934 resultados para Rat-brain


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Grafts of favorable axonal growth substrates were combined with transient nerve growth factor (NGF) infusions to promote morphological and functional recovery in the adult rat brain after lesions of the septohippocampal projection. Long-term septal cholinergic neuronal rescue and partial hippocampal reinnervation were achieved, resulting in partial functional recovery on a simple task assessing habituation but not on a more complex task assessing spatial reference memory. Control animals that received transient NGF infusions without axonal-growth-promoting grafts lacked behavioral recovery but also showed long-term septal neuronal rescue. These findings indicate that (i) partial recovery from central nervous system injury can be induced by both preventing host neuronal loss and promoting host axonal regrowth and (ii) long-term neuronal loss can be prevented with transient NGF infusions.

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Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

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The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoletic development and phenotypic variation. BioEssays 25:930-939,2003. (C) 2003 Wiley Periodicals, Inc.

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Apomorphine is a dopamine receptor agonist that was recently licensed for the treatment of erectile dysfunction. However, although sexual activity can be stressful, there has been little investigation into whether treatments for erectile dysfunction affect stress responses. We have examined whether a single dose of apomorphine, sufficient to produce penile erections (50 mug/kg, i.a.), can alter basal or stress-induced plasma ACTH levels, or activity of central pathways thought to control the hypothalamic-pituitary-adrenal axis in rats. An immune challenge (interleukin-1beta, 1 mug/kg, i.a.) was used as a physical stressor while sound stress (100 dB white noise, 30 min) was used as a psychological stressor. Intravascular administration of apomorphine had no effect on basal ACTH levels but did substantially increase the number of Fos-positive amygdala and nucleus tractus solitarius catecholamine cells. Administration of apomorphine prior to immune challenge augmented the normal ACTH response to this stressor at 90 min and there was a corresponding increase in the number of Fos-positive paraventricular nucleus corticotropin-releasing factor cells, paraventricular nucleus oxytocin cells and nucleus tractus solitarius catecholamine cells. However, apomorphine treatment did not alter ACTH or Fos responses to sound stress. These data suggest that erection-inducing levels of apomorphine interfere with hypothalamic-pituitary-adrenal axis inhibitory feedback mechanisms in response to a physical stressor, but have no effect on the response to a psychological stressor. Consequently, it is likely that apomorphine acts on a hypothalamic-pituitary-adrenal axis control pathway that is unique to physical stressors. A candidate for this site of action is the nucleus tractus solitarius catecholamine cell population and, in particular, A2 noradrenergic neurons. (C) 2003 Elsevier Science Ltd. All rights reserved.

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Previous studies have shown that the medial prefrontal cortex can suppress the hypothalamic-pituitary-adrenal axis response to stress. However, this effect appears to vary with the type of stressor. Furthermore, the absence of direct projections between the medial prefrontal cortex and corticotropin-releasing factor cells at the apex of the hypothalamic-pituitary-adrenal axis suggest that other brain regions must act as a relay when this inhibitory mechanism is activated. In the present study, we first established that electrolytic lesions involving the prelimbic and infralimbic medial prefrontal cortex increased plasma adrenocorticotropic hormone levels seen in response to a physical stressor, the systemic delivery of interleukin-1beta. However, medial prefrontal cortex lesions did not alter plasma adrenocorticotropic hormone levels seen in response to a psychological stressor, noise. To identify brain regions that might mediate the effect of medial prefrontal cortex lesions on hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta, we next mapped the effects of similar lesions on interleukin-1beta-induced Fos expression in regions previously shown to regulate the hypothalamic-pituitary-adrenal axis response to this stressor. It was found that medial prefrontal cortex lesions reduced the number of Fos-positive cells in the ventral aspect of the bed nucleus of the stria terminalis. However, the final experiment, which involved combining retrograde tracing with Fos immunolabelling, revealed that bed nucleus of the stria terminalis-projecting medial prefrontal cortex neurons were largely separate from medial prefrontal cortex neurons recruited by systemic interleukin-1beta, an outcome that is difficult to reconcile with a simple medial prefrontal cortex-bed nucleus of the stria terminalis-corticotropin-releasing factor cell control circuit.

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Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [H-3]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V-max for uptake of [H-3]-D-aspartate, with no change in the K-m value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [H-3]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation. of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder. (C) 2003 Wiley-Liss, Inc.

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Numerous mRNA molecules are localized in regions of the dendrites of neurons, some moving along dendrites in response to synaptic activity. The proteins encoded by these RNAs have diverse functions, including participation in memory formation and long-term potentiation. Recent experiments have shown that a cytoplasmic RNA trafficking pathway described for oligodendrocytes also operates in neurons. Transported RNAs possess a cis-acting element that directs them to granules, which are transported along microtubules by the motor proteins kinesin and dynein. These RNA molecules are recruited to the cytoplasmic transport granules by cooperative interaction with a cognate trans-acting factor. mRNAs containing the 11-nucleotide A2RE11 or 21-nucleotide A2RE sequences bind heterogeneous nuclear ribonucleoproteins A2 and A3, which are abundant in the brain. Mutations in this cis-acting element that weaken its interaction with hnRNP A2 also interfere with RNA trafficking. Several dendritically localized mRNAs, including those encoding calcium-calmodulin-dependent protein kinase 11 a subunit and neurogranin, possess A2RE-like sequences, suggesting that they may be localized by interaction with these heterogeneous nuclear ribonucleoproteins. Calcium-calmodulin-dependent protein kinase 11 a subunit is of particular interest: Its RNA is transported in depolarized neurons, and the protein it encodes is essential for establishing long-term memory. Several other cis-acting sequences and trans-acting factors that participate in neuronal RNA localization have been discovered.

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A benzothiazole-derived compound (4a) designed to mimic the C-alpha-C-beta bond vectors and terminal functionalities of Lys2, TyrI3 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low PM binding affinity to N-type VGCCs (IC50 = 1.9 muM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50 = 4.1 muM) showed a greater than 25-fold selectivity for the N-type channel. (C) 2004 Elsevier Ltd. All rights reserved.

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The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

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Pyramidal neurons in the lateral amygdala discharge trains of action potentials that show marked spike frequency adaptation, which is primarily mediated by activation of a slow calcium-activated potassium current. We show here that these neurons also express an alpha-dendrotoxin- and tityustoxin-Kalpha-sensitive voltage-dependent potassium current that plays a key role in the control of spike discharge frequency. This current is selectively targeted to the primary apical dendrite of these neurons. Activation of mu-opioid receptors by application of morphine or D-Ala(2)-N-Me-Phe(4)-Glycol(5)-enkephalin (DAMGO) potentiates spike frequency adaptation by enhancing the alpha-dendrotoxin-sensitive potassium current. The effects of mu-opioid agonists on spike frequency adaptation were blocked by inhibiting G-proteins with N-ethylmaleimide (NEM) and by blocking phospholipase A(2). Application of arachidonic acid mimicked the actions of DAMGO or morphine. These results show that mu-opioid receptor activation enhances spike frequency adaptation in lateral amygdala neurons by modulating a voltage-dependent potassium channel containing Kv1.2 subunits, through activation of the phospholipase A(2)-arachidonic acid-lipoxygenases cascade.

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Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN. (C) 2004 Wiley-Liss, Inc.

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Using Fos immunolabelling as a marker of neuronal activation, we investigated the role of the parabrachial nucleus in generating central neuronal responses to the systemic administration of the proinflarnmatory cytokine interleukin-1beta (1 mug/kg, i.a.). Relative to intact animals, parabrachial nucleus lesions significantly reduced the number of Fos-positive cells observed in the central amygdala (CeA), the bed nucleus of the stria terminalis (BNST), and the ventrolateral medulla (VLM) after systemic interleukin-1beta. In a subsequent experiment in which animals received parabrachial-directed deposits of a retrograde tracer, it was found that many neurons located in the nucleus tractus solitarius (NTS) and the VLM neurons were both retrogradely labelled and Fos-positive after interleukin-1beta administration. These results suggest that the parabrachial nucleus plays a critical role in interleukin-1beta-induced Fos expression in CeA, BNST and VLM neurons and that neurons of the NTS and VLM may serve to trigger or at least influence changes in parabrachial nucleus activity that follows systemic interleukin-1beta administration. (C) 2004 Elsevier B.V. All rights reserved.

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Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties. (c) 2005 Elsevier Inc. All rights reserved.

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Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

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The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.