Jacqueline Fischer

CB1-cannabinoid and mu-opioid receptor co-localization on postsynaptic target in the rat dorsal horn.

Cannabinoids and opioids interact in the control of nociception at the spinal level. Likely, several mechanisms are involved, with one of them being co-localization of cannabinoid and opioid receptors. In order to validate this hypothesis, a double labeling study of CB1 cannabinoid receptors and μ-opioid receptors in the dorsal horn of the rat spinal cord was performed. A strong co-localization of CB1 and μ-opioid receptors was observed in lamina II interneurons at the ultrastructural level. The physiological consequences of the co-localization are discussed.

Pre- and postsynaptic localizations of the CB1 cannabinoid receptor in the dorsal horn of the rat spinal cord.

Several lines of evidence show that endogenous and exogenous cannabinoids modulate pain transmission at the spinal level through specific cannabinoid-1 (CB1) receptors. Since anatomical data concerning spinal CB1 receptors are rather contradictory, we studied the cellular and subcellular localizations of the CB1 receptors by immunocytochemistry. Results show a dual pre- and postsynaptic localization of CB1 receptors. Presynaptic receptors are evidenced by the labeling of (1) heterogeneous dorsal root ganglion neurons and (2) axons of Lissauers tract. Postsynaptic receptors are shown by the labeling of numerous interneurons in the outer part of lamina II. Double immunolabelings show that lamina II outer CB1 neurons, probably islet cells, may also contain GABA or nitric oxide synthase. Numerous CB1-containing neurons in lamina X are also immunostained with anti-nitric oxide synthase (NOS) antibody. Under the electron microscope, CB1 immunoreactivity is exclusively localized postsynaptically in both somatic and dendritic compartments. The absence of labeling on primary afferent axon terminals is discussed and compared to the absence of labeling on terminals or vesicle-containing dendrites of islet cells, where a presynaptic localization was expected according to data of the literature.

Neuronal and astrocytic localization of the cannabinoid receptor-1 in the dorsal horn of the rat spinal cord.

Cannabinoids are involved in the control of pain at the spinal level through the cannabinoid receptor-1 (CB1) localized pre- and postsynaptically on primary afferent fibres and dorsal horn interneurones, respectively. Using immunocytochemistry, we show that in addition to its neuronal localization, CB1 is also expressed in numerous astrocytes in laminae I and II of the rat dorsal horn. This ubiquitous localization may account for the complex role played by cannabinoids in antinociception. CB1 receptors in astrocytes may be involved in the anti-hyperalgesic action of exogenous cannabinoids.

The vanilloid receptor-1 is expressed in rat spinal dorsal horn astrocytes

The vanilloid receptor-1 (TRPV1), expressed by nociceptive fibers, is a transducer of thermal and chemical nociceptive messages. However, endogenous ligands excite TRPV1 receptors localized on central nociceptive terminals and interneurons. Using immunocytochemistry at the ultrastructural level, we show that TRPV1 is also expressed in spinal glial cells characterized as astrocyte by double labeling with glial fibrillary acid protein. Quantification of the labeling shows that the most numerous labeling is neuronal and that 7% of the total TRPV1 labeling is localized in astrocytes. The total absence of staining in TRPV1 knock out mice strongly suggests that true TRPV1 protein is present in astrocytes. The localization of TRPV1-containing astrocytes apposed to nociceptive C-terminals suggests that they may be involved in the control of pain transmission.

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

Abstract 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 (0.1 mg/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 Aβ 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.

&NA; 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 100 mg/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.

The 5-HT2A receptor is mainly expressed in nociceptive sensory neurons in rat lumbar dorsal root ganglia.

Several lines of evidence indicate that peripheral 5-HT2A receptors are involved in the development of inflammatory and neuropathic pain. However, their localization in sensory cell bodies is not accurately known. We therefore studied 5-HT2A receptor distribution in rat lumbar dorsal root ganglia using immunocytochemistry. Forty percent of L3 lumbar dorsal root ganglion cells were immunoreactive for 5-HT2A receptor. Most were small- to medium-sized cell bodies. Double-labeled experiments revealed that they expressed various chemical phenotypes. The smaller 5-HT2AR cell bodies often bind the isolectin B4 although some 5-HT2AR cell bodies also express substance P (SP). Many 5-HT2A-positive small dorsal root ganglion cells expressed the capsaicin receptor transient receptor potential vanilloid type 1 receptor (TRPV1), confirming their nociceptive nature. In addition, a few large cell bodies were labeled for 5-HT2A, and they also expressed NF200 suggesting that they were at the origin of Adelta or Abeta fibers. A total absence of double labeling with parvalbumin showed that they were not proprioceptors. 5-HT2A immunoreactivity in dorsal root ganglia cells was found in the cytoplasm and along the plasma membrane at the interface between sensory cell and the adjacent satellite cells; this distribution was confirmed under the electron microscope, and suggested a functional role for the 5-HT2A receptor at these sites. We therefore investigated the presence of 5-HT and 5-HIAA in lumbar dorsal root ganglia by high performance liquid chromatography. There were 5.75+/-0.80 ng 5-HT and 3.19+/-0.37 ng 5-hydroxyindoleacetic acid (5-HIAA) per mg of protein with a ratio 5-HIAA/5-HT of 0.67+/-0.10, similar to values typically observed in brain tissues. These findings suggest that 5-HT, via the 5-HT2AR, may be involved in the peripheral control of sensory afferents, mainly unmyelinated nociceptors and to a lesser extent neurons with Adelta or Abeta fibers, and in the control of cellular excitability of some dorsal root cell bodies through a paracrine mechanism of action.

Expression and G-protein coupling of μ-opioid receptors in the spinal cord and dorsal root ganglia of polyarthritic rats

Although chronic inflammatory pain is known to be associated with hypersensitivity to mu opioid receptor agonists, no evidence for changes in the expression and/or characteristics of central mu opioid receptors has yet been reported in relevant models of this type of pain. In the present study, both immunohistochemical and autoradiographic approaches were used to address this question in polyarthritic rats, on the 4th week after intradermal injection of complete Freunds adjuvant, when inflammatory pain was at its maximum. Immunohistochemical labeling with specific anti-mu opioid receptor antibodies and autoradiographic labeling with [3H]DAMGO showed an upregulation of mu opioid receptors in the dorsal root ganglia but no changes in the density of these receptors in the dorsal horn at the level of L4-L6 segments in polyarthritic compared to age-paired control rats. On the other hand, autoradiographic quantification of the concentration-dependent increase in [35S]GTP-gamma-S binding by the mu-opioid receptor agonist DAMGO did not show any significant differences within the lumbar dorsal horn between polyarthritic and control rats. These data indicate that chronic inflammatory pain caused by polyarthritis was associated with an increased expression of mu-opioid receptors in dorsal root ganglion sensory neurones that did not result in an increased spinal density of these receptors, in spite of their well established axonal transport in the central portion of primary afferent fibres to the dorsal horn. In contrast, axonal transport of mu-opioid receptors in the peripheral portion of these fibres probably accounts for the increased receptor density in inflamed tissues already reported in the literature.

The vanilloid receptor-1 (TRPV1) is expressed in some rat dorsal horn NK1 cells.

The vanilloid receptor-1 (TRPV1), the capsaicin receptor, a transducer of several nociceptive stimuli, is principally expressed by nociceptive fibers that specifically target supraspinal-projecting neurons expressing the NK1 receptor. TRPV1 is also expressed by intraspinal neurons. Using double immunocytochemistry, we show that 14.2% of these TRPV1 dorsal horn cell bodies also express the NK1 receptor suggesting that endogenous vanilloids may directly modulate second-order ascending neurons.

Ultrastructural study of δ-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies

Abstract The ultrastructural localization of δ-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti- d -Ala2- d -Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, δ or μ, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3–4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10−9 M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for δ-opioids in the spinal cord, providing that these receptors are functional. δ-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.