- Email: [email protected]
Molecular and biochemical characterization of observed CNS developmental alterations in Canavan Disease
Poster Abstracts ISDN 2012 / Int. J. Devl Neuroscience 30 (2012) 672–692
ISDN2012 0230 Endogenous repair mechanism following motor cortex lesion in adult mice Bhaskar Saha, Sophie Péron, Mohamed Jaber, Afsaneh Gaillard Institut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Université de Poitiers, France The SubVentricular Zone (SVZ) and the Dentate Gyrus of the Hippocampus are the two main proliferative niches in adult mammalian brain. In the rodent adult brain, neuroblasts generated in the SVZ migrate along the rostral migratory stream (RMS) to the olfactory bulb. Previous studies have demonstrated that in several diseases and in brain injury, newly generated neuroblasts from the SVZ can migrate ectopically to the affected areas, which might constitute an endogenous repair mechanism. The aim of our study is to investigate changes happen in the SVZ following aspiration lesion in the motor cortex of adult mice and to study the ectopic migration of neuroblasts from the SVZ and their fate. We have observed that there is a transient increase in the cellular proliferation in the SVZ, which peaks at day 7, following lesion. Neuroblasts start to migrate out of the RMS to the lesion site after 3 days. This migration appears to be multimodal as we found migrating cells in close association with either blood vessels or glial cells. Some cells also migrate without any such association. We have not observed any ‘chain’ formation; a hallmark of neuroblasts migration along the RMS. This ectopic migration is possibly regulated by SDF1-CXCR4 signaling. The morphology of the glial tube, which restricts neuroblasts within the RMS, is altered and appeared to be more ‘open’ following lesion. Ongoing study is focused on detail characterization of the cell types those are generated following lesion and migrated to the affected area. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.043 ISDN2012 0232 Molecular mechanisms of somatosensory cortex patterning during development Gabrielle Pouchelon, Bruno Golding, Denis Jabaudon Dept. of Basic Neurosciences, and Clinic of Neurology, University of Geneva, Switzerland The rodent somatosensory cortex has a characteristic cytoarchitecture in which individual whiskers are represented by clusters of layer IV neurons called “barrels”. Individual barrels receive input from a single main whisker via a specific subtype of thalamocortical (TC) neurons; adjacent barrels represent adjacent whiskers and together they form a point-to-point map of the whisker pad in the somatosensory cortex. Although this topographical mapping is critical for normal somatosensory function, the molecular mechanisms that control the target specificity of distinct TC neurons and cognate clustering of layer IV neurons during development are still poorly understood. The somatosensory cortex receives peripheral input via two main classes of TC neurons with distinctive laminar target specificities: while VPM TC neurons project mainly to layer IV, where their axons branch locally to generate barrels, POm TC neurons instead avoid the barrels and project to layer I where their axons branch tangentially over extended distances. Taking advantage of this area-specific, mutually exclusive projection pattern, here we investigate the molecular determinants of VPM and POm TC neuron target specificity during development. To this end, we compared gene expression in pure populations of VPM and POm neurons at key developmental stages of S1 thalamocortical innervation, using microarray analysis after retrograde labelling and
685
targeted microdissection. This approach has identified several gene candidates with highly specific expression in VPM or POm neurons, providing novel functional insights into the mechanisms controlling the formation of precise topographic maps during development. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.044 ISDN2012 0235 Proteomic analysis of APE1/Ref-1 regulation of A(23-35)induced neurotoxicity in cultured PC12 and SH-SY5Y cells Anil K. Mantha 1,3,∗ , Monisha Dhiman 2 , Sankar Mitra 1 , Regino J. Perez-Polo 1,3 1
Dept. of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, United States 2 Dept. of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, United States 3 Dept. of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, United States E-mail address: [email protected] (A.K. Mantha). The genotoxic extracellular accumulation of amyloid beta (A) protein and subsequent neuronal cell death is associated with Alzheimer’s disease (AD). APE1/Ref-1, the predominant apurinic/apyrimidinic (AP) endonuclease, essential in eukaryotic cells, is rate-limiting in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single-strand breaks (SSBs) in DNA. APE1/Ref-1 is also involved in the redox activation of several trans-acting factors (TFs) in various cell types but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref-1’s role in neuronal cells vulnerable in AD and other neurodegenerative disorders, as reflected in its nuclear accumulation in the AD brains. Increase of APE1/Ref1 has been shown to enhance neuronal survival after oxidative stress. We asked whether APE1/Ref-1 levels or its association with other proteins is responsible for these protective effects. We used 2D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha-3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N-acetyl transferase, sulfotransferase 1c), proteins involved in stress response (i.e., antiNGF30 antibody, lucine-rich and death domain) and heterogeneous nuclear ribonucleoprotien-H (hnRNP-H) as being associated with APE1/Ref-1 in A(25-35)-treated rat pheochromocytoma PC12 and human neuroblastoma SH-SY5Y cell lines, two common neural lines used in A neurotoxicity studies. Some of these proteins are affected in the brains of AD patients, in agreement with a neuro-protective role for APE1/Ref-1 via its association with various proteins known to alter cellular functions during A-mediated neurotoxicity. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.045 ISDN2012 0237 Molecular and biochemical characterization of observed CNS developmental alterations in Canavan Disease Shalini Kumar 1 , Reuben Matalon 2 , Jeande Vellis 1 1
Department of Neurobiology, Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behaviour, University of California, Los Angeles, CA 90095-7332, USA 2 Division of Genetics, Department of Paediatrics, University of Texas Medical Branch, Galveston, TX 77555, USA We are studying molecular mechanisms involved in early brain development in a mouse model of Canavan Disease (CD), a
686
Poster Abstracts ISDN 2012 / Int. J. Devl Neuroscience 30 (2012) 672–692
hereditary fatal childhood leukodystrophy, caused by mutation in aspartoacylase (ASPA) gene. In normal brain ASPA gene expression occurs in oligodendrocytes (OLs) and its first expression coincides with appearance of OL progenitors at embryonic stage E12.5 in the forebrain. A sign of hypomyelination, which correlates with pathophysiology of CD in young children, is detected in ASPA KO mouse brain during peak of myelination. In these mutant brains a continued proliferation of immature OLs, presence of highly acetylated histones H3 associated with epigenetic regulation of cell proliferation is observed. The process of myelin synthesis and myelination normally proceeds with repression of proliferation and initiation of differentiation/maturation of OLs. In order to understand developmental changes in gene expression and involvement of biological mechanisms in devastating demyelinating disease, we performed gene array analysis, microRNA and Metabolomics studies of normal and ASPA KO mouse cortical white matter (WM) tissue at P20, at the peak of myelination. Many studies have revealed the involvement of a set of required genes that play critical role in the process of normal OL maturation and myelination. However, alteration in regulatory mechanisms involving WM disorders is not understood at this time. We have identified a set of 331 negative and one of144 positive, differentially expressed genes between KO vs. wt WM. The gene ontology report shows down-regulation of a number of key genes belonging to the myelin family, as well as embryonic and postnatal developmental genes, which we are currently validating. Most significant observation includes increased oxidative stress during early developmental stages. Among several relevant molecular pathways, a cluster of histone genes are identified that are involved in nucleosome assembly or disassembly, modification of histones with indicated epigenetic role in the leukodystrophy. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.046 ISDN2012 0238 Reelin upregulation and impaired Purkinje cell functional organization following maternal exposure of deltamethrin in rat K. Kumar 1 , N. Patro 1 , I.K. Patro 1,2
ISDN2012 0239 Understanding neurotransmitter-neuropeptide regulation through neural circuits controlling innate behaviour Mahendra Wagle, Su Guo Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, United States Innate behaviours are known to be hard-wired in the nervous system, and are best suited for studying the influence of genetic and environmental factors on functioning of neural circuits. Camouflage is an innate behaviour displayed by many aquatic and terrestrial animals. Zebrafish larvae display a simple camouflage response according to the background lighting conditions. It manifests as a cellular behaviour of bi-directional movement of pigment granules (melanosomes) in melanocytes and is regulated by complex neuro and endocrine systems. The neuropeptidecorticotrophin-releasing factor (CRF), a critical component of stress response, is also important for camouflage behaviour. We recently demonstrated that ethanol, a highly addictive substance, robustly modulates camouflage response by acting upstream of the CRFProopiomelanocortin (POMC) pathway, which has been previously implicated in the development of human alcoholism. We show that CRF expression is modulated by lighting conditions and acute ethanol treatment. Activity of both adenylyl cyclase 5 and extracellular signal-regulated kinase (ERK) is required for such ethanolor light-induced plasticity of CRF expression. These results reveal that CRF act as an important node in camouflage neural circuit and ethanol acts through the CRF-POMC neuropeptide system to regulate camouflage in zebrafish. Our recent results demonstrate that the neurotransmitter dopamine inhibits CRF-POMC pathway in a receptor subtype-specific manner. Thus we demonstrate a novel system for genetically dissecting neural circuitry involving neuropeptide and neurotransmitter systems that control an innate behaviour. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.048 ISDN2012 0248
1
School of Studies in Neuroscience, Jiwaji University, Gwalior 474011, India 2 School of Studies in Zoology, Jiwaji University, Gwalior 474011, India We have evaluated the prenatal neurotoxicity of deltamethrin (DLT), a pyrethroid insecticide in rats that has long been considered safe to humans. DLT (0.75 mg/kg body weight, intraperitonialy, dissolved in dimethylsulfoxide) was administered in timed pregnant rats during two different gestational time periods, i.e., gestational days 7–10 and 11–14. Reelin is a large extracellular glycoprotein secreted by glutamatergic granule cells during cerebellar development and plays a key role in instructing neurons to achieve normal differentiation when they reach their final destination at the end of the radial migration. A sharp upregulation of reelin specifically in the cells of external granular layer (EGL) and the granule cells of the internal granule cell layer (IGL), suggested an impaired granule cell migration following DLT exposure. The disorganized and hypertrophied morphology of Bergmann glial fibers along with hindered granule cell migration could be seen in GFAP immunolabeled sections. Reelin overexpression also resulted in the misalignment of the Purkinje cells and also inhibited the neuritis growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbor. A significantly impaired motor coordination was also evaluated following DLT exposure. DLT is thus proposed to affect the structural and functional development of cerebellum via reelin overexpression. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.047
A fine balance between inhibition and excitation and the clusters of hyper/hypoactive neurons in sensory barrel cortex after traumatic brain injury Manoj Kumar Jaiswal 1,2,∗ , Fritz W. Lischka 1 , Zygmunt Galdzicki 1,2 1
Center for Neuroscience and Regenerative Medicine, Jones Bridge Road, Bethesda, MD, USA 2 Department of Anatomy, Physiology and Genetics, USUHS, School of Medicine, Bethesda, MD, USA Traumatic brain injury (TBI) causes a wide range of pathophysiological changes, affecting sensory and motor functions. The barrel cortex with its well-characterized somatotropic organization is an established model of neuronal circuitry in the sensory cortex that can be utilized to study functional and molecular phenotypes of mild TBI. A balance exists within healthy neuronal networks between excitation and inhibition and major neurological afflictions, such as epilepsy, anxiety disorder, schizophrenia and autism have been linked to the disruption of this balance. Understanding how this balance is disrupted after TBI is critical. We hypothesize that TBI causes a shift in the inhibitory and excitatory balance by strengthening inhibitory circuits, which impairs the ability of the neocortex to recover from injury. In this study we focused on the assessment of network activity by two-photon calcium imaging in combination with whole-cell patch clamp recordings. This approach enabled us to monitor calcium changes and electrical activity in astrocytes, excitatory
ISDN2012 0230 Endogenous repair mechanism following motor cortex lesion in adult mice Bhaskar Saha, Sophie Péron, Mohamed Jaber, Afsaneh Gaillard Institut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Université de Poitiers, France The SubVentricular Zone (SVZ) and the Dentate Gyrus of the Hippocampus are the two main proliferative niches in adult mammalian brain. In the rodent adult brain, neuroblasts generated in the SVZ migrate along the rostral migratory stream (RMS) to the olfactory bulb. Previous studies have demonstrated that in several diseases and in brain injury, newly generated neuroblasts from the SVZ can migrate ectopically to the affected areas, which might constitute an endogenous repair mechanism. The aim of our study is to investigate changes happen in the SVZ following aspiration lesion in the motor cortex of adult mice and to study the ectopic migration of neuroblasts from the SVZ and their fate. We have observed that there is a transient increase in the cellular proliferation in the SVZ, which peaks at day 7, following lesion. Neuroblasts start to migrate out of the RMS to the lesion site after 3 days. This migration appears to be multimodal as we found migrating cells in close association with either blood vessels or glial cells. Some cells also migrate without any such association. We have not observed any ‘chain’ formation; a hallmark of neuroblasts migration along the RMS. This ectopic migration is possibly regulated by SDF1-CXCR4 signaling. The morphology of the glial tube, which restricts neuroblasts within the RMS, is altered and appeared to be more ‘open’ following lesion. Ongoing study is focused on detail characterization of the cell types those are generated following lesion and migrated to the affected area. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.043 ISDN2012 0232 Molecular mechanisms of somatosensory cortex patterning during development Gabrielle Pouchelon, Bruno Golding, Denis Jabaudon Dept. of Basic Neurosciences, and Clinic of Neurology, University of Geneva, Switzerland The rodent somatosensory cortex has a characteristic cytoarchitecture in which individual whiskers are represented by clusters of layer IV neurons called “barrels”. Individual barrels receive input from a single main whisker via a specific subtype of thalamocortical (TC) neurons; adjacent barrels represent adjacent whiskers and together they form a point-to-point map of the whisker pad in the somatosensory cortex. Although this topographical mapping is critical for normal somatosensory function, the molecular mechanisms that control the target specificity of distinct TC neurons and cognate clustering of layer IV neurons during development are still poorly understood. The somatosensory cortex receives peripheral input via two main classes of TC neurons with distinctive laminar target specificities: while VPM TC neurons project mainly to layer IV, where their axons branch locally to generate barrels, POm TC neurons instead avoid the barrels and project to layer I where their axons branch tangentially over extended distances. Taking advantage of this area-specific, mutually exclusive projection pattern, here we investigate the molecular determinants of VPM and POm TC neuron target specificity during development. To this end, we compared gene expression in pure populations of VPM and POm neurons at key developmental stages of S1 thalamocortical innervation, using microarray analysis after retrograde labelling and
685
targeted microdissection. This approach has identified several gene candidates with highly specific expression in VPM or POm neurons, providing novel functional insights into the mechanisms controlling the formation of precise topographic maps during development. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.044 ISDN2012 0235 Proteomic analysis of APE1/Ref-1 regulation of A(23-35)induced neurotoxicity in cultured PC12 and SH-SY5Y cells Anil K. Mantha 1,3,∗ , Monisha Dhiman 2 , Sankar Mitra 1 , Regino J. Perez-Polo 1,3 1
Dept. of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, United States 2 Dept. of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, United States 3 Dept. of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, United States E-mail address: [email protected] (A.K. Mantha). The genotoxic extracellular accumulation of amyloid beta (A) protein and subsequent neuronal cell death is associated with Alzheimer’s disease (AD). APE1/Ref-1, the predominant apurinic/apyrimidinic (AP) endonuclease, essential in eukaryotic cells, is rate-limiting in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single-strand breaks (SSBs) in DNA. APE1/Ref-1 is also involved in the redox activation of several trans-acting factors (TFs) in various cell types but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref-1’s role in neuronal cells vulnerable in AD and other neurodegenerative disorders, as reflected in its nuclear accumulation in the AD brains. Increase of APE1/Ref1 has been shown to enhance neuronal survival after oxidative stress. We asked whether APE1/Ref-1 levels or its association with other proteins is responsible for these protective effects. We used 2D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha-3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N-acetyl transferase, sulfotransferase 1c), proteins involved in stress response (i.e., antiNGF30 antibody, lucine-rich and death domain) and heterogeneous nuclear ribonucleoprotien-H (hnRNP-H) as being associated with APE1/Ref-1 in A(25-35)-treated rat pheochromocytoma PC12 and human neuroblastoma SH-SY5Y cell lines, two common neural lines used in A neurotoxicity studies. Some of these proteins are affected in the brains of AD patients, in agreement with a neuro-protective role for APE1/Ref-1 via its association with various proteins known to alter cellular functions during A-mediated neurotoxicity. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.045 ISDN2012 0237 Molecular and biochemical characterization of observed CNS developmental alterations in Canavan Disease Shalini Kumar 1 , Reuben Matalon 2 , Jeande Vellis 1 1
Department of Neurobiology, Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behaviour, University of California, Los Angeles, CA 90095-7332, USA 2 Division of Genetics, Department of Paediatrics, University of Texas Medical Branch, Galveston, TX 77555, USA We are studying molecular mechanisms involved in early brain development in a mouse model of Canavan Disease (CD), a
686
Poster Abstracts ISDN 2012 / Int. J. Devl Neuroscience 30 (2012) 672–692
hereditary fatal childhood leukodystrophy, caused by mutation in aspartoacylase (ASPA) gene. In normal brain ASPA gene expression occurs in oligodendrocytes (OLs) and its first expression coincides with appearance of OL progenitors at embryonic stage E12.5 in the forebrain. A sign of hypomyelination, which correlates with pathophysiology of CD in young children, is detected in ASPA KO mouse brain during peak of myelination. In these mutant brains a continued proliferation of immature OLs, presence of highly acetylated histones H3 associated with epigenetic regulation of cell proliferation is observed. The process of myelin synthesis and myelination normally proceeds with repression of proliferation and initiation of differentiation/maturation of OLs. In order to understand developmental changes in gene expression and involvement of biological mechanisms in devastating demyelinating disease, we performed gene array analysis, microRNA and Metabolomics studies of normal and ASPA KO mouse cortical white matter (WM) tissue at P20, at the peak of myelination. Many studies have revealed the involvement of a set of required genes that play critical role in the process of normal OL maturation and myelination. However, alteration in regulatory mechanisms involving WM disorders is not understood at this time. We have identified a set of 331 negative and one of144 positive, differentially expressed genes between KO vs. wt WM. The gene ontology report shows down-regulation of a number of key genes belonging to the myelin family, as well as embryonic and postnatal developmental genes, which we are currently validating. Most significant observation includes increased oxidative stress during early developmental stages. Among several relevant molecular pathways, a cluster of histone genes are identified that are involved in nucleosome assembly or disassembly, modification of histones with indicated epigenetic role in the leukodystrophy. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.046 ISDN2012 0238 Reelin upregulation and impaired Purkinje cell functional organization following maternal exposure of deltamethrin in rat K. Kumar 1 , N. Patro 1 , I.K. Patro 1,2
ISDN2012 0239 Understanding neurotransmitter-neuropeptide regulation through neural circuits controlling innate behaviour Mahendra Wagle, Su Guo Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, United States Innate behaviours are known to be hard-wired in the nervous system, and are best suited for studying the influence of genetic and environmental factors on functioning of neural circuits. Camouflage is an innate behaviour displayed by many aquatic and terrestrial animals. Zebrafish larvae display a simple camouflage response according to the background lighting conditions. It manifests as a cellular behaviour of bi-directional movement of pigment granules (melanosomes) in melanocytes and is regulated by complex neuro and endocrine systems. The neuropeptidecorticotrophin-releasing factor (CRF), a critical component of stress response, is also important for camouflage behaviour. We recently demonstrated that ethanol, a highly addictive substance, robustly modulates camouflage response by acting upstream of the CRFProopiomelanocortin (POMC) pathway, which has been previously implicated in the development of human alcoholism. We show that CRF expression is modulated by lighting conditions and acute ethanol treatment. Activity of both adenylyl cyclase 5 and extracellular signal-regulated kinase (ERK) is required for such ethanolor light-induced plasticity of CRF expression. These results reveal that CRF act as an important node in camouflage neural circuit and ethanol acts through the CRF-POMC neuropeptide system to regulate camouflage in zebrafish. Our recent results demonstrate that the neurotransmitter dopamine inhibits CRF-POMC pathway in a receptor subtype-specific manner. Thus we demonstrate a novel system for genetically dissecting neural circuitry involving neuropeptide and neurotransmitter systems that control an innate behaviour. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.048 ISDN2012 0248
1
School of Studies in Neuroscience, Jiwaji University, Gwalior 474011, India 2 School of Studies in Zoology, Jiwaji University, Gwalior 474011, India We have evaluated the prenatal neurotoxicity of deltamethrin (DLT), a pyrethroid insecticide in rats that has long been considered safe to humans. DLT (0.75 mg/kg body weight, intraperitonialy, dissolved in dimethylsulfoxide) was administered in timed pregnant rats during two different gestational time periods, i.e., gestational days 7–10 and 11–14. Reelin is a large extracellular glycoprotein secreted by glutamatergic granule cells during cerebellar development and plays a key role in instructing neurons to achieve normal differentiation when they reach their final destination at the end of the radial migration. A sharp upregulation of reelin specifically in the cells of external granular layer (EGL) and the granule cells of the internal granule cell layer (IGL), suggested an impaired granule cell migration following DLT exposure. The disorganized and hypertrophied morphology of Bergmann glial fibers along with hindered granule cell migration could be seen in GFAP immunolabeled sections. Reelin overexpression also resulted in the misalignment of the Purkinje cells and also inhibited the neuritis growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbor. A significantly impaired motor coordination was also evaluated following DLT exposure. DLT is thus proposed to affect the structural and functional development of cerebellum via reelin overexpression. http://dx.doi.org/10.1016/j.ijdevneu.2012.10.047
A fine balance between inhibition and excitation and the clusters of hyper/hypoactive neurons in sensory barrel cortex after traumatic brain injury Manoj Kumar Jaiswal 1,2,∗ , Fritz W. Lischka 1 , Zygmunt Galdzicki 1,2 1
Center for Neuroscience and Regenerative Medicine, Jones Bridge Road, Bethesda, MD, USA 2 Department of Anatomy, Physiology and Genetics, USUHS, School of Medicine, Bethesda, MD, USA Traumatic brain injury (TBI) causes a wide range of pathophysiological changes, affecting sensory and motor functions. The barrel cortex with its well-characterized somatotropic organization is an established model of neuronal circuitry in the sensory cortex that can be utilized to study functional and molecular phenotypes of mild TBI. A balance exists within healthy neuronal networks between excitation and inhibition and major neurological afflictions, such as epilepsy, anxiety disorder, schizophrenia and autism have been linked to the disruption of this balance. Understanding how this balance is disrupted after TBI is critical. We hypothesize that TBI causes a shift in the inhibitory and excitatory balance by strengthening inhibitory circuits, which impairs the ability of the neocortex to recover from injury. In this study we focused on the assessment of network activity by two-photon calcium imaging in combination with whole-cell patch clamp recordings. This approach enabled us to monitor calcium changes and electrical activity in astrocytes, excitatory