Search

Reactive astrogliosis is a common pathological hallmark of central nervous system (CNS) injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. TheseSrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains ofSrfknockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases,SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce β-amyloid plaques in mouse models of Parkinson’s and Alzheimer’s disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

Major Depressive Disorder (MDD) is a debilitating neuropsychiatric illness affecting over 20% of the population worldwide. Despite its prevalence, our understanding of its pathophysiology is severely limited, thus hampering the development of novel therapeutic strategies. Recent advances have clearly established astrocytes as major players in the pathophysiology, and plausibly pathogenesis, of major depression. In particular, astrocyte density in the hippocampus is severely diminished in MDD patients and correlates strongly with the disease outcome. Moreover, astrocyte densities from different subfields of the hippocampus show varying trends in terms of their correlation to the disease outcome. Given the central role that hippocampus plays in the pathophysiology of depression and in the action of antidepressant drugs, changes in hippocampal astrocyte density and physiology may have a significant effect on behavioral symptoms of MDD. In this study, we used Chronic Mild Unpredictable Stress (CMUS) in mice, which induces a depressive-like state, and examined its effects on astrocytes from different subfields of the hippocampus. We used S100β immunostaining to estimate the number of astrocytes per mm2 from various hippocampal subfields. Furthermore, using confocal images of fluorescently labeled GFAP-immunopositive hippocampal astrocytes, we quantified various morphology-related parameters and performed Sholl analysis. We found that CMUS exerts differential effects on astrocyte cell density, ramification, cell radius, surface area, and process width of hippocampal astrocytes from different hippocampal subfields. Taken together, our study reveals that chronic stress doesn’t uniformly affect all hippocampal astrocytes; but exerts its effects differentially on different astrocytic subpopulations within the hippocampus.

Early-life stress (ELS), including chronic deprivation of maternal care, exerts persistent life-long effects on animal physiology and behavior, and is associated with several neurodevelopmental disorders. Long-lasting changes in neuronal plasticity and electrophysiology are documented extensively in the animal models of ELS. However, the role of astroglia in the lasting effects of ELS remains elusive. Astrocytes are intricately involved in the regulation of synaptic physiology and behavior. Moreover, astrocytes play a major role in the innate and adaptive immune responses in the central nervous system (CNS). The role of immune responses and neuroinflammation in the altered brain development and persistent adverse effects of ELS are beginning to be explored. Innate immune response in the CNS is characterized by a phenomenon called astrogliosis, a process in which astrocytes undergo hypertrophy, along with changes in gene expression and function. While the immune activation and neuroinflammatory changes concomitant with ELS, or in juveniles and young adults have been reported, it is unclear whether mice subjected to ELS exhibit astrogliosis-like alterations well into late-adulthood. Here, we subjected mice to maternal separation from postnatal day 2 to day 22 and performed comprehensive morphometric analysis of hippocampal astrocytes during late-adulthood. We found that the astrocytes in the stratum radiatum region of the CA1 hippocampal subfield from maternally separated mice exhibit significant hypertrophy as late as 8 months of age, revealing the crucial changes in astrocytes that manifest long after the cessation of ELS. This study highlights the persistence of neuroinflammatory changes in mice exposed to ELS.

Nervous system development and plasticity involve changes in cellular morphology, making morphological analysis a valuable exercise in the study of nervous system development, function and disease. Morphological analysis is a time-consuming exercise requiring meticulous manual tracing of cellular contours and extensions. We have developed a software tool, called SMorph, to rapidly analyze the morphology of cells of the nervous system. SMorph performs completely automated Sholl analysis. It extracts 23 morphometric features based on cell images and Sholl analysis parameters, followed by principal component analysis (PCA). SMorph was tested on neurons, astrocytes and microglia and reveals subtle changes in cell morphology. Using SMorph, we found that chronic 21-day treatment with the antidepressant desipramine results in a significant structural remodeling in hippocampal astrocytes in mice. Given the proposed involvement of astroglial structural changes and atrophy in major depression in humans, our results reveal a novel kind of structural plasticity induced by chronic antidepressant administration.

Nervous system development and plasticity involves changes in cellular morphology, making morphological analysis a valuable exercise in the study of nervous system development, function and disease. Morphological analysis is a time-consuming exercise requiring meticulous manual tracing of cellular contours and extensions. We have developed a software tool, called SMorph, to rapidly analyse the morphology of cells of the nervous system. SMorph performs completely automated Sholl analysis. It extracts 23 morphometric features based on cell images and Sholl analysis parameters, followed by Principal Component Analysis. SMorph is tested on neurons, astrocytes and microglia and reveals subtle changes in cell morphology. Using SMorph, we found that chronic 21-day treatment with antidepressant desipramine results in a significant structural remodeling in hippocampal astrocytes. Given the proposed involvement of astroglial structural changes and atrophy in major depression in humans, our results reveal a novel kind of structural plasticity induced by chronic antidepressant administration.

Forced swim test (FST) and tail suspension test (TST) are commonly used behavioral tests for screening antidepressant drugs with a high predictive validity. These tests have also proved useful to assess the non-motor symptoms in the animal models of movement disorders such as Parkinson's disease and Huntington's disease. Manual analysis of FST and TST is a time-consuming exercise, and has large observer-to-observer variability. Automation of behavioral analysis alleviates these concerns, but there are no easy-to-use open source tools for such analysis. Here, we describe the development of DBscorer, an open source program installable on Windows®, with an intuitive graphical user interface, that helps in accurate quantification of immobility behavior in FST and TST from video analysis. Several calibration options allow customization of various parameters to suit the experimental requirements. Apart from the readout of time spent immobile, DBscorer also provides additional data and graphics of immobility/mobility states across time revealing the evolution of behavioral despair over the duration of the test and allows the analysis of additional parameters. Such comprehensive analysis allows a more nuanced understanding of the expression of behavioral despair in FST and TST. We believe that DBscorer would make analysis of behavior in FST and TST unbiased, automated and rapid, and hence prove to be helpful to the wider neuroscience community.Significance StatementFST and TST are commonly used to rapidly screen novel antidepressant compounds. They are also used to assess the non-motor behaviors in models of Parkinson's and Huntinton's disease. However, the manual analysis of behavior is time-consuming and subject to observer-to-observer variability. The tools available for automation are either difficult to use, are expensive, require special apparatus, or not comprehensively validated. DBscorer, described here, is an open source software for Windows® with a user-friendly GUI, which we have extensively validated against the performance of highly trained human scorers. We believe that the ease of installation and use, as well as the high accuracy would lead to a widespread adoption of DBscorer by the neuroscience community.

Adjunct α2-adrenoceptor antagonism is a potential strategy to accelerate the behavioral effects of antidepressants. Co-administration of the α2-adrenoceptor antagonist yohimbine hastens the behavioral and neurogenic effects of the antidepressant imipramine. We examined the transcriptional targets of short duration (7days), combination treatment of yohimbine and imipramine (Y+I) within the adult rat hippocampus. Using microarray and qPCR analysis we observed functional enrichment of genes involved in intracellular signaling cascades, plasma membrane, cellular metal ion homeostasis, multicellular stress responses and neuropeptide signaling pathways in the Y+I transcriptome. We noted reduced expression of the α2A-adrenoceptor (Adra2a), serotonin 5HT2C receptor (Htr2c) and the somatostatin receptor 1 (Sstr1), which modulate antidepressant action. Further, we noted a regulation of signaling pathway genes like inositol monophosphatase 2 (Impa2), iodothyronine deiodinase 3 (Dio3), regulator of G-protein signaling 4 (Rgs4), alkaline ceramidase 2 (Acer2), doublecortin-like kinase 2 (Dclk2), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (Nfkbia) and serum/glucocorticoid-regulated kinase 1 (Sgk1), several of which are implicated in the pathophysiology of mood disorders. Comparative analysis revealed an overlap in the hippocampal regulation of Acer2, Nfkbia, Sgk1 and Impa2 between Y+I treatment, the fast-acting electroconvulsive seizure (ECS) paradigm, and the slow-onset chronic (21days) imipramine treatment. Further, Y+I treatment enhanced the quiescent neural progenitor pool in the hippocampal neurogenic niche similar to ECS, and distinct from chronic imipramine treatment. Taken together, our results provide insight into the molecular and cellular targets of short duration Y+I treatment, and identify potential leads for the development of rapid-action antidepressants.

Memory is a temporally evolving molecular and structural process, which involves changes from local synapses to complex neural networks. There is increasing evidence for an involvement of developmental pathways in regulating synaptic communication in the adult nervous system. Notch signaling has been implicated in memory formation in a variety of species. Nevertheless, the mechanism of Notch in memory consolidation remains poorly understood. In this commentary, besides offering an overview of the advances in the field of Notch in memory, we highlight some of the weaknesses of the studies and attempt to cast light on some of the apparent discrepancies on the role of Notch in memory. We believe that future studies, employing high-throughput technologies and targeted Notch loss and gain of function animal models, will reveal the mechanisms of Notch-dependent plasticity and resolve whether this signaling pathway is implicated in the cognitive deficit associated with dementia.

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that β3-adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective β3-adrenergic receptor agonistin vivoincreases the number of proliferating cells in the SGZ. Similarly, systemic injection of the β-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursorsin vivo. Finally, using a novelex vivo“slice-sphere” assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via β-adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via β3-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants.

Major depressive disorder (MDD) is one of the most common neuropsychiatric disorders affecting over one-fifth of the population worldwide. Owing to our limited understanding of the pathophysiology of MDD, the quest for finding novel antidepressant drug targets is severely impeded. Monoamine hypothesis of MDD provides a robust theoretical framework, forming the core of a large jigsaw puzzle, around which we must look for the vital missing pieces. Growing evidence suggests that the glial loss observed in key regions of the limbic system in depressed patients, at least partly, accounts for the structural and cognitive manifestations of MDD. Studies in animal models have subsequently hinted at the possibility that the glial atrophy may play a causative role in the precipitation of depressive symptoms. Antidepressants as well as monoamine neurotransmitters exert profound effects on the gene expression and metabolism in astrocytes. This raises an intriguing possibility that the astrocytes may play a central role alongside neurons in the behavioral effects of antidepressant drugs. In this article, we discuss the gene expression and metabolic changes brought about by antidepressants in astrocytes, which could be of relevance to synaptic plasticity and behavioral effects of antidepressant treatments.

Slow-onset adaptive changes that arise from sustained antidepressant treatment, such as enhanced adult hippocampal neurogenesis and increased trophic factor expression, play a key role in the behavioral effects of antidepressants. α2-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of α2-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that α2-adrenoceptor agonists, clonidine and guanabenz, decrease adult hippocampal neurogenesis through a selective effect on the proliferation, but not the survival or differentiation, of progenitors. These effects persist in dopamine β-hydroxylase knock-out (Dbh−/−) mice lacking norepinephrine, supporting a role for α2-heteroceptors on progenitor cells, rather than α2-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitorsin vitroexpress all the α2-adrenoceptor subtypes, and decreased neurosphere frequency and BrdU incorporation indicate direct effects of α2-adrenoceptor stimulation on progenitors. Furthermore, coadministration of the α2-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation, the morphological maturation of newborn neurons, and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally, short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test, which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that α2-adrenoceptors, expressed by progenitor cells, decrease adult hippocampal neurogenesis, while their blockade speeds up antidepressant action, highlighting their importance as targets for faster acting antidepressants.

Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. Highlights In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade. Notch1 regulates both NMDAR expression and composition. Notch1 influences a cascade of cellular events culminating in CREB activation.

Memory is a temporally evolving molecular and structural process which involves changes from local synapses to complex neural networks. There is increasing evidence for an involvement of developmental pathways in regulating synaptic communication in the adult nervous system. Notch signaling has been implicated in memory formation in a variety of species. Nevertheless the mechanism of Notch in memory consolidation remains poorly understood. In this commentary besides offering an overview of the advances in the field of Notch in memory we highlight some of the weaknesses of the studies and attempt to cast light on some of the apparent discrepancies on the role of Notch in memory. We believe that future studies employing high throughput technologies and targeted Notch loss and gain of function animal models will reveal the mechanisms of Notch dependent plasticity and resolve whether this signaling pathway is implicated in the cognitive deficit associated with dementia. © 2015 Wiley Periodicals Inc.

Several laboratories have developed genetic methods to monitor Notch activity in developing and adult mice. These approaches have been useful in identifying Notch signaling with high temporal and spatial resolution. This research has contributed substantially to our understanding of the role of Notch in cell specification and cellular physiology. Here, we present two protocols to monitor Notch activity in the mouse brain: (1) by intraventricular electroporation and (2) by intracranial viral injections of Notch reporter constructs. These methods allow monitoring of Notch signaling in specific brain regions from development to adulthood. In addition, using the appropriate modifications, the Notch reporter systems can also be used to monitor Notch activity in other organs of the mouse such as retina, skin, skeletal muscle, and cancer cells.

Background: Adult neurogenesis and hippocampal function are both regulated by neuronal activity, particularly by GABAergic signals. Recently, it has been reported that an imbalance of the GABAergic ~a transmission impairs adult neurogenesis in Alzheimer’s disease (AD). This GABAergic signal is modulated by several endogenous molecules. Diazepam binding inhibitor is a small cytosolic polypeptide which is an inverse agonist of GABA A receptor. Thus, we researched the expression and the effect of diazepam binding inhibitor (DBI) on neurogenesis in the hippocampus by using AD model mice. Methods: In this study, we used AD model mice which the early onset of the symptoms of AD was induced. Our recent study showed that feeding with High Fat Diet is a viable method for inducing the early onset of the symptoms of AD in B6C3-Tg (APPswe/PSEN1dE9)85Dbo/J Alzheimer’s Disease Model Transgenic mice. Using these mice, we performed DBI knockdown experiment and immunostaing of DBI to investigate a functional role of DBI in dentate gyrus. To evaluate the effect of DBI knockdown for differentiation we observed the number and morphology of doublecortin (DCX; immature neuron marker) positive cells. Results: We immunostained DBI and dyed senile plaques to compare the expression of DBI in AD model and control mice. As a result, we observed a tendency of increased DBI expression in the hippocampus in AD model mice. Commonly, DBI is expressed in neural stem cells, however, it was also expressed in glial cell in AD model mice. Furthermore, expression of DBI was observed in astrocytes surrounding senile plaques in AD model mice. This result corroborates the report that the Ab peptide stimulates DBI biosynthesis in cultured rat astrocytes. The impairment of maturation of immature neuron in AD model mice was repaired by the inhibition of DBI expression in the dentate gyrus. Conclusions: These results suggest that increased DBI in the hippocampus inhibit normal maturation in AD model mice. This increased DBI is derived from astrocyte activated by Ab. DBI produced by astrocyte and released in the dentate gyrus cause disorder in neurogenesis in AD mice via GABA A receptor.

Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.

Several laboratories have developed genetic methods to monitor Notch activity in developing and adult mice. These approaches have been useful in identifying Notch signaling with high temporal and spatial resolution. This research has contributed substantially to our understanding of the role of Notch in cell specification and cellular physiology. Here, we present two protocols to monitor Notch activity in the mouse brain: (1) by intraventricular electroporation and (2) by intracranial viral injections of Notch reporter constructs. These methods allow monitoring of Notch signaling in specific brain regions from development to adulthood. In addition, using the appropriate modifications, the Notch reporter systems can also be used to monitor Notch activity in other organs of the mouse such as retina, skin, skeletal muscle, and cancer cells.

Notch signaling is an evolutionarily conserved pathway, which is fundamental for neuronal development and specification. In the last decade, increasing evidence has pointed out an important role of this pathway beyond embryonic development, indicating that Notch also displays a critical function in the mature brain of vertebrates and invertebrates. This pathway appears to be involved in neural progenitor regulation, neuronal connectivity, synaptic plasticity and learning/memory. In addition, Notch appears to be aberrantly regulated in neurodegenerative diseases, including Alzheimer's disease and ischemic injury. The molecular mechanisms by which Notch displays these functions in the mature brain are not fully understood, but are currently the subject of intense research. In this review, we will discuss old and novel Notch targets and molecular mediators that contribute to Notch function in the mature brain and will summarize recent findings that explore the two facets of Notch signaling in brain physiology and pathology.

Neurological disorders such as Alzheimer's disease stroke and epilepsy are currently marred by the lack of effective treatments to prevent neuronal death. Erroneous cell cycle reentry (CCR) is hypothesized to have a causative role in neurodegeneration. We show that forcing S phase reentry in cultured hippocampal neurons is sufficient to induce neurodegeneration. We found that kainic acid treatment in vivo induces erroneous CCR and neuronal death through a Notch dependent mechanism. Ablating Notch signaling in neurons provides neuroprotection against kainic acid induced neuronal death. We further show that kainic acid treatment activates Notch signaling which increases the bioavailability of CyclinD1 through Akt/GSK3ß pathway leading to aberrant CCR via activation of CyclinD1 Rb E2F1 axis. In addition pharmacological blockade of this pathway at critical steps is sufficient to confer resistance to kainic acid induced neurotoxicity in mice. Taken together our results demonstrate that excitotoxicity leads to neuronal death in a Notch dependent manner through erroneous CCR.Cell Death and Differentiation advance online publication 27 March 2015; doi:10.1038/cdd.2015.23.