RESUMEN
In the brain, most synapses are formed on minute protrusions known as dendritic spines. Unlike their artificial intelligence counterparts, spines are not merely tuneable memory elements: they also embody algorithms that implement the brain's ability to learn from experience and cope with new challenges. Importantly, they exhibit structural dynamics that depend on activity, excitatory input and inhibitory input (synaptic plasticity or 'extrinsic' dynamics) and dynamics independent of activity ('intrinsic' dynamics), both of which are subject to neuromodulatory influences and reinforcers such as dopamine. Here we succinctly review extrinsic and intrinsic dynamics, compare these with parallels in machine learning where they exist, describe the importance of intrinsic dynamics for memory management and adaptation, and speculate on how disruption of extrinsic and intrinsic dynamics may give rise to mental disorders. Throughout, we also highlight algorithmic features of spine dynamics that may be relevant to future artificial intelligence developments.
Asunto(s)
Encéfalo/fisiología , Espinas Dendríticas/fisiología , Trastornos Mentales/fisiopatología , Modelos Neurológicos , Redes Neurales de la Computación , Algoritmos , Animales , Inteligencia Artificial , Encéfalo/citología , Espinas Dendríticas/ultraestructura , Dopamina/fisiología , Humanos , Aprendizaje Automático , Memoria a Corto Plazo/fisiología , Procesos Mentales/fisiología , Plasticidad Neuronal , Neurotransmisores/fisiología , Optogenética , Receptores Dopaminérgicos/fisiología , Recompensa , Especificidad de la Especie , Sinapsis/fisiologíaRESUMEN
Anti-NMDA receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder associated with autoantibodies against NMDARs, which cause a variety of symptoms from prominent psychiatric and cognitive manifestations to seizures and autonomic instability. Previous studies mainly focused on hippocampal effects of these autoantibodies, helping to explain mechanistic causes for cognitive impairment. However, antibodies' effects on higher cortical network function, where they could contribute to psychosis and/or seizures, have not been explored in detail until now. Here, we employed a patient-derived monoclonal antibody targeting the NR1 subunit of NMDAR and tested its effects on in vitro cultures of rodent cortical neurons, using imaging and electrophysiological techniques. We report that this hNR1 antibody drives cortical networks to a hyperexcitable state and disrupts mechanisms stabilizing network activity such as Npas4 signaling. Network hyperactivity is in part a result of a reduced synaptic output of inhibitory neurons, as indicated by a decreased inhibitory drive and levels of presynaptic inhibitory proteins, specifically in inhibitory-to-excitatory neuron synapses. Importantly, on a single-cell level hNR1 antibody selectively impairs NMDAR-mediated currents and synaptic transmission of cortical inhibitory neurons, yet has no effect on excitatory neurons, which contrasts with its effects on hippocampal neurons. Together, these findings provide a novel, cortex-specific mechanism of antibody-induced neuronal hyperexcitability, highlighting regional specificity underlying the pathology of autoimmune encephalitis.SIGNIFICANCE STATEMENT It is increasingly appreciated that the inadvertent activation of the immune system within CNS can underlie pathogenesis of neuropsychiatric disorders. Although the exact mechanisms remain elusive, autoantibodies derived from patients with autoimmune encephalitis pose a unique tool to study pathogenesis of neuropsychiatric states. Our analysis reveals that autoantibody against the NMDA receptor (NMDAR) has a distinct mechanism of action in the cortex, where it impairs function of inhibitory neurons leading to increased cortical network excitability, in contrast to previously described hippocampal synaptic mechanisms of information encoding, highlighting brain regional specificity. Notably, similar mechanism of NMDAR-mediated inhibitory hypofunction leading to cortical disinhibition has been suggested to underlie pathology of schizophrenia, hence our data provide new evidence for common mechanisms underlying neuropsychiatric disorders.
Asunto(s)
Encefalitis , Receptores de N-Metil-D-Aspartato , Autoanticuerpos/metabolismo , Enfermedad de Hashimoto , Humanos , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/metabolismoRESUMEN
Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood. Here, using a patient-derived monoclonal NR1 autoantibody (hNR1-mAb) and a co-culture system of microglia and neurons, we could show that hNR1-mAb bound to hippocampal neurons led to microglia-mediated removal of hNR1-mAb bound NMDARs. These complexes were found to accumulate inside endo-lysosomal compartments of microglia. Utilizing another patient isolated monoclonal autoantibody, against the α1-subunit of GABAA receptors (α1-GABAA -mAb), such removal of receptors was found to be specific to the antibody-bound receptor targets. Interestingly, along with receptor removal, we also observed a reduction in synapse number, more specifically in the numbers of post-synaptic proteins like PSD95 and Homer 1, when microglia were present in the culture. Importantly, mutations in the Fc region of hNR1-mAb, blocking its Fcγ receptor (FcγR) and complement binding, attenuated hNR1-mAb driven loss of NMDARs and synapses, indicating that microglia engagement by bound hNR1-mAb is critical for receptor and synapse loss. Our data argues for an active involvement of microglia in removal of NMDARs and other receptors in individuals with autoimmune encephalitis, thereby contributing to the etiology of these diseases.
Asunto(s)
Autoanticuerpos , Receptores de N-Metil-D-Aspartato , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Autoanticuerpos/metabolismo , Técnicas de Cocultivo , Microglía/metabolismo , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.
Asunto(s)
Proteína Huntingtina , Enfermedad de Huntington , Ubiquitinación/fisiología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Muerte Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Lisina/química , Lisina/metabolismo , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal , Agregación Patológica de Proteínas/metabolismo , Ratas , Ratas TransgénicasRESUMEN
The extraordinary diversity of excitatory synapse sizes is commonly attributed to activity-dependent processes that drive synaptic growth and diminution. Recent studies also point to activity-independent size fluctuations, possibly driven by innate synaptic molecule dynamics, as important generators of size diversity. To examine the contributions of activity-dependent and independent processes to excitatory synapse size diversity, we studied glutamatergic synapse size dynamics and diversification in cultured rat cortical neurons (both sexes), silenced from plating. We found that in networks with no history of activity whatsoever, synaptic size diversity was no less extensive than that observed in spontaneously active networks. Synapses in silenced networks were larger, size distributions were broader, yet these were rightward-skewed and similar in shape when scaled by mean synaptic size. Silencing reduced the magnitude of size fluctuations and weakened constraints on size distributions, yet these were sufficient to explain synaptic size diversity in silenced networks. Model-based exploration followed by experimental testing indicated that silencing-associated changes in innate molecular dynamics and fluctuation characteristics might negatively impact synaptic persistence, resulting in reduced synaptic numbers. This, in turn, would increase synaptic molecule availability, promote synaptic enlargement, and ultimately alter fluctuation characteristics. These findings suggest that activity-independent size fluctuations are sufficient to fully diversify glutamatergic synaptic sizes, with activity-dependent processes primarily setting the scale rather than the shape of size distributions. Moreover, they point to reciprocal relationships between synaptic size fluctuations, size distributions, and synaptic numbers mediated by the innate dynamics of synaptic molecules as they move in, out, and between synapses.SIGNIFICANCE STATEMENT Sizes of glutamatergic synapses vary tremendously, even when formed on the same neuron. This diversity is commonly thought to reflect the outcome of activity-dependent forms of synaptic plasticity, yet activity-independent processes might also play some part. Here we show that in neurons with no history of activity whatsoever, synaptic sizes are no less diverse. We show that this diversity is the product of activity-independent size fluctuations, which are sufficient to generate a full repertoire of synaptic sizes at correct proportions. By combining modeling and experimentation we expose reciprocal relationships between size fluctuations, synaptic sizes and synaptic counts, and show how these phenomena might be connected through the dynamics of synaptic molecules as they move in, out, and between synapses.
Asunto(s)
Modelos Neurológicos , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Corteza Cerebral/fisiología , Femenino , Masculino , RatasRESUMEN
Synaptic function crucially depends on uninterrupted synthesis and degradation of synaptic proteins. While much has been learned on synaptic protein synthesis, little is known on the routes by which synaptic proteins are degraded. Here we systematically studied how inhibition of the ubiquitin-proteasome system (UPS) affects the degradation rates of thousands of neuronal and synaptic proteins. We identified a group of proteins, including several proteins related to glutamate receptor trafficking, whose degradation rates were significantly slowed by UPS inhibition. Unexpectedly, however, degradation rates of most synaptic proteins were not significantly affected. Interestingly, many of the differential effects of UPS inhibition were readily explained by a quantitative framework that considered known metabolic turnover rates for the same proteins. In contrast to the limited effects on protein degradation, UPS inhibition profoundly and preferentially suppressed the synthesis of a large number of synaptic proteins. Our findings point to the importance of the UPS in the degradation of certain synaptic proteins, yet indicate that under basal conditions most synaptic proteins might be degraded through alternative pathways.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sinapsis/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Células Cultivadas , Femenino , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oligopéptidos/farmacología , Inhibidores de Proteasoma/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Ratas WistarRESUMEN
The idea that synaptic properties are defined by specific pre- and postsynaptic activity histories is one of the oldest and most influential tenets of contemporary neuroscience. Recent studies also indicate, however, that synaptic properties often change spontaneously, even in the absence of specific activity patterns or any activity whatsoever. What, then, are the relative contributions of activity history-dependent and activity history-independent processes to changes synapses undergo? To compare the relative contributions of these processes, we imaged, in spontaneously active networks of cortical neurons, glutamatergic synapses formed between the same axons and neurons or dendrites under the assumption that their similar activity histories should result in similar size changes over timescales of days. The size covariance of such commonly innervated (CI) synapses was then compared to that of synapses formed by different axons (non-CI synapses) that differed in their activity histories. We found that the size covariance of CI synapses was greater than that of non-CI synapses; yet overall size covariance of CI synapses was rather modest. Moreover, momentary and time-averaged sizes of CI synapses correlated rather poorly, in perfect agreement with published electron microscopy-based measurements of mouse cortex synapses. A conservative estimate suggested that ~40% of the observed size remodeling was attributable to specific activity histories, whereas ~10% and ~50% were attributable to cell-wide and spontaneous, synapse-autonomous processes, respectively. These findings demonstrate that histories of naturally occurring activity patterns can direct glutamatergic synapse remodeling but also suggest that the contributions of spontaneous, possibly stochastic, processes are at least as great.
Asunto(s)
Ácido Glutámico/metabolismo , Modelos Teóricos , Sinapsis/metabolismo , Animales , Ratas , Procesos EstocásticosRESUMEN
Synapses are dynamic molecular assemblies whose sizes fluctuate significantly over time-scales of hours and days. In the current study, we examined the possibility that the spontaneous microscopic dynamics exhibited by synaptic molecules can explain the macroscopic size fluctuations of individual synapses and the statistical properties of synaptic populations. We present a mesoscopic model, which ties the two levels. Its basic premise is that synaptic size fluctuations reflect cooperative assimilation and removal of molecules at a patch of postsynaptic membrane. The introduction of cooperativity to both assimilation and removal in a stochastic biophysical model of these processes, gives rise to features qualitatively similar to those measured experimentally: nanoclusters of synaptic scaffolds, fluctuations in synaptic sizes, skewed, stable size distributions and their scaling in response to perturbations. Our model thus points to the potentially fundamental role of cooperativity in dictating synaptic remodeling dynamics and offers a conceptual understanding of these dynamics in terms of central microscopic features and processes.
Asunto(s)
Neuronas/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Biología Computacional , Modelos Neurológicos , Tamaño de la Partícula , Unión Proteica , Ratas , Procesos EstocásticosRESUMEN
Glutamatergic synapse size remodeling is governed not only by specific activity forms but also by apparently stochastic processes with well-defined statistics. These spontaneous remodeling processes can give rise to skewed and stable synaptic size distributions, underlie scaling of these distributions and drive changes in glutamatergic synapse size "configurations". Where inhibitory synapses are concerned, however, little is known on spontaneous remodeling dynamics, their statistics, their activity dependence or their long-term consequences. Here we followed individual inhibitory synapses for days, and analyzed their size remodeling dynamics within the statistical framework previously developed for glutamatergic synapses. Similar to glutamatergic synapses, size distributions of inhibitory synapses were skewed and stable; at the same time, however, sizes of individual synapses changed considerably, leading to gradual changes in synaptic size configurations. The suppression of network activity only transiently affected spontaneous remodeling dynamics, did not affect synaptic size configuration change rates and was not followed by the scaling of inhibitory synapse size distributions. Comparisons with glutamatergic synapses within the same dendrites revealed a degree of coupling between nearby inhibitory and excitatory synapse remodeling, but also revealed that inhibitory synapse size configurations changed at considerably slower rates than those of their glutamatergic neighbors. These findings point to quantitative differences in spontaneous remodeling dynamics of inhibitory and excitatory synapses but also reveal deep qualitative similarities in the processes that control their sizes and govern their remodeling dynamics.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Modelos Neurológicos , Red Nerviosa/fisiología , Animales , Células Cultivadas , Biología Computacional , Hipocampo/citología , Hipocampo/fisiología , RatasRESUMEN
Long-term, repeated measurements of individual synaptic properties have revealed that synapses can undergo significant directed and spontaneous changes over time scales of minutes to weeks. These changes are presumably driven by a large number of activity-dependent and independent molecular processes, yet how these processes integrate to determine the totality of synaptic size remains unknown. Here we propose, as an alternative to detailed, mechanistic descriptions, a statistical approach to synaptic size dynamics. The basic premise of this approach is that the integrated outcome of the myriad of processes that drive synaptic size dynamics are effectively described as a combination of multiplicative and additive processes, both of which are stochastic and taken from distributions parametrically affected by physiological signals. We show that this seemingly simple model, known in probability theory as the Kesten process, can generate rich dynamics which are qualitatively similar to the dynamics of individual glutamatergic synapses recorded in long-term time-lapse experiments in ex-vivo cortical networks. Moreover, we show that this stochastic model, which is insensitive to many of its underlying details, quantitatively captures the distributions of synaptic sizes measured in these experiments, the long-term stability of such distributions and their scaling in response to pharmacological manipulations. Finally, we show that the average kinetics of new postsynaptic density formation measured in such experiments is also faithfully captured by the same model. The model thus provides a useful framework for characterizing synapse size dynamics at steady state, during initial formation of such steady states, and during their convergence to new steady states following perturbations. These findings show the strength of a simple low dimensional statistical model to quantitatively describe synapse size dynamics as the integrated result of many underlying complex processes.
Asunto(s)
Modelos Neurológicos , Sinapsis/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Biología Computacional , Femenino , Masculino , Neuronas/fisiología , Ratas , Procesos EstocásticosRESUMEN
BACKGROUND: Prolonged neuromodulatory regimes, such as those critically involved in promoting arousal and suppressing sleep-associated synchronous activity patterns, might be expected to trigger adaptation processes and, consequently, a decline in neuromodulator-driven effects. This possibility, however, has rarely been addressed. RESULTS: Using networks of cultured cortical neurons, acetylcholine microinjections and a novel closed-loop 'synchrony-clamp' system, we found that acetylcholine pulses strongly suppressed network synchrony. Over the course of many hours, however, synchrony invariably reemerged, even when feedback was used to compensate for declining cholinergic efficacy. Network synchrony also reemerged following its initial suppression by noradrenaline, but this did not occlude the suppression of synchrony or its gradual reemergence following subsequent cholinergic input. Importantly, cholinergic efficacy could be restored and preserved over extended time scales by periodically withdrawing cholinergic input. CONCLUSIONS: These findings indicate that the capacity of neuromodulators to suppress network synchrony is constrained by slow-acting, reactive processes. A multiplicity of neuromodulators and ultimately neuromodulator withdrawal periods might thus be necessary to cope with an inevitable reemergence of network synchrony.
Asunto(s)
Adaptación Fisiológica/fisiología , Neurotransmisores/farmacología , Animales , Células Cultivadas , Medios de Cultivo/química , Fenómenos Electrofisiológicos , Modelos Animales , Modelos Teóricos , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Synapses undergo substantial activity-dependent and independent remodeling over time scales of minutes, hours, and days. Presumably, changes in presynaptic properties should be matched by corresponding changes in postsynaptic properties and vice versa. Wherever measured, presynaptic and postsynaptic molecular properties tend to correlate, yet these correlations are often quite imperfect, raising questions as the origins of such mismatches: Are these the outcome of "single snapshot" analyses of asynchronous remodeling processes? Alternatively, do these indicate that synapses genuinely vary in the "stoichiometries" of their presynaptic and postsynaptic molecular contents? If so, are these "stoichiometries" preserved over time? To address these questions, we followed the matching dynamics of the presynaptic active-zone molecule Munc13-1 and the postsynaptic molecule PSD-95 in networks of cultured cortical mouse neurons. We find that presynaptic and postsynaptic remodeling were generally well correlated, but the degree of this correlation was highly variable, with little and even negative correlation observed at some synapses. No evidence was found that remodeling in one compartment consistently preceded remodeling in the other. Interestingly, even though the Munc13-1 and PSD-95 contents of individual synapses changed considerably over 15-22 h, Munc13-1/PSD-95 ratios, which varied over a fourfold range, were well conserved over these durations. These findings indicate that the "stoichiometries" of presynaptic and postsynaptic molecules can genuinely differ among synapses and that synapses can maintain their specific stoichiometries even in face of extensive presynaptic and postsynaptic remodeling.
Asunto(s)
Neuronas/citología , Dinámicas no Lineales , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Transformada , Células Cultivadas , Corteza Cerebral/citología , Homólogo 4 de la Proteína Discs Large , Femenino , Proteínas Fluorescentes Verdes/genética , Guanilato-Quinasas/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Factores de Tiempo , TransfecciónRESUMEN
Huntington's disease (HD) is caused by a glutamine repeat expansion in the protein huntingtin. Mutated huntingtin (mHtt) forms aggregates whose impacts on neuronal survival are still debated. Using weeks-long, continual imaging of cortical neurons, we find that mHtt is gradually sequestrated into peripheral, mainly axonal aggregates, concomitant with dramatic reductions in cytosolic mHtt levels and enhanced neuronal survival. in-situ pulse-chase imaging reveals that aggregates continually gain and lose mHtt, in line with these acting as mHtt sinks at equilibrium with cytosolic pools. Mutating two N-terminal lysines found to be ubiquitinated in HD animal models suppresses peripheral aggregate formation and reductions in cytosolic mHtt, promotes nuclear aggregate formation, stabilizes aggregates and leads to pervasive neuronal death. These findings demonstrate the capacity of aggregates formed at peripheral locations to sequester away cytosolic, presumably toxic mHtt forms and support a crucial role for N-terminal ubiquitination in promoting these processes and delaying neuronal death.
Asunto(s)
Muerte Celular , Proteína Huntingtina , Enfermedad de Huntington , Neuronas , Ubiquitinación , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Animales , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , HumanosRESUMEN
Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.
Asunto(s)
Aparato de Golgi/metabolismo , Neuronas/ultraestructura , Terminales Presinápticos/metabolismo , Vesículas Transportadoras/metabolismo , Factores de Edad , Animales , Anticuerpos/farmacología , Autoantígenos/metabolismo , Axones/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Proteínas del Citoesqueleto/metabolismo , Embrión de Mamíferos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Sinaptofisina/metabolismo , Factores de Tiempo , Transfección , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP rab , Red trans-Golgi/metabolismoRESUMEN
Recent studies indicate that synaptic vesicles (SVs) are continuously interchanged among nearby synapses at very significant rates. These dynamics and the lack of obvious barriers confining synaptic vesicles to specific synapses would seem to challenge the ability of synapses to maintain a constant amount of synaptic vesicles over prolonged time scales. Moreover, the extensive mobilization of synaptic vesicles associated with presynaptic activity might be expected to intensify this challenge. Here we examined the ability of individual presynaptic boutons of rat hippocampal neurons to maintain their synaptic vesicle content, and the degree to which this ability is affected by continuous activity. We found that the synaptic vesicle content of individual boutons belonging to the same axons gradually changed over several hours, and that these changes occurred independently of activity. Intermittent stimulation for 1 h accelerated rates of vesicle pool size change. Interestingly, however, following stimulation cessation, vesicle pool size change rates gradually converged with basal change rates. Over similar time scales, active zones (AZs) exhibited substantial remodeling; yet, unlike synaptic vesicles, AZ remodeling was not affected by the stimulation paradigms used here. These findings indicate that enhanced activity levels can increase synaptic vesicle redistribution among nearby synapses, but also highlight the presence of forces that act to restore particular set points in terms of SV contents, and support a role for active zones in preserving such set points. These findings also indicate, however, that neither AZ size nor SV content set points are particularly stable, questioning the long-term tenacity of presynaptic specializations.
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Neuronas/citología , Terminales Presinápticos/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Animales Recién Nacidos , Proteínas Bacterianas , Células Cultivadas , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Ratas Sprague-Dawley , Vesículas Sinápticas/metabolismo , Factores de Tiempo , Transducción GenéticaRESUMEN
At the synapse, vesicles stably dock at the active zone. However, in cellular membranes, proteins undergo a diffusive motion. It is not known how the motion of membrane proteins involved in vesicle exocytosis is compatible with both vesicle docking and the dynamic remodeling of the plasma membrane imposed by cycles of exocytosis and endocytosis. To address this question, we studied the motion of the presynaptic membrane protein syntaxin1A at both the population and single-molecule levels in primary cultures of rat spinal cord neurons. Syntaxin1A was rapidly exchanged between synaptic and extrasynaptic regions. Changes in syntaxin1A mobility were associated with interactions related to the formation of the exocytotic complex. Finally, we propose a reaction-diffusion model reconciling the observed diffusive properties of syntaxin at the population level and at the molecular level. This work allows us to describe the diffusive behavior and kinetics of interactions between syntaxin1A and its partners that lead to its transient stabilization at the synapse.
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Exocitosis/fisiología , Neuronas/metabolismo , Proteínas SNARE/metabolismo , Sinapsis/metabolismo , Sintaxina 1/metabolismo , Animales , Axones/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Endocitosis/fisiología , Modelos Biológicos , Neuronas/citología , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Biological networks are ubiquitously modular, a feature that is believed to be essential for the enhancement of their functional capacities. Here, we have used a simple modular in vitro design to examine the possibility that modularity enhances network functionality in the context of input representation. We cultured networks of cortical neurons obtained from newborn rats in vitro on substrate-integrated multi-electrode arrays, forcing the network to develop two well-defined modules of neural populations that are coupled by a narrow canal. We measured the neural activity, and examined the capacity of each module to individually classify (i.e. represent) spatially distinct electrical stimuli and propagate input-specific activity features to their downstream coupled counterpart. We show that, although each of the coupled modules maintains its autonomous functionality, a significant enhancement of representational capacity is achieved when the system is observed as a whole. We interpret our results in terms of a relative decorrelation effect imposed by weak coupling between modules.
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Potenciales de Acción/fisiología , Corteza Cerebral/fisiología , Red Nerviosa/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Corteza Cerebral/citología , Red Nerviosa/citología , Vías Nerviosas/citología , Neuronas/citología , Cultivo Primario de Células , Ratas , Ratas Sprague-DawleyRESUMEN
Synaptic plasticity is widely believed to constitute a key mechanism for modifying functional properties of neuronal networks. This belief implicitly implies, however, that synapses, when not driven to change their characteristics by physiologically relevant stimuli, will maintain these characteristics over time. How tenacious are synapses over behaviorally relevant time scales? To begin to address this question, we developed a system for continuously imaging the structural dynamics of individual synapses over many days, while recording network activity in the same preparations. We found that in spontaneously active networks, distributions of synaptic sizes were generally stable over days. Following individual synapses revealed, however, that the apparently static distributions were actually steady states of synapses exhibiting continual and extensive remodeling. In active networks, large synapses tended to grow smaller, whereas small synapses tended to grow larger, mainly during periods of particularly synchronous activity. Suppression of network activity only mildly affected the magnitude of synaptic remodeling, but dependence on synaptic size was lost, leading to the broadening of synaptic size distributions and increases in mean synaptic size. From the perspective of individual neurons, activity drove changes in the relative sizes of their excitatory inputs, but such changes continued, albeit at lower rates, even when network activity was blocked. Our findings show that activity strongly drives synaptic remodeling, but they also show that significant remodeling occurs spontaneously. Whereas such spontaneous remodeling provides an explanation for "synaptic homeostasis" like processes, it also raises significant questions concerning the reliability of individual synapses as sites for persistently modifying network function.
Asunto(s)
Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción/fisiología , Algoritmos , Animales , Animales Recién Nacidos , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Homólogo 4 de la Proteína Discs Large , Potenciales Postsinápticos Excitadores/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Modelos Neurológicos , Redes Neurales de la Computación , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Synaptic integrity and function depend on myriad proteins - labile molecules with finite lifetimes that need to be continually replaced with freshly synthesized copies. Here we describe experiments designed to expose synaptic (and neuronal) properties and functions that are particularly sensitive to disruptions in protein supply, identify proteins lost early upon such disruptions, and uncover potential, yet currently underappreciated failure points. We report here that acute suppressions of protein synthesis are followed within hours by reductions in spontaneous network activity levels, impaired oxidative phosphorylation and mitochondrial function, and, importantly, destabilization and loss of both excitatory and inhibitory postsynaptic specializations. Conversely, gross impairments in presynaptic vesicle recycling occur over longer time scales (days), as does overt cell death. Proteomic analysis identified groups of potentially essential 'early-lost' proteins including regulators of synapse stability, proteins related to bioenergetics, fatty acid and lipid metabolism, and, unexpectedly, numerous proteins involved in Alzheimer's disease pathology and amyloid beta processing. Collectively, these findings point to neuronal excitability, energy supply and synaptic stability as early-occurring failure points under conditions of compromised supply of newly synthesized protein copies.