Substance P immunoreactive neurons following neonatal administration of capsaicin

Summary Neonatal administration of capsaicin on the days 2, 10 or 20 leads to a long-lasting loss of substance P immunoreactive material in fibers of primary sensory neurons in the spinal cord and medulla oblongata. The degree of depletion examined 6 months after treatment was related to the day of injection. Injections on the second day produced dramatic losses of substance P in fibers of the substantia gelatinosa and the marginal layer of the spinal cord and the spinal nucleus of the trigeminal nerve, although these losses were never complete. The observed depletion of substance P immunoreactive material was homogenous throughout the superficial layers of the dorsal horn and the spinal nucleus of the trigeminal nerve. No changes were observed for the immunoreactivity of Leu-enkephalin in the substantia gelatinosa and the marginal layer of the spinal cord in consecutive sections from the same treated animals. In the medulla oblongata a reduction of substance P immunofluorescent fibers was found in the nucleus tractus solitarii and the spinal nucleus of the trigeminal nerve. Other areas of the central nervous system with a rich innervation of substance P immunoreactive fibers were not affected by capsaicin treatment.     

Analgesic doses of morphine do not reduce noxious stimulus-evoked release of immunoreactive neurokinins in the dorsal horn of the spinal cat.

1. Antibody microprobes were used to detect immunoreactive neurokinin A release in the dorsal spinal cord of barbiturate-anaesthetized spinal cats. 2. Noxious mechanical stimulation of the ipsilateral hind paw and electrical stimulation (suprathreshold for unmyelinated primary afferent fibres) of the ipsilateral tibial nerve evoked immunoreactive neurokinin A release. 3. Systemic morphine, 5 mg kg-1, i.v., did not block immunoreactive neurokinin A release in response to these stimuli. 4. Subsequent naloxone administration, 0.5 mg kg-1, i.v., did not alter this stimulus-evoked release. 5. Basal levels of immunoreactive neurokinin A were unaltered by morphine or naloxone. 6. These results suggest that the analgesic effects of morphine at the spinal cord level are not brought about by activation of presynaptic opiate receptors on neurokinin A containing afferent terminals.     

Capsaicin-like effect of (6)-shogaol on substance P-containing primary afferents of rats: a possible mechanism of its analgesic action.

The effects of (6)-shogaol, a pungent component of dried ginger with a capsaicin-like chemical structure, on the release of immunoreactive substance P from the spinal dorsal horn were examined by in vitro superfusion of the dorsal-half slices of the spinal cord of the rat. (6)-Shogaol (30 microM to 1 mM) increased dose-dependently the release of immunoreactive substance P. The maximum effect of (6)-shogaol was observed at a concentration of 100 microM and less than a half of the effect of 10 microM capsaicin. The effect of (6)-shogaol (100 microM) was attenuated in slices from rats with dorsal rhizotomy and abolished by elimination of calcium ions from the perfusion medium. Pretreatment with (6)-shogaol in vitro inhibited the capsaicin-evoked release of immunoreactive substance P. On the other hand, systemic administration of (6)-shogaol (160 mg/kg) produced antinociception in rats, with a peak effect between 15 and 30 min and a smaller dose of 80 mg/kg was without effect. Treatment of rats with (6)-shogaol, at a dose of 160 mg/kg but not at 80 mg/kg, for 20 min significantly decreased release of immunoreactive substance P, evoked by capsaicin (10 microM), from the slices of cord. These data suggest that (6)-shogaol shares the sites of action with capsaicin, on the terminals of substance P-containing primary afferents, to release of the neuropeptide and inhibit the release of substance P, by subsequent stimulation of the primary afferents. The latter action of (6)-shogaol might be relevant to its analgesic effect.     

坐骨神经慢性压迫损伤性疼痛与脊髓背角P物质关系研究

目的观察大鼠坐骨神经慢性压迫性损伤（CCI）后脊髓背角P物质表达的变化,探讨P物质在疼痛发生机制中的作用。方法SD雄性大鼠60只,随机分为：A组：CCI组（30只）;B组：对照组（30只）。术前及术后3、7、14、28 d分别测定大鼠热痛阈值、机械痛阈值和行为学评分。术后3、7、14、28d每组取4只,麻醉后用4%多聚甲醛灌注固定,取L4-6段脊髓,以备免疫组化,测定SP的变化。结果所有CCI动物从术后第3天起,出现明显的疼痛行为学改变和热痛阈值、机械痛阈值的降低,与对照组比较差异有统计学意义（P〈0.05或P〈0.01）。免疫组织化学结果表明,A组术后术侧明显高于B组（P〈0.05或P〈0.01）;A组术侧明显高于健侧（P〈0.05或P〈0.01）;而B组仅在第4天术侧高于健侧（P〈0.05）。结论慢性坐骨神经损伤后,脊髓背角SP的表达增加,而且表达增加与CCI大鼠的痛觉过敏、行为变化在时相上基本一致,说明CCI大鼠痛觉过敏与脊髓背角SP的表达增加有关。     

Noradrenaline synthesis and utilization: Control by nerve impulse flow under normal conditions and after treatment with alpha-adrenoreceptor blocking agents

The changes in the synthesis and utilization or noradrenaline cranial and caudal to an acute section of the rat spinal cord have been used to investigate the importance of nerve impulses for these processes. 1. Cranial to a lesion of the spinal cord, the alpha-methyltyrosine-induced disappearance of noradrenaline was accelerated by the alpha-adrenoreceptor blocking agents yohimbine (10 mg/kg), piperoxan (60 mg/kg) and tolazoline (50 mg/kg). In the absence of nerve impulses caudal to a lesion of the spinal cord, this disappearance was decelerated as compared to that cranial to the lesion and it was not influenced by the three alpha-adrenoreceptor blocking agents. 2. The nialamide-induced accumulation of normetanephrine in the whole brain was increased by phenoxybenzamine (20 mg/kg) and yohimbine whereas it was decreased by the alpha-adrenoreceptor stimulating agent clonidine (0.1 mg/kg). The effect of clonidine was completely antagonized by yohimbine, but not by phenoxybenzamine, giving further evidence for the view that clonidine and yohimbine have a stronger effect than phenoxybenzamine on the alpha-adrenoreceptors regulating the release of noradrenaline induced by nerve impluses. 3. The accumulation of Dopa after decarboxylase inhibition cranial to a lesion of the spinal cord was accelerated by yohimbine, piperoxan and tolazoline, but not significantly affected by phenoxybenzamine and haloperidol (10 mg/kg). In the absence of nerve impulses caudal to a lesion of the spinal cord, the popa accumulation was decelerated as compared to that cranial to the lesion and it was not influenced by the former three alpha--adrenoreceptor blocking agents as well as by clonidine. 4. The results show that the synthesis and the utilization noradrenaline normally, as well as the accelerations of these processes by alpha-adrenoreceptor blocking agents, are dependent on nerve impulses. The stimulation of the synthesis and utilization of noradrenaline by nerve impulses might by influenced via the activity of teh alpha-adrenoreceptors located either on the nerve terminals or on the cell bodies or on both parts of the noradrenergic neurones. In the absence of nerve impulses, a receptor-mediated feedback mechanism similar to that described for the synthesis of dopamine does not appear to regulate the synthesis of noradrenaline.     

Effect of acute and chronic treatment with practolol on cardiovascular responses in the pithed rat

Chronic administration of practolol to the pithed rat produced a reduction in the pressor responses to electrical stimulation of the spinal cord and potentiation of pressor responses to high doses of (—)-noradrenaline compared to control animals. Acute administration of practolol caused an increase in the pressor responses to both electrical stimulation and high doses of noradrenaline. Heart rate responses to both forms of stimulation were less than control values after both acute and chronic dosage with practolol. It is possible that practolol reduces the release of noradrenaline at the sympathetic nerve ending after chronic administration.     

Capsaicin and nociception in the rat and mouse

Summary Newborn or adult rats and mice were treated with capsaicin. The effect of systemic or intrathecal treatment on thermonociception, chemonociception, content and release of immunoreactive substance P (I-SP) was investigated.Treatment of two day old rats caused a small, but life-long elevation of the hot plate or tail withdrawal latency. Treatment of adult rats led to a large increase in the reaction time on the hot plate for 4–10 days but the tail withdrawal latency was only slightly elevated for not more than 1–2 days.Mice treated on the 2nd day of life had normal reaction times on the hot plate and a small and inconsistent prolongation of the tail withdrawal latency. In contrast, mice treated on day 7, 10 or as adults had greatly prolonged latencies in both tests for at least 3 months. The changes in latencies were not affected by naloxone or methysergide.Responses to noxious chemical stimuli were moderately inhibited in mice treated on the 2nd day of life, but almost abolished in mice treated on day 7, 10 or as adults.Neonatal capsaicin treatment of rats resulted in a depletion of I-SP in spinal cord and sciatic nerve for 20 months. Capsaicin-evoked release of I-SP from rat spinal cord was reduced by 93% after neonatal treatment, but only by 69% 2 weeks after adult treatment.Treatment of mice on day 2 caused a similar decrease of the I-SP content in spinal cord and of the capsaicin-evoked I-SP release (88%) as treatment on day 4 or 7 although behavioural changes were different. After treatment of adult mice release of I-SP was reduced by 93%.Capsaicin administered intrathecally to rats or mice depleted I-SP in the spinal cord but not in the sciatic nerve. The animals were almost insensitive to noxious heat (tail withdrawal test) and to local application of mustard oil or capsaicin to the hindpaw. Chemosensitivity of the eye, however, remained unchanged.The experiments indicate that systemic or intrathecal capsaicin treatment of rats or mice affects thermo- and chemonociception but species differences were found. It appears, furthermore, that changes in substance P alone cannot explain all the observed behavioral effects after capsaicin treatment.     

Presynaptic alpha 2-adrenoceptors modulate the release of [3H]noradrenaline from rat spinal cord dorsal horn neurones

Slices of the dorsal half of the rat spinal cord were used to investigate the existence of a noradrenergic feedback modulation of noradrenaline release. After crude preparation of the vertebral column, the spinal cord was ejected by hydraulic pressure and transverse slices were cut. These were preincubated with [3H]noradrenaline during 0.1 Hz electrical stimulation and then superfused and stimulated electrically for two periods. The stimulation-evoked release of [3H]noradrenaline was Ca2+-dependent and tetrodotoxin-sensitive. Pretreatment of the animals with the noradrenergic neurotoxin, DSP-4, reduced the tritium content in the slices and the stimulation-evoked release to less than 10% of the controls. Clonidine (0.01-1 microM) inhibited the evoked overflow by 60% maximally and yohimbine (0.1-1 microM) enhanced it by 160% maximally. The effects of clonidine were antagonized by yohimbine. These results provide evidence that noradrenaline release from spinal cord slices is controlled by an alpha 2-adrenoceptor-mediated, negative feedback mechanism.     

Opioid control of the function of primary afferent substance P fibres

Lofentanil, a very potent and long-acting opiate agonist, was used to evaluate the opioid control of substance P release from primary afferents. Substance P release from the central terminals of primary afferents was studied in the superfused isolated dorsal half of the rat spinal cord. Substance P release as initiated by electrical field stimulation and by capsaicin was found to be diminished by 50% by lofentanil (1 microM) in a naloxone-reversible manner. Substance P release from peripheral terminals of primary afferents was induced by antidromic saphenous nerve stimulation. Release was measured indirectly by its effect on blood flow and plasma extravasation in the rat hind paw. Both antidromic vasodilatation and plasma extravasation were dose dependently inhibited by lofentanil. The inhibition of antidromic vasodilatation by 10 micrograms X kg-1 lofentanil i.p. was completely prevented by 1 mg X kg-1 naloxone. On the other hand, vasodilatation and plasma extravasation induced by infusion of 3.7 pmol X min-1 substance P remained unaffected by lofentanil. It is concluded that lofentanil inhibits the release of substance P from central as well as peripheral terminals of substance P-containing primary afferent neurons.     

Activation of 5-HT2 receptors inhibits the evoked release of [3H]noradrenaline in the rat spinal cord.

1. The participation of 5-HT2 receptors in the modulation of the evoked release of [3H]noradrenaline from rat spinal cord slices has been examined. 2. In rat spinal cord slices preincubated with [3H]noradrenaline, the alpha 2-receptor agonist clonidine (10(-6) mol/l) decreased the release of tritium evoked by field stimulation (600 pulses at 5 Hz, 20 mA, 2 msec), while the alpha 2-antagonist yohimbine (10(-6) mol/l) increased it. 3. The 5-HT2/5-HT1c agonist DOI (3 x 10(-7) mol/l) decreased the evoked release of tritium, an effect which was prevented by ketanserin (10(-7) mol/l). 4. It is suggested that in addition to presynaptic alpha 2-adrenoceptors, there are 5-HT2 receptors which modulate the release of noradrenaline in the rat spinal cord.     

Apparent vascular to cardiac sympatholytic selectivity of omega-conotoxin GVIA in the pithed rat

The effects of omega-conotoxin GVIA (omega-CTX), a blocker of N-type voltage-operated calcium channels (VOCCs), were investigated in the pithed rat, omega-CTX (1.6 and 3.2 micrograms/kg i.v.) did not alter resting diastolic pressure or heart rate nor the pressor and chronotropic responses to noradrenaline injections (0.1-10 micrograms/kg). In contrast, the pressor responses to electrical stimulation of the whole spinal cord (0.2-6.4 Hz) were dose dependently reduced by omega-CTX whereas the concomitant tachycardia was less affected. When selective stimulation of the cardiac sympathetic outflow was applied, the resulting chronotropic response was more sensitive to omega-CTX. This result is discussed in the light of the possible interference of adrenal catecholamine release during whole spinal cord stimulation which is not sensitive to omega-CTX. These results provide in vivo evidence that omega-CTX is able to reduce sympathetic neurotransmission to the vasculature and the heart, presumably by blocking N-type VOCCs on pre- and post-ganglionic nerve terminals.     

Further evidence for the possible relationship between neuropeptides and serotonergic neurones in rat spinal cord

The relationship between neuropeptides and serotonergic neurones was investigated in rat spinal cord, in vivo, using a subarachnoidal perfusion technique. The 5-hydroxytryptamine (5-HT) release from the spinal cord could be evoked by substance P and peripheral pain stimulation (tail pinch), but not by methionine-enkephalin (met-EK). The substance P-evoked 5-HT release was slightly potentiated by met-EK. The tail pinch-evoked 5-HT release was inhibited by met-EK and baclofen, but potentiated by naloxone. These findings may infer a possible mechanism by which neuroactive peptides regulate 5-HT release from serotonergic neurones in the spinal cord.     

In vitro release of substance P from spinal cord slices by capsaicin congeners

The in vitro release of immunoreactive substance P (I-SP) by capsaicin and three congeners was studied on slices of rat spinal cord upper dorsal horn. Capsaicin and its congeners were all able to stimulate I-SP release, indicating that they act on chemosensitive primary afferents terminating in this region. A positive correlation was found between the I-SP releasing and pain-producing potencies of these compounds. This is in agreement with the concept that primary afferents containing substance P (SP) are involved in the transmission of nociceptive information.     

Modification of responses to sympathetic nerve stimulation by the renin-angiotensin system in rats

1 Angiotensin I (AI) and AII elicited a dose-dependent potentiation of contractions by rat vas deferens produced by low frequency nerve stimulation without enhancing the contraction produced by exogenous noradrenaline. The AII-induced presynaptic potentiation was blocked by the specific antagonist cysteine(8)-AII.2 The vasoconstrictor response to periarterial stimulation of rat isolated perfused kidney was potentiated by AII and there was a lesser enhancement of the effect of exogenous noradrenaline.3 The response to stimulation of complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat was enhanced by angiotensin or vasopressin in direct proportion to the increase in prestimulus muscular tone. The blood pressure in the pithed rats is primarily maintained by the renin-angiotensin system since the converting-enzyme inhibitor (SQ-20881) or bilateral nephrectomy caused further substantial lowering of systemic blood pressure after spinal cord destruction and after treatment with curare and atropine.     

Excessive release of [3H]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.     

Evidence for an excitatory action of the benzodiazepine receptor inverse agonist FG 7142 on C-fibre afferents

Summary (1) The stimulation of adrenocorticotrophic hormone (ACTH) release by FG 7142 was found to be 50% inhibited in capsaicin-desensitized rats indicating that the excitatory effect of this inverse benzodiazepine agonist is partly mediated through capsaicin-sensitive afferents. (2) The in vitro release of substance P from spinal cord slices was stimulated in a tetrodotoxin-resistant manner by FG 7142. No stimulation of the substance R release by FG 7142 was observed when spinal cord slices were taken from capsaicin-pretreated rats which lack afferent C-fibres. (3) Ro 15-1788 antagonized the in vivo as well as the in vitro stimulatory effects of FG 7142 indicating that they were brought about by interaction with benzodiazepine receptors. (4) It is concluded that FG 7142 has a direct excitatory effect on central terminals of capsaicin-sensitive afferents and that neurotransmitters released from primary afferents are involved in inducing ACTH release following administration of FG 7142 in vivo.     

A new dopamine- -hydroxylase inhibitor: effects on the noradrenaline concentration and on the action of L-DOPA in the spinal cord

1. The dopamine-beta-hydroxylase inhibitor bis(4-methyl-1-homopiperazinyl-thiocarbonyl) disulphide (FLA-63; 25 mg/kg i.p.) caused within 4 h a 65% loss of noradrenaline throughout the intact rat spinal cord and also cranial to a transection of the cut spinal cord. Caudal to the lesion, there was only an insignificant depletion of 17% indicating the importance of nerve impulses for the disappearance of noradrenaline.2. Dopamine accumulated in the spinal cord after treatment with FLA-63 although the amounts were not sufficient to replace the missing noradrenaline. Even after treatment with L-3,4-dihydroxyphenylalanine (L-DOPA), the catecholamine store was incompletely replenished by dopamine.3. After a large depletion of the noradrenaline stores, induced by repeated doses of FLA-63 or by reserpine plus FLA-63, the L-DOPA-induced increase in flexor reflex activity of the hind limbs of spinal rats was inhibited much more than after pretreatment with alpha-methyl-tyrosine or reserpine. FLA-63 blocked the formation of noradrenaline but not of dopamine from L-DOPA.4. The increase in flexor reflex activity induced by the noradrenaline receptor stimulating agent clonidine was not changed by FLA-63, indicating that the noradrenaline receptor sensitivity was not influenced.5. After depletion of the noradrenaline stores, the small formation of noradrenaline from L-DOPA may be of greater functional significance for the noradrenaline receptor stimulation than the greater formation of dopamine, but the dopamine formed also has a slight action. With intact noradrenaline stores, displacement of endogenous noradrenaline by newly formed dopamine contributes, at least after monoamine oxidase inhibition, to the increase in the flexor reflex activity caused by L-DOPA.     

Adenosine analogs do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices

Summary Adenosine agonists produce antinociception when injected directly onto the spinal cord of rats and mice. One mechanism to account for this effect could be inhibition of neurotransmitter release from nociceptive sensory neurons. Consequently, we studied whether these agents could inhibit the potassium stimulated release of one such transmitter, substance P, from rat spinal cord slices. A 2 cm section of lumbar spinal cord was dissected from male Sprague-Dawley rats, chopped into 0.5 × 0.5 mm sections and perfused at 37°C with a modified Krebs bicarbonate buffer containing either 3.5 mM, 30 mM, or 50 mM KCl in the presence and absence of various adenosine analogs. Perfusates, collected every 2 min, were assayed for substance P by radioimmunoassay. Exposure of tissue to 50 mM KC1 produced an approximate three-fold increase in the release of substance P over basal release. This increase in release was calcium dependent. Perfusion of spinal cord tissues with either adenosine (10^–3 M), N⁶-cyclohexyladenosine (10^–5 M or 5 × 10^–5 M), 5-N-ethylcarboxamide adenosine (10^–5 M) or L-N⁶-phenylisopropyladenosine (10^–5 M) did not significantly alter basal or potassium-stimulated release of SP when compared to controls. In contrast to the adenosine agonists, exposure of the spinal cord tissue to 10^–5 M morphine significantly reduced the potassium-stimulated release of substance P. Pretreatment of the slices with 10^–5 M theophylline or 8-phenyltheophylline did not significantly attenuate the inhibition of substance P release produced by morphine. Theophylline alone (10^–5 M) had no significant effect on either basal or potassium-stimulated release of SP. These studies demonstrate that adenosine does not inhibit the release of SP from spinal cord slices and does not appear to mediate the morphine-induced inhibition of SP release. The results suggest that the mechanism of the antinociceptive effects of adenosine at the level of the spinal cord is not via inhibition of substance P release. Send offprint requests to M. R. Vasko at the above address     

Capsaicin in adult frogs: Effects on nociceptive responses to cutaneous stimuli and on nervous tissue concentrations of immunoreactive substance P,somatostatin and cholecystokinin

Summary Adult frogs (Rana esculenta) were given subcutaneous injections of 10, 20, 30, 50 and 100 mg/kg capsaicin in sequential order over 5 days, or the vehicle only. The nociceptive thresholds to electrical, thermal and chemical stimuli were measured before, and 1, 5 and 24 h after each injection. Capsaicin was followed by a dose-related reduction of nociceptive responses to all stimuli, but these effects lasted for only 1–5 h after the given injection. Water/acetic extracts of undivided brains and spinal cords were prepared at the corresponding time periods for the radioimmunoassay of peptides. Spinal cord concentrations of immunoreactive substance P were essentially unaffected by capsaicin, while those of immunoreactive somatostatin were significantly increased after the second for fourth injections (20, 30 and 50 mg/kg) of capsaicin. Brain extracts showed an increase of somatostatin and substance P concentrations after the dose of 50 mg/kg. In an additional experiment, immunoreactive substance P, somatostatin and cholecystokinin were measured in tissue samples taken at 2 and 10 min, and 1, 5 and 24 h after a single dose of either 50 mg/kg capsaicin or the vehicle. The only signficant effect of capsaicin was an increase of immunoreactive somatostatin concentration in brain homogenates at 5 h, while the vehicle in itself elicited major variations of all three peptides in spinal cord and/or brain. These results indicate that capsaicin reduces the nociceptive responses to cutaneous stimuli in adult frogs. This effect is transient, and bears no clear relationship to the variations of spinal cord nor of brain concentrations of immunoreactive substance P, somatostatin and cholecystokinin. In the present experimental conditions, the effects of the vehicle injection to neuropeptides far exceeded those of capsaicin itself.