Long-term treatment with the antioxidant drug EGb 761 at senescence restored some neurobehavioral effects of chronic ultramild stress exposure seen in young mice.

In this study, we compared the effects of chronic ultramild stress (CUMS) exposure on decision-making behavior in a validated test, and on the stress responsive serotoninergic and dopaminergic systems in four age groups of B6D2F1 female mice (5-6, 11-12, 17-18 and 23-24 months old). The levels of serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid (5-HIAA) were measured in the brain stem, the cortex, the striatum and the hippocampus; the levels of dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) were measured in the brain stem and the striatum. The influence of a long-term treatment with the extract of Ginkgo biloba leaves EGb 761 (Tanakan) on age- and stress-related changes was also investigated in the two oldest age groups. In the absence of drug treatment, middle-age mice were the least efficient in making a decision, and senescent mice exhibited reduced levels of both 5-HT and DA and their metabolites in all the brain areas examined. CUMS facilitated evaluation and choice behavior in all age groups, but induced age-dependent reduction of hesitation, acceleration of information processing and reduction in serotoninergic neurotransmission. In senescent mice, EGb 761 reduced the impact of stress on evaluation and hesitation, and restored some stress-related neurobehavioral changes that were only seen in young mice, i.e. acceleration of information processing and reduction in brain 5-HIAA levels. Restoration of some plasticity of the serotoninergic systems might contribute to the stress alleviating influence of EGb 761 in old age.

Excessive activation of serotonin (5-HT) 1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-ht transporter knock-out mice.

Deficiency in the monoamine degradation enzyme monoamine oxidase A (MAOA) or prenatal exposure to the monoamine uptake inhibitor cocaine alters behavior in humans and rodents, but the mechanisms are unclear. In MAOA knock-out mice, inhibiting serotonin synthesis during development can prevent abnormal segregation of axons in the retinogeniculate and somatosensory thalamocortical systems. To investigate this effect, we crossed MAOA knock-outs with mice lacking the serotonin transporter 5-HTT or the 5-HT1B receptor, two molecules present in developing sensory projections. Segregation was abnormal in 5-HTT knock-outs and MAOA/5-HTT double knock-outs but was normalized in MAOA/5-HT1B double knock-outs and MAOA/5-HTT/5-HT1B triple knock-outs. This demonstrates that the 5-HT1B receptor is a key factor in abnormal segregation of sensory projections and suggests that serotonergic drugs represent a risk for the development of these projections. We also found that the 5-HT1B receptor has an adverse developmental impact on beam-walking behavior in MAOA knock-outs. Finally, because the 5-HT1B receptor inhibits glutamate release, our results suggest that visual and somatosensory projections must release glutamate for proper segregation.

Altered opioid-mediated control of the spinal release of dynorphin and met-enkephalin in polyarthritic rats.

Previous studies showed that spinal opioidergic neurotransmission is markedly altered in the polyarthritic rat, a model of chronic inflammatory pain. Present investigations aimed at assessing possible changes in opioid-mediated control of the spinal outflow of met-enkephalin (ME) and dynorphin (DYN) in these animals. Intrathecal (i.t.) perfusion under halothane anesthesia showed that polyarthritis was associated with both a 40% decrease in the spinal outflow of ME-like material (MELM) and a 90% increase in that of DYNLM. Local treatment with the mu-opioid agonist DAGO (10 microM i.t.) inhibited equally (-30%) the MELM outflow in polyarthritic and control rats, whereas the delta agonist DTLET (10 microM i.t.) also reduced the peptide outflow in controls (-27%) but enhanced it in polyarthritic animals (+56%). On the other hand, both DAGO (10 microM i.t.) and DTLET (10 microM i.t.) decreased (-40 and -49%) DYNLM outflow in polyarthritic rats, but were inactive in controls. Finally, neither MELM outflow nor that of DYNLM were affected by the kappa-agonist U50488H (10 microM i.t.) in both groups of rats. In all cases, the changes due to active agonists could be prevented by specific antagonists which were inactive on their own except the kappa antagonist nor-binaltorphimine (10 microM i.t.) that decreased (-38%) DYNLM outflow in polyarthritic rats. These data indicate that functional changes in spinal opioid receptors may promote enkephalinergic neurotransmission and reduce dynorphinergic neurotransmission in polyarthritic rats, thereby contributing to the analgesic efficacy of opioids in inflammatory pain.

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.

The "orphan" Na+/Cl(-)-dependent transporter, Rxt1, is primarily localized within nerve endings of cortical origin in the rat striatum.

Previous studies have shown that the striatum expresses very low levels of Na+/Cl(-)-dependent "orphan" transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6-hydroxydopamine-induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin-dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one-third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1-positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.

Identification and characterization of a zinc finger gene (ZNF213) from 16p13.3.

During our search for the familial Mediterranean fever (FMF) gene, we identified by cDNA selection a 1.2 kb cDNA fragment representing a novel human gene that is expressed in a wide variety of tissues. This gene spans approx. 8.0 kb genomic DNA and has seven exons. Its 3' untranslated region contains a long tandem repeat that gives rise to a polymorphism with two alleles of approx. 1.1 kb and 1.0 kb, with the 1.1 kb allele in strong linkage disequilibrium with FMF in patients of different ethnic backgrounds. However, both genetic and mutational analyses have excluded this gene as the one responsible for FMF. The predicted 424 amino acid protein, designated ZNF213, contains three C2H2 zinc fingers, a Kruppel associated A box and a leucine rich motif (LeR domain/SCAN box), strongly suggestive of a transcription factor.

Construction of an approximately 700-kb transcript map around the familial Mediterranean fever locus on human chromosome 16p13.3.

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the approximately 700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron-exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (between D16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.

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.

Stress- and yohimbine-induced release of cholecystokinin in the frontal cortex of the freely moving rat: prevention by diazepam but not ondansetron.

The in vivo release of cholecystokinin (CCK)-like material (CCKLM) was measured in the frontal cortex of freely moving rats using the microdialysis technique combined with a sensitive radioimmunoassay. Local perfusion of K+ (100 mM)-enriched artificial CSF resulted in a 10-fold increase in CCKLM outflow, as compared with that occurring under basal resting (K+ = 3.0 mM) conditions, and this effect could be completely prevented by removal of Ca2+ in the perfusing fluid. Chromatographic analyses demonstrated that CCK-8S contributed to 70% of CCKLM. Stressful stimuli such as a 2-min exposure to diethyl ether and a 30-min restraint produced a marked but transient increase in cortical CCKLM release. In addition, anxiety-like behavior induced by the systemic administration of yohimbine (5 mg/kg i.p.) was associated with a long-lasting enhancement in the peptide outflow. Pretreatment with the potent anxiolytic drug diazepam (5 mg/kg i.p., 5 min before each condition), which exerted no effect on its own, completely prevented CCKLM overflow due to diethyl ether, restraint, or yohimbine administration. In contrast, neither the systemic injection (0.1 mg/kg i.p.) nor the local application (100 microM through the microdialysis probe) of the serotonin 5-HT3 antagonist ondansetron affected the increased release of CCKLM in rats restrained for 30 min or treated with yohimbine. These results indicate that cortical CCKergic neurotransmission is increased during stress or anxiety-like behavior in rats. Prevention of this effect by diazepam suggests that an inhibitory influence of benzodiazepines on cortical CCKergic neurons might participate in the anxiolytic action of these drugs.

Immunocytochemical localization of serotonin1A receptors in the rat central nervous system.

Specific anti-rat 5-hydroxytryptamine1A (serotonin1A) receptor antibodies raised in a rabbit injected with a synthetic peptide corresponding to a highly selective portion of the third intracellular loop of the receptor protein (El Mestikawy et al. [1990] Neurosci. Lett. 118:189-192) were used for immunohistochemical mapping of serotonin1A receptors in the brain and spinal cord of adult rats. The highest density of immunostaining was found in limbic areas (lateral septum, CA1 area of Ammon's horn and dentate gyrus in the hippocampus, and frontal and entorhinal cortices), in the anterior raphe nuclei, and in the interpeduncular nucleus, in agreement with previous autoradiographic studies with selective radioligands showing the enrichment of these regions in serotonin1A receptor binding sites. Serotonin1A receptor-like immunoreactivity was also present, but at a moderate level, in the neocortex, in some thalamic and hypothalamic nuclei, in the nucleus of the solitary tract, in the dorsal tegmentum, in the nucleus of the spinal tract of the trigeminal nerve, and in the superficial layers of the dorsal horn in the spinal cord. In contrast, extrapyramidal areas, including the caudate putamen, the globus pallidus, and the substantia nigra as well as the cerebellum, exhibited very low to no immunostaining by antiserotonin1A receptor antibodies. At the cellular level, both the plasma membrane of neuronal perikarya and fine neuronal processes probably corresponding to dendritic fields were found to bind antiserotonin1A receptor antibodies. Regional differences were noted regarding these two types of immunostaining, because only dendrites bound antibodies within the hippocampus and the lateral septum, whereas both dendrites and neuronal cell bodies were immunoreactive in the medial septum, in the diagonal band of Broca, and in the dorsal and median raphe nuclei. Therefore, differential addressing of serotonin1A receptors could occur from one neuron to another. In general, the distribution and density of serotonin1A receptor-like immunoreactivity in the whole brain and in spinal cord were consistent with the mapping of serotonin1A receptor binding sites and serotonin1A receptor mRNA previously established by immunoautoradiographic and in situ hybridization procedures.

Glucocorticoid receptor-mediated inhibition by corticosterone of 5-HT1A autoreceptor functioning in the rat dorsal raphe nucleus.

In the rat brain, the dorsal raphe nucleus contains a large proportion of serotoninergic neurons, which are mostly regulated by somato-dendritic 5-HT1A autoreceptors. This nucleus also possesses intracellular glucocorticoid receptors (GR), which may be involved in the well established modulation of serotonin (5-hydroxytryptamine, 5-HT) metabolism by glucocorticoids. Control by corticosteroids of 5-HT1A receptor-mediated inhibitory control of the firing of serotoninergic neurons in the dorsal raphe nucleus was investigated using an in vitro electrophysiological approach. The spontaneous firing rate of serotoninergic neurons recorded in brain stem slices and its inhibition due to 5-HT1A autoreceptor stimulation by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) were similar in adrenalectomized rats and sham-operated animals. In vitro pretreatment with corticosterone (30-100 nM) significantly reduced 8-OH-DPAT-induced inhibition of the 5-HT cell discharge in slices from adrenalectomized rats. This effect could be prevented by the GR antagonist, 11 beta-(4-dimethyl-amino-phenyl)- 17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one (RU) 38486, 30 nM), and mimicked by the GR agonist, 11 beta, 17 beta-dihydroxy-6-methyl-17 alpha (prop-1-ynyl) androsta-1,4,6-trien-3-one (RU 28362, 500 nM). In contrast, the mineralocorticoid receptor (MR) agonist, aldosterone (10 nM), did not alter 8-OH-DPAT-induced inhibition in tissues from adrenalectomized animals. Complementary autoradiographic experiments showed that [3H]8-OH-DPAT specific binding to 5-HT1A sites in the dorsal raphe nucleus (and the hippocampus) was not significantly altered following adrenalectomy and exposure of brain stem slices to corticosterone. These data suggest that GR are involved in the suppressive effects of high levels of corticosterone on the 5-HT1A receptor-dependent regulation of 5-HT neuronal activity in the rat dorsal raphe nucleus.

Long-term oral administration of L-arginine reduces intimal thickening and enhances neoendothelium-dependent acetylcholine-induced relaxation after arterial injury.

BACKGROUND: Nitric oxide (NO), in addition to its potent vasorelaxant properties, may participate in growth regulation of cultured smooth muscle cells. It was recently demonstrated that in vivo endothelial injury induces the production of NO from L-arginine in the arterial wall. METHODS AND RESULTS: We studied the effects of long-term administration of L-arginine, the precursor of NO, on neointimal thickening and on neoendothelium-dependent vasorelaxation 4 weeks after balloon denudation of normocholesterolemic rabbit iliac arteries. Rabbits were fed with either a standard diet or a diet supplemented with L-arginine (2.25%) in their drinking water 3 days before and during 4 weeks after balloon denudation. The effectiveness of L-arginine supplementation was confirmed by measurement of plasma arginine levels. L-Arginine had no effect on hemodynamic parameters. All animals were killed 4 weeks after balloon denudation, and a digital histomorphometric analysis of three serial nonconsecutive histological cross sections per iliac artery was performed. Intimal thickening was reduced (P < .05) from 0.43 +/- 0.08 (SE) mm2 in controls (n = 8) to 0.24 +/- 0.02 mm2 in treated animals (n = 8). Ten animals (n = 5 in each group) were used for in vitro vasoreactivity assessment 4 weeks after balloon denudation. Neoendothelium-dependent acetylcholine-induced relaxation (10(-8) mol/L to 3.10(-5) mol/L) in treated animals (Emax = -24.1 +/- 5.5%) was significantly greater than in controls (Emax = -8.9 +/- 2.2%). Endothelium-independent relaxation did not differ between groups (Emax = -58.1 +/- 6.5% in L-arginine-supplemented animals versus -52.9 +/- 6.8% in controls). CONCLUSIONS: Our results demonstrate that L-arginine, a precursor of NO, reduces neointimal thickening after balloon denudation and improves neoendothelial-dependent acetylcholine-induced relaxation.

Neuronal localization of 5-HT1A receptor mRNA and protein in rat embryonic brain stem cultures.

The aim of the present work was to study the cellular localization of 5-HT1A receptor protein and mRNA in rat embryonic brain cultures. Primary cultures of the whole brain from rat fetuses at embryonic day (ED) 12 and of the brain stem at ED 14-ED 16 were stained with specific anti-5-HT1A receptor antibodies or a 40-mer biotin-labelled deoxyoligonucleotide complementary to the 5-HT1A receptor mRNA. The use of a biotinylated probe allowed the morphology of the cells to be preserved. 5-HT1A receptor mRNA was already detected in primary cultures from the brain of ED 12 embryos whereas the receptor protein first appeared two days later, at ED 14. Both 5-HT1A receptor mRNA and protein were found within neuron-like cells (labelled with antibodies against neuron specific enolase, microtubule-associated protein 2 or aromatic L-amino acid decarboxylase) but not in glial cells (specifically labelled with antibodies against glial fibrillary acidic protein, myelin basic protein or carbonic anhydrase II). Double staining with the 5-HT1A receptor mRNA probe and anti-5-HT antibodies suggests that 5-HT1A (auto)receptors are expressed by serotoninergic neurons during early ontogenesis.

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.

Dopamine increases the expression of tyrosine hydroxylase and aromatic amino acid decarboxylase in primary cultures of fetal neurons.

Previous studies, aimed at identifying which diffusible signals may influence the differentiation of embryonic neurons towards the monoaminergic phenotypes during brain development, have shown that serotonin itself could promote the 'serotoninergic-like properties' of hypothalamic cells from mouse embryos. We presently reinvestigated such 'autocrine/paracrine' regulatory mechanisms by exposing dissociated cell cultures from embryonic rat hypothalamus and brain stem to dopamine--or related agonists--in an attempt to influence their differentiation towards the catecholaminergic phenotype. Chronic treatment of cells by dopamine or apomorphine (a mixed D1/D2 agonist), but not selective D1 and D2 agonists, significantly increased the number of cells that expressed tyrosine hydroxylase (TH: as assessed with a specific anti-TH antiserum) and the activity of aromatic L-amino acid decarboxylase (AADC) in the cultures. Furthermore, apomorphine treatment also decreased the levels of cholecystokinin-like material in primary cultures from the brainstem (but not the hypothalamus) where both dopamine and cholecystokinin are--partly--colocalized in mesencephalic dopaminergic neurons. The maximal effects of both dopamine and apomorphine on TH expression and AADC activity occurred earlier in the brainstem (on cells from 14- to 15-day-old embryos) than in the hypothalamus (on cells from 15- to 16-day-old embryos), in line with the well established caudo-rostral maturation of the rat brain. Furthermore both the expression and the dopamine-induced modulation of AADC activity and TH immunoreactivity appeared to occur independently of each other. Present and previous data are in agreement with a possible autocrine/paracrine action of dopamine and serotonin during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Opposite effects of delta and mu opioid receptor agonists on the in vitro release of substance P-like material from the rat spinal cord.

Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs-Henseleit medium supplemented with 30 microM bacitracin allowed the collection of substance P-like immunoreactive material (SPLI), which was released at a rate of approximately 10 pg/4 min. Tissue depolarization by an excess of K+ (30-60 mM) or veratridine (50 microM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+- or veratridine-induced SPLI overflow could be modulated in opposite directions by mu and delta opioid receptor agonists. Thus, the two preferential mu agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and Tyr-D-Ala-Gly-MePhe-Met(O)5-OH (FK-33824; 0.1 microM) enhanced SPLI overflow from depolarized tissues, whereas the selective delta agonists Tyr-D-Thr-Gly-Phe-Leu-Thr (deltakephalin; 3 microM) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (50 microM) reduced it. The effect of DAGO was antagonized by a low concentration (1 microM) of naloxone but not by the selective delta antagonist ICI-154129 (50 microM). In contrast, the latter drug prevented the inhibitory influence of delta agonists on K+-induced SPLI release. Complementary experiments with morphine (10 microM) and [2-D-alanine, 5-D-leucine]enkephalinamide (3 microM), in combination with 1 microM naloxone or 50 microM ICI-154129 for the selective blockade of mu or delta receptors, respectively, confirmed that the stimulation of mu receptors increased, whereas the stimulation of delta receptors reduced, SPLI overflow. The results suggest that, at the spinal level, and antinociceptive action of delta but not mu agonists might involve a presynaptic inhibition of substance P-containing primary afferent fibers.