Comparative proteomic analysis of hypothalamus tissue from Huoyan geese between pre-laying period and laying period using an iTRAQ-based approach.

The hypothalamus plays a central role in controlling poultry endocrine and reproductive activities. So far there is limited information focused on the proteome profiles of the hypothalamus from geese during different stages of the egg-laying cycle. In order to identify proteins regulating the egg-laying process of Huoyan geese, we investigated the proteome profiles of the hypothalamus from Huoyan geese during the laying period and pre-laying period by applying an isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic technology. A total number of 3,337 were identified and quantified, of which 18 were significantly up-regulated and 16 were significantly down-regulated. These differentially expressed proteins were subjected to bioinformatics analyses based on the Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway. Some of these were revealed to be involved in hormone and neurotransmitter secretion, exocytosis, calcium ion transport and synaptic transmission. Subsequently, excitatory amino acid transporter 2, complexin-1 and inositol 1,4,5-trisphosphate receptor, type 3 were confirmed at the messenger RNA level using quantitative real-time RT-PCR. Then, the abundance change of these proteins was verified further using Western blotting analysis. These data may aid in elucidating the molecular mechanism of higher laying performance in Huoyan geese.

Astrocytes modulate a postsynaptic NMDA-GABAA-receptor crosstalk in hypothalamic neurosecretory neurons.

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABA(A)ergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABA(A) receptors (GABA(A)Rs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABA(A)Rs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR-GABA(A)R crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate-GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate-GABA interactions in MNCs, which involves crosstalk between NMDA and GABA(A) postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABA(A)Rs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.

Exon 4-skipping GLT-1: a new form of an abundantly expressed glutamate transporter.

GLT-1 (EAAT2) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles, especially in the termination of neurotransmitter signals at excitatory synapses in grey matter. In normal brain, alternative splicing of GLT-1 has been described, where exons in the GLT-1 gene are skipped or intronic sequences spliced in to generate new sequences. This study describes the isolation of a cDNA clone encoding a new splice variant of GLT-1 where exon 4 is skipped. This novel variant was isolated by RT-PCR cloning from adult rat brain and encodes a protein of 500 amino acids (MW ~54.5 kDa). RT-PCR analysis showed that mRNA was readily detectable in various brain regions of rat, primary astrocyte cultures and in tissues such as testis, but little mRNA was detectable in retina and liver. An antibody that selectively recognizes exon-4 skipping GLT-1 revealed strong signals in Western blots and labelled grey matter astrocytes. We conclude that exon-4 skipping GLT 1 is abundantly expressed in the brain and may represent either a functional glutamate transporter or a modulator of glutamate transporter function.

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 increases glutamate uptake through overexpression of GLT1 and EAAC1 glutamate transporter subtypes in rat frontal cerebral cortex.

Prenatal exposure to the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone) mesylate (WIN) at a daily dose of 0.5 mg/kg, and Delta9-tetrahydrocannabinol (Delta9-THC) at a daily dose of 5 mg/kg, reduced dialysate glutamate levels in frontal cerebral cortex of adolescent offspring (40-day-old) with respect to those born from vehicle-treated mothers. WIN treatment induced a statistically significant enhancement of Vmaxl-[3H]glutamate uptake, whereas it did not modify glutamate Km, in frontal cerebral cortex synaptosomes of adolescent rats. Western blotting analysis, performed either in membrane proteins derived from homogenates and in proteins extracted from synaptosomes of frontal cerebral cortex, revealed that prenatal WIN exposure enhanced the expression of glutamate transporter 1 (GLT1) and excitatory amino acid carrier 1 (EAAC1). Moreover, immunocytochemical analyses of frontal cortex area revealed a more intense GLT1 and EAAC1 immunoreactivity (ir) distribution in the WIN-treated group. Collectively these results show that prenatal exposure to the cannabinoid CB1 receptor agonist WIN increases expression and functional activity of GLT1 and EAAC1 glutamate transporters (GluTs) associated to a decrease of cortical glutamate outflow, in adolescent rats. These findings may contribute to explain the mechanism underlying the cognitive impairment observed in the offspring of mothers who used marijuana during pregnancy.