Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Altered expression of neuronal nitric oxide synthase in weaver mutant mice

The weaver mouse represents the only genetic animal model of gradual nigrostriatal dopaminergic neurodegeneration which is proposed as a pathophysiological phenotype of Parkinson`s disease. The aim of the present study was to analyze the nitric oxide and dopaminergic systems in selected brain regions of homozygous weaver mice at different postnatal ages corresponding to specific stages of the dopamine loss. Structural deficits were evaluated by quantification of tyrosine hydroxylase and neuronal nitric oxide synthase-immunostaining in the cortex, striatum, accumbens nuclei, subthalamic nuclei, ventral tegmental area, and substantia nigra compacta of 10-day, 1- and 2-month-old wildtype and weaver mutant mice. The results confirmed the progressive loss of dopamine during the postnatal development in the adult weaver mainly affecting the substantia nigra pars compacta, striatum, and subthalamic nucleus and slightly affecting the accumbens nuclei and ventral tegmental area. A general decrease in neuronal nitric oxide synthase-immunostaining with age was revealed in both the weaver and wild-type mice, with the decrease being most pronounced in the weaver. In contrast, there was an increase in the substantia nigra pars compacta nitric oxide synthase-immunostaining and a decrease mainly in the subthalamic and accumbens nuclei of the 2-month-old weaver mutant. The decrease in the expression of nNOS may bear functional significance related to the process of aging. DA neurons from the substantia nigra directly modulate the activity of subthalamic nucleus neurons, and their loss may contribute to the abnormal activity of subthalamic nucleus neurons. Although the functional significance of these changes is not clear, it may represent plastic compensating adjustments resulting from the loss of dopamine innervation, highlighting a possible role of nitric oxide in this process. (C) 2010 Elsevier B.V. All rights reserved.

Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ER alpha (alpha ERKO) or ER beta (beta ERKO) knockout mice, and their wild-type (alpha WT and beta WT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ER beta was more abundant. Estradiol benzoate (EB) decreased ER alpha-positive cells in WT and beta ERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ER beta expression. ER beta deletion increased ER alpha while ER alpha deletion did not alter ER beta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alpha ERKO animals but to a lesser extent, suggesting that ER alpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in beta ERKO mice were similar to those in the alpha ERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alpha ER, beta ER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint. (C) 2007 Elsevier Inc. All rights reserved.

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Muller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions. (C) 2001 Wiley-Liss, Inc.

Amino acids and their transporters in the retina

This review provides an overview of the distributions, properties and roles of amino acid transport systems in normal and pathological retinal tissues and discusses the roles of specific identified transporters in the mammalian retina. The retina is used in this context as a vehicle for describing neuronal and glial properties. which are in semi, but not all cases comparable to those found elsewhere an the brain. Where significant departures are noted, these are discussed in the context of functional specialisations of the retina and its relationship to adjacent supporting tissues such as the retinal pigment epithelium. Specific examples are given where immunocytochemical labelling for amino acid transporters may yield inaccurate results, possibly because of activity-dependent conformation changes of epitopes in these proteins which render the epitopes more or less accessible to antibodies. (C) 2001 Elsevier Science Ltd. All rights reserved.

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha -internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.

Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and in medullary noradrenergic cell groups

It has been hypothesized that the brain categorizes stressors and utilizes neural response pathways that vary in accordance with the assigned category. If this is true, stressors should elicit patterns of neuronal activation within the brain that are category-specific. Data from previous Immediate-early gene expression mapping studies have hinted that this is the case, but interstudy differences in methodology render conclusions tenuous. In the present study, immunolabelling for the expression of c-fos was used as a marker of neuronal activity elicited in the rat brain by haemorrhage, immune challenge, noise, restraint and forced swim. All stressors elicited c-fos expression in 25-30% of hypothalamic paraventricular nucleus corticotrophin-releasing-factor cells, suggesting that these stimuli were of comparable strength, at least with regard to their ability to activate the hypothalamic-pituitary-ad renal axis. In the amygdala, haemorrhage and immune challenge both elicited c-fos expression in a large number of neurons in the central nucleus of the amygdala, whereas noise, restraint and forced swim primarily elicited recruitment of cells within the medial nucleus of the amygdala. In the medulla, all stressors recruited similar numbers of noradrenergic (A1 and A2) and adrenergic (C1 and C2) cells. However, haemorrhage and immune challenge elicited c-fos expression In subpopulations of A1 and A2 noradrenergic cells that were significantly more rostral than those recruited by noise, restraint or forced swim. The present data support the suggestion that the brain recognizes at least two major categories of stressor, which we have referred to as 'physical' and 'psychological'. Moreover, the present data suggest that the neural activation footprint that is left in the brain by stressors can be used to determine the category to which they have been assigned by the brain.

Application of DNA microarrays to study human alcoholism

An emerging idea is that long-term alcohol abuse results in changes in gene expression in the brain and that these changes are responsible at least partly for alcohol tolerance, dependence and neurotoxicity, The overall goal of our research is to identify genes which are differentia[ly expressed in the brains of well-characterized human alcoholics as compared with non-alcoholics. This should identify as-yet-unknown alcohol-responsive genes, and may well confirm changes in the expression of genes which have been delineated in animal models of alcohol abuse. Cases were carefully selected and samples pooled on the basis of relevant criteria; differential expression was monitored by microarray hybridization. The inherent diversity of human alcoholics can be exploited to identify genes associated with specific pathological processes, as well as to assess the effects of concomitant disease, severity of brain damage, drinking behavior, and factors such as gender and smoking history. initial results show selective changes in gene expression in alcoholics; of particular importance is a coordinated reduction in genes coding for myelin components, Copyright (C) 2001 National Science Council, ROC and S. Karger AG, Basel.

Glutamate(NMDA) receptor NR1 subunit mRNA expression in Alzheimer's disease

We analyzed the expression profile of two NMDAR1 mRNA isoform subsets. NR1(0xx) and NR1(1xx), in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21-amino acid N-terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimer's disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR1(0xx)) and 405 bp (NR1(1xx)), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR1(1xx) transcript expression was calculated as a proportion of the NR1(1xx) + NR1(0xx) total. Values were significantly lower in AD cases than in controls in mid-cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p similar to 0.05. Cortical proportionate NR1(1xx) transcript expression was invariant over the range of ages acid areas of controls tested, at similar to 50%. This was also true for AD motor and occipital cortex. Proportionate NR1(1xx) expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N-terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41 -, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Contribution of cholinergic systems to state-dependent modulation of respiratory control

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, Such as obstructive sleep apnea. (C) 2002 Elsevier Science B.V. All rights reserved.

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P

Systemic administration of interleukin-1 beta activates select populations of central amygdala afferents

The central nucleus of the amygdala (CeA) is activated robustly by an immune challenge such as the systemic administration of the proinflammatory cytokine interleukin-1beta (IL-1beta). Because IL-1beta is not believed to cross the blood-brain barrier in any significant amount, it is likely that IL-1beta elicits CeA cell recruitment by means of activation of afferents to the CeA. However, although many studies have investigated the origins of afferent inputs to the CeA, we do not know which of these also respond to IL-1beta. Therefore, to identify candidate neurons responsible for the recruitment of CeA cells by an immune challenge, we iontophoretically deposited a retrograde tracer, cholera toxin b-subunit (CTb), into the CeA of rats 7 days before systemic delivery of IL-1beta (1 mug/kg, i.a.). By using combined immunohistochemistry, we then quantified the number of Fos-positive CTb cells in six major regions known to innervate the CeA. These included the medial prefrontal cortex, paraventricular thalamus (PVT), ventral tegmental area, parabrachial nucleus (PB), nucleus tractus solitarius, and ventrolateral medulla. Our results show that after deposit of CTb into the CeA, the majority of double-labeled cells were located in the PB and the PVT, suggesting that CeA cell activation by systemic IL-1beta is likely to arise predominantly from cell bodies located in these regions. These findings may have significant implications in determining the central pathways involved in generating acute central responses to a systemic immune challenge. J. Comp. Neurol. 452:288-296, 2002. (C) 2002 Wiley-Liss, Inc.