Masanori Otsuka

Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons

The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [3H]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75--80% depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both the peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred 1 month after nerve section. Dorsal rhizotomy produced an 80% depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 40%. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.

Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section

Chemical properties of substance P in the spinal cord and dorsal roots of the cat were examined by gel chromatography and paper electrophoresis. The results suggest that the substance is identical with the undecapeptide, hypothalamic substance P, recently isolated from bovine hypothalamus. Distribution of substance P in the cat spinal cord (L5-S1) was investigated. Substance P was highly concentrated in the dorsal horn, where the highest level was found in the dorsal part. After the unilateral ligation and/or section of the dorsal roots, the level of substance P in the dorsal horn, particularly in its dorsal part, was markedly lowered. In the ligated and sectioned dorsal root, substance P was highly accumulated on the ganglion side, whereas its level was lowered on the central side of the ligature. These results suggest that hypothalamic substance P synthesized in the spinal ganglia is transported in the dorsal root toward their intraspinal axon terminals, where the substance is concentrated and serves as a neurotransmitter.

Capsaicin-evoked release of substance P from primary sensory neurons

The presence of the peptide substance P in small unmyelinated primary sensory neurons 5 appears to be related to the transmission of nociceptive impulses at the level of the spinal cord. Release of substance P has been demonstrated from the rat spinal cord, in vitro, in response to electrical stimulation of the dorsal roots 1~. Dorsal horn neurons that respond to noxious peripheral stimuli are excited by the iontophoretic application of substance p3,ts and the peptide also produces a depolarization of ventral roots in the isolated rat spinal cord preparation 12,t6. Substance P-containing sensory neurons may then represent one target for the pharmacological modification of pain transmission within the spinal cord. The analgesic properties of the opiates, in particular, may be mediated by the inhibition of substance P release from primary sensory neuronsg, ~3. Although it has been known for many years that repeated administration of the homovanillylamide derivative, capsaicin, renders animals insensitive to painful chemogenic stimuli 8, the mechanism of action of this compound has remained obscure. Recently chronic capsaicin treatment has been shown to produce a dramatic and specific depletion of substance P within primary sensory neurons in the rat dorsal horn 10 suggesting a functional association between the effects of capsaicin and substance P. Moreover, the acute effects of capsaicin administration in vivo are characterized by intense pain which might be consistent with a capsaicin-evoked stimulation of substance P release from the central terminals of primary sensory neurons. To investigate this possibility we have examined the effects of capsaicin in the isolated rat spinal cord preparation 14 and on the release of substance P from spinal cord tissue. Spinal cords were removed from 0-4-day-old Wistar rats according to the method of Otsuka and Konishi is. Hemisected cords were superfused with artificial cerebrospinal fluid (CSF) at 27 °C. The composition of artificial CSF was as follows (mM): NaCI 138.6 ; KCI 3.35 ; CaC12 1.26 ; MgClz 1.16 ; NaHCO3 21.0 ; NaH2PO4 0.58 ; glucose 10 ; gassed with 95 ~o 02--5 ~o CO2. Potential changes generated in mote-

The effects of substance P and other peptides on spinal neurons of the frog.

The effects of substance P and other peptides were studied on the neurons of isolated spinal cord of the frog. Substance P and certain related peptides, which have a common C-terminal sequence, -Phe-X-Gly-Leu-Met-NH2 (where X=Ile, Tyr or Phe), exerted a remarkably strong excitant action on spinal motoneurons. On a molar basis, substance P was about 200 times, physalaemin 1500 times, and eledoisin 2000 times more active thanl-glutamate in depolarizing the spinal motoneuron in 0.4 mM Ca-Ringers solution. Since the depolarizing action of substance P and related peptides persisted after the synaptic transmission was blocked by Ca-deficient (0–0.2 mM) Ringers solution or by tetrodotoxin (10−8−10−7 g/ml), it was concluded that these peptides have a direct action on the motoneurons to induce the depolarization. Another group of peptides, bradykinin, angiotensins I and II, caused the depolarization of the spinal motoneurons by a trans-synaptic mechanism. These peptides probably activated the interneurons and thus caused the release of the excitatory transmitter from the nerve terminals synapsing with motoneurons. The possibility that substance P may be an excitatory transmitter of primary sensory neurons was discussed.

Substance P as an excitatory transmitter of primary afferent neurons in guinea-pig sympathetic ganglia

Electrophysiological and neurochemical experiments were carried out to examine a possible transmitter role substance P in the prevertebral ganglia of the guinea-pig. When potentials were recorded intracellularly from neurons of the isolated ganglia, stimulation of the pre- or postganglionic nerves elicited a non-cholinergic slow excitatory postsynaptic potential (EPSP). This synaptic potential was compared with the effects of substance P. Brief application of substance P caused a depolarization of the ganglion cells with a similar time course to that of the non-cholinergic slow EPSP. Changes in membrane resistance during the substance P-induced depolarization resembled those associated with the non-cholinergic slow EPSP. During the substance P-induced depolarization the non-cholinergic slow EPSP was markedly depressed. Attempts were made to determine the origin of the fibers eliciting the non-cholinergic slow EPSP. In the inferior mesenteric ganglia isolated together with preganglionic nerves that retained intact connections with spinal nerve roots, dorsal root stimulation evoked a non-cholinergic slow EPSP but not a cholinergic fast EPSP in the ganglion cells, whereas ventral root stimulation caused only cholinergic fast EPSPs. Following the prolonged treatment with capsaicin, the non-cholinergic slow EPSP was greatly depressed or abolished. Radioimmunoassay revealed that after ligation or section of pre- or postganglionic nerves an accumulation of substance P occurred in the proximal stumps of the interrupted nerves. Stimulation with high potassium medium evoked a release of immunoreactive substance P from the prevertebral ganglia and the release was calcium-dependent. The present findings suggests that axon collaterals of certain visceral primary efferents form synapses with principal cells in the prevertebral ganglia and release substance P as a transmitter for the non-cholinergic slow EPSP.

Antagonism between Lioresal and substance P in rat spinal cord

There is considerable evidence implicating the undecapeptide substance P (see ref. 1) in the primary afferent transmission in the spinal cord. Substance P is present in the dorsal root of bovine and feline spinal nerves in a concentration 9-27 times higher than that in the ventral root6, 9,10. Substance P is highly concentrated in the dorsal part of dorsal horn of cat spinal cord, and its level in this region is greatly reduced after the section of dorsal roots10. Synthetic substance P exerts a strong depolarizing action on the spinal motoneurons of the frog and the rat3,4, 6. Lioresal (Ciba-Geigy, fl-(4-chlorophenyl)-~-aminobutyric acid) has recently been reported to suppress the spinal monosynaptic and polysynaptic reflexes without affecting the electrical properties of spinal motoneurons in the cat 7,8. If substance P or a related peptide is a transmitter of primary afferent neurons, a possible mechanism of the action of Lioresal is its antagonism toward the peptide transmitter inducing the depolarization of spinal neurons. This possibility was examined in the present study. The spinal cord of 0-7-day-old Wistar rats was isolated, hemisected sagittally and placed in a bath perfused with Krebs solution (27 ~: 1 °C). The solution was equilibrated with a mixture of 95 ~, 02 and 5 ~o COz. Potentials were recorded from the ventral or dorsal root (L3-L5) with a suction electrode of the type described in a previous paperL The recording electrode was connected through a preamplifier to an oscilloscope or a pen-recorder. The bath was grounded through a reference calomel electrode. When a dorsal root was stimulated by a single shock, typical monoand polysynaptic reflexes were recorded from the corresponding ventral root (Fig. I A~). The application of synthetic substance P or L-glutamic acid produced a depolarization of motoneurons, this being recorded as a positive potential at the electrode recording from the cut end of the ventral root (Fig. IB and C). After the bath was flushed with normal Krebs solution, the potential returned to the original level. From the comparison of the dose-response curves of substance P and e-glutamate at the level of 0.5

Substance P — the first peptide neurotransmitter?

Abstract This article reviews the evidence for substance P (SP) as a neurotransmitter in primary afferent neurons in the spinal cord and sympathetic ganglia. There is strong evidence indicating that SP released from peripheral branches of visceral primary afferent neurons produces non-cholinergic slow excitatory postsynaptic potentials (EPSPs) in the inferior mesenteric ganglion cells of the guinea-pig. It is also probable that SP released from central terminals of certain primary afferent neurons produces slow EPSPs in the dorsal-horn neurons which are involved in certain types of slow reflex in the neonatal rat spinal cord.

Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal cord‐tail preparation of the newborn rat.

1. The pharmacological profile of Spantide, [D‐Arg1, D‐Trp7,9, Leu11] substance P, as a substance P (SP) antagonist was examined in isolated spinal cords of newborn rats. Potential changes were recorded extracellularly from a lumbar ventral root (L1‐L5). Application of SP to the perfusion bath with a brief pressure pulse of 0.05‐0.8 s duration produced a dose‐dependent depolarization of the ventral root. Spantide in concentrations of 2‐16 microM depressed the depolarizing responses of the ventral root to SP in a concentration‐dependent manner. The log dose‐response curve of SP was shifted to the right in the presence of 16 microM‐Spantide by log 5. The responses to neurokinin A (NKA) and bombesin were similarly depressed by 16 microM‐Spantide whereas the responses to noradrenaline, gamma‐aminobutyric acid (GABA), neurotensin and thyrotrophin‐releasing hormone were not affected by 16 microM‐Spantide. 2. In an isolated spinal cord‐tail preparation of the newborn rat, brief pulse injection of capsaicin into the perfusion solution of the tail induced a depolarizing response in a lumbar ventral root (L3‐L5). This response probably represents a nociceptive C fibre reflex. 3. The capsaicin‐induced nociceptive reflex was markedly depressed by 16 microM‐Spantide and the reflex recovered its original amplitude and shape 30‐60 min after removal of Spantide. 4. The capsaicin‐induced nociceptive reflex was depressed by morphine (2 microM) and dynorphin (1‐13) (0.2 microM), and these effects were reversed by 1 microM‐naloxone. 5. In an isolated spinal cord preparation of the newborn rat, stimulation of a dorsal root with single or double shocks induced depolarizing responses of slow time course in both ipsilateral and contralateral ventral roots of the same segment. These slow depolarizing responses were also depressed by 16 microM‐Spantide. In contrast the monosynaptic reflex was not affected by 16 microM‐Spantide. 6. The present results suggest that SP and NKA are involved as neurotransmitters in the capsaicin‐induced nociceptive reflex in the isolated spinal cord‐tail preparation of the newborn rat.

The role of substance P as a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord.

1 In order to reveal the spinal reflexes involving the transmitter action of substance P (SP), the effects of capsaicin and an SP antagonist on the isolated spinal cord of the neonatal rat were studied. 2 When a single shock stimulus was given to a dorsal root (L3–L5) or a sciatic nerve, depolarizing responses of various time courses were recorded extracellularly from both ipsi‐ and contra‐lateral ventral roots of the corresponding segments. The reflex response recorded from the contralateral ventral root consisted of fast and slow components, which will be referred to as contralateral fast and slow ventral root potentials (v.r.ps). The latter contralateral slow v.r.p. had a time‐to‐peak of 2–5 s and lasted 10–30 s. 3 The threshold for the contralateral slow v.r.p. was about two times higher than that for the monosynaptic reflex, and it coincided with the threshold for activating the slow‐conducting afferent fibres. 4 The contralateral slow v.r.p. was abolished after the spinal cord was treated with capsaicin (1 μm for 30 min) in vitro. The contralateral slow v.r.p. was absent in the spinal cord derived from 4 day‐old rats that had received capsaicin (50 mg kg−1, s.c.) on the 2nd day of life. The contralateral fast v.r.p. and other reflexes of fast time courses remained unaltered after treatment with capsaicin in vitro or in vivo. 5 Administration of an SP antagonist, [d‐Arg1, d‐Pro2, d‐Trp7,9 Leu11]‐SP in concentrations of 5–16 μm depressed the contralateral slow v.r.p., but did not affect the monosynaptic reflex, the dorsal root potential and the contralateral fast v.r.p. [d‐Arg1, d‐Pro2, d‐Trp7,9, Leu11]‐SP (5 μm) markedly depressed the SP‐induced depolarizing response recorded from the ventral root whereas the responses to noradrenaline, 5‐hydroxytryptamine, neurotensin and thyrotrophin releasing hormone (TRH) were unaffected by the SP antagonist. The response of the ventral root to acetylcholine was slightly depressed by the antagonist. The SP antagonist at 5–10 μm did not exert any agonist action on the motoneurones. 6 The present results in conjunction with those of previous studies support the hypothesis that SP released from certain primary afferent fibres acts as a neurotransmitter, producing in dorsal horn neurones slow excitatory postsynaptic potentials which lead to the generation of the contralateral slow v.r.p.

Actions of vasopressin, gastrin releasing peptide and other peptides on neurons of newborn rat spinal cord in vitro

The actions of various peptides were studied using isolated spinal cord preparation of newborn rat. Vasopressin, substance P, thyrotropin releasing hormone, bombesin, gastrin releasing peptide, oxytocin, neurotensin, cholecystokinin-octapeptide and angiotensin II produced marked depolarizing responses of motoneurons with threshold concentrations of 5 X 10(-10)--8 X 10(-9) M. After the elimination of transsynaptic action by tetrodotoxin, the actions of these peptides were depressed to various extents, the former 5 peptides producing relatively large responses. Somatostatin and enkephalin depressed the dorsal root potential and produced slight hyperpolarization of dorsal root fibers. It is suggested that many of these peptides play important roles in synaptic transmission in mammalian spinal cord.