RESUMO
Glial cells such as astrocytes can modulate neuronal signaling. Astrocytes can also acquire a reactive phenotype that correlates with cognitive impairments in brain diseases. A study in PLOS Biology shows that prolonged activation of astrocytes can trigger both cognitive impairments and a reactive astrocyte phenotype.
Assuntos
Astrócitos , Cognição , Astrócitos/fisiologia , Animais , Humanos , Cognição/fisiologia , Disfunção Cognitiva/fisiopatologia , Neurônios/fisiologiaRESUMO
Astrocytes functionally interact with neurons and with other brain cells. Although not electrically excitable, astrocytes display a complex repertoire of intracellular Ca2+ signalling that evolves in space and time within single astrocytes and across astrocytic networks. Decoding the physiological meaning of these dynamic changes in astrocytic Ca2+ activity has remained a major challenge. This Review describes experimental preparations and methods for recording and studying Ca2+ activity in astrocytes, focusing on the analysis of Ca2+ signalling events in single astrocytes and in astrocytic networks. The limitations of existing experimental approaches and ongoing technical and conceptual challenges in the interpretation of astrocytic Ca2+ events and their spatio-temporal patterns are also discussed.
Assuntos
Astrócitos/fisiologia , Encéfalo/fisiologia , Sinalização do Cálcio/fisiologia , Neurônios/fisiologia , Animais , Células Cultivadas , Humanos , Imagem ÓpticaRESUMO
Long-term modifications of astrocyte function and morphology are well known to occur in epilepsy. They are implicated in the development and manifestation of the disease, but the relevant mechanisms and their pathophysiological role are not firmly established. For instance, it is unclear how quickly the onset of epileptic activity triggers astrocyte morphology changes and what the relevant molecular signals are. We therefore used two-photon excitation fluorescence microscopy to monitor astrocyte morphology in parallel to the induction of epileptiform activity. We uncovered astrocyte morphology changes within 10-20 min under various experimental conditions in acute hippocampal slices. In vivo, induction of status epilepticus resulted in similarly altered astrocyte morphology within 30 min. Further analysis in vitro revealed a persistent volume reduction of peripheral astrocyte processes triggered by induction of epileptiform activity. In addition, an impaired diffusion within astrocytes and within the astrocyte network was observed, which most likely is a direct consequence of the astrocyte remodeling. These astrocyte morphology changes were prevented by inhibition of the Rho GTPase RhoA and of the Rho-associated kinase (ROCK). Selective deletion of ROCK1 but not ROCK2 from astrocytes also prevented the morphology change after induction of epileptiform activity and reduced epileptiform activity. Together these observations reveal that epileptic activity triggers a rapid ROCK1-dependent astrocyte morphology change, which is mechanistically linked to the strength of epileptiform activity. This suggests that astrocytic ROCK1 signaling is a maladaptive response of astrocytes to the onset of epileptic activity.
Assuntos
Epilepsia , Estado Epiléptico , Humanos , Astrócitos , Quinases Associadas a rho , HipocampoRESUMO
Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.
Assuntos
Dendritos , Epilepsia , Camundongos , Animais , Dendritos/fisiologia , Hipocampo/fisiologia , Acetamidas/metabolismo , Células Piramidais/metabolismo , Epilepsia/metabolismo , Potenciais de Ação/fisiologiaRESUMO
Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.
Assuntos
Isquemia Encefálica/metabolismo , Neurônios/metabolismo , Imagem Óptica/métodos , Sódio/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Isquemia Encefálica/patologia , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/química , Técnicas de Cultura de Órgãos , Canais de Cátion TRPV/análiseRESUMO
NG2 glia represents a distinct type of macroglial cells in the CNS and is unique among glia because they receive synaptic input from neurons. They are abundantly present in white and gray matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of gray matter NG2 glia and their synaptic input are still ill defined. Here, we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Kir4.1 was deleted at postnatal day 23-26 (recombination efficiency about 75%) and mice were investigated 3-8 weeks later. Notably, these mice with dysfunctional NG2 glia demonstrated improved spatial memory as revealed by testing new object location recognition while working and social memory remained unaffected. Focussing on the hippocampus, we found that loss of Kir4.1 potentiated synaptic depolarizations of NG2 glia and stimulated the expression of myelin basic protein while proliferation and differentiation of hippocampal NG2 glia remained largely unaffected. Mice with targeted deletion of the K+ channel in NG2 glia showed impaired long-term potentiation at CA3-CA1 synapses, which could be fully rescued by extracellular application of a TrkB receptor agonist. Our data demonstrate that proper NG2 glia function is important for normal brain function and behavior.
Assuntos
Neuroglia , Proteoglicanas , Camundongos , Animais , Proteoglicanas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Plasticidade Neuronal , Antígenos/metabolismoRESUMO
Astrocytes are integral components of brain circuits, where they sense, process, and respond to surrounding activity, maintaining homeostasis and regulating synaptic transmission, the sum of which results in behavior modulation. These interactions are possible due to their complex morphology, composed of a tree-like structure of processes to cover defined territories ramifying in a mesh-like system of fine leaflets unresolved by conventional optic microscopy. While recent reports devoted more attention to leaflets and their dynamic interactions with synapses, our knowledge about the tree-like "backbone" structure in physiological conditions is incomplete. Recent transcriptomic studies described astrocyte molecular diversity, suggesting structural heterogeneity in regions such as the hippocampus, which is crucial for cognitive and emotional behaviors. In this study, we carried out the structural analysis of astrocytes across the hippocampal subfields of Cornu Ammonis area 1 (CA1) and dentate gyrus in the dorsoventral axis. We found that astrocytes display heterogeneity across the hippocampal subfields, which is conserved along the dorsoventral axis. We further found that astrocytes appear to contribute in an exocytosis-dependent manner to a signaling loop that maintains the backbone structure. These findings reveal astrocyte heterogeneity in the hippocampus, which appears to follow layer-specific cues and depend on the neuro-glial environment.
Assuntos
Astrócitos , Hipocampo , Animais , Camundongos , Astrócitos/fisiologia , Região CA1 Hipocampal , Neuroglia , Transmissão SinápticaRESUMO
Astrocytes play a dual role in the brain. On the one hand, they are active signaling partners of neurons and can for instance control synaptic transmission and its plasticity. On the other hand, they fulfill various homeostatic functions such as clearance of glutamate and K+ released from neurons. The latter is for instance important for limiting neuronal excitability. Therefore, an impairment or failure of glutamate and K+ clearance will lead to increased neuronal excitability, which could trigger or aggravate brain diseases such as epilepsy, in which neuronal hyperexcitability plays a role. Experimental data indicate that astrocytes could have such a causal role in epilepsy, but the role of astrocytes as initiators of epilepsy and the relevant mechanisms are under debate. In this overview, we will discuss the potential mechanisms with focus on K+ clearance, glutamate uptake and homoeostasis and related mechanisms, and the evidence for their causative role in epilepsy.
Assuntos
Astrócitos , Epilepsia , Humanos , Astrócitos/fisiologia , Encéfalo , Transmissão Sináptica , Ácido GlutâmicoRESUMO
NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron-glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABAA receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron-glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABAA receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity.
Assuntos
Receptores de GABA-A , Animais , Camundongos , Ácido gama-Aminobutírico , Hipocampo , Interneurônios , NeurogliaRESUMO
Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca2+ activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca2+ activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca2+ activity recordings, hindering global comparisons. Here, we present a biophysically-based analytical concept for deciphering the complex spatio-temporal changes of Ca2+ biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel-based multi-threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca2+ activity across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. We identified extended Ca2+ activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long-lasting astrocytic Ca2+ activity. Our MTED strategy, as a parameter-free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.
Assuntos
Astrócitos , Sinalização do Cálcio , Animais , Astrócitos/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Camundongos , Neurônios/metabolismoRESUMO
Astrocytes are an important component of the multipartite synapse and crucial for proper neuronal network function. Although small GTPases of the Rho family are powerful regulators of cellular morphology, the signaling modules of Rho-mediated pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin receptor 4 (5-HT4 R) is expressed in hippocampal astrocytes, both in vitro and in vivo. Through fluorescence microscopy, we established that 5-HT4 R activation triggered RhoA activity via Gα13 -mediated signaling, which boosted filamentous actin assembly, leading to morphological changes in hippocampal astrocytes. We investigated the effects of these 5-HT4 R-mediated changes in mixed cultures and in acute slices, in which 5-HT4 R was expressed exclusively in astrocytes. In both systems, 5-HT4 R-RhoA signaling changed glutamatergic synaptic transmission: It increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic 5-HT4 R-Gα13 -RhoA signaling is a previously unrecognized molecular pathway involved in the functional regulation of excitatory synaptic circuits.
Assuntos
Astrócitos , Serotonina , Potenciais Pós-Sinápticos Excitadores , Hipocampo , Receptores de Serotonina/genética , Transmissão SinápticaRESUMO
Astrocytes form large networks, in which individual cells are connected via gap junctions. It is thought that this astroglial gap junction coupling contributes to the buffering of extracellular K+ increases. However, it is largely unknown how the control of extracellular K+ by astroglial gap junction coupling depends on the underlying activity patterns and on the magnitude of extracellular K+ increases. We explored this dependency in acute hippocampal slices (CA1, stratum radiatum) by direct K+ -sensitive microelectrode recordings and acute pharmacological inhibition of gap junctions. K+ transients evoked by synaptic and axonal activity were largely unaffected by acute astroglial uncoupling in slices obtained from young and adult rats. Iontophoretic K+ -application enabled us to generate K+ gradients with defined spatial properties and magnitude. By varying the K+ -iontophoresis position and protocol, we found that acute pharmacological uncoupling increases the amplitude of K+ transients once their initial amplitude exceeded ~10 mM. Our experiments demonstrate that the contribution of gap junction coupling to buffering of extracellular K+ gradients is limited to large and localized K+ increases.
Assuntos
Astrócitos/metabolismo , Região CA1 Hipocampal/metabolismo , Junções Comunicantes/metabolismo , Neurônios/metabolismo , Potássio/metabolismo , Sinapses/metabolismo , Animais , Potenciais da Membrana/fisiologia , Ratos , Ratos WistarRESUMO
Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.
Assuntos
Corantes Fluorescentes/química , Glicina/análise , Hipocampo/química , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Células HEK293 , Humanos , Masculino , Imagem Óptica , Ratos , Ratos WistarRESUMO
In the central nervous system, several forms of experience-dependent plasticity, learning and memory require the activity-dependent control of synaptic efficacy. Despite substantial progress in describing synaptic plasticity, mechanisms related to heterogeneity of synaptic functions at local circuits remain elusive. Here we studied the functional and molecular aspects of hippocampal circuit plasticity by analyzing excitatory synapses at basal and apical dendrites of mouse hippocampal pyramidal cells (CA1 region) in acute brain slices. In the past decade, activity of metalloproteinases (MMPs) has been implicated as a widespread and critical factor in plasticity mechanisms at various projections in the CNS. However, in the present study we discovered that in striking contrast to apical dendrites, synapses located within basal dendrites undergo MMP-independent synaptic potentiation. We demonstrate that synapse-specific molecular pathway allowing MMPs to rapidly upregulate function of NMDARs in stratum radiatum involved protease activated receptor 1 and intracellular kinases and GTPases activity. In contrast, MMP-independent scaling of synaptic strength in stratum oriens involved dopamine D1/D5 receptors and Src kinases. Results of this study reveal that 2 neighboring synaptic systems differ significantly in extracellular and intracellular cascades that control synaptic gain and provide long-searched transduction pathways relevant for MMP-dependent synaptic plasticity.
Assuntos
Dendritos/fisiologia , Líquido Extracelular/fisiologia , Hipocampo/fisiologia , Líquido Intracelular/fisiologia , Células Piramidais/fisiologia , Potenciais Sinápticos/fisiologia , Animais , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Sinapses/fisiologiaRESUMO
The ventral posterior nucleus of the thalamus plays an important role in somatosensory information processing. It contains elongated cellular domains called barreloids, which are the structural basis for the somatotopic organization of vibrissae representation. So far, the organization of glial networks in these barreloid structures and its modulation by neuronal activity has not been studied. We have developed a method to visualize thalamic barreloid fields in acute slices. Combining electrophysiology, immunohistochemistry, and electroporation in transgenic mice with cell type-specific fluorescence labeling, we provide the first structure-function analyses of barreloidal glial gap junction networks. We observed coupled networks, which comprised both astrocytes and oligodendrocytes. The spread of tracers or a fluorescent glucose derivative through these networks was dependent on neuronal activity and limited by the barreloid borders, which were formed by uncoupled or weakly coupled oligodendrocytes. Neuronal somata were distributed homogeneously across barreloid fields with their processes running in parallel to the barreloid borders. Many astrocytes and oligodendrocytes were not part of the panglial networks. Thus, oligodendrocytes are the cellular elements limiting the communicating panglial network to a single barreloid, which might be important to ensure proper metabolic support to active neurons located within a particular vibrissae signaling pathway.
Assuntos
Junções Comunicantes/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Núcleos Ventrais do Tálamo/fisiologia , Animais , Eletroporação , Corantes Fluorescentes , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Neuroglia/citologia , Neurônios/citologia , Técnicas de Patch-Clamp , Técnicas de Cultura de Tecidos , Núcleos Ventrais do Tálamo/citologiaRESUMO
Chemokines are important signaling molecules in the immune and nervous system. Using a fluorescence reporter mouse model, we demonstrate that the chemokine CCL17, a ligand of the chemokine receptor CCR4, is produced in the murine brain, particularly in a subset of hippocampal CA1 neurons. We found that basal expression of Ccl17 in hippocampal neurons was strongly enhanced by peripheral challenge with lipopolysaccharide (LPS). LPS-mediated induction of Ccl17 in the hippocampus was dependent on local tumor necrosis factor (TNF) signaling, whereas upregulation of Ccl22 required granulocyte-macrophage colony-stimulating factor (GM-CSF). CCL17 deficiency resulted in a diminished microglia density under homeostatic and inflammatory conditions. Further, microglia from naïve Ccl17-deficient mice possessed a reduced cellular volume and a more polarized process tree as assessed by computer-assisted imaging analysis. Regarding the overall branching, cell surface area, and total tree length, the morphology of microglia from naïve Ccl17-deficient mice resembled that of microglia from wild-type mice after LPS stimulation. In line, electrophysiological recordings indicated that CCL17 downmodulates basal synaptic transmission at CA3-CA1 Schaffer collaterals in acute slices from naïve but not LPS-treated animals. Taken together, our data identify CCL17 as a homeostatic and inducible neuromodulatory chemokine affecting the presence and morphology of microglia and synaptic transmission in the hippocampus.
Assuntos
Quimiocina CCL17/metabolismo , Hipocampo/imunologia , Neuroimunomodulação/fisiologia , Neurônios/imunologia , Animais , Quimiocina CCL17/genética , Quimiocina CCL22/metabolismo , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/patologia , Homeostase/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/imunologia , Microglia/patologia , Monócitos/imunologia , Monócitos/patologia , Neurônios/patologia , Receptores CCR4/metabolismo , Transmissão Sináptica/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Heparan sulfate (HS) proteoglycans represent a major component of the extracellular matrix and are critical for brain development. However, their function in the mature brain remains to be characterized. Here, acute enzymatic digestion of HS side chains was used to uncover how HSs support hippocampal function in vitro and in vivo. We found that long-term potentiation (LTP) of synaptic transmission at CA3-CA1 Schaffer collateral synapses was impaired after removal of highly sulfated HSs with heparinase 1. This reduction was associated with decreased Ca2+ influx during LTP induction, which was the consequence of a reduced excitability of CA1 pyramidal neurons. At the subcellular level, heparinase treatment resulted in reorganization of the distal axon initial segment, as detected by a reduction in ankyrin G expression. In vivo, digestion of HSs impaired context discrimination in a fear conditioning paradigm and oscillatory network activity in the low theta band after fear conditioning. Thus, HSs maintain neuronal excitability and, as a consequence, support synaptic plasticity and learning.
Assuntos
Discriminação Psicológica/fisiologia , Heparitina Sulfato/fisiologia , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Animais , Anquirinas/biossíntese , Anquirinas/genética , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/fisiologia , Sinalização do Cálcio/fisiologia , Condicionamento Psicológico , Medo/fisiologia , Heparina Liase/farmacologia , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/fisiologia , Ritmo TetaRESUMO
The hippocampus is a key brain region in the pathophysiology of mesial temporal lobe epilepsy. Long-term changes of its architecture and function on the network and cellular level are well documented in epilepsy. Astrocytes can control many aspects of neuronal function and their long-term alterations over weeks, months and years play an important role in epilepsy. However, a pathophysiological transformation of astrocytes does not seem to be required for astrocytes to contribute to epileptic activity. Some of the properties of physiological astrocyte-neuron communication could allow these cells to exacerbate or synchronize neuronal firing on shorter time scales of milliseconds to minutes. Therefore, these astrocyte-neuron interactions are increasingly recognized as potential contributors to epileptic activity. Fast and reciprocal communication between astrocytes and neurons is enabled by a diverse set of mechanisms that could both amplify and counteract epileptic activity. They may thus promote or cause development of epileptic activity or inhibit it. Mechanisms of astrocyte-neuron interactions that can quickly increase network excitability involve, for example, astrocyte Ca2+ and Na+ signalling, K+ buffering, gap junction coupling and metabolism. However, rapid changes of astrocyte neurotransmitter uptake and morphology may also underlie or support development of network hyperexcitability. The temporal characteristics of these interactions, their ability to synchronize neuronal activity and their net effect on network activity will determine their contribution to the emergence or maintenance of epileptic activity.
Assuntos
Astrócitos/fisiologia , Epilepsia/fisiopatologia , Neurônios/fisiologia , Animais , Astrócitos/citologia , Cálcio/fisiologia , Junções Comunicantes/fisiologia , Transdução de Sinais , Sódio/fisiologiaRESUMO
Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca2+ rises, the dopamine sensitivity of astroglia in situ and its physiological roles remain unknown. To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca2+ in cells connected via gap junctions to astrocytes loaded whole-cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca2+ ] measurements, we also employed life-time imaging of the Ca2+ indicator Oregon Green BAPTA-1. We found that dopamine triggered a dose-dependent, bidirectional Ca2+ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below-baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca2+ storage and removal whereas the dopamine-induced [Ca2+ ] decrease involved D2 receptors only and was sensitive to Ca2+ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher-threshold dopamine-induced Ca2+ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter-dopamine-could either elevate or decrease astrocyte [Ca2+ ] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca2+ ] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447-459.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Dopamina/farmacologia , Hipocampo/citologia , Sinapses/efeitos dos fármacos , Animais , Astrócitos/citologia , Dopamina/metabolismo , Dopaminérgicos/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Técnicas In Vitro , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurotransmissores/farmacologia , Imagem Óptica , Técnicas de Patch-Clamp , RatosRESUMO
Dysfunctional astrocytes are increasingly recognized as key players in the development and progression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-µm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.