Colocalization of CGRP with 5-HT1B/1D receptors and substance P in trigeminal ganglion neurons in rats.

Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis.

Distribution of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) 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.

Expression of messenger RNAs for glutamic acid decarboxylase, preprotachykinin, cholecystokinin, somatostatin, proenkephalin and neuropeptide Y in the adult rat superior colliculus.

The mammalian superior colliculus is an important subcortical integrator of sensorimotor behaviours. It is multi-layered, each layer containing specific neuronal types and possessing distinct input/output relationships. Here we use in situ hybridisation methods to map the distribution of seven neurotransmitters/neuromodulator systems in adult rat superior colliculus. Coronal sections were probed for preprotachykinin, cholecystokinin, somatostatin, proenkephalin, neuropeptide Y and the enzymes glutamic acid decarboxylase and choline acetyltransferase, markers for GABA and acetylcholine respectively. Cells expressing glutamic acid decarboxylase messenger RNA were the most abundant, the highest density being found in the superficial layers. Many cells containing proprotachykinin messenger RNA were found in stratum zonale and the upper two-thirds of stratum griseum superficiale; cells were also located in deeper tectal laminae, particularly caudomedially. Most cholecystokinin messenger RNA expressing cells were located in the superficial layers with a prominent band in the middle third of stratum griseum superficiale. Cells expressing moderate to high levels of somatostatin messenger RNA formed a dense band in the lower third of stratum griseum superficiale/upper stratum opticum; two less distinct tiers of labelling were seen in deeper layers. These in situ hybridisation data reveal three distinct sub-laminae in rat stratum griseum superficiale. Cells expressing moderate to low levels of proenkephalin messenger RNA were located in lower stratum griseum superficiale/upper stratum opticum and intermediate laminae. A cluster of enkephalinergic cells was located medially in the deep tectal laminae. Expression of neuropeptide Y messenger RNA was relatively low and mostly confined to cells in stratum griseum superficiale and stratum opticum. No choline acetyltransferase messenger RNA was detected. This in situ analysis of seven different neurotransmitters/neuromodulator systems sheds new light on the neurochemical organisation of the rat superior colliculus. The data are related to what is known anatomically and physiologically about intrinsic and extrinsic tectal circuitry, and the potential involvement of different neuropeptides in these circuits is discussed. The work forms the basis for future developmental studies examining the effects of transplantation and visual deprivation/deafferentation on tectal neurochemistry and function.

Expression of GABA(A) receptor alpha5 subunit-like immunoreactivity in human hippocampus.

To investigate the distribution of GABA(A) receptor alpha5 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 alpha5 subunit.

CEP-1347 increases ChAT activity in culture and promotes cholinergic neurone survival following fimbria-fornix lesion.

Recent evidence suggests that the activation of the Jun N-terminal kinase (JNK) signal transduction pathway may be important in neuronal responses to stresses such as trophic factor deprivation. Preventing the activation of JNK and expression of c-Jun may, therefore, be neuroprotective. Here, we report that the small molecule CEP-1347, which has been shown to inhibit the JNK signalling pathway, promotes cholinergic activity in cultured embryonic septal neurones. In vivo, we have shown that CEP-1347, administered either by sub-cutaneous (s.c.) injection or by continuous infusion, is partially neuroprotective, for cholinergic neurones in the medial septum, following fimbria-fornix transection. These data suggest that small molecules such as CEP-1347 may have beneficial effects in treating neurodegenerative diseases.

Localization of 5-ht(5A) 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.

Basal expression of bradykinin B1 receptor in peripheral sensory ganglia in the rat.

The bradykinin B1 receptor is thought to be induced by tissue injury and inflammation. In the present study, we have investigated whether there is a basal expression of B1 receptor in dorsal root ganglion (DRG) and trigeminal ganglion neurons in rats. A substantial number of neurons in both DRGs and trigeminal ganglia were found to be B1-immunoreactive in rats. Both small and medium-sized DRG neurons were B1-immunoreactive, suggesting that they are likely to be Adelta- or C-fibre neurons which are involved in nociceptive transmission. These results support a possible role for B1 receptors in the modulation of nociceptive sensory transmission.

From basic research on neuropeptide receptors to clinical benefit.

The present article provides a concise overview of progress in our understanding of neuropeptides, their receptors and the current and potential ways in which they may be targeted for clinical use. Neuropeptide systems offer certain characteristics that distinguish them from those utilizing classic neurotransmitters and thus make them increasingly attractive for drug targeting. A key example of the highly successful use of neuropeptide receptor ligands is that of opioid receptor agonists for the treatment of pain. More recently, classically identified neuropeptide receptors, including those for substance P, cholecystokinin and neurotensin, have been singled out for development of antagonists by several pharmaceutical companies, for various therapeutic indications. These examples can be categorized as having been identified using classic methods; that is, the neuropeptide(s) and/or their function(s) were established prior to molecular identification of their receptor(s). Modern methodologies being widely applied in the industry today have somewhat reversed the need for this ordering in drug discovery. Technologies based upon bioinformatics, molecular, proteomic and transgenic approaches have accelerated the identification of putative receptor targets and the generation of information on their involvement in physiological and pathophysiological processes. Now research and development teams are in a position to best select targets from this rapidly expanding catalogue. This article seeks to outline aspects of these procedures and additionally summarize recent data which have important implications for drug development in this field. Consideration has been given to potential targets and how these may ultimately lead to clinical benefit.

Cortical and nigral deafferentation and striatal cholinergic markers in the rat dorsal striatum: different effects on the expression of mRNAs encoding choline acetyltransferase and muscarinic m1 and m4 receptors.

The regulation of the striatal m1 and m4 muscarinic receptor mRNA as well as the choline acetyltransferase (ChAT) mRNA expression by nigral dopaminergic and cortical glutamatergic afferent fibres was investigated using quantitative in situ hybridization histochemistry. The effects induced by a unilateral lesion of the medial forebrain bundle and a bilateral lesion of the sensorimotor (SM) cortex were analysed in the dorsal striatum 3 weeks after the lesions. Dopaminergic denervation of the striatum resulted in a marked decrease in the levels of m4 mRNA throughout the striatum, while the levels of muscarinic m1 mRNA and ChAT mRNA in cholinergic neurons were unaffected by the lesion. In contrast, following bilateral cortical ablation, the levels of the muscarinic m1 mRNA were significantly increased in the striatal projection area of the SM cortex, whereas the expression of m4 mRNA remained unchanged. Single cholinergic cell analysis by computer-assisted grain counting revealed a decreased labelling for ChAT mRNA per neuron following cortical ablation. However, in contrast to the topographical m1 mRNA changes, the decreased ChAT mRNA expression was evenly distributed within the striatum, suggesting an indirect cortical control upon striatal cholinergic interneurons. Altogether, these data suggest that dopaminergic nigral and glutamatergic cortical afferents modulate differentially cholinergic markers, at the pre- and post-synaptic levels. Beside the fact that nigral and cortical inputs exert an opposite control on cholinergic neurotransmission, our study further shows that this control involved different muscarinic receptor subtypes: the m4 and m1 receptors, respectively.

Increased expression of GAP-43 in small sensory neurons after stimulation by NGF indicative of neuroregeneration in capsaicin-treated rats.

Intraplantar injections of human recombinant nerve growth factor (rhNGF-beta) into the hind paw of capsaicin-treated adult rats are known to lead to a recovery of depleted peptide transmitter substances, to the immunohistochemical reappearance of peptidergic innervation in the skin and in the dorsal horn of the spinal cord, as well as to a recovery of the function of capsaicin-lesioned neurons. In the present study a marker peptide for neuronal regeneration and outgrowth, growth associated protein 43 (GAP-43), was investigated in lumbar dorsal root ganglia (DRGs) and in the hindpaw skin, in order to differentiate which population of the sensory neurons responds with a neuroregenerative behaviour. In situ hybridization histochemistry (ISH) revealed that at day 8 after the capsaicin treatment GAP-43 expression was significantly increased in small DRG cells as compared to control animals, and treatment with NGF in capsaicinized rats lead to an even more pronounced increase of GAP-43 expression in the small-sized cell population. Intraepidermal labelling of GAP-43 peptide was observed in the skin of control animals, but was markedly reduced in the animals that were treated with capsaicin alone. However, intraepidermal GAP-43 immunoreactive (GAP-43-IR) fibres nearly fully recovered in the capsaicin + NGF-treated group. These results indicate that the population of small DRG cells shows spontaneous regenerative activity after a capsaicin lesion which does not lead to a successful recovery of nerve terminals in the skin. Only after an additional NGF treatment small DRG cells show an even stronger regenerative response which now also involves structural reorganization of neuron membranes and axogenesis in the periphery.

theta, a novel gamma-aminobutyric acid type A receptor subunit.

gamma-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the theta subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the beta1 subunit. Within the rat striatum, this subunit coassembles with alpha2, beta1, and gamma1, suggesting that gamma-aminobutyric acid type A receptors consisting of arrangements other than alpha beta + gamma, delta, or epsilon do exist. Expression of alpha2beta1gamma1theta in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for gamma-aminobutyric acid compared with alpha2beta1gamma1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.

Molecular and functional diversity of the expanding GABA-A receptor gene family.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn.

The present study investigated the regional distribution of the N-methyl-D-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibers and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02-1 mg/kg i.p., L-687,414 10-300 mg/kg i.p., and L-701,324 1-10 mg/kg i.p.) and the anticonvulsant, gabapentin (10-500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (+/-)-CP-101,606 (1-100 mg/kg p.o.) and (+/-)-Ro 25-6981 (3-100 mg/kg i.p.) showed a significant dose window. (+/-)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (+/-)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3-500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.

Mechanisms contributing to the deficits in hippocampal synaptic plasticity in mice lacking amyloid precursor protein.

Abnormal processing of amyloid precursor protein (APP), in particular the generation of beta-amyloid (Abeta) peptides, has been implicated in the pathogenesis of Alzheimer's disease. This study examined the consequences of deleting the APP gene on hippocampal synaptic plasticity, and upon the biophysical properties of morphologically identified neurones in APP-null mice. The hippocampus of APP-null mice had a characteristic increase in gliosis throughout the CA1 region and a disruption of staining for the dendritic marker MAP2 and the presynaptic marker synaptophysin. The disruption of MAP2 staining was associated with a significant reduction in overall dendritic length and projection depth of biocytin labeled CA1 neurones. In two groups of APP-null mice that were examined at 8-12 months, and 20-24 months of age, there was an impairment in the formation of long-term potentiation (LTP) in the CA1 region compared to isogenic age matched controls. This LTP deficit was not associated with an alteration in the amplitude of EPSPs at low stimulus frequencies (0.033 Hz) or facilitation during a 100 Hz stimulus train, but was associated with a reduction in post-tetanic potentiation. Paired-pulse depression of GABA-mediated inhibitory post-synaptic currents was also attenuated in APP-null mice. These data demonstrate that the impaired synaptic plasticity in APP deficient mice is associated with abnormal neuronal morphology and synaptic function within the hippocampus.

Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein.

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimer's disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimer's disease.

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.

Mouse cortical neurones lacking APP show normal neurite outgrowth and survival responses in vitro.

We have cultured neurones from the developing cortex of mice that have had the amyloid precursor protein gene deleted (APP-null). Neurones cultured for a period of 24 h show similar neurite outgrowth and survival responses to wild-type neurones. Similar neurite outgrowth responses were also seen when neurones from APP-null mice were treated with a neurotrophic peptide derived from the APP sequence and compared with wild-type neurones. Finally, cortical cultures derived from APP-null mice showed similar survival responses to the toxic amyloid-beta peptide.

Development and characterisation of human 5-HT1B- or 5-HT1D-receptor specific antibodies as unique research tools.

The serotonin 5-HT1B/1D-receptor family comprises of two closely related receptors encoded by two distinct genes. There are no pharmacological ligands which can adequately distinguish between these two receptor subtypes in human tissues. Therefore, we have developed human 5-HT1B- and 5-HT1D-receptor subtype specific polyclonal antibodies. Rabbits were immunised with synthetic peptides identical to unique amino acid sequences located in the third intracellular loops of these receptors. Polyclonal antibodies were subjected to immunoaffinity purification and were characterised using ELISA, dot blot analysis and immunostaining of stably-transfected CHO cell lines expressing either human 5-HT1B-receptors or 5-HT1D-receptors and in human trigeminal ganglia. The antibodies were specific for either the 5-HT1B- or 5-HT1D-receptors and did not cross-react. Both 5-HT1B- and 5-HT1D-immunoreactivities were detected on cell bodies in human trigeminal ganglia. In the absence of selective pharmacological agents, these antibodies represent unique and essential research tools to study the anatomical distribution of 5-HT1B/1D-receptor subtypes in human tissue.

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.