RESUMEN
Dendrites receive excitatory synaptic inputs from upstream cell ensembles to trigger action potentials at the cell body. The efficiency of excitatory synaptic inputs on neuronal output depends on the spatiotemporal pattern of synaptic inputs. However, technical limitations still make it unclear how synaptic inputs are organized along dendrites in both space and time. Spine calcium imaging, which records synaptic inputs as calcium transients at individual spines using calcium ion-sensitive fluorophores, is a unique method for studying the spatiotemporal patterns of synaptic input. We developed a functional multiple-spine calcium imaging (fMsCI) that combines whole-cell patch-clamp recording and spinning-disk confocal imaging to observe hundreds of synaptic inputs simultaneously. Using this method, we discovered sequential synaptic inputs that accompanied sharp wave ripple oscillations. In this review, I will discuss the function of sequential synaptic inputs and the potential uses of fMsCI to better understand neurological disorders.
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Calcio , Colorantes Fluorescentes , Potenciales de AcciónRESUMEN
Cerebral edema is a major contributor to the mortality associated with ischemic stroke and traumatic brain injuries; however, limited therapeutic strategies are available for cerebral edema. Aquaporin-4 (AQP4), the main water channel in the brain plays a key role in water homeostasis and edema formation in the central nervous system. Therefore, regulation of AQP4 function or expression is considered a possible target for treatment of edema. Despite extensive research over several decades, AQP4 inhibitors have not been approved for the treatment of edema in humans. Further studies are warranted to gain a deeper understanding of the exact properties and functions of AQP4, to facilitate the development of newer therapeutic approaches for cerebral edema.
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Edema Encefálico , Acuaporina 4 , Encéfalo/metabolismo , Sistema Nervioso Central , Humanos , Agua/metabolismoRESUMEN
Most excitatory inputs arrive at dendritic spines in a postsynaptic neuron. To understand dendritic information processing, it is critical to scrutinize the spatiotemporal dynamics of synaptic inputs along dendrites. This protocol combines spinning-disk confocal imaging with whole-cell patch-clamp recording to perform wide-field, high-speed optical recording of synaptic inputs in a neuron loaded with a calcium indicator in ex vivo cultured networks. Our protocol enables simultaneous detection of synaptic inputs as calcium signals from hundreds of spines in multiple dendritic branches. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2012, 2016), Kobayashi et al. (2019), and Ishikawa and Ikegaya (2020).
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Encéfalo/diagnóstico por imagen , Calcio/metabolismo , Técnicas de Placa-Clamp/métodos , Terminales Presinápticos/metabolismo , Potenciales de Acción , Encéfalo/metabolismo , Dendritas/metabolismo , Espinas Dendríticas/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Neuronas/metabolismo , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Two-photon imaging is a major recording technique used in neuroscience. However, it suffers from several limitations, including a low sampling rate, the nonlinearity of calcium responses, the slow dynamics of calcium dyes and a low SNR, all of which severely limit the potential of two-photon imaging to elucidate neuronal dynamics with high temporal resolution. We developed a hyperacuity algorithm (HA_time) based on an approach that combines a generative model and machine learning to improve spike detection and the precision of spike time inference. Bayesian inference was performed to estimate the calcium spike model, assuming constant spike shape and size. A support vector machine using this information and a jittering method maximizing the likelihood of estimated spike times enhanced spike time estimation precision approximately fourfold (range, 2-7; mean, 3.5-4.0; 2SEM, 0.1-0.25) compared to the sampling interval. Benchmark scores of HA_time for biological data from three different brain regions were among the best of the benchmark algorithms. Simulation of broader data conditions indicated that our algorithm performed better than others with high firing rate conditions. Furthermore, HA_time exhibited comparable performance for conditions with and without ground truths. Thus HA_time is a useful tool for spike reconstruction from two-photon imaging.
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Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells.
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Calcio , Colorantes Fluorescentes , Iones , Lisosomas , RodaminasRESUMEN
The sequential reactivation of memory-relevant neuronal ensembles during hippocampal sharp-wave (SW) ripple oscillations reflects cognitive processing. However, how a downstream neuron decodes this spatiotemporally organized activity remains unexplored. Using subcellular calcium imaging from CA1 pyramidal neurons in ex vivo hippocampal networks, we discovered that neighboring spines are activated serially along dendrites toward or away from cell bodies. Sequential spine activity was engaged repeatedly in different SWs in a complex manner. In a single SW event, multiple sequences appeared discretely in dendritic trees, but overall, sequences occurred preferentially in some dendritic branches. Thus, sequential replays of multineuronal spikes are distributed across several compartmentalized dendritic foci of a postsynaptic neuron, with their spatiotemporal features preserved.
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Potenciales de Acción/fisiología , Hipocampo/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Espinas Dendríticas/fisiología , Femenino , Masculino , Células Piramidales/fisiología , RatasRESUMEN
BACKGROUND: A method that promotes the retrieval of lost long-term memories has not been well established. Histamine in the central nervous system is implicated in learning and memory, and treatment with antihistamines impairs learning and memory. Because histamine H3 receptor inverse agonists upregulate histamine release, the inverse agonists may enhance learning and memory. However, whether the inverse agonists promote the retrieval of forgotten long-term memory has not yet been determined. METHODS: Here, we employed multidisciplinary methods, including mouse behavior, calcium imaging, and chemogenetic manipulation, to examine whether and how the histamine H3 receptor inverse agonists, thioperamide and betahistine, promote the retrieval of a forgotten long-term object memory in mice. In addition, we conducted a randomized double-blind, placebo-controlled crossover trial in healthy adult participants to investigate whether betahistine treatment promotes memory retrieval in humans. RESULTS: The treatment of H3 receptor inverse agonists induced the recall of forgotten memories even 1 week and 1 month after training in mice. The memory recovery was mediated by the disinhibition of histamine release in the perirhinal cortex, which activated the histamine H2 receptor. Histamine depolarized perirhinal cortex neurons, enhanced their spontaneous activity, and facilitated the reactivation of behaviorally activated neuronal ensembles. A human clinical trial revealed that treatment of H3 receptor inverse agonists is specifically more effective for items that are more difficult to remember and subjects with poorer performance. CONCLUSIONS: These results highlight a novel interaction between the central histamine signaling and memory engrams.
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Agonistas de los Receptores Histamínicos/farmacología , Trastornos de la Memoria/tratamiento farmacológico , Recuerdo Mental/efectos de los fármacos , Corteza Perirrinal/efectos de los fármacos , Adulto , Animales , Betahistina , Cognición/efectos de los fármacos , Método Doble Ciego , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Apego a Objetos , Piperidinas , Procesos Estocásticos , Adulto JovenRESUMEN
The effect of excitatory synaptic input on the excitation of the cell body is believed to vary depending on where and when the synaptic activation occurs in dendritic trees and the spatiotemporal modulation by inhibitory synaptic input. However, few studies have examined how individual synaptic inputs influence the excitability of the cell body in spontaneously active neuronal networks mainly because of the lack of an appropriate method. We developed a calcium imaging technique that monitors synaptic inputs to hundreds of spines from a single neuron with millisecond resolution in combination with whole-cell patch-clamp recordings of somatic excitation. In rat hippocampal CA3 pyramidal neurons ex vivo, a fraction of the excitatory synaptic inputs were not detectable in the cell body against background noise. These synaptic inputs partially restored their somatic impact when a GABAA receptor blocker was intracellularly perfused. Thus, GABAergic inhibition reduces the influence of some excitatory synaptic inputs on the somatic excitability. Numerical simulation using a single neuron model demonstrates that the timing and locus of a dendritic GABAergic input are critical to exert this effect. Moreover, logistic regression analyses suggest that the GABAergic inputs sectionalize spine activity; that is, only some subsets of synchronous synaptic activity seemed to be preferably passed to the cell body. Thus, dendrites actively sift inputs from specific presynaptic cell assemblies.
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Calcio/metabolismo , Espinas Dendríticas/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/metabolismo , Receptores de GABA-A/metabolismo , Potenciales de Acción , Animales , Espinas Dendríticas/efectos de los fármacos , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/metabolismo , Potenciales Postsinápticos Excitadores , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas , Ratas WistarRESUMEN
The specific effects of sleep on synaptic plasticity remain unclear. We report that mouse hippocampal sharp-wave ripple oscillations serve as intrinsic events that trigger long-lasting synaptic depression. Silencing of sharp-wave ripples during slow-wave states prevented the spontaneous down-regulation of net synaptic weights and impaired the learning of new memories. The synaptic down-regulation was dependent on the N-methyl-d-aspartate receptor and selective for a specific input pathway. Thus, our findings are consistent with the role of slow-wave states in refining memory engrams by reducing recent memory-irrelevant neuronal activity and suggest a previously unrecognized function for sharp-wave ripples.
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Hipocampo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Memoria/fisiología , Sinapsis/fisiología , Animales , Regulación hacia Abajo , Aprendizaje/fisiología , Ratones , Ratones Endogámicos C57BL , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiología , Sueño/fisiologíaRESUMEN
Functional multineuron calcium imaging (fMCI) provides a useful experimental platform to simultaneously capture the spatiotemporal patterns of neuronal activity from a large cell population in situ. However, fMCI often suffers from low signal-to-noise ratios (S/N). The main factor that causes the low S/N is shot noise that arises from photon detectors. Here, we propose a new denoising procedure, termed the Okada filter, which is designed to reduce shot noise under low S/N conditions, such as fMCI. The core idea of the Okada filter is to replace the fluorescence intensity value of a given frame time with the average of two values at the preceding and following frames unless the focused value is the median among these three values. This process is iterated serially throughout a time-series vector. In fMCI data of hippocampal neurons, the Okada filter rapidly reduces background noise and significantly improves the S/N. The Okada filter is also applicable for reducing shot noise in electrophysiological data and photographs. Finally, the Okada filter can be described using a single continuous differentiable equation based on the logistic function and is thus mathematically tractable.
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Calcio/metabolismo , Diagnóstico por Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Células Piramidales/metabolismo , Potenciales de Acción/fisiología , Algoritmos , Animales , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Diagnóstico por Imagen/instrumentación , Procesamiento de Imagen Asistido por Computador/instrumentación , Microscopía Fluorescente , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/fisiología , Ratas Wistar , Reproducibilidad de los Resultados , Relación Señal-RuidoRESUMEN
The dynamic interactions between synaptic excitation and inhibition (E/I) shape membrane potential fluctuations and determine patterns of neuronal outputs; however, the spatiotemporal organization of these interactions within a single cell is poorly understood. Here, we investigated the relationship between local synaptic excitation and global inhibition in hippocampal pyramidal neurons using functional dendrite imaging in combination with whole-cell recordings of inhibitory postsynaptic currents. We found that the sums of spine inputs over dendritic trees were counterbalanced by a proportional amount of somatic inhibitory inputs. This online E/I correlation was maintained in dendritic segments that were longer than 50 µm. However, at the single spine level, only 22% of the active spines were activated with inhibitory inputs. This inhibition-coupled activity occurred mainly in the spines with large heads. These results shed light on a microscopic E/I-balancing mechanism that operates at selected synapses and that may increase the accuracy of neural information.
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Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Hipocampo/citología , Técnicas de Placa-Clamp , Células Piramidales/citología , Ratas , Ratas Wistar , Análisis de la Célula Individual , Sinapsis/ultraestructura , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Calcium imaging has become a fundamental modality for studying neuronal circuit dynamics both in vitro and in vivo. However, identifying calcium events (CEs) from spectral data remains laborious and difficult, especially since the signal-to-noise ratio (SNR) often falls below 2. Existing automated signal detection methods are generally applied at high SNRs, leaving a large need for an automated algorithm that can accurately extract CEs from fluorescence intensity data of SNR 2 and below. NEW METHOD: In this work we develop a Matched filter for Multi-unit Calcium Event (MMiCE) detection to extract CEs from fluorescence intensity traces of simulated and experimentally recorded neuronal calcium imaging data. RESULTS: MMiCE reached perfect performance on simulated data with SNR ≥ 2 and a true positive (TP) rate of 98.27% (± 1.38% with a 95% confidence interval), and a false positive(FP) rate of 6.59% (± 2.56%) on simulated data with SNR 0.2. On real data, verified by patch-clamp recording, MMiCE performed with a TP rate of 100.00% (± 0.00) and a FP rate of 2.04% (± 4.10). COMPARISON WITH EXISTING METHOD(S): This high level of performance exceeds existing methods at SNRs as low as 0.2, which are well below those used in previous studies (SNR ≃ 5-10). CONCLUSION: Overall, the MMiCE detector performed exceptionally well on both simulated data, and experimentally recorded neuronal calcium imaging data. The MMiCE detector is accurate, reliable, well suited for wide-spread use, and freely available at sites.uci.edu/aggies or from the corresponding author.
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Calcio/metabolismo , Hipocampo/citología , Neuronas/citología , Imagen Óptica/métodos , Relación Señal-Ruido , Animales , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Activity patterns of neural population are constrained by underlying biological mechanisms. These patterns are characterized not only by individual activity rates and pairwise correlations but also by statistical dependencies among groups of neurons larger than two, known as higher-order interactions (HOIs). While HOIs are ubiquitous in neural activity, primary characteristics of HOIs remain unknown. Here, we report that simultaneous silence (SS) of neurons concisely summarizes neural HOIs. Spontaneously active neurons in cultured hippocampal slices express SS that is more frequent than predicted by their individual activity rates and pairwise correlations. The SS explains structured HOIs seen in the data, namely, alternating signs at successive interaction orders. Inhibitory neurons are necessary to maintain significant SS. The structured HOIs predicted by SS were observed in a simple neural population model characterized by spiking nonlinearity and correlated input. These results suggest that SS is a ubiquitous feature of HOIs that constrain neural activity patterns and can influence information processing.
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Red Nerviosa/fisiología , Neuronas/fisiología , Animales , Femenino , Hipocampo/fisiología , Masculino , Modelos Neurológicos , Ratas , Ratas WistarRESUMEN
Crosstalk between neurons and glia may constitute a significant part of information processing in the brain. We present a novel method of statistically identifying interactions in a neuron-glia network. We attempted to identify neuron-glia interactions from neuronal and glial activities via maximum-a-posteriori (MAP)-based parameter estimation by developing a generalized linear model (GLM) of a neuron-glia network. The interactions in our interest included functional connectivity and response functions. We evaluated the cross-validated likelihood of GLMs that resulted from the addition or removal of connections to confirm the existence of specific neuron-to-glia or glia-to-neuron connections. We only accepted addition or removal when the modification improved the cross-validated likelihood. We applied the method to a high-throughput, multicellular in vitro Ca2+ imaging dataset obtained from the CA3 region of a rat hippocampus, and then evaluated the reliability of connectivity estimates using a statistical test based on a surrogate method. Our findings based on the estimated connectivity were in good agreement with currently available physiological knowledge, suggesting our method can elucidate undiscovered functions of neuron-glia systems.
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Región CA3 Hipocampal/citología , Calcio/metabolismo , Biología Computacional/métodos , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Región CA3 Hipocampal/metabolismo , Modelos Neurológicos , Modelos Estadísticos , Ratas , Ratas WistarRESUMEN
Spontaneous neuronal activity is present in virtually all brain regions, but neither its function nor spatiotemporal patterns are fully understood. Ex vivo organotypic slice cultures may offer an opportunity to investigate some aspects of spontaneous activity, because they self-restore their networks that collapsed during slicing procedures. In hippocampal networks, we compared the levels and patterns of in vivo spontaneous activity to those in acute and cultured slices. We found that the firing rates and excitatory synaptic activity in the in vivo hippocampus are more similar to those in slice cultures compared to acute slices. The soft confidence-weighted algorithm, a machine learning technique without human bias, also revealed that hippocampal slice cultures resemble the in vivo hippocampus in terms of the overall tendency of the parameters of spontaneous activity.
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Red Nerviosa/fisiología , Neuronas/fisiología , Animales , Células Cultivadas , Femenino , Hipocampo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratas Wistar , Sinapsis/fisiologíaRESUMEN
Hippocampal sharp wave (SW)/ripple complexes are thought to contribute to memory consolidation. Previous studies suggest that behavioral rewards facilitate SW occurrence in vivo. However, little is known about the precise mechanism underlying this enhancement. Here, we examined the effect of dopaminergic neuromodulation on spontaneously occurring SWs in acute hippocampal slices. Local field potentials were recorded from the CA1 region. A brief (1 min) treatment with dopamine led to a persistent increase in the event frequency and the magnitude of SWs. This effect lasted at least for our recording period of 45 min and did not occur in the presence of a dopamine D1/D5 receptor antagonist. Functional multineuron calcium imaging revealed that dopamine-induced SW augmentation was associated with an enriched repertoire of the firing patterns in SW events, whereas the overall tendency of individual neurons to participate in SWs and the mean number of cells participating in a single SW were maintained. Therefore, dopaminergic activation is likely to reorganize cell assemblies during SWs.
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Dopamina/metabolismo , Hipocampo/efectos de los fármacos , Memoria/fisiología , Neuronas/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Dopamina/administración & dosificación , Hipocampo/metabolismo , Hipocampo/fisiología , Memoria/efectos de los fármacos , Ratones , Neuronas/fisiología , Receptores de Dopamina D5/antagonistas & inhibidoresRESUMEN
Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level.
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Astrocitos/metabolismo , Región CA3 Hipocampal/fisiología , Señalización del Calcio , Células Piramidales/fisiología , Transmisión Sináptica , Animales , Astrocitos/fisiología , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Ratas , Ratas WistarRESUMEN
Cortical microcircuits are nonrandomly wired by neurons. As a natural consequence, spikes emitted by microcircuits are also nonrandomly patterned in time and space. One of the prominent spike organizations is a repetition of fixed patterns of spike series across multiple neurons. However, several questions remain unsolved, including how precisely spike sequences repeat, how the sequences are spatially organized, how many neurons participate in sequences, and how different sequences are functionally linked. To address these questions, we monitored spontaneous spikes of hippocampal CA3 neurons ex vivo using a high-speed functional multineuron calcium imaging (fMCI) technique that allowed us to monitor spikes with millisecond resolution and to record the location of spiking and non-spiking neurons. Multineuronal spike sequences (MSSs) were overrepresented in spontaneous activity compared to the statistical chance level. Approximately 75% of neurons participated in at least one sequence during our observation period. The participants were sparsely dispersed and did not show specific spatial organization. The number of sequences relative to the chance level decreased when larger time frames were used to detect sequences. Thus, sequences were precise at the millisecond level. Sequences often shared common spikes with other sequences; parts of sequences were subsequently relayed by following sequences, generating complex chains of multiple sequences.