Sergio Ovalle

Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury

ABSTRACT Sigma‐1 receptor (σ1R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of σ1R in modulating nerve injury‐evoked pain. For this purpose, wild‐type mice and mice lacking the σ1R gene were exposed to partial sciatic nerve ligation and neuropathic pain‐related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal‐regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous σ1R knockout mice did not differ from wild‐type mice. Baseline values obtained in σ1R knockout mice before nerve injury in the plantar, cold‐plate and von Frey tests were also indistinguishable from those obtained in wild‐type mice. However, cold and mechanical allodynia did not develop in σ1R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking σ1R showed reduced wind‐up responses respect to wild‐type mice, as evidenced by a reduced number of action potentials induced by trains of C‐fiber intensity stimuli. In addition, in contrast to wild‐type mice, σ1R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind‐up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify σ1R as a constituent of the mechanisms modulating activity‐induced sensitization in pain pathways and point to σ1R as a new potential target for drugs designed to alleviate neuropathic pain.

Pharmacological activation of 5-HT7 receptors reduces nerve injury-induced mechanical and thermal hypersensitivity

&NA; The involvement of the 5‐HT7 receptor in nociception and pain, particularly chronic pain (i.e., neuropathic pain), has been poorly investigated. In the present study, we examined whether the 5‐HT7 receptor participates in some modulatory control of nerve injury‐evoked mechanical hypersensitivity and thermal (heat) hyperalgesia in mice. Activation of 5‐HT7 receptors by systemic administration of the selective 5‐HT7 receptor agonist AS‐19 (1 and 10 mg/kg) exerted a clear‐cut reduction of mechanical and thermal hypersensitivities that were reversed by co‐administering the selective 5‐HT7 receptor antagonist SB‐258719. Interestingly, blocking of 5‐HT7 receptors with SB‐258719 (2.5 and 10 mg/kg) enhanced mechanical (but not thermal) hypersensitivity in nerve‐injured mice and induced mechanical hypersensitivity in sham‐operated mice. Effectiveness of the treatment with a 5‐HT7 receptor agonist was maintained after repeated systemic administration: no tolerance to the antiallodynic and antihyperalgesic effects was developed following treatment with the selective 5‐HT7 receptor agonist E‐57431 (10 mg/kg) twice daily for 11 days. The 5‐HT7 receptor co‐localized with GABAergic cells in the dorsal horn of the spinal cord, suggesting that the activation of spinal inhibitory GABAergic interneurons could contribute to the analgesic effects of 5‐HT7 receptor agonists. In addition, a significant increase of 5‐HT7 receptors was found by immunohistochemistry in the ipsilateral dorsal horn of the spinal cord after nerve injury, suggesting a “pain”‐triggered regulation of receptor expression. These results support the idea that the 5‐HT7 receptor subtype is involved in the control of pain and point to a new potential use of 5‐HT7 receptor agonists for the treatment of neuropathic pain.

Immunohistochemical localization of the σ1-receptor in oligodendrocytes in the rat central nervous system

Abstract By using a new polyclonal antibody raised against a 21-amino acid peptide sequence corresponding to the fragment 138–157 of the cloned rat σ1-receptor, we demonstrated by immunoperoxidase and double immunofluorescence techniques, that rat oligodendrocytes express the σ1-receptor. Experiments in vivo and in vitro showed that σ1-receptor colocalized with specific markers of progenitor (A2B5) and mature oligodendrocytes (GalC, RIP). These results suggest that σ1-receptor in oligodendrocytes might be involved in myelination by direct implication in cholesterol biosynthesis or by interaction with endogenous ligands such as neurosteroids.

Molecular Cloning of α1d-Adrenergic Receptor and Tissue Distribution of Three α1-Adrenergic Receptor Subtypes in Mouse

Abstract: A partial cDNA encoding most of the third intracellular loop of the mouse α1d‐adrenergic receptor subtype was amplified from hippocampus by reverse transcription‐polymerase chain reaction (RT‐PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an α1d‐adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0‐kb transcript. Partial cDNA fragments of the α1b‐ and α1a‐adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT‐PCR indicated that the α1‐adrenergic receptors are widely distributed in mouse tissues. The α1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The α1a subtype is also expressed in many tissues, whereas the α1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.

Up-regulation of sigma1 receptor mRNA in rat brain by a putative atypical antipsychotic and sigma receptor ligand

Sigma(1) (sigma(1)) receptor mRNA expression was studied in the prefrontal cortex, striatum, hippocampus and cerebellum of rat brain by northern blot and in situ hybridization. The effects of a chronic treatment with antipsychotic drugs (haloperidol and clozapine), and with E-5842, a sigma(1) receptor ligand and putative atypical antipsychotic on sigma(1) receptor expression were examined. A significant increase in the levels of sigma(1) receptor mRNA in the prefrontal cortex and striatum after E-5842 administration was observed, while no apparent changes were seen with either haloperidol or clozapine. Our results suggest a long-term adaptation of the sigma(1) receptor at the level of mRNA expression in specific areas of the brain as a response to a sustained treatment with E-5842.

Fibroblast growth factor-2 is selectively modulated in the rat brain by E-5842, a preferential sigma-1 receptor ligand and putative atypical antipsychotic.

Fibroblast growth factor‐2 (FGF‐2) is a member of a large family of trophic factors whose expression is regulated under several conditions in different areas of the brain. The goal of our experiments was to determine whether the administration of 4‐(4‐fluorophenyl)‐1,2,3,6‐tetrahydro‐1‐[4‐(1,2,4‐triazol‐1‐il)butyl] pyridine citrate (E‐5842), a sigma‐1 receptor ligand and putative atypical antipsychotic, could regulate the expression of FGF‐2. After chronic treatment with E‐5842 (21 days, and the animals killed 24 h after the last administration), an up‐regulation was observed of the expression of FGF‐2 mRNA in the prefrontal cortex and the striatum, and a down‐regulation of the expression of FGF‐2 mRNA in the hypothalamus of the rat brain. Acute treatment with E‐5842 (one single administration and animals killed 6 h later) up‐regulated FGF‐2 expression in the prefrontal cortex, the striatum, the hypothalamus and the hippocampus in a dose‐dependent manner. The acute up‐regulation was transient and disappeared 24 h after E‐5842 administration. The induction of FGF‐2 in the striatum after repeated administration has been described for clozapine, but our data concerning regulation in the prefrontal cortex suggest that this effect is unique to E‐5852 among other antipsychotics. Given the neuroprotective activity of FGF‐2, the data presented here might be relevant to the deficit in cognition and other symptoms that appear in schizophrenia.

Regulation of Ionotropic Glutamate Receptor Subunits in Different Rat Brain Areas by a Preferential Sigma1 Receptor Ligand and Potential Atypical Antipsychotic

The effect of chronic administration of the putative atypical antipsychotic E-5842, a preferential sigma1 receptor ligand, on ionotropic glutamate receptor subunit levels of mRNA and protein, was studied. The repeated administration of E-5842 differentially regulated levels of the NMDA-2A and of GluR2 subunits in a regionally specific way. Levels of immunoreactivity for the NMDA-2A subunit were up-regulated in the medial prefrontal cortex, the frontoparietal cortex, the cingulate cortex, and in the dorsal striatum, while they were down-regulated in the nucleus accumbens. Levels of the GluR2 subunit of the AMPA receptor were up-regulated in the medial prefrontal cortex and the nucleus accumbens and down-regulation was observed in the dorso-lateral striatum. Regulation of the levels of mRNA for the different subunits was also observed in some cases. The results show that E-5842, through a mechanism still unknown, is able to modify levels of several glutamate receptor subunits and these changes could be related to its antipsychotic activity in pre-clinical tests.

Immunodetection of serotonin transporter from mouse brain

A DNA fragment encoding amino acid sequences from the amino terminal region of the mouse serotonin transporter was isolated and sequenced. This transporter is widely distributed throughout the mouse brain, as deduced by heminested reverse transcription-polymerase chain reaction (RT-PCR) assays. To identify the serotonin transporter protein, we have developed specific antibodies against a fusion protein containing its amino terminal region, a domain which shows a low degree of homology between the different neurotransmitter transporters. Western blot analysis of mouse brain membranes detected the serotonin transporter as a 71 kDa polypeptide.

Phosphorylation of the Third Intracellular Loop of the Mouse α1b-Adrenergic Receptor by cAMP-dependent Protein Kinase

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. alpha1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse alpha1a-, alpha1b-, and alpha1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the alpha1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.

Immunodetection of the large form of the γ2 subunit of mammalian GABAA receptor

Abstract Two forms of the GABAA receptor γ2-subunit, named short (γ2S) and long (γ2L), and generated by alternative RNA splicing, have been identified in mammalian brain by molecular cloning techniques. We have produced antibodies against a synthetic peptide containing the 8-amino acid insertion present in the long form but not in the short one. Using the antipeptide serum, we have identified the γ2L subunit in membrane preparations of GABAA receptors from rat and mouse cerebellum and its cellular location in cerebellum.