RESUMO
Numerous well-defined classes of retinal ganglion cells innervate the thalamus to guide image-forming vision, yet the rules governing their convergence and divergence remain unknown. Using two-photon calcium imaging in awake mouse thalamus, we observed a functional arrangement of retinal ganglion cell axonal boutons in which coarse-scale retinotopic ordering gives way to fine-scale organization based on shared preferences for other visual features. Specifically, at the â¼6 µm scale, clusters of boutons from different axons often showed similar preferences for either one or multiple features, including axis and direction of motion, spatial frequency, and changes in luminance. Conversely, individual axons could "de-multiplex" information channels by participating in multiple, functionally distinct bouton clusters. Finally, ultrastructural analyses demonstrated that retinal axonal boutons in a local cluster often target the same dendritic domain. These data suggest that functionally specific convergence and divergence of retinal axons may impart diverse, robust, and often novel feature selectivity to visual thalamus.
Assuntos
Axônios/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Tálamo/fisiologia , Animais , Análise por Conglomerados , Dendritos/fisiologia , Lógica Fuzzy , Corpos Geniculados/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Movimento (Física) , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Visão Ocular , Vias VisuaisRESUMO
Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. Here, we report that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, our results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Produtos do Gene gag/genética , Corpos Multivesiculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , RNA Mensageiro/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Produtos do Gene gag/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/metabolismo , Plasticidade Neuronal , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Terminações Pré-Sinápticas/fisiologia , Ligação Proteica , Domínios Proteicos , Retroelementos/genéticaRESUMO
Changes in the amount of daylight (photoperiod) alter physiology and behaviour1,2. Adaptive responses to seasonal photoperiods are vital to all organisms-dysregulation associates with disease, including affective disorders3 and metabolic syndromes4. The circadian rhythm circuitry is implicated in such responses5,6, yet little is known about the precise cellular substrates that underlie phase synchronization to photoperiod change. Here we identify a brain circuit and system of axon branch-specific and reversible neurotransmitter deployment that are critical for behavioural and sleep adaptation to photoperiod. A type of neuron called mrEn1-Pet17 in the mouse brainstem median raphe nucleus segregates serotonin from VGLUT3 (also known as SLC17A8, a proxy for glutamate) to different axonal branches that innervate specific brain regions involved in circadian rhythm and sleep-wake timing8,9. This branch-specific neurotransmitter deployment did not distinguish between daylight and dark phase; however, it reorganized with change in photoperiod. Axonal boutons, but not cell soma, changed neurochemical phenotype upon a shift away from equinox light/dark conditions, and these changes were reversed upon return to equinox conditions. When we genetically disabled Vglut3 in mrEn1-Pet1 neurons, sleep-wake periods, voluntary activity and clock gene expression did not synchronize to the new photoperiod or were delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing, we delineated a preoptic area-to-mrEn1Pet1 connection that was responsible for decoding the photoperiodic inputs, driving the neurotransmitter reorganization and promoting behavioural synchronization. Our results reveal a brain circuit and periodic, branch-specific neurotransmitter deployment that regulates organismal adaptation to photoperiod change.
Assuntos
Adaptação Fisiológica , Axônios , Ritmo Circadiano , Neurotransmissores , Fotoperíodo , Animais , Feminino , Camundongos , Adaptação Fisiológica/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Ritmo Circadiano/fisiologia , Proteínas CLOCK/genética , Escuridão , Núcleo Dorsal da Rafe/citologia , Núcleo Dorsal da Rafe/metabolismo , Vias Neurais/fisiologia , Neurotransmissores/metabolismo , Área Pré-Óptica/citologia , Área Pré-Óptica/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Vírus da Raiva , Serotonina/metabolismo , Sono/fisiologia , Vigília/fisiologiaRESUMO
The selectivity of neuronal responses arises from the architecture of excitatory and inhibitory connections. In the primary visual cortex, the selectivity of a neuron in layer 2/3 for stimulus orientation and direction is thought to arise from intracortical inputs that are similarly selective1-8. However, the excitatory inputs of a neuron can have diverse stimulus preferences1-4,6,7,9, and inhibitory inputs can be promiscuous10 and unselective11. Here we show that the excitatory and inhibitory intracortical connections to a layer 2/3 neuron accord with its selectivity by obeying precise spatial patterns. We used rabies tracing1,12 to label and functionally image the excitatory and inhibitory inputs to individual pyramidal neurons of layer 2/3 of the mouse visual cortex. Presynaptic excitatory neurons spanned layers 2/3 and 4 and were distributed coaxial to the preferred orientation of the postsynaptic neuron, favouring the region opposite to its preferred direction. By contrast, presynaptic inhibitory neurons resided within layer 2/3 and favoured locations near the postsynaptic neuron and ahead of its preferred direction. The direction selectivity of a postsynaptic neuron was unrelated to the selectivity of presynaptic neurons, but correlated with the spatial displacement between excitatory and inhibitory presynaptic ensembles. Similar asymmetric connectivity establishes direction selectivity in the retina13-17. This suggests that this circuit motif might be canonical in sensory processing.
Assuntos
Vias Neurais , Células Piramidais/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores , Feminino , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos , Inibição Neural , Técnicas de Rastreamento Neuroanatômico , Terminações Pré-Sinápticas/fisiologia , Vírus da Raiva/metabolismo , Receptores Virais/metabolismo , Retina/citologia , Retina/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9â weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100â Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9â weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to â¼25â mV and short durations of â¼0.5â ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.
Assuntos
Potenciais de Ação , Células-Tronco Pluripotentes Induzidas , Neurônios , Terminações Pré-Sinápticas , Sinapses , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Potenciais de Ação/fisiologia , Sinapses/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Transmissão Sináptica/fisiologia , Células Cultivadas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologiaRESUMO
Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential (AP) across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle (SV) fusing (Pv) and in the number of vesicles available for immediate release, known as the readily releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of APs. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses. Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release.
Assuntos
Terminações Pré-Sinápticas , Transmissão Sináptica , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiologia , Animais , Transmissão Sináptica/fisiologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/metabolismo , Masculino , Ratos , Feminino , Ácido Glutâmico/metabolismo , Camundongos , Ratos Sprague-DawleyRESUMO
Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.
Assuntos
Proteínas de Drosophila , Sistema de Sinalização das MAP Quinases , Neuroglia , Neurônios , Terminações Pré-Sinápticas , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Sinapsinas , Transmissão Sináptica , Vesículas Sinápticas , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação , Neuroglia/metabolismo , Neuroglia/fisiologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Sinapsinas/metabolismo , Sinapsinas/genética , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Chemical synapses are heterogeneous junctions formed between neurons that are specialized for the conversion of electrical impulses into the exocytotic release of neurotransmitters. Voltage-gated Ca2+ channels play a pivotal role in this process as they are the major conduits for the Ca2+ ions that trigger the fusion of neurotransmitter-containing vesicles with the presynaptic membrane. Alterations in the intrinsic function of these channels and their positioning within the active zone can profoundly alter the timing and strength of synaptic output. Advances in optical and electron microscopic imaging, structural biology and molecular techniques have facilitated recent breakthroughs in our understanding of the properties of voltage-gated Ca2+ channels that support their presynaptic functions. Here we examine the nature of these channels, how they are trafficked to and anchored within presynaptic boutons, and the mechanisms that allow them to function optimally in shaping the flow of information through neural circuits.
Assuntos
Canais de Cálcio/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Humanos , Transporte ProteicoRESUMO
Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca2+ influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, we show that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. Our results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release.
Assuntos
Dopamina , Proteínas de Drosophila , Drosophila melanogaster , Memória , Proteínas do Tecido Nervoso , Animais , Dopamina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão SinápticaRESUMO
During prolonged trains of presynaptic action potentials (APs), synaptic release reaches a stable level that reflects the speed of replenishment of the readily releasable pool (RRP). Determining the size and filling dynamics of vesicular pools upstream of the RRP has been hampered by a lack of precision of synaptic output measurements during trains. Using the recent technique of tracking vesicular release in single active zone synapses, we now developed a method that allows the sizes of the RRP and upstream pools to be followed in time. We find that the RRP is fed by a small-sized pool containing approximately one to four vesicles per docking site at rest. This upstream pool is significantly depleted by short AP trains, and reaches a steady, depleted state for trains of >10 APs. We conclude that a small, highly dynamic vesicular pool upstream of the RRP potently controls synaptic strength during sustained stimulation.
Assuntos
Potenciais Sinápticos/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Potenciais de Ação/fisiologia , Animais , Masculino , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologiaRESUMO
Neurons in the thalamic reticular nucleus (TRN) are a primary source of inhibition to the dorsal thalamus and, as they are innervated in part by the cortex, are a means of corticothalamic regulation. Previously, cortical inputs to the TRN were thought to originate solely from layer 6 (L6), but we recently reported the presence of putative synaptic terminals from layer 5 (L5) neurons in multiple cortical areas in the TRN [J. A. Prasad, B. J. Carroll, S. M. Sherman, J. Neurosci. 40, 5785-5796 (2020)]. Here, we demonstrate with electron microscopy that L5 terminals from multiple cortical regions make bona fide synapses in the TRN. We further use light microscopy to localize these synapses relative to recently described TRN subdivisions and show that L5 terminals target the edges of the somatosensory TRN, where neurons reciprocally connect to higher-order thalamus, and that L5 terminals are scarce in the core of the TRN, where neurons reciprocally connect to first-order thalamus. In contrast, L6 terminals densely innervate both edge and core subregions and are smaller than those from L5. These data suggest that a sparse but potent input from L5 neurons of multiple cortical regions to the TRN may yield transreticular inhibition targeted to higher-order thalamus.
Assuntos
Córtex Cerebral , Núcleos Ventrais do Tálamo , Animais , Córtex Cerebral/fisiologia , Córtex Cerebral/ultraestrutura , Camundongos , Microscopia Eletrônica , Inibição Neural , Neurônios/fisiologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Núcleos Ventrais do Tálamo/fisiologia , Núcleos Ventrais do Tálamo/ultraestruturaRESUMO
VEGF was initially discovered due to its angiogenic activity and therefore named "vascular endothelial growth factor." However, its more recently discovered neurotrophic activity may be evolutionarily more ancient. Our previous work showed that all the changes produced by axotomy on the firing activity and synaptic inputs of abducens motoneurons were completely restored after VEGF administration. Therefore, we hypothesized that the lack of VEGF delivered by retrograde transport from the periphery should also affect the physiology of otherwise intact abducens motoneurons. For VEGF retrograde blockade, we chronically applied a neutralizing VEGF antibody to the lateral rectus muscle. Recordings of extracellular single-unit activity and eye movements were made in alert cats before and after the application of the neutralizing antibody. Our data revealed that intact, noninjured abducens motoneurons retrogradely deprived of VEGF exhibited noticeable changes in their firing pattern. There is a general decrease in firing rate and a significant reduction in eye position and eye velocity sensitivity (i.e., a decrease in the tonic and phasic components of their discharge, respectively). Moreover, by means of confocal immunocytochemistry, motoneurons under VEGF blockade showed a marked reduction in the density of afferent synaptic terminals contacting with their cell bodies. Altogether, the present findings demonstrate that the lack of retrogradely delivered VEGF renders abducens motoneurons into an axotomy-like state. This indicates that VEGF is an essential retrograde factor for motoneuronal synaptic drive and discharge activity.
Assuntos
Movimentos Oculares , Neurônios Motores , Terminações Pré-Sinápticas , Fator A de Crescimento do Endotélio Vascular , Animais , Anticorpos Neutralizantes , Axotomia , Gatos , Movimentos Oculares/efeitos dos fármacos , Movimentos Oculares/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Músculos Oculomotores/efeitos dos fármacos , Músculos Oculomotores/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.
Assuntos
Proteínas de Drosophila , Memória , Corpos Pedunculados , Terminações Pré-Sinápticas , Proteínas rab3 de Ligação ao GTP , Animais , Corpos Pedunculados/fisiologia , Corpos Pedunculados/metabolismo , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas de Drosophila/metabolismo , Memória/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Proteínas do Tecido Nervoso/metabolismo , Drosophila , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiologiaRESUMO
Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2 subtype levels are largely unsolved. Because the CaV 2 α1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1 subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1 subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1 subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1 subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2 subtype abundance and preference remain enigmatic. The CaV α1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2 subtype abundance but not preference, whereas the CaV 2.2 α1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2 subtype abundance or preference. The CaV 2 α1 subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.
Assuntos
Canais de Cálcio Tipo N , Transmissão Sináptica , Animais , Canais de Cálcio Tipo N/genética , Transmissão Sináptica/fisiologia , Sinapses/fisiologia , Terminações Pré-Sinápticas/fisiologia , Neurônios/metabolismo , Mamíferos/metabolismoRESUMO
Neurons in the central nervous system communicate with each other by activating billions of tiny synaptic boutons distributed along their fine axons. These presynaptic varicosities are very crowded environments, comprising hundreds of synaptic vesicles. Only a fraction of these vesicles can be recruited in a single release episode, either spontaneous or evoked by action potentials. Since the seminal work by Fatt and Katz, spontaneous release has been modelled as a memoryless process. Nevertheless, at central synapses, experimental evidence indicates more complex features, including non-exponential distributions of release intervals and power-law behaviour in their rate. To describe these features, we developed a probabilistic model of spontaneous release based on Brownian motion of synaptic vesicles in the presynaptic environment. To account for different diffusion regimes, we based our simulations on fractional Brownian motion. We show that this model can predict both deviation from the Poisson hypothesis and power-law features in experimental quantal release series, thus suggesting that the vesicular motion by diffusion could per se explain the emergence of these properties. We demonstrate the efficacy of our modelling approach using electrophysiological recordings at single synaptic boutons and ultrastructural data. When this approach was used to simulate evoked responses, we found that the replenishment of the readily releasable pool driven by Brownian motion of vesicles can reproduce the characteristic binomial release distributions seen experimentally. We believe that our modelling approach supports the idea that vesicle diffusion and readily releasable pool dynamics are crucial factors for the physiological functioning of neuronal communication. KEY POINTS: We developed a new probabilistic model of spontaneous and evoked vesicle fusion based on simple biophysical assumptions, including the motion of vesicles before they dock to the release site. We provide closed-form equations for the interval distribution of spontaneous releases in the special case of Brownian diffusion of vesicles, showing that a power-law heavy tail is generated. Fractional Brownian motion (fBm) was exploited to simulate anomalous vesicle diffusion, including directed and non-directed motion, by varying the Hurst exponent. We show that our model predicts non-linear features observed in experimental spontaneous quantal release series as well as ultrastructural data of synaptic vesicles spatial distribution. Evoked exocytosis based on a diffusion-replenished readily releasable pool might explain the emergence of power-law behaviour in neuronal activity.
Assuntos
Transmissão Sináptica , Vesículas Sinápticas , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Animais , Transmissão Sináptica/fisiologia , Modelos Neurológicos , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , DifusãoRESUMO
Exocytosis is a fundamental membrane fusion process by which the soluble or membrane-associated cargoes of a secretory vesicle are delivered to the extracellular milieu or the cell surface. While essential for all organs, the brain relies on a specialized form of exocytosis to mediate information flow throughout its vast circuitry. Neurotransmitter-laden synaptic vesicles fuse with the plasma membrane on cue with astonishing speed in a probabilistic process that is both tightly regulated and capable of a fascinating array of plasticities. Here, we examine progress in the molecular understanding of synaptic vesicle fusion and its control.
Assuntos
Exocitose/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Membrana Celular/metabolismo , Fusão de Membrana/fisiologia , Plasticidade Neuronal/fisiologiaRESUMO
Alzheimer's disease is linked to increased levels of amyloid beta (Aß) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aß. Using an optical readout method in cultured hippocampal neurons, we show that acute Aß42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aß transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aß is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aß-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value.
Assuntos
Peptídeos beta-Amiloides , Vesículas Sinápticas , Vesículas Sinápticas/fisiologia , Peptídeos beta-Amiloides/metabolismo , Terminações Pré-Sinápticas/fisiologia , Neurônios/metabolismo , Hipocampo/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
The precise mechanism behind the supply of adenosine triphosphate (ATP) to approximately half of the presynaptic release sites in axons that lack a stationary mitochondrion is not fully understood. This paper presents a mathematical model designed to simulate the transient ATP concentration in presynaptic en passant boutons. The model is utilized to investigate how the ATP concentration responds to increased ATP demand during neuronal firing in boutons with a stationary mitochondrion and those without one. The analysis suggests that neuron firing may cause oscillations in the ATP concentrations, with peak-to-peak amplitudes ranging from 0.06% to 5% of their average values. However, this does not deplete boutons lacking a mitochondrion of ATP; for physiologically relevant values of model parameters, their concentration remains approximately 3.75 times higher than the minimum concentration required for synaptic activity. The variance in average ATP concentrations between boutons containing a stationary mitochondrion and those lacking one ranges from 0.3% to 0.8%, contingent on the distance between the boutons. The model indicates that diffusion-driven ATP transport is rapid enough to adequately supply ATP molecules to boutons lacking a stationary mitochondrion.
Assuntos
Trifosfato de Adenosina , Mitocôndrias , Neurônios , Terminações Pré-Sinápticas , Trifosfato de Adenosina/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Modelos Neurológicos , Animais , Potenciais de Ação , Fatores de TempoRESUMO
Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.
Assuntos
Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Vesículas Sinápticas/ultraestrutura , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Citoesqueleto , Tomografia com Microscopia Eletrônica/métodos , Hipocampo/metabolismo , Potenciação de Longa Duração/genética , Masculino , Neurotransmissores , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Long-Evans , Sinapses/fisiologia , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/fisiologiaRESUMO
Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.