Pain and neurotransmitters

1. To study physiological roles of substance P (SP), gamma-aminobutyric acid (GABA), enkephalins and other endogenous substances, we developed several kinds of isolated spinal cord preparations of newborn rats. 2. In these preparations, various slow responses of spinal neurons evoked by stimulation of primary afferent C fibers were depressed by a tachykinin antagonist, spantide. These results together with many other lines of evidence suggest that SP and neurokinin A serve as pain transmitters in a subpopulation of primary afferent C fibers. 3. Some C-fiber responses in various isolated spinal cord preparations were depressed by GABA, muscimol, and opioid peptides. In contrast, bicuculline (GABA antagonist) and naloxone (opioid antagonist) potentiated the "tail pinch potential," i.e., a nociceptive response of the ventral root evoked by pinch stimulation of the tail in isolated spinal cord-tail preparation of the newborn rat. The latter results support the hypothesis that some primary afferents activate inhibitory spinal interneurons which release GABA and enkephalins as transmitters to modulate pain inputs.     

Substance P release from spinal cord slices by capsaicin

The release of substance P (SP) from slices of hypothalamus, substantia nigra and spinal cord of the rat was studied. In contrast to 47 mM potassium, capsaicin induced SP release from spinal cord slices only. The SP release was not reduced by 10^?6 M tetrodotoxin but was abolished by a Ca²⁺ free medium containing 3 mM ethylene-glycol-bis- (β-aminoethyl-ether) N,N-tetraacetic acid (EGTA). Capsaicin induced SP release was dose-dependent and exhibited tachyphylaxis. The ability of capsaicin to release SP supports the hypothesis that SP is involved in pain transmission and in neurogenic plasma extravasation.     

Tachykinin Systems in the Spinal Cord and Basal Ganglia: Influence of Neonatal Capsaicin Treatment or Dopaminergic Intervention on Levels of Peptides, Substance P–Encoding mRNAs, and Substance P Receptor mRNA

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Distribution of Calcitonin Gene-Related Peptide-, Substance P-, Cholecystokinin-, Met5-Enkephalin-, and Dynorphin A (1–8)-Like Materials in the Spinal Cord and Dorsal Root Ganglia of Adult Rats: Effects of Dorsal Rhizotomy and Neonatal Capsaicin

Biochemical mapping of five different peptide-like materials--calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1-8) (DYN)--was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c.). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.     

Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.     

The central consequences of the application of capsaicin to one peripheral nerve in adult rat

This paper reviews the central consequences of local application of capsaicin to one nerve in adult animals. 1) Marked chemical changes occur in the central terminals of C fibres. These include depletion of the enzyme FRAP and the peptides SP, CCK, somatostatin, CGRP and an increase of VIP. Maximal depletions occur if the nerve is soaked with capsaicin solutions with a concentration higher than 3 mM. The depletion begins by 7 days and is complete by 11. Recovery begins at about 110 days and is largely complete by 200. Our studies have concentrated on the effects of 40 mM capsaicin examined 14 days after the application. 2) Capsaicin treatment of a peripheral nerve decreased the ability of C fibres in that nerve to excite or to inhibit spinal cord cells. It produces a marked expansion of receptive fields of some cells in the dorsal horn which respond to A fibre stimulation. It is proposed that this change is not due to anatomical changes but to disinhibition. A further example of receptive field expansion is seen after treatment of the mouse infraorbital nerve which defocuses the normally precise projection of individual whiskers onto single cells in the barrel field of the somatosensory cortex. 3) Behavioural consequences follow the treatment of one adult nerve with capsaicin. In the area subserved by the treated nerve, there is a raised threshold to response to chemical and thermal stimuli, no change in the response to mechanical stimuli and an increase of autotomy following nerve section. 4) The aim of the experiments was to determine the role of C fibres in producing the changes seen in spinal cord following peripheral nerve section. Capsaicin treatment of nerve imitates the central effect of complete nerve section in certain important ways. Both result in a marked expansion of the receptive field of some cells. The effect is produced by a change of chemical transport. The results show that C fibres influence the connection of A fibres onto spinal cord cells.     

Tachykinin release from rat spinal cord in vitro and in vivo in response to various stimuli

The aim of the present study was to establish an experimental model, previously used in cat, for studying tachykinin release from the rat spinal cord in vivo and to compare the results with those obtained in vitro. Stimulation with pulses of 40 mM potassium or 10 microM capsaicin in the spinal cord superfusion fluid increased the release of substance P (SP)- and neurokinin A (NKA)-like immunoreactivity (LI) both in vivo and in vitro. The amounts of SP-LI and NKA-LI released by potassium in vitro were 1.02 +/- 0.12 and 1.17 +/- 0.22 fmol/mg tissue, respectively. Also the ratio between the amounts released by two consecutive potassium stimulations were similar for SP-LI and NKA-LI. Reversed-phase high performance liquid chromatography of the NKA-LI released in vitro by potassium or capsaicin revealed a major immunoreactive component coeluting with synthetic NKA. Despite the use of highly sensitive radioimmunoassays, basal release of SP-LI and NKA-LI was found only in 9 of 31 in vivo experiments. In these, peripheral electrical stimulation of the sciatic nerves (50 Hz, 50 V and 0.05 ms or 10 Hz, 10 V and 5 ms) induced an increase of the SP-LI and NKA-LI levels in the superfusates. This increase persisted for more than 40 min after a 2 min stimulation. In most experiments, however, no SP-LI or NKA-LI could be detected in the superfusates, neither at basal conditions nor following electrical nerve stimulation. Similarly, no release of SP-LI could be detected in response to various noxious mechanical, thermal or chemical stimuli applied to the skin. The present results demonstrate that the superfused rat spinal cord may be used to study in vivo release of tachykinins in response to intense chemical stimulation of the entire spinal cord. However, the method seems to be less suitable for studies of tachykinin release in response to electrical activation engaging only a few spinal segments or in response to natural noxious stimuli. The results obtained in vitro suggest that SP and NKA are released in equimolar amounts from the spinal cord upon stimulation with potassium.     

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP-1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP-1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP-1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co-localized with pain-related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium-dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2-induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.     

P物质对大鼠骶髓后连合核神经元甘氨酸激活的氯电流的调控

采用制霉菌素穿孔膜片箍技术,研究了Ｐ物质对急性分离的大鼠骶髓后的核神经元士的宁敏感性甘氨酸反应的调控作用。在箍制电压为－４０ｍＶ时,ＳＰ时１ｍｍｏｌ／Ｌ－１μｍｏｌ／Ｌ之间呈浓度依赖性地增强３０μｍｏｌ／Ｌ甘氨酸激活的氯电流。ＳＰ既不改变ＩＧｌｙ的翻转电位,也不是影响Ｇｌｙ与其受体的亲和力。Ｓｐａｎｔｉｄｅ和选择性Ｎ中受体拮抗剂,Ｌ－６６８,１６９,可阻断ＳＰ的增强作用,而选择性ＮＫ２受体拮抗剂,     

鞘内注射神经激肽-1受体激动剂Sar-SP增强大鼠脊髓一氧化氮合酶表达和一氧化氮生成

探讨P物质(substance P,SP)对脊髓一氧化氮合酶(nitric oxide synthase,NOS)表达和一氧化氮(nitric oxide,NO)生成的影响。实验用热甩尾法测定大鼠痛阈的变化,分别应用NADPH-d组织化学法和硝酸还原法测定大鼠脊髓内NOS表达和NO生成的变化。结果显示,鞘内注射神经激肽-1受体(neurokinin-1 receptor,NK-1)激动剂[Sar^9,Met(O2)^11]-substance P(Sar-SP)可使大鼠痛阈降低,脊髓后角浅层和中央管周围灰质内NOS表达增强,脊髓腰膨大部位NO生成增多;预先鞘内注射非选择性NK-1受体拮抗剂[D—Arg^1,D-Trp^7,9,Leu^11]-substance P(spantide)可抑制上述变化。结果表明,SP可促进脊髓内NOS表达和NO生成。     

Effects of substance P analogues on spinal dorsal horn neurons

The effects of iontophoretically applied (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP on the spontaneous and evoked activity of functionally identified cat spinal dorsal horn neurons have been investigated in vivo by means of extracellular single unit recording technique. In addition, the rat spinal cord slice preparation has been used to study the actions of (D-Pro2, D-Trp7,9)-SP and (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP on the resting membrane potential of dorsal horn neurons and also on their responses to dorsal root stimulation and exogenous SP application. We have observed that both (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP produced an excitation of about 15% of all neurons tested and had a weak antagonistic effect against SP in the cat spinal cord. (D-Pro2, D-Trp7,9)-SP suppressed the SP-induced excitation in 63% of examined cells. In addition, depression of the glutamate-induced excitation and spontaneous activity was evident in 10% and 19% of the cat dorsal horn neurons tested, respectively. In the spinal cord slice preparation (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP proved to be a more potent antagonist of the SP-induced depolarization and the dorsal root-elicited slow depolarization, if compared with (D-Pro2, D-Trp7,9)-SP.     

Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P

Calcitonin gene-related peptide (CGRP) is cleaved by an endopeptidase, also known to hydrolyze substance P (SP). The enzyme which was isolated from human cerebrospinal fluid, converted rCGRP into two products, clearly separable on HPLC. Amino acid analysis showed cleavage to occur at Leu16-Ser17. The carboxy-terminal fragment, rCGRP-(17-37), was weakly active in inhibiting 125I-rCGRP binding to a rat medulla oblongata membrane preparation, but it showed no binding to spinal cord membranes. The N-terminal fragment, rCGRP-(1-16), had very low or no affinity. Autoradiography with 125I-rCGRP showed distinct labelling of rat dorsal spinal cord, while there was no consistent pattern with 125I-rCGRP-(1-16). In the isolated guinea pig ileum preparation, the two fragments showed no CGRP-like activity. The ability of CGRP to interfere with SP degradation is offered as the explanation why CGRP has been reported to potentiate several biologic actions of SP.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.     

Effect of neuroactive peptides on labeled 5-hydroxytryptamine release from rat spinal cord

The effects of neuroactive peptides on the release of 5-HT were studied. The 5-HT released from the spinal cord was significantly increased by somatostatin, substance P and peripheral pain stimulation (tail pinch), but not affected by neurotensin, beta-endorphin and met-enkephalin. The somatostatin-evoked 5-HT release was inhibited by baclofen and met-enkephalin in vivo but not in vitro. The substance P-evoked 5-HT release was strongly inhibited by baclofen, and slightly potentiated by met-enkephalin in vivo but not in vitro. The tail pinch-induced 5-HT release was inhibited by met-enkephalin and baclofen, but potentiated by naloxone. These findings provide further evidence on the important role of neuropeptides and suggest that the descending serotonergic neurones are modulated by neuropeptide interneurones in the spinal cord.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Different SNP combinations in the GCH1 gene and use of labor analgesia

Recent research has proposed a pathway in which sensory neurons expressing the capsaicin activated ion channel TRPV1 are required for histamine-induced itch and subsequent scratching behavior. We examined histamine-induced itch in the African naked mole-rat (Heterocephalus glaber) and found that although naked mole-rats display innate scratching behavior, histamine was unable to evoke increased scratching as is observed in most mouse strains. Using calcium imaging, we examined the histamine sensitivity of naked mole-rat dorsal root ganglia (DRG) neurons and identified a population of small diameter neurons activated by histamine, the majority of which are also capsaicin-sensitive. This suggested that naked mole-rat sensory neurons are activated by histamine, but that spinal dorsal horn processing of sensory information is not the same as in other rodents. We have previously shown that naked mole-rats naturally lack substance P (SP) in cutaneous C-fibers, but that the neurokinin-1 receptor is expressed in the superficial spinal cord. This led us to investigate if SP deficiency plays a role in the lack of histamine-induced scratching in this species. After intrathecal administration of SP into the spinal cord we observed robust scratching behavior in response to histamine injection. Our data therefore support a model in which TRPV1-expressing sensory neurons are important for histamine-induced itch. In addition, we demonstrate a requirement for active, SP-induced post-synaptic drive to enable histamine sensitive afferents to drive itch-related behavior in the naked mole-rat. These results illustrate that it is altered dorsal horn connectivity of nociceptors that underlies the lack of itch and pain-related behavior in the naked mole-rat.     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.     

Myocardial ischemia induces the release of substance P from cardiac afferent neurons in rat thoracic spinal cord

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.