Molecular Brain Research最新文献

GABA and glutamate are the major neurotransmitters in the human central nervous system. Disturbances in these transmitter systems have been suggested to influence a variety of neurological and psychiatric diseases. Human platelets have been used as a model for neural amino acid transport, although it has not been known exactly which transporters participate in the transport process. In this study, we identify with reverse transcription-polymerase chain reaction (RT-PCR) BGT-1 and EAAT3 as transporters for GABA and glutamate, respectively. We also show that platelets contain transporters for dopamine, taurine and creatine. The cloning of these transporters confirms that blood platelets can be used as a model for neurotransmitter transport in the CNS.

The genetically dystonic rat (SD-dt:JFL) is an autosomal recessive model of generalized dystonia. Without cerebellectomy, the dt rat dies prior to Postnatal Day 40. The dt locus was mapped to a 4.2 Mb region on Chr 7q11 and candidate genes were screened with semi-quantitative RT-PCR. Then, Southern blotting and genomic DNA sequencing identified the 3′-long terminal repeat portion of an intracisternal A particle element inserted into Intron 1 of Atcay, the gene which encodes caytaxin. Northern and Western blotting and quantitative real-time RT-PCR defined the Atcay allele in dt rats (Atcay^dt) as hypomorphic. To establish a framework for functional studies of caytaxin, the developmental expression of rat Atcay transcript was analyzed with Northern blotting, relative quantitative multiplex real-time RT-PCR (QRT-PCR) and in situ hybridization. With a multiple tissue Northern blot, three Atcay transcripts were identified in brain but none were present in heart, spleen, lung, liver, muscle, kidney or testis. With a multiple time-point Northern blot, the same three transcripts were present in cerebellum at Embryonic Day (E15), Postnatal Day 1 (P1), P7, P14, P36 and 8 months. During early development (E15 to P14), the relative proportion of the smallest transcript was increased. QRT-PCR was performed with total RNA from cerebral cortex, striatum, thalamus, hippocampus and cerebellum. Transcript levels peaked at P7 in hippocampus, increased linearly from P1 to P36 in cerebellum, and showed minimal developmental regulation in cerebral cortex. Radioactive in situ hybridization localized Atcay transcript to seemingly all neuronal populations in brain. In cerebellum, Atcay transcript was present in the molecular, Purkinje and granular layers; transcript density in the molecular layer peaked at P14. In the background of previous biochemical, behavioral and electrophysiological studies in the dt rat, our data are compatible with a vital role for caytaxin in the development and neurophysiology of cerebellar cortex.

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 μg/rat) and the ODN antagonist to metabotropic receptor, cyclo_1–8[Dleu⁵]OP (20 μg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo_1–8[Dleu⁵]OP. When cyclo_1–8[Dleu⁵]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo_1–8[Dleu⁵]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo_1–8[Dleu⁵]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

We have tested the effect of deprenyl on the neurotoxicity induced by the injection of quinolinic acid within the striatum. Deprenyl was unable to prevent these quinolinic acid-induced damages, but enhanced the loss of several gamma-aminobutyric acid (GABA) positive subpopulations, the loss of the astroglial population and the activation of microglia produced by quinolinic acid. These effects are produced by deprenyl potentiation of dopamine actions since dopamine depletion produced by previous injection of the dopaminergic toxin 6-hydroxydopamine within the medial forebrain bundle overcomes deprenyl effects and the involvement of dopamine in the quinolinic acid-induced toxicity in striatum. In these conditions, quinolinic acid toxic action in striatum is significantly lower and similar in the animals treated with or without deprenyl. All these data justify why deprenyl worsen some pathological signals of disorders involving excitotoxicity. This also may be involved in other secondary effects described for deprenyl.

The response to stress is influenced by prior experience with the same or different stressor. For example, exposure of cold pre-stressed rats to heterotypic (novel) stressors, such as immobilization (IMO), triggers an exaggerated release of catecholamines and increase in gene expression for adrenomedullary tyrosine hydroxylase (TH), the rate limiting catecholamine biosynthetic enzyme. To study the mechanism, we examined induction or phosphorylation of several transcription factors, which are implicated in IMO-triggered regulation of TH transcription, in rats exposed to cold (4 °C) for up to 28 days and then subjected to IMO. Levels of c-fos increased transiently after 2–6 h and returned to basal levels after 1–28 days cold stress. Fra-2, was unaffected by short term cold, but was induced about 2-fold by 28 days continual cold. In contrast, there were no significant changes in CREB phosphorylation or Egr1 induction. Rats, with and without pre-exposure to 28 days cold, were subjected to single IMO for up to 2 h. Phosphorylation of CREB after 30 min IMO was greater in cold pre-exposed rats. Induction of Egr1 was three times higher in cold pre-exposed rats and remained significantly elevated even 3 h after cessation of IMO. Exposure to IMO triggered a 10–20-fold elevation in Fra-2 in both groups, which was even higher 3 h after the IMO. However, Fra-2 was more heavily phosphorylated following IMO stress in cold pre-exposed animals. The results reveal that sensitization to novel stress in cold pre-exposed animals is manifested by exaggerated response of several transcription factors.

Recent studies have identified a new family of gap junction-forming proteins in vertebrates, called pannexins. Although their function in vivo is still not known, studies in Xenopus oocytes have indicated that pannexin1 (Px1) and pannexin2 (Px2) can form functional gap junction channels and can contribute to functional hemichannels. In this study, we have utilized a combination of radioactive and non-radioactive in situ hybridization experiments to characterize the expression pattern of the two pannexin genes during development and maturation of the rat brain. Expression analysis revealed a widespread and similar mRNA distribution for both genes, but indicated that Px1 and Px2 are inversely regulated during the development of the rat brain. Px1 is expressed at a high level in the embryonic and young postnatal brain and declines considerably in the adult, whereas Px2 mRNA is low in the prenatal brain but increases substantially during subsequent postnatal development. Immunohistochemical studies using different antibodies confirm the neuronal origin of pannexin-expressing cells and ascertain the presence of both pannexins in the majority of pyramidal cells and in GABAergic interneurons. The abundant presence of both pannexins in most neurons suggests that they may play a role in intercellular communication in many neuronal circuits. Furthermore, the temporal difference in the expression of the two genes indicates that the relative contribution of the two pannexins in immature and mature neuronal circuits may vary.

Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-α (TNF-α) plus interferon-γ (IFN-γ). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IκBα (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (±)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-α/IFN-γ. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-κB-dependent expression of NOS2.

Brn-3a is a transcription factor expressed in a subset of neurons of the peripheral nervous system. Its role encompasses the activation of genes involved in neuronal differentiation and survival. While a lot of data have been produced on Brn-3a target promoters, very little is known about the upstream regulatory signals that mediate its activation in response to differentiation. In this work, we describe for the first time that Brn-3a is phosphorylated in IMR-32 neuroblastoma cells in response to differentiation induced by retinoic acid treatment and that its post-translational modification is potentially mediated by the activation of the MAPK/ERK pathway. Furthermore, we show that the mutation of a putative phosphorylated amino acid strongly reduces the ability of Brn-3a to mediate the differentiation of IMR-32 cells.