Powerful new approaches to study molecular variation in distinct neuronal populations have recently been developed enabling a more precise investigation of the control of neural circuits involved in complex behaviors such as wake and sleep. We applied laser capture microdissection (LCM) to isolate precise brain nuclei from rat CNS at opposing circadian time points associated with wake and sleep. Discrete anatomical and temporal analysis was performed to examine the extent of variation in the transcriptional control associated with both identifiable anatomical nuclei and with light/dark cycle. Precise isolation of specific brain nuclei regulating sleep and arousal, including the LC, SCN, TMN, VTA, and VLPO, demonstrated robust changes in gene expression. Many of these differences were not observed in previous studies where whole brain lysates or gross dissections were used to probe for changes in gene expression. The robust and differential profiles of genomic data obtained from the approaches used herein underscore the requirement for careful anatomical refinement in CNS gene expression studies designed to understand genomic control within behaviorally-linked, but functionally isolated brain nuclei.

The surging obesity epidemic calls for a deeper understanding of central nervous system (CNS) mechanisms underlying the biologically defended level of body weight. Here, we analyzed global gene expression in four hypothalamic and two brainstem nuclei involved in energy homeostatic control of body weight in diet-induced obese (DIO) and lean rats. Male Sprague-Dawley rats were offered ad libitum chow, or a two-choice diet consisting of a high palatable high sugar/fat diet and chow for 40 weeks. At termination, the hypothalamic arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), paraventricular nucleus (PVN) and lateral hypothalamus area (LHA), as well as the brainstem area postrema (AP) and nucleus of the solitary tract (NTS), were isolated by laser capture microdissection (LCM) followed by mRNA sequencing. Global gene expression analyses revealed a total of 88 differentially expressed genes (DEGs) in DIO rats. Transcriptome changes were mainly observed in the DMH and NTS and associated with neuropeptide signaling and regulation of signaling transduction pathways, suggesting a key role of these brain regions in body weight regulation.

Polyglutamine (PolyQ) diseases have common features that include progressive selective neurodegeneration and the formation of protein aggregates. There is growing evidence to suggest that critical nuclear events lead to transcriptional alterations in PolyQ diseases such as spinocerebellar ataxia type 7 (SCA7) and Huntington's disease (HD), conditions which share a cerebellar degenerative phenotype. Taking advantage of laser capture microdissection technique, we compared the Purkinje cell (PC) gene expression profiles of two transgenic polyQ mouse models (HD: R6/2; SCA7: P7E) by microarray analysis that was validated by real time quantitative PCR. A large number of transcriptional alterations were detected in the R6/2 transgenic model of HD. Similar decreases in the same mRNAs, such as phospholipase C, β 3, purkinje cell protein 2 (Pcp2) and aldolase C, were found in both models. A decrease in aldolase C and phospholipase C, β 3, may lead to an increase in the vulnerability of PCs to excitotoxic events. Furthermore, downregulation of mRNAs mediated by the Pcp2-promoter is common in both models. Thus, our data reveal shared molecular abnormalities in different polyQ disorders.

The outcome of dopaminergic signaling and effectiveness of dopaminergic drugs depend on the relative preponderance of each of the five dopamine receptors in a given brain region. The separate contribution of each receptor to overall dopaminergic tone is difficult to establish at a functional level due to lack of receptor subtype specific pharmacological agents. A surrogate for receptor function is receptor protein or mRNA expression. We examined dopamine receptor mRNA expression by quantitative reverse transcription real-time PCR in the striatum, globus pallidus, frontal cortex and cingulate cortex of embryonic and postnatal mice. Samples of each region were collected by laser capture microdissection. D1- and D2-receptor mRNAs were the most abundant in all the regions of the mature brain. The D1-receptor was predominant over the D2-receptor in the frontal and cingulate cortices whereas the situation was reversed in the striatum and globus pallidus. In the proliferative domains of the embryonic forebrain, D3-, D4- and D5-receptors were predominant. In the corpus striatum and cerebral cortex, the D3- and D4-receptors were the only receptors that showed marked developmental regulation. By analyzing D1 receptor protein expression, we show that developmental changes in mRNA expression reliably translate into changes in protein levels, at least for the D1-receptor.

Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.

Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague-Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42-45 mm Hg). In a separate group of animals, a mild (32 degrees C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from approximately 500 brain vessels (20-100 microm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using (3)H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p<0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for (3)H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.