RESUMO
Complex retinal circuits process visual information and deliver it to the brain. Few molecular determinants of synaptic specificity in this system are known. Using genetic and optogenetic methods, we identified two types of bipolar interneurons that convey visual input from photoreceptors to a circuit that computes the direction in which objects are moving. We then sought recognition molecules that promote selective connections of these cells with previously characterized components of the circuit. We found that the type II cadherins, cdh8 and cdh9, are each expressed selectively by one of the two bipolar cell types. Using loss- and gain-of-function methods, we showed that they are critical determinants of connectivity in this circuit and that perturbation of their expression leads to distinct defects in visually evoked responses. Our results reveal cellular components of a retinal circuit and demonstrate roles of type II cadherins in synaptic choice and circuit function.
Assuntos
Caderinas/metabolismo , Retina/fisiologia , Células Bipolares da Retina/metabolismo , Vias Visuais , Animais , Axônios/metabolismo , Caderinas/genética , Técnicas de Introdução de Genes , Camundongos , Retina/citologia , SinapsesRESUMO
Recent advances in microscopy, genetics, physiology, and data processing have expanded the scope and accelerated the pace of discovery in visual neuroscience. However, the pace of discovery and the ever increasing number of published articles can present a serious issue for both trainees and senior scientists alike: with each passing year the fog of progress thickens, making it easy to lose sight of important earlier advances. As part of this special issue of the Journal of Neuroscience commemorating the 50th anniversary of SfN, here, we provide a variation on Stephen Kuffler's Oldies but Goodies classic reading list, with the hope that by looking back at highlights in the field of visual neuroscience we can better define remaining gaps in our knowledge and thus guide future work. We also hope that this article can serve as a resource that will aid those new to the field to find their bearings.
Assuntos
Neurociências/história , Percepção Visual/fisiologia , Potenciais de Ação , Animais , Mapeamento Encefálico , Córtex Cerebral/fisiologia , Conectoma , Percepção de Forma/fisiologia , Corpos Geniculados/fisiologia , História do Século XX , História do Século XXI , Humanos , Modelos Neurológicos , Percepção de Movimento/fisiologia , Retina/citologia , Retina/fisiologia , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/fisiologia , Percepção Espacial/fisiologia , Córtex Visual/fisiologia , Vias Visuais/fisiologiaRESUMO
In the mammalian retina, processes of approximately 70 types of interneurons form specific synapses on roughly 30 types of retinal ganglion cells (RGCs) in a neuropil called the inner plexiform layer. Each RGC type extracts salient features from visual input, which are sent deeper into the brain for further processing. The specificity and stereotypy of synapses formed in the inner plexiform layer account for the feature-detecting ability of RGCs. Here we analyse the development and function of synapses on one mouse RGC type, called the W3B-RGC. These cells have the remarkable property of responding when the timing of the movement of a small object differs from that of the background, but not when they coincide. Such cells, known as local edge detectors or object motion sensors, can distinguish moving objects from a visual scene that is also moving. We show that W3B-RGCs receive strong and selective input from an unusual excitatory amacrine cell type known as VG3-AC (vesicular glutamate transporter 3). Both W3B-RGCs and VG3-ACs express the immunoglobulin superfamily recognition molecule sidekick 2 (Sdk2), and both loss- and gain-of-function studies indicate that Sdk2-dependent homophilic interactions are necessary for the selectivity of the connection. The Sdk2-specified synapse is essential for visual responses of W3B-RGCs: whereas bipolar cells relay visual input directly to most RGCs, the W3B-RGCs receive much of their input indirectly, via the VG3-ACs. This non-canonical circuit introduces a delay into the pathway from photoreceptors in the centre of the receptive field to W3B-RGCs, which could improve their ability to judge the synchrony of local and global motion.
Assuntos
Imunoglobulina G/metabolismo , Proteínas de Membrana/metabolismo , Percepção de Movimento/fisiologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Vias Visuais/fisiologia , Células Amácrinas/citologia , Células Amácrinas/fisiologia , Animais , Feminino , Imunoglobulina G/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Movimento (Física) , Mutação , Sinapses/genética , Sinapses/metabolismoRESUMO
Axons develop in a series of steps, beginning with specification, outgrowth, and arborization, and terminating with formation and maturation of presynaptic specializations. We found previously that the SAD-A and SAD-B kinases are required for axon specification and arborization in subsets of mouse neurons. Here, we show that following these steps, SAD kinases become localized to synaptic sites and are required within presynaptic cells for structural and functional maturation of synapses in both peripheral and central nervous systems. Deleting SADs from sensory neurons can perturb either axonal arborization or nerve terminal maturation, depending on the stage of deletion. Thus, a single pair of kinases plays multiple, sequential roles in axonal differentiation.
Assuntos
Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Terminações Nervosas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Diferenciação Celular , Eletrofisiologia , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Sinapses/fisiologiaRESUMO
Characterizing neurons by their electrophysiological phenotypes is essential for understanding the neural basis of behavioral and cognitive functions. Technological developments have enabled the collection of hundreds of neural recordings; this calls for new tools capable of performing feature extraction efficiently. To address the urgent need for a powerful and accessible tool, we developed ElecFeX, an open-source MATLAB-based toolbox that (1) has an intuitive graphical user interface, (2) provides customizable measurements for a wide range of electrophysiological features, (3) processes large-size datasets effortlessly via batch analysis, and (4) yields formatted output for further analysis. We implemented ElecFeX on a diverse set of neural recordings; demonstrated its functionality, versatility, and efficiency in capturing electrical features; and established its significance in distinguishing neuronal subgroups across brain regions and species. ElecFeX is thus presented as a user-friendly toolbox to benefit the neuroscience community by minimizing the time required for extracting features from their electrophysiological datasets.
Assuntos
Fenômenos Eletrofisiológicos , Análise de Célula Única , Software , Fenômenos Eletrofisiológicos/fisiologia , Animais , Análise de Célula Única/métodos , Neurônios/fisiologia , Humanos , Encéfalo/fisiologia , Camundongos , RatosRESUMO
Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.
Assuntos
Astrócitos , Gliose , Animais , Gliose/patologia , Gliose/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Camundongos , Cromatina/metabolismo , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Montagem e Desmontagem da Cromatina , Microglia/metabolismo , Microglia/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos Endogâmicos C57BL , Lipopolissacarídeos/farmacologia , Humanos , Camundongos Knockout , Masculino , Proliferação de CélulasRESUMO
Visual attention allows the brain to evoke behaviors based on the most important visual features. Mouse models offer immense potential to gain a circuit-level understanding of this phenomenon, yet how mice distribute attention across features and locations is not well understood. Here, we describe a new approach to address this limitation by training mice to detect weak vertical bars in a background of dynamic noise while spatial cues manipulate their attention. By adapting a reverse-correlation method from human studies, we linked behavioral decisions to stimulus features and locations. We show that mice deployed attention to a small rostral region of the visual field. Within this region, mice attended to multiple features (orientation, spatial frequency, contrast) that indicated the presence of weak vertical bars. This attentional tuning grew with training, multiplicatively scaled behavioral sensitivity, approached that of an ideal observer, and resembled the effects of attention in humans. Taken together, we demonstrate that mice can simultaneously attend to multiple features and locations of a visual stimulus.
Assuntos
Encéfalo , Sinais (Psicologia) , Humanos , Animais , Camundongos , Modelos Animais de Doenças , Campos VisuaisRESUMO
An intriguing feature of several nicotinic acetylcholine receptors (nAChRs) on neurons is that their subunits contain a highly conserved cysteine residue located near the intracellular mouth of the receptor pore. The work summarized in this review indicates that α3ß4-containing and α4ß2-containing neuronal nAChRs, and possibly other subtypes, are inactivated by elevations in intracellular reactive oxygen species (ROS). This review discusses a model for the molecular mechanisms that underlie this inactivation. In addition, we explore the implications of this mechanism in the context of complications that arise from diabetes. We review the evidence that diabetes elevates cytosolic ROS in sympathetic neurons and inactivates postsynaptic α3ß4-containing nAChRs shortly after the onset of diabetes, leading to a depression of synaptic transmission in sympathetic ganglia, an impairment of sympathetic reflexes. These effects of ROS on nAChR function are due to the highly conserved Cys residues in the receptors: replacing the cysteine residues in α3 allow ganglionic transmission and sympathetic reflexes to function normally in diabetes. This example from diabetes suggests that other diseases involving oxidative stress, such as Parkinson's disease, could lead to the inactivation of nAChRs on neurons and disrupt cholinergic nicotinic signalling.
Assuntos
Cisteína/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Nicotínicos/metabolismo , Diabetes Mellitus/metabolismo , Gânglios Simpáticos/metabolismo , Humanos , Transmissão SinápticaRESUMO
Nearly 50 different mouse retinal ganglion cell (RGC) types sample the visual scene for distinct features. RGC feature selectivity arises from their synapses with a specific subset of amacrine (AC) and bipolar cell (BC) types, but how RGC dendrites arborize and collect input from these specific subsets remains poorly understood. Here we examine the hypothesis that RGCs employ molecular recognition systems to meet this challenge. By combining calcium imaging and type-specific histological stains, we define a family of circuits that express the recognition molecule Sidekick-1 (Sdk1), which include a novel RGC type (S1-RGC) that responds to local edges. Genetic and physiological studies revealed that Sdk1 loss selectively disrupts S1-RGC visual responses, which result from a loss of excitatory and inhibitory inputs and selective dendritic deficits on this neuron. We conclude that Sdk1 shapes dendrite growth and wiring to help S1-RGCs become feature selective.
Assuntos
Sinalização do Cálcio , Dendritos/metabolismo , Imunoglobulina G/metabolismo , Proteínas de Membrana/metabolismo , Plasticidade Neuronal , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Visão Ocular , Percepção Visual , Animais , Fator II de Transcrição COUP/genética , Fator II de Transcrição COUP/metabolismo , Potenciais Pós-Sinápticos Excitadores , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunoglobulina G/genética , Potenciais Pós-Sinápticos Inibidores , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Inibição Neural , Estimulação Luminosa , Sinapses/genética , Fatores de Tempo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismo , Vias Visuais/metabolismoRESUMO
During development, neurons navigate a tangled thicket of thousands of axons and dendrites to synapse with just a few specific targets. This phenomenon termed wiring specificity, is critical to the assembly of neural circuits and the way neurons manage this feat is only now becoming clear. Recent studies in the mouse retina are shedding new insight into this process. They show that specific wiring arises through a series of stages that include: directed axonal and dendritic growth, the formation of neuropil layers, positioning of such layers, and matching of co-laminar synaptic partners. Each stage appears to be directed by a distinct family of recognition molecules, suggesting that the combinatorial expression of such family members might act as a blueprint for retinal connectivity. By reviewing the evidence in support of each stage, and by considering their underlying molecular mechanisms, we attempt to synthesize these results into a wiring model which generates testable predictions for future studies. Finally, we conclude by highlighting new optical methods that could be used to address such predictions and gain further insight into this fundamental process.
Assuntos
Vias Neurais/citologia , Optogenética , Neurônios Retinianos/citologia , Sinapses , Orientação de Axônios/fisiologia , Humanos , Vias Neurais/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Retina/citologia , Retina/fisiologia , Neurônios Retinianos/fisiologiaRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels implicated in a variety of cognitive, motor, and sensory behaviours, are targeted to compartments rich in mitochondria, particularly postsynaptic domains and presynaptic terminals, exposing these receptors to reactive oxygen species (ROS) generated by oxidative phosphorylation. In addition, these receptors can become exposed to ROS during the progression of certain neurodegenerative diseases. Because ROS are known to modify several membrane proteins, including some types of ion channels, it raises the question of whether elevations in cytosolic ROS alter the function of nAChRs. To address this, we elevated ROS in cultured sympathetic neurons, directly by perfusing neurons intracellularly with ROS, indirectly by blocking the mitochondrial electron transport chain, or noninvasively by transient NGF removal; we then simultaneously measured changes in cytosolic ROS levels and whole-cell ACh-evoked currents. In addition, we elevated cytosolic ROS in postganglionic neurons in intact ganglia and measured changes in nerve-evoked EPSPs. Our experiments indicate that mild elevations in cytosolic ROS, including that produced by transient interruption of NGF signaling, induce a use-dependent, long-lasting rundown of ACh-evoked currents on cultured sympathetic neurons and a long-lasting depression of fast nerve-evoked EPSPs. We show that these effects of cytosolic ROS are specific to nAChRs on neurons and do not cause rundown of ACh-evoked currents on muscle. Our results demonstrate that elevations in cytosolic ROS inactivate neuronal nAChRs in a use-dependent manner and suggest that mild oxidative stress impairs mechanisms mediated by cholinergic nicotinic signaling at neuronal-neuronal synapses.
Assuntos
Mitocôndrias/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Potenciais Evocados , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismoRESUMO
We report here the construction of a novel knock-in mouse expressing chimeric alpha3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to alpha-bungarotoxin (alphaBTX). Sensitivity was generated by substituting five amino acids in the loop C (beta9-beta10) region of the mouse alpha3 subunit with the corresponding residues from the alpha1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed alpha3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic alpha3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated alphaBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the alphaBTX-labeled puncta colocalized with a marker for synaptic varicosities. Following in vivo deafferentation, these puncta persisted but with significant changes in intensity and distribution that varied with the length of the recovery period. Compound action potentials and excitatory postsynaptic potentials recorded from SCG of mice homozygous for alpha3[5] were abolished by 100 nmalphaBTX, even in an alpha7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the alpha3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of alpha3[5]-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As alphaBTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to alphaBTX, the findings described here should be applicable to many other receptors.
Assuntos
Bungarotoxinas/farmacologia , Camundongos Knockout , Neurônios/efeitos dos fármacos , Receptores Nicotínicos/deficiência , Gânglio Cervical Superior/citologia , Acetilcolina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Denervação Autônoma/métodos , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Bungarotoxinas/metabolismo , Células Cultivadas , Colinérgicos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neurofilamentos/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Fenótipo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Fatores de Tempo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Distinct neuronal types connect in complex ways to generate functional neural circuits. The molecular diversity required to specify this connectivity could be supplied by multigene families of synaptic recognition molecules, but most studies to date have assessed just one or a few members at a time. Here, we analyze roles of cadherins (Cdhs) in formation of retinal circuits comprising eight neuronal types that inform the brain about motion in four directions. We show that at least 15 classical Cdhs are expressed by neurons in these circuits and at least 6 (Cdh6-10 and 18) act individually or in combinations to promote specific connectivity among the cells. They act in part by directing the processes of output neurons and excitatory interneurons to a cellular scaffold formed by inhibitory interneurons. Because Cdhs are expressed combinatorially by many central neurons, similar interactions could be involved in patterning circuits throughout the brain.
Assuntos
Caderinas/metabolismo , Dendritos/fisiologia , Interneurônios/fisiologia , Neurônios Retinianos/fisiologia , Sinapses/fisiologia , Animais , Camundongos , Retina/fisiologia , Células Ganglionares da Retina/fisiologiaRESUMO
Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1+ RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Dendritos/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Caderinas/fisiologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteínas de Ligação a DNA/genética , Eletroporação , Feminino , Perfilação da Expressão Gênica , Interneurônios/fisiologia , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Neuritos/fisiologia , Gravidez , Receptores de Superfície Celular/fisiologia , Proteínas com Domínio TRESUMO
The size and shape of dendritic arbors are prime determinants of neuronal connectivity and function. We asked how ON-OFF direction-selective ganglion cells (ooDSGCs) in mouse retina acquire their bistratified dendrites, in which responses to light onset and light offset are segregated to distinct strata. We found that the transcriptional regulator Satb1 is selectively expressed by ooDSGCs. In Satb1 mutant mice, ooDSGC dendrites lack ON arbors, and the cells selectively lose ON responses. Satb1 regulates expression of a homophilic adhesion molecule, Contactin 5 (Cntn5). Both Cntn5 and its co-receptor Caspr4 are expressed not only by ooDSGCs, but also by interneurons that form a scaffold on which ooDSGC ON dendrites fasciculate. Removing Cntn5 from either ooDSGCs or interneurons partially phenocopies Satb1 mutants, demonstrating that Satb1-dependent Cntn5 expression in ooDSGCs leads to branch-specific homophilic interactions with interneurons. Thus, Satb1 directs formation of a morphologically and functionally specialized compartment within a complex dendritic arbor.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Contactinas/metabolismo , Dendritos/metabolismo , Retina/citologia , Células Ganglionares da Retina/citologia , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
In vertebrates, synaptic activity exerts an important influence on the formation of neural circuits, yet our understanding of its role in directing presynaptic and postsynaptic differentiation during synaptogenesis is incomplete. This study investigates how activity influences synaptic differentiation as synapses mature during early postnatal life. Specifically, we ask what happens to presynaptic terminals when synapses develop without functional postsynaptic receptors and without fast synaptic transmission. To address this issue, we investigated cholinergic nicotinic synapses in sympathetic ganglia of mice with a null mutation for the alpha3 nicotinic ACh receptor gene. Disrupting the alpha3 gene completely eliminates fast excitatory synaptic potentials on postganglionic sympathetic neurons, establishing a crucial role for alpha3-containing postsynaptic receptors in synaptic transmission. Interestingly, the preganglionic nerve terminals form morphologically normal synapses with sympathetic neurons, and these synapses persist without activity in postnatal animals. Surprisingly, when stimulating the preganglionic nerve at physiological rates, we discovered a significant decrease in ACh output from the presynaptic terminals in these alpha3(-/-) sympathetic ganglia. We show that this decrease in ACh output from the presynaptic terminals results, in part, from a lack of functional high-affinity choline transporters. We conclude the following: (1) fast synaptic transmission in mammalian SCG requires alpha3 expression; (2) in the absence of activity, the preganglionic nerve forms synapses that appear morphologically normal and persist for several weeks; and (3) to sustain transmitter release, developing presynaptic terminals require an activity-dependent retrograde signal.
Assuntos
Axônios/fisiologia , Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/fisiologia , Animais , Modelos Animais de Doenças , Genótipo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores Nicotínicos/genética , Degeneração RetrógradaRESUMO
Mutations in the gene encoding the immunoglobulin-superfamily member cell adhesion molecule contactin1 (CNTN1) cause lethal congenital myopathy in human patients and neurodevelopmental phenotypes in knockout mice. Whether the mutant mice provide an accurate model of the human disease is unclear; resolving this will require additional functional tests of the neuromuscular system and examination of Cntn1 mutations on different genetic backgrounds that may influence the phenotype. Toward these ends, we have analyzed a new, spontaneous mutation in the mouse Cntn1 gene that arose in a BALB/c genetic background. The overt phenotype is very similar to the knockout of Cntn1, with affected animals having reduced body weight, a failure to thrive, locomotor abnormalities, and a lifespan of 2-3 weeks. Mice homozygous for the new allele have CNTN1 protein undetectable by western blotting, suggesting that it is a null or very severe hypomorph. In an analysis of neuromuscular function, neuromuscular junctions had normal morphology, consistent with previous studies in knockout mice, and the muscles were able to generate appropriate force when normalized for their reduced size in late stage animals. Therefore, the Cntn1 mutant mice do not show evidence for a myopathy, but instead the phenotype is likely to be caused by dysfunction in the nervous system. Given the similarity of CNTN1 to other Ig-superfamily proteins such as DSCAMs, we also characterized the expression and localization of Cntn1 in the retinas of mutant mice for developmental defects. Despite widespread expression, no anomalies in retinal anatomy were detected histologically or using a battery of cell-type specific antibodies. We therefore conclude that the phenotype of the Cntn1 mice arises from dysfunction in the brain, spinal cord or peripheral nervous system, and is similar in either a BALB/c or B6;129;Black Swiss background, raising a possible discordance between the mouse and human phenotypes resulting from Cntn1 mutations.
Assuntos
Contactina 1/genética , Mutação/genética , Animais , Cromossomos de Mamíferos/genética , Contactina 1/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Estudos de Associação Genética , Padrões de Herança/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Contração Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/citologia , Junção Neuromuscular/metabolismo , Fenótipo , Transporte Proteico , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismoRESUMO
Most people with diabetes develop severe complications of the autonomic nervous system; yet, the underlying causes of many diabetic-induced dysautonomias are poorly understood. Here we explore the idea that these dysautonomias results, in part, from a defect in synaptic transmission. To test this idea, we investigated cultured sympathetic neurons and show that hyperglycemia inactivates nAChRs through a mechanism involving an elevation in reactive oxygen species and an interaction with highly conserved cysteine residues located near the intracellular mouth of the nAChR channel. Consistent with this, we show that diabetic mice have depressed ganglionic transmission and reduced sympathetic reflexes, whereas diabetic mice expressing mutant postsynaptic nAChRs that lack the conserved cysteine residues on the alpha3 subunit have normal synaptic transmission in sympathetic ganglia and normal sympathetic reflexes. Our work suggests a new model for diabetic-induced dysautonomias and identifies ganglionic nAChRs as targets of hyperglycemia-induced downstream signals.
Assuntos
Cisteína/metabolismo , Diabetes Mellitus Experimental/patologia , Receptores Nicotínicos/metabolismo , Células Receptoras Sensoriais/fisiologia , Gânglio Cervical Superior/patologia , Transmissão Sináptica/fisiologia , Acetilcolina/farmacologia , Adenoviridae , Fatores Etários , Aldeídos/metabolismo , Animais , Animais Recém-Nascidos , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Células Cultivadas , Cisteína/genética , Diabetes Mellitus Experimental/induzido quimicamente , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Glucose/farmacologia , Guanidinas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Leptina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo , Receptores para Leptina/genética , Receptores Nicotínicos/deficiência , Transmissão Sináptica/efeitos dos fármacosRESUMO
A well-accepted view of developing circuits is that synapses must be active to mature and persist, whereas inactive synapses remain immature and are eventually eliminated. We question this long-standing view by investigating nonfunctional cholinergic nicotinic synapses in the superior cervical ganglia (SCG) of mice with a disruption in the alpha3 nicotinic receptor (nAChR) subunit gene, a gene essential for fast synaptic transmission in sympathetic ganglia. Using imaging and electrophysiology, we show that synapses persist for at least 2-3 months without postsynaptic activity; however, the presynaptic terminals lack high-affinity choline transporters (CHTs), and as a result, they are quickly depleted of transmitter. Moreover, we demonstrate with rescue experiments that CHT is induced by signals downstream of postsynaptic activity, converting immature terminals to mature terminals capable of sustaining transmitter release in response to high-frequency or continuous firing. Importantly, postsynaptic neurons must be continually active to maintain CHT in presynaptic terminals.