Serotonin 5-HT2A receptor involvement and Fos expression at the spinal level in vincristine-induced neuropathy in the rat.

We recently showed that peripheral and spinal 5-HT2A receptors (5-HT2AR) are involved in a rodent model of neuropathy induced by a nucleoside analogue reverse transcriptase inhibitor. In this paper, we show that 5-HT2AR are also involved in neuropathy induced by an anti-neoplasic drug, vincristine. Vincristine-treated rats (0.1mg/kg, daily i.p. administration for two 5-day cycles) developed thermal allodynia and mechanical hypersensitivity, which decreased in a dose-related manner after epidural injection a 5-HT2A receptor antagonist. Moreover, 5-HT2A-/- mice did not develop vincristine-induced neuropathy contrarily to their 5-HT2A+/+ littermates. In vincristine-treated rats, the number of nociceptive dorsal root ganglion cells expressing the 5-HT2AR was increased by 38%, and 5-HT2AR immunolabelling was enhanced in layers I-IV of the dorsal horn. At the EM level, a 76.3% increase in the density of 5-HT2AR immunopositive axon terminals within superficial layers of the dorsal horn was noted after vincristine treatment. Immunocytochemical study of Fos expression in vincristine-treated rats revealed a significant increase in the number of Fos-positive neurons not only in regions where nociceptive fibres terminate superficial (I-II) and deep layers (V-VI) of the spinal cord, but also in intermediate layers, suggesting that Abeta fibres could be involved in the spinal sensitization observed in this model. Double labelling experiments showed that Fos-positive neurons were endowed with 5-HT2AR immunolabelling in the dorsal horn of vincristine-treated rats. These data provide support to the idea that, in vincristine-induced neuropathy, 5-HT2AR are involved in the sensitization of peripheral nociceptors and spinal nociceptive processing.

Role of spinal serotonin 5-HT2A receptor in 2',3'-dideoxycytidine-induced neuropathic pain in the rat and the mouse.

Several lines of evidence suggest that descending serotoninergic facilitatory pathways are involved in neuropathic pain. These pathways may involve 5-HT2A receptors known to play a role in spinal and peripheral sensitization. The implication of this receptor in neuropathy was investigated in a model of peripheral neuropathy induced by 2',3'-dideoxycytidine, a nucleoside analogue with reverse transcriptase inhibitory properties used in HIV/AIDS therapy. Four days after a single 100mg/kg i.v. administration in the tail vein, mitochondrial alterations in nociceptive and non-nociceptive dorsal root ganglion cells were observed at the lumbar level. These alterations were not associated with TUNEL labelling or with modification of the total number of dorsal root ganglion cells. At the same time point, 5-HT2A receptor immunolabelling was increased throughout the dorsal horn (by 49.5% in layer II and 57.8% in layer III). The number of 5-HT2A receptor immunoreactive neurons in the dorsal root ganglion was also increased by 30.7%. Four days after 2',3'-dideoxycytidine administration, rats had developed thermal allodynia as well as mechanical hyperalgesia and allodynia, which dose-dependently decreased after epidural injection of MDL 11,939, a 5-HT2A receptor antagonist. Moreover, 5-HT2A receptor knock-out mice did not develop 2',3'-dideoxycytidine-induced neuropathy whereas their control littermates displayed a neuropathy comparable to that observed in rats. Our data show that 2',3'-dideoxycytidine-induced neuropathy is associated with alterations of nociceptive and non-nociceptive peripheral cells and that the 5-HT2A receptor is involved in the peripheral sensitization of nociceptors as well as in a wide central sensitization of dorsal horn neurons.

Serotonin 5-HT2A and 5-HT5A receptors are expressed by different motoneuron populations in rat Onuf's nucleus.

Motoneurons of Onuf's nucleus innervate the pelvic striated muscles, which play a crucial role in erection, ejaculation, and urinary continence. Serotonergic descending projections from the brain are involved in the modulation of Onuf's motoneuron activity. However, conflicting results regarding the effects of spinal serotonin (5-HT) on pelvi-perineal functions have been reported. They may be partly accounted for by the multiplicity of neuronal targets and receptor subtypes on which 5-HT is acting. In order to provide comparative data regarding 5-HT receptor expression in various groups of Onuf's motoneurons, we used retrograde tracing techniques from different pelvic muscles combined with immunocytochemistry of 5-HT2A and 5-HT5A receptors in male and female rats. In males, 5-HT2A receptor immunolabeling was very dense in motoneurons innervating the ischiocavernosus muscle. By contrast, in female rats, 5-HT2A receptor expression in Onuf's nucleus was very weak. In both genders, 5-HT5A receptor immunoreactivity was found in motoneurons innervating the external urethral sphincter. In males, a moderate or low 5-HT5A immunolabeling was observed in motoneurons innervating the bulbospongiosus and ischiocavernosus muscles, respectively. These data show a preferential localization of 5-HT2A and 5-HT5A receptors to motoneurons controlling the striated muscles located at the penile crus and sphincter muscles, respectively, suggesting a specific serotoninergic control on different pelvic functions. In addition, the subcellular distribution of receptors suggests a different mode of action of 5-HT, paracrine at 5-HT2A receptors and synaptic at 5-HT5A receptors. This might have implications for pharmacological research targeting different pelvic functions e.g., micturition and ejaculation.

Pre- and postsynaptic localization of the 5-HT7 receptor in rat dorsal spinal cord: immunocytochemical evidence.

Several lines of evidence indicate that 5-HT7 receptors are involved in pain control at the level of the spinal cord, although their mechanism of action is poorly understood. To provide a morphological basis for understanding the action of 5-HT on this receptor, we performed an immunocytochemical study of 5-HT7 receptor distribution at the lumbar level. 5-HT7 immunolabelling is localized mainly in the two superficial laminae of the dorsal horn and in small and medium-sized dorsal root ganglion cells, which is consistent with a predominant role in nociception. In addition, moderate labelling is found in the lumbar dorsolateral nucleus (Onuf's nucleus), suggesting involvement in the control of pelvic floor muscles. Electron microscopic examination of the dorsal horn revealed three main localizations: 1) a postsynaptic localization on peptidergic cell bodies in laminae I-III and in numerous dendrites; 2) a presynaptic localization on unmyelinated and thin myelinated peptidergic fibers (two types of axon terminals are observed, large ones, presumably of primary afferent origin, and smaller ones partially from intrinsic cells; this presynaptic labelling represents 60% and 22% of total labelling in laminae I and II, respectively); and 3) 16.9% of labelling in lamina I and 19.8% in lamina II are observed in astrocytes. Labeled astrocytes are either intermingled with neuronal elements or make astrocytic "feet" on blood vessels. In dendrites, the labelling is localized on synaptic differentiations, suggesting that 5-HT may act synaptically on the 5-HT7 receptor. This localization is compared with other 5-HT receptor localizations, and their physiological consequences are discussed.

Rapid up-regulation of the neuronal serotoninergic phenotype by brain-derived neurotrophic factor and cyclic adenosine monophosphate: relations with raphe astrocytes.

Up-regulation of the neuronal serotoninergic phenotype in relation to astrocytic population was studied in primary cultures of rat embryonic rostral raphe. Short treatments (18 hr at day in vitro 4) with brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) increased the number of serotoninergic neurons by approximately 80% and approximately 40%, respectively, and markedly enhanced the branching (by 11-fold and 5-fold, respectively) and total length (by 4-fold and 2.5-fold, respectively) of their neurites. Concomitantly, under BDNF treatment, the astrocyte population was decreased by half and became mostly protoplasmic-like. In contrast, dBcAMP treatment also reduced the astrocytic cell density (by one-third) but induced a stellate morphology. Similar short treatment with the astrocyte-derived S100beta factor induced no modification of the serotonin (5-HT) neuronal phenotype nor of astrocytes morphology. Both BDNF- and cAMP-induced effects were abolished by simultaneous treatment with the specific tyrosine kinase inhibitor genistein, suggesting a role for the high-affinity BDNF receptor tyrosine kinase (TrkB). These data suggest that BDNF and cAMP, but not S100beta, rapidly induce both an up-regulation of the 5-HT neuronal phenotype and modifications of the neighboring astrocytes in a TrkB-dependent manner.

5-HT5A receptor localization in the rat spinal cord suggests a role in nociception and control of pelvic floor musculature.

The 5-HT5A receptor is a seven-transmembrane receptor negatively coupled to adenylate cyclase, whose activation opens K+ channels. The 5-HT5A receptor may thus exert an inhibitory effect on neuronal activity. However, the function of this receptor is still largely unknown, in particular at the spinal level, and this is partly due to lack of specific ligands. Immunocytochemistry using specific anti-5-HT5A antibodies reveals a particularly dense labeling in the two superficial layers of the dorsal horn, suggesting that the 5-HT5A receptor may be involved in the spinal modulation of pain. In addition, a very intense staining in the lumbar dorsolateral nucleus (Onuf nucleus) in both males and females suggests that the 5-HT5A receptor is also involved in micturition through the control of urethral sphincter muscles. Colchicine pretreatment allows the staining of numerous cell bodies in lamina II. Fewer labeled cell bodies are seen in laminae I and III-VI, in the lateral spinal nucleus, and in lamina X. Electron microscope examination of 5-HT5A receptor immunoreactivity in spinal cords from untreated animals confirmed the postsynaptic labeling in all regions studied (dorsal horn, dorsolateral nucleus, and lamina X). The morphological heterogeneity of labeled dorsal horn cell bodies suggests that they belong to functionally distinct neurons (projection neurons and interneurons). In the lumbar dorsolateral nucleus, the labeling is preferentially localized on dendrites, suggesting that in this nucleus 5-HT preferentially acts at the dendritic level. Finally, the dense labeling of postsynaptic specializations suggests that the receptor may be in stock before being addressed to the synaptic differentiation.

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons.

Serotonin (5-HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5-HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5-HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5-HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I-II) and ventral horn (lamina IX). At both levels, 5-HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the "nonsynaptic" plasma membrane localization of 5-HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre- and postsynaptic) for serotonin on these receptors on various cellular targets.

Adaption of the serotoninergic neuronal phenotype in the absence of 5-HT autoreceptors or the 5-HT transporter: involvement of BDNF and cAMP.

Serotonin 5-HT1A and 5-HT1B receptors and the 5-HT transporter are key regulators of the serotoninergic neuronal phenotype. We show here that genetic deletion of any of these elements differentially regulates 5-HT neuronal number in rostral raphe cultures from E14 mice. Serotonin neuronal number was increased by almost four-fold and 1.8-fold in cultures from 5-HT1AR-/- and 5-HT1BR-/- mice, respectively. In contrast, the lack of serotonin transporter expression was associated with a 50% decrease in 5-HT neuronal number. In raphe cultures from the rat, BDNF and cAMP have been shown to up-regulate the neuronal serotoninergic phenotype through TrkB-dependent mechanisms [Rumajogee et al. (2002) J. Neurochem., 83, 1525-1528]. Similar tyrosine kinase-dependent up-regulating effects, in the absence of serotoninergic key-elements are reported here, on both 5-HT neuronal number and neurites length. However, the extents of BDNF-triggered and cAMP-triggered effects on serotoninergic neuritic length were approximately 1.5-fold higher in 5-HT1AR-/- mutants. These findings show that the up-regulatory mechanisms triggered by BDNF on serotoninergic neuronal number and neurite extension are different and that the latter are partially linked to 5-HT, probably through 5-HT1A autoreceptors. Together, these data suggest that serotonin autoreceptors, mainly 5-HT1A but also 5-HT1B, may be responsible for a tonic auto-inhibitory effect of 5-HT itself on the serotoninergic neuronal phenotype during embryonic development, particularly marked in the absence of the 5-HT transporter.

Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry.

Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway.