A study of the role of neuro-glial remodeling in the oxytocin system at lactation.

Under conditions of strong secretion of neurohypophysial hormone, such as during parturition, lactation and dehydration, the hypothalamic oxytocin-system displays a remarkable morphological plasticity such that astrocytic coverage of its neurones diminishes, their surfaces become directly juxtaposed and contacted by an increased number of synapses. A growing body of evidence indicates that these anatomical changes have an impact on glutamatergic neurotransmission in the supraoptic nucleus, and may be therefore of physiological consequence. We here evaluated the consequences of the inhibition of such plasticity on the overall activity of the oxytocin system during lactation. Remodeling was prevented by performing hypothalamic microinjections in gestating rats of endoneuraminidase, an enzyme that removes polysialic acid from the neural cell adhesion molecule. Our earlier studies established that the presence of polysialic acid is a prerequisite for remodeling of the oxytocin system in the supraoptic and paraventricular nuclei. In dams in which polysialic acid was absent in all magnocellular nuclei after bilateral endoneuraminidase injections, parturition was normal and neither the frequency nor the amplitude of suckling-induced reflex milk ejections was different from vehicle-treated dams. The weight gain of pups was also normal as was water intake by the dams. We then assessed the electrical activity of antidromically identified magnocellular neurones in the polysialic acid-free supraoptic nucleus of isoflurane-anesthetized lactating rats. Basal and bursting activity characteristic of oxytocin neurones before each reflex milk ejection was not significantly different from that recorded in the supraoptic nucleus of rats with normal levels of polysialic acid. Our results indicate that neuro-glial remodeling, despite its role on fine modulation of oxytocin neuronal activity, is not essential to parturition and lactation.

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study.

The aim was to study the regulation of &mgr; and delta opioid binding sites in the superficial layers (laminae I - II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [3H]Tyr*-d-Ala-Gly-NMe-Phe-Gly-ol ([3H]DAMGO) (&mgr; sites) and [3H]Tyr*-d-Thr-Gly-Phe-Leu-Thr ([3H]DTLET) (delta sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [3H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [3H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of &mgr; and delta binding sites than in cases of short-term deafferentation (1 - 2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining &mgr; and delta binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of &mgr; and delta binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.

The Distribution of &mgr; and delta Opioid Binding Sites Belonging to a Single Cervical Dorsal Root in the Superficial Dorsal Horn of the Rat Spinal Cord: A Quantitative Autoradiographic Study.

Numerous studies have demonstrated a dense concentration of opioid receptors in the superficial layers (laminae I - II) of the spinal cord. These receptors are located both pre- and postsynaptically at this level. The purpose of this study was to assess the distribution of opioid receptors belonging to a single (C7) dorsal root. Thus, quantitative autoradiography of &mgr; ([3H]Tyr-d-Ala-Gly-NMe-Phe-Gly-ol; [3H]DAMGO) and delta ([3H]Tyr-d-Thr-Gly-Phe-Leu-Thr; [3H]DTLET) opioid binding sites was performed for several experimental groups: control rats with intact dorsal roots and lesioned rats with a unilateral dorsal rhizotomy of (a) the C7 root alone, (b) the three successive roots rostral and caudal to the spared C7 root, and (c) the seven roots C4 - Th2. By subtracting results of the 'C7 cut' group from the 'intact' group or by subtracting results of the C4 - Th2 cut group from the C7 spaced group, it was possible to measure the distribution of &mgr; and delta opioid binding sites belonging to the C7 root. The combination of these two methods of calculation allowed us to demonstrate a significant distribution over two segments rostral and one segment caudal to the segment of entry. For [3H]DAMGO, the distribution was 10% (P < 0.05) in the C5, 27%, (P < 0.001) in the C6, 38% (P < 0.001) in the C7 and 14% (P < 0.05) in the C8 segment. For [3H]DTLET, the distribution was 11% (P=0.05) in the C5, 27%, (P < 0.01) in the C6, 37% (P < 0.001) in the C7 and 18% (P < 0.05) in the C8 segment. It is also noted that rostral distributions spread more densely and further than the caudal ones.

Potent and long lasting antinociceptive effects after injection of low doses of a mu-opioid receptor agonist, fentanyl, into the brachial plexus sheath of the rat.

The effect of administering low doses (0.5-1.5 micrograms) of the mu-opioid receptor agonist fentanyl into the right brachial plexus sheath of the rat was examined using the vocalization threshold to paw pressure test. Both forepaws were tested in each rat. Fentanyl injected into the right brachial plexus sheath at 0.5-1.5 micrograms/kg produced a localized, dose-dependent, potent and long lasting antinociceptive effect, as gauged on the right forepaw. At the lower dose used (0.5 microgram/kg of fentanyl), the antinociceptive effect was restricted to the right forepaw and lasted for more than 2 h. Increasing doses of fentanyl (1 and 1.5 micrograms/kg) induced potent effects, lasting up to 5-6 h or even longer. In complete contrast, fentanyl administered i.v. at the dose of 1 microgram/kg had a very transient effect, only lasting up to 25 min. The results of injection of low doses of the opioid antagonist naloxone when administered either i.v. or locally into the paw, on the effect of fentanyl suggest the involvement of a peripheral site of action of the opioid. The present findings suggest that, as already observed in patients in clinical situations, low doses of opiates delivered using this administration route may provide prolonged regional analgesia, with the potential of avoiding centrally mediated side effects.

Regulation of opioid binding sites in the superficial dorsal horn of the rat spinal cord following loose ligation of the sciatic nerve: comparison with sciatic nerve section and lumbar dorsal rhizotomy.

The aim of the present study was to quantify time-related modifications in mu and delta opioid binding sites in the superficial layers (laminae I and II) of the L4 lumbar segment in a rat model of mononeuropathy induced by loose ligation of the sciatic nerve. We have shown a 28% (P < 0.01) and 24% (P < 0.01) decrease in ipsi/contralateral side binding ratios for tritiated (Tyr*-D-Ala-Gly-NMe-Phe-Gly-ol) ([3H]DAMGO) and tritiated (Tyr*-D-Thr-Gly-Phe-Leu-Thr) ([3H]DTLET) respectively, at two weeks postlesion which correspond to the delay of maximal hyperalgesia and of maximal alteration of fine diameter primary afferent fibers. In contrast, no change in [3H]U.69593 specific binding could be detected at this postlesion delay. For longer survival delays (four, eight and 15 weeks postlesion), mu and delta binding ratios return towards control values (approximately equal to 1), probably reflecting the occurrence of a long-term neuroplasticity (i.e. a new equilibrium in the metabolism of primary neurons, or collateral sprouting from intact primary afferents) following loose nerve ligation. In addition, a comparison of the results obtained in this model with those measured after sciatic nerve section and lumbar dorsal rhizotomy was performed in order to compare the degree of loss in opioid binding sites in these three types of lesion. The section of the sciatic nerve induced at eight days postlesion an 18% (P < 0.01) and 28% (P < 0.01) decrease in binding ratio for [3H]DAMGO and [3H]DTLET, respectively. At two weeks postlesion the loss was 24% (P < 0.01) for the two ligands, and at longer delays (four and 12 weeks), a progressive recovery in binding ratio was observed. Thus, it appears that both sciatic nerve lesions we have studied result in mu and delta binding modifications which have similar intensity and similar time course from two to 12-15 weeks postlesion. In contrast, the unilateral rhizotomy of nine consecutive dorsal roots (T13-S2), which is known to induce a massive degeneration of fine diameter primary afferent fibers, is followed by a dramatic decrease in binding ratios for [3H]DAMGO (53%, P < 0.001) and [3H]DTLET (45%, P < 0.001) at two weeks postlesion. These data suggest that the more deprived the dorsal horn is of fine diameter primary afferent fibers, the more dramatic is the opioid binding loss in the ipsilateral side as compared to the contralateral side.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of neuropeptide FF (FLFQPQRFamide) receptors in the adult rat spinal cord: effects of dorsal rhizotomy and neonatal capsaicin.

By using quantitative autoradiography and highly selective iodinated ligands, we quantified modifications in neuropeptide FF binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8 segments) and lumbar (L3-L5 segments) enlargements in two models: (i) rats neonatally treated with capsaicin; (ii) rat submitted 15 days before to unilateral dorsal rhizotomies. We comparatively analysed the distribution of mu-opioid binding sites in the same animals. We have shown that the [125I]YLFQPQRFamide (neuropeptide FF sites) labelling is not significantly modified following selective damage of fine afferent fibres by neonatal capsaicin treatment. In the cervical and lumbar enlargements, capsaicin-treated/control binding ratios for [125I]YLFQPQRFamide were 0.90 and 0.86, respectively. While unilateral dorsal rhizotomy induced a drastic decrease in [125I]FK-33-824 labelling in the side ipsilateral to the lesion as compared to the intact side of (yielding ratios of 0.29 and 0.31 for cervical and lumbar levels, respectively), [125I]YLFQPQRFamide labelling was not significantly modified, yielding ratios of 0.98 and 0.91 for cervical and lumbar levels, respectively. These data suggest that, in contrast with a majority of mu-opioid receptors, neuropeptide FF receptors are not located on fine primary afferent fibers carrying nociceptive information from the fore- or hindlimb in the rat. This preferential postsynaptic localization, together with the reported "morphine modulating" action of this peptide, support the proposal of a role for neuropeptide FF in intraspinal modulation of nociceptive input.

Characterization and localization of a putative morphine-modulating peptide, FLFQPQRFamide, in the rat spinal cord: biochemical and immunocytochemical studies.

The bovine octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (FLFQPQRFamide), originally detected by antisera raised against the invertebrate peptide, Phe-Met-Arg-Phe-NH2, is a neuropeptide which antagonizes the actions of endogenous and exogenous opiates. Using a sensitive radioreceptor assay, we show that rat spinal cord extracts were able to inhibit binding of FLFQPQRFamide, suggesting that a biologically active FLFQPQRFamide-like material exists in the rat spinal cord. We also raised antibodies against the peptide and used them, together with radioimmunological and immunohistochemical methods, to characterize this material further and analyse its cellular and subcellular localization in this area of the central nervous system. Radioimmunoassay showed that extracts from cervical and thoracolumbar levels contained measurable amounts of FLFQPQRFamide-immunoreactive material (about 3 ng/g tissue), present essentially in the dorsal horn. Analytical reverse-phase chromatography revealed that this material existed in several molecular forms. One of these fractions (about 20% of the total immunoreactivity) had the elution characteristics of synthetic FLFQPQRFamide. Light microscopic immunohistochemistry showed FLFQPQRFamide immunoreactivity at all spinal levels, localized mainly in a dense plexus of fibers in the superficial layers of the dorsal horn. Immunoreactive profiles were also seen in the lateral funiculi and around the central canal at all levels and in the intermediolateral columns; some rare immunoreactive fibers were also noted in the ventral horn at cervical and thoracic levels. FLFQPQRFamide-positive cell bodies were never detected in any of our sections. Electron microscopy of ultrathin sections of the dorsal horn and central gray treated with our antisera and a post-embedding immunogold procedure revealed that the immunoreactivity, at least within these areas, was restricted to dense-cored vesicles (90-120 nm in diameter) in axonal and terminal profiles. As seen by radioimmunoassay and immunohistochemistry, unilateral rhizotomy of all dorsal roots between segments C4 and T2 did not change the levels of FLFQPQRFamide immunoreactivity in the ipsilateral C6-C8 segments. Taken together with our recent data showing the existence of specific FLFQPQRFamide receptors at the spinal cord level, our present observations suggest that the dorsal horn of the rat spinal cord may be a site where vertebrate Phe-Met-Arg-Phe-like peptides, and in particular, FLFQPQRFamide, may exert opiate modulating activities.

Distribution pattern and ultrastructural localization of Rxt1, an orphan Na+/Cl(-)-dependent transporter, in the central nervous system of rats and mice.

The cellular and subcellular localization of Rxt1 protein, an orphan Na+/Cl(-)-dependent transporter, was investigated in the central nervous system of rats and mice, with rabbit polyclonal antibodies specifically directed against its C-terminal region. At the light microscope level, the distribution of Rxt1, visualized by the immunoperoxidase method, was found to be similar in rats and mice. Labelled elements were present in numerous gray matter regions of the central nervous system, from the olfactory bulb to the spinal cord. In all labelled regions, immunoreactivity was confined to the neuropil where both a diffuse labelling of low intensity and an intense punctate staining were noted. To further identify the nature of the cellular elements bearing the punctate staining, possible changes in this labelling pattern were investigated: (i) in deep cerebellar nuclei and lateral vestibular nucleus of the Lurcher mutant mouse, in which all cerebellar Purkinje cells are missing and (ii) in the rat cervical spinal cord, 10 days after multiple resections of dorsal roots. The vast majority of the punctate structures, delineating the neuronal perikaryal and stem dendritic contours, had disappeared in the mutant mouse, providing evidence that they belong to Purkinje cell axon terminals. In rhizotomized rats, the intense labelling in laminae I and III had disappeared, demonstrating that it occurred in subclasses of axonal projections of primary afferent fibres. These results strongly suggest that Rxt1 is present in presynaptic axon terminals. The electron microscopic study was carried out in the hippocampus, cerebellum and lateral vestibular nucleus of control mice, where Rxt1-labelled punctate structures were found to be abundant. Immunostaining was confined to axon terminals, particularly in hippocampal and cerebellar mossy fibres and in Purkinje cell axonal terminations of the cerebellar deep nuclei and lateral vestibular nucleus. In the cerebellar cortex, axon terminals belonging to inhibitory local circuit neurons (basket and Golgi cells), were free of labelling. The observations reported in this study have shown that: (1) The Rxt1 transporter is neuron-specific, and is expressed by only some classes or even subclasses of neuronal systems. (2) This transporter can be encountered in excitatory axons using glutamate as neurotransmitter (hippocampal and cerebellar mossy fibres: primary afferent fibres), as well as in inhibitory axons known by their GABAergic nature (Purkinje cell axon terminals) where it might be involved in the re-uptake process of one or several molecules released from corresponding terminals.

Fos-like immunoreactivity in the rat spinal cord induced by formalin injection in the forelimb to gauge possible plasticity of primary afferent fibers following partial deafferentation.

The aim of this study was to gauge the possible sprouting of primary afferent fibers following dorsal rhizotomies using Fos-like immunoreactivity (Fos-LI) in order to label neurons activated by formalin injection into the rat forepaw. To assess the functional consequences of possible sprouting of fine diameter primary afferents, we monitored the behavioral responses and to visualize fine afferent fiber sprouting, we examined calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the superficial laminae of the dorsal horn. Two types of dorsal rhizotomies and two delays post-rhizotomy were used: one dorsal root, C7 (C7 cut) or three consecutive dorsal roots, C6, C7 and C8 (C6, C7, C8 cut), were sectioned on the right side three months or 7 days before the day when formalin was injected in the extremity of the ipsilateral forelimb. Control animals had no rhizotomy but received the formalin injection. In control animals, following the formalin injection the greatest number of Fos-LI neurons was encountered in the superficial laminae and in the neck of the dorsal horn of segments C5-T1. In the C7 cut group, the number of Fos-LI neurons was slightly decreased in all segments 7 days after the lesion whereas it was slightly increased 3 months after the lesion as compared to 7 days. In C6, C7 C8 cut group, the number of Fos-LI significantly decreased (90% of the control values) 7 days after the lesion, but after three months, it significantly increased in segments C7 and C8 as compared to 7 days. In parallel in this latter group, a marked depletion of CGRP-LI fibers was observed in the medial part of the superficial laminae at 7 days whereas a clear increase in CGRP-LI occured in the same region at 3 months. Behavioral observations showed a slight decrease in the licking time induced by the formalin injection in the C7 cut group both at 7 days and 3 months after the lesion as compared to the control group. The significant decrease of this behavior observed in C6, C7, C8 cut group at 7 days was not changed after 3 months. The increase in the number of Fos-LI neurons after 3 months in the C6, C7, C8 cut group is discussed in terms of collateral sprouting of thin primary afferent fibers and/or central compensatory mechanisms in response to peripheral deafferentation. Our data favor the first hypothesis, and in addition, support the use of the Fos-LI technique to assess the functional post-synaptic changes at the dorsal horn level.

Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization.

Specific antibodies raised against a fusion protein containing the amino acid sequence of the putative second intracellular loop of the cloned 5-HT3-A receptor subunit were used for the immunohistochemical visualization of 5-HT3 receptors in the rat spinal cord. A dense 5-HT3-like immunoreactivity was found in the superficial layers of the dorsal horn, which closely matched the labelling of 5-HT3 binding sites by [125I]iodo-zacopride. This immunostaining was markedly decreased following unilateral rhizotomy, consistently with a preferential location of 5-HT3 receptors on terminals of primary afferent fibres, and with the presence of 5-HT3 mRNA in dorsal root ganglia. However, a significant proportion of 5-HT3 receptors persisted after rhizotomy, and the corresponding mRNA was found in the dorsal horn of the spinal cord. 5-HT3 receptors are therefore also located on intrinsic neurones of the spinal cord.

Evidence for neuropeptide FF (FLFQRFamide) in rat dorsal root ganglia.

By using specific antibodies and radioimmunological and immunohistochemical methods, we here show that neuropeptide FF (NPFF) occurs in cervical and lumbar dorsal root ganglia cells. Levels in the ganglia were low because they were detectable only after colchicine treatment or after unilateral dorsal rhizotomy. Similar high-performance liquid chromatography profiles were obtained from dorsal root ganglia and spinal cord extracts, indicating that the NPFF-immunoreactivity in the dorsal root ganglia represented similar molecular forms to that in the spinal cord. Immunocytochemistry localized NPFF-immunoreactivity in small- and medium-sized cells. These data suggest that low levels of NPFF present in fine diameter primary afferent fibers could be involved in the treatment of nociceptive information from fore- or hindlimb.

Opioid control of the in vitro release of calcitonin gene-related peptide from primary afferent fibres projecting in the rat cervical cord.

In vitro superfusion of slices from the dorsal half of the rat cervical enlargement allowed the measurement of spontaneous, K+ (30 mM)- and capsaicin (0.5 microM)-evoked release of calcitonin gene-related peptide-like immunoreactive material (CGRPLI). The greater part of this immunoreactive material originated in primary afferent fibres since dorsal rhizotomy from C4 to Th2 (8 days before sacrifice) resulted in a 85-90% decrease in CGRPLI release. CGRPLI outflow which persisted after dorsal rhizotomy could still be enhanced by K+-induced depolarization but was no longer sensitive to the stimulatory effect of 0.5 microM capsaicin. Both delta (DTLET, D-Pen2-D-Pen5-enkephalin) and mu (DAGO, PL 017) opioid receptor agonists reduced the K+ evoked release of CGRPLI from the dorsal half of the cervical enlargement. Morphine was also inhibitory but the selective K opioid agonist U 69593 was inactive. As expected from the involvement of delta and mu receptors, the selective opioid antagonist ICI 174864 and naloxone prevented the inhibitory effects of DTLET and DAGO, respectively. These data suggest that opioid-induced presynaptic inhibiton of CGRP-containing primary afferent fibres may be involved in the analgesic effect of intrathecally injected delta and mu opioid agonists in rats.

Electrophysiological evidence for a tonic activity of the spinal cord intrinsic opioid systems in a chronic pain model.

The aim of this electrophysiological investigation was to evaluate the activity of the spinal endogenous opioid systems in a chronic pain model, the arthritic rat. The activity of nociceptive non-specific dorsal horn neurons (n = 23) were recorded in 23 spinal unanesthetized decerebrated rats. Naloxone (1 mg/kg i.v.) induced a highly significant increase in the spontaneous firing rate of these neurons. This observation is in favor of a tonic activity of spinal opioid endogenous systems in such a disease. In addition, the same dose of naloxone facilitates the transmission of noxious messages at the spinal level as revealed by the large enhancement of the responses of these neurons to C-fiber stimulation. These results are in good agreement with behavioral data showing that such a relatively high dose of naloxone induced well-reproducible hyperalgesia and with some biochemical observations showing an increase of levels and biosynthesis of endogenous opioids in the spinal cord of the arthritic rat.

Time-related decreases in mu and delta opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy.

The aim of the present study was to measure the time-related modifications of mu and delta opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for mu sites and [3H]DTLET for delta sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of mu receptors (71-74%) is significantly more pronounced than the loss of delta receptors (57-62%) and (d) affinities of postsynaptic mu and delta receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of mu and delta opioid binding sites on fine diameter primary afferent fibers.

Autoradiographic distribution of mu, delta and kappa opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord.

The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of mu, delta and kappa opioid binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8), thoracic (T5-T7), lumbar (L3-L5) and sacral (S2-S3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for mu, delta, kappa sites, respectively. The physiological relevance of these data is discussed.

Fos-like immunoreactivity in the rat superficial dorsal horn induced by formalin injection in the forepaw: effects of dorsal rhizotomies.

As previously described at the lumbar spinal level, we found that 2 h after subcutaneous formalin injection in the distal part of the fore-limb, Fos-like immunoreactivity (FLI) was induced in the ipsilateral cervical enlargement. Not surprisingly, as the injection site corresponds to the distal part of the C6-C8 dorsal root dermatomes, maximal labelling which predominated in the superficial laminae, was observed in the C6-C8 segments and to a lesser extent in C5. Similar experiments were performed on rats which underwent various types of unilateral dorsal rhizotomies (DRh) 7 days before formalin injection. In animals with C4, C5, T1 and T2 DRh sparing C6-C8 the rostrocaudal distribution was similar to the intact one. But, in animals having C4-T2 DRh sparing one single root, C7, the segmental FLI distribution was modified: it was slightly increased in C7, decreased in C6 and significantly decreased in C8. As expected, no FLI was found in animals with C4 to T2 DRh. The spared root model provides information about the segmental distribution in the cervical spinal cord of the input brought by a single root following stimulation of the distal forelimb, i.e., maximal distribution in the entry segment, but also in the two rostral and one caudal segments.

Unilateral joint inflammation induces bilateral and time-dependent changes in neuropeptide FF binding in the superficial dorsal horn of the rat spinal cord: implication of supraspinal descending systems.

Using quantitative autoradiography, the effects of acute and chronic inflammation on specific 125I-1DMethyl-FLFQPQRFamide binding were investigated in the rat spinal cord dorsal horn superficial layers, at 6 and 24 h and 2, 4, 6 and 12 weeks after induction of monoarthritis produced by injection of killed Mycobacterium butyricum suspended in Freund adjuvant in one tibio-tarsal joint. Six hours after monoarthritis induction, no modification in specific 125I-1DMethyl-FLFQPQRFamide binding was observed, whereas a significant bilateral increase occurred after 24 h and 2 weeks in L4/L5 dorsal horns, with a return to control values at 4, 6 and 12 weeks. Specific 125I-1DMethyl-FLFQPQRFamide binding was also investigated 24 h after monoarthritis induction in rats submitted 4 days before the induction to spinal cord lesions at the thoracic level (T9-T10). Hemisection of the spinal cord contralateral to the affected ankle prevented the transient bilateral increase in specific 125I-1DMethyl-FLFQPQRFamide binding, whereas total spinal cord section induced a significant bilateral decrease. All of these modifications were restricted to the spinal segments receiving afferent input from the arthritic ankle (L4/L5); no modifications were found at the levels L1 or C6-C8. These data suggest that FLFQPQRFamide is involved in spinal nociceptive processing during sustained peripheral nociceptor activation. The effects of spinal cord lesions in monoarthritic rats indicate that the modifications seen in the FLFQPQRFamide system activity, during sustained peripheral inflammation, depend on afferent fiber activation as well as on supraspinal controls.

Pre- and postsynaptic distribution of mu, delta and kappa opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord.

Highly selective tritiated ligands and quantitative autoradiography have been used to study mu, delta and kappa binding sites in the dorsal horn of the rat spinal cord. We have measured the proportions of the 3 main types of opioid binding sites in the superficial layers of the cervical dorsal horn (laminae I and II). The proportions of mu, delta and kappa sites were 70 +/- 4%, 23 +/- 2% and 7 +/- 1%, respectively, over the whole C4-T2 extent. Similar percentages were encountered at the level of each individual segment from C4 to T2. Eight days after a unilateral dorsal rhizotomy C4-T2, dramatic decreases were seen on the ipsilateral side to the lesion by comparison to the intact side. In the C7 segment, these decreases were 76 +/- 1%, 61 +/- 1% and 53 +/- 3% for mu, delta and kappa binding sites, respectively. The C7 segment can be considered as completely deafferented, so we attribute the residual values to postsynaptic binding whereas the decrease can be attributed to a loss of the presynaptic sites. These results are discussed with respect to the contribution of pre- and postsynaptic depressive effects of opiates on the transmission of noxious messages at the level of the dorsal horn.

Autoradiographic study of mu and delta opioid binding sites and neutral endopeptidase-24.11 in rat after dorsal root rhizotomy.

Highly selective tritiated ligands and autoradiography have been used to study mu and delta binding sites as well as neutral endopeptidase-24.11 in the dorsal horn of the rat spinal cord. Two weeks and 4 months after dorsal root rhizotomy, both opioid binding sites were dramatically decreased (congruent to 60%). In contrast, the level of neutral endopeptidase-24.11 remained unaltered.

5-HT3 receptors in the rat central nervous system are mainly located on nerve fibres and terminals.

Autoradiographic and membrane binding studies with [3H](R,S)- or [3H](S)-zacopride were performed in combination with lesions using various neurotoxins in an attempt to identify which neuronal cell types are endowed with 5-HT3 receptors in the rat central nervous system. Lesions of noradrenergic (by DSP-4), dopaminergic (by 6-hydroxydopamine) and serotonergic (by 5,7-dihydroxytryptamine) systems had little effect generally on the density of 5-HT3 receptors labelled with [3H](R,S)- or [3H](S)-zacopride in various regions of the brain and the spinal cord. The only exception was the amygdala where a significant loss (approximately -20%) of 5-HT3 receptors labelled by [3H](R,S)-zacopride was associated with the selective lesion of serotonergic fibres by 5,7-dihydroxytryptamine. Microinjection of kainic or ibotenic acid into the dorsal and ventral hippocampus reduced the density of 5-HT1A receptors labelled with [3H]8-OH-DPAT (approximately -45%) as expected from their known location on intrinsic neuronal cell bodies and/or dendrites. In contrast, the same lesion did not affect 5-HT3 receptors, suggesting their location on fibres 'en passage'. At the spinal level, 5-HT3 receptors were found to exist on primary afferent fibres terminating within the superficial layers of the dorsal horn, as shown by the marked reduction in the local autoradiographic labelling by [3H](S)-zacopride after either dorsal rhizotomy (-81%) or neonatal capsaicin treatment (-72%). These data suggest that 5-HT3 receptors in the central nervous system are generally located presynaptically on nerve terminals or fibres of non-monoaminergic neurones.