Only one neomort had received,  disease, ventilated  intraopera-  a drug (dopamine) which was used to maintain good peripheral perfuLumbar sympathectomies were performed (by Dr. E. Puil) within 1-2 min  after bilateral nephrectomy, i . e . , protocol for organ retrieval.  the f i r s t procedure in the operating room  An excised chain of 4 ganglia was immersed in  cold (~4°C) oxygenated (95% 0 , 5% C0 ) a r t i f i c i a l cerebrospinal fluid ?  ?  SPIGELMAN  25  Fig. 1. Schematic diagram of the transport chamber. The plexiglass chamber was fastened to a gas tank (Linde, size E) containing the gas mixture which was used to oxygenate the a r t i f i c i a l cerebrospinal fluid (ACSF). The two layers of nylon mesh were separated by a space of ~1.5 mm.  SPIGELMAN (ACSF).  In an adjacent  laboratory, each ganglion with the  nerve  26  stumps  (~1 cm total) was dissected free of the connective sheaths with the aid of a microscope and sliced longitudinally with a manual chopper, within ~5 min after the sympathectomy.  The slices (300-500 um thick) were transported in  a specially designed chamber (Fig. 1) with oxygenated ACSF to the electrophysiological  laboratory where they were maintained with constant  tion until needed for recording.  oxygena-  In one case, the intact ganglia, as above,  were stored overnight (~8 hr) at 4°C before slice preparation.  2.5  Electrophysiological recordings All  electrophysiological  studies of  neurons in the different prepara-  tions were performed in a recording chamber (Fig. 2). volume) had a single  inlet for inflow of solutions.  chamber was by capillary action through a strip of adjacent  chamber,  where  it  was  removed  by  This chamber (1ml The outflow from the filter  aspiration.  paper into an A nylon mesh  prevented floating movement of slices in the chamber during perfusion with artificial  cerebrospinal fluid (ACSF) at rates of  were visualized with a compound microscope modulation contrast Faraday cage (to  optics.  1-3 ml/min.  (Leitz)  Preparations  equipped with Hoffman  The recording assembly was housed in a wire  prevent 60 Hz electrical  interference)  on a vibration-  damping table (Micro G). The control ACSF had the following chemical composition (in mM): NaCl, 124; 2.0;  NaHC0 , 3  26;  and dextrose,  KC1, 10.  5.0;  KH P0 , 2  4  1.25;  CaCl , 2  2.0;  Continuous bubbling with a 95/5% 0 / C 0  ensured adequate oxygenation and a pH of 7.4.  2  MgS0 , 4  2  mixture  Bath fluid temperature was  SPIGELMAN 27  perfusion barrels  Fig.2. Schematic representation of the recording chamber. A heating device was installed underneath the glass bottom of the chamber. Short (~15 cm) sections of sylastic tubing connected the perfusion barrels to the polyethylene perfusion lines (inside diameter, 0.86 mm). These lines terminated in a single outlet ~8 cm from the recording chamber. Vascular clamps were used to switch between perfusates from different barrels.  SPIGELMAN  28  maintained at 32-34°C with a heating device fitted underneath the floor of the  recording chamber and by additional  (Fig.  heating  of  the  perfusion  lines  2).  2.5.1  Ion  substitutions.  Low-Na  +  solutions  substitution of NaCl with choline chloride.  were  made by equimolar  Changes in [K ] of the ACSF  were made by equimolar substitution of NaCl with KC1. CaCl  2  was replaced  2+ with CoCl in low-Ca solutions. MgCl was omitted from Mg -deficient solutions. In several experiments, another 2 mM of CaCl were added 2+  2  2  2  2+  to Mg -deficient solutions. 2.5.2 Drugs.  All drugs were obtained from Sigma.  Aliquots of substance  P and bradykinin were measured from stock solutions (0.1 mM) that were kept frozen until required.  The other solutions were prepared on the day of the  experiment, or the night before,  from drugs that were stored according to  drug company recommendations. 2.5.3 Electrodes.  The recording electrodes were pulled on a microelec-  trode puller (Narishige PA-81) from borosilicate Frederick Haer,  1-1.2 mm outside  with 3 M KC1, 3 M CsCl,  diameter).  4M K S0 2  tances ranging from 12 to 35 Mn.  4  glass  pipettes  These electrodes  or 3 M Cs S0 2  4>  (WPI or  were f i l l e d  and had tip  resis-  Voltage-clamp experiments were performed  using electrodes with tip resistances  no greater than 25 Mfi, whereas elec-  trodes with higher tip resistances were used in current-clamp experiments. Penetration of neurons was achieved (Burleigh Instruments). used to  facilitate  ground electrode. trodes central  Brief  with the aid of a micropositioner  bursts of capacitance overcompensation were  penetration.  A chlorided silver wire  served as  the  In some experiments, concentric bipolar stimulating elec-  (Rhodes Medical  Instruments)  stumps of TRG slices.  were inserted  into  Applied voltages of  the peripheral  or  0.5-6 V were used to  SPIGELMAN  29  e l i c i t action potentials recorded in the cell body. 2.5.4 Recording equipment.  In several early experiments, the microprobe  amplifier system (WP Instruments M701) which was used to measure potentials, allowed injection of current via a bridge-balance circuit for conventional measurement of membrane resistance with constant current pulses.  In subse-  quent experiments, specified voltage or current steps were injected through the recording electrode with the Axoclamp-2A amplifier (Axon Instruments) in either  the  current-clamp  frequency 2.5-3.5 kHz) mode. continuously with 7404A).  a chart  or  single  electrode  voltage-clamp  (sampling  Voltage and current recordings were monitored recorder  (Gould  Brush  220  or Hewlett-Packard  Amplified potentials were stored on FM tape (Sony or Scotch) using  a tape recorder (Hewlett-Packard 3968A) that had a frequency response to 5 KHz at 7.5 ips.  For detailed data analysis,  flat  intracellular recordings  were played back via a waveform recorder (Biomation 805) and reproduced on paper with an X-Y pen  recorder  (Hewlett-Packard 7015B).  signals were averaged by computer (MINK-23;  PDP 11/44).  Alternatively,  SPIGELMAN  30  3 RESULTS 3.1  Dependence of resting membrane potential on extracellular cations. Data were obtained from TRG neurons that showed stable resting poten-  tials more negative than -50 mV and spike amplitudes greater than 50 mV for periods of 5-180 min.  In all  cases,  the extracellular  voltage level was  determined following withdrawal of an electrode from the c e l l .  Recordings  were performed using KCl-filled electrodes, unless otherwise indicated. input  resistance of  injections  of  neurons was estimated  small  (0.1-0.5 nA)  from the  hyperpolarizing  voltage  The  responses to  current  pulses  (50 ms  duration). 3.1.1  Changes in extracellular  extracellular  concentration  of  neurons,  extracellular  [K ]  the  K +  containing 20, 30, or 40 mM K . +  [K ]. +  +  were  was  The effects studied  varied  10  neurons.  changing  to  In  the 8  perfusates  In each case, a steady level of membrane  potential was attained at 3 min of perfusion. potential  by  in  of changes in  The mean values of resting  for these 8 neurons are plotted against the concentration of K  in the perfusate (Fig. 3). complete removal of K  +  to -79 mV and -104 mV.  +  In separate experiments on two other neurons,  from the perfusate resulted in a hyperpolarization The depolarizations in high [K ] +  20 mM) were accompanied by decreases in input resistance.  solutions (e.g. In 8 neurons the  mean input resistance was decreased from 12 ±2.4 to 5.9 ±0.7 Mn S. E. M.  It  is predicted from the constant field equation that for 5 mV hyperpolarizing test pulses the K -conductance ratio +  the 1.47. [K ] +  Cl"  is  passively  distributed,  increases by a factor the  of  Cl"-conductance ratio  In 6 of 7 cells the conductance ratios for 20 mM [K ] +  were  greater  than  the  1.69.  conductance  ratios  expected  If  would be  and 6.25 mM from  the  SPIGELMAN  31  - 2 0  > - 4 0  CD  "o CL  60  +  CD C  o  _Q  E - 8 0 + CD  E 100  H  1  1  1  1  l i l t  10  e x t r a c e l l u l a r [K  1  •  1 1 1 | | ) |  100  +]•( m M )  +  Fig. 3. Dependence of membrane potential on extracellular [ K ] . Each point on the graph represents the mean and S. E . M. of membrane potential measurements for 8 TRG neurons obtained at different concentrations of K in the perfusate. The extrapolated best line f i t to the points in the graph intersects the ordinate at -77 mV. +  SPIGELMAN constant field equation (assuming 100% membrane potential and' unaltered  K -permeabil ity).  However,  +  significant  (student  t-test).  increased  K -permeability  Therefore, (i.e.,  +  the  differences  these  no  dependence  data  opening  of  on K  were  do not  32 +  not  support an  channels  with  depolarization in this range). 3.1.2  Changes  in  extracellular  [Na ].  Perfusion  +  of  neurons  with  solutions where choline chloride was substituted for NaCI resulted in a 2 mV hyperpolarization  of  the  resting  potential  in  8/10  neurons.  These  hyperpolarizations could be recorded in the presence of TTX (1 uM, 5 min), suggesting that the for  action  potential  depolarization perfusate. potential  effects  to  its  ~10 min  to  control  control  In  solution  values. +  ±2.5 Mf2 S.  E.  M.)  In  depolarizing current pulses,  most  following  observed on perfusion with low-[Na ] 10.1  responsible  +  generation.  ensued  Return  were not due to the Na -channels  all  neurons  the  a  slow  switch  usually  to  restored  Increases  in  solutions  in 5 neurons  cells  tested  input  with  a the  membrane low-Na  +  membrane  resistance (8.8  supra-  were  ±2.2 to threshold  the evoked action potentials were abolished by  low-Na perfusion, although a local membrane response remained (Fig. 4). 2+ 2+ 3.1.3 Changes in extracellular [Ca ] and Co -application. 2+ 2+ +  Perfusion  of  low-[Ca  ],  Co -containing  influence on the membrane potential hyperpolarization Na -deficient +  was  media  hyperpolarization  only (2.6  frequently  solutions  had a hyperpolarizing  of TRG neurons.  slightly ±0.5 mV declined  greater S.  E. despite  than M.,  The amplitude of the that n= 7).  continued  observed  in  Also,  the  perfusion  and  reverted into a depolarization that could be halted by returning to control perfusate.  SPIGELMAN  33  Fig. 4. Effects of 1ow-[Na ] perfusate on the voltage responses of a TRG neuron. Perfusion with solution containing 1ow-[Na ] resulted in suppression of the evoked spike. The resting membrane potential was -53 mV in control and -55 mV in the low-[Na ] perfusates, respectively. Note the slight reduction of the voltage sag in response to hyperpolarizing current pulses in Na -deficient perfusate. +  +  +  +  low - Na 20 ms  > E o  CO  SPIGELMAN 3.2  35  Membrane properties of TRG neurons  All TRG neurons exhibited varying degrees of time-dependent membrane rectification;  this was evident from their 'sagging' voltage responses to hyper-  polarizing current pulse injections.  Examples are shown in Figs. 4 and 22.  Due to presence of a sag at large hyperpolarizations, estimates of input resistance were obtained from voltage responses where sag behavior was not prominent, i.e.  responses of <10 mV amplitude.  was 8.9 ±0.5 Mfi S. E. M. (n = 83). resistance were impaled (e.g.  The average input resistance  Many neurons with high values of input  20-35 Mn), but stable recordings could not be  maintained for long periods in such cases, presumably because of cell damage by the relatively coarse electrode tips. probably  resulted  in an underestimate  Therefore electrode of  the  number of  sampling bias  cells  with high  values of input resistance. 3.2.1 potential  Membrane potential  oscillations.  A tendency  to  observed  neurons  oscillate  was  intracellular current injections  (Figs.  when  7 and 8).  for  were  the membrane  depolarized by  The oscillations  were  evident in both the sub- and suprathreshold responses of most neurons, and were prominent in neurons that could discharge repetitively  in response to  depolarizing current pulses. 3.2.2  Action  potentials  and  afterhyperpolarizations.  Two types  of  action potentials could be distinguished by an absence or presence of a hump on  the  repolarization  depolarizing exhibited 5A-C). similar  current  spikes  with  phase  pulse  (Fig.  a 5).  a continuously  spike  evoked  by  The majority smooth  to  that  observed  in  TRG neurons  an  after  intracellular  (>85%)  repolarizing  The slowing of repolarization in humped spikes  K -conductances (see below). +  of  of  neurons  phase  (Figs.  (Fig. 5D) was very interference  with  SPIGELMAN 36  Fig. 5. A and D: two types of action potentials observed in TRG neurons. Arrows indicate onset of i n t r a c e l l u l a r ^ injected depolarizing current pulses. Note the long spike duration and the presence of a hump during late repolarization just below 0 mV (dashed line) of spike in D. B and C: examples of a single spike and repetitive firing in another neuron exhibiting fast spikes as in A. E and F: examples of a single spike and repetitive firing in another neuron exhibiting humped spikes as in D.  SPIGELMAN Some characteristics  for these spikes are summarized in Table I.  37 The  maximum amplitude and duration of spikes including the AHPs were significantly greater in neurons where the repolarization of the spike was biphasic. Mean resting potentials did not differ significantly for the two groups of neurons. Spike  discharge  at  high  neurons that exhibited either  frequencies  (100-240/s)  was  elicited  humped or non-humped spikes.  from  However, the  apparent threshold for spike generation was higher in cells with non-humped spikes;  in addition, the maximal rate of spike discharge was greater than  in cells possessing humped spikes (cf.  Fig. 5C and F).  Long-lasting post-  spike AHPs, comparable in peak magnitude and duration to those following repetitive 1976;  spike  discharge  in other  sensory  ganglia  (Jaffe  and Sampson,  Weinreich, 1983), were not prominent in the TRG neurons.  AHPs (3-10 mV peak amplitude;  However,  cf. Fig. 5C and F) often followed a repetitive  discharge evoked by injections (e.g., 1/s for 30 s) of intracellular depolarizing current pulses.  Such AHPs lasted about 100-150 ms on termination of  a current pulse. 3.2.3 Effects of ionic channel blockade on spikes. a differential complete  sensitivity  blockade of  starting TTX-perfusion.  to  action  tetrodotoxin potentials  was  TRG neurons exhibited  (TTX, 1 uM). observed  In 24 neurons,  within  1 min  after  A return of a neuron's ability to discharge spikes  of control amplitude was slow, and the amount of depolarizing current needed to evoke a spike remained elevated for more than 20 min after discontinuing the TTX application.  The spikes  in 15 other neurons were not blocked by  similar applications of TTX, even after more than 10-20 min of perfusion. Both TTX-sensitive and TTX insensitive spikes were blocked after 5 min of  Table I.  Summary of spike and afterhyperpolarization (AHP) characteristics resting  Action potential  AHP  membrane  1  potential  amplitude  (mV)  overshoot  duration  (mV)  (ms)  (mV)  amplitude  duration  (mV)  (ms)  Fast spikes Range  -50 to -75  55 to 104  Mean * SE -60.0 ± 0.5 Number  106  4 - 45  0.3 - 1.3  1-18  78.7 ± 1.0  17.6 * 1.0  0.6 ± 0.1  11.9 ± 0.4  118  106  113  1-20 4.7 ± 0.3  106  105  of cells Humped spikes Range  -53 to -70  70 to 101 "k  ick  Mean * SE -59.4 ± 1.7 Number  12  8-43  0.9-4 ~k  5-25 ~k  4-20 ~k  "k  82.7 ± 1.9  23.9 ± 2.3  1.8 ± 0.2  13.9 ± 1.6  8.2 ± 1.1  13  12  13  12  11  of cells *  at one-half peak spike amplitude.  *  p < 0.05 (significant difference between means of fast and humped spike parameters were determined with Student's t-test).  **  p > 0.05.  SPIGELMAN perfusion with solutions unaffected 2+ Co  -  during  containing  2+ Ca -channel  presumed  containing perfusates  low-[Na ]  (n = 7).  (Fig. 4).  The spikes  blockade  with  However, a slight  39  were  low-[Ca  2  +  ],  deterioration of  spike amplitude was observed after prolonged periods (>5 min) of perfusion with the low-[Ca 3.3  ] solutions.  Membrane properties of human sympathetic neurons. Stable recordings were obtained from 16 neurons that had resting membrane  potentials in the range of -28 to -65 mV (mean, -45 ± 2.8 mV S. E . M.), for stable periods of at least 10 min to ~12 hr. ~-10 to -20 mV were recorded in the stored  overnight  at  4°C, but  Several resting potentials of  slices of the ganglia that had been  spontaneous  activity  spikes could not be evoked by current injections.  was  not observed and  Spontaneous activity also  was not observed in most neurons of the other "acute" preparations, although positive transients of ~5-6 mV amplitude were suggestive of synaptic activity in 1 neuron. The  neurons  exhibited  average  input  resistance  of  28.7 ±  (S. E. M.) when injected with hyperpolarizing current pulses.  4.6 Mn  The responses  to these pulses generally were 8-10 mV in amplitude and usually followed an approximately exponential  time course  (Figs.  6A, 15A and C).  A membrane  time constant of 13.2 ± 1.0 ms (S. E . M.) was calculated for 3 neurons by measuring the time taken to reach 68.8% of peak voltage responses (<20 mV) to  hyperpolarizing current  amplitude sometimes sagged, voltage-sensitive  pulses.  Hyperpolarizing  indicative of  responses  time-dependent  of  activation  >20 mV of a  conductance (Fig. 6C).  Anodal break responses often consisted of a spike of >60 mV amplitude on the depolarizing rebound, followed by a slow, pronounced hyperpolarization.  SPIGELMAN  40  FIG. 6. Responses of human neurons to injections of square wave current pulses. A-C: anodal break responses, including spikes displaying prominent afterhyperpolarizations in 3 human sympathetic neurons (same neuron in C and D). Hyperpolarizing current pulses (100 ms) were 0.2, 0.3 and 1.7 nA in A, B and C, respectively. D: depolarizing current injections (100 ms) were 0.3, 0.8 and 1.3 nA for Dj, D2 and D3, respectively. Resting membrane potential was -65 mV. Upper dashed lines represent 0 mV level.  SPIGELMAN 41  SPIGELMAN With graded injections  of current steps, a progressive delay in the appear-  ance of the spike could be demonstrated (Fig. 15A and C). afterhyperpolarizations smaller test pulses.  42  (AHPs)  could  be  more  easily  Such spikes and  activated  with  the  In some neurons, the anodal spike disappeared entirely  with large current pulse injections  (Fig. 14C), unlike the anodal spikes in  TRG neurons which were more easily  evoked by large test pulses.  had large amplitudes (e.g.,  The AHPs  >20 mV in Fig. 6A) and long durations (50-400  ms) even in neurons with resting potentials of ~-50 to 65 mV. In general,  and compared with the spikes that were part of the anodal  break responses, spikes were less readily evoked with intracellular injections of depolarizing current pulses.  Such pulses generated  mV amplitude in many of these neurons (Fig. 6D). spikes cells.  to  Na -channel +  spikes of >40  The sensitivities of the  blockade with TTX application  were observed  in 2  The anodal break and the directly evoked spikes were blocked after  2.5 minutes of perfusion with TTX (1 pM).  Recovery from TTX blockade was  observed 8 minutes after return to control solution. In some neurons, repetitive firing could be generated with large current pulses such as  in the case of Fig. 6D.  current injections  was followed  Termination of the depolarizing  by slow hyperpolarizations.  and duration of these "AHPs" increased with the  The amplitude  number of  spikes in the  train (Fig. 6D-j-D^)-  3.4  Effects of K*-channe1 blockers 3.4.1  potential s.  Current In  administration of  clamp: contrast  effects to  the  of  tetraethylammonium effects  TEA (5-10 mM) blocked the  of  TTX  on in  subthreshold  subthreshold TRG neurons,  oscillations  injection of depolarizing current pulses (Figs. 7D and 8A) and did not  on  SPIGELMAN  43  F i g . 7. Effects of combined application of TTX (1 M ) and TEA (10 mM) on o s c i l l a t o r y membrane potential responses and spikes evoked by i n t r a c e l l u l a r depolarizing current pulses. Spikes of this TRG neuron were unaltered by TTX applied for 20 min. A-D: superimposed oscilloscope traces show membrane responses to sub- and suprathreshold s t i m u l i . Note that membrane potential o s c i l l a t i o n s persist in TTX perfusate and are blocked by TEA application. E: suprathreshold response shows reduction in repetitive f i r i n g a b i l i t y compared with C. F: partial recovery from the effects of TEA was evident 5 min after return to control perfusate as an increase in evoked repetitive f i r i n g and a reduction in spike duration compared with E. I n i t i a l resting membrane potential was -52 mV. The amplitudes of suprathreshold current pulses were approximately 2.0 nA in A and D, 2.2 nA in B, C and E, and 2.1 n A in F. U  Table II.  Effects of tetraethylammonium (TEA; resting  . input  membrane  resistance  10 mM) on electrical properties  Action potential _  AHP  amplitude  overshoot  duration  amplitude  potential (mV)  (M )  (mV)  (mV)  (ms)  (mV)  -57.4 * 1.8  12.1 ± 1.5  74 ± 2.8  16 * 3.4  1.1 ± 0.3  11 ± 2.0  -54.7 ± 1.8  16.9 ± 1.9  78 ± 3.6  20 ± 3.6  2.4 ± 0 . 6  3 ± 1.0  10  8  8  8  Control mean ± S.E.  TEA mean ± S.E. number  9  6  SPIGELMAN affect  the  sag  in the  hyperpolan'zng responses  to  current pulses.  46  TEA  (10 mM) application resulted in 47% increase in the mean input resistance of 10 neurons (Table II). Typically this increase was associated with a 2-5 mV depolarization. 3.4.2 Effects of TEA on spikes and repetitive discharge.  The effects of  external TEA (10 mM) application were assesed on the spike characteristics of  10 neurons.  The rising phase of evoked spikes was not affected.  An  increase in the spike overshoot was evident after TEA application, and was attributable, at least in part, to an increased input resistance.  A pro-  longation of the falling phase of spikes and a reduction in peak amplitude of  the  spike AHPs were observed in all  cases.  The prolonged mean spike  duration and the reduction in the mean amplitude of the AHPs measured at one-half peak amplitude were 1.3 ms and 8 mV, respectively, after 3-5 min of perfusion with TEA (10 mM). During TEA administration, enhancement of repetitive spike discharge was not observed induced input  during  suprathreshold current pulse  depolarization  resistance.  (2-5 mV), reduced  In fact,  spike  injections  threshold,  despite and  increased  TEA application produced a decrease  ability of neurons to discharge repetitively (Fig. 7).  the  in  the  Recovery from a TEA  application was rapid and usually complete after 5-6 min. The  effects  of  TEA (10 mM) on  spikes  evoked  in  human sympathetic  ganglion neurons by current injection were studied in 4 neurons. increase  in input resistance  amplitude (cf.  A slight  (6.6%; range, 0-17%) and an increased spike  Fig. 9) was observed during TEA (10 mM) perfusion.  Spikes  were broadened, sometimes approaching 10 ms in duration and the repolarization phase was composed of two distinct slopes during TEA perfusion (Fig. 9).  SPIGELMAN  47  FIG. 8. Comparison of TEA and 4-AP effects on spikes and subthreshold oscillations. A: responses to sub- and suprathreshold injections of current pulses in the presence and absence of TEA (5 mM) and 4-AP (5mM). During drug application, the membrane potential was hyperpolarized with direct current (DC) injection to control levels. TEA application (at 2 min) abolished the subthreshold oscillations, reduced spike threshold, increased spike duration and reduced AHP amplitude. Following recovery from the effects of TEA (not shown), application of 4-AP (at 2 min) produced a large increase in the subthreshold oscillations, a decrease in spike threshold and a slight increase in spike duration, but did not reduce spike AHPs. B: responses of another neuron to application of 4-AP (1 mM) in the absence of DC injection. The resting potential was depolarized by several millivolts and a large increase in repetitive spike discharge was observed.  4-AP  ms  > E  -T  1 2  nA  control  SPIGELMAN  49  Fig. 9. Effects of TEA (10 mM for 3 min) and 4-AP (1 mM for 6 min) on directly evoked spikes from a human sympathetic neuron. Current pulses were 0.9 nA in control, 0.7 nA in TEA and 4-AP perfusates. Membrane potential was maintained at -65 mV using DC injection. Input resistance was 32 Mn, 39 Mn and 43 Mn in control, TEA and 4-AP perfusates, respectively.  SPIGELMAN  50  Also, TEA applications produced a slight reduction in the amplitude of the spike AHPs. 3.4.3 neurons.  Effects  of  4-aminopyridine  to  subthreshold  potentials  of TRG  Applications of 4-AP (1 mM) usually depolarized neurons by 3-9 mV  (4.9 ± 0 . 9 mV S. E. M . , n = 6). ±0.3  on  11  The input resistance  ±0.6 Mn S . E . M .  (n=4).  The  was increased from 7  subthreshold  potential  oscillations resulting from intracellular injections of depolarizing current pulses were greatly increased (Fig. 8A, B), in contrast to the effects of TEA.  These  increased  oscillations  were  not  a  result  of  membrane  depolarization as they were observed even when the membrane potential was compensated with hyperpolarizing direct current injection (Fig. 8A). 3.4.4 Effects of 4-AP on spikes and repetitive discharge.  Injections of  suprathreshold depolarizing current pulses into many neurons often  elicited  multiple spike firing (cf. Figs. 7, 8, 10) that was facilitated by an application of 4-AP.  In other neurons,  spike;  in  however,  the  presence  such injections of  4-AP, these  repetitive spike firing as shown in Fig. 8B. the oscillations. not reduced.  evoked only a single neurons  also  exhibited  Spikes tended to sit on top of  Spike duration was slightly  increased but the AHPs were  Furthermore, applications of 4-AP (1-5 mM) elicited spontaneous  spike activity in 4 neurons (cf.  Figs. 10, 24).  The enhancement of repeti-  tive discharge ability and the lack of major effects on the AHPs were in direct  contrast  to  the  effects  of  TEA applications.  Some recovery was  observed in most cases at 5-30 min after termination of a 4-AP application. Full recovery sometimes was observed at 30-40 min after a low dose application (<0.1 mM) in several neurons.  SPIGELMAN  51  Fig. 10. K -channel blockade results in repetitive spike discharge and burst activity in a TRG neuron. A: In control conditions, discharges elicited with intracellular injections of depolarizing current pulses of 3 different amplitudes shows phenomenon similar to spike accommodation. Note oscillation at the termination of spike activity. A maximal discharge rate of ~240 Hz was obtained with the pulse illustrated at the righthand side. B, left: continuous spiking was evident at ~3.5 min after starting 4-AP perfusion (1 mM). B, right: repetitive activity was replaced by bursts at ~10s intervals on hyperpolarizing the neuron with direct current (DC) injection. Note long interspike interval before termination of the burst. C, left: application of TEA (10 mM) concomitantly with 4-AP led to irregular spikes in bursts at ~5-20s intervals and long afterhyperpolarizations. Injection of DC to potentials more negative than rest (e.g., <-75 mV) did not arrest bursts, but prolonged "spontaneous" depolarizing potentials and afterhyperpolarizations. +  20 ms > E  o CO  "|_  I  1nA  200 ms  >  DC 400 ms  SPIGELMAN Application of 4-AP to human sympathetic neurons also slightly the duration of the spikes,  and unlike TEA applications,  affect their shapes or AHPs (cf. Fig. 9).  53  increased  did not greatly  With washout, the effects of 4-AP  persisted for more than 20 min, whereas the recovery from the effects of TEA usually was complete within 5-8 min. 3.4.5  Combined applications  of  4-AP and TEA.  One of  the  prominent  features of a concomitant application of 4-AP (1-5 mM) and TEA (5-10 mM) was the increase presumably  in the ability of neurons (n = 5) to discharge slow spikes, 2+ mediated by Ca , in response to current pulse injections.  These spikes were evoked in cases where prior applications  of TTX (1 uM)  blocked the evoked spikes (Fig. 11). 2+  An increase  in the duration of these  Ca  cells  discharged  -  spikes  also  was  observed  following application of 4-AP.  in  that  spontaneously  As shown for the neuron in Fig. IOC, the  combined application of 4-AP (5 mM) and TEA (5 mM) transformed the continuous spike activity into a burst-like pattern of spikes followed by a long hyperpolarization at regular interburst intervals of  5-20 s.  When this neuron  was hyperpolarized by ~20 mV using DC injection, the bursts of spikes induced by the combined application of 4-AP and TEA were replaced with i n i t i a l few spikes  and  oscillations  depolarization  similar  that to  rode  cardiac  depolarizations were followed  the action  crest  of  a  potentials.  by long hyperpolarizations  single, Also,  prolonged these  slow  resembling AHPs.  In the cell of Fig. 10, the spontaneous depolarizations including spikes and bursts were completely suppressed by a 3 min application of TTX (1 uM).  Fig. 11. Effects of Ca -channel blockade on the spikes evoked during the combined application of 4-AP (5 mM), TEA (10 mM) and TTX (1 M). The membrane potential was maintained with DC injection at -55 mV , -53 mV and -52 mV in control, 0 [ C a ] , Co -containing and recovery perfusates, respectively. Note that the hyperpolarization that follows the large depolarization in the absence of a Ca^ -spike is of a smaller amplitude than that of AHPs following spikes. 2+  M  2+  2+  SPIGELMAN 3.4.6  Effects  of  intracellular Cs*.  Intracellular injections  (1-4 nA, 50-100 ms depolarizing pulses delivered at 1/s) leakage  from the  recording pipette  input resistance, spikes.  resulted  +  increase  in  depolarization, as well as a reduction in the amplitude of  Neurons studied  with Cs -containing electrodes +  theless, when the membrane potential direct current injection,  were depolarized  impalement (n = 12).  the amplitude of spikes was greater compared to  tion of evoked spikes was prolonged and the +  in spike  overshoot  As with TEA administra-  was observed with  Cs  probably a consequence of the increased input resistance. both types of K -channel +  spikes to humped spikes.  The dura-  amplitude of AHPs following  spikes was reduced by Cs -application (Fig. 12). an increase  Never-  was compensated with hyperpolarizing  the amplitude of spikes evoked at the early stages of recording.  to  of Cs  or application by  in a progressive  by 22 to 35 mV within 8 min after the i n i t i a l  tion,  55  +  application,  An effect common  blockade was a transformation of non-humped  The i n i t i a l  part (10-20 mV of repolarization (cf.  Figs. 7, 12) was more "resistant" than the rest of the falling phase ( i . e . , toward the resting potential) to the actions of TEA or Cs . +  In contrast to the effects of combined applications Cs  +  applications  resulted  in  spike afterhyperpolarizations. blockade  of  the  Perfusion  progressive  Indeed,  spikes  afterhyperpolarizations  electrodes were followed C).  the  with  blockade  of 4-AP and TEA, of  ],  prolonged  recorded a short time  with  CsCl  or  by prolonged afterdepolarizations  low-[Ca  the  Co -containing  solutions  blockade of the spikes including the afterdepolarizations.  after  Cs S0 -filled 2  (Fig.  4  13B and  resulted  in  SPIGELMAN  56  CsCI recording  Fig. 12. Action potentials evoked in a TRG neuron at different times after i n i t i a l impalement of a neuron with a 3 M CsCI electrode. Top traces represent differentiated traces of lower records. Blockade of the AHPs as well as spike prolongation was evident after 9 min. Note that the repolarization phase in the second action potential is greately prolonged relative to that of the spike evoked at the onset of depolarizing current pulse. Dashed lines represent 0 mV potential.  SPIGELMAN  57  Fig. 13. Spike afterpotentials in TRG neurons. A: depolarizing current pulse evoked a Ca -spike in the presence of 4-AP (5mM), TEA (5 mM) and TTX (1 LIM). Such spikes were blocked in a low-[Ca ], Co -containing medium. Note the long lasting afterhyperpolarization that follows the termination of the current pulse. B: in another neuron, a C a - s p i k e evoked by a depolarizing current pulse injected through a CsCl-containing electrode is followed by a prolonged afterdepolarization. C: depolarizing current pulse injected through a Cs2S04-containing microelectrode also produces an afterdepolarization. The membrane resting potential was held at -50 mV, -60 mV and -62 mV in A, B and C, respectively. 2  + 2  SPIGELMAN 58  Cs S0 2  4  electrode  SPIGELMAN 3.5  59  Voltage-clamp analysis TRG neurons (n = 35) were investigated with single electrode  voltage-  clamp techniques during perfusion with TTX (1 uM) in order to identify the +  specific  currents affected  by the K -channel  blockers.  These experiments  required a trade-off between the current passing abilities of the recording electrodes and minimization of damage to impaled neurons. input resistances neurons  in  the  of TRG neurons CNS or  resulted  sympathetic  voltage-clamp was imperfect;  in further complications  ganglia).  an effective  However, the low  For these  reasons,  (cf. the  space-clamp could not always be  achieved despite the approximately spherical perikarya, and further resulted in a sampling bias that yielded rather low numbers of successfully clamped neurons.  The use of K -channel +  blockers resulted in an improvement of the  clamp characteristics due to the increase in neuronal input resistance. 3.5.1 Transient outward currents. from the  others  by simple  membrane  potential  was  voltage  held  at  One outward current could be isolated conditioning.  about  -40  For example,  mV, hyperpolarizing  when the voltage  commands evoked inward currents that were followed by outward currents at the termination of the commands (Fig. 14A).  These transient currents had a  rapid onset (<5 ms) and were inactivated within ~50 ms.  The mean decay time  constant  for 5 neurons was  of  the  transient  19.3 ± 6 . 3 ms S. E. M.  outward currents estimated  These estimates were obtained from 4-7 averages of  current traces that were fitted with a single exponential ("0.9 correlation). The presence of inward (anomalous)  rectification did not allow the use  of hyperpolarizing voltage commands greater than 35 mV because activation of an inward rectifier current interfered with the transient outward currents (cf.  Fig. 14A, bottom), and was the likely cause for a slight deviation in  the transient current decay from a single exponential.  SPIGELMAN  60  A reduction in the inactivation of the transient outward currents could be achieved with hyperpolarizing commands as potentials near -40 mV.  small  as 5 mV from holding  The time constant for the removal of inactivation  was determined by varying the duration of hyperpolarizing voltage commands of  constant  amplitude  (Fig.  14B).  average decay time constant  The use  of  this  paradigm yielded an  value of 29 ± 7 ms in 3 neurons.  limitations of the single electrode  Due to the  voltage clamp, no attempt was made to  investigate steady-state activation or inactivation of the transient outward currents. Human sympathetic neurons also exhibited transient outward currents. two neurons that could be successfully  voltage-clamped,  termination of the  larger hyperpolarizing voltage commands from a holding potential was accompanied by transient  outward currents  15B).  small  The durations  of  the  transient  <40 ms) than the latencies of the anodal  of  of -40 mV  1-3 nA amplitude  (Fig.  currents were briefer  spike  In  responses to the  (eg.  injected  pulses using bridge-balance techniques. 3.5.2 increased  Effects to  of ionic  20 mM,  a reduction  currents of TRG neurons. only  slightly  i<25%)  substitution. was  When [K ] in the perfusate +  observed  in  the  transient  was  outward  The peak amplitudes and decay of the currents were  reduced by perfusion  CoCl2 had been used as a substitute  with  solutions  for CaC^ (n = 2).  in which 2 mM A small outward  shift in steady-state holding current usually was observed on perfusion with the  low-[Ca  shifted  ],  Co -containing  in an outward direction  solutions. during  The holding current also was perfusion  with  low-[Na ] +  media.  These shifts corresponded to the small membrane hyperpolarizations that were observed in ionic substitution experiments using bridge-balance techniques.  SPIGELMAN  61  Fig. 14. Transient outward currents in TRG neurons. A: hyperpolarizing voltage commands (upper traces) and the corresponding currents (lower traces) evoked from a holding potential of -40 mV. A transient outward current was evoked at the termination of the commands. The decay of this current was approximately exponential and nearly complete by ~50 ms. Large hyperpolarizing commands activated a slow inward current that contributed to a small reduction of the tail current. The graph (below) represents the peak amplitude of the transient current plotted against the amplitude of the command step. B: changes in the duration of constant amplitude hyperpolarizing voltage command steps produced changes in the peak amplitude of. the outward current. The peak outward current amplitude is plotted against the duration of the voltage command step in the lower raph. The time constant for the removal of inactivation was ~35 ms obtained from the best l i n e - f i t to the points in the graph).  SPIGELMAN 62  SPIGELMAN  63  Fig. 15. Transient outward current in human sympathetic ganglion neurons. A: responses of a human sympathetic neuron to hyperpolarizing current pulse injections. Note the approximately exponential time course at onset of voltage responses, and not on termination of the current pulses. Spike appearance was delayed by pulses of larger amplitude (cf. A and C). B: single electrode voltage clamp recording in another neuron revealed outward currents (lower record) following the two largest voltage command steps (upper record). Holding potential, -40 mV. C: voltage traces selected from A illustrate the progressive delay of spike generation with larger hyperpolarizations. The callibration in A also applies to traces in B and C.  SPIGELMAN 3.5.3  Ineffectiveness  of  muscarinic  agents.  Applications  64 of  acetylcholine (100 uM, n = 2) and muscarine (20 uM, n = 1) had no effect on the  transient  current-clamp muscarine changes constant  outward current. techniques,  (50 uM, n = 2)  in the  resting  current pulse  In experiments  applications  of  and methacholine potentials  on  other  using  acetylcholine  (100 uM, n = 3),  (20 uM, n = 1)  did not produce  or input resistance  injections.  neurons  Likewise,  as  assessed from  no changes  in  the  spike  characteristics were observed following application of these compounds. 3.5.4 Effects of 4-AP and TEA.  Applications of 4-AP had only minor and  mostly unspecific effects on the transient outward current;  at a relatively  high concentration of 5 mM (n = 3), the transient current was unaffected and a decrease  in the  steady-state current was observed  (Fig.  16A, B).  In  contrast, TEA applications (5 mM) produced a much greater blockade of the transient outward current (n = 3),  and elicited a smaller inward shift  in  the steady-state current (Fig 16C, D). The possibility  that the effects  would be dose-specific  was assessed  from the dose-response relationships for both drugs (Fig. 17). The percentage of inhibition of the transient outward current was determined in the presence and  absence  of  the  these relationships.  K -channel +  blockers,  from  the  slope  conductance  in  The dose-response curves in Fig. 17 show that TEA was  more potent than 4-AP as a blocker of the transient outward current.  In  this comparison, the blockade of the current by 4-AP application was less specific because the maximum inhibition of slope conductance that could be obtained was less than 40%. No inhibition was observed on application of 4-AP in 5 mM doses.  This decrease in blocking effectiveness may be attribu-  table to membrane breakdown phenomena observed at higher doses of 4-AP (cf. Puil et a l . , 1988).  SPIGELMAN  65  Fig. 16., A: single electrode voltage clamp records during control and 4-AP (5 mM) application. Holding potential was -40 mV. Top traces are voltage commands and bottom traces, evoked currents. Transient outward currents were evoked at the termination of the command steps. 4-AP application produced a large decrease in the steady-state and evoked inward currents but had l i t t l e effect on the transient currents. B: plot of the peak outward current amplitudes against the amplitudes of the voltage commands. Open circles joined by solid lines are control responses and solid triangles with dashed lines, responses obtained during 4-AP application. C: blockade of tail currents by TEA application. Note that TEA produced only a small decrease in the steady-state current. D: graph shows the large TEA-induced reduction in the peak outward currents (solid circles, dashed lines) compared to control (open circles, solid lines).  SPIGELMAN  SPIGELMAN  67  Fig. 17. The reduction of the peak amplitude of the transient outward current by TEA (open circles) and 4-AP (closed circles). Inhibition of the slope conductance was obtained from I/V relationships in the absence and after 2-3 min of perfusion with a K^channel blocker. Individual points represent the mean of several cells. The number of cells tested is indicated near the points. For several points the standard errors were very small. Curves are computer generated lines (2nd order regression) fitted to the points.  SPIGELMAN 3.5.5  Effects  of  internal  Cs .  +  that  internal  blocked  In view of the earlier  +  Cs -blockade  of  K -channels  fast  AHPs  (cf.  applications  also  were  examined  techniques.  A progressive  Fig.  prolonged  12),  the  with  membrane  the  following  effects  depolarization  penetration  electrodes (Fig. 18, A and C).  spike  of  duration and  of  internal  current- and  blockade of the AHPs as well as a time-dependent observed  observations  +  the  were  68  (up  Cs  +  voltage-clamp to  35 mV) and  increase in spike duration  neurons  with  Cs -containing +  During the first 10 min after the i n i t i a l  impalement, there also was a progressive diminution in the transient outward currents (Fig. 18, B and D).  A complete blockade of the AHPs and transient  outward currents often was observed after 8 min of Cs application. + + applications of Cs also blocked other K -currents generating resting membrane potentials  as well  application  depolarize  did not  rectification.  The  always  blocking  as  inward rectification, neurons  actions  of  +  the  whereas TEA  and did not Cs  The  block inward  therefore  were  more  generalized but were similar to the effects of TEA.  in  3.5.6 Other outward currents.  The effects of 4-AP and TEA were examined  5  membrane  neurons  delivering currents.  by  clamping  depolarizing  the  voltage  potentials  commands  in  close  to  to  evoke  order  rest  and  outward  The transient outward currents could be distinguished from other  outward currents because of their more rapid decay  (Fig.  19).  The time  course of this decay was similar to that of the tail currents evoked with the  hyperpolarizing voltage  command paradigm.  Application of  TEA (5 mM)  reduced the envelope  size of the outward currents including the  current  (Fig.  The application  envelope  of  19). the  currents (Fig. 19).  evoked  outward  of  4-AP (5 mM) also  currents,  except  for  transient  diminished  faster  the  transient  SPIGELMAN  CsCI  recording  electrode  c  1 min  „  9 mm  20 ms 40 mV  -.  B  D  4 min  I 2 nA  11 min 15 mV  1 nA  40 ms  F i g . 18. E f f e c t s o f Cs a p p l i c a t i o n on a c t i o n p o t e n t i a l s and t r a n s i e n t outward c u r r e n t . The i l l u s t r a t e d t r a c e s were obtained a t s p e c i f i e d time i n t e r v a l s f o l l o w i n g penetration o f the neuron. Recordings i n B, C and D were obtained during p e r f u s i o n with a TTX-containing low-[Na ] medium. Note the absence o f an AHP i n C and t r a n s i e n t outward current i n D. +  SPIGELMAN  70  Fig. 19. Voltage-clamp responses of a neuron to depolarizing command steps. A transient current in addition to other outward currents was evoked from a holding potential of -50 mV. TEA applica- tion (at 2 min) blocked the transient current and reduced the overall envelope of currents. Following partial recovery from TEA, 4-AP application (at 3 min) also reduced the current envelope but did not block the transient current. TTX (1 pM) was present throughout.  SPIGELMAN In 2 neurons where the membrane potential transient currents were not observed.  was  held  at  -50 mV, fast  Under such conditions,  TEA applica-  tion increased the slope of decay in the outward currents, blocking more slowly  72  activating currents.  presumably by  On the other hand, 4-AP was a  potent blocker of the outward currents evoked at the onset of the voltage commands (Fig. 20). 3.5.7  Combined  applications  of  indicated that the 4-AP applications onset  and  a  relatively  4-AP-actions for the  slow  4-AP  TEA.  The  above  results  blocked outward currents with a fast  decay.  fast-activating,  and  Therefore, slowly  the  inactivating  specificity  outward current  was assessed by applying 4-AP in combination with 10 mM TEA (n = 4). procedure  permitted  suppression  of  improvement of the voltage-clamp.  the  fast  transient  of  current  This  and an  The effects of 4-AP application on the  evoked outward currents where neurons were clamped at holding potentials of -70 mV in the presence of TEA and are shown in Fig. 21; commands elicited  a fast-activating,  sustained  (>1 s)  here, depolarizing current.  Separation  of the 4-AP sensitive currents then was obtained by subtraction from the currents observed in the control condition (cf. Fig. 21C).  SPIGELMAN  73  Fig. 20. Outward currents evoked by depolarizing voltage commands from a holding potential of -50 mV in a neuron that did not exhibit a transient outward current. At 2 min of TEA application the decay of the outward current was increased, presumably due to the blockade of the delayed rectifier. Following a 3.4 min return to control perfu- sate, a 2 min application of 4-AP did not block the delayed rectifier and produced a large decrease in the outward currents evoked at the onset of the command steps.  control  TEA 5 mM  4-AP 5 mM  SPIGELMAN  75  B  3 0 ms 3 nA 29 mV  Fig. 21. A: currents evoked in the presence of TEA (10 mM) and TTX (1 uM) with voltage commands from a holding potential of -70 mV. B: currents evoked on application of a perfusate containing TEA (10 mM), TTX (1 pM) and 4-AP (5 mM). C: 4-AP-sensitive component of the outward current was obtained by computer subtraction of currents in B and A. Each trace was averaged from two sweeps.  SPIGELMAN 3.6  76  Studies on the perikaryal invasion of spikes in the TRG Electrical  stimulation of  axons was performed in  several  experiments  because this paradigm made i t possible to observe the characteristics of the postspike AHPs in the absence of current injection.  Secondly, axonal stimu-  lation facilitated the study of the modulation of spike invasion by changes in  the  perikaryal  potential  resting  membrane potential.  were induced by injections  Changes  in  the membrane  of DC and/or by application of  the  K -channel blocker, 4-AP. +  3.6.1  Changes  injections  in  on the  resting  membrane  spike characteristics  potential.  The  were examined  effects  of DC  in 6 TRG neurons.  Generally, the amplitudes of evoked action potentials were greater at more hyperpolarized membrane potentials.  The amplitudes  of  the  AHPs,  other hand, were greater at more depolarized membrane potentials and 23). the  on the  (Figs. 22  In four neurons, extrapolated values of the reversal potential for  AHPs averaged  followed  -79 ±7 mV S. E. M.  by afterdepolarizations  In one case,  (ADPs) that  the  fast  had a relatively  AHPs were long  time  course (Fig. 22). The time course of ADPs was similar to that of the spike afterdepolarizations Fig.  observed during experiments  with Cs  +  injections  (cf.  13). Hyper- or depolarization of the membrane with DC injections  had marked  effects on the invasion of spikes and on the repetitive discharge  abilities  of  responses  the  neuronal  perikarya.  Only  small  (5-10 mV) depolarizing  could be evoked with axonal stimulation in 3 neurons that were depolarized by 5-15 mV from rest. membrane potentials invasion  of  spikes  current pulses  On the other hand, hyperpolarization of the resting  to levels of -85 to -104 mV did not prevent perikaryal in  these  neurons.  Also,  injections  of  depolarizing  SPIGELMAN  77  Fig. 22. Comparison of responses to axonal and perikaryal stimulation. A: responses of a TRG neuron to injection of depolarizing and hyperpolarizing current pulses. A sag was evident in the voltage responses to the two largest hyperpolarizing pulses. B: action potential evoked with stimulation (arrow; 1.9 V, 0.5 ms) of the peripheral axonal f i e l d , in the same neuron as in A. Note the fast afterhyperpolarization (AHP) followed by an afterdepolarization (ADP) with a much longer duration. C: action potentials evoked with axonal stimulation at various membrane potentials. Polarization of the membrane was achieved by injection of direct currents. D: A plot of the AHP and ADP amplitudes against the membrane potential. The extrapolated reversal potentials for the AHP and ADP were -85 and -54 mV respectively. Values of AHP and ADP obtained at polarized potentials more positive than -65 mV were not included because spikes evoked at these membrane potentials did not overshoot 0 mV.  SPIGELMAN  M e m b r a n e P o t e n t i a l (mV)  78  SPIGELMAN  79  evoked action potentials in the perikarya at resting potentials where axonal stimulation failed to e l i c i t a spike.  In two neurons, depolarization of the  membrane by 15-20 mV did not result in failure of spike invasion. depolarization of discharge  the membrane led to the  following  stimulation  the  (Fig. 23).  invasion  of  induction of  a single  These observations  changes in the resting potential  affect  spike  However,  repetitive  elicited  provided direct  spike  by axonal  evidence  the ability of axonally  that  generated  spikes to invade the perikaryon and that spikes may be generated within the cell body. 3.6.2  Effects of 4-AP.  The changes in membrane properties of neurons  following applications of 4-AP (1-5 mM) were similar to the effects  observed  in  slightly  the  absence of  axonal  stimulation.  4-AP application  (ImM)  increased the duration of evoked spikes and also increased the amplitude of the spike overshoot (Fig. 24).  Similarly, the amplitude and duration of the  spike AHPs was increased, which was at least in part due to the input resistance. producing spikes  Electrical stimulation of the axons was more effective in in the perikaryon following  during perfusion with the control solution. 4-AP  perfusion  increased  in  two  neurons,  was  the  administration of 4-AP, than Another observed event during  spontaneous  appearance  of  (10-15 ms) depolarizations that often reached spike threshold (Fig. 24C).  fast  SPIGELMAN  80  Fig. 23. Effect of changes in membrane resting potential on the suprathreshold responses evoked by electrical stimulation of axons. Upper traces show the intracellular responses to the brief stimuli (4.3 V; 0.5 ms) indicated by the arrows. The stimulating electrode was positioned in the axonal field distal to the recording electrode. Middle traces correspond to the positive direct current applied through the recording electrode. Response at the left was obtained in the absence of membrane polarization. Note the additional spikes that followed the evoked spike when the resting potential was depolarized to -49 mV.  SPIGELMAN 81  SPIGELMAN  B  evoked  A  A  82  evoked  t  4-AP  control  spontaneous  A .  4-AP  20 m s  > E o  Fig. 24. Effects of 4-AP on the perikarya! invasion of spikes. A: control responses of a TRG neuron to the extracellular stimulation of the peripheral axonal f i e l d . A brief stimulus (3.5 V; 0.5 ms) evoked a local response or an action potential in the perikaryon. B: application of 4-AP (1 mM) led to spike discharge following every stimulus (3.5 V; 0.5 ms). C: in the absence of stimulation spontaneous depolarizing responses and spikes were evident. In A and B upper dashed lines indicate 0 mV and lower dashed lines correspond to the cell membrane potential. Note that the resting membrane potential is maintained at -60 mV with DC injection in all figure traces.  SPIGELMAN  83  Studies of autacoid effects in TRG neurons  3.7  3.7.1 Bradykinin.  Applications of this autacoid in concentrations of 5  to 10 mM for 2-5 min did not produce significant membrane potentials of 4 TRG neurons.  changes in the  resting  Similarly, no change was observed in  their input resistances or the characteristics of evoked spikes. 3.7.2 Histamine.  Histamine was applied to 9 TRG neurons in concentra-  tions of 10 and 100 uM.  In 7 of these neurons, histamine did not signifi-  cantly affect the subthreshold membrane properties or the characteristics of evoked spikes.  However, applications of histamine (100 uM) elicited a large  depolarization in two neurons (Fig. 25).  The time course and duration of  these depolarizations were similar to those observed on perfusion with substance P (see  below).  The histamine-induced  by a decrease in input resistance the  depolarizations,  injections  depolarization was accompanied  (75% reduction at peak response). of  depolarizing  current  pulses  During elicited  spikes that were reduced in amplitude, presumably due excessive inactivation of  Na channels. +  The depolarization in Fig. 25 was followed by a hyper-  polarization to a new resting potential that persisted for 8 min. Subsequent applications of histamine (100 uM) produced smaller (<9 mV) depolarizations in only one of these neurons.  Applications of cimetidine  (1 mM) for 3 min  followed by histamine (100 uM for 1 min) did not block the small depolarizations. 3.7.3 Substance P. 3.7.3.1 Effects on subthreshold membrane properties. of  substance  P were determined on 50 TRG neurons with resting  more negative than -50 mV and spike  amplitudes  >50 mV.  The effects potentials  In 35 of  neurons applications of substance P (0.1 uM-10 uM) elicited depolarizing  these  Fig. 25. Effects of histamine on a TRG neuron. The resting membrane potential was -58 mV. The large depolarization induced by histamine (100 uM) application in this neuron was followed by a long-lasting hyperpolarization (~8 min) to a new resting membrane potential of -70 mV.  SPIGELMAN responses such as those illustrated in Fig. 26.  85  The responses were slow in  onset, often requiring tens of seconds to develop, large in amplitude (up to 45 mV), and fully reversible on termination of the application.  In several  neurons, the depolarizations were preceded by small (1-2 mV) hyperpolarizations.  In 11 neurons, applications of substance P (2 pM) produced depolar-  izations that ranged from 5-37 mV with a mean peak amplitude of 14.4 ± 3.4 mV (S. E. M.). were  The effects could be detected with doses as low as 0.1 K M and  insensitive  Construction  of  to  the  dose-  neurons varied greatly desensitized,  after  inclusion  of  TTX (1 LIM)  in  the  perfusates.  response  relationships  was  difficult  in  sensitivities  to,  and frequently  their  an application of  substance  P.  because  the  became  The "desensitization"  was evident from a slow waning of a response despite continued application, and from the reduced responses, to subsequent lasting  or even a total unresponsiveness  administrations of the peptide.  and apparent even  after  of a c e l l ,  Desensitization was long-  long recovery periods  (e.g.,  1 hr).  In  addition, increases in the responses to the second application of substance P were observed in 5 neurons.  Additional applications resulted in comparable  or reduced amplitudes of responses. As illustrated in Fig. 26A, a depolarization evoked by perfusion with substance  P was  associated  with  a decrease  in  input  resistance  (72%).  However, when the peak depolarization was compensated by injecting hyperpolarizing DC, input resistance was increased in 4/4 cells (37 ± 22% S. E. M., range 4-100%) at resting potentials near control levels (Fig. 26B).  substance  P  2JJM  0  Fig. 26. Depolarizing responses to substance P application in 2 cells (A and B). Fast vertical deflections are voltage responses to intracellular injections of constant current pulses (50 ms) monitored in the bottom trace, and represent tests for input resistance. Note that when the membrane potential was returned to the initial resting level using direct current injection (B), the responses to hyperpolarizing test pulses had amplitudes similar to those of control. Suprathreshold responses to depolarizing current pulses are truncated.  Single substance current  electrode  voltage-clamp  analyses  in  2  neurons  P application produced a large inward shift (Fig.  27B).  However,  inward currents  voltage commands from a holding potential  SPIGELMAN  87  revealed  that  in the steady-state  evoked  by hyperpolarizing  near rest were increased margin-  a l l y , and the outward currents that developed on termination of the commands were reduced slightly,  during the peak shift  in the  steady-state current  (Fig. 27B). 3.7.3.2 Effects of substance P on spikes and repetitive  discharge.  Perfusion with substance P also increased the excitability of TRG neurons in other ways.  Although "spontaneous" spikes were not initiated, a facilitation  of stimulus-evoked spike discharge was observed near the peak of P-depolarizations (Fig. 27A).  substance  Increases in excitability were detected in 16  of 20 cells that were injected i n t r a c e l l u l a r ^ with suprathreshold depolarizing  current pulses  during  the  depolarizations.  In 4 cells  where  the  peptide-induced depolarizations had exceeded 25 mV in amplitude, generation of spikes was impeded, presumably by excessive Na -inactivation. +  3.8  Studies on the ionic mechanism of substance P actions In light of the above observations of substance P effects on TRG neurons,  the possible  ionic mechanism(s)  generating  these responses were examined  using substitutions of cations in the extracellular medium. 3.8.1  Effects  Na -deficient +  of  solution  changes  in  produced  extracellular membrane  iNa*].  Perfusion  hyperpolarizations  neurons (cf. section on membrane potential dependence on [Na ]). +  in  with 8/10  SPIGELMAN  control  88  substance P  25 ms > E o  1 | 2 nA  B c  o  n  t  r  o  1  V = .65nW  substance P  h  1  1  15 mV  1  1  Y  2 nA  60 ms  h  Fig. 27. A: substance P application (10 uM) facilitates development of repetitive spikes. Repetitive discharge was evoked with depolarizing current injection near the peak of a substance P-induced depolarization, but could not be elicited in control conditions. Dashed lines indicate 0 mV. B: voltage clamped response of the same cell to a second application of substance P (10 uM). A large inward shift (right bottom) in the steady-state current was observed. Note also the slight increase in the evoked inward current and the reduction in the outward currents.  SPIGELMAN  89  Fig. 28. Reduction of the response to substance P (3 uM) in a neuron by perfusion with a Na -deficient solution. Resting potentials were -53, -54, and -53 mV in control, 1ow-[Na ], and recovery conditions, respectively. Spikes recorded in control ACSF had amplitudes of ~85 mV and were blocked in the Na -deficient medium (not shown). Ten minutes were allowed for recovery prior to each application of the peptide.  recovery substance P  SPIGELMAN Stimulus-evoked spikes (cf.  F i g . 4).  were abolished  in conditions  of  low-Na  91  perfusate  +  When substance P was applied in the low-Na perfusate,  the  +  depolarizing responses  were reduced or blocked in 3/4  These data indicated that  the  substance  P effects  neurons  (Fig. 28).  depend partly on the  +  presence of extracellular Na . 3.8.2, Effects  of  2+  changes  in  extracellular  [Mg _].  The effects  of  2+  removing Mg from the extracellular medium were examined in 5 neurons. The resting membrane potential was not significantly affected affected by 2+  perfusion with a Mg -deficient not affected.  solution.  No obvious changes  The input resistance  also was  were observed in the action  potentials  2+  evoked by current pulse injections in the absence of Mg . The  depolarizing  Mg*- -deficient  responses  conditions.  to  Fig.  substance  P were  greatly  reduced  in  29  chart  recorder  traces  of  shows  recordings obtained from a neuron in which the response to substance P in a 2+  low-[Mg ] solution was reduced to ~25 of the peak response obtained in the control perfusate. In other neurons similarly tested, the responses to substance  P  application  were  completely  and  reversibly  abolished  in  2+  Mg -deficient solutions (n = 4). 3.8.3  Effects  2+  of  Ca -channel  blockade.  The  low-Ca  2+  ?+  ,  Co -  containing perfusates were inconsistent in their ability to affect, cantly, the responses to substance P application. +  the  readily  observed  effects  P-induced depolarizations. substance  P-induced  of  signifi-  This was in contrast to  2+  Na - or Mg -removal  on the  substance  In 2 neurons, decreases in the peak amplitude of  depolarizations  were  observed  in  low-Ca  ,  Co -  containing perfusates, whereas in 3 other neurons increases in the responses were evident.  SPIGELMAN  92  Fig. 29. Reduction in the response to substance P (2 pM) in a TRG neuron by perfusion with a Mg -deficient solution. Resting membrane potential was -75 mV during control conditions. Spontaneous cell depolarization that followed recovery from the control application of substance P required injection of hyperpolarizing direct current to maintain resting potential at -75 mV. Ten minutes were allowed for recovery prior to each application of the peptide. Action potentials were not affected by the Mg -deficient perfusate. 2  2+  MMIWMBMIMIIIIBIMMIIIBIIJM  <4D  CO  SPIGELMAN  94  3.8.4 Voltage-clamp studies of substance P actions in the presence of + + K -channel blockers. The results of Na -substitution experiments are consistent  with  the  possibility  that  depolarizing responses to substance P.  Na -influx +  was  involved  Therefore, the effects of  in  the  substance  P applications (2 uM) were examined in the presence of K -channel blockers, +  with voltage-clamp analysis techniques. Combined applications  of 4-AP (5 mM) and TEA (10 mM) produced large  increases in the input resistance which allowed an improvement in the voltage clamp.  Also,  i t was reasoned that the blockade of certain K -conductances +  that may be involved in the responses to substance P would allow to separate the peptide-induced activation of a proposed inward current.  Two effects of  substance P were observed following its application (2 uM) to neurons that were clamped near resting potentials in the presence of K -channel blockers +  (n = 2).  First,  there  was an inward shift  in the  steady-state current  (Fig. 30) that was similar to the steady-state current shifts observed with substance  P  Fig. 27).  Secondly, an increase in the inward currents evoked by hyperpol-  arizing  applications  voltage  commands  in  the  absence  (Fig. 30A and B)  of  K -channel  could  be  observed  current-voltage relationships obtained near the peak shift state current.  These results  in part to  the  from  (cf.  the  in the  steady-  that  substance  of  an inward  provided additional evidence  P-induced responses may be due current.  blockers  +  activation  SPIGELMAN  95  Fig. 30. Effects of substance P application during K -channel blockade. A: substance P evoked an inward current in the presence of K -channel blockers. The responses to substance P were obtained during the peak change in the steady-state current. The membrane potential was clamped at -60 mV. TEA (10 mM) and 4-AP (5 mM) were present throughout. B: current-voltage relationship for the responses exemplified in (A). The measurements were made at ~10 ms following the onset of the voltage commands. The lines (lst-order regression) through the points were computer generated. The regression line for the recovery was the same as for control and therefore omitted for clarity. +  +  SPIGELMAN  control  substance P  i  r  recovery  ~  1  -  1 1  40 mV  0.9 nA 40 ms  SPIGELMAN  97  4 DISCUSSION These actions well  as  investigations  of  the  have  selective  certain  revealed  K -channel +  autacoids,  on the  many  significant  blockers  --  features  in  the  TEA, 4-AP and C s ,  as  +  membrane properties  of  TRG neurons.  Because the main purpose of these studies was to determine the ionic mechanisms in the excitability of TRG neurons, particularly those mediated by K , +  the most pertinent findings in the investigations  will  be summarized accor-  ding to this theme before commencing a general discussion in the context of signal transmission within sensory ganglia. The resting membrane potential  of TRG neurons was found to be dependent  on extracellular [K ] and to a lesser extent on extracellular 2+ +  [Ca  ].  various  Injections  subthreshold  voltage- and time-dependent  oscillations pulses.  of  intracellular responses  and sags in the voltage  current  [Na ] and +  pulses  evoked  such as membrane potential  responses to hyperpolarizing current  Suprathreshold current pulses evoked spikes that could be differen-  tiated into two groups, based on their repolarization phase characteristics. Differences  in the  sensitivities  demonstrated with applications  of  spikes  to  of tetrodotoxin  Na -channel +  blockade were  (1 uM), a specific  channel  blocker. The sub- and suprathreshold membrane responses to current pulses were greatly  affected  by the three K -channel +  blockers  - - TEA, 4-AP and Cs . +  The membrane of TRG neurons was depolarized following applications of each of  the  above  agents,  internal  Cs  +  being  the  most  effective.  External  applications of TEA (5-10 mM) produced a reduction in the spike afterhyperpolarizations neurons.  The  and also latter  decreased effect  was  the  repetitive  evident  discharge  despite  abilities  increases  in  of  input  SPIGELMAN resistance,  depolarization,  amplitude.  In contrast,  decreased  spike  threshold  and enhanced  98 spike  applications of 4-AP (0.5-5 mM) did not block the  AHPs and greatly increased the repetitive discharge ability of TRG neurons, sometimes  resulting  in  spontaneous  discharge  of  spikes.  Combined  applications of 4-AP (1-5 mM) and TEA (5-10 mM) evoked long-duration spikes  2+ that were insensitive  to TTX applications, but were blocked in low-[Ca  ],  2+ Co  -containing perfusates.  Such spikes were followed  by prolonged AHPs.  +  In  the  experiments  performed  AHPs were not observed; tions.  These  ?+  Ca  spikes  with  indeed, and  Cs -containing  electrodes,  spikes were followed  afterdepolarizations  2+  -deficient solutions that contained Co  2+  prolonged  by afterdepolariza-  also  were  blocked  in  , a Ca -channel blocker.  Voltage-clamp analyses revealed that a transient outward current in TRG neurons was susceptible was not affected current  also  to blockade with TEA in a dose-dependent manner and  by applications of ACh and other muscarinic agents.  was  little  affected  by 4-AP applications.  However,  This such  applications were effective in blocking another outward current that had a relatively long time course of inactivation.  Because this current could not  be blocked by high doses of TEA, another distinct K -channel is inferred. +  Applications of 4-AP enhanced the ability of spikes evoked by electrical stimulation of the axons to invade the perikaryon. fast  depolarizations  that  often  following 4-AP applications,  reached  spike  In addition, spontaneous threshold  were  observed  suggesting that these phenomena may result in  the generation of spontaneous discharges within perikarya of TRG neurons. Experiments  using  electrical  hyperpolarization of  the  invasion  into  of  spikes  resting the  stimulation  of  axons  membrane potential  perikaryon.  However,  also  did not  showed prevent  perikaryal  that the  membrane  depolarization could result in the failure of spike invasion, or in other  SPIGELMAN cases, in the generation of additional  99  spikes following the invasion of a  single spike in the perikaryon. Studies  on the actions  of autacoids  in TRG neurons demonstrated  that  bradykinin (5-10 uM) or histamine (100 pM) applications did not significantly affect  the electrical  membrane properties in most neurons,  although large  membrane depolarizations were evoked by histamine in 2 of 9 neurons.  Subse-  quent applications of histamine to these neurons evoked only small membrane depolarizations, since  they  possibly  could  not  attributable  to  abolished  with  be  desensitization application  at H^-receptors  of  cimetidine,  an  ^-receptor antagonist. The majority of neurons (35 of 50) exhibited reversible depolarization following application of substance P (0.1-10 uM).  Repetitive spike discharge  ability was enhanced during such depolarizations, except when the amplitude of an evoked depolarization exceeded 25 mV. Substance P-induced depolarizations were accompanied by decreases in input resistance.  However, when the  peak depolarization was compensated by hyperpolarizing DC-injections, input resistance was increased, suggesting that a net blockade of conductances was involved in the  responses to substance  P.  Voltage-clamp analyses of  the  substance P effects revealed a large inward shift in the steady-state current at  holding  potentials  near  rest.  Also,  the  inward currents  evoked by  voltage command steps were increased slightly, whereas the outward currents evoked on termination of the voltage commands were reduced, during the time of peak responses to substance P application.  Voltage-clamp studies in the  presence of 4-AP (1 mM) and TEA (10 mM), provided additional evidence that substance P applications evoked an inward current in addition to blockade of a conductance, as inferred from input resistance current-clamp  experiments.  The substance  increases  P-induced  observed during  depolarizations  were  SPIGELMAN greatly  reduced in conditions  of  lowered  extracellular  [Na ]  and [Mg  were  not  affected  significantly  during  perfusion  with  ]  2 -, +  r  and  100  low-LCa  J,  Co -containing solutions. c  These data suggest that substance P may produce its excitatory  effects  in TRG neurons by decreasing the resting conductance of ions, possibly K , +  concomitantly with an increase in conductance to other ions, possibly, Na . +  4.1  Membrane potential dependence on extracellular cations The  results  of  these experiments  employing  changes  in  extracellular  [K ] indicate that TRG neurons are similar to other neurons in the CNS and +  PNS.  Specifically,  external  [K ]. +  their  resting  The deviation  of  membrane the  potentials  experimentally  are  dependent  derived  fit  to  on the  points in F i g . 3 from the staight line expected for a single ionic species + + (K ), suggests that other ions as well as K contribute to the resting potentials. This suggestion is corroborated by results of the experiments + Na -substitution  using  and  ?+ Ca -channel  blockade  by  incorporation  of  ?+  Co  in the extracellular media.  During perfusion, +  observed  consistently,  inferring  that  Na  ficantly to the resting membrane potential + of  Na  ?  and/or  Ca  +  contribute  of TRG neurons.  signi-  Indeed, influx  2+ and/or Ca  blockade with Co 4.2  hyperpolarization was  2+  may occur through channels  that  are  susceptible  to  (Konnerth, 1987).  Electrical membrane properties of TRG neurons In general, the electrical properties of TRG neurons appear to be similar  to those of other cerebrospinal ganglion cells,  particularly DRG neurons.  The offstream anatomical position of neuronal perikarya with respect to their axonal processes has led to the limited electrophysiological  view that the cell  significance  in the intact animal (Lieberman, 1976).  bodies  are of rather  for primary afferent  transmission  However, certain membrane properties  SPIGELMAN  101  of TRG neurons, to be discussed below, are consistent with more interesting interpretations  of the functions  sensory system.  Also, since it is not yet possible to record intracellular^  from the central  of their perikarya within the trigeminal  (or peripheral) terminations of TRG fibers,  these results  on TRG perikarya may be a preview of membrane electrical and pharmacological properties of their terminations (cf.  Feltz and Rasminsky, 1974;  Deschenes  et a l . , 1976). 4.2.1  Subthreshold responses.  exhibited  time-dependent  responses to  The TRG neurons in these  rectification  intracellular injections  described in frog DRG neurons (Ito,  in of  their  hyperpolarizing  current pulses;  of  removal  (Czeh et a l . , 1977).  of  partial  involvement  was of  a  has been  2+  Na - and/or  Ca -channel  inactivation  Mayer and Westbrook (1983), who performed voltage-  clamp analyses on cultured mouse DRG neurons, rectification  this  voltage  1957), and later was suggested to be a +  consequence  investigations  consequence  external  Na ,  of  possibly  +  "mixed"  found that Na - and +  mediating  this  form of  K -currents.  An  +  such currents,  also  is  supported by our observations that the rectification is reduced in low-Na  +  solutions, or by applications of tetrodotoxin. nation of the  rectification  tetrodotoxin present well  In addition, complete elimi-  has been observed in Na -free +  (Gallego,  1983).  solution,  with  A reduction in Na -inactivation as +  as a presumed inward Na -current that +  is  turned on by the  induced  hyperpolarization, thereby would contribute to the anodal break response on termination of the hyperpolarizing current pulse (cf.  Fig. 5).  The voltage  responses of TRG neurons to intracellular injections of depolarizing current pulses usually were characterized by damped oscillations that were unaffected by prolonged applications of tetrodotoxin,  indicating that a TTX-sensitive,  Na -conductance was not involved in the subthreshold membrane oscillations. +  SPIGELMAN 4.2.2  Action  potentials.  (Spigelman, 1986;  In  these,  as  in  previous  102  investigations  Puil and Spigelman, 1988), two types of action potentials  could be distinguished on the basis of an absence or presence (hump) on the falling phase of evoked spikes.  of a plateau  Although we were not able to  identify electrophysiologically different types of TRG neurons for technical reasons, DRG cells that discharge humped spikes of long duration have been classified  as  slowly  conducting,  unmyelinated  C-neurons  and  myelinated  A6-neurons which transmit nociceptive information (Czeh et a l . , 1977; and Pierau, 1980; 2+  of Ca  ions,  Harper and Lawson, 1985; +  in addition to Na  long duration spikes  ions,  in ganglionic  Rose et a l . , 1986).  Gorke  An influx  may participate in the genesis of  neurons (Ito,  1982).  For example,  the  Na -conductance in such humped spikes is only partly sensitive to blockade +  with TTX, whereas fast spikes without a hump are blocked completely by TTX administration (Gallego, 1983; 1978).  Stansfeld and Wailis, 1985;  Yoshida et a l . ,  The present results on TRG neurons suggest that a main component in  the development of either type of action potential in some cases, is TTX-insensitive.  including the fast afterhyperpolar-  ization, could be blocked with either external K  +  probably contributes  to  +  Because the falling phase, particularly  the later part of TRG action potentials  cation,  is a Na -current which,  most  of  TEA or internal Cs the  +  appli-  repolarization phase  of  humped and non-humped spikes. 4.2.3  Postspike  afterhyperpolarizations.  Although the peak amplitudes  of AHPs of TRG neurons were similar to those observed in other mammalian sensory  neurons, the durations were longer  Gbrke and Pierau, 1980; In addition,  the  AHPs of  Holz et a l . , 1985; TRG neurons  that  (Gallego and Eyzaguirre, 1978; Stansfeld exhibited  and Wallis, humped spikes  1985). were  larger in amplitude and longer in duration than the AHPs of sensory neurons  SPIGELMAN  103  with short spike durations.  In the DRG of the pigeon (Gorke and Pierau,  1980)  al.,  and the frog  (Holz et  1985),  action potentials  of  identified  C-neurons are accompanied by AHPs with amplitudes and durations that exceed the AHPs of A-neurons. of  In the rabbit nodose ganglion, the peak amplitudes  the AHPs are similar in both C- and A-neurons, but the AHP duration is  much longer in the case of C-neurons (Stansfeld and Wallis, 1985). tent  afterhyperpolarizations lasting  several  hundreds of milliseconds  seconds in duration like those observed in nodose ganglion neurons and  Sampson,  1976)  were  not  commonly observed  discharge evoked by current pulse long-lasting  (<150  ms)  AHPs  injection into TRG neurons.  The small  be  +  sensory  neurons  (Jaffe  repetitive  may  (Weinreich,  a  following  or  the  K -conductance activated by the repetitive other  Persis-  result  of  a  Ca -activated  discharge in TRG neurons as in  1986).  However,  the  fast  AHPs  accompanying humped and non-humped spikes in TRG neurons are likely to be a consequence  of  activation of  a K -conductance, as  inferred from the AHP  +  blockade by external TEA or internal Cs applications. +  4.3  Membrane electrical responses of human sympathetic neurons Although human ganglia have been employed as a source of primary cell  cultures (Scott et a l . , 1979; have shown that slice  Fukuda et a l . , 1983), these  preparations of sympathetic  investigations  ganglia of peripherally  perfused, brain-dead humans can be kept viable under in vitro conditions for more than 12 hr.  A well-known clinical observation is that,  during lower  limb surgical procedures that involve an aortic clamp, an interruption of blood supply to the ganglia for several hours is not associated with clinical sequelae.  This apparent resistance to anoxia could not be confirmed experi-  mentally in slices of ganglia that had been kept in cold, oxygenated ACSF  SPIGELMAN for  ~8 hr, or in similar preparations of  human trigeminal  excised 4 hr postmortem, in the present investigations.  104  root ganglia  However, intracell-  ular recordings could be obtained from neurons in in vitro slice preparations of  acutely  excised  human sympathetic  ganglia,  for  electrophysiological  analyses of membrane properties and pharmacology. The resting membrane potentials  and average value of  input  resistance  (~29 Mfi) in the human neurons were mostly in the same ranges reported for various  lumbar sympathetic  1973).  The wide  neurons  variation of  probably reflects differences  in  input  guinea  pigs  resistance  and cats  values  Skok,  human neurons  in the diameters of the perikarya, as well as  electrode sampling bias, particularly since successful the penetrability of the strong, fibrous connective cells.  in  (cf.  recording depends on  tissue surrounding the  When neurons have been isolated from human sympathetic ganglia prior  to tissue culture, their diameters have been found to be ~40-60 um (Fukuda et at., 1983).  The above data and the long membrane time constants measured  in 3 neurons are consistent with certain remarkable properties of sympathetic neurons in other mammals.  For example, the high input resistance values and  low threshold for spikes generated on current injections neurons  of  rodents  have  resistance rather than  been  attributed  to  into  sympathetic  a high membrane  (specific)  to their size (cf. Perri et a l . , 1970).  An interesting result here was the frequent appearance of TTX-sensitive spikes on the  anodal  break responses as well  currents revealed by the voltage step commands.  as the  small  outward tail  These responses, like those  observed in rat sympathetic neurons (Galvan, 1982), suggest that an outward current may modulate neuronal spike genesis during the depolarizing rebound of the anodal break response.  SPIGELMAN In general,  the pharmacological sensitivities  of  the  spikes  105  of human  neurons appear to be similar to sympathetic neurons in other mammals (cf. McAfee and Yarowsky, 1979).  The observed alterations in spike amplitude and  duration in human neurons during TEA applications voltage-sensitive  K -conductance. +  On the  other  suggest blockade of a  hand, the  postspike AHPs  were slightly reduced by the TEA applications indicating the existence of a distinct K -conductance which also was unaffected by 4-AP applications. +  4.4  Differences in the actions of K*-channel blockers on TRG neurons The observations that applications of 4-AP had excitatory effects in TRG  neurons in contrast  to  the  ineffectiveness  of  TEA applications,  suggest  separate sites of blocking action on the outward currents. Presumably, the amphipathic property of 4-AP confers ready access of 4-AP molecules to the K -channel sites within the neuronal membranes, whereas the more hydrophil i c TEA is  likely to gain access to the blocking sites from the external  membrane surface i . e . , An important difference greater efficacy  from the aqueous phase (Thompson and Aldrich, 1980). observed in their actions  on TRG neurons was the  of TEA in blocking the transient outward currents whereas  outward currents that had much longer time courses of inactivation were much more readily blocked by 4-AP applications. The actions  of  another  K -channel +  blocker,  internal  Cs , +  on  spikes  and the transient outward current were similar to those of TEA. However, unlike 4-AP and/or TEA, Cs  +  injections produced a greater degree of block-  ade of the conductances that presumably contribute to the resting membrane potential.  In addition, Cs  +  blocked the  afterhyperpolarizations observed  during spontaneous or stimulus-evoked spiking in the presence of 4-AP and TEA.  Indeed,  prolonged afterdepolarizations  were associated  with  spikes  SPIGELMAN during internal Cs activation  of  +  applications.  a Cl"-current.  DRG neurons is  These probably were a consequence For example,  higher than the extracellular  the  intracellular  [Cl ]  (Nishi et  -  106  of an  [ C T ] in  al.,  1974).  In TRG neurons, y-aminobutyrate evokes depolarizations during intracellular recording with I^SO^-containing electrodes  and these  blockade with bicuculline (Spigelman, 1986;  Puil and Spigelman, 1988).  present  observations  Cs2S0 -filled  of  spike  high intracellular content of Cl" in TRG neurons. 2+ Ca -activated Cl -conductance has been described (Mayer, 1985)  2+ Co -containing  studies, perfusion with low-[Ca ], 2+ Ca -spikes and the afterdepolarizations. 2+  neurons. Cl"  would  obtained  the  presence  A likely be  to  of  a  in cultures  physiological reduce  role  membrane  for  The with  of DRG  In the present  media  abolished  These observations  Ca -activated  to  suggestion of a  and spinal cord neurons (Owen et a l . , 1984). 2+  with  susceptible  afterdepolarizations  electrodes provide further support for the  4  tent  are  are  Cl -conductance  the  consisin TRG  an inward current mediated by  excitability  by  a  shunting  action.  Assuming similar currents exist in the terminals of sensory neurons, activation of a Cl "-conductance also may serve to limit transmitter release,  as  proposed previously in the mechanism of presynaptic inhibition in the CNS (Eccles, 1964; 4.4.1  Nicoll and Alger, 1980;  Membrane potential  Padjen and Hashiguchi, 1983).  oscillations.  potentials of TRG neurons to oscillate  The tendency  for  the  resting  on depolarization with subthreshold  amounts of intracellularly-injected current pulses was suppressed during TEA applications. repetitive injections.  The TEA-actions  spike discharge  that  were  accompanied  could be evoked  by  a  reduction  in  the  by depolarizing current  The oscillations observed at resting potential levels that were  subthreshold for spike genesis, are likely to be a consequence  of the same  SPIGELMAN  107  membrane properties giving rise to the resonant behavior in the impedance magnitude functions  of TRG neurons  (Puil  et  al.,  1987,  1988;  Puil  and  Spigelman, 1988). The importance of this behavior or its ionic generation in the entrapment of neuronal discharge is related particularly to the observations that excitable cells are most likely to develop a repetitive discharge in response to depolarizing inputs at the natural frequency of oscillation in the membrane, i . e . ,  at potentials where the resonant behavior becomes prominent in  the impedance magnitude function (Clapham and DeFelice, 1976, 1982; a l . , 1987).  Puil et  Therefore, the findings that TEA blocks resonance (Puil et a l . ,  1988) and the oscillations of membrane potential as well as the tendency in some TRG neurons to discharge repetitive spikes, take on special  significance  in view of the other concomitant effects of TEA. The small depolarization induced by TEA, the reduction of spike threshold which was at least partly due to a TEA-evoked increase in input resistance,  and blockade of postspike  afterhyperpolarization, would represent favorable conditions for repetitive spike  genesis.  associated  These  observations  with oscillations  of  the  suggest  that  the  membrane potential  K -conductance( s) +  may modulate  the  level of Na -inactivation in TRG neurons. +  The above  effects  of  an agent which  varying degrees (Armstrong and Hille, of  blocks  various  +  1972) suggest that more than one type  K -conductance may be involved in the excitabi1ities +  Because several  K -channels to  of TRG neurons.  neurons were only slightly depolarized by TEA applications  in these experiments, the strong reduction of oscillatory behavior cannot be explained by the observed changes in the resting potentials The simplest explanation for the depression is  (cf.  Fig. 8A).  that TEA blocks the time-  SPIGELMAN dependent K -conductances (Armstrong and Hille,  1972)  +  which are  108  manifest  as oscillations in the time-domain and as resonance in the frequency-domain (Puil et a l . , 1988). An interesting enhancement  of  feature of 4-AP actions on most TRG perikarya  their  subthreshold  oscillations  development of repetitive spike firing.  which  may  lead  is  the  to  the  This feature is opposite to that  observed with TEA administrations to TRG neurons, but was evident with TEA administrations to spinal root axons in the rat  (Baker et a l . , 1987).  The  oscillations which also were induced by 4-AP application and blocked when TEA was additionally applied, were presumably unmasked by the 4-AP blockade of a slow outward current.  Each oscillation probably results from a fast,  TTX-insensitive inward current  and a repolarizing outward K -current +  is sensitive to blockade by TEA. 4-AP)  If  the inward current (in the presence of  2+  were carried partly  that  by Ca  (cf.  Rogawski  and Barker,  1983),  the  slow depolarizing shift on which the oscillations are superimposed would be 2+  generated  by  intracellular  outward Cl~-current  Ca  -accumulation  (Mayer, 1985).  and/or  activation  of  an  On reaching threshold, these oscilla-  tions could facilitate repetitive spike discharge and give rise to a burst pattern. 4.4.2  Repetitive  spike f i r i n g .  electrically silent (cf. synaptic  excitation  (cf.  Usually, most TRG neurons in situ are  INTRODUCTION), presumably because of an absence of Lieberman,  1976)  and  as  a  result  of  various  voltage- and time-dependent outward currents such as those observed during voltage-clamp experiments. of ~-60 to -70 mV.  These currents are active near resting potentials  Indeed, many TRG neurons do not discharge  repetitively,  i.e.,  more than one spike, during intracellular injections of suprathreshold  step  currents  (cf. Fig. 8B).  In  the  presence  of  4-AP  this  situation  SPIGELMAN changes, and with imposed depolarizing step currents, continuous firing may be observed in TRG neurons. after  4-AP  application  to  neurons  109  repetitive  Similar phenomena have been observed of  sympathetic  ganglia  (Galvan and  Sedlemeir, 1984) and dorsal root axons (Kocsis et a l . , 1986)  or after TEA  application to spinal root myelinated axons (Baker et a l . , 1987). The facilitation of firing by 4-AP may be a result of blockade of a slow K -current that produces a decrease +  in spike accommodation.  Note that in  the control conditions for the cell of Fig. 8B, only a single spike could be evoked with a range of suprathreshold depolarizing current stimuli.  Indeed,  subsequent experiments show that a current with such characteristics exists in TRG neurons. The observations of spontaneous  fast  depolarizations that occur during  appications of 4-AP in some TRG neurons suggest that such depolarizations may initiate the spontaneous repetitive spike discharge in sensory neurons. These responses may result from spike activities in the axon and be detected in the impaled perikaryon only after increases induced by 4-AP.  An alternative hypothesis  is  in membrane resistance were that 4-AP may activate an  inward current that produces the transient membrane depolarizations.  The ?+  latter  possibility  is  supported  by the  observed  enhancement  of  Ca -  currents by 4-AP in spinal neurons (Rogawski and Barker, 1983). 4.5  Comparison of transient outward current L"I( )] with 1^ T  The TEA-sensitive transient current observed in TRG neurons has kinetics that are qualitatively similar to those of the transient A-current U ) in A  central and sympathetic neurons of mammals.  A major difference is that the  inactivation of the current in TRG neurons can be reduced with relatively small hyperpolarizing voltage commands (cf. Figs. 14A and 15B).  In some TRG  SPIGELMAN neurons,  this  probably accounts  outward current may be  for  activated  holding potentials near rest (e.g. of  this  the  ease  with  which the  by depolarizing voltage -50 to -60 mV).  TEA-sensitive current in TRG neurons to I  110  transient  commands from  Despite some similarity in other neurons, the  A  two currents are most likely mediated by ionic channels in the membrane that have  dissimilar  sensitivities  infrastructures.  Because  of  these  to 4-AP and TEA applications, as well  tence of a 4-AP-sensitive vertebrates  I  distinctions  as a possible  in  coexis-  observed in many other types of neurons in  A  and invertebrates,  the TEA-sensitive outward transient current  in TRG neurons will be refered to as The  applications  of  muscarinic  membrane properties or I(y)-  agonists  did not  The ineffectiveness  affect  the  resting  suggests an absence  of  receptors for acetylcholine on the perikarya of TRG neurons, as found in other primary sensory neurons (cf. Lieberman, 1976), although nodose neurons in culture can be depolarized by acetylcholine applications (Baccaglini and Cooper, 1982). 4.5.1 in  I^ j  Ionic is  T  external  species mediating I (•[•)•  .  The slight  reduction  with  2  conditions  in  dependencies A  (1984)  m a i n  species involved  1 0 n i c  nodose  on Ca  in  I^  that  was  observed  during  solutions  was  similar to  +  3, Co -containing  low-[Ca  modest diminution in the  I  e  +  2+  of  n  likely K , whereas the current has only a small dependency on  Ca  perfusion  T  the  transient outward currents observed during such  ganglion  neurons  (Stansfeld  et  al.,  1986).  These  in TRG and nodose neurons are intermediate to those  of mammalian sympathetic ganglion neurons where Gal van and Sedlmeyer have  reported almost  complete  blockade  of  I  ft  by C d  2+  and Mn  2+  2+ applications,  whereas  an  insensitivity  observed by Belluzzi et a l . (1985).  of  I  A  to  Cd  applications  was  SPIGELMAN 2+ In TRG neurons, perfusion with the low-[Ca ]- or containing solutions also produced an outward shift in the currents,  suggesting  that  + 2+ Na and Ca  contribute  111  + low-[Na ]steady-state  significantly  to  the  +  steady  state currents in TRG neurons at rest.  proton-transformed  Ca -channels  can be blocked 2+  (DRG)  neurons  cations  by applications  (Konnerth et  al.,  of  1986).  in  dorsal  root  ganglion  other  divalent  2+  Cd ,  Co  or certain  Indeed,  proton-sensitive  have been demonstrated in cultured TRG neurons 1980).  A Na -current mediated by  (Krishtal  Na -currents +  and Pidoplichko,  However, i t is unlikely that the outward shift or 1 ^  by Na -dependent, +  K -channels similar to +  those observed  neurons because  of their differing kinetics  sensitivity  these channels  of  is mediated  in cultured TRG  of activation as well  in cultured neurons  to  as the  blockade with TTX  (Bader et a l . , 1985). 4.5.2  Membrane repolarization.  applications blocked both I( ) T  IJJJ  The observations  that  and the AHPs in TRG neurons  internal  Cs  suggest  that  +  contributes to the slow repolarization of TRG neuronal membranes, at  least in the subthreshold regions depolarized from the i n i t i a l resting state. Although  the  time  course  of  1^^  activation  cannot  be  determined with  single electrode voltage-clamp techniques, this current may contribute partly to the genesis of AHPs which have similar time courses. ting  the  discharge  AHP is  well-suited  by indirect actions  temporally on the  to  voltage  The current genera-  influence dependent  repetitive  spike  Na -channels. +  A  steady-state inactivation of the TTX-sensitive Na -current is half-maximal +  at ~ -80 mV, and complete at -40 mV in rat sensory neurons (Kostyuk, 1981). In this  resting potential  range,  the AHPs would remove Na  +  inactivation  SPIGELMAN thereby  allowing  the  reactivation  promoting spike discharge.  of  voltage-dependent  112  Na -channels and +  In this scheme, the blocking actions of TEA (on  the generation of I( ) and the AHPs) would inhibit repetitive discharge. T  An involvement of I  A  in spike repolarization has been demonstrated in  sympathetic ganglion neurons (Belluzzi et a l . , 1985). I( )  Since the kinetics of  activation are unknown, i t would be premature to exclude a contribu-  T  tion of I( ) T  to the mechanism of spike repolarization in TRG neurons  Bader et a l . , 1985). tions  of a slight  This reservation receives emphasis from the observa-  blockade of 1 ^  applications produce small  increases  during administration of 4-AP; in the  spikes, suggesting a coexistence of an I 4.5.3  (cf.  Other outward currents.  A  durations of  such  directly evoked  in TRG neurons.  These investigations  have demonstrated  that 4-AP applications blocked an outward current that had a fast onset and a relatively slow time course of inactivation.  The combined applications of  4-AP and TEA allowed a separation of this current from I^jj as well as the TEA-sensitive delayed rectifier current. Although the fast onset of the 4-AP-sensitive current is indicative of its participation in spike repolarization, this current may be better suited than I ^ J J  to exert a steady  l i t i e s of TRG neurons.  influence on the repetitive  The rather long time course of inactivation suggests  an explanation for the general ineffectiveness to evoke repetitive  of current pulse  injections  spike discharge in a majority of TRG neurons in these  and previous studies (Spigelman, 1986). fast-activating,  discharge capabi-  The stabilizing influence  of the  slowly-inactivating current is removed by 4-AP application  and this allows repetitive firing to occur.  SPIGELMAN The 4-AP-sensitive outward current in TRG neurons resembles  113  the  fast,  sustained current in nodose ganglion neurons which is blocked by 4-AP, and dendrotoxin (Stansfeld et a l . , 1986;  1987), as well as the non-inactivating  K -current in DRG neurons which is blocked by e-bungarotoxin (Peterson a l . , 1986).  et  This pharmacological similarity ( i . e . , sensitivity to 4-AP) may  be common to primary sensory neurons.  In contrast  to other neurons,  the  putative 1^ in nodose neurons is not affected by 4-AP or TEA administration (Stansfeld  et  a l . , 1986).  Another difference  is  that  both I( )  and the  T  fast, sustained current can be observed in the same TRG neurons, whereas the analogous currents are present in separate A- and C-populations of  nodose  neurons. 4.5.4  Significance.  The duration of action potentials  in rat primary  afferent axons can be increased by 4-AP, but not by TEA applications (Grafe et a l . , 1985;  Kocsis et a l . , 1987).  The present investigations demonstrated  only minor effects of 4-AP applications on spike shape compared to TEA, but a pronounced enhancement of  repetitive  spike  firing  was observed;  these  effects were opposite to those obtained with TEA applications. The findings physiological of  significance  K -channels +  Although early  in TRG neurons  in  raise  of differences  sensory  investigations  myelinated  an intriguing question in pharmacological axons  and  perikarya  of mammalian myelinated axons  absence of K-currents at the nodes (Horackova et a l . , 1968; 1979;  about  the  sensitivities of  mammals.  indicated an Chiu et a l . ,  Brismar, 1980), at least two pharmacologically separable K -currents +  have been subsequently described (Grafe et a l . , 1985;  Baker et a l . , 1987).  These currents are more commonly observed in the axons of younger animals and also are present at the internodal segments of demyelinated fibers (Chiu and Ritchie, 1980).  The perikaryal  membranes of primary sensory  neurons  SPIGELMAN  114  exhibit far greater diversity of outward currents in a comparison to their axons.  It seems unlikely that these currents are simply a vestigial repre-  sentation  of ontogeny.  Indeed, they may have important functions  in the  adult sensory nervous system. Perikarya!  outward  outward current  currents,  in TRG neurons,  such  as  provide  the  4-AP-sensitive  a plausible  sustained  mechanism for  the  prevention of excessive spike discharge in trigeminal sensory transmission. In circumstances where this  braking  influence  has  been  removed,  somatic  invasion by a single action potential may initiate spike discharge in bursts (cf.  Fig. 23)  terminations.  which would be propagated to  the  central  and peripheral  The generation of somatic spike bursts in the TRG may occur  in certain conditions  such as trigeminal neuralgia where a brief  stimulus can induce paroxysmal attacks of pain.  Also,  may cause a release of neuroactive autacoids (e.g.  sensory  certain conditions  histamine, substance P)  in the TRG that would affect, either directly or indirectly, the excitabilities of perikarya. 4.5.5 Bursts and ionic mechanisms.  The 4-AP induced train of repetitive  activity in the cell of Fig. 10B could be transformed into a recurrent burst pattern by imposed hyperpolarization to a background potential where a slow outward current may have developed [cf. successive increase in the amplitudes of the spike afterhyperpolarizations in Fig. 10B, right]. A second increase  type 2+  of burst  in Ca -influx)  and TEA (cf.  Fig. IOC).  (or modification  of  the  first  type  due  to  was evident with a combined application of 4-AP These "spontaneous"  bursts of action  potentials  were observed in several TRG neurons and are similar to those reported in neurons of olfactory cortical slice preparations (Galvan et a l . , 1982) and in  peripheral  sensory  axons  (Kocsis  et  al.,  1987)  after  application of  SPIGELMAN 4-AP.  115  Here, one may presume that a fast inward current sensitive to blockade  with TTX produced the spikes and a much more slowly activated inward current, possibly 2+ Ca  due to unopposed, or 4-AP enhanced (Rogawski and Barker,  -influx  burst.  gave  rise  to  the  background  depolarization  of  the  1983), spike  The Na -inactivation and a slower repolarizing current, insensitive +  to blockade with 4-AP or TEA, presumably reduced the spike amplitudes and terminated each burst. This termination may be a direct result of a 2+ + Ca -dependent, outward K -current, as suggested by the cardiac-like spikes that had prolonged AHPs when the neuron was hyperpolarized by DC-current injection (Fig. IOC; cf. Heyer and Macdonald, 1982; Calabresi et 2+ al.,  1987).  The prolonged AHPs that were observed  (Fig. 13A) provide further support for this 4.5.6  Significance.  The tendencies  following  Ca  -spikes  suggestion. of  TRG neurons  to  discharge  in  bursts, given an appropriate stimulus such as a small abrupt depolarization, are a consequence in  the  of membrane characteristics that manifest as  responses  physiological  to  injected  conditions,  such  depolarizing stimuli  may  current  include  oscillations  pulses.  Under  electrical  impulse  activity from the trigeminal axons that invades the TRG perikarya and, slow depolarizing shifts in their membrane potentials as a result of the actions of  a chemical  electrical  mediator such as  substance  P,  in  the  TRG.  This  complex  behavior of the TRG perikarya would likely modulate transmission  of afferent activity along the trigeminal nerve. It is possible that the above tendencies of TRG neurons may be exaggerated  in  pathophysiological  paresthesias, firing  cf.  Kocsis  conditions et  al.,  including transformation  such  1986).  into  as  trigeminal  For example,  bursts  neuralgia  continuous  can occur when there  (or spike  is  an  interference with, or a defect in K -channel function, as observed in the +  SPIGELMAN present  experiments  with 4-AP applications  to  the  TRG neurons.  116  Further  +  impairment of the K -channel  system,  as in the combined blockade by 4-AP  and TEA application, may result in the second type of burst activity  (cf.  above section on Bursts and ionic mechanisms). 4.6  Spike initiation in the TRG Previous  several  electrophysiological  species  have  revealed  investigations that  action  of  sensory  potentials  neurons  travelling  orthodromic direction always invade the neuronal perikaryon (cf. 1976).  The present  investigations  in  in an  Lieberman,  in guinea pig TRG neurons showed that  changes in resting membrane potential may exert a profound influence on the invasion  of  perikarya  by spikes  generated  in  the  axons.  In  neurons, experimental depolarization of the membrane to potentials to resting potential presumably  due  Na -channels  to  results  positive  in the inability to support spike generation,  excessive  and a shunting  some TRG  inactivation action  of  the  of  voltage- and  increased  time-dependent  K -conductance.  In  other TRG neurons, membrane depolarization may lead to the generation of action potentials within perikarya. quence of the partial persistence  of  I^jj  This phenomenon possibly  is  a conse-  inactivation of the 4-AP sensitive current and the  at depolarized membrane potentials.  This may result  in a heightened state of excitability of some TRG neurons which manifests as increased membrane resonance observed in frequency-domain studies (cf. et a l . , 1987; that  1988).  The present  under certain conditions,  investigations  modulation of  Puil  provide direct evidence  sensory  impulses  may occur  prior to the first synaptic junction in the CNS, in the perikarya of sensory neurons  SPIGELMAN 4.7  117  Membrane responses to autacoids The unresponsiveness  surprising, especially  of TRG neurons to applications of bradykinin  since the majority of TRG neurons in tissue culture  have been observed to respond to applications of this and Hogan, 1983).  was  peptide (Baccaglini  Moreover, a recent study has provided evidence  for the  presence of receptors for bradykinin on a subset of small diameter neurons within  trigeminal  and spinal  ganglia  of  1988).  The small sample of TRG neurons tested with bradykinin applications  did not exhibit spikes with inflections  guinea  pigs  et  al.,  in the repolarization phase sugges-  ting that these cells were not nociceptive C-neurons. vations that bradykinin applications  (Steranka  specifically  In view of the obser-  affect  only C-neurons in  the nodose ganglia (Weinreich, 1983), this also may be the case in the TRG where  neurons  which  may  belong  to  encountered in these investigations. our previous  investigations,  this  category  were  infrequently  A similar situation was encountered in  in which  high  doses  of  5-hydroxytryptamine  failed to produce responses during intracellular recording in TRG neurons (Spigelman,  1986;  Puil  recordings  from  cells  presumably  because  the  and  Spigelman,  with  long  small  diameter  1988).  duration of  The  spikes  C-cells  number  was makes  of  quite  stable limited,  them much more  difficult to investigate using microelectrode techniques. The results  obtained with  histamine  demonstration of the effects of this  applications  represent  autacoid on TRG neurons.  the  first  The large  depolarizations observed in 2 of 9 TRG neurons were similar to the depolarizations evoked by substance  P applications.  Histamine has been shown to  produce depolarizations in ~25% of C-neurons in the rabbit nodose ganglion, but was without effect  on the A-neurons (Higashi  et  al.,  1982).  It  is  possible that histamine at relatively high doses is capable of causing the  SPIGELMAN release  of  substance  P within  producing the observed response.  the  TRG, the  latter  tachykinin  in  118 turn  Alternatively, the depolarizations may be  evoked directly by histamine acting on distinct receptors.  The inability to  block the histamine-induced depolarizations with a high dose of cimetidine indicates that these receptors probably are not of the ^-variety.  In the  rat sympathetic ganglion and the frog neuromuscular junction, histamine may facilitate or inhibit transmitter release by acting presynaptically on the Hj- and  H -receptors, 2  respectively  (Snow,  et  al.,  1980).  A further  pharmacological characterization was not attempted because of the unspecific actions  of available H^-antagonists.  In addition, the  low percentage  of  neurons that responded to histamine made difficult an extensive characterization of such responses as opposed to the large number of TRG neurons that responded to substance P applications.  4.8  Ionic mechanism of substance P action The synthesis and release of substance P from the central and peripheral  processes of sensory neurons have been ascertained in several (cf.  Introduction).  investigations  However, the results described here represent the first  demonstration of the profound depolarizing actions  of the peptide on the  perikaryal membranes of primary sensory neurons in mammals.  The responses  to substance P in many TRG neurons were subject to desensitization following multiple applications of the peptide.  Such desensitization has been observed  in the receptor-mediated responses to substance P in the rat acinar cells (McMillian et a l . , 1987).  In addition, several neurons exhibited  increases  in the amplitude of the response to a second application of the peptide, indicating  possible  receptor  sensitization.  The  possibility  that  the  SPIGELMAN increases  in the  substance  P-responses  were  membrane properties is unlikely because the  due to  improvements  input resistance  119  of  the  and membrane  potential remained stable throughout the recording periods. The actions of substance P were observed in the voltage-clamp studies on TRG neurons as a slight increase in the evoked inward currents as well as a small but consistent decrease in the outward currents.  Because such outward  currents in TRG neurons are mostly a result of an efflux and Puil,  1987), the possibility arises  that  depolarization by blocking K -current(s).  substance  conductance during a response to substance P (cf. However, a small  +  P may produce the  The slight decrease  +  suggestion.  of K (Spigelman  reduction  in  in membrane  Fig. 26B) supports this  K -conductance alone  is  +  not  sufficient to account for the large amplitude of the depolarization, unless the  actions  Macdonald  of  substance  1982).  An  combination with the  P are  increase  decrease  highly in  voltage-dependent  membrane  conductance  Nowak and  for  Na ,  in  +  in outward currents could give  significant increase in net inward (steady-  rise  to a  state) current resulting in a  large depolarization.  This interpretation is  observed  substance  reduction in  (cf.  P-responses  likely of  in view of:  neurons  bathed  (1)  the  in Na +  deficient media and (2) activation of an inward current by substance P in the presence of K -channel blockers. +  The suggestion that the mechanism(s) for the substance P-induced depolarization  in TRG neurons  of  conductance and a concomitant  guinea pigs decrease  involves  an increase  in K -conductance is  in Na +  unlike  +  that  proposed for mouse spinal neurons in culture (Nowak and Macdonald 1982), rat dorsal  horn neurons  (Murase et  neurons (Dun and Mo, 1988).  al.,  1986)  However, the  or preganglionic dependencies  of  the  sympathetic substance  SPIGELMAN P-induced  effects  on  both  Na  and  +  K  in  +  TRG  neurons  are  120  similar  to  observations in i n f e r i o r mesenteric ganglia of guinea pigs where substance P is a l i k e l y  transmitter  In the present  (Dun and Minota, 1981).  studies, the depolarizations 2+  not  blocked  (1987)  in  have  current  shown that  2+ Co -containing  ],  of  the  solutions. 2+  substance P augments  in voltage clamped spinal  experiments, tions  low-[Ca  dorsal  evoked by substance P were  a Ca  Murase  -sensitive  et  slow  horn neurons of the rat.  inward  In  substance P-induced responses also were reduced in  reduced  (36 mM)  extracellular  [Na ].  Therefore  +  the  al.  their condi-  possibility  exists that the inward current evoked by substance P in the rat  spinal cord + 2+ and in TRG neurons may result from a combined influx of Na and Ca . This current would l i k e l y be mediated by channels that are d i s t i n c t from 2+ 2+ those involved in the generation of the Co - s e n s i t i v e , Ca -spikes observed during  combined applications  responses  not  of r  were  possibility the  in  • supported by the  is  resting  blocked  conductance  of  low-[Ca  4-AP and TEA because substance P 2+j  2+ , Co -containing media. This + 2+ that Na and Ca contribute to  findings  TRG neurons.  Substance  P may  augment  these  conductances, resulting in membrane depolarization. In  the  present  investigations,  the  effects  of  substance P applications +2+  on TRG neurons  were  greatly  perfusates. 2+ of Mg , may  reduced  or  deficient  These observations  influx  produce  applications, internal  [Mg  mediated  by  However,  the  2+  the  extracellular  ].  this  2+ Ca -channels  that  intracellular  [Mg  are  current  [Mg  mechanism,  abolished  have several  inward  assuming In  completely  ]  was  the  Mg  susceptible  interpretations.  evoked high  2+  by  An  substance  relative  -influx  to  in Mg  would  blockade  P  to  the  not  be  with  2+ Co .  2+ ]  has  not  been  estimated  in  mammalian  SPIGELMAN sensory  neurons.  skeletal  muscle  Estimates  of  121  2+ internal [Mg ] have been made in frog 2+ [Mg ] was found to be higher than the  where internal 2+ extracellular Mg content (Alvarez-Leefmans et a l . , 1986). 2+ Secondly, Mg that utilize substrates  is well known to be a required cofactor of all enzymes  adenosine  triphosphate  (Mudge, 1987).  and other nucleotide  activity  the  extracellular  of  Mg  as  If the substance P effects on TRG neurons are at  least in part due to decreased 2+ removal  triphosphates  from the  of  an electrogenic  Na /K +  medium could  pump,  +  inhibit  this  effect. 2+ Also, external Mg may serve to modulate the substance P-induced responses by direct interactions with the channels mediating the inward 2+ current.  The  modulation  of  membrane  responsiveness  by  external  Mg  acting directly on the channels activated by N-methyl-D-aspartate (NMDA) has been observed in neurons of the CNS (Ascher and Nowak, 1988).  However, the  increases in the NMDA-evoked inward current in the absence of extracellular 2+ Mg in central neurons are in contrast to the observed decreases in the 2+ responses to substance P in the absence of external Mg in the TRG. 2+ Therefore, it is unlikely that the mechanism by which external  Mg  modu-  lates the responses to NMDA is applicable directly to the responses evoked 2+ by substance P in TRG neurons. However, external Mg may be required for the interaction of substance P with its presumed membrane receptors on TRG +  neurons,  in  a manner  similar  to  the  Na -dependent  receptor-binding  of  opioid agonists (Snyder, 1978). 4.8.1  Implications  neurons of guinea pigs sensory  information  for  sensory  transmission.  imply a role for substance through  craniospinal  The  findings  P in the  ganglia.  The  properties of TRG somatic membranes as revealed by previous  in TRG  transfer of specialized  (Puil et a l . ,  SPIGELMAN 1986;  1987a;  1987b;  122  1988) and these investigations may serve to modulate  transmission of afferent impulses along the trigeminal nerve.  For example,  a release of  of Katz and  substance  P is  inferred from the observations  Karten (1980) who described the envelopment of sensory neurons by substance P-positive varicose fibers in nodose anglia.  Pericellular arborizations in  the nodose and trigeminal ganglia of the cat have been extensively described in the classical treatises by Cajal (1909). enhance  the  membranes to  excitability potentials  "amplification" action  of  potentials  of  TRG neurons  where  excitability  Such release of the peptide may by  slowly  membrane resonance may result  which would travel  central and/or peripheral terminations.  along  comes  in the the  depolarizing into  play.  production of  trigeminal  their  ectopic  nerve  Assuming that release of  The  to  the  substance  P occurs within the TRG, the depolarizations evoked by such release also may provide a negative peptide.  feedback  system  for regulating the  synthesis of  this  Kesseler et a l . (1983) have shown that a reduction in mRNA content  and synthesis  of  substance  P occur following  veratridine- or K -induced +  depolarizations of neurons within sensory ganglia. Secondly, the somatic effects of neuroactive substances  in craniospinal  neurons of mammals may mimic putative transmitter effects on the membrane receptors  of  the  primary  afferent  terminals.  For example,  applications have been shown to modify the excitabilities identified 1982).  primary  afferent  fibers  in  the  spinal  cord  substance P  of terminals of (Randic et  al.,  Furthermore, depolarizations of nerve terminals have been observed  following substance P applications in chick sympathetic ganglia (Dryer and Chiappinel1i, of  1985).  the central  Hence, substance P may act directly on the membranes  and peripheral terminals by a mechanism similar to  described here for TRG perikarya.  Thus, an involvement of substance  that P in  SPIGELMAN a f f e r e n t s i g n a l transmission i s i n d i c a t e d f o r at l e a s t two cranial  nerve,  as well as f o r the  second  order  123  sites in this  neurons of the c e n t r a l  nervous system (Andersen et a l . , 1977, 1978).  4.9  D i r e c t i o n s f o r f u t u r e research The  outward  i n v e s t i g a t i o n s i n TRG currents  with  neurons have revealed the presence  different sensitivities  for  K -channel +  of  two  blockers.  Because the k i n e t i c s of these c u r r e n t s were only p a r t i a l l y resolved with the s i n g l e e l e c t r o d e voltage-clamp TRG  neurons using  two  techniques i t would be of i n t e r e s t to examine  e l e c t r o d e voltage-clamp,  i n order  to obtain  the  steady-state a c t i v a t i o n and i n a c t i v a t i o n k i n e t i c s f o r the two c u r r e n t s . Another i n t r i g u i n g f i n d i n g was  the appearance of prolonged  followed spikes evoked during the concomitant  a p p l i c a t i o n s of 4-AP  AHPs that and  TEA.  I t would be of i n t e r e s t to see whether K -channel blockers s e l e c t i v e f o r 2+ + the Ca - a c t i v a t e d K - c u r r e n t s (e.g., apamin) could block the prolonged AHPs. Such experiments should a l s o be performed without adding 4-AP and TEA 2+ +  to the media i n order to estimate  the c o n t r i b u t i o n of the Ca  -activated  K - c u r r e n t ( s ) to the e x c i t a b i l i t i e s of TRG neurons. +  The  r e s u l t s of the i n v e s t i g a t i o n s on substance  P a c t i o n s i n the  r a i s e the p o s s i b i l i t y that t h i s peptide i s r e l e a s e d w i t h i n the This  hypothesis  could  be  i n v e s t i g a t e d i n j_n v i t r o  TRG  ganglion.  preparations,  using  e s t a b l i s h e d methodology f o r substance P d e t e c t i o n ( O l g a r t et a l . , 1977). Extensive pharmacological  i n v e s t i g a t i o n s of autacoid a c t i o n s i n the  TRG  are d e s i r a b l e . Tachykinins other than substance P (e.g. a and 8 neurokinins) should  be examined f o r t h e i r e f f e c t s on t r i g e m i n a l  neurons i n order  to  e s t a b l i s h whether more than one t a c h y k i n i n receptor i s capable of mediating the observed  depolarizations.  A v a r i e t y of antagonists  f o r substance  P  SPIGELMAN effects have been described,  although the specificity  has been questioned (cf. Jessell,  1983).  of these  124  antagonists  It would be of interest to inves-  tigate their responses in TRG neurons excited by substance P applications. The relatively slow onset of substance cates  a possible  involvement  of  P-induced depolarizations  intracellular  secondary  indi-  messenger.  For  example, a role for inositol phosphates has been postulated in the responses of rat acinar cells to substance P (McMillian et a l . , 1987). Another important area of investigation prostaglandins  and endorphins with  is the possible  receptors  for  interaction of  substance  P.  Although  prostaglandins are not algogenic, except at very high doses (Horton, 1963; Crunkchorn and Willis, 1971), they can augment nociception. action is antagonized by cyclo-oxygenase  This sensitizing  inhibitors (Ferreira et a l . , 1973;  Lembeck and Juan, 1974) and is considered to be a mechanism for the analgesic effects 1987). to  of  drugs  such as  aspirin and indomethacin  (cf.  Flower et  al.,  Furthermore, prostaglandins have been shown to enhance the responses  substance  P released  from the  trigeminal  nerve  (Ueda et  al.,  1985).  Therefore, i t would be of interest to see whether prostaglandins are capable of  sensitizing  endogenous  substance  opioid  P receptors  peptides  are  very  in the potent  TRG. Unlike prostaglandins, analgesics  which  have  been  proposed, to inhibit the release of excitatory transmitters from terminals of nerves conveying nociceptive possible  interference  with  information (cf. substance  Duggan and North,  P-induced  responses  by  1983).  A  enkephalins  should be explored. The electrophysiological (e.g.  by conduction velocity)  neurons.  identification of various sensory neuron types is difficult in j_n vitro preparations of TRG  In order to estimate the conduction velocity of nerve fibers with  reasonable accuracy, the required length of fibers exceeds that of axons in  SPIGELMAN TRG slices.  The identification  of  neurons therefore  125  can be more easily  accomplished in in vivo experiments where stimulation of the axons may be accomplished peripherally in the skin or the tooth pulp. matching of  the  perikaryal  velocities of their axons.  membrane characteristics  This would allow  with  the  As a result of these and other  conduction  investigations,  i t is especially important to determine which subsets of neuronal populations are  responsive  to  substance  receptor binding techniques. able  to  examine  the  P.  This  information may be obtained  For example,  detailed  kinetics  membrane receptors in the rat brain.  Hanley et a l . of  using  (1980) have been  [ H]-substance  P binding  to  Such studies should be performed using  relatively short periods of incubation with substance order to avoid receptor desensitization  P or its  agonists in  and the resulting underestimation of  the number of substance P-receptors (cf.  McMillian et a l . , 1987).  Another  way of determining i f receptors for substance P are located on neurons other than those mediating nociception  is  to perform binding studies  following  neonatal pretreatment of animals with capsaicin, thus eliminating the small diameter C-neurons from the ganglionic a l . , 1977).  neuronal population (cf.  Jancso  et  SPIGELMAN  126  5 SUMMARY AND CONCLUSIONS 1.  The electrical  and pharmacological membrane properties of neurons  were studied with current-clamp and single electrode voltage-clamp techniques in ui vitro slices of trigeminal root ganglia of guinea pigs and in human sympathetic ganglia. similar to, neurons.  The trigeminal neurons had some properties that were  and others  which distinguished  them from other craniospinal  A much smaller sample of human sympathetic neurons showed proper-  ties similar to certain reported characteristics of sympathetic neurons in experimental animals. 2.  Human neurons exhibited  resting  potentials  and input  resistances  mostly in the ranges reported for sympathetic neurons in other mammals. The sensitivities of these neurons to specific ionic channel blockers (e.g. TEA, 4-AP, TTX) are similar to those in other vertebrate neurons. gations demonstrate  for the  first  neurons can be studied successfully  time,  that  These investi-  human sympathetic  ganglion  in j_n vitro preparations, and hence are  valuable for direct relevance to the human condition. 3.  Two groups of trigeminal neurons could be distinguished on the basis  of a presence or absence of a plateau (hump) on the falling phase of evoked spikes.  Differences were observed in the sensitivities of non-humped spikes  to TTX, indicating similarities in ionic mechanisms of spike generation in trigeminal and other craniospinal neurons.  However, the durations of AHPs  in both types of neurons were greater than those observed in other mammalian sensory neurons, suggesting stronger inhibition of the postspike excitabilities in trigeminal neurons. 4. the  Bath applications of TEA and 4-AP, or Cs  recording electrode,  decrease  in  threshold  produced an increase for  spike  generation  +  applied internally from  in input resistance in  all  neurons.  and a Also,  SPIGELMAN applications potential  of  4-AP increased  and enhanced the repetitive  injections of current pulses, contrast, AHPs,  subthreshold oscillations  the  and  TEA or Cs  did  not  investigations  +  the membrane  spike firing evoked by intracellular  without major effects on the spike AHPs.  applications  exaggerate  suggested  of  127  blocked the  repetitive  that  several  oscillations  spike  In  and the  discharges.  pharmacologically  These distinct  K -currents contribute to the control of excitability of TRG neurons. +  5.  During combined applications of 4-AP and TEA, long duration spikes  and AHPs were evoked with depolarizing current pulses.  These were resistant  to blockade with TTX but were abolished in media where CoCl^ was tuted for CaCl -  Similar  2  with  CsCI- or  spikes  CsgSO^-fi11ed  substi-  were evoked during prolonged recordings  electrodes.  However  in  these  conditions  spikes were followed by afterdepolarizations, suggesting that ionic currents in addition to K may contribute to the postspike events in TRG neurons. +  6.  During perfusion with TTX, transient outward currents were elicited  at the termination of hyperpolarizing voltage commands from holding potentials  near -40 mV.  inactivation range.  The activation of  (T=19 ms)  was complete  such currents was rapid at  potentials  the  activation  The amplitudes of these currents were reduced in conditions of high  extracellular  [K ] and were only  (2 mM) in low-[Ca  ] perfusates.  slightly  affected  The observations  + specific  within  (<5ms) and  K -channel  by inclusion of Co that applications of a +  blocker,  TEA, and  internal  Cs  produced  dose-  dependent reductions in the amplitudes of the transient outward currents, whereas administrations of 4-AP or muscarinic agonists at high doses did not greatly affect these currents, suggest a fundamental distinction from similar  SPIGELMAN outward currents observed previously in other invertebrates,  and hence this  neurons of  128  vertebrates and  transient outward current in TRG neurons  is  referred to as l(j)7.  The kinetics of I^jj are indicative of its  in the spike AHPs.  Therefore, blockade of I( )  possible participation  by TEA may interfere with  T  the re-activation of voltage-dependent Na -channels, leading to an observed +  decrease in the ability of TRG neurons to discharge spikes repetitively. 8.  During combined application of TTX (1 uM) and TEA (10 mM), fast  activating,  sustained  outward currents (>1 s)  commands from holding potentials additional  were evoked by depolarizing  near -70 mV.  inclusion of  These currents were blocked  completely  by the  solution.  The TEA-insensitive sustained outward currents presumably have a  braking influence on repetitive  discharge.  4-AP (5 mM) in the  perfusing  Conditions that interfere with  these currents, such as blockade of K -channels by 4-AP application which +  does  not  produce  a  significant  blockade  of  I(y)>  strongly  favour  the  generation of repetitive spike firing in TRG neurons. 9.  The investigations  using electrical  stimulation of axons revealed  that changes in the perikaryal resting potential may result in the inhibition of spike invasion into the perikarya, or facilitate the generation of ectopic spike discharges.  Applications of 4-AP facilitated the perikaryal invasion  of spikes evoked by axonal stimulation, and also induced the appearance of spontaneous,  fast  depolarizations  absence of electrical  stimulation.  that  reached  spike  These investigations  threshold  in  the  provided evidence  that spike generation may occur within the perikarya of sensory neurons, and suggest that such behavior may be operative during normal or pathophysiological conditions.  SPIGELMAN 10.  129  Applications of substance P in micromolar doses produced reversible  depolarizations in majority of neurons, whereas other autacoids did not have consistent effects.  Increases in the repetitive discharge ability of neurons  were evident during such depolarizations. substance  P-action  revealed  that  Studies on the ionic mechanism of  peptide  applications  resulted  in  the  activation of inward currents as well as a blockade of outward currents. +  also  was  shown  that  It  2+  Na and Mg  were  involved  in  the  mechanism  of  substance P-actions. 11.  The above findings represent the first demonstration of the profound  depolarizing actions of substance P on the perikaryal membranes of sensory neurons in mammals. give  rise  The excitatory actions of this endogenous peptide also  to the possibility  of physiological  multiple sites in the trigeminal system.  actions  of  substance  P at  SPIGELMAN  130  6 REFERENCES ADAMS, P. R. and GALVAN, M. 1986. V o l t a g e dependent c u r r e n t s of v e r t e b r a t e neurons and t h e i r r o l e on membrane e x c i t a b i l i t y . In: Advances i n N e u r o l o g y , Ward, A. A. 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Spigelman, Electrical and chemical responsiveness of trigeminal root anglion neurons in vitro. Proceedings of the Canadian Federation of Biological ocieties, Vol. 29, p. 1/8, 1986. S. U. Kim, I. Spigelman, E. Puil and A. Eisen, Morphological and electrophyiological characteristics of human spinal cord neurons Tn culture. International Union 6T Physiological Sciences, XXXth Congress, 1986. E. Puil, B. Gimbarzevsky and I. Spigelman, Ionic mechanism of "membrane resonance" behavior in trigeminal root ganglion neurons in vitro. Canadian Journal of Physiology and Pharmacology, Vol. 65(5), Axvvii, 198/. S. U. Kim, I. Spigelman and E. Puil, Academy of Neurology, 1987.  Human Retina Neurons in Culture.  American  E. Puil, I. Spigelman and S. U. Kim, Transmitter Effects on Human Cultured Neurons. Canadian Neurological Sciences XXIIth Congress, 1987. I. Spigelman and E. Puil, Characterization of a Transient K -Current in Trigeminal Root Ganglion Sensory Neurons. Society for Neuroscience Abstracts Vol. 13(1), p. +  531 , 1987.  E. Puil, B. Gimbarzevsky and I. Spigelman, Membrane Resonance in Trigeminal Root Ganglion Neurons: Involvement of K -Conductances. Society for Neuroscience Abstracts, Vol. 13(1), p. 531, 198/. +  E. Puil, H. El-Beheiry and I. Spigelman,. Excitable .membrane properties of human sympathetic ganglion neurons. Proceedings of the Canadian Federation of Biological Societies, Vol. 31, 1988. I. Spigelman and E. Puil, Substance P excitation of trigeminal ganglion neurons. Proceedings of the Canadian Federation ot Biological Societies, Vol. 31, 1988 I. Spigelman and E. Puil, Ionic basis for substance P excitation ganglion (TRG) neurons. Society for Neuroscience, Vol. 14, 1988.  in trigeminal root  Articles: Puil, E . , I. Spigelman, A. Eisen and S.U. Kim, Electrophysiological responses of human spinal neurons in culture. Neuroscience Letters, 71: 77-82, 1986. Puil, E. and I. Spigelman, Electrophysiological responses of trigeminal ganglion neurons in vitro. Neuroscience, 24(2): 635-646, 1988.  root  Puil, E . , Gimbarzevsky, B., and I. Spigelman, Primary involvement of K -conductance in membrane resonance of trigeminal root ganglion neurons. Journal of Neurophysiology, 59(1): 77-89, 1988. +  Kim, S.U., D. Osborne, M. Kim, I. Spigelman, E. Puil, D. Shin and A. Eisen, Long-term culture of human spinal cord neurons: morphological, immunocytochemical and electrophysiological characteristics. Neuroscience (in press). Puil, E , R. M. Miura and I. Spigelman, Consequences of applications to trigeminal root ganglion neurons (submitted).  4-aminopyridine  Puil, E. H. El-Beheiry and I. Spigelman, Excitable membrane properties of human sympathetic ganglion neurons. Canadian Journal of Physiology and Pharmacology (accepted for publication). Spigelman, I. and E. Puil, Excitatory resonses of trigeminal neurons Substance P suggest involvement in sensory transmission. Canadian Journal Physiology and Pharmacology (accepted for publication). Spigelman, I. and E. Puil, K -channel blockade in trigeminal neurons: effects on the membrane voltage responses and outward (submitted). +  to of  ganglion currents  

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