Anna Wainwright

Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature

Calcitonin gene-related peptide and adrenomedullin belong to a structurally related neuropeptide family and are potent vasodilators expressed in the trigeminovascular system. The molecular identity of receptors for these proteins has only recently been elucidated. Central to functional binding of these neuropeptides is the G-protein–coupled receptor, the calcitonin receptor–like receptor (CRLR), whose cell surface expression and pharmacology is determined by coexpression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP1 binds calcitonin gene-related peptide with high affinity, whereas CRLR coexpression with RAMP2 or −3 confers high-affinity binding of adrenomedullin. The authors investigated the expression of these receptor components in human cerebral vasculature to further characterize neuropeptide receptor content and the potential functions of these receptors. Localization has been carried out using specific antisera raised against immunogenic peptide sequences that were subsequently applied using modern immunohistochemical techniques and confocal microscopy. The results are the first to show the presence of these receptor component proteins in human middle meningeal, middle cerebral, pial, and superficial temporal vessels, and confirm that both calcitonin gene-related peptide and adrenomedullin receptors may arise from the coassembly of RAMPs with CRLR in these vessel types. These novel data advance the understanding of the molecular function of the trigeminovascular system, its potential role in vascular headache disorders such as migraine, and may lead to possible ways in which future synthetic ligands may be applied to manage these disorders.

Localization of 5-ht5A receptor-like immunoreactivity in the rat brain

5-Hydroxytryptamine exerts modulatory physiological effects on both the central and peripheral nervous systems by activation of discrete receptor families. 5-ht(5A) is among the recently cloned, novel 5-HT receptors and currently under investigation to identify its pharmacological characteristics and potential physiological function(s). In this study, antibodies raised to a 5-ht(5A)-specific peptide were characterized using dot blot, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry, and the distribution of 5-ht(5A)-like immunoreactive material determined in rat brain. A major band of 41 kDa was observed following SDS-PAGE, corresponding to the predicted size of this receptor. 5-ht(5A)-like immunoreactivity was detected in areas known to have significant serotonergic input, such as hypothalamic and amygdaloid nuclei. Interestingly, 5-ht(5A)-like immunoreactivity showed a predilection for the suprachiasmatic nucleus, suggesting its possible role in the regulation of circadian rhythms, a function previously ascribed to 5-HT(1A) and 5-HT(7) receptors.

Distribution of 5-ht5A, 5-ht5B, 5-ht6 and 5-HT7 receptor mRNAs in the rat brain

The precise involvement of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht(5A) mRNA was widely expressed while 5-ht(5B) mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht(6) mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT(7) mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.

Distribution of novel CGRP1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

Discrete expression of TRPV2 within the hypothalamo-neurohypophysial system: Implications for regulatory activity within the hypothalamic-pituitary-adrenal axis

Transient receptor potential channel proteins (TRPs) constitute a steadily growing family of ion channels with a range of purported functions. It has been demonstrated that TRPV2 is activated by moderate thermal stimuli and, in the rat, is expressed in medium to large diameter dorsal root ganglion neurons. In this study, antisera specific for the human TRPV2 homologue were raised and characterized for immunohistochemical use. Subsequently, thorough investigation was made of the localization of this cation channel in the macaque primate brain. TRPV2‐immunoreactive material was highly restrictively localized to hypothalamic paraventricular, suprachiasmatic, and supraoptic nuclei. Confocal double‐ and triple‐labeling studies demonstrated that TRPV2 immunoreactivity is preferentially localized to oxytocinergic and vasopressinergic neurons. Few, if any, cells in these regions expressed TRPV2 immunoreactivity in the absence of oxytocin immunoreactivity or vasopressin immunoreactivity. Expression in the paraventricular and supraoptic nuclei suggests that TRPV2 is likely to play a fundamental role in mediating cation transport in neurohypophysial neurons. TRPV2 has been shown to be translocated upon cell activation and neurons expressing TRPV2 immunoreactivity in vivo are among those known to engage in sporadic, intense activity. Taken together, these data suggest that this channel may play a vital role in mediating physiological activities associated with oxytocin and vasopressin release such as parturition, lactation, and diuresis. These data may also implicate the involvement of TRPV2 in disorders of the hypothalamic‐pituitary‐adrenal axis, including anxiety, depression, hypertension, and preterm labor. J. Comp. Neurol. 474:24–42, 2004.

Autoradiographic localization of 125I[Tyr14] nociceptin/ orphanin FQ binding sites in macaque primate CNS

Nociceptin/orphanin FQ (N/OFQ) is a recently identified neuropeptide that has been implicated in a multitude of CNS functions. These include nociception, feeding, cognition, locomotion, stress and neuroendocrine control. The endogenous receptor for this ligand is the nociceptin/orphanin FQ peptide (NOP) receptor. The distribution of NOP in rodent has been widely reported by the use of in situ hybridization, immunohistochemistry and autoradiographic radioligand binding but less is known of its localization in higher species. We have therefore sought to optimize and determine the distribution of (125)I[Tyr(14)]N/OFQ binding sites in macaque primate brain and spinal cord. Highest levels of binding were observed in neocortical areas, hippocampus, amygdala, caudate nucleus and putamen, medial thalamic nuclei and superficial laminae of the superior colliculus. These novel data present for the first time, the distribution of N/OFQ receptors in non-human primate CNS and, by comparison with localization in the rat, reveal that species differences may exist in the distribution of this neuropeptide receptor. These data have important implications regarding the roles of N/OFQ across species and may have ramifications in the interpretation of preclinical pharmacological studies.

Expression of GABAA receptor α5 subunit-like immunoreactivity in human hippocampus

Abstract To investigate the distribution of GABAA receptor α5 subunit expression in brain, polyclonal antisera were raised, characterised and applied to human and rat brain sections. The resultant antibodies detected a major band of 53 kDa in sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblots. Abundant immunostaining was demonstrated in the hippocampal formation in multiple cell types, although predominantly in pyramidal neurons. These data are supportive of GABA-ergic involvement in cognition, and suggest that this influence may be mediated through receptors containing the α5 subunit.

Regional and cellular localization of calcitonin gene-related peptide-receptor component protein mRNA in the guinea-pig central nervous system

Recent cloning studies have isolated proteins which confer responsiveness to calcitonin gene-related peptide (CGRP). In this study, we have determined the central nervous system (CNS) distribution of the mRNA of one such protein, termed CGRP-receptor component protein (RCP), by in situ hybridization. CGRP-RCP mRNA was widely expressed in the guinea-pig CNS, being particularly abundant in cerebellum and hippocampus. These data should assist in the determination of the potential physiological function(s) of this protein in the CNS.