Opposite changes in dopamine metabolites and met-enkephalin levels in the ventricular CSF of patients subjected to thalamic electrical stimulation.

High-frequency electrical stimulations of thalamic nuclei are currently used for the suppression of parkinsonian or essential tremor and for the relief of some types of intractable pain in man. However, the mechanisms by which such stimulations exert their therapeutic effects are essentially unknown. Attempts were made to provide some insight into these mechanisms by measuring the levels of the dopamine metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and met-enkephalin-like immunoreactivity in ventricular cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) or multiple sclerosis (MS) after a 30-minute therapeutic electrical stimulation of the ventralis intermedius nucleus of the thalamus. In nonstimulated control patients, the levels of these compounds did not significantly differ in two CSF samples taken 30 minutes apart. In stimulated patients, a decrease in dopamine metabolite levels associated with a relative increase in met-enkephalin-like immunoreactivity were observed in the CSF sample taken after the 30-minute stimulation as compared to the sample taken immediately before the stimulation. In contrast, the levels of 5-HIAA remained unaffected by the stimulation. These data confirmed the existence of negative interactions between dopaminergic and enkephalinergic systems in man similar to those previously demonstrated in rats. In addition, they suggest that alterations in dopaminergic or enkephalinergic neurotransmission might be involved in the therapeutic action of thalamic electrical stimulation in patients with parkinsonian symptoms and other patients.

Polyarthritis-associated changes in the opioid control of spinal CGRP release in the rat.

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.

Increased in vivo release of calcitonin gene-related peptide-like material from the spinal cord in arthritic rats.

Possible alterations in spinal systems containing calcitonin gene-related peptide (CGRP) due to polyarthritis were assessed in rats 3-4 weeks after an intradermal injection of Freund's adjuvant in the low back. The tissue levels of CGRP-like material (CGRPLM) were approximately 50% higher in the dorsal zone of the spinal cord and dorsal root ganglia at both the cervical and lumbar (but not thoracic) segments in polyarthritic rats than in age-paired control animals. In addition the rate of the spinal release of CGRPLM determined through an intrathecal perfusion procedure in halothane-anaesthetized animals was approximately 15-fold higher in polyarthritic rats than in controls. The blockade of mu-opioid receptors by intrathecal perfusion with 10 microM naloxone produced a larger increase in the spontaneous CGRPLM outflow in polyarthritic rats than in age-paired controls. Furthermore, the stimulation of mu-opioid receptors by intrathecal perfusion with 10 microM DAGO significantly inhibited the spinal outflow of CGRPLM only in polyarthritic rats. These data indicate that CGRP-containing primary afferent fibres are markedly activated in chronic suffering polyarthritic rats. This activation occurs in spite of an increased tonic inhibitory control by endogenous opioids acting at mu receptors.

Acupuncture-like stimulation induces a heterosegmental release of Met-enkephalin-like material in the rat spinal cord.

In order to investigate the effects induced by acupuncture on the activity of enkephalinergic neurons in the spinal cord, either the lumbar or the cervico-trigeminal area was perfused with artificial cerebrospinal fluid (CSF) (0.1 ml/min) in halothane-anaesthetized rats, and Met-enkephalin-like material (MELM) was measured in 0.5 ml fractions of the perfusates. The effects of manual acupuncture performed by a traditional Chinese acupuncturist at the 'Zusanli' point on the right hind limb were compared to the effects induced by acupuncture applied at a non-acupoint next to 'Zusanli.' The manipulation of needles either at the 'Zusanli' point or at the non-acupoint had no effect on the release of MELM from the lumbar area but significantly increased the release from the cervico-trigeminal zone. It is concluded that manual acupuncture triggers a heterosegmental activation of enkephalinergic neurones within the spinal cord and that this effect is non-specific in terms of the location of the stimulated point.

Neonatal capsaicin treatment abolishes the modulations by opioids of substance P release from rat spinal cord slices.

The possible modulation by opioids of substance P (SP) release at the spinal level was studied using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Capsaicin (0.5 microM) selectively evoked a Ca2+-dependent overflow of SP-like material (SPLI) from primary afferent fibers which was enhanced in the presence of mu-opioid agonists (DAGO, FK 33824, sufentanyl, morphine), reduced by the delta-opioid agonist DTLET, and unaltered by the kappa-opioid agonist U 50488 H. Selective antagonists (naloxone, ICI 154129) prevented the effects of mu- and delta-opioid agonists. Neonatal capsaicin (50 mg/kg) abolished the stimulatory effect of in vitro capsaicin (0.5 microM) but not that of 30 mM K+ on SPLI outflow. This K+-induced SPLI release was unaffected by opioids. Presynaptic inhibitory control of SPLI release from capsaicin-sensitive primary afferent fibers might account for the analgesic effect of delta- but not mu- and kappa-opioid agonists at the spinal level.

Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo.

Perfusion of the intrathecal space with artificial CSF was achieved in control and arthritic rats under halothane anaesthesia in order to collect the met-enkephalin-like material (MELM) released from the whole spinal cord. On the fourth week following the intradermal injection of Freund's adjuvant to induce arthritis, a marked reduction (-56%) in the spontaneous outflow of MELM was noted in arthritic rats. This effect did not involve changes in the degradation process of MELM, since it persisted when kelatorphan was added to the perfusing fluid in order to inhibit completely the peptidases acting on met-enkephalin. Raising the K+ concentration in the perfusing fluid from 2.4 to 40 mM, as well as moving the hind paws, produced a significant enhancement of MELM release which was (at least) as pronounced in arthritic as in control rats. These results suggest that the basal activity of spinal enkephalinergic neurones, but not that triggered by various stimuli, is reduced in arthritic rats.

Opioid receptors and neuropeptides in the CNS in rats treated chronically with amoxapine or amitriptyline.

The central mechanism responsible for the potentiation by antidepressant drugs of analgesia induced by morphine, was explored by measuring the levels of various neuropeptides (met-enkephalin, leu-enkephalin, dynorphin, substance P and cholecystokinin-like materials) and the density of delta and mu opioid binding sites in the spinal cord of rats treated for 14 days with amoxapine (10 mg/kg i.p., daily) or amitriptyline (10 mg/kg i.p., daily). Similar measurements were made in the hypothalamus and cerebral cortex for comparison. Chronic treatment with amoxapine or amitriptyline did not affect the levels of dynorphin, substance P and cholecystokinin, but markedly enhanced the levels of leu-enkephalin in the three structures examined. The levels of met-enkephalin were also increased after treatment with amitriptyline but only in the spinal cord and hypothalamus. No changes in opioid receptors were found in the cerebral cortex, but the densities of delta and mu opioid binding sites were increased in the spinal cord, and decreased in the hypothalamus of rats treated with amoxapine or amitriptyline. These changes induced by antidepressants in opioidergic markers at the spinal level might account for the potentiation of the action of morphine in amoxapine- or amitriptyline-treated rats. In addition, the observed alterations in the same markers in the hypothalamus could be associated with changes induced by antidepressants in neuroendocrine regulation.

Distribution of Met-enkephalin immunoreactive fibres in the thalamus of the cat.

The Met-enkephalin-like immunoreactivity was studied in the thalamus of the cat using an indirect immunoperoxidase method. The densest network of immunoreactive fibres and terminals was observed in the epithalamus and the intralaminar nuclei, particularly those located along the midline nuclei interanteromedialis, submedius, rhomboidens and reuniens. The nuclei parafascicularis and centrum medianum contained also numerous immunoreactive fibres and terminals, whereas the lamina medullaris externa had a lower density of immunoreactive terminals. Enkephalin fibres were almost totally absent in the lateral nuclei of the thalamus, and in the posterior group only the magnocellular part of the corpus geniculatum mediale contained some immunoreactive fibres.

Local and remote effects of intra-caudate administration of GABA-related drugs on Met-enkephalin release in the basal ganglia.

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.

Midbrain raphe lesion in the newborn rat: II. Biochemical alterations in serotoninergic innervation.

Electrolytic raphe lesion was performed in 4-6-day-old rats and the resulting changes of 5HT 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 5HT 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 pronounce decrease in the Vmax of synaptosomal KHT 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).

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices.

The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized 3H-5-HT were examined in rat hypothalamic slices. 3H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with L-3H-tryptophan in the presence of the various drugs tested. ACh (10(-5) M) in the presence of eserine (2 X 10(-4) M), and carbachol (10(-5) M) stimulated the release of 3H-5-HT. In contrast, oxotremorine (10(-5) M) reduced the 3H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10(-6) M) and d-tubocurarine (10(-6) M). It was not reduced by the muscarinic antagonists, atropine (10(-6) M) and scopolamine (10(-6) M). In fact, each of two antagonists added alone to the incubating medium enhanced 3H-5-HT release. The scopolamine (10(-6) M) stimulating effect on 3H-5-HT release was suppressed by d-tubocurarine (10(-6) M). Finally, the inhibiting effect of oxotremorine on 3H-5-HT release was not prevented by d-tubocurarine (10(-6) M) but was in the presence of atropine (10(-6) M) or scopolamine (10(-6) M). In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of 3H-5-HT and 3H-5-HIAA from L-3H-tryptophan and on the accumulation of L-3H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous 3H-5-HT in hypothalamic synaptosomes (P2 fraction). These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of 3H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of 3H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.