RESUMO
Parvalbumin (PV)-positive interneurons modulate cortical activity through highly specialized connectivity patterns onto excitatory pyramidal neurons (PNs) and other inhibitory cells. PV cells are autoconnected through powerful autapses, but the contribution of this form of fast disinhibition to cortical function is unknown. We found that autaptic transmission represents the most powerful inhibitory input of PV cells in neocortical layer V. Autaptic strength was greater than synaptic strength onto PNs as a result of a larger quantal size, whereas autaptic and heterosynaptic PV-PV synapses differed in the number of release sites. Overall, single-axon autaptic transmission contributed to approximately 40% of the global inhibition (mostly perisomatic) that PV interneurons received. The strength of autaptic transmission modulated the coupling of PV-cell firing with optogenetically induced γ-oscillations, preventing high-frequency bursts of spikes. Autaptic self-inhibition represents an exceptionally large and fast disinhibitory mechanism, favoring synchronization of PV-cell firing during cognitive-relevant cortical network activity.
Assuntos
Interneurônios/fisiologia , Neocórtex/fisiologia , Sinapses , Transmissão Sináptica , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BLRESUMO
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 thalamus is the primary gateway that relays sensory information to the cerebral cortex. While a single recipient cortical cell receives the convergence of many principal relay cells of the thalamus, each thalamic cell in turn integrates a dense and distributed synaptic feedback from the cortex. During sensory processing, the influence of this functional loop remains largely ignored. Using dynamic-clamp techniques in thalamic slices in vitro, we combined theoretical and experimental approaches to implement a realistic hybrid retino-thalamo-cortical pathway mixing biological cells and simulated circuits. The synaptic bombardment of cortical origin was mimicked through the injection of a stochastic mixture of excitatory and inhibitory conductances, resulting in a gradable correlation level of afferent activity shared by thalamic cells. The study of the impact of the simulated cortical input on the global retinocortical signal transfer efficiency revealed a novel control mechanism resulting from the collective resonance of all thalamic relay neurons. We show here that the transfer efficiency of sensory input transmission depends on three key features: i) the number of thalamocortical cells involved in the many-to-one convergence from thalamus to cortex, ii) the statistics of the corticothalamic synaptic bombardment and iii) the level of correlation imposed between converging thalamic relay cells. In particular, our results demonstrate counterintuitively that the retinocortical signal transfer efficiency increases when the level of correlation across thalamic cells decreases. This suggests that the transfer efficiency of relay cells could be selectively amplified when they become simultaneously desynchronized by the cortical feedback. When applied to the intact brain, this network regulation mechanism could direct an attentional focus to specific thalamic subassemblies and select the appropriate input lines to the cortex according to the descending influence of cortically-defined "priors".
Assuntos
Córtex Cerebral/fisiologia , Processos Estocásticos , Tálamo/fisiologia , Potenciais de Ação , Humanos , Sinapses/fisiologiaRESUMO
The thalamic output during different behavioral states is strictly controlled by the firing modes of thalamocortical neurons. During sleep, their hyperpolarized membrane potential allows activation of the T-type calcium channels, promoting rhythmic high-frequency burst firing that reduces sensory information transfer. In contrast, in the waking state thalamic neurons mostly exhibit action potentials at low frequency (i.e., tonic firing), enabling the reliable transfer of incoming sensory inputs to cortex. Because of their nearly complete inactivation at the depolarized potentials that are experienced during the wake state, T-channels are not believed to modulate tonic action potential discharges. Here, we demonstrate using mice brain slices that activation of T-channels in thalamocortical neurons maintained in the depolarized/wake-like state is critical for the reliable expression of tonic firing, securing their excitability over changes in membrane potential that occur in the depolarized state. Our results establish a novel mechanism for the integration of sensory information by thalamocortical neurons and point to an unexpected role for T-channels in the early stage of information processing.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo T/fisiologia , Neocórtex/fisiologia , Neurônios/fisiologia , Tálamo/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Neocórtex/citologia , Tálamo/citologia , Vigília/fisiologiaRESUMO
Optimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.
Assuntos
Mesencéfalo , Neurônios , Animais , Mamíferos , Camundongos , Neurônios/fisiologiaRESUMO
Absence epilepsy, characterized by spike-wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses-consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures.
Assuntos
Epilepsia Tipo Ausência/genética , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Animais , Eletroencefalografia , Epilepsia Tipo Ausência/fisiopatologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Dados de Sequência Molecular , Sinapses/fisiologia , Tálamo/fisiologiaRESUMO
In the neocortex, synaptic inhibition shapes all forms of spontaneous and sensory evoked activity. Importantly, inhibitory transmission is highly plastic, but the functional role of inhibitory synaptic plasticity is unknown. In the mouse barrel cortex, activation of layer (L) 2/3 pyramidal neurons (PNs) elicits strong feedforward inhibition (FFI) onto L5 PNs. We find that FFI involving parvalbumin (PV)-expressing cells is strongly potentiated by postsynaptic PN burst firing. FFI plasticity modifies the PN excitation-to-inhibition (E/I) ratio, strongly modulates PN gain, and alters information transfer across cortical layers. Moreover, our LTPi-inducing protocol modifies firing of L5 PNs and alters the temporal association of PN spikes to γ-oscillations both in vitro and in vivo. All of these effects are captured by unbalancing the E/I ratio in a feedforward inhibition circuit model. Altogether, our results indicate that activity-dependent modulation of perisomatic inhibitory strength effectively influences the participation of single principal cortical neurons to cognition-relevant network activity.
Assuntos
Neocórtex/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Feminino , Ritmo Gama/efeitos da radiação , Luz , Potenciação de Longa Duração/fisiologia , Potenciação de Longa Duração/efeitos da radiação , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Inibição Neural/efeitos da radiação , Plasticidade Neuronal/efeitos da radiação , Células Piramidais/fisiologia , Células Piramidais/efeitos da radiação , Sinapses/efeitos da radiação , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.
Assuntos
Opsinas/genética , Optogenética , Animais , Animais Geneticamente Modificados , Calibragem , Cloretos/metabolismo , Reação de Fuga , Neurônios Motores/fisiologia , Bombas de Próton/fisiologia , Rodopsina/fisiologia , Gânglio Trigeminal/embriologia , Peixe-Zebra/embriologiaRESUMO
In the neocortex, critical periods (CPs) of plasticity are closed following the accumulation of perineuronal nets (PNNs) around parvalbumin (PV)-positive inhibitory interneurons. However, how PNNs tune cortical function and plasticity is unknown. We found that PNNs modulated the gain of visual responses and γ-oscillations in the adult mouse visual cortex in vivo, consistent with increased interneuron function. Removal of PNNs in adult V1 did not affect GABAergic neurotransmission from PV cells, nor neuronal excitability in layer 4. Importantly, PNN degradation coupled to sensory input potentiated glutamatergic thalamic synapses selectively onto PV cells. In the absence of PNNs, increased thalamic PV-cell recruitment modulated feed-forward inhibition differently on PV cells and pyramidal neurons. These effects depended on visual input, as they were strongly attenuated by monocular deprivation in PNN-depleted adult mice. Thus, PNNs control visual processing and plasticity by selectively setting the strength of thalamic recruitment of PV cells.
Assuntos
Moléculas de Adesão Celular/metabolismo , Matriz Extracelular/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Proteoglicanas/metabolismo , Vias Visuais/fisiologia , Animais , Camundongos , Tálamo/fisiologia , Córtex Visual/fisiologiaRESUMO
DEP domain-containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid-sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit - brain somatic and germline - mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.
Assuntos
Epilepsias Parciais , Proteínas Ativadoras de GTPase , Mutação em Linhagem Germinativa , Malformações do Desenvolvimento Cortical , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Repressoras , Animais , Sistemas CRISPR-Cas , Dendritos/metabolismo , Dendritos/patologia , Epilepsias Parciais/genética , Epilepsias Parciais/metabolismo , Epilepsias Parciais/patologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Masculino , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Neurônios/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Coluna Vertebral/metabolismo , Coluna Vertebral/patologiaRESUMO
GABAergic neurons of the thalamic reticular nucleus (nRt) provide thalamocortical relay neurons with feedback inhibition that influences sensory processing and thalamocortical rhythm generation. Mutual interactions between reticular neurons coordinate oscillatory activities developed within the network during normal sleep and in absence epilepsy, but the chemical versus electrical nature of these connections and their functional influence remain controversial. Here, we investigated the incidence and spatial extent of intra-nRt connectivity in vitro in horizontal and coronal thalamic slices from rat. Laser scanning photostimulation activated presynaptic nRt cells during patch-clamp recordings of postsynaptic neurons. Photolysis of caged glutamate evoked GABAergic IPSCs and/or depolarizing events (spikelets, mediated via electrical coupling) in a large proportion of neurons, thus indicating connectivity with presynaptic cell(s). Synaptic inputs were organized along the major axis of the nucleus in the same orientation as, but commonly exceeding the extent of, dendritic arborization of the postsynaptic neuron. In the anteroposterior (horizontal) plane, chemical connectivity had higher incidence (60% of recorded neurons vs 40% in vertical plane) and longer spatial extent, whereas in the dorsoventral (vertical) plane, electrical coupling dominated (47% incidence vs 37% in horizontal plane) and was more widely distributed. These data demonstrate that both electrical and chemical synapses are prominent within nRt and suggest different roles for the two types of connections. We thus propose that, along the vertical plane, electrical connectivity will promote coordinated rhythmic activity of sleep and/or thalamocortical epilepsy, whereas along the horizontal plane, chemical connectivity will oppose widespread thalamocortical synchronization and modulate sensory throughput.
Assuntos
Mapeamento Encefálico , Sincronização Cortical , Sensação/fisiologia , Núcleos Talâmicos/fisiologia , Animais , Condutividade Elétrica , Eletrofisiologia , Técnicas In Vitro , Lasers , Rede Nervosa/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Estimulação Luminosa , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Sinapses/fisiologia , Núcleos Talâmicos/metabolismoRESUMO
A reason why the thalamus is more than a passive gateway for sensory signals is that two-third of the synapses of thalamocortical neurons are directly or indirectly related to the activity of corticothalamic axons. While the responses of thalamocortical neurons evoked by sensory stimuli are well characterized, with ON- and OFF-center receptive field structures, the prevalence of synaptic noise resulting from neocortical feedback in intracellularly recorded thalamocortical neurons in vivo has attracted little attention. However, in vitro and modeling experiments point to its critical role for the integration of sensory signals. Here we combine our recent findings in a unified framework suggesting the hypothesis that corticothalamic synaptic activity is adapted to modulate the transfer efficiency of thalamocortical neurons during selective attention at three different levels: First, on ionic channels by interacting with intrinsic membrane properties, second at the neuron level by impacting on the input-output gain, and third even more effectively at the cell assembly level by boosting the information transfer of sensory features encoded in thalamic subnetworks. This top-down population control is achieved by tuning the correlations in subthreshold membrane potential fluctuations and is adapted to modulate the transfer of sensory features encoded by assemblies of thalamocortical relay neurons. We thus propose that cortically-controlled (de-)correlation of subthreshold noise is an efficient and swift dynamic mechanism for selective attention in the thalamus.
Assuntos
Atenção/fisiologia , Córtex Cerebral/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Tálamo/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Simulação por Computador , Retroalimentação , Cobaias , Teoria da Informação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vias Neurais/fisiologia , Técnicas de Patch-Clamp , Percepção/fisiologia , Ratos Wistar , Técnicas de Cultura de Tecidos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismoRESUMO
Fast synaptic inhibition sculpts all forms of cortical activity by means of a specialized connectivity pattern between highly heterogeneous inhibitory interneurons and principal excitatory cells. Importantly, inhibitory neurons connect also to each other extensively, following a detailed blueprint, and, indeed, specific forms of disinhibition affect important behavioral functions. Here we discuss a peculiar form of cortical disinhibition: the massive autaptic self-inhibition of parvalbumin-(PV) positive basket cells. Despite being described long ago, autaptic inhibition onto PV basket cells is rarely included in cortical circuit diagrams, perhaps because of its still elusive function. We propose here a potential dual role of autaptic feedback inhibition in temporally coordinating PV basket cells during cortical network activity.
Assuntos
Neurônios GABAérgicos/fisiologia , Neocórtex/citologia , Inibição Neural/fisiologia , Sinapses/fisiologia , Animais , Neurônios GABAérgicos/classificação , Neurônios GABAérgicos/ultraestrutura , Humanos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Parvalbuminas/metabolismo , Sinapses/ultraestruturaRESUMO
Variations of excitatory and inhibitory conductances determine the membrane potential (V(m)) activity of neurons, as well as their spike responses, and are thus of primary importance. Methods to estimate these conductances require clamping the cell at several different levels of V(m), thus making it impossible to estimate conductances from "single trial" V(m) recordings. We present here a new method that allows extracting estimates of the full time course of excitatory and inhibitory conductances from single-trial V(m) recordings. This method is based on oversampling of the V(m). We test the method numerically using models of increasing complexity. Finally, the method is evaluated using controlled conductance injection in cortical neurons in vitro using the dynamic-clamp technique. This conductance extraction method should be very useful for future in vivo applications.
Assuntos
Potenciais de Ação/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Modelos Neurológicos , Inibição Neural/fisiologia , Técnicas de Patch-Clamp/métodos , Algoritmos , Animais , Humanos , Neurônios/fisiologiaRESUMO
Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na(+) current is supported by Nav1.2 and/or Nav1.3 alpha-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Nav beta 2.1 transcript was not detected, whereas the Nav beta 1.1 transcript was present. Even in the absence of Nav beta 2.1, alpha-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K(+) currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K(+) current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv alpha-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K(+) current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na(+) and K(+) channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.
Assuntos
Embrião de Mamíferos/química , Embrião de Mamíferos/inervação , Neurônios Motores/química , Neurônios Motores/fisiologia , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia , Processamento Alternativo , Animais , Células Cultivadas , Estimulação Elétrica , Eletrofisiologia , Feminino , Imunofluorescência , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/genética , Gravidez , RNA/análise , RNA/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Tetrodotoxina/farmacologiaRESUMO
Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.