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We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.
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Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized connectivity following macroscopic axon tracts to individual axon target-finding and synapse formation. Identifying mechanisms that enable entire subpopulations of related neurons to project their axons with regional specificity within stereotyped tracts to form appropriate long-range connectivity is key to understanding brain development, organization, and function. Here, we investigate how axons of the cerebral cortex form precise connections between the two cortical hemispheres via the corpus callosum. We identify topographic principles of the developing trans-hemispheric callosal tract that emerge through intrinsic guidance executed by growing axons in the corpus callosum within the first postnatal week in mice. Using micro-transplantation of regionally distinct neurons, subtype-specific growth cone purification, subcellular proteomics, and in utero gene manipulation, we investigate guidance mechanisms of transhemispheric axons. We find that adhesion molecule levels instruct tract topography and target field guidance. We propose a model in which transcallosal axons in the developing brain perform a "handshake" that is guided through co-fasciculation with symmetric contralateral axons, resulting in the stereotyped homotopic connectivity between the brain's hemispheres.
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Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.
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Astrócitos , Permeabilidade da Membrana Celular , Conexina 43 , Ubiquitina-Proteína Ligases Nedd4 , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Humanos , Camundongos , Conexina 43/genética , Mutação de Sentido Incorreto , Proteostase , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ubiquitina-Proteína Ligases Nedd4/genética , EpilepsiaRESUMO
Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient's clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient's clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.
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BACKGROUND: Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. METHODS: We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. RESULTS: Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. CONCLUSIONS: These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.
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Dr. Deepak "Dee" Pandya spent his career as an internal medicine physician as well as in his respective laboratories at the Bedford, Massachusetts Veterans Administration Hospital and at Boston University School of Medicine. His achievements mapping out the cytoarchitecture and connectivity of areas all over the nonhuman primate brain and small mammals are unparalleled. Dee made numerous discoveries and created painstakingly detailed reports, which impacted the field of neuroanatomy and expanded our perceptions of the many diverse inputs and suggestive functions of specific brain regions. The "old school" methods employed from microscopic work to detailed analyses yielded a product that was accurate and exciting all at the same time. We will all miss Dee's smile and tender manner, but more so, we will miss his wonderful and patient mentorship during the precious time we all spent with him. His mentorship resulted in all of his trainees becoming better scientists and left us with the understanding that people like Dee only come by once in a lifetime. In this tribute article for this special issue in the Journal of Comparative Neurology (JCN), the authors describe some of the tedious methods that were used to present our work as a way to provide insight into the extraordinary time and effort it took to produce and publish our articles with Dee in JCN. Dee's work with his colleagues set the stage for more modern methods of counting and mapping neuronal populations presented here, paving the way for such technologies as artificial intelligence and light sheet imaging to advance the field forward to reach new and exciting discoveries.
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Inteligência Artificial , Neurologia , Humanos , NeuroanatomiaRESUMO
Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.
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Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Reparo do DNA/genética , Quebras de DNA de Cadeia DuplaRESUMO
Transcriptomic approaches are powerful strategies to map the molecular diversity of cells in the brain. Single-cell genomic atlases have now been compiled for entire mammalian brains. However, complementary techniques are only just beginning to map the subcellular transcriptomes from distal cellular compartments. We review single-cell datasets alongside subtranscriptome data from the mammalian brain to explore the development of cellular and subcellular diversity. We discuss how single-cell RNA-seq misses transcripts localized away from cell bodies, which form the 'dark transcriptome' of the brain: a collection of subtranscriptomes in dendrites, axons, growth cones, synapses, and endfeet with important roles in brain development and function. Recent advances in subcellular transcriptome sequencing are beginning to reveal these elusive pools of RNA. We outline the success stories to date in uncovering the constituent subtranscriptomes of neurons and glia, as well as present the emerging toolkit that is accelerating the pace of subtranscriptome discovery.
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RNA , Transcriptoma , Animais , Neurônios , Neuroglia , Encéfalo , Mamíferos/genéticaRESUMO
Genetic mosaic analysis, in which mutant cells reside intermingled with wild-type cells, is a powerful experimental approach, but has not been widely used in mice because existing genome-based strategies require complicated and protracted breeding schemes. We have developed an alternative approach termed BEAM (for Binary Expression Aleatory Mosaic) that relies on sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Following delivery of DNA constructs by transfection or viral transduction, combinatorial recombinase activity generates two distinct populations of cells labeled with either green or red fluorescent protein. Any gene of interest can be mis-expressed or deleted in one population for comparison with intermingled control cells. We have extensively optimized and characterized this system both in vitro and in vivo , and demonstrate its power for investigating cell autonomy, identifying temporally or spatially aberrant phenotypes, revealing changes in cell proliferation or death, and controlling for procedural variability.
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The kinase mTOR is a signaling hub for pathways that regulate cellular growth. In neurons, the subcellular localization of mTOR takes on increased significance. Here, we review findings on the localization of mTOR in axons and offer a perspective on how these may impact our understanding of nervous system development, function, and disease. We propose a model where mTOR accumulates in local foci we term mTOR outposts, which can be found in processes distant from a neuron's cell body. In this model, pathways that funnel through mTOR are gated by local outposts to spatially select and amplify local signaling. The presence or absence of mTOR outposts in a segment of axon or dendrite may determine whether regional mTOR-dependent signals, such as nutrient and growth factor signaling, register toward neuron-wide responses. In this perspective, we present the emerging evidence for mTOR outposts in neurons, their putative roles as spatial gatekeepers of signaling inputs, and the implications of the mTOR outpost model for neuronal protein synthesis, signal transduction, and synaptic plasticity.
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Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mTOR pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signalling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in foetal mouse brain in vivo was used to assess the effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signalling, cell morphology and aggregation, cerebral cortical cytoarchitecture and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies, i.e. focal or generalized onset, brain imaging abnormalities, i.e. polymicrogyria, focal cortical dysplasia or normal imaging, and EEG findings, e.g. focal, multi-focal or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 knockout cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of ribosomal S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid-free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 knockout in foetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose results from mTOR-dependent effects on cell structure, cortical development and network organization.
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Epilepsia , Malformações do Desenvolvimento Cortical , Animais , Humanos , Camundongos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Malformações do Desenvolvimento Cortical/genética , Proteínas Ativadoras de GTPase/genética , Epilepsia/genética , Neurônios/metabolismo , Convulsões/genética , SirolimoRESUMO
During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.
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Cones de CrescimentoRESUMO
The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection1. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA-proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR2, together with mRNAs that contain mTOR-dependent motifs3,4. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration.
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Axônios/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteoma/metabolismo , Transcriptoma/genética , Animais , Axônios/enzimologia , Processos de Crescimento Celular , Movimento Celular , Separação Celular , Feminino , Cones de Crescimento/enzimologia , Cones de Crescimento/metabolismo , Masculino , Camundongos , Proteoma/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
The claustrum is a narrow subcortical brain structure that resides between the striatum and insular cortex. The function of the claustrum is not fully described, and while our previous work supports a role for the claustrum in top-down cognitive control of action, other evidence suggests the claustrum may be involved in detecting salient changes in the external environment. The anterior cingulate cortex (ACC) and the anterior insular (aINS) are the two major participants in the salience network of human brain regions that activate in response to salient stimuli. While bidirectional connections between the ACC and the claustrum exist from mouse to non-human primate, the aINS connectivity with claustrum remains unclear, particularly in mouse. Here, we explored structural connections of the aINS with the claustrum and ACC through adeno-associated virus neuronal tract tracer injections into the ACC and aINS of the mouse. We detected sparse projections from the claustrum to the aINS and diffuse projections from the aINS to the borders of the claustrum were observed in some cases. In contrast, the insular cortex and endopiriform nucleus surrounding the claustrum had rich interconnectivity with aINS. Additionally, we observed a modest interconnectivity between ACC and the aINS. These data support the idea that claustrum neuron responses to salient stimuli may be driven by the ACC rather than the aINS.
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The development of reproducible folding in the gyrencephalic cerebral cortex is a topic of great interest to neuroscientists. In a recent paper in Cell, del Toro et al. (2017) show that changing the adhesive properties of neurons in the normally lissencephalic mouse cortex leads to the formation of stereotyped folding.
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Córtex Cerebral , Neurônios , Animais , Dobramento de ProteínaRESUMO
The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.
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Regulação Alostérica , Proteínas de Transporte/análise , Proteínas de Membrana/análise , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sinapses/química , Sinapses/fisiologia , Animais , Membrana Celular/química , Células Cultivadas , Cristalografia por Raios X , Camundongos , Microscopia de Força Atômica , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo ÂnguloRESUMO
Neuroligins are postsynaptic adhesion proteins involved in the establishment of functional synapses in the central nervous system. In rodents, four genes give rise to neuroligins that function at distinct synapses, with corresponding neurotransmitter and subtype specificities. In the present study, we examined the interactions between the different neuroligins by isolating endogenous oligomeric complexes using in situ cross-linking on primary neurons. Examining hippocampal, striatal, cerebellar and spinal cord cultures, we found that neuroligins form constitutive dimers, including homomers and, most notably, neuroligin 1/3 heteromers. Additionally, we found that neuroligin monomers are specifically retained in the secretory pathway through a cellular quality control mechanism that involves the neuroligin transmembrane domain, ensuring that dimerization occurs prior to cell surface trafficking. Lastly, we identified differences in the dimerization capacity of autism-associated neuroligin mutants, and found that neuroligin 3 R471C mutants can form heterodimers with neuroligin 1. The pervasive nature of neuroligin dimerization indicates that the unit of neuroligin function is the dimer, and raises intriguing possibilities of distinct heterodimer functions, and of interactions between native and mutant neuroligins contributing to disease phenotypes.
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Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Substituição de Aminoácidos , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Células COS , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Reagentes de Ligações Cruzadas/química , Dimerização , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
The postsynaptic adhesion protein neuroligin-2 (NL2) is selectively localized at inhibitory synapses. Here, we studied network activity in the dentate gyrus of NL2-deficient mice following perforant path (PP) stimulation in vivo. We found a strong increase in granule cell (GC) excitability. Furthermore, paired-pulse inhibition (PPI) of the population spike, a measure for γ-aminobutyric acid (GABA)ergic network inhibition, was severely impaired and associated with reduced GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents recorded from NL2-deficient GCs. In agreement with these functional data, the number of gephyrin and GABA(A)R clusters was significantly reduced in the absence of NL2, indicating a loss of synaptic GABA(A)Rs from the somata of GCs. Computer simulations of the dentate network showed that impairment of perisomatic inhibition is able to explain the electrophysiological changes observed in the dentate circuitry of NL2 knockout animals. Collectively, our data demonstrate for the first time that deletion of NL2 increases excitability of cortical neurons in the hippocampus of intact animals, most likely through impaired GABA(A)R clustering.
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Potenciais de Ação/fisiologia , Moléculas de Adesão Celular Neuronais/deficiência , Giro Denteado/fisiologia , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/genética , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Simulação por Computador , Giro Denteado/citologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/genética , Técnicas In Vitro , Inibição Psicológica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Neurológicos , Técnicas de Patch-Clamp/métodos , Receptores de GABA-A/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Estatísticas não Paramétricas , Tetrodotoxina/farmacologia , Valina/análogos & derivados , Valina/farmacologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Synapses between nerve cells in the mammalian brain are not only extremely numerous but also very diverse with respect to their structural and functional characteristics. This heterogeneity arises despite the fact that a set of common basic protein 'building blocks' is shared by many synapses. Among these, postsynaptic scaffolding proteins play a key role. They have the ability to assemble into membrane-tethered lattices and to adopt unique conformational states in different postsynaptic microenvironments, which may represent a key prerequisite of synapse heterogeneity. Analyses of such synaptic superstructures, rather than individual proteins and their interactions, are required to develop a mechanistic understanding of postsynaptic differentiation, synapse diversity, and dynamics.
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Neurogênese/fisiologia , Sinapses/fisiologia , Animais , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Sinapses/ultraestruturaRESUMO
In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.