RESUMO
A spinal cord injury interrupts the communication between the brain and the region of the spinal cord that produces walking, leading to paralysis1,2. Here, we restored this communication with a digital bridge between the brain and spinal cord that enabled an individual with chronic tetraplegia to stand and walk naturally in community settings. This brain-spine interface (BSI) consists of fully implanted recording and stimulation systems that establish a direct link between cortical signals3 and the analogue modulation of epidural electrical stimulation targeting the spinal cord regions involved in the production of walking4-6. A highly reliable BSI is calibrated within a few minutes. This reliability has remained stable over one year, including during independent use at home. The participant reports that the BSI enables natural control over the movements of his legs to stand, walk, climb stairs and even traverse complex terrains. Moreover, neurorehabilitation supported by the BSI improved neurological recovery. The participant regained the ability to walk with crutches overground even when the BSI was switched off. This digital bridge establishes a framework to restore natural control of movement after paralysis.
Assuntos
Interfaces Cérebro-Computador , Encéfalo , Terapia por Estimulação Elétrica , Reabilitação Neurológica , Traumatismos da Medula Espinal , Medula Espinal , Caminhada , Humanos , Encéfalo/fisiologia , Terapia por Estimulação Elétrica/instrumentação , Terapia por Estimulação Elétrica/métodos , Quadriplegia/etiologia , Quadriplegia/reabilitação , Quadriplegia/terapia , Reprodutibilidade dos Testes , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/reabilitação , Traumatismos da Medula Espinal/terapia , Caminhada/fisiologia , Perna (Membro)/fisiologia , Reabilitação Neurológica/instrumentação , Reabilitação Neurológica/métodos , MasculinoRESUMO
Complex interactions between brain regions and the spinal cord (SC) govern body motion, which is ultimately driven by muscle activation. Motor planning or learning are mainly conducted at higher brain regions, whilst the SC acts as a brain-muscle gateway and as a motor control centre providing fast reflexes and muscle activity regulation. Thus, higher brain areas need to cope with the SC as an inherent and evolutionary older part of the body dynamics. Here, we address the question of how SC dynamics affects motor learning within the cerebellum; in particular, does the SC facilitate cerebellar motor learning or constitute a biological constraint? We provide an exploratory framework by integrating biologically plausible cerebellar and SC computational models in a musculoskeletal upper limb control loop. The cerebellar model, equipped with the main form of cerebellar plasticity, provides motor adaptation; whilst the SC model implements stretch reflex and reciprocal inhibition between antagonist muscles. The resulting spino-cerebellar model is tested performing a set of upper limb motor tasks, including external perturbation studies. A cerebellar model, lacking the implemented SC model and directly controlling the simulated muscles, was also tested in the same. The performances of the spino-cerebellar and cerebellar models were then compared, thus allowing directly addressing the SC influence on cerebellar motor adaptation and learning, and on handling external motor perturbations. Performance was assessed in both joint and muscle space, and compared with kinematic and EMG recordings from healthy participants. The differences in cerebellar synaptic adaptation between both models were also studied. We conclude that the SC facilitates cerebellar motor learning; when the SC circuits are in the loop, faster convergence in motor learning is achieved with simpler cerebellar synaptic weight distributions. The SC is also found to improve robustness against external perturbations, by better reproducing and modulating muscle cocontraction patterns.
Assuntos
Cerebelo , Medula Espinal , Humanos , Cerebelo/fisiologia , Medula Espinal/fisiologia , Simulação por Computador , Extremidade Superior , Aprendizagem/fisiologiaRESUMO
It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.
Assuntos
Potenciais de Ação/fisiologia , Axônios/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Células de Purkinje/metabolismo , Cálcio/metabolismo , Cerebelo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Purinérgicos P2Y1 , Fosfolipases Tipo C/metabolismoRESUMO
Human post-natal neurodevelopmental delay is often associated with cerebral alterations that can lead, by themselves or associated with peripheral deficits, to premature death. Here, we report the clinical features of 10 patients from six independent families with mutations in the autosomal YIF1B gene encoding a ubiquitous protein involved in anterograde traffic from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus morphology. The patients displayed global developmental delay, motor delay, visual deficits with brain MRI evidence of ventricle enlargement, myelination alterations and cerebellar atrophy. A similar profile was observed in the Yif1b knockout (KO) mouse model developed to identify the cellular alterations involved in the clinical defects. In the CNS, mice lacking Yif1b displayed neuronal reduction, altered myelination of the motor cortex, cerebellar atrophy, enlargement of the ventricles, and subcellular alterations of endoplasmic reticulum and Golgi apparatus compartments. Remarkably, although YIF1B was not detected in primary cilia, biallelic YIF1B mutations caused primary cilia abnormalities in skin fibroblasts from both patients and Yif1b-KO mice, and in ciliary architectural components in the Yif1b-KO brain. Consequently, our findings identify YIF1B as an essential gene in early post-natal development in human, and provide a new genetic target that should be tested in patients developing a neurodevelopmental delay during the first year of life. Thus, our work is the first description of a functional deficit linking Golgipathies and ciliopathies, diseases so far associated exclusively to mutations in genes coding for proteins expressed within the primary cilium or related ultrastructures. We therefore propose that these pathologies should be considered as belonging to a larger class of neurodevelopmental diseases depending on proteins involved in the trafficking of proteins towards specific cell membrane compartments.
Assuntos
Cílios/genética , Complexo de Golgi/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas de Transporte Vesicular/genética , Animais , Células Cultivadas , Cílios/patologia , Feminino , Complexo de Golgi/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/diagnóstico por imagemRESUMO
While high threshold voltage-dependent Ca2+ channels (VDCCs) of the N and P/Q families are crucial for evoked neurotransmitter release in the mammalian CNS, it remains unclear to what extent L-type Ca2+ channels (LTCCs), which have been mainly considered as acting at postsynaptic sites, participate in the control of transmitter release. Here, we investigate the possible role of LTCCs in regulating GABA release by cerebellar molecular layer interneurons (MLIs) from rats. We found that BayK8644 (BayK) markedly increases mIPSC frequency in MLIs and Purkinje cells (PCs), suggesting that LTCCs are expressed presynaptically. Furthermore, we observed (1) a potentiation of evoked IPSCs in the presence of BayK, (2) an inhibition of evoked IPSCs in the presence of the LTCC-specific inhibitor Compound 8 (Cp8), and (3) a strong reduction of mIPSC frequency by Cp8. BayK effects are reduced by dantrolene, suggesting that ryanodine receptors act in synergy with LTCCs. Finally, BayK enhances presynaptic AP-evoked Ca2+ transients and increases the frequency of spontaneous axonal Ca2+ transients observed in TTX. Taken together, our data demonstrate that LTCCs are of primary importance in regulating GABA release by MLIs.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Cerebelo/fisiologia , Interneurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Cerebelo/citologia , Feminino , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/patologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Butadienos/farmacologia , Cálcio/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Técnicas de Cocultura/métodos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Comportamento Exploratório/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Gânglios Espinais/citologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Células Musculares/efeitos dos fármacos , Células Musculares/fisiologia , N-Metilaspartato/farmacologia , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Proteína 2 de Sobrevivência do Neurônio Motor/deficiênciaRESUMO
PCP4/PEP19 is a modulator of Ca(2+)-CaM signaling. In the brain, it is expressed in a very specific pattern in postmitotic neurons. In particular, Pcp4 is highly expressed in the Purkinje cell, the sole output neuron of the cerebellum. PCP4, located on human chromosome 21, is present in three copies in individuals with Down syndrome (DS). In a previous study using a transgenic mouse model (TgPCP4) to evaluate the consequences of 3 copies of this gene, we found that PCP4 overexpression induces precocious neuronal differentiation during mouse embryogenesis. Here, we report combined analyses of the cerebellum at postnatal stages (P14 and adult) in which we identified age-related molecular, electrophysiological, and behavioral alterations in the TgPCP4 mouse. While Pcp4 overexpression at P14 induces an earlier neuronal maturation, at adult stage it induces increase in cerebellar CaMK2alpha and in cerebellar LTD, as well as learning impairments. We therefore propose that PCP4 contributes significantly to the development of Down syndrome phenotypes through molecular and functional changes.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Animais Recém-Nascidos , Cerebelo/citologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Técnicas In Vitro , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/genética , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Quinoxalinas/farmacologia , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Valina/análogos & derivados , Valina/farmacologiaRESUMO
In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra- or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²âº transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre- and post-synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²âº transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²âº transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²âº transients at varicosities located more than 5 µm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²âº stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high-gain glutamate detectors.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Receptores Pré-Sinápticos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Axônios/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Cerebelo/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Luz , Masculino , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos da radiação , Rianodina/farmacologiaRESUMO
In central synapses, spontaneous transmitter release observed in the absence of action potential firing is often considered as a random process lacking time or space specificity. However, when studying miniature glutamatergic currents at cerebellar synapses between parallel fibers and molecular layer interneurons, we found that these currents were sometimes organized in bursts of events occurring at high frequency (about 30 Hz). Bursts displayed homogeneous quantal size amplitudes. Furthermore, in the presence of the desensitization inhibitor cyclothiazide, successive events within a burst displayed quantal amplitude occlusion. Based on these findings, we conclude that bursts originate in individual synapses. Bursts were enhanced by increasing either the external potassium concentration or the external calcium concentration, and they were strongly inhibited when blocking voltage-gated calcium channels by cadmium. Bursts were prevalent in elevated potassium concentration during the formation of the molecular layer but were infrequent later in development. Since postsynaptic AMPA receptors are largely calcium permeant in developing parallel fiber-interneuron synapses, we propose that bursts involve presynaptic calcium transients implicating presynaptic voltage-gated calcium channels, together with postsynaptic calcium transients implicating postsynaptic AMPA receptors. These simultaneous pre- and postsynaptic calcium transients may contribute to the formation and/or stabilization of synaptic connections.
Assuntos
Cálcio , Receptores de AMPA , Cálcio/metabolismo , Receptores de AMPA/fisiologia , Sinapses/metabolismo , Cerebelo/fisiologia , Canais de Cálcio/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
The role of the axonal membrane compartment in synaptic integration is usually neglected. We show here that in interneurons of the cerebellar molecular layer, where dendrites are so short that the somatodendritic domain can be considered isopotential, the axonal membrane contributes a significant part of the cell input capacitance. We examine the impact of axonal membrane on synaptic integration by cutting the axon with two-photon illumination. We find that the axonal compartment acts as a sink for signals generated at fast conductance synapses, thus increasing the initial decay rate of corresponding synaptic potentials over the value predicted from the resistance-capacitance (RC) product of the cell membrane; signals generated at slower synapses are much less affected. This mechanism sharpens the spike firing precision of fast glutamatergic inputs without resorting to multisynaptic pathways.
Assuntos
Axônios/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Axotomia/métodos , Cálcio/metabolismo , Cerebelo/citologia , Dendritos/fisiologia , Dendritos/efeitos da radiação , Impedância Elétrica , Estimulação Elétrica/métodos , Técnicas In Vitro , Condução Nervosa/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Sinapses/fisiologiaRESUMO
Little is known about the generation of slow rhythms in brain neuronal circuits. Nevertheless, a few studies, both from reconstituted systems and from hippocampal slices, indicate that activation of metabotropic glutamate receptors (mGluRs) could generate such rhythms. Here we show in rat cerebellar slices that after either release of glutamate by repetitive stimulation, or direct stimulation of type 1 mGluRs, molecular layer interneurons exhibit repetitive slow Ca(2+) transients. By combining cell-attached patch-clamp recording with Ca(2+) imaging, we show that the regular Ca(2+) transients (mean frequency, 35 mHz induced by 2 microm quisqualate in the presence of ionotropic glutamate receptor blockers) are locked with bursts of action potentials. Nevertheless, the Ca(2+) transients are not blocked by tetrodotoxin, indicating that firing is not necessary to entrain oscillations. The first Ca(2+) transient within a train is different in several ways from subsequent transients. It is broader than the subsequent transients, displays a different phase relationship to associated spike bursts, and exhibits a distinct sensitivity to ionic and pharmacological manipulations. Whereas the first transient appears to involve entry of Ca(2+) ions through transient receptor potential channel-like channels and secondarily activated L-type Ca(2+) channels, subsequent transients rely mostly on an exchange of Ca(2+) ions between the cytosol and D-myo-inositol-1,4,5-triphosphate-sensitive intracellular Ca(2+) stores. The slow, highly regular oscillations observed in the present work are likely to drive pauses in postsynaptic Purkinje cells, and could play a role in coordinating slow oscillations involving the cerebello-olivar circuit loop.
Assuntos
Cálcio/metabolismo , Cerebelo/fisiologia , Ácido Glutâmico/metabolismo , Interneurônios/fisiologia , Periodicidade , Receptores de Glutamato Metabotrópico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cerebelo/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Estimulação Elétrica , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interneurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ácido Quisquálico/administração & dosagem , Ratos , Receptores de Glutamato Metabotrópico/agonistas , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Bloqueadores dos Canais de Sódio/administração & dosagem , Tetrodotoxina/administração & dosagemRESUMO
Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions.
Assuntos
Cerebelo/metabolismo , Interneurônios/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica , Animais , Técnicas Biossensoriais , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cerebelo/ultraestrutura , Feminino , Interneurônios/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Atividade Motora , CaminhadaRESUMO
Intracellular calcium concentration rises have been reported following activation of GABA(A) receptors in neonatal preparations and attributed to activation of voltage-dependent Ca(2+) channels. However, we show that, in cerebellar interneurons, GABA(A) agonists induce a somatodendritic Ca(2+) rise that persists at least until postnatal day 20 and is not mediated by depolarization-induced Ca(2+) entry. A local Ca(2+) elevation can likewise be elicited by repetitive stimulation of presynaptic GABAergic afferent fibers. We find that, following GABA(A) receptor activation, bicarbonate-induced Cl(-) entry leads to cell depolarization, Cl(-) accumulation, and osmotic tension. We propose that this tension induces the intracellular Ca(2+) rise as part of a regulatory volume decrease reaction. This mechanism introduces an unexpected link between activation of GABA(A) receptors and intracellular Ca(2+) elevation, which could contribute to activity-driven synaptic plasticity.
Assuntos
Cálcio/metabolismo , Interneurônios/fisiologia , Osmose/fisiologia , Receptores de GABA-A/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Cerebelo/fisiologia , Agonistas GABAérgicos/farmacologia , Líquido Intracelular/química , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Muscimol/farmacologia , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , RatosRESUMO
The inositol 1,4,5-trisphosphate (InsP3) receptor type I (InsP3R-I) is the principle channel for intracellular calcium (Ca2+) release in many cell types, including central neurons. It is regulated by endogenous compounds like Ca2+ and ATP, by protein partners, and by posttranslational modification. We report that the InsP3R-I is modified by O-linked glycosylation of serine or threonine residues with beta-N-acetylglucosamine (O-GlcNAc). The level of O-GlcNAcylation can be altered in vitro by the addition of the enzymes which add [OGT (O-GlcNActransferase)] or remove (O-GlcNAcase) this sugar or by loading cells with UDP-GlcNAc. We monitored the effects of this modification on InsP3R function at the single-channel level and on intracellular Ca2+ transients. Single-channel activity was monitored with InsP3R incorporated into bilayers; Ca2+ signaling was monitored using cells loaded with a Ca2+-sensitive fluorophore. We found that channel activity was decreased by the addition of O-GlcNAc and that this decrease was reversed by removal of the sugar. Similarly, cells loaded with UDP-GlcNAc had an attenuated response to uncaging of InsP3. These results show that O-GlcNAcylation is an important regulator of the InsP3R-I and suggest a mechanism for neuronal dysfunction under conditions in which O-GlcNAc is high, such as diabetes or physiological stress.
Assuntos
Glucosamina/análogos & derivados , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interneurônios/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/química , Cerebelo/metabolismo , Glucosamina/química , Glucosamina/metabolismo , Glicosilação , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Interneurônios/efeitos dos fármacos , Bicamadas Lipídicas/química , Camundongos , Microssomos/química , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ratos , Raios Ultravioleta , Uridina Difosfato N-Acetilglicosamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/farmacologia , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input from vestibular afferents in the form of a unique excitatory synapse on their dendritic brush. UBCs constitute independent relay lines for vestibular signals, and their inherent properties most likely determine how vestibular activity is encoded by the cerebellar cortex. We now demonstrate that UBCs are bimodal cells; they can either fire high-frequency bursts of action potentials when stimulated from hyperpolarized potentials or discharge tonically during sustained depolarizations. The two functional states can be triggered by physiological-like activity of the excitatory input and are encoded by distinct Ca2+-signaling systems. By combining complementary strategies, consisting of molecular and electrophysiological analysis and of ultrafast acousto-optical deflector-based two-photon imaging, we unraveled the identity and the subcellular localization of the Ca2+ conductances activating in each mode. Fast inactivating T-type Ca2+ channels produce low-threshold spikes, which trigger the high-frequency bursts and generate powerful Ca2+ transients in the brush and, to a much lesser extent, in the soma. The tonic firing mode is encoded by a signalization system principally composed of L-type channels. Ca2+ influx during tonic firing produces a linear representation of the spike rate of the cell in the form of a widespread and sustained Ca2+ concentration increase and regulates cellular excitability via BK potassium channels. The bimodal firing pattern of UBCs may underlie different coding strategies of the vestibular input by the cerebellum, thus likely increasing the computational power of this structure.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo T/fisiologia , Cerebelo/fisiologia , Interneurônios/fisiologia , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Interneurônios/citologia , Interneurônios/ultraestrutura , Microvilosidades/fisiologia , Ratos , Ratos WistarRESUMO
Axons of cerebellar molecular layer interneurones (MLIs) bear ionotropic glutamate receptors. Here, we show that these receptors elicit cytosolic [Ca2+] transients in axonal varicosities following glutamate spillover induced by stimulation of parallel fibres (PFs). A spatial profile analysis indicates that these transients occur at the same locations when induced by PF stimulation or trains of action potentials. They are not affected by the NMDAR antagonist AP-V, but are abolished by the AMPAR inhibitor GYKI-53655. Mimicking glutamate spillover by a puff of AMPA triggers axonal [Ca2+]i transients even in the presence of TTX. Addition of specific voltage-dependent Ca2+ channel (VDCC) blockers such as omega-AGAIVA and omega-conotoxin GVIA or broad range inhibitors such as Cd2+ did not significantly inhibit the signal indicating the involvement of Ca2+-permeable AMPARs. This hypothesis is further supported by the finding that the subunit specific AMPAR antagonist IEM-1460 blocks 75% of the signal. Bath application of AMPA increases the frequency and mean peak amplitude of GABAergic mIPSCs, an effect that is blocked by philanthotoxin-433 (PhTx) and reinforced by facilitating concentrations of ryanodine. By contrast, a high concentration of ryanodine or dantrolene reduced the effects of AMPA on mIPSCs. Single-cell RT-PCR experiments show that all GluR1-4 subunits are potentially expressed in MLI. Taken together, the results suggest that Ca2+-permeable AMPARs are colocalized with VDCCs in axonal varicosities and can be activated by glutamate spillover through PF stimulation. The AMPAR-mediated Ca2+ signal is amplified by Ca2+-induced Ca2+ release from intracellular stores, leading to GABA release by MLIs.
Assuntos
Cálcio/metabolismo , Cerebelo/citologia , Interneurônios/fisiologia , Receptores de AMPA/metabolismo , Potenciais de Ação/fisiologia , Animais , Axônios/fisiologia , Regulação da Expressão Gênica/fisiologia , Interneurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Subunidades Proteicas , Células de Purkinje , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Rianodina/farmacologiaRESUMO
Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.
Assuntos
Cálcio/metabolismo , Modelos Neurológicos , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Potenciação de Longa Duração/fisiologia , Neurotransmissores/metabolismo , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Certain interneurons contain large concentrations of specific Ca2+-binding proteins (CBPs), but consequences on presynaptic Ca2+ signaling are poorly understood. Here we show that expression of the slow CBP parvalbumin (PV) in cerebellar interneurons is cell specific and developmentally regulated, leading to characteristic changes in presynaptic Ca2+ dynamics (Ca(i)). Using whole-cell recording and fluorescence imaging, we studied action potential-evoked Ca(i) transients in axons of GABA-releasing interneurons from mouse cerebellum. At early developmental stages [postnatal days 10-12 (P10-P12)], decay kinetics were significantly faster for basket cells than for stellate cells, whereas at P19-P21 both interneurons displayed fast decay kinetics. Biochemical and immunocytochemical analysis showed parallel changes in the expression levels and cellular distribution of PV. By comparing wild-type and PV(-/-) mice, PV was shown to accelerate the initial decay of action potential-evoked Ca(i) signals in single varicosities and to introduce an additional slow phase that summates during bursts of action potentials. The fast initial Ca(i) decay accounts for a previous report that PV elimination favors synaptic facilitation. The slow decay component is responsible for a pronounced, PV-dependent, delayed transmitter release that we describe here at interneuron-interneuron synapses after presynaptic bursts of action potentials. Numerical simulations account for the effect of PV on Ca(i) kinetics, allow estimates for the axonal PV concentration (approximately 150 microm), and predict the time course of volume-averaged Ca(i) in the absence of exogenous buffer. Overall, PV arises as a major contributor to presynaptic Ca(i) signals and synaptic integration in the cerebellar cortex.
Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebelar/crescimento & desenvolvimento , Parvalbuminas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Potenciais de Ação/fisiologia , Animais , Córtex Cerebelar/citologia , Córtex Cerebelar/metabolismo , Córtex Cerebelar/fisiologia , Ácido Glutâmico/fisiologia , Imuno-Histoquímica , Técnicas In Vitro , Interneurônios/fisiologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Inibição Neural/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Phospholipase C (PLC)-coupled metabotropic receptors trigger the release of intracellular Ca2+ through activation of IP3 receptors (IP3Rs). Increasing evidence suggests that they can also and perhaps more efficiently mobilize Ca2+ through ryanodine receptors (RyRs). We constructed a model allowing a variable PLC stimulation level (via the parameter gamma) as well as a variable involvement of RyRs (via the parameter A). The sole presence of RyRs (A not = 0) affected the basal Ca2+ concentration [Ca2+]i. To keep Ca2+ below 160 nM, we fixed the upper limit of A to 0.2, a value that is compatible with the numerical ratio between RyRs and IP3Rs in cerebellar Purkinje neurons. Metabotropic responses were simulated by abruptly raising the value of gamma to various levels. In the absence of RyRs, the model starts to oscillate with gamma=0.4. For lower levels of PLC stimulation (gamma< or =0.3), the presence of RyR is capable of triggering an oscillatory signal. When A< or =0.18, the frequency of the Ca2+ oscillations augments from 0.1 to 1.3 Hz as a function of gamma. Conversely, as the frequency increases, the amplitude of the oscillations is reduced from 1 microM to 50 nM. With higher values of A, the oscillating pattern is definitively inhibited. It is concluded that RyRs have the potentiality to strikingly affect the temporal pattern of the Ca2+ signalling triggered by IP3-related metabotropic responses.
Assuntos
Relógios Biológicos/fisiologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Modelos Neurológicos , Neurônios/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Simulação por Computador , Humanos , Receptores de Inositol 1,4,5-TrifosfatoRESUMO
Consequences of 5-HT(1/2) systems blockade by methysergide on newborn rats respiratory drive were evaluated in vivo with unrestrained animals and in vitro using brainstem-spinal cord preparations. A decrease in respiratory frequency until a plateau level was observed under both in vivo (82.8 +/- 0.6% of control values) and in vitro (76.8 +/- 0.8% of control values) conditions whereas an increase in inspiratory amplitude (135.1 +/- 2.1% of control values) was only retrieved in vivo. By the use of the c-fos expression analysis, we correlated these effects with neuronal activity changes, particularly, in vivo in two key structures between the respiratory ponto-medullary network and the peripheral or suprapontine afferences, namely the commissural subnucleus of the nucleus of the solitary tract and the lateral parabrachial nucleus. Thus, peripheral and suprapontine inputs seem to be of a primeval importance in the respiratory influence of endogenous 5-HT. Besides, as 5-HT is involved in the respiratory perturbations that occur in sudden infant death syndrome (SIDS), our results suggest a participation of peripheral and suprapontine inputs in these disorders.