Depressive behavior and activation of the orexin/hypocretin system.

The orexin/hypocretin peptide signaling system plays a neuromodulatory role in motivation and stress; two critical components of depression. Although work has been done to identify links between orexin and depression, few specific neuroanatomical associations have been made. These studies have not investigated the relationship between orexin and orexin receptor expression in specific brain regions associated with this disorder. To address this, we examined immobility during the forced swim test (FST) in mice, a commonly used measure of depressive behavior. We analyzed the variation in FST immobility with the distribution of orexin and its receptor mRNA. We found that animals that exhibited more robust depressive behavior had greater or lesser orexin system expression that depended on the limbic brain region analyzed. In the hippocampus there was a negative correlation between orexin expression and FST immobility. Animals that displayed relatively more depressive behavior had lower hippocampal expression of Orexin A (OrxA). In the amygdala, there was a curvilinear relationship between OrxA and FST performance. In addition there was a positive correlation with amygdalar Type I orexin receptor (Orx1) mRNA and depressive behavior. Despite the differences in limbic orexin expression, there was no correlation between immobility and hypothalamic orexin neuron activation as measured by c-Fos. Overall, more severe depressive behavior was associated with reduced hippocampal orexin expression, contrasted with increased orexin plus Orx1 receptor mRNA expression in the amygdala. This divergent pattern between the hippocampus and amygdala mirrors a neurobiological theme seen in depression resulting from reduced hippocampal, but increased amygdalar, size and function.

Contrasting hippocampal and amygdalar expression of genes related to neural plasticity during escape from social aggression.

Social subjugation has widespread consequences affecting behavior and underlying neural systems. We hypothesized that individual differences in stress responsiveness were associated with differential expression of neurotrophin associated genes within the hippocampus and amygdala. To do this we examined the brains of hamsters placed in resident/intruder interactions, modified by the opportunity to escape from aggression. In the amygdala, aggressive social interaction stimulated increased BDNF receptor TrK(B) mRNA levels regardless of the ability to escape the aggressor. In contrast, the availability of escape limited the elevation of GluR(1) AMPA subunit mRNA. In the hippocampal CA(1), the glucocorticoid stress hormone, cortisol, was negatively correlated with BDNF and TrK(B) gene expression, but showed a positive correlation with BDNF expression in the DG. Latency to escape the aggressor was also negatively correlated with CA(1) BDNF expression. In contrast, the relationship between amygdalar TrK(B) and GluR(1) was positive with respect to escape latency. These results suggest that an interplay of stress and neurotrophic systems influences learned escape behavior. Animals which escape faster seem to have a more robust neurotrophic profile in the hippocampus, with the opposite of this pattern in the amygdala. We propose that changes in the equilibrium of hippocampal and amygdalar learning result in differing behavioral stress coping choices.

A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR)-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR). This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

A rapid method for the quantification of mouse hippocampal neurogenesis in vivo by flow cytometry. Validation with conventional and enhanced immunohistochemical methods.

Neural stem cells reside in the subventricular zone and the dentate gyrus of the hippocampus in adult mammalian brain. In the hippocampus, a number of factors are reported to modulate the rate of neural progenitor proliferation in the hippocampus, such as exercise, corticosteroids, and many pharmacological agents including several classes of antidepressants. It is currently unclear whether this increased proliferation is physiologically relevant, but it provides a potentially useful biomarker to assess novel antidepressant compounds. Changes in neurogenesis are typically quantified by administration of bromodeoxyuridine (BrdU) in vivo, and subsequent quantification of labelled nuclei. A robust and rapid means of quantifying BrdU labelling in adult hippocampus in vivo would allow higher throughput screening of potential antidepressant compounds. In this study we describe a FACS-based method for quantification of BrdU labelled cells in fixed cell suspensions from BrdU-treated adult mouse hippocampus. A variety of experimental conditions known to modulate proliferation were tested, including administration of corticosterone and the antidepressants imipramine and fluoxetine. The robust changes compared to control groups observed in these models were similar to previously reported studies, thus offering a more rapid and streamlined means to quantify effects of compounds on hippocampal proliferation.

Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components.

The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin-R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin-R and HA. Whereas both net-bearing neurons and glial cells were the sources of CSPGs and tenascin-R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE-1. No immunolabelling for HA receptors on the membrane of net-bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability.

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.

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 RAMP 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.

Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists.

The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity-modifying protein (RAMP) 1 (McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339). Several non-peptide CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species (Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K., Engel, W., and Eberlein, W. (2000) Br. J. Pharmacol. 129, 420-423; Edvinsson, L., Sams, A., Jansen-Olesen, I., Tajti, J., Kane, S. A., Rutledge, R. Z., Koblan, K. S., Hill, R. G., and Longmore, J. (2001) Eur. J. Pharmacol. 415, 39-44). This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a > or =100-fold increase in antagonist affinities, similar to the K(i) values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.