Expression of P2 nucleotide receptors varies with age and sex in murine brain microglia

Microglia are implicated in multiple neurodegenerative disorders, many of which display sexual dimorphisms and have symptom onsets at different ages. P2 purinergic receptors are critical for regulating various microglial functions, but little is known about how their expression varies with age or sex. Therefore, comprehensive information about purinergic receptor expression in normal microglia, in both sexes, over age is necessary if we are to better understand their roles in the healthy and diseased CNS. We analyzed the expression of all fourteen rodent P2X and P2Y receptors in CD11b+ cells freshly-isolated from the brains of C57Bl/6 mice at five different ages ranging from postnatal day 3 to 12 months, in males and females, using quantitative RT-PCR. We also compared purinergic receptor expression in microglia freshly-isolated from 3 day-old pups to that in primary neonatal microglial cultures created from mice of the same age. We observed patterns in P2 receptor expression with age, most notably increased expression with age and age-restricted expression. There were also several receptors that showed sexually dimorphic expression. Lastly, we noted that in vitro culturing of neonatal microglia greatly changed their P2 receptor expression profiles. These data represent the first complete and systematic report of changes in purinergic receptor expression of microglia with age and sex, and provide important information necessary for accurate in vitro modeling of healthy animals

Increased GABAA Receptor ε-Subunit Expression on Ventral Respiratory Column Neurons Protects Breathing during Pregnancy

GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABAARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABAAR expression on brainstem neurons of the ventral respiratory column (VRC). In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC) were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABAAR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABAAR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors) in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABAAR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life) despite increased neurosteroid levels during pregnancy

Efficient isolation of live microglia with preserved phenotypes from adult mouse brain

Abstract Background Microglial activation plays a key role in the neuroinflammation associated with virtually all CNS disorders, although their role in normal CNS physiology is becoming increasingly appreciated. Neuroinflammation is often assessed by analyzing pro-inflammatory mediators in CNS tissue homogenates, under the assumption that microglia are the main source of these molecules. However, other cell types in the CNS can also synthesize inflammatory molecules. Hence, to enable direct analysis of microglial activities ex vivo, an efficient, reliable, and reproducible method of microglial isolation is needed. Methods After enzymatic digestion of brain tissues and myelin removal, CD11b+ cells were isolated using immunomagnetic separation, yielding highly purified microglia without astrocyte or neuronal contamination. We used three methods of myelin removal (30% Percoll, 0.9 mol/l sucrose and anti-myelin magnetic beads), and compared their effects on microglial viability and yield. To determine whether the isolation procedure itself activates microglia, we used flow cytometry to examine microglial properties in brain-tissue homogenates and isolated microglia from control and lipopolysaccharide (LPS) -treated mice. Results This method yielded a highly purified CD11b+ cell population with properties that reflected their in vivo phenotype. The viability and yield of isolated cells were significantly affected by the myelin removal method. Although the microglial phenotype was comparable in all methods used, the highest viability and number of CD11b+ cells was obtained with Percoll. Microglia isolated from LPS-treated mice displayed a pro-inflammatory phenotype as determined by upregulated levels of TNF-α, whereas microglia isolated from control mice did not. Conclusions Immunomagnetic separation is an efficient method to isolate microglia from the CNS, and is equally suitable for isolating quiescent and activated microglia. This technique allows evaluation of microglial activities ex vivo, which accurately reflects their activities in vivo. Microglia obtained by this method can be used for multiple downstream applications including qRT-PCR, ELISA, Western blotting, and flow cytometry to analyze microglial activities in any number of CNS pathologies or injuries.</p

Cell-Type-Specific Jumonji Histone Demethylase Gene Expression in the Healthy Rat CNS: Detection by a Novel Flow Cytometry Method

Our understanding of how histone demethylation contributes to the regulation of basal gene expression in the brain is largely unknown in any injury model, and especially in the healthy adult brain. Although Jumonji genes are often regulated transcriptionally, cell-specific gene expression of Jumonji histone demethylases in the brain remains poorly understood. Thus, in the present study we profiled the mRNA levels of 26 Jumonji genes in microglia (CD11b + ), neurons (NeuN + ) and astrocytes (GFAP + ) from the healthy adult rat brain. We optimized a method combining a mZBF (modified zinc-based fixative) and FCM (flow cytometry) to simultaneously sort cells from non-transgenic animals. We evaluated cell-surface, intracellular and nuclear proteins, including histones, as well as messenger- and micro-RNAs in different cell types simultaneously from a single-sorted sample. We found that 12 Jumonji genes were differentially expressed between adult microglia, neurons and astrocytes. While JMJD2D was neuron-restricted, PHF8 and JMJD1C were expressed in all three cell types although the expression was highest in neurons. JMJD3 and JMJD5 were expressed in all cell types, but were highly enriched in microglia; astrocytes had the lowest expression of UTX and JHDM1D. Levels of global H3K27 (H3 lysine 27) methylation varied among cell types and appeared to be lowest in microglia, indicating that differences in basal gene expression of specific Jumonji histone demethylases may contribute to cell-specific gene expression in the CNS (central nervous system). This multiparametric technique will be valuable for simultaneously assaying chromatin modifications and gene regulation in the adult CNS

Chronic intermittent hypoxia exerts CNS region-specific effects on rat microglial inflammatory and TLR4 gene expression.

Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O2) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions

Intermittent hypoxia-induced inflammatory gene expression differs temporally in brainstem microglia and brainstem tissue homogenates.

<p>iNOS, COX-2, TNFα, IL-1α and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the brainstem of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ^#p = 0.06.</p