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Using in vivo voltammetric detection of nitric oxide (NO) a previous study demonstrated an increased NO release at the lumbar dorsal horn level of the spinal cord by peripheral inflammatory processes in decerebrated-spinalized rats. This study concerns the effects of acute peripheral stimulations. Gentle non-noxious or isolated nociceptive stimulation did not modify the oxidation current due to NO. However, transient transcutaneous repetitive electrical stimulation of the hindpaw at mild to high intensities (1, 3, and 6 mA) provoked increases in the voltammograms, which were prolonged after stimulation ceased. These effects were absent in animals pretreated with the N-methyl-D-aspartate channel blocker MK-801. The data provide novel direct evidence that NO participates, at least during repetitive stimulation, in the transmission of noxious afferent messages in the dorsal horn of the spinal cord.

The paper describes in vivo voltammetric detection of nitric oxide with carbon fibre microelectrodes at the lumbar spinal dorsal horn level of decerebrated-spinalized rats during peripheral noxious inflammatory processes. At the lumbar (L3-L4) dorsal horn level, a nitric oxide dependent peak of oxidation current (650 mV), remaining stable for up to 4h ((92 +/- 5)% of control) could be detected indicating that significant amounts of nitric oxide are produced continuously. Following subcutaneous injection in the hindpaw of 50 microl of 0.5% formalin the oxidation current rapidly increased ((115 +/- 5)% of control at 25 min) and reached (120 +/- 6)% of control 1h later. Subsequently the voltammograms stabilized for up to 90 min and decreased ((107 +/- 4)% at 124 min). After an injection in the hindpaw of 150 microl of 4% carrageenan, the voltammograms remained at control level for 1h and then the oxidation current increased continuously for up to 4h ((145 +/- 16)% of control at 240 min); such an increase was reversed by ketamine. In these two models of inflammation, the delay in onset and the duration of the increases in NO release within the dorsal horn relate, to some extent, to the time course of the peripheral inflammatory processes, since they are shorter after formalin than after carrageenan. The results provide a direct in vivo demonstration that the intercellular messenger nitric oxide participates in the transmission of noxious afferent messages within the dorsal horn of the spinal cord following peripheral inflammation., (Copyright 2002 European Federation of Chapters of the International Association for the Study of Pain)

Glutamate acts as a neurotransmitter of primary afferent messages in the spinal cord. Through glutamatergic mechanisms nitric oxide (NO) is also a potential intermediary in the transmission of sensory messages, particularly nociceptive, at the spinal level. The aim of the present study was, by using electrochemical monitoring of NO, to determine if the activation of glutamatergic transmission, particularly through NMDA receptors, could increase NO production within the dorsal horn of the lumbar spinal cord in the rat. 30 micrometers diameter treated carbon fiber electrodes coated with nickel-porphyrine and Nafion(R), and associated with differential normal pulse voltammetry, have been used in vivo to monitor NO within the dorsal horn of the lumbar spinal cord of decerebrated-spinalized rats. A NO-dependent peak of oxidation current (650 mV vs. Ag-AgCl), remaining stable for up to 3 h (+/-5%) could be detected under basal conditions, which indicates that significant amounts of NO are produced continuously. The non-competitive N-methyl-d-aspartate (NMDA) channel blockers, Ketamine (100 mg kg-1 i.p.) and MK-801 (10 mg kg-1 i.p.), decreased the voltammograms to 70+/-5% and 69+/-2% of controls at 120 min, respectively. Glutamate (10 mM), when directly superfused upon the spinal cord (20 min at 50 microliters min-1) induced a rapid and significant increase of the 650 mV peak, with a maximum at around 90 min (148+/-6% of control) followed by a slow decay (138+/-4% of control at 150 min). This increase could be totally reversed or blocked by i.p. injection of 100 mg kg-1 of Ketamine. NMDA (30 mg kg-1 i.p.) induced a long-lasting increase in the peaks (149+/-11% at 90 min and 162+/-20% at 120 min), which was also fully reversed by Ketamine or MK-801. These results provide in vivo direct evidence of a glutamate- and/or NMDA-induced release of NO at the spinal level, and is discussed in relation to the glutamatergic transmission of primary afferent messages., (Copyright 1999 Elsevier Science B.V.)

NO synthase (NOS) is largely distributed in the superficial and deep laminae of the dorsal horn as well as in dorsal root ganglion cells. It has been proposed that nitric oxide (NO) participates in the transmission of sustained, and possibly brief, nociceptive, inputs at the spinal level. The aim of this study was to check the ability of in vivo electrochemical monitoring of NO within the dorsal horn of the lumbar spinal cord (L3-L4 level) of chloral hydrate anesthetized or decerebrated spinalized rats. 30 microm diameter and 450 microm length treated carbon fiber electrodes coated with nickel(II) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrine and NafionR, and associated with differential normal pulse voltammetry, gave a peak of oxidation current around 650 mV (vs. Ag-AgCl) in vitro in NO solutions between 0.125 and 1.25 microM. In vivo, a 650 mV peak appeared which was stable (recording interval 2 min) for up to 3 h (+/-6%). Comparison between in vitro calibration and in vivo voltammograms gave an estimated in vivo extracellular concentration of 0.50 microM. In vivo, peaks decreased by 95% at 90 min and for up to 3 h after an i.p. injection of 100 mg/kg of the NOS inhibitor (NOSI) L-arginine-p-nitroanilide (L-ANA). At the same dose i.p., N(G)-nitro-L-arginine methyl ester (L-NAME) was almost ineffective after 90 min in animals paralyzed with pancuronium bromate or gallamine trethiodide. However, in non-curarized decerebrated spinalized animals, L-NAME depressed the voltammograms by 36% at 90 min. S-Ethylthiourea (80 mg/kg i.p.), also decreased the voltammograms by 45% at 140 min, and finally, 7-nitroindazole (7-NI, 90 mg/kg i.p), induced a important decrease of the 650 mV peak (23% of control) at 120 min. These results are in agreement with biochemical data showing the decrease of NOS activity within the lumbar spinal cord by L-NAME (45% of control at 90 min) and 7-NI (20% of control at 90 min). The NO donor hydroxylamine (30 mg/kg i.p.) significantly increased the peaks (140% at 90 min), and sodium nitroprusside (SNP, 20 mM) when directly superfused upon the spinal cord (200-300 microl min(-1)) induced a large increase in the peak (300% at 90 min). Moreover, SNP 60 min after L-ANA, or 90 min after L-NAME, rapidly restored the 650 mV peak up to control values. These results demonstrate the validity of electrochemical monitoring of NO within the dorsal horn of the spinal cord. The in vivo electrochemical detection of NO is in progress to study the implication of this messenger in the transmission of nociceptive messages at the spinal level.

Biosensors sensitive for in vivo monitoring of serotonin (5-HT) in the CNS by differential normal pulse voltammetry were constructed by coating treated multicarbon fiber electrodes (mCFEs) with Nafion (N-mCFE). In vitro sensitivities of mCFE and N-mCFE were compared in solutions ranging from 5 nM to 20 microM of uric acid (UA), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. The mCFEs were three to seven times less sensitive for 5-HIAA or UA than for 5-HT. Nafion treatment dramatically decreased sensitivity for 5-HIAA and UA of N-mCFEs (approximately 10(3) times), whereas it remained in the nanomolar range for 5-HT. In vivo, in the dorsal horn of the lumbar spinal cord of anesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 +/- 3% at 180 min) of peak 3 of oxidation current (characteristic of 5-hydroxyindoles) monitored with mCFEs, but with N-mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 +/- 5% at 180 min. The 5-HT release-inducer p-chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 +/- 3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with N-mCFEs PCA induced a rapid increase of peak 3N (137 +/- 6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 +/- 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N-mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 +/- 6% at 90 min) in peak 3N.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of i.p. administration of the preferential 5-HT1B agonist 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU 24969) (10 mg/kg) has been investigated by in vivo 5-hydroxyindole electrochemical (peak 3) detection in the medullary dorsal horn (MDH) of acute anesthetized and unanesthetized freely moving rats. RU 24969 induced a significant decrease in peak 3 in the MDH of anesthetized rats. In freely moving animals, RU 24969 induced a biphasic effect. Thus, after the injection the curve remained above that of the saline group and returned to control levels up to 60 min. Subsequently the curve decayed to below the control values and rapidly plateaued for up to 180 min. The initial increase and the decrease thereafter were both statistically significant vs. saline. With reference to similar in vivo studies demonstrating the responsiveness of ascending serotonergic systems to RU 24969, it is concluded that the 5-HT metabolism in the serotonergic NMR-dorsal horn system is affected by this 5-HT1B agonist. However, the biphasic effect reported here in unanesthetized animals suggests that RU 24969 could act by two different ways on 5-HT metabolism and indicates that there could be a primary interaction of RU 24969 on the 5-HT uptake system (inhibition) which could, at first, prevail over the interaction with terminal autoreceptors.

The combination of electrochemically treated carbon-fiber electrodes with DPV, DNPV or DPA represents a wide range of possibilities. As shown in this review, the choice of treatment and measurement technique depends on the purpose. As regards in vivo monitoring of 5-HIAA or DOPAC from very small brain nuclei, electrochemically treated carbon-fiber electrodes appear very potent and inexpensive. The main limitation of the established electrochemical techniques, including those discussed here, is that the unequivocal measurement of the basal extracellular neurotransmitter level cannot be achieved unless animals are treated with pargyline. On the other hand, this monitoring is feasible with in vivo dialysis. Therefore, electrochemical techniques, on the one hand, and in vivo dialysis, on the other hand, present different advantages. The former are much more potent than the latter in two respects. First, due to the much smaller size of the sensor, electrochemical techniques are more suitable for studying small brain nuclei. Second, since electrochemical techniques exhibit a better temporal resolution, they are recommended for investigating the relationship between impulse flow and neurotransmitter release. However, when high anatomical or temporal resolution is not required, in vivo dialysis is more suitable for recording the basal monoamine release.

The present study, by the use of in vivo electrochemical detection of 5-hydroxyindole (peak '3') in the bulbo spinal serotonergic system at the medullary dorsal horn (MDH) level, investigated the effects of the new tricyclic antidepressant (TCA) tianeptine, which has been shown to be a specific serotonin (5-HT) uptake enhancer. It was found that acutely administered tianeptine (10 mg/kg, i.p.) induced a marked significant increase in peak 3 within the dorsal horn, an in vivo observation which is in accordance with the biochemical properties of tianeptine as studied in forebrain structures. In addition, the effect of tianeptine on the morphine-induced increase in 5-HT metabolism was investigated, by comparison with the previous data obtained with the specific 5-HT uptake inhibitor femoxetine in the MDH. It was shown that tianeptine can display additive effect with morphine (10 mg/kg, i.p.) on 5-HT metabolism at the MDH level. These results are discussed in relation to the effects of classical TCAs and the particular properties of tianeptine.

The effect of subcutaneous administration of the chemical algogen formalin, on serotonin (5-HT) metabolism in the nucleus raphe magnus (NRM) and the medullary dorsal horn (MDH) has been investigated using in vivo 5-hydroxyindole electrochemical (peak '3') detection with treated, multi-carbon fiber electrodes and differential pulse, or normal pulse, voltammetry in freely moving rats. The subcutaneous (s.c.) injection of 50 microliters of 10% formalin in the left forepaw was followed, at the NRM level, by a significant increase in the voltammograms as compared to controls (50 microliters of saline 0.9% s.c. in left forepaw) for about 70 min after the injection, before a return to control values. At the MDH level, the formalin injection induced no significant effect on peak 3, as compared to controls, during the first 70 min. After that, the voltammograms significantly increased and remained above controls for up to 180 min. Thus, the time-courses of NRM and MDH effects appear markedly different. These findings suggest that, depending on the anatomical level (NRM or MDH) and/or the period of observation, one can measure differences in the time-course of the increase in 5-HT metabolism in the NRM-dorsal horn serotonergic system by tonic noxious stimuli, such as the formalin test.

The effect of i.p. administration of the selective 5-HT1A agonist 8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) (100 micrograms kg-1) has been investigated by in vivo 5-hydroxyindole electrochemical (peak 3) detection in the nucleus raphe magnus (NRM) and medullary dorsal horn (MDH) of acute anaesthetized and unanaesthetized freely moving rats. 8-OH-DPAT induced a small but significant decrease in peak 3 in the NRM and MDH of anaesthetized rats. In freely moving animals, a similar small effect was observed at both NRM and MDH levels. With reference to similar in vivo studies demonstrating differential responsiveness of ascending serotonergic systems to 8-OH-DPAT, it is concluded that the serotonergic NRM-dorsal horn system is slightly affected by this 5-HT1A agonist.

Tricyclic antidepressants, when administered acutely, are known to potentiate morphine-induced antinociception. Systemic administration of morphine has been shown to increase the metabolism of serotonin (5-HT) at the level of the nucleus raphe magnus, as measured by in vivo electrochemistry, in freely-moving rats. Using a similar electrochemical detection of 5-hydroxyindole (peak "3") in the nucleus raphe magnus, the present study investigated the effect of the specific 5-HT uptake inhibitor, femoxetine, on peak 3 and on changes in the metabolism of 5-HT, induced by morphine. Acutely administered femoxetine (40 mg/kg i.p.) induced a significant decrease in peak 3 and completely abolished the effect of morphine (10 mg/kg i.p.) on the metabolism of 5-HT. These data do not support the contention that potentiation of morphine-induced analgesia, by tricyclic depressants results from an interaction between the tricyclic antidepressants and the morphine-induced increase in metabolism of 5-HT, at the level of the nucleus raphe magnus.

Acute administration of tricyclic antidepressants (TCAs) is known to potentiate morphine antinociception. At the medullary dorsal horn (MDH) level systemic morphine has been shown to increase serotonin (5-HT) metabolism as measured by in vivo electrochemistry in freely moving rats. Using similar electrochemical detection of 5-hydroxyindole (peak '3') within the MDH, the present study investigated the effect of the specific 5-HT uptake inhibitor femoxetine on peak 3 and the effects of this TCA on changes in 5-HT metabolism induced by morphine. Acutely administered femoxetine (40 mg/kg i.p.) (i) induced a small but significant increase in peak 3 and (ii) strongly potentiated the effect of morphine (10 mg/kg i.p.) on 5-HT metabolism, this potentiation being opiate specific since simultaneous injection of naloxone (1 mg/kg i.p.) abolished the effect of morphine. These findings provide an in vivo neurochemical basis for the potentiation of morphine antinociception by TCAs. They further emphasize the importance of 5-HT bulbospinal descending pathways in morphine antinociception.

The aim of the present experiments was to test whether adrenal chromaffin cells implanted into the striatum of rats could exert a functional effect through a release of catecholamines. A cell suspension obtained from bovine adrenal medulla was implanted unilaterally into the striatum. The striatal dopaminergic input was extensively destroyed beforehand to preclude the possibility of reinnervation of the striatum by endogenous dopaminergic neurons. The functional influence of the implant was tested through the measurement of drug-induced rotation, while catecholamine release was measured subsequently in the same animals by in vivo electrochemistry. Transplant survival, as shown by the immunohistochemical analysis performed at the end of the in vivo experiments, was highly variable. Surviving chromaffin cells maintained their endocrine morphology and no reinnervation of the host striatum could be detected. Rotation of the animals evoked by apomorphine (0.1 mg/kg, sc) or amphetamine (5.0 mg/kg, ip) following the lesion was left uninfluenced following transplantation, even when a large transplant was recovered. On the other hand, nicotine (0.5 mg/kg, sc) evoked a strong contraversive rotational response in the transplant-bearing animals. This response could not be ascribed to the central effect of substances released peripherally and entering the nervous system through the blood-brain barrier opened by the implantation procedure, as it could not be found in animals bearing implants of other peripheral endocrine tissue, viz, pituitary. The effect of nicotine was not blocked by the pretreatment of the animals with either the opiate antagonist naloxone (2.5 mg/kg, 10 min) or the dopamine receptor blocker pimozide (0.5 mg/kg, 1 h), although the latter pretreatment blocked the amphetamine-evoked rotation. No spontaneous catecholamine release could be detected from the implanted chromaffin cells by in vivo electrochemistry, while treatment with amphetamine or nicotine did evoke a release. The results suggest that the functional effects of such intrastriatal grafts of chromaffin cells, reported in previous studies, cannot be explained by the secretion from the grafted cells of catecholamines into the denervated striatum. On the other hand the results obtained following the pharmacological stimulation of these cells indicate that adrenal grafts can, under suitable conditions, influence the functioning of the host nervous system.

In intact rats anesthetized with chloralose, the effects of naloxone were studied on the responses of spinal cord dorsal horn neurons to C fiber stimulation and upon the inhibition induced on these responses by stimulation of the nucleus raphé magnus (NRM). (1) A mean 44% facilitatory effect on responses to C fibers was observed for 12/19 units. (2) A mean 30% reduction of the inhibitory effects of NRM was found for 14/29 units. (3) However there is no clear relationship between these facilitatory effects and the diminution of the efficiency of NRM stimulation. These results demonstrate a facilitatory effect of naloxone upon the transmission of noxious messages at the spinal level and confirm that opiate endogenous substances are implicated in the inhibitory mechanisms activated by stimulation of NRM.

Treated carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vitro and in vivo determination of indoleamines. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Treated carbon fiber microelectrodes respond in vitro linearly over a large range of concentrations of 5-hydroxytryptamine (5-HT) and of 5-hydroxyindoleacetic acid (5-HIAA), but are 5-8 times more sensitive to 5-HT than to 5-HIAA. In vivo, the question remains as to the exact nature of the peak because the oxidation potentials of 5-HT and 5-HIAA are close together and cannot be monitored separately. Pharmacological investigations were hence carried out in order to characterize the electrochemical signal detected at 300 mV in the dorsal horn of the lumbar spinal cord of chloral hydrate-anesthetized rats. Using 250 micron long carbon fiber microelectrodes, the electrochemical signal stabilizes at 30-90 min and the peak remains constant for up to 210 min. Administration of the monoamine oxidase inhibitor (MAOI) clorgyline produced a progressive decrease of the signal which reached a decrease of 33% of control at 180 min after injection. At this time biochemical measures demonstrated a 117% increase in 5-HT and a 32% decrease in 5-HIAA in the dorsal half of the spinal cord. Reserpine provoked an increase of 20% in the electrochemical peak and the 5-HIAA outflow blocker probenecid gave rise to a sustained plateau of about 60% above control values.(ABSTRACT TRUNCATED AT 250 WORDS)

1. In the rat under N2O-halothane anesthesia, stimulation of the nucleus raphe magnus (NRM) with stimulus parameters similar to those used to obtain analgesia in freely moving animals strongly inhibits the responses of dorsal horn convergent neurons due to A-delta- and C-afferents. 2. Responses to noxious radiant heat were also depressed, and pronounced post-effects were frequently observed. 3. Comparison between coupled sites of stimulation in NRM and in adjacent bulbar reticular formation (BRF) on responses to C-fibers revealed the preeminent effects of NRM; these were systematically encountered (93% of neurons), much more pronounced, and of longer duration. 4. The latency of these inhibitory effects (around 20 ms) suggests the participation of myelinated axons in such descending action and, consequently, we question the involvement of unmyelinated serotonergic fibers. 5. However, descending inhibitory influences from NRM on responses to C-fibers are reduced after 5-hydroxytryptamine (5-HT) depletion by p-chlorophenylalinine (pCPA), thus demonstrating the implication of both serotonergic and nonserotonergic pathways. 6. In addition, after pCPA pretreatment, long-lasting and sustained excitatory effects from NRM were observed in 35% of convergent neurons; their possible origin is discussed.

Differential pulse voltammetry was used for the detection of 5-hydroxyindoles in the cerebral cortex of rats anaesthetized with urethane. The stimulation of the lateral hypothalamus or of the dorsal raphe nucleus induced a 10-40% increase in the amplitude of the signal. The signal recorded from p-chlorophenylalanine (pCPA)-pretreated animals was much smaller than in normal animals and could be increased by 5-HTP administration. The stimulation of the serotonergic pathways was ineffective in the pCPA-pretreated animals.

Treated carbon fiber electrodes were used with differential normal pulse voltammetry (DNPV) for in vivo determination of the relative participation of uric acid (UA) to peak 3 derived between 250-300 mV in the dorsal horn of the spinal cord of anesthetized rats. In vitro, treated carbon fiber electrodes respond linearly over a large range of concentrations of UA (oxidation potential around 250 mV) and 5-hydroxyindoleacetic acid (5-HIAA, oxidation potential around 280-290 mV), but are 3 to 4 times more sensitive to 5-HIAA than to UA. In vivo the question remains as to the exact nature of peak 3 because the difference between oxidation potentials of UA and 5-HIAA is not great enough to permit a separate monitoring of the two compounds. In normal rats, administration of the xanthine oxidase inhibitor allopurinol, produced a progressive decrease of the signal, which reached 64.3% of controls at 120 min (35.6% diminution) after injection, and then plateaued around this value for up to 2 h. The administration of the monoamine oxidase inhibitor (MAOI) clorgyline, produced a classical decay in the voltammograms due to a diminution of endogenous 5-HIAA; however, allopurinol injected 3 h after MAOI gave an additional decrease of peak 3 of about 28%. Finally, in rats pretreated with parachlorophenylalanine (pCPA), the residual peak (32.48% as compared to peak 3 of normal rats taken as 100%), the potential of which is shifted to near that of UA, could be decreased by allopurinol to a level of 9.6% of the peak in control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Intensely noxious peripheral stimuli of the anaesthetized rat produce two changes in the activity of convergent dorsal horn units: the segmental neuronal pool is activated, while all other convergent neurones are inhibited. These Diffuse Noxious Inhibitory Controls (DNIC) are highly potent (60-80% inhibition) and suppress all convergent neuronal activity, whether spontaneous or evoked by noxious or nonnoxious stimuli. On the other hand, they have no effect on other dorsal horn cell types, including noxious-only and proprioceptive units. The "DNIC" circuits include at least one supraspinal relay since DNIC is not seen in spinal animals. Furthermore, they are greatly reduced by lesions of the Nucleus Raphé Magnus (NRM). It has been shown that this nucleus massively projects onto the spinal cord, in particular onto the dorsal horn, and that stimulation of the NRM results in convergent unit inhibition of the same degree of magnitude as with DNIC. The role of serotonergic mechanisms in DNIC can be demonstrated pharmacologically: pCPA pre-treatment (3 daily I.P. injections, 300 mg/kg) or cinanserin (4 mg/kg I.V.) both result in a potent decrease (50-80%). We have proposed that the nociceptive message from the convergent units could result in a contrast between activity of the activated segmental pool and silence of the remaining convergent units. If this hypothesis can be verified, then some raphé nuclei and brain stem serotonergic pathways may function as filters in the detection of nociceptive messages, allowing extraction of information from somatic background activity including the firing from peripheral mechanoreceptors. While superficially paradoxical in fact our hypothesis fits well with the observation of profound analgesia following NRM stimulation: indeed, this hypothetical contrast would be completely eliminated by NRM stimulation since both neuronal pools would then be inhibited.

The purpose of this study was to evaluate in freely moving animals the effect of morphine on the 5-hydroxyindole oxidation current recorded in the nucleus raphe magnus (NRM) which is the origin of serotonergic control systems modulating the transmission of noxious inputs at the spinal level. A current recorded at 270-290 mV (peak 3), characteristic of 5-hydroxyindoleacetic acid (5-HIAA), was measured with treated multi-fiber carbon electrodes, using differential pulse (DPV) or differential normal pulse (DNPV) voltammetry. In control rats the amplitude of the peak remains constant for many hours. Morphine (10 mg/kg i.p.) caused a very significant increase which plateaued between 60 and 80 min (mean increase: 142 +/- 7% of control values); recovery was complete by about 3 h. Simultaneous injection of naloxone (1 mg/kg i.p.) completely abolished the effect of morphine. The peak 3 augmentation was still observed (151 +/- 5%) in rats pretreated with the xanthine oxidase inhibitor, allopurinol (12 mg/kg i.p.), but did not occur when animals were given an anaesthetic dose (450 mg/kg i.p.) of chloral hydrate. It is concluded that morphine clearly increases the metabolism of serotonin (5-HT) in the NRM, and one could speculate that the increase in 5-HIAA results from 5-HT release. Such a release could be due either to 5-HT terminals originating in the periaqueductal gray, or to somato-dendritic mechanisms. Thus the question remains as to the relationship between the activation of 5-HT metabolism in the NRM and previous neurochemical evidence for morphine-induced augmentation of 5-HT metabolism within the terminal area of serotonergic raphe-spinal pathways.

In vivo differential pulse voltammetry with electrochemically treated carbon fiber microelectrodes has been used to investigate the anatomical nature of the GABAergic influence on striatal serotonergic transmission in the rat. Lesion studies and pharmacological treatments demonstrated that the electrochemical signal recorded at 300 mV in the striatum probably corresponds to the oxidation of extracellularly released 5-hydroxyindoleacetic acid. Thus, dorsal raphé lesions or systemic administration of alpha-propyldopacetamide, NSD 1015, pargyline and MK212 decreased, whereas reserpine injection increased the amplitude of the signal. Moreover, L-5-hydroxytryptophan administration caused an increase in the signal which was almost completely prevented by pargyline pretreatment. Acute administration of dipropylacetamide (150 mg/kg i.p.) reduced the amplitude of the signal from the striatum, while injection of gamma-acetylenic GABA (200 mg/kg i.p.) was without effect. Repeated (but not acute) treatment with the GABA receptor agonist, progabide (400 mg/kg i.p.b.i.d. for 14 days), led to a pronounced decrease in the amplitude of the signal from the striatum. A similar effect was observed after intradorsal raphé infusion of GABA (10 and 100 micrograms), gamma-vinyl GABA (100 micrograms) and SL 75102 (10 micrograms), a principal metabolite of progabide. In contrast, local injection of the GABA receptor antagonists, bicuculline (1 and 10 micrograms) or R5135 (0.05 microgram), failed to affect the peak amplitude in the striatum. When infused into the dorsal raphé, R5135 (0.05-0.1 microgram) antagonized the diminution of the signal induced by intradorsal raphé infusion of GABA (100 micrograms) or SL 75102 (10 micrograms). Finally, electrolytic lesion of the habenular nuclei completely blocked the diminution of the signal from striatum induced by an intradorsal raphé infusion of GABA (100 micrograms). These results indicate that the inhibitory GABAergic control of striatal serotonergic transmission is exerted at the level of the dorsal raphé cells and depends upon the integrity of the habenulo-dorsal pathway.

In vivo differential pulse voltammetry was used for the detection of indoleamines during vertical electrode penetrations in rat first somatosensory cortex, for studying the laminar distribution of serotonin and/or its metabolites in that part of the cortex. The peak of current corresponding to 5-hydroxyindoles was maximum in the most superficial part of the cortex and diminished gradually in the deeper layers. These results suggest that the cortical serotonergic innervation is predominant in the superficial layers.

Carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vivo determination of indolamines within the extracellular space of the dorsal horn of the spinal cord or chloral hydrate-anesthetized rats. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Stimulation of the nucleus raphé magnus (NRM) with stimulation parameters comparable to those used to elicit analgesia in freely moving animals produced marked alterations in the voltammograms: (1) stimulation of the NRM for 10 min induced an immediate and sustained increase in the peak amplitude; (2) post-effects of variable duration were observed; (3) the increase in the 5-hydroxyindolaminergic signal was significantly reduced during a second series of NRM stimulations indicating some degree of tolerance to central stimulation. The accuracy of these observations is strengthened by the fact that the basal 5 hydroxyindolaminergic signal is strongly depressed after pretreatment of the animal with p-chlorophenylalanine; in addition, under these conditions, NRM stimulation is totally inefficient. We suggest that these results reflect the in vivo modification of 5-HT metabolism. This represents the first evidence for an in vivo release of 5-HT during stimulation of brain stem areas which induces powerful analgesia in freely moving animals.

In this study cutaneous nociceptive messages are followed at different levels of the CNS, from the periphery to the cortex. A brief summary is given concerning the role of the fine myelinated and unmyelinated fibres which are specifically activated by noxious stimuli. A more extensive review considers the spinal mechanisms which sustain the transmission of nociceptive messages; the electrophysiological properties of interneurones located in laminae VIII, V, and I of the dorsal horn are described in detail. At the same time, the problem of the ascending projections of those cells activated by nociceptive stimuli is discussed. Particular attention is paid to the controls acting at the spinal level: segmental controls are described first and lead to discussion of the "gate control theory"; descending inhibitory controls are then discussed and their importance emphasized. The complexity of pain mechanisms at the supra-spinal level is underlined and a brief review considers the role of various bulbar, mesencephalic and thalamic structures involved in transmission of noxious messages. Among these structures, the PO group of nuclei seem to have a particular role in pain processes. Although the importance of the cortex for final integration of nociceptive messages is discussed, a brief summary is also given of investigations into the role of somatic area SII.

The trigeminal nucleus caudalis is considered the equivalent of the orofacial nociceptive system of the dorsal horn of the spinal cord. At the level of this trigeminal area (i.e. medullary dorsal horn) the transmission of noxious inputs is strongly modulated by a descending, serotonergic system mainly originating from the nucleus raphe magnus (NRM). The present study in freely moving animals reports the effect of morphine on the 5-hydroxyindole oxidation current recorded in the medullary dorsal horn. Complementary data from recordings in spinal dorsal horn in acutely anesthetized rats are also presented. A current recorded at 270-290 mV (peak '3'), characteristic of 5-hydroxyindoleacetic acid (5-HIAA), was measured with treated multi-fiber carbon electrodes, using differential pulse voltammetry (DPV) or differential normal pulse voltammetry (DNPV). In control rats, the amplitude of the peak remained constant for many hours. Morphine (10 mg/kg i.p.) caused a significant increase which plateaued between 35 and 80 min (mean increase: 127 +/- 5% of control values); recovery was complete by about 3 h. Simultaneous injection of naloxone (1 mg/kg i.p.) totally abolished the effect of morphine. By contrast, morphine was without effect on peak 3 recorded in the spinal dorsal horn of chloral hydrate (450 mg/kg i.p.) anesthetized rats. It is concluded that in non-anesthetized freely moving animals morphine clearly increases the metabolism of serotonin (5-HT) in the medullary dorsal horn. This finding confirms previous neurochemical data showing an increased synthesis or release of 5-HT in the spinal cord after systemic morphine or its microinjection into either the periaqueductal gray matter or the NRM, and underlines the value of in vivo electrochemistry in monitoring changes in 5-HT metabolism directly and continuously during various physiological and pharmacological procedures.

In the anaesthetized Rat, the entire population of dorsal horn convergent neurones is differentially affected by a noxious stimulus: while exciting the segmental pool, it strongly inhibits the remaining population. The inhibitory effects, which involve supraspinal mechanisms, are reduced to a great extent in parachlorophenylalanine pretreated animals. The role of raphé-spinal serotonergic pathways in nociception is discussed.

Differential pulse voltammetry associated with carbon fiber microelectrodes was used to detect the 300 mV signal which is known to reflect the concentration of 5-hydroxyindoles in the spinal cord and cerebral neocortex of rats anesthetized with urethane or chloral hydrate. The intraperitoneal injection of p-chloroamphetamine resulted in an increase in the amplitude of the signal in the neocortex but not in the spinal cord. Administration of clorgyline did not consistently modify the signal monitored in the neocortex whereas it decreased in the spinal cord. Probenecid induced a larger increase in 5-hydroxyindoles in the neocortex than in the spinal cord. These results demonstrate that different parts of the serotonergic system might be differentially sensitive to drugs affecting serotonin metabolism.

Diffuse Noxious Inhibitory Controls (DNIC) were investigated in anaesthetized intact rats, with or without p-chlorophenylalanine (pCPA) pretreatment. Dorsal horn convergent neurones responding to both noxious and non-noxious stimuli applied to their excitatory receptive field located on the distal part of the hindlimb, were recorded in the lumbar spinal cord. These cells received A alpha and C fibre inputs as shown by electrical stimulation of their receptive field. In control animals, the evoked responses to C fibre inputs could be strongly inhibited by various noxious stimuli applied to widespread areas of the body: the inhibitory effects induced by intraperitoneal administration of bradykinin, pinch applied to the tail or muzzle and noxious heat applied to the tail were of 77%, 87%, 83% and 61% respectively. Long-lasting post-effects were seen in most cases after cessation of the application of the conditioning stimulus. Pretreatment with pCPA (300 mg/kg, i.p., 3 days) resulted in a strong reduction of DNIC. The inhibitory effects induced by intraperitoneal administration of bradykinin, pinch applied to the tail or muzzle and noxious heat applied to the tail were reduced by 47%, 63%, 87% and 63%, respectively. The post-effects were also reduced both in terms of magnitude and duration. These results strongly suggest that serotonergic pathways partially involved in DNIC. They are discussed with reference to the descending control systems, originating from the caudal raphé, which modulate the transmission and/or the integration of nociceptive messages at the spinal level. The possible involvement of DNIC and 5-HT mechanisms to the hypo-algesic phenomena induced by hyper-stimulation is also suggested.

In the anaesthetized rat the intravenous injection of 5-HT antagonists, cinanserin and methysergide, induces in two-thirds of neurones studied, an increase in the responsiveness of dorsal horn convergent neurones to C-fibre stimulation. These results are in favour of the existence of serotonergic tonic descending inhibitory effects on the spinal transmission of noxious messages.

The effect of systemic morphine on serotonin (5-HT) metabolism within the dorsal raphe nucleus (DRN) has been investigated by in vivo 5-hydroxyindole electrochemical (peak '3') detection in freely moving rats. Morphine caused a weak and delayed, but naloxone-reversible, increase in peak '3'. This increase was poorly, if at all, correlated with the morphine-induced analgesia. Finally, stress and/or noxious stimulation had no effect on this signal. These results are compared with our previous studies using the same methodological approaches and show that morphine caused a significant and specific increase in 5-HT metabolism at the levels of nucleus raphe magnus (NRM) and medullary dorsal horn. Furthermore, as shown in the present paper, there was also a good correlation between the time course of such increases and the analgesic effect of morphine. These findings are discussed with reference to the involvement of 5-HT mechanisms in the so-called DRN-NRM-dorsal horn 'intrinsic analgesic system'.

(1) The effects of morphine upon the transmission of nociceptive messages at the spinal level have been investigated in the spinal rat. The responses of dorsal horn cells induced by the activation of C fibres were depressed in all cases in a dose-dependent fashion, this effect being reversed by the opiate antagonist naloxone. An estimation of the ED50 at the cellular level leads to the value of 6.3 mg/kg. The responses to A delta fibres were also depressed dose-dependently whereas the responses to A alpha fibres were unaffected. This is a confirmation in the rat of the differential effects of morphine on responses of convergent units elicited by the stimulation of different fibres, as previously described in the cat. (2) The hypothesis of the participation of serotonergic terminals in these effects has been checked by comparing the preceding results to those obtained in pCPA pretreated animals. Two populations of units were observed in the latter group: two-thirds of cells showed a dose-response curve similar to that of the non-pretreated group whereas the remaining one-third were unaffected either by morphine or naloxone. It is concluded that, at least, two mechanisms are involved in the depressive effects of morphine at the spinal level, serotonergic terminals being implicated in one of these. (3) The lowering of spinal cord serotonin content was associated with a decrease of both the size of the excitatory receptive field (34%) and the activities related to C fibre input (36%) of the recorded dorsal horn cells. This result is discussed with reference to the excitatory or sensitizatory effect of serotonin upon chemoreceptors related to pain.