A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior.

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model.

Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne granule cells. Particularities relating to performing electrophysiological recordings from small cells, such as immature adult-borne granule cells, are also discussed. The present protocol may be complemented by multi-modal investigations (behavioral, morpho-structural, biochemical), to hopefully facilitate research and advances into the long-term sequelae of stroke and the discovery of new therapeutic opportunities.

Tissue-specific Gene Expression Changes Are Associated with Aging in Mice.

Aging is a complex process that can be characterized by functional and cognitive decline in an individual. Aging can be assessed based on the functional capacity of vital organs and their intricate interactions with one another. Thus, the nature of aging can be described by focusing on a specific organ and an individual itself. However, to fully understand the complexity of aging, one must investigate not only a single tissue or biological process but also its complex interplay and interdependencies with other biological processes. Here, using RNA-seq, we monitored changes in the transcriptome during aging in four tissues (including brain, blood, skin and liver) in mice at 9 months, 15 months, and 24 months, with a final evaluation at the very old age of 30 months. We identified several genes and processes that were differentially regulated during aging in both tissue-dependent and tissue-independent manners. Most importantly, we found that the electron transport chain (ETC) of mitochondria was similarly affected at the transcriptome level in the four tissues during the aging process. We also identified the liver as the tissue showing the largest variety of differentially expressed genes (DEGs) over time. Lcn2 (Lipocalin-2) was found to be similarly regulated among all tissues, and its effect on longevity and survival was validated using its orthologue in Caenorhabditis elegans. Our study demonstrated that the molecular processes of aging are relatively subtle in their progress, and the aging process of every tissue depends on the tissue's specialized function and environment. Hence, individual gene or process alone cannot be described as the key of aging in the whole organism.

Sigma 1 receptor activation modifies intracellular calcium exchange in the G93AhSOD1 ALS model.

Aberrations in intracellular calcium (Ca2+) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca2+ cycle (ERMCC). The ER supplies Ca2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca2+ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca2+ homeostasis in the presence of G93AhSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca2+ clearance following a) AMPAR activation by kainate and b) IP3R-mediated ER Ca2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca2+. Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca2+ homeostasis.

Transcriptional profiling reveals protective mechanisms in brains of long-lived mice.

The brain plays a central role in organismal aging but is itself most sensitive to aging-related functional impairments and pathologies. Insights into processes underlying brain aging are the basis to positively impact brain health. Using high-throughput RNA sequencing and quantitative polymerase chain reaction (PCR), we monitored cerebral gene expression in mice throughout their whole lifespan (2, 9, 15, 24, and 30 months). Differentially expressed genes were clustered in 6 characteristic temporal expression profiles, 3 of which revealed a distinct change between 24 and 30 months, the period when most mice die. Functional annotation of these genes indicated a participation in protection against cancer and oxidative stress. Specifically, the most enriched pathways for the differentially expressed genes with higher expression at 30 versus 24 months were found to be glutathione metabolism and chemokine signaling pathway, whereas those lower expressed were enriched in focal adhesion and pathways in cancer. We therefore conclude that brains of very old mice are protected from certain aspects of aging, in particular cancer, which might have an impact on organismal health and lifespan.

Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures.

Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4ß3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors.

Pro-apoptotic function of GABA-related transcripts following stroke.

Following cerebral injuries such as stroke, a structural and functional reorganization of the impaired tissue occurs, which is often accompanied by a re-expression of developmental genes. During brain development, embryonic splice variants of the GABA-synthesizing GAD67 gene (collectively termed EGAD) participate in cell proliferation, migration, and neuronal differentiation. We thus hypothesized an involvement of EGAD in post-ischemic plasticity. EGAD transcripts were up-regulated at early reperfusion times in the injured area following transient middle cerebral artery occlusion (with a peak expression of 4.5-fold at 6h in C57BL/6 mice). Cell-specific analysis by a combination of radioactive in situ hybridization and immunolabeling revealed EGAD up-regulation in TUNEL-positive neurons. This unexpected cell death-associated expression of EGAD was confirmed in cell culture models of ischemia (combined oxygen-glucose deprivation) and apoptosis (staurosporine). Staurosporine-mediated cell death led to cleaved Caspase-3 activation, a key regulator of apoptosis following stroke. Blocking of staurosporine-associated EGAD expression via antisense RNA treatment reduced cleaved Caspase-3 activation by ~30%. In addition to the involvement of EGAD in proliferative processes during brain development, we found here that EGAD participates in cell death under pathophysiological conditions in the adult brain. Re-expression of EGAD in neurons following stroke may play a role in aberrant cell cycle activation, consequently being pro-apoptotic. Our observation of a new GABA related pro-apoptotic mechanism and its successful modification might be of significant clinical relevance.

Impact of indomethacin on neuroinflammation and hippocampal neurogenesis in aged mice.

Age-induced neuroinflammation could be a contributing factor to the restricted neurogenesis in aged mice. Indomethacin, a common non-steroidal anti-inflammatory drug, has been demonstrated to partially restore neurogenesis under pathophysiological inflammation-associated conditions in adult C57BL/6 mice. This study investigated whether indomethacin is able to decrease age-related neuroinflammation in the hippocampus (24-month-old mice) and thereby stimulate neurogenesis. During hippocampal aging, the transcript expression of pro-inflammatory cytokines (Tnfα, Il-1α, Il-1ß), the chemokine Mip-1α, and markers for activated astrocytes (Gfap, Lcn2, but not Vim and Serpina3n) and microglia (Iba1, F4/80, Cd68, Cd86) significantly increased. Treatment with indomethacin significantly decreased COX-1 and COX-2 transcript expression. Of the age-related inflammatory mediators, only Gfap and Iba1 were affected by indomethacin treatment in the hippocampus, with a significantly reduced transcript expression being detected for both markers. Neurogenesis was unaffected by indomethacin. Thus, our data reveal that administration of indomethacin to aged mice is not able to effectively decrease neuroinflammation and promote neurogenesis.

Age-specific transcriptional response to stroke.

Increased age is a major risk factor for stroke incidence and post-ischemic mortality. To develop age-adjusted therapeutic interventions, a clear understanding of the complexity of age-related post-ischemic mechanisms is essential. Transient occlusion of the middle cerebral artery--a model that closely resembles human stroke--was used to induce cerebral infarction in mice of 4 different ages (2, 9, 15, 24 months). By using Illumina cDNA microarrays and quantitative PCR we detected a distinct age-dependent response to stroke involving 350 differentially expressed genes. Our analyses also identified 327 differentially expressed genes that responded to stroke in an age-independent manner. These genes are involved in different aspects of the inflammatory and immune response, oxidative stress, cell cycle activation and/or DNA repair, apoptosis, cytoskeleton reorganization and/or astrogliosis, synaptic plasticity and/or neurotransmission, and depressive disorders and/or dopamine-, serotonin-, GABA-signaling. In agreement with our earlier work, aged brains displayed an attenuated inflammatory and immune response (Sieber et al., 2011) and a reduced impairment of post-stroke synaptic plasticity. Our data also revealed a distinct age-related susceptibility for post-ischemic depression, the most common neuropsychiatric consequence of stroke, which has a major influence on functional outcome.

Astrocytic Cx43 and Cx30 differentially modulate adult neurogenesis in mice.

In addition to their well known function in astrocyte coupling, gap junction forming connexins are also important for cell proliferation, migration and differentiation during brain development. The aim of this study was to determine whether loss of the main astrocytic connexins, connexin 43 (Cx43) or connexin 30 (Cx30), influences various stages of adult hippocampal neurogenesis. To that end, mice with a conditional Cx43 deletion in astrocytes and mice with a conventional knockout of Cx30 were used. We assessed cell proliferation based on Ki67-immunoreactive cell number and cell survival based on BrdU-immunoreactive cell number in the subgranular zone (SGZ) and the granular cell layer (GCL) of the dentate gyrus. The neuronal phenotype of surviving cells was analyzed following immunofluorescent co-localization of BrdU-positive cells with the neuronal markers doublecortin (DCX) and neuronal nuclear antigen (NeuN). Ablation of Cx43 in astrocytes significantly diminished proliferation and reduced the overall survival of newborn cells. In contrast, knockout of Cx30 showed a tendency towards increased proliferation and significantly enhanced the overall survival of newborn cells. The differentiation of surviving cells into neurons is unaffected following Cx43 or Cx30 knockout. Our data reveal that Cx43 promotes the survival of newborn neurons in the adult mouse hippocampus whereas Cx30 restricts their survival.

Attenuated inflammatory response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice following stroke.

BACKGROUND: Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. METHODS AND RESULTS: As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1ß, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. CONCLUSIONS: Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.