RESUMO
Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA sequencing of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glial aging was particularly accelerated in white matter compared with cortical regions, whereas specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.
Assuntos
Envelhecimento , Disfunção Cognitiva , Substância Branca , Animais , Humanos , Camundongos , Disfunção Cognitiva/genética , Perfilação da Expressão Gênica , Núcleo Solitário , Substância Branca/patologia , Análise da Expressão Gênica de Célula Única , Encéfalo/patologiaRESUMO
The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.
Assuntos
Núcleo Dorsal da Rafe/anatomia & histologia , Núcleo Dorsal da Rafe/fisiologia , Serotonina/fisiologia , Adaptação Psicológica/fisiologia , Tonsila do Cerebelo/fisiologia , Animais , Ansiedade/fisiopatologia , Encéfalo/fisiologia , Núcleo Dorsal da Rafe/metabolismo , Feminino , Lobo Frontal/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Serotonina/metabolismoRESUMO
Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.
Assuntos
Transtorno Autístico , Síndrome do QT Longo , Oligonucleotídeos Antissenso , Sindactilia , Animais , Feminino , Humanos , Masculino , Camundongos , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/genética , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Movimento Celular/efeitos dos fármacos , Dendritos/metabolismo , Éxons/genética , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/genética , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Organoides/efeitos dos fármacos , Organoides/metabolismo , Prosencéfalo/metabolismo , Prosencéfalo/citologia , Sindactilia/tratamento farmacológico , Sindactilia/genética , Interneurônios/citologia , Interneurônios/efeitos dos fármacosRESUMO
Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.
Assuntos
Vias Neurais , Organoides , Animais , Animais Recém-Nascidos , Transtorno Autístico , Humanos , Síndrome do QT Longo , Motivação , Neurônios/fisiologia , Optogenética , Organoides/citologia , Organoides/inervação , Organoides/transplante , Ratos , Recompensa , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Células-Tronco/citologia , SindactiliaRESUMO
Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.
Assuntos
Transformação Celular Neoplásica/genética , Genes da Neurofibromatose 1 , Mutação , Neurofibromina 1/genética , Neurônios/metabolismo , Glioma do Nervo Óptico/genética , Glioma do Nervo Óptico/patologia , Animais , Astrocitoma/genética , Astrocitoma/patologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos da radiação , Nervo Óptico/citologia , Nervo Óptico/efeitos da radiação , Estimulação Luminosa , Retina/citologia , Retina/efeitos da radiaçãoRESUMO
For the human brain to operate, populations of neurons across anatomical structures must coordinate their activity within milliseconds. To date, our understanding of such interactions has remained limited. We recorded directly from the hippocampus (HPC), posteromedial cortex (PMC), ventromedial/orbital prefrontal cortex (OFC), and the anterior nuclei of the thalamus (ANT) during two experiments of autobiographical memory processing that are known from decades of neuroimaging work to coactivate these regions. In 31 patients implanted with intracranial electrodes, we found that the presentation of memory retrieval cues elicited a significant increase of low frequency (LF < 6 Hz) activity followed by cross-regional phase coherence of this LF activity before select populations of neurons within each of the four regions increased high-frequency (HF > 70 Hz) activity. The power of HF activity was modulated by memory content, and its onset followed a specific temporal order of ANTâHPC/PMCâOFC. Further, we probed cross-regional causal effective interactions with repeated electrical pulses and found that HPC stimulations cause the greatest increase in LF-phase coherence across all regions, whereas the stimulation of any region caused the greatest LF-phase coherence between that particular region and ANT. These observations support the role of the ANT in gating, and the HPC in synchronizing, the activity of cortical midline structures when humans retrieve self-relevant memories of their past. Our findings offer a fresh perspective, with high temporal fidelity, about the dynamic signaling and underlying causal connections among distant regions when the brain is actively involved in retrieving self-referential memories from the past.
Assuntos
Memória Episódica , Humanos , Masculino , Feminino , Adulto , Hipocampo/fisiologia , Córtex Pré-Frontal/fisiologia , Córtex Pré-Frontal/diagnóstico por imagem , Encéfalo/fisiologia , Encéfalo/diagnóstico por imagem , Rememoração Mental/fisiologia , Mapeamento Encefálico , Pessoa de Meia-Idade , Neurônios/fisiologia , Núcleos Anteriores do Tálamo/fisiologiaRESUMO
Hyperexcitability of brain circuits is a common feature of autism spectrum disorders (ASDs). Genetic deletion of a chromatin-binding protein, retinoic acid induced 1 (RAI1), causes Smith-Magenis syndrome (SMS). SMS is a syndromic ASD associated with intellectual disability, autistic features, maladaptive behaviors, overt seizures, and abnormal electroencephalogram (EEG) patterns. The molecular and neural mechanisms underlying abnormal brain activity in SMS remain unclear. Here we show that panneural Rai1 deletions in mice result in increased seizure susceptibility and prolonged hippocampal seizure duration in vivo and increased dentate gyrus population spikes ex vivo. Brain-wide mapping of neuronal activity pinpointed selective cell types within the limbic system, including the hippocampal dentate gyrus granule cells (dGCs) that are hyperactivated by chemoconvulsant administration or sensory experience in Rai1-deficient brains. Deletion of Rai1 from glutamatergic neurons, but not from gamma-aminobutyric acidergic (GABAergic) neurons, was responsible for increased seizure susceptibility. Deleting Rai1 from the Emx1Cre-lineage glutamatergic neurons resulted in abnormal dGC properties, including increased excitatory synaptic transmission and increased intrinsic excitability. Our work uncovers the mechanism of neuronal hyperexcitability in SMS by identifying Rai1 as a negative regulator of dGC intrinsic and synaptic excitability.
Assuntos
Síndrome de Smith-Magenis , Camundongos , Animais , Síndrome de Smith-Magenis/genética , Transativadores/genética , Transativadores/metabolismo , Fenótipo , Modelos Animais de Doenças , Cromatina , Hipocampo/metabolismo , Convulsões/genética , TretinoínaRESUMO
The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
Assuntos
Neurônios/citologia , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Esferoides Celulares/citologia , Transtorno Autístico/genética , Transtorno Autístico/patologia , Linhagem Celular , Movimento Celular , Células Cultivadas , Feminino , Neurônios GABAérgicos/citologia , Ácido Glutâmico/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/patologia , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Masculino , Modelos Biológicos , Neurogênese , Neurônios/patologia , Células-Tronco Pluripotentes/citologia , Prosencéfalo/anatomia & histologia , Sinapses/fisiologia , Sindactilia/genética , Sindactilia/patologiaRESUMO
CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Células CHO , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Hipocampo/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.
Assuntos
Organoides/crescimento & desenvolvimento , Engenharia Tecidual , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Prosencéfalo/fisiologia , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Análise de Célula Única/métodosRESUMO
The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/fisiologia , Células-Tronco Pluripotentes/citologia , Astrócitos/citologia , Células Cultivadas , Córtex Cerebral/citologia , Humanos , Esferoides Celulares , Sinapses/fisiologiaRESUMO
The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.
Assuntos
Editoração/normas , Projetos de Pesquisa/normas , Animais , Editoração/tendências , Distribuição Aleatória , Tamanho da Amostra , Estatística como AssuntoRESUMO
Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.
Assuntos
Ciclídeos/fisiologia , Sinapses Elétricas , Hormônio Liberador de Gonadotropina/metabolismo , Animais , Conexinas/metabolismo , Feminino , Junções Comunicantes , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Masculino , Ácido Meclofenâmico/química , Modelos Neurológicos , Neurônios/fisiologia , Hipófise/metabolismo , Transmissão Sináptica , TransgenesRESUMO
Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem's potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery.
Assuntos
Sistemas de Liberação de Medicamentos , Agonistas de Receptores de GABA-A/administração & dosagem , Inibição Neural/fisiologia , Piridinas/administração & dosagem , Receptores de GABA-A/fisiologia , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Sistemas de Liberação de Medicamentos/tendências , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/efeitos dos fármacos , Neocórtex/fisiologia , Inibição Neural/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia , ZolpidemRESUMO
Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.
Assuntos
Modelos Neurológicos , Inibição Neural/fisiologia , Neurônios/metabolismo , Córtex Pré-Frontal/fisiologia , Córtex Pré-Frontal/fisiopatologia , Comportamento Social , Animais , Transtorno Autístico/fisiopatologia , Modelos Animais de Doenças , Células HEK293 , Hipocampo/citologia , Humanos , Aprendizagem , Transtornos Mentais/fisiopatologia , Camundongos , Atividade Motora , Opsinas/metabolismo , Esquizofrenia/fisiopatologiaRESUMO
The modulation of gamma power (25-90 Hz) is associated with attention and has been observed across species and brain areas. However, mechanisms that control these modulations are poorly understood. The midbrain spatial attention network in birds generates high-amplitude gamma oscillations in the local field potential that are thought to represent the highest priority location for attention. Here we explore, in midbrain slices from chickens, mechanisms that regulate the power of these oscillations, using high-resolution techniques including intracellular recordings from neurons targeted by calcium imaging. The results identify a specific subtype of neuron, expressing non-α7 nicotinic acetylcholine receptors, that directly drives inhibition in the gamma-generating circuit and switches the network into a primed state capable of producing high-amplitude oscillations. The special properties of this mechanism enable rapid, persistent changes in gamma power. The brain may employ this mechanism wherever rapid modulations of gamma power are critical to information processing.
Assuntos
Atenção , Neurônios Colinérgicos/fisiologia , Ritmo Gama , Mesencéfalo/fisiologia , Animais , Células Cultivadas , Galinhas , Neurônios Colinérgicos/metabolismo , Feminino , Masculino , Mesencéfalo/citologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
The thalamic reticular nucleus (nRt), composed of GABAergic cells providing inhibition of relay neurons in the dorsal thalamus, receives excitation from the neocortex and thalamus. The two excitatory pathways promoting feedback or feedforward inhibition of thalamocortical neurons contribute to sensory processing and rhythm generation. While synaptic inhibition within the nRt has been carefully characterized, little is known regarding the biophysics of synaptic excitation. To characterize the functional properties of thalamocortical and corticothalamic connections to the nRt, we recorded minimal electrically evoked excitatory postsynaptic currents from nRt cells in vitro. A hierarchical clustering algorithm distinguished two types of events. Type 1 events had larger amplitudes and faster kinetics, largely mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, whereas type 2 responses had more prominent N-methyl-d-aspartate (NMDA) receptor contribution. Type 1 responses showed subnormal axonal propagation and paired pulse depression, consistent with thalamocortical inputs. Furthermore, responses kinetically similar to type 1 events were evoked by glutamate-mediated activation of thalamic neurons. Type 2 responses, in contrast, likely arise from corticothalamic inputs, with larger NMDA conductance and weak Mg(2+)-dependent block, suggesting that NMDA receptors are critical for the cortical excitation of reticular neurons. The long-lasting action of NMDA receptors would promote reticular cell burst firing and produce powerful inhibitory output to relay neurons proposed to be important in triggering epilepsy. This work provides the first complete voltage-clamp analysis of the kinetics and voltage dependence of AMPA and NMDA responses of thalamocortical and corticothalamic synapses in the nRt and will be critical in optimizing biologically realistic neural network models of thalamocortical circuits relevant to sensory processing and thalamocortical oscillations.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/fisiologia , Sinapses/classificação , Sinapses/fisiologia , Núcleos Talâmicos/citologia , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Análise por Conglomerados , Estimulação Elétrica , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.
Assuntos
Núcleos Intralaminares do Tálamo/metabolismo , Receptores de GABA-A/metabolismo , Animais , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Potenciais Pós-Sinápticos Inibidores , Núcleos Intralaminares do Tálamo/citologia , Núcleos Intralaminares do Tálamo/crescimento & desenvolvimento , Núcleos Intralaminares do Tálamo/fisiologia , Camundongos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genéticaRESUMO
The chromatin-remodeling protein Satb2 plays a role in the generation of distinct subtypes of neocortical pyramidal neurons. Previous studies have shown that Satb2 is required for normal development of callosal projection neurons (CPNs), which fail to extend axons callosally in the absence of Satb2 and instead project subcortically. Here we conditionally delete Satb2 from the developing neocortex and find that neurons in the upper layers adopt some electrophysiological properties characteristic of deep layer neurons, but projections from the superficial layers do not contribute to the aberrant subcortical projections seen in Satb2 mutants. Instead, axons from deep layer CPNs descend subcortically in the absence of Satb2. These data demonstrate distinct developmental roles of Satb2 in regulating the fates of upper and deep layer neurons. Unexpectedly, Satb2 mutant brains also display changes in gene expression by subcerebral projection neurons (SCPNs), accompanied by a failure of corticospinal tract (CST) formation. Altering the timing of Satb2 ablation reveals that SCPNs require an early expression of Satb2 for differentiation and extension of the CST, suggesting that early transient expression of Satb2 in these cells plays an essential role in development. Collectively these data show that Satb2 is required by both CPNs and SCPNs for proper differentiation and axon pathfinding.
Assuntos
Axônios/fisiologia , Diferenciação Celular , Córtex Cerebral/embriologia , Corpo Caloso/embriologia , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Neurônios/fisiologia , Fatores de Transcrição/fisiologia , Animais , Axônios/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Corpo Caloso/metabolismo , Feminino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos Transgênicos , Vias Neurais/embriologia , Vias Neurais/metabolismo , Neurônios/metabolismo , Córtex Somatossensorial/embriologia , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition.