Brain Research, 166 (1979) 273-282 © Elsevier/North-Holland Biomedical Press
273
EFFECTS OF SUBSTANCE P O N N E U R O N E S IN T H E D O R S A L H O R N OF T H E SPINAL CORD OF T H E CAT
W. ZIEGLG.~NSBERGER and I. F. TULLOCH Max-Planck-lnstitut fiir Psychiatrie, Department of Neuropharmacology, Kraepelinstrasse 2, 8000 Munich 40 ( G.F.R.)
(Accepted August 17th, 1978)
SUMMARY The activity of single, physiologically identified neurones has been recorded both extra- and intracellularly in the 6th and 7th lumbar segments of the pentobarbitoneanaesthetized cat. In the majority of dorsal horn neurones studied (laminae 4 and 5 of Rexed) microiontophoretically applied synthetic substance P and the active fragment, substance P (4-I 1), were found to cause a slow and prolonged increase of the spontaneous firing rate and/or an enhancement of L-glutamate induced activity. Intracellular studies revealed that substance P caused a reversible depolarization of both dorsal horn neurones and motoneurones without a detectable alteration of the membrane resistance, antidromic action potential or postsynaptic potentials. These results are compatible with a possible role of substance P in sensory transmission in the spinal cord o f the cat.
INTRODUCTION Substance P is widely but unevenly distributed in the central nervous system. The active principle of the peptide has been purified 4 and the amino acid sequence determined 5. Its presence in areas receiving sensory afferent fibres has encouraged speculation that it may be a neurotransmitter in primary sensory systems, in particular, the dorsal horn of the spinal cord24,2a,29,31,46. Synthetic substance p42 has a slow onset but long-lasting, potent excitatory effect on neurones in a variety of structures in the mammalian central nervous system 8,11-13, 22,25,32,37-40. These findings are consistent with the depolarizing action of substance P on frog, rat and cat motoneuronestg-2L This evidence, taken together with the fact that substance P release in the spinal cord appears to be a Ca2+-dependent process 28-3°, and is influenced by opiate analgesicsXa, 49 serves to underline the possibility
274 that it may act as a neurotransmitter or may play a role in long-term modulation of neuronal activity2L Immunohistochemical studies utilizing antibodies to synthetic substance P have convincingly demonstrated the presence of substance P in dense terminal networks in the dorsal spinal cord, in particular in lamina 1-3 of Rexed where neurones from lamina 4 and 5 project their dendrites15-1L Furthermore this activity has been shown to be localized in large vesicles in axons terminating in lamina 1 and 22a. Ligation or sectioning of the dorsal roots is known to cause a pronounced decrease of substance P activity in the dorsal horn 16,41 and the development of supersensitivity to this peptide 45. This evidence favours substance P subserving a possible role in synaptic transmission in the spinal cord, prompting us to further investigate the action of microiontophoretically applied synthetic substance P and its active fragment, substance P(4-11), on the activity of functionally identified neurones, recorded extra- and intracellularly, in this region. METHODS The experiments were performed on 25 adult cats of either sex (body weight 2.0-4.0 kg). All the surgical procedures were performed following the induction of anaesthesia by sodium pentobarbitone (35 mg/kg, intraperitoneally). Thereafter, during both the later surgical stages and recordings, anaesthesia was maintained by regular intravenous infusion of sodium pentobarbitone (5 mg/kg/h). The arterial blood pressure was monitored, and experiments were terminated when the blood pressure dropped below 80 mm Hg. The rectal temperature of the cat was maintained within the range of 37.8-38.2 °C by means of thermostatically controlled electric heating devices. The animals were immobilized with gallamine triethiodide and artificially ventilated. A bilateral pneumothorax was routinely made. Plasma expanders (2.25 mg/h), gallamine triethiodide (8 mg/h) and small amounts (0.25 ml/h) of dextrose (20 ~, w/v) were intravenously infused. The spinal cord was exposed by laminectomy at the lumbar-sacral level (L3-$2) and covered with a pool of paraffin oil which had been preheated to body temperature. The dura mater was then dissected and pinned back to the adjacent muscles. A small tear was made in the underlying pia mater to facilitate the penetration of the twin electrode assembly. Dorsal and ventral roots of the segments L5 and L6 were stimulated, in situ, employing bipolar Ag-AgC1 electrodes and appropriate stimulation parameters (0.5-5.0 V; duration 0.02-0.2 msec). The electrode assemblies consisted of a single-barrelled micropipette for recording and a triple- or four-barrelled micropipette, constructed from single, glass fibre containing (omega dot) barrels for microiontophoretic drug application. These twin assemblies were aligned and then fixed together by a fast-setting epoxy resin so that the tip of the recording microelectrode projected beyond the application electrode (intertip distance, 30-40 #m for extracellular recording and 100-160/~m for intracellular recording). For extracellular recording a glass-coated tungsten electrode was
275 used27. The microelectrodes for intracellular recording were filled with a 1/10 mixture of KC1 (1 M) and potassium citrate (1.6 M) and showed ohmic resistances in the range of 5-20 Mr2 (tip size 0.5-1.0/~m). The solutions used for microiontophoretic drug application were as follows. Substance P or the active octapeptide fragment, substance P(4-11) (Beckman), were dissolved in 20 mM acetic acid to give a final concentration of 1.9 mM, at pH 5.0-5.5; monosodium L-glutamate (0.5 M, pH 8.0); NaC1 (1.0 M, pH 5.0), acetic acid (20 mM; pH 5.0). All compounds, with the exception of L-glutamate, were applied by means of cationic currents. The drug application electrodes were always filled immediately before use, thus minimizing the degradation of the peptide 9. Prior to filling the multibarrelled pipettes were broken back to a total tip diameter of 3 4 #m. The ohmic resistance of each barrel was in the range of 30-60 MfL Electrodes were rarely used for periods of more than 5 h in any one experiment. Extra- and intracellular recordings, monitoring and display of single unit activity, as well as intracellular current injection for membrane resistance measurements, were performed using conventional techniques. Drug containing electrodes were discarded when either the applied current or the voltage recorded fluctuated. Current neutralization was used throughout and conventional current controls from acetic acid filled pipettes were occasionally performed. Dorsal horn cells were identified functionally, in a comparable manner to that described by Wall43,44, by studying their responsiveness to an array of different kinds of cutaneous stimulation, applied to their receptive fields in the ipsilateral hindlimb. Motoneurones were identified by antidromic invasion following stimulation of the ventral roots.
RESULTS The present results were obtained from 45 neurones recorded extracellularly and from 18 neurones recorded intracellularly, all from the 6th and 7th lumbar segments. All extracellular recordings were performed on dorsal horn neurones in laminae 4 and 5, which received sensory input from the ipsilateral hindlimb. The intracellular recordings were obtained from 8 dorsal horn cells and 10 motoneurones. Only neurones with stable resting potentials above --60 mV and spike amplitudes ranging between 70 and 90 mV were considered suitable for testing. In this study only neurones that were excited by microiontophoretically applied L-glutamate (GLU)are included. The data from the experiments with substance P and its active fragment, substance P(4-11) were pooled since no differences could be discerned between the actions of these two peptides.
(a) Extracellular recordings The majority of neurones were spontaneously active (range 2-25 spikes/sec) and/or could be synaptically driven by both gentle and heavy mechanical stimulation applied to the hair and skin folds of the hindlimb. Those cells referred to as lamina 5
276
Substance PSO
5 5eC
Fig. 1. Excitatory response of a spontaneously active lamina 5 type neurone to microiontophoretie application of substance P. In this and in subsequent figures the microiontophoretic applications are indicated by bars and currents are indexed and expressed in nA (10-a A).
type cells were characterized by their fairly large receptive fields, as well as by their dynamic responsiveness to increasing intensities of stimulation. In 10 out of 16 spontaneously active lamina 5 type neurones microiontophoretically applied substance P (5-150 hA), administered for 1-4 min, increased the discharge activity 20-250 ~ . The firing rate of these neurones began to increase after a latency of 5-40 sec and the excitation outlasted the application in a dose-dependent manner (range 0.5-5.0 rain). Of the remaining cells, 5 were unaffected and 1 was slightly depressed. Usually peptide applications were repeated 3-4 times employing identical phoretic currents and application periods of 10-180 see, spaced at intervals such that the firing rate in each case had returned to its pretest level. Substance P responses were found to be fairly dose-related and reproducibly elicited (in some neurones up to 10 times) without any apparent change ( < I0 ~ ) in the magnitude of the response. Figure 1 illustrates a typical example of an excitatory response elicited by substance P. A further 25 lamina 5 type neurones were recorded which were either quiescent or which displayed slow or erratic firing rates ( < 1 spike/see). These neurones were activated by regular, mieroiontophoretic applications of G L U (30-80 nA/5-20 sec) spaced at 1 min intervals in order to reveal any changes in subthreshold excitation elicited by substance P. In 7 of 12 neurones with a slow background firing ( < 1 spike/see) and 9 out of 13 quiescent neurones substance P (20-100 nA/30-300 sec) caused a dose-related enhancement of both the spontaneous firing rate and/or the response to G L U (20-150 ~). Figure 2 depicts a typical ratemeter record of such a neurone. The remainder of neurones in this group were not influenced by substance P. Four neurones were classified as lamina 4 type cells on the basis of their brief burst-like responses to light tactile stimulation (hair movement or gentle pressure) applied to their restricted peripheral receptive field. They were either quiescent or displayed only slow spontaneous discharge rates ( < 1 spike/see). Mieroiontophoretically administered substance P (60-100 nA/20-360 see) basically had the same effect as
277
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Fig. 2. Ratemeter record of a lamina 5 type neurone displayingexcitatory effects to the application of substance P. The cell is excited by regular pulses of L-glutamate(g, 10 hA). Substance P (50 and 100 nA) enhanced both the spontaneous and the GLU-evoked activity of this neurone in a dose-related manner. on lamina 5 type cells; increasing the spontaneous firing rate of these neurones and/or potentiating the G L U induced activation.
(b) Intracellular recording A relatively slowly increasing depolarization (5-20 mV, reached within 1-3 min) was the most consistent and reproducible effect elicited by microiontophoretically applied substance P (50-400 nA/1-4 min) on the resting membrane potential of both lamina 5 type neurones (6 out of 8) and motoneurones (8 out of 10). The remainder of neurones in this sample were not influenced by substance P. No apparent differences were observed in the time course of the depolarizing responses between these two cell groups. Following substance P application at a distance of 100-160 ffm from the recording site - - presumably the cell soma - - the cell membrane began to depolarize within 2-15 sec after the start of the drug application. Recovery was generally observed within 3-10 min after termination of the application. Figure 3 shows a recording from a lamina 5 type neurone. With the highest ejection current employed
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Fig. 3. DC record showingdepolarizing action of substance P upon the membrane potential of a lamina 5 type neurone. Application by the highest ejection current (100 nA) depolarized the cell beyond the firing threshold (arrow, action potentials are not shown).
278
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i~ I
i
~5 current injection (nA) -20 -10
otO
~20
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.
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Fig. 4. The lack of effect of substance P on neuronal membrane input resistance. The upper trace shows the depolarizingeffect of substance P (100 nA) on a lamina 5 type neurone (resting potential, 65-70 mV). The substance P application lasted 1 min. Potential deflections (damped) in the hyperpolarizing and depolarizing directions indicate the intracellular application of negative and positive current pulses which were used to construct the current/voltage plots shown in the graph below (amplitudes measured from oscilloscopetrace). The slope of the current/voltage plot during the substance P-induced depolarization (filled squares) was not significantly different from those obtained either before (dots) or after (open squares) the depolarization. Duration of the test pulse was 20 msec. The current/voltage curves show non-linearity. Substance P was applied 3 times and mean values of membrane deflections following identical current pulses are plotted. The electrode resistance (8.0 MI2) did not change during the measurement (testpulse, 2 msec, 5 hA, hyperpolarizing preceding the intracellular current injection).
t h e t h r e s h o l d m e m b r a n e p o t e n t i a l for spike i n i t i a t i o n was passed (arrow) a n d t h e n e u r o n e d i s p l a y e d a sustained b u r s t o f discharge activity. A s in the e x t r a c e l l u l a r recordings no significant desensitization to the d e p o l a r i z i n g effect was observed. D e p o l a r i z a t i o n s i n d u c e d b y s h o r t G L U a p p l i c a t i o n s (50-100 n A / 5 - 2 0 sec) were additive to the substance P i n d u c e d changes in m e m b r a n e p o t e n t i a l . Close e x a m i n a t i o n of the a n t i d r o m i c a l l y e v o k e d a c t i o n p o t e n t i a l s b y d v / d t analysis in the m o t o n e u r o n e s studied revealed t h a t neither the spike o v e r s h o o t n o r t h e a f t e r h y p e r p o l a r i z a t i o n o f the spike were d e t e c t a b l y influenced d u r i n g the course o f substance P - e v o k e d d e p o l a r i z a t i o n . F u r t h e r m o r e , no influence o f substance P could be
279 detected on either excitatory or inhibitory postsynaptic potentials evoked in these neurones. Input resistance measurements, employing either hyperpolarizing current pulses (2-20 nA, 20 msec duration, repetition rate 1 Hz) or pulses of variable current to obtain current/voltage curves (24 tests on 14 neurones, 7 lamina 5 type cells, 7 motoneurones), revealed no significant change in membrane resistance associated with depolarizations (5-15 mV) induced by substance P, even when excessive doses were applied (up to 400 nA). This is illustrated in Fig. 4 which shows the depolarizing effect of substance P on a lamina 5 type neurone and a current/voltage plot obtained before, during and after substance P-induced depolarization (mean values from 3 consecutive tests). Neurones which displayed marked ( > 40 ~ ) non-linearity (anomalous rectification) in their current/voltage curves were not included in this sample. DISCUSSION In the present study the predominant effect of microiontophoretically applied synthetic substance P and the active fragment substance P(4-11) on neurones in lamina 4 and 5 of the dorsal horn was a slow and relatively long-lasting increase of neuronal discharge activity. These effects are in good agreement with previous findings, including data from other brain regions (see Introduction). The most frequently encountered cells in lamina 5 of the dorsal horn of the spinal cord are activated by a wide range of peripheral cutaneous stimulation and are likely to play, along with neurones in Rexed's lamina 16, a key role in nociception 1,3,10,14,34,35,44,~o(and refs. therein). Most lamina 4 type cells are optimally excited by innocuous tactile stimuli and are thus not considered to play a major role in relaying information from mechanical nociceptive stimuli. However, these neurones are activated by pronounced heating or cooling of the limb 2,3,1° and therefore might be implicated in the transmission of information from thermosensitive receptors activated by painful thermal stimuli frequently used for testing the analgesic actions of drugs. Recently it has been suggested that substance P causes a selective activation of dorsal horn neurones receiving nociceptive thermal information 11,37. The localization of substance P in unmyelinated axons 16, most probably C-fibres, support such a contention, although recent histological evidence suggests that this peptide is also present in cell bodies intrinsic to the dorsal hornlL Thus, in addition to its role in primary afferent transmission substance P could be involved in the segmental control of neurones giving rise to ascending tracts. The intracellular recordings performed on lamina 5 type neurones and motoneurones showed that substance P causes a depolarization of the neuronal membrane with a time course of action that paralleles the increase in the firing rate observed with extracellular recording. The depolarization was not associated with a detectable change in membrane conductance at the recording site - - most probably the cell soma. Previous intracellular studies on the effect of substance P on motoneurones have yielded conflicting results. In the isolated frog spinal cord preparation, application of substance P onto the organ bath causes a depolarization of the cell membrane
280 associated with an increase in membrane conductance and a reversal potential close to zero (mV) membrane potential 29. In contrast, microiontophoretically applied substance P causes a depolarization of cat motoneurones with an accompanying decrease in membrane conductance and a reversal potential more negative than the resting potential ~1,36. From this latter finding it was proposed that substance P might cause a selective decrease of the membrane conductance for ions with a highly negative equilibrium potential, such as K ÷ and C1- ions. In this respect it bears a striking resemblance to the conductance changes associated with the (muscarinic) effect of acetylcholine on cortical ~3 and spinal neurones 4s. The present intracellular results were obtained from neurones displaying fairly linear current/voltage characteristics. Substance P was applied approximately 100-160/~m distant from the recording site and it is likely that changes in conductance elicited at such a site would be detectable by the currently employed technique of resistance measurement 47. Employing smaller tip separation ~1 and releasing substance P at quite close proximity to the soma could increase the likelihood that a membrane conductance change would be detected, assuming that the receptive sites for substance P are located near the soma. In view of the slow and quite prolonged effect of substance P on neurones it is obvious that some objections can be raised against it subserving a role as a fast neurotransmitter in primary afferents. In this connection G L U which has both a fast excitatory effect and an efficient uptake system 7 is a much better candidate for a primary sensory transmitter. However, it should be emphasized that a contributory factor to the slow onset of action of substance P could be the problem of low iontophoretic transfer number and of diffusion to the receptive site and its inactivation in the extracellular space. Histochemical electron microscopic studies have demonstrated that substance P immunoreactivity is present in large vesicles, located in axon terminals in the dorsal horn 38. These axons also contain smaller vesicles which were interpreted as storage sites for other neurotransmitters. In the light of this finding it might be speculated that substance P could be released, together with a fast-acting transmitter, with the result that the action of this transmitter, e.g. GLU, is potentiated. This kind of a modulatory effect may be important in producing more prolonged membrane depolarizations such that superimposed depolarizations evoked by fast acting transmitters become more effective in eliciting spike discharge 22. ACKNOWLEDGEMENTS I. F. Tulloch gratefully acknowledges the financial support of the Royal Society of Great Britain.
REFERENCES 1 Beck, P. W., Handwerker, H. O. and Zimmermann, M., Nervous outflow from the cat's foot during noxious radiant heat stimulation, Brain Research, 67 (1974) 373-386. 2 Brown, A. G. and Franz, D. N., Responses of spinocervical tract neurones to natural stimulation of identified cutaneous receptors, Exp. Brain Res., 7 (1969) 231-249.
281 3 Cervero, F., Iggo, A. and Moiony, V., Responses ofspinocervical tract neurones to noxious stimulation of the skin, J. Physiol. (Lond.), 267 (1977) 537-558. 4 Chang, M. M. and Leeman, S. E., Isolation o f a sialogogic peptide from bovine hypothalamic tissue and its characterization as Substance P, J. biol. Chem., 245 (1970) 4784-4790. 5 Chang, M. M., Leeman, S. E. and Niall, H. D., Amino acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 6 Christensen, B. N. and Perl, E. R., Spinal neurones, specifically excited by noxious or thermal stimuli: marginal zone of the dorsal horn, J. Neurophysiol., 33 (1970) 293-307. 7 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system, Rev. Physiol., 69 (1974) 97-188. 8 Davies, J. and Dray, A., Substance P in the substantia nigra, Brain Research, 107 (1976) 623-627. 9 Gozlan, H., Le Gal La Salle, G., Michelot, R. and Ben-Ari, Y., Rapid degeneration of Substance P and related peptides during microiontophoretic experiments, Neurosci. Lett., (1977) 26-33. 10 Handwerker, H. O., Iggo, A. and Zimmermann, M., Segmental and supraspinal actions on dorsal horn neurones responding to noxious and non-noxious skin stimuli, Pain, 1 (1975) 147-165. 11 Henry• J. L.• E•ects •f substance P •n functi•na••y identi•ed units in cat spina• c•rd• Brain Research• 114 (1976) 431-451. 12 Henry, J. L. and Ben-Ari, Y., Actions of the p-chlorophenyl derivative of GABA, Lioresal, on nociceptive and non-nociceptive units in the spinal cord of the cat, Brain Research, 117 (1976) 540--544. 13 Henry, J. L., Krfijevic, K. and Morris, M. E., Substance P and spinal neruones, Canad. J. PhysioL PharmacoL, 53 (1975) 423-432. 14 Hillman, P. and Wall, P. D., Inhibitory and excitatory factors influencing the receptive fields of lamina 5 spinal cord cells, Exp. Brain Res., 9 (1969) 284-306. 15 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Substance P: Localization in the central nervous system and in some primary sensory neurones, Science, 190 (1975) 889-890. 16 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurones, Brain Research, 100 (1975) 235-252. 17 H6kfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3081-3085. 18 Jessel, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Land.), 268 (1977) 549-551. 19 Konishi, S. and Otsuka, M., The effects of substance P and other peptides on spinal neurones of the frog, Brain Research, 65 (1974) 397-410. 20 Konishi, S. and Otsuka, M., Excitatory action of hypothalamic substance P on spinal motoneurons of new-born rats, Nature (Lond.), 252 (1974) 734-735. 21 Krfijevic, K., Effects of substance P on central neurons in cats. In U. S. von Euler and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 217-230. 22 Krfijevic, K. and Morris, M. E., An excitatory action of substance P on cuneate neurones, Canad. J. Physiol. PharmacoL, 52 (1974) 736-744. 23 Krfijevic, K., Pumain, R. and Renaud, L., The mechanism of excitation by acetylcholine in the cerebral cortex, J. Physiol. (Lond.), 215 (1971) 247-268. 24 Leeman, S. E. and Mroz, E. A., Minireview: Substance P, Life Sci., 15 (1974) 2033-2044. 25 Le Gal la Salle, G. and Ben-Ari, Y., Microiontophoretic effects of substance P on neurones of the medial amygdala and putamen of the rat, Brain Research, 135 (1977) 174-179. 26 Lembeck, F., Zur Frage der Zentralen 12bertragung afferenter Impulse. IlL Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Riickenmarks, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 107-213. 27 Levick, W. R., Another tungsten electrode, Med. Biol. Eng., 10 (1972) 510-515. 28 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of new-born rat, Nature (Lond.), 264 (1976) 83-84. 29 Otsuka, M. and Konishi, S., Electrophysiological and neurochemical evidence for Substance P as a transmitter of primary sensory neurones. In U. S. von Euler and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 207-214. 30 Otsuka, M., Konishi, S. and Takahashi, T., The presence o f a motoneurone-depolarizing peptide in bovine dorsal roots of spinal nerve, Proc. Jap. Acad., 48 (1972) 342-346.
282 31 Pernow, B., Studies on substance P. Purification, occurrence and biological actions, Actaphysiol. seand., 29, Suppl. 105 (1953) 1-90. 32 Phillis, J. W. and Limacher, J. J., Substance P excitation of cerebral cortex Betz cells, Brain Research, 69 (1974) 158-163. 33 Pickel, V. M., Reis, D. J. and Leeman, S. E., Ultrastructural localization of substance P in neurones of rat spinal cord, Brain Research, 122 (1977) 534-540. 34 Price, D. D. and Dubner, R., Neurones that subserve the sensory-discrimative aspects of pain, Pain, 3 (1977) 307-338. 35 Price, D. D. and Mayer, D. J., Neurophysiological characterization of the anterolateral quadrant neurones subserving pain in M. mulatta, Pain, 1 (1975) 59-72. 36 Puil, E. A. and Krfijevic, K., Neurotransmitter actions and interactions, Symposium on Interactions of Various Transmitter Systems, Proc. of the Xth CINP Congress., Pergamon Press, Oxford, 1977. 37 Randic, M. and Miletic, V., Effects of substance P in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 38 Sastry, B. R., Effect of acetylcholine, Substance P and stimulation of habenular nuclei on rat interpeduncular neuronal activity, Proe. Neurosci., (1977) 205. 39 Saito, K., Konishi, S. and Otsuka, M., Antagonism between Lioresal and Substance P in rat spinal cord, Brain Research, 97 (1975) 177-180. 40 Sessle, B. J., Hu, T. W., Lucier, G. E. and Henry, J. E., Differential effect of substance P on trigeminal brain stem units responding to tooth pulp or innocuous orefacial stimuli, Proc. Neurosci., (1977) 415. 41 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11. 42 Tregar, G. W., Niall, H. D., Potts, J. T. Jr., Leeman, S. E. and Chang, M. M., Synthesis of Substance P, Nature New Biol., 232 (1971) 87-89. 43 Wall, P. D., The laminar organization of the dorsal horn and effects of descending impulses, J. Physiol. (Lond.), 188 (1967)423-443. 44 Wall, P. D., Dorsal horn electrophysiology. In A. Iggo (Ed.), Handbook of Sensory Physiology, Vol. 2, Springer, Berlin, 1973, pp. 253-270. 45 Wright, D. M. and Roberts, M. H. T., Supersensitivity to a Substance P analog following dorsal root section, Life Sci., 22 (1978) 19-24. 46 Zetler, G., Biologically active peptides (Substance P). In A. Lajtha (Ed.), Handbook of Neuroehemistry, Vol. 4, Plenum Press, New York, 1970, pp. 135-148. 47 Zieglg~insberger, W. and Champagnat, J., L-Glutamate and glycine receptive sites on the somadendritic membrane of lumbar motoneurones of the cat. In R. W. Ryall and O. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier/NorthHolland Biomedical Press, Amsterdam, 1978. 48 Zieglg~insberger, W. and Reiter, C., A cholinergic mechanism in the spinal cord of cats, Neuropharmacology, 13 (1974) 519-527. 49 Zieglg~insberger, W. and Fry, J., Actions of opioids on single neurones. In A. Herz (Ed.), Developments in Opiate Research, Dekker, New York, 1978. 50 Zimmermann, M., Neurophysiology of nociception. In R. Porter (Ed.), Int. Rev. of Physiol., Neurophysiol., II. Vol. 10, University Park Press, Baltimore, Md., 1976, pp. 179-221.