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As a model of chronic inflammatory pain, Freund's adjuvant-induced polyarthritis has been shown to be associated with marked alterations in the activity of opioid- and calcitonin gene-related peptide (CGRP)-containing neurons in the dorsal horn of the spinal cord in rats. Possible changes in the interactions between these two peptidergic systems in chronic inflammatory pain were investigated by comparing the effects of various opioid receptor ligands on the spinal outflow of CGRP-like material (CGRPLM) in polyarthritic and age-paired control rats. Intrathecal perfusion of an artificial cerebrospinal fluid in halothane-anaesthetized animals allowed the collection of CGRPLM released from the spinal cord and the application of opioid receptor ligands. The blockade of kappa-opioid receptors similarly increased CGRPLM release in both groups of rats as expected of a kappa-mediated tonic inhibitory control of CGRP-containing fibres in control, as well as in polyarthritic rats. In contrast, the higher increase in CGRPLM outflow due to the preferential blockade of mu opioid receptors by naloxone in polyarthritic rats as compared to non-suffering animals supports the idea of a reinforced mu opioid receptor-mediated tonic inhibitory control of CGRP-containing fibres in rats suffering from chronic pain. Even more strikingly, the differences observed in the effects of delta-opioid receptor ligands on CGRPLM outflow suggest that delta receptors are functionally shifted from a participation in a phasic excitatory control in non-suffering rats to a tonic inhibitory control in polyarthritic rats. These data indicate that agonists acting at the three types of opioid receptors all exert a tonic inhibitory influence on CGRP-containing nociceptive primary afferent fibres within the spinal cord of polyarthritic rats. Such a convergence probably explains why morphine and other opioids are especially potent to reduce pain in subjects suffering from chronic inflammatory diseases.

Complex and contradictory data have been reported regarding the changes in spinal opioidergic systems associated with chronic inflammatory pain in the rat. In an attempt to solve these discrepancies, the in vivo release of met-enkephalin and dynorphin and the expression of the corresponding propeptide genes were investigated at the spinal level in arthritic rats and paired controls. A dramatic increase in the concentration of prodynorphin mRNA (+300–550%) and a less pronounced elevation of that of dynorphin-like material (+40–50%) were found in the dorsal part of cervical and lumbar segments of the spinal cord in rats rendered arthritic by an intradermal injection of Freund's adjuvant four weeks prior to these measurements. In addition, the spinal release of dynorphin-like material (assessed through an intrathecal perfusion procedure in halothane-anaesthetized animals) was approximately twice as high in arthritic rats as in controls. In spite of significant elevations in the levels of both met-enkephalin (+30–70%) and proenkephalin A mRNA (+40−50%) in the dorsal part of cervical and lumbar segments, the spinal release of met-enkephalin-like material was decreased (−50%) in arthritic rats as compared to paired controls. Proenkephalin A mRNA (but not prodynorphin mRNA) could be measured in dorsal root ganglia, and its levels were dramatically reduced in ganglia at the lumbar segments in arthritic rats. Such parallel reductions in the spinal release of met-enkephalin-like material and the levels of proenkephalin A mRNA in dorsal root ganglia of arthritic rats support the idea that the activity of primary afferent enkephalinergic fibres decreases markedly during chronic inflammatory pain.

The possible control by opioids of the spinal release of calcitonin gene-related peptide-like material (CGRPLM) was investigated in halothane-anaesthetized rats whose intrathecal space was perfused with an artificial cerebrospinal fluid. Morphine (20 mg/kg i.v.; or at 10-100 microM added to the perfusing fluid), the mu selective agonist DAGO (10 microM) and the kappa selective agonist U 50488 H (10 microM) did not affect the spontaneous outflow of the CGRPLM. In contrast, the selective delta agonist DTLET (10 microM) significantly increased CGRPLM release. The latter effect could be prevented by the selective delta antagonist naltrindole (10 microM) as expected from the involvement of this class of opioid receptors. However, the addition of naltrindole alone to the perfusing fluid did not modify CGRPLM outflow, indicating that endogenous opioids do not exert a tonic control of CGRP-containing fibers through the stimulation of delta receptors. In contrast, intrathecal perfusion with naloxone (10 microM) or nor-binaltorphimine (10 microM), a selective antagonist of kappa receptors, produced a marked increase in spinal CGRPLM release, suggesting that endogenous opioids acting at mu and kappa receptors, respectively, exert a tonic inhibitory control of CGRP-containing fibers. Indeed, a significant decrease in the spinal release of CGRPLM release could be evoked by the combined addition of U 50488 H (10 microM) plus DAGO (10 microM) to the perfusing medium, indicating that the simultaneous stimulation of both kappa and mu receptors is required for this negative control to occur. This could notably be achieved with morphine (10 microM) in the presence of naltrindole (10 microM) which also produced a significant reduction in the spinal release of CGRPLM. In conclusion, morphine per se did not change CGRPLM release because this drug triggers opposite positive (through the stimulation of delta receptors) and negative (through the concomitant stimulation of both kappa and mu receptors) control mechanisms within the rat spinal cord.

Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca(2+)-dependent release could be evoked by tissue depolarization with 30 mM K+. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABAA agonist, muscimol, and the GABAB agonist, baclofen, inhibited the K(+)-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed -40% with either agonist. Furthermore, the effects of GABAA and GABAB receptor stimulation were not additive. Whereas the effects of muscimol (10 microM) and baclofen (1 microM) could be completely antagonized by bicuculline (1 microM) and phaclofen (10 microM), respectively, complete blockade of the inhibition by GABA (1 microM) could only be achieved in the presence of both antagonists. These data indicate that both GABAA and GABAB receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K(+)-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 microM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.

Superfusion of slices of the dorsal zone of the lumbar enlargement with an artificial cerebrospinal fluid was used to investigate the possible modulation by GABA receptor ligands of the in vitro release of calcitonin gene-related peptide- and substance P-like materials (CGRPLM and SPLM) from the rat spinal cord. Whereas the spontaneous outflow of both peptides remained unaffected, the K+ (30 mM)-evoked overflow of CGRPLM could be partially inhibited (approx. -30%) by GABA (1 microM-0.1 mM) and muscimol (10 microM-0.1 mM) but not by baclofen (1-10 microM). Bicuculline methiodide (1 microM) completely prevented the inhibition by GABA (1 microM) and muscimol (10 microM) as expected from an action through GABAA receptors. By contrast, the K(+)-evoked SPLM overflow was altered neither by GABA nor muscimol and baclofen. These data further support that GABA exerts a presynaptic inhibitory control of (CGRP-containing) primary afferent fibres within the rat dorsal horn.

The possible control by opioids of the release of Met-enkephalin-like material (MELM) from the rat spinal cord was investigated in vitro and in vivo. Superfusion of slices of the dorsal zone of the lumbar enlargement with the mu selective agonists DAGO or PL 017 or the delta selective agonist DTLET produced a significant reduction in the K(+)-evoked MELM release from these tissues. These effects persisted in the presence ot tetrodotoxin, as expected from their mediation through presynaptically located opioid autoreceptors. Furthermore, the inhibitory effect of DAGO and PL 017, but not that of DTLET, was prevented by the preferential mu antagonist naloxone. Conversely, the effect of DTLET was prevented by the delta antagonist naltrindole but not by naloxone. In vivo experiments performed in halothane-anaesthetized rats have shown that the intrathecal perfusion of DAGO and DTLET significantly depressed the spontaneous MELM outflow from the whole spinal cord. In contrast to these mu and delta agonists, the kappa selective agonist U 50488 H did not affect the in vivo- and only slightly reduced (at a very high concentration: 50 microM) the in vitro-release of MELM from the rat spinal cord. These data indicate that both mu and delta opioid autoreceptors are involved in a local presynaptic autoinhibitory control of MELM release in the rat dorsal horn.

The possible modulations by morphine and various opioids of the spinal release of cholecystokinin-like material (CCKLM) evoked by 30 mM K+ was studied in vitro, using slices of the dorsal part of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Addition of the mu agonist, DAGO (0.1-10 microM), to the perfusing fluid produced a concentration-dependent decrease in the peptide release, which could be prevented by the preferential mu antagonist, naloxone. Complex modulations were induced by the delta agonist, DTLET, as this drug inhibited CCKLM release when added at 10 nM-3 microM to the perfusing fluid, but enhanced it at 10 microM. Both effects were preventable by the delta antagonists naltrindole and ICI 154129, suggesting that delta receptors, possibly of different subtypes, mediated the inhibition and stimulation by DTLET. Morphine also exerted a biphasic effect, as the alkaloid decreased CCKLM release at 0.01-0.1 microM and enhanced it at 10 microM. Morphine-induced inhibition was preventable by naloxone, whereas its stimulatory effect could be blocked by naltrindole and ICI 154129. Although inactive on its own on CCKLM release, the selective kappa 1 agonist U 50488H (1 microM) prevented the inhibitory effects of both DAGO (10 microM) and morphine (0.1 microM), suggesting the existence of interactions between kappa 1 and mu receptors within the dorsal zone of the rat spinal cord. These data indicate that low concentrations of morphine exert an inhibitory influence on spinal CCKergic neurons that depends on the stimulation of mu opioid receptors. The excitatory influence of 10 microM morphine likely results from the simultaneous stimulation of mu, delta and kappa receptors, as the inhibitory effect of mu receptor stimulation can be masked by that of kappa 1 receptors, allowing only the expression of a delta-dependent excitatory effect similar to that induced by 10 microM DTLET.

The topographical distribution of 5-HT in the spinal cord of the cat was established by the combined use of a microdissection technique and an enzymic microassay for the indoleamine. The procedure consisted of determining the 5-HT content in microdiscs of tissue (0.07 cu.mm) punched out in various zones of frontal sections of the spinal cord at the level of the cervical and lumbar enlargements. In both cases, the concentration of 5-HT in the grey matter was at least twice as high as that in the white matter. The motoneuron area of the ventral horn contained the highest level of 5-HT (402–472 pg) in both enlargements. In the dorsal horn 5-HT was particularly concentrated in the lateral part of the substantia gelatinosa, where its level (∼-320pg) reached about 75 % that found in the motoneuron area. A close value for 5-HT concentration (337–357 pg) was measured in the central area bordering the ependymal canal. A thoracic transection (Th9–10) induced a progressive decay in 5-HT concentrations below the lesion without altering that found in cervical enlargement. A time-course study has shown that 5-HT was almost undetectable in the lumbar enlargement on the 21st day after the thoracic transection. The selective electrolytic lesion of the nucleus raphe dorsalis did not alter the levels and distribution of 5-HT in the lumbar enlargement. In contrast, a partial lesion of the nucleus raphe magnus (40%) induced a significant decrease in 5-HT levels in this region. This effect was particularly pronounced in the dorsal horns where the 5-HT levels in the substantia gelatinosa were reduced by 44%.

The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat was established owing to the combined use of microdissection techniques and biochemical microassays. The density of 5-HT terminals in various areas of both structures was quantified first by measuring 5-HT levels in microdiscs of frozen tissue. Since the high affinity uptake process for 5-HT appeared undamaged in isotonic homogenates of previously frozen (--5 degrees C) tissues, it was possible to confirm the findings obtained with the measurement of 5-HT levels by also determining 5-HT uptake activity in these microdiscs. However, in the rat neostriatum, but not in the cat caudate nucleus, [3H]5-HT even at a very low extracellular concentration (4.4 -x 10(-8) M) was taken up not only by serotoninergic terminals but also to a significant extent by dopaminergic terminals. In presence of benztropine, this second component was suppressed and [3H]5-HT uptake activity could then be considered as a specific marker of serotoninergic terminals also in the neostriatum of the rat. In both species, 5-HT terminals were mainly localized in the ventrocaudal area of the structure. In this area, 5-HT levels were among the highest values found in the brain (17 ng/mg protein). The density of 5-HT terminals decreased progressively from the acudal to the rostral planes of the neostriatum in rats or the caudate nucleus in cats. The poorest area, i.e. the dorsorostral zone, contained about 4 times less 5-HT than the ventrocaudal zone of the structure. Electrolytic lesion of the dorsalis (B7) and centralis superior (B8) raphe nuclei during early life resulted in a large decrease of 5-HT levels (--90%) in various parts of the neostriatum of adult rats. The present findings might be of interest to further analyze the role of serotoninergic neurons in extrapyramidal functions.

Summary Intraventricular 6-OHDA was injected in kittens at different stages of development, and the subsequent sleep polygram was analyzed, in order to determine the role of the catecholaminergic system in the ontogenesis of sleep regulations during the first and the second postnatal months. 6-OHDA, used with or without previous chlorimipramine treatment, led within a 10-day period to drastic reductions of the endogenous monoamines in the forebrain of all age groups. Although the neurotoxicity of 6-OHDA was almost constant among the different age groups, the effects on sleep depended on the age of the animals at the time of the injection. In the 5-week-old injected kittens, 6-OHDA affected PS according to an adult-like pattern. In the 3-week-old kittens, 6-OHDA alone (leading to both catecholamines and serotonin decreases) induced the same PS deficit as in the adult cat. In the 1- and 2-week-old kittens, neither 6-OHDA alone, nor 6-OHDA with previous chlorimipramine treatment, distrubed the sleep regulations. These data are compared to similar experiments performed in the adult cat. They are discussed in terms of sleep control ontogenesis. It is concluded that the catecholaminergic system plays no important role in the mechanisms of sleep regulation in the early postnatal period in the kitten, whereas its regulatory influence on PS is confirmed in the juvenile animal. The functional maturation of the catecholaminergic system in terms of sleep regulation is achieved between the third and the fifth week of postnatal life.

Using push-pull cannulae, the release of endogenously synthesized [ 3 H]serotonin was estimated in both substantia nigra and caudate nuclei of ‘encephale isole’ cats. The unilateral nigral application of dopamine (10 −7 M) reduced [ 3 H]serotonin release in ipsilateral structures whereas α-methylparatyrosine (10 −4 M) induced opposite effects. Both treatments decreased [ 3 H]serotonin release in the contralateral caudate nucleus but not in the contralateral substantia nigra. As a working hypothesis it is suggested that the effects of observed are related to changes in the activity of nigroraphe neurons regulated by dopamine released from dendrites of the nigrostriatal dopaminergic neurons. However it cannot vet be excluded that the local changes in [ 3 H]serotonin release induced by the nigral application of dopamine or α-methylparatyrosine result from presynaptic modulation.

In order to investigate possible changes induced by noxious thermal stimuli in the activity of enkephalinergic neurones at various levels of the spinal cord, either the whole cord, the cervicotrigeminal area or the lumbar area were perifused with artificial CSF at a rate of 0.1 ml/min in halothane anaesthetized rats, and Met-enkephalin-like material (MELM) was measured in 0.5 ml fractions collected from the perifusates. Immersing the muzzle of intact rats in water at 52°C produced an significant enhancement of MELM content in perifusates from both the whole spinal cord and the cervicotrigeminal area but not from the lumbar area. Heating the tail resulted in an increase in MELM release from the whole spinal cord of intact as well as of cervically transected rats. It is concluded that noxious thermal stimuli can induce a segmental release of MELM, i.e., only within spinal zones receiving the nociceptive inputs.

Although the physiological functions of the endogenous opioid systems are not yet clearly established, it is widely accepted that they exert an inhibitory control on pain transmission. However, the well-documented hypoalgesic effects of low doses of the opiate antagonist naloxone both in animals and humans do not fit in with this concept. The present investigations, at two different spinal/medullary levels (viz. cervicotrigeminal and lumbar) demonstrate that, in the rat, a noxious mechanical stimulus does not alter the release of Met-enkephalin-like material (MELM) from neural segments related to the stimulated area of the body, but does increase its release from other segments. Electrophysiological studies have already demonstrated the existence of such heterosegmental mechanisms, notably ‘diffuse noxious inhibitory controls’ (DNIC), which are naloxone-reversible and could play an important role in pain perception. The involvement of spinal enkephalins in DNIC would seem to mean that the heterosegmental spinal release of MELM triggered by noxious stimuli participates in pain processes.

Summary The direct electrical stimulation (with biphasic pulses of 1 msec, 10 pulses/sec, 200 μA, for 30 min) of the nucleus raphe magnus in chloral hydrate anaesthesized rats produced a significant acceleration (+ 50%) of 5-HT synthesis in the spinal cord as revealed by the increased rate of 5-HTP accumulation occurring at this level after the blockade of central 5-HTP decar☐ylase with benserazid. In contrast, no change was detected in 5-HT metabolism in the forebrain of stimulated rats. The acceleration of 5-HT synthesis was likely not due to an increased availability of tryptophan for the rate-limiting enzyme, tryptophan hydroxylase, since the concentration of this amino acid was changed neither in the spinal cord, nor in the forebrain of stimulated rats. The measurement of tryptophan hydroxylase activity in soluble extracts from the spinal cord of control and stimulated rats revealed that the acceleration in 5-HT synthesis produced by the electrical stimulation of the nucleus raphe magnus was not associated with a persisting activation of this enzyme. Although one cannot completely exclude that a short-lasting activation of tryptophan hydroxylase, no longer detectable in soluble extracts, has occurred in the spinal cord of stimulated rats, the present findings rather suggest that the rate of 5-HT synthesis can be controlled by factors other than only the concentration of tryptophan and the intrinsic activity of tryptophan hydroxylase in serotoninergic neurons. The demonstration of an acceleration of 5-HT synthesis in bulbospinal serotoninergic neurons under stimulating conditions close to those producing analgesia in rats further supports the role of these neuronal systems in the physiological mechanisms of pain control.

A new isotopic approach has been developed to study the in vivo release of serotonin (5-HT). 'Encéphale isolé' cats were implanted with a push-pull cannula in the ventrocaudal part of the head of the caudate nucleus to estimate the release of [3H]5-HT continuously synthesized from L-[3H]tryptophan. Both [3H]5-HT and [3H]tryptamine were found in superfusates. Resting steady state in the release of [3H]indoleamines was observed as soon as 20 min after the beginning of the superfusion with L-[3H]tryptophan; the levels of [3H]5-HT in superfusates were 2.5 times those of [3H]tryptamine and about 6 times the blank value. They were markedly enhanced in the presence of fluoxetine (5 x 10(-6)M), a blocker of the 5-HT uptake process. A marked increase in the release of [3H]5-HT was seen during the local depolarization of 5-HT terminals with potassium chloride (60 mM) or batrachotoxin (10(-6)M) or during the stimulation of 5-HT cell bodies in the nucleus raphe dorsalis with L-glutamic acid (5 x 10(-5)M). These treatments did not enhance the efflux of [3H]tryptamine. The potassium-evoked release of [3H]5-HT was reduced by LSD (10(-5)M). LSD added alone in the superfusing fluid was without effect. The batrachotoxin-evoked release of [3H]5-HT was inhibited in the presence of tetrodotoxin (9 x 10(-6)M). The spontaneous release of [3H]5-HT and [3H]tryptamine was markedly reduced in the presence of a calcium-free medium containing cobalt (10 mM). A transient slight reduction in the spontaneous release of [3H]5-HT was observed in the presence of tetrodotoxin (9 x 10(-6)M). The local cooling of 5-HT cell bodies with a cryoelectrode induced a slight reversible decrease in [3H]5-HT release. These last two treatments were without significant effect on [3H]tryptamine efflux in superfusates. These results indicate that the release of [3H]5-HT endogenously formed from [3H]tryptophan is dependent on nerve activity and that this is not the case for [3H]tryptamine. The advantages of the isotopic approach for in vivo studies on the release of 5-HT are discussed.

Summary The intraventricular administration of 6-hydroxydopamine (6-OHDA) to kittens between 5 days and 4 months of age induced marked changes in the endogenous levels of DA, NE and 5-HT in various brain areas. In contrast to the well-known selective effect of 6-OHDA against catecholaminergic neurones in the rat, serotoninergic neurones were also markedly affected by 6-OHDA treatment in kittens; particularly in the hippocampus and the colliculi, 5-HT levels were markedly and permanently decreased after the intraventricular administration of 6-OHDA. A significant but less pronounced reduction in 5-HT levels was also noted in other areas such as the piriform cortex, the cerebral neocortex, the cerebellum and the septum. Only very discrete changes were detected in the caudate nucleus, the olfactory tubercle and the raphe area. The administration of chlorimipramine (10 mg/kg, i.p.) 1 h before 6-OHDA treatment completely prevented the effects of the neurotoxic agent on serotoninergic innervation. Marked regional differences were also noted concerning the effects of 6-OHDA treatment on dopaminergic neurones. Whereas DA levels in the raphe area and the hypothalamus were almost unaffected, they were permanently reduced by about 50% in the caudate nucleus and the olfactory tubercle after the intraventricular administration of 6-OHDA. In the caudate nucleus, the reduction was even much more pronounced (−90%) when 6-OHDA was administered during the first 3 postnatal weeks. In most forebrain areas (hippocampus, piriform cortex and cerebral neocortex) and in the cerebellum, NE levels were permanently reduced to about 10% of those of control kittens as soon as the third day following the intraventricular injection of 6-OHDA. In contrast, after a transient drop, NE levels in the lateral brain stem (containing the locus coeruleus) of 6-OHDA-treated kittens returned and even surpassed (+100%) those found in age-paired controls. Analyses of the characteristics of l -[ 3 H]NE uptake in synaptosomes indicated that 6-OHDA treatment resulted in both a striking loss of specific uptake sites in forebrain areas and a significant increase in the V max of the NE uptake process in synaptosomes from the lateral brain stem. However, in contrast to the rapid increase in NE levels, this change in V max occurred much later, since it was first detected at more than one month after 6-OHDA treatment. This delay suggests that the doubling in NE levels occurring for the first month following the intraventricular administration of 6-OHDA simply resulted from an increased accumulation of the catecholamine in noradrenergic terminals in the lateral brain stem. Later on, the change in V max might indicate a sprouting of new noradrenergic terminals in the vicinity of the locus coeruleus area. The intraventricular administration of 6-OHDA resulted in a significant increase in the maximal stimulatory effect of l -isoproterenol on adenylate cyclase activity in homogenates of the cerebral cortex and the lateral brain stem. Therefore, not only the degeneration (in the cerebral cortex) but also the proliferation (in the lateral brain stem) of noradrenergic terminals were associated with an increase in the density of beta-adrenergic receptors. These results are discussed in relation to the possible function of the noradrenergic sprouts in the lateral brain stem.

The effects of LSD on 5-hydroxytryptamine (5-HT) metabolism were studied in rat hippocampal and striatal slices. One hour after in vivo treatment with the drug (1 mg/kg i.p.) the synthesis of [3H]5-HT from l -[3H]tryptophan ([3]Try) was only decreased in the striatum. This effect was associated with a significant reduction in the accumulation of [3H]Try in tissues, whereas endogenous levels of Try in the striatum and hippocampus were significantly increased by this treatment. Such modifications of 5-HT metabolism could not be reproduced by adding LSD directly into the incubating medium of control slices. The release of [3H]5-HT, synthetized from [3H]Try or taken up by tissues, was very much increased when concentrations of K+ in the incubating medium reached 30 or 50 m M. This effect was counteracted by LSD, the drug being administered previously in vivo or added in vitro. Finally, high concentrations of LSD (0.1 m M) added in vitro induced a reserpine-like effect on newly synthetized as well as exogenous [3H]5-HT: the amine was markedly inactivated in its acidic metabolite.

Tooth pulp stimulation in halothane-anaesthetized cats induced a long lasting (greater than or equal to 3 h) increase in the levels of Met-enkephalin-like material (MELM) in the cisternal CSF. Chromatographic analyses (gel filtration, HPLC) revealed that most of the immunoreactivity was attributable to high molecular weight (mol. wt. greater than or equal to 4000) compounds; in non-stimulated cats, Met-enkephalin (largely in the form of the sulfoxide derivative) only accounted for about 10% of total MELM. In contrast, following tooth pulp stimulation, a large increase in Met-enkephalin (plus Met-Ox5-enkephalin) levels was noted so that the pentapeptide thus represented more than 50% of total MELM. No evidence was obtained for the presence of Met-enkephalin-Arg6-Phe7 in the cisternal CSF of halothane-anaesthetized cats. These data strongly suggest that the activity of enkephalinergic neurons was increased following nociceptive stimulation. This indirectly supports the possible physiological role of enkephalinergic systems in modulating nociceptive inputs.

The lumbar spinal cord was superfused with artificial CSF at a rate of 0.1 ml/min in halothane anaesthetized rats. Under resting conditions, Met-enkephalin-like material (MELM) found in superfusates corresponded to a spinal release of 4.2 ± 1.4pg Met-enkephalin equivalents per 5 min. During a 30-min period in which pinches were applied to the muzzle, the MELM content in the superfusates increased markedly (by 120.5 ± 32.9% ). This effect was totally suppressed following bilateral lesions of the dorsolateral funiculi (DLF), under both chronic and acute conditions. It is concluded that strong mechanical stimuli applied in the trigeminal region can induce the release of MELM within the lumbar spinal cord via mechanisms involving the DLF. This heterosegmental release of Met-enkephalin may participate in the management of pain by methods involving high intensity stimulation.

l -Tryptophan uptake was studied in C6 glial cells and found to be a very rapid and temperature dependent process. Kinetic analysis shows the presence of two saturable transport systems acting at different tryptophan concentrations. Both these high- and low-affinity processes were found in 3T3 fibroblast cultured cells, as well as in synaptosomes and cell suspensions obtained from rat cortex. No Na + requirement could be demonstrated in a wide range of tryptophan concentrations. The data presented here suggest that tryptophan accumulation in glial cells could be dependent on allosteric changes of the carrier, which might be relevant to the understanding of 5-HT synthesis regulation.

In the present study, using neonatal intraventricular injections of 6-hydroxydopamine (6-OHDA) and the fluorescence histochemical method for monoamines, it is observed that an extensive plexus of noradrenaline (NA) fibres develops in the pontine region of the cat brain subsequently to the neonatal destruction of the ascending NA bundles and of the NA innervation in the cerebral cortex by the neurotoxin. This plexus is only partly conserved 10 months after the 6-OHDA treatment. Generally, only a limited number of NA perikarya degenerate in the region of the locus coeruleus, the others (nucleus subcoeruleus senso lato) exhibiting the same strong fluorescence as the new NA fibres. Using the radioautographic method after intraventricular injections of [3H]NA, our work demonstrates also the transient disappearance (at least one month) of the uptake of [3H]NA in the pons, whose NA cell bodies and nerve terminals are no longer labeled in the same number as in control animals. The possibility of again labeling significantly NA perikarya and numerous nerve terminals occurred between 3 and 5 months of age, probably indicating both a re-establishment of normal uptake properties in the preserved NA perikarya and nerve terminals and some maturation of the uptake mechanisms in the abnormal NA fibres of the pons. This last observation is at variance with data from newborn animals showing that the uptake of NA develops in parallel with the accumulation of endogenous NA in catecholamine nerve terminals. The present results, however, do corroborate and complement previous biochemical data obtained in the cat after neonatal injection of 6-OHDA.

The influence of the intrapallidal application of GABA-related compounds on the release of Met-enkephalin in the globus pallidus and the caudate nucleus in the two hemispheres was investigated in vivo in the cat. For this purpose, the 4 structures were continuously superfused with an artificial CSF through implanted push-pull cannulae and Met-enkephalin released in superfusates was determined using a specific radioimmunoassay. GABA (10-500 microM) reduced the local release of Met-enkephalin during its application but once the amino acid was removed from the superfusing fluid, an increase in the peptide release was observed. Diazepam (10 microM) induced only an inhibitory effect whereas muscimol (1 microM) stimulated Met-enkephalin release. Opposite changes in Met-enkephalin release were also seen with the GABA antagonists, bicuculline methiodide (1 microM) and picrotoxin (10 microM), suggesting that the local regulation of Met-enkephalin release by GABA related compounds may be mediated by at least two types of GABA receptors. In several cases, the unilateral pallidal application of GABA agonists and antagonists induced significant changes in Met-enkephalin release at distant structures. The most striking effect was observed with diazepam which markedly reduced the peptide release in both caudate nuclei and pallida. These data suggest that GABAergic systems can contribute to some bilateral regulation of striato-pallidal enkephalinergic neurones.

The possible influence of GABAergic systems on the activity of enkephalinergic neurones within the basal ganglia was examined by measuring the release of Met-enkephalin in the caudate nuclei and pallida of halothane-anesthesized cats treated by intra-caudate applications of GABA-related drugs. Depending on the concentration used, GABA exerted local stimulatory (at 10 microM of the amino acid) or inhibitory (at 0.5 mM) action on Met-enkephalin release in the cat caudate nucleus. Only the inhibition was reproduced by the GABA agonists muscimol (1 microM) and (-)-balcofen(10 microM) and by diazepam 10 microM). Conversely, the intra-caudate application of the GABA antagonist bicuculline enhanced markedly the local release of the pentapeptide. Complementary studies using slices of the rat striatum (caudate nucleus + putamen) revealed that a low concentration of GABA (10 microM) tended to increase the K+-evoked efflux of Met-enkephalin, whereas a high concentration of the amino acid exerted a strong inhibitory effect on the peptide release. Such in vivo and in vitro findings suggest that the GABA-induced inhibition of Met-enkephalin release took place via the stimulation of specific GABA A and GABA B receptors within the caudate nucleus, whereas the GABA-induced increase of the peptide release might involve some intracellular regulatory processes in striatal neurones containing both GABA and enkephalins. In addition to altering the local release of Met-enkephalin, intra-caudate applications of GABA-related drugs affected the peptide release in the ipsilateral globus pallidus and contralateral basal ganglia. The observed changes suggest that GABA A, but not GABA B, receptors participated in some tonic inhibitory influence of striatal GABAergic neurones on the striato-pallidal enkephalinergic system. Furthermore, the present results confirmed previous studies (Bourgoin et al.) showing that GABAergic neurones can contribute to some bilateral modulation of enkephalinergic neurones within the basal ganglia.

Summary Electrolytic raphe lesion was performed in 4–6-day-old rats and the resulting changes of 5-HT metabolism within the central nervous system were analyzed up to 9 months later. As soon as the 2nd day following the selective destruction of B7 and B8 nuclei, forebrain 5-HT levels were decreased by more than 75%. This reduction persisted for at least 9 months with no sign of recovery. The time course of 5-HIAA decrease was parallel to that of the indoleamine so that the ratio of 5-HIAA over 5-HT levels in the forebrain of lesioned rats was similar to that estimated in controls, whatever their age. This result would suggest that the remaining serotoninergic neurons in the lesioned rats did not develop a compensatory hyperactivity. The raphe lesion induced no change in MAO activity and synaptosomal tryptophan uptake but a pronounced decrease in the Vmax of synaptosomal 5-HT uptake process in various forebrain areas occurred. The serotonin sensitive adenylate cyclase activity in colliculi homogenate was not altered by the lesion suggesting that this enzyme was probably located in postsynaptic membranes. In addition, this observation would indicate that 5-HT receptors which are linked to this adenylate cyclase did not become supersensitive following the selective degeneration of serotoninergic neurons. Animals without forebrain serotoninergic innervation might be of great interest to analyse the role of serotoninergic neurons in various functions (sleep, analgesia, thermoregulation).

In vivo perfusion of the subarachnoid space with an artificial cerebrospinal fluid (CSF) in paralyzed halothane-anesthetized rats allowed the collection of methionine-enkephalin (Met-Enk)-like material (MELM) released from the spinal cord. Bio-Gel P2 chromatography and high-performance liquid chromatography showed that 65% of this material corresponded to authentic Met-Enk. Under resting conditions, about 1 pg of MELM per minute was regularly released for at least 3 h; for Met-Enk, this value corresponded to a fractional rate constant of 0.002% (i.e. tissue content of the pentapeptide which was released per minute from the whole spinal cord). Perfusion with K+-enriched (40–60 mM) CSF resulted in a marked enhancement(+ 150–200%) of spinal MELM release. Similarly, calibrated pinches of the muzzle and i.p. administration of acetic acid, two strong noxious stimuli in awake animals, induced a significant increase (+ 75–150%) in spinal MELM release. In contrast, pinches applied to the tail did not enhance but instead slightly reduced (−35%) MELM release from the rat spinal cord. These data suggest that mecchanisms other than segmental controls could be involved in the activation of spinal enkephalinergic neurons by some nociceptive stimuli.

Electrolytic lesions of the anterior raphe nuclei in the 4-6 day puppy rat were performed stereotaxically. The subsequent development of the sleep parameters in these animals was followed up to the second month of life. No difference could be found between the lesioned and control rats, neither in the qualitative nor in the quantitative characteristics of slow wave sleep (SWS) and paradoxical sleep (PS). However, when performing identical lesions on 3-5 week-old animals, a relative insomnia was obtained, more severe for PS than for SWS. The correlation of the neurophysiological data with the biochemical analysis of monoamines in the forebrain revealed that the serotoninergic (5-HT) system of the anterior raphe in the early lesioned animals was almost totally destroyed, with no recovery up to 9 months of age. Our results indicate that the anterior raphe nuclei are not functional during the first week of age, in regard to sleep control, whereas they do play an important role in sleep regulation after 3 weeks of age. Furthermore, some compensatory mechanisms which develop in the early lesioned animal as a consequence of the lesion, could explain the exhibition of normal sleep ontogenesis. The possible nature of these mechanisms is discussed in light of the monoaminergic theory of sleep control.

The in vivo release of 5-HT was examined in the rat brain. For this purpose, the left lateral ventricle was perfused at a constant rate with an artificial CSF for several hours in animals anaesthetized with halothane. 5-HT was estimated in serial 1-h collected fractions. The amine was first isolated by adsorption on a Sephadex G-10 column and then assayed using the radioenzymatic method of Saavedra et al.37, slightly modified to improve its sensitivity. The quantity of 5-HT released spontaneously during the first hour fraction was 296 pg, it was lower (99 pg/h) in the following fractions. 5-HT released into the CSF may in great part originate from serotoninergic terminals localized in structures surrounding the ventricle. This was suggested by experiments in which exogenous [3H]5-HT or [3H]tryptophan were perfused through the lateral ventricle during a few hours. [3H]5-HT taken up or synthetized was mainly localized in structures surrounding the ventricular space. The acute injection of 5-hydroxytryptophan (100 mg/kg) induced an immediate important and long lasting increase of 5-HT release. In contrast the acute injection of tryptophan (100 mg/kg) led to a transient and moderate elevation of 5-HT release which was only detected during the second hour of perfusion. Curiously a similar pattern of transmitter release was observed following the constant intravenous infusion of the amino acid (70 mg/kg/h) except that the increase in 5-HT release was much more pronounced during the second hour than after the acute injection. Parallel experiments were made to determine the time course of the changes of free and total tryptophan levels in plasma and of those of tryptophan, 5-HT, and 5-hydroxyindoleacetic-acid (5-HIAA) in brain tissues, induced by the acute and long term administrations of tryptophan. Moreover the rate of 5-HT synthesis was estimated using the monoamine oxidase inhibition method 2 and 5 h after both tryptophan treatments in halothane anaesthetized rats. 5-HT levels and the synthesis rate of the transmitter were increased at 2 h (when both tryptophan treatments stimulated 5-HT release). Despite the presence of high tryptophan levels in plasma and tissues and of high 5-HT and 5-HIAA levels in tissues, the synthesis rate of 5-HT (as the 5-HT release) was similar to that of controls 5 h after the onset of tryptophan infusion. These results suggest that some relationships occurred between the changes in 5-HT SYNTHESIs and release after the first hour of perfusion. The absence of effects of tryptophan treatments on 5-HT release during the first hour of perfusion are also discussed.