Michèle Allard

Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a Kd value of 0.09-0.1 nM and a maximal capacity Bmax of 14.5 +/- 2 fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that mu, delta and kappa opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that is capacity to reduce opiate-induced analgesia is supported by specific binding sites.

Autoradiographic distribution of receptors to FLFQPQRFamide, a morphine-modulating peptide, in rat central nervous system

The neuropeptide FLFQPQRFamide is a structure related to FMRFamide which is able to inhibit the effects of both endogenous and exogenous opiates. This morphine-modulating activity is mediated via the stimulation of specific FLFQPQRFamide receptors, different from opiate receptors. In vitro quantitative receptor autoradiography was performed on frozen sections of rat central nervous system to characterize binding properties and visualize FLFQPQRFamide receptors using the specific ligand [125I]YLFQPQRFamide, a radio-iodinated analogue of FLFQPQRFamide. [125I]YLFQPQRFamide appeared to interact reversibly with a single class of binding sites (KD = 0.2 nM). The specific binding represented 80% of the total binding at 0.05 nM, the FLFQPQRFamide concentration used in this mapping study. Sites labelled with [125I]YLFQPQRFamide were distributed heterogeneously within the brain and spinal cord. A high density of FLFQPQRFamide binding sites was detected in the most external layers of the dorsal horn of spinal cord and various nuclei of pons and medulla including trigeminal, dorsal tegmental and reticular nuclei. Nucleus of solitary tract, parabrachial, ambiguous and facial nuclei are also intensively labelled. Some structures of mesencephalon and diencephalon exhibited a high density of FLFQPQRFamide binding sites: central gray, raphe nuclei and thalamic nuclei such as parafascicular, laterodorsal, central median, paratenial and paraventricular nuclei. Suprachiasmatic and mammillary nuclei, lateral, posterior and anterior areas of hypothalamus and medial preoptic area exhibited high labelling. FLFQPQRFamide binding sites were also seen in some structures of the dopaminergic meso-cortico-limbic system including ventral tegmental area, cingulate cortex, lateral septum and the head of the caudate-putamen. Dense labelling appeared in the presubiculum of hippocampus. The dissimilar mapping of FLFQPQRFamide and opiate brain receptors confirms our previous pharmacological findings in FLFQPQRFamide binding studies on rat spinal cord membranes, showing that FLFQPQRFamide receptors are different from opiate receptors. There was a good correspondence between localization of binding sites and that of the putative endogenous peptide. Both occur in brain areas previously associated with analgesic action of opiates. However, the mapping of FLFQPQRFamide receptors in the central nervous system suggests that the FLFQPQRFamide system could be implicated in other physiological functions.

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.

Mobilization of the cell adhesion glycoprotein F3/contactin to axonal surfaces is activity dependent.

F3/contactin is a cell adhesion/recognition molecule of the immunoglobulin superfamily implicated in axonal growth. We examined its subcellular distribution and mobilization to the cell surface in oxytocin‐ (OT‐) secreting neurons, which express it throughout life and the axons of which undergo activity‐dependent remodelling. This was performed in hypothalamic organotypic slice cultures containing OT neurons with properties of adult neurosecretory cells. Immunocytochemistry and immunoblot analysis confirmed that OT neurons express high levels of F3/contactin in vitro. Light and confocal microscopy of cultures that underwent double immunofluorescence after fixation showed F3/contactin immunoreactivity throughout the cytoplasm of OT somata, dendrites and axons, and also in non‐OT axons and in putative synaptic boutons which contacted OT neurons. By contrast, after treatment of live cultures with anti‐F3/contactin antibodies followed by double immunofluorescence for the glycoprotein and OT, F3/contactin immunoreactivity was visible only on the surface of axons, whether or not OT‐immunoreactivity was present. Because of its glycosylphosphatidyl‐inositol (GPI) linkage, F3/contactin can occur in a membrane‐bound or soluble form. As seen from immunocytochemistry of live cells and immunoblot analysis, treatment of cultures with a GPI‐specific phospholipase C (GPI‐PLC) resulted in loss of F3/contactin immunoreactivity from all cell surfaces. F3/contactin immunoreactivity reappeared on axonal surfaces within 5 h after enzyme washout. Such re‐expression was accelerated by neuronal activity facilitation (by K+ depolarization or γ‐aminobutyric acid (GABA)‐A receptor blockade with bicuculline) and inhibited by neuronal activity repression [by blockade of Ca2+ channels with Mn2+, Na+ channels with tetrodotoxin (TTX) or excitatory inputs with glutamate antagonists]. Our observations establish therefore that F3/contactin surface expression in hypothalamic neurons is polarized to the axons where it occurs mainly in a GPI‐linked form. We also provide direct evidence that externalization of F3/contactin depends on Ca2+ entry and neuronal electrical activity. Taken together with our earlier finding that the glycoprotein is localized in neurosecretory granules, we demonstrate that F3/contactin is mobilized to the axonal surface via the activity‐dependent regulated pathway, thus arriving at the correct place and time to intervene in activity‐dependent remodelling of axons.

Evidence for neuropeptide FF (FLFQPQRFamide) 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.

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.