RESUMEN
The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABAA receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.
Asunto(s)
Relojes Circadianos , Ritmo Circadiano , Neuronas/metabolismo , Núcleo Supraquiasmático/metabolismo , Vasopresinas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Conducta Animal , Calcio/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica , Locomoción , Ratones , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Factores de Tiempo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/deficiencia , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
The suprachiasmatic nucleus (SCN), the master circadian clock in mammals, sends major output signals to the subparaventricular zone (SPZ) and further to the paraventricular nucleus (PVN), the neural mechanism of which is largely unknown. In this study, the intracellular calcium levels were measured continuously in cultured hypothalamic slices containing the PVN, SPZ, and SCN. We detected ultradian calcium rhythms in both the SPZ-PVN and SCN regions with periods of 0.5-4.0 hours, the frequency of which depended on the local circadian rhythm in the SPZ-PVN region. The ultradian rhythms were synchronous in the entire SPZ-PVN region and a part of the SCN. Because the ultradian rhythms were not detected in the SCN-only slice, the origin of ultradian rhythm is the SPZ-PVN region. In association with an ultradian bout, a rapid increase of intracellular calcium in a millisecond order was detected, the frequency of which determined the amplitude of an ultradian bout. The synchronous ultradian rhythms were desynchronized and depressed by a sodium channel blocker tetrodotoxin, suggesting that a tetrodotoxin-sensitive network is involved in synchrony of the ultradian bouts. In contrast, the ultradian rhythm is abolished by glutamate receptor blockers, indicating the critical role of glutamatergic mechanism in ultradian rhythm generation, while a GABAA receptor blocker increased the frequency of ultradian rhythm and modified the circadian rhythm in the SCN. A GABAergic network may refine the circadian output signals. The present study provides a clue to unraveling the loci and network mechanisms of the ultradian rhythm.
Asunto(s)
Ondas Encefálicas/fisiología , Señalización del Calcio/fisiología , Relojes Circadianos/fisiología , Neuronas GABAérgicas/metabolismo , Núcleo Hipotalámico Paraventricular/fisiología , Animales , Ondas Encefálicas/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Relojes Circadianos/efectos de los fármacos , Antagonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/citología , Ratones , Núcleo Hipotalámico Paraventricular/citología , Tetrodotoxina/farmacologíaRESUMEN
The suprachiasmatic nucleus (SCN), the master circadian clock, contains a network composed of multiple types of neurons which are thought to form a hierarchical and multioscillator system. The molecular clock machinery in SCN neurons drives membrane excitability and sends time cue signals to various brain regions and peripheral organs. However, how and at what time of the day these neurons transmit output signals remain largely unknown. Here, we successfully visualized circadian voltage rhythms optically for many days using a genetically encoded voltage sensor, ArcLightD. Unexpectedly, the voltage rhythms are synchronized across the entire SCN network of cultured slices, whereas simultaneously recorded Ca2+ rhythms are topologically specific to the dorsal and ventral regions. We further found that the temporal order of these two rhythms is cell-type specific: The Ca2+ rhythms phase-lead the voltage rhythms in AVP neurons but Ca2+ and voltage rhythms are nearly in phase in VIP neurons. We confirmed that circadian firing rhythms are also synchronous and are coupled with the voltage rhythms. These results indicate that SCN networks with asynchronous Ca2+ rhythms produce coherent voltage rhythms.
Asunto(s)
Calcio/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Ritmo Circadiano , Femenino , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Núcleo Supraquiasmático/citologíaRESUMEN
The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.
Asunto(s)
Factores de Transcripción ARNTL/biosíntesis , Conducta Animal , Señalización del Calcio , Ritmo Circadiano , Proteínas Circadianas Period/biosíntesis , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Calcio/metabolismo , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Proteínas Circadianas Period/genéticaRESUMEN
The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a â¼24 h clock not only in the living animal, but also when isolated in culture. This "clock in a dish" can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca(2+)]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.
Asunto(s)
Relojes Circadianos/genética , Relojes Circadianos/fisiología , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Neuronas/fisiología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/fisiología , Animales , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Expresión Génica/fisiología , Mamíferos/fisiología , Neuroglía/fisiología , Fotoperiodo , TiempoRESUMEN
Arginine vasopressin (AVP), a major neuropeptide in the suprachiasmatic nucleus (SCN), is postulated to mediate the output of the circadian oscillation. Mice carrying a reporter gene of AVP transcription (AVP(ELuc)) were produced by knocking-in a cDNA of Emerald-luciferase (ELuc) in the translational initiation site. Homozygous mice did not survive beyond postnatal day 7. Using the heterozygous (AVP(ELuc/+)) mice, a bioluminescence reporter system was developed that enabled to monitor AVP transcription through AVP-ELuc measurement in real time for more than 10 cycles in the cultured brain slice. AVP(ELuc/+) mice showed circadian behaviour rhythms and light responsiveness indistinguishable from those of the wild-type. Robust circadian rhythms in AVP-ELuc were detected in the cultured SCN slice at a single cell as well as tissue levels. The circadian rhythm of the whole SCN slice was stable, with the peak at the mid-light phase of a light-dark cycle, while that of a single cell was more variable. By comparison, rhythmicity in the paraventricular nucleus and supraoptic nucleus in the hypothalamus was unstable and damped rapidly. Spatiotemporal profiles of AVP expression at the pixel level revealed significant circadian rhythms in the entire area of AVP-positive cells in the SCN, and at least two clusters that showed different circadian oscillations. Contour analysis of bioluminescence intensity in a cell-like region demonstrated the radiation area was almost identical to the cell size. This newly developed reporter system for AVP gene expression is a useful tool for the study of circadian rhythms.
Asunto(s)
Arginina Vasopresina/genética , Ritmo Circadiano/genética , Neuronas/metabolismo , Núcleo Supraquiasmático/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Femenino , Técnicas de Sustitución del Gen , Genes Reporteros , Mediciones Luminiscentes , Masculino , Ratones , Ratones TransgénicosRESUMEN
The circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multioscillator system in which neuronal networks play crucial roles in expressing coherent rhythms in physiology and behavior. However, our understanding of the neuronal network is still incomplete. Intracellular calcium mediates the input signals, such as phase-resetting stimuli, to the core molecular loop involving clock genes for circadian rhythm generation and the output signals from the loop to various cellular functions, including changes in neurotransmitter release. Using a unique large-scale calcium imaging method with genetically encoded calcium sensors, we visualized intracellular calcium from the entire surface of SCN slice in culture including the regions where autonomous clock gene expression was undetectable. We found circadian calcium rhythms at a single-cell level in the SCN, which were topologically specific with a larger amplitude and more delayed phase in the ventral region than the dorsal. The robustness of the rhythm was reduced but persisted even after blocking the neuronal firing with tetrodotoxin (TTX). Notably, TTX dissociated the circadian calcium rhythms between the dorsal and ventral SCN. In contrast, a blocker of gap junctions, carbenoxolone, had only a minor effect on the calcium rhythms at both the single-cell and network levels. These results reveal the topological specificity of the circadian calcium rhythm in the SCN and the presence of coupled regional pacemakers in the dorsal and ventral regions. Neuronal firings are not necessary for the persistence of the calcium rhythms but indispensable for the hierarchical organization of rhythmicity in the SCN.
Asunto(s)
Calcio/metabolismo , Ritmo Circadiano/fisiología , Red Nerviosa/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Carbenoxolona/farmacología , Ritmo Circadiano/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Humanos , Ratones , Red Nerviosa/efectos de los fármacos , Núcleo Supraquiasmático/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450â µm from the surface. In vivo volumetric Ca2+ imaging at a volume rate of 1.5â Hz within a depth range of 130-200â µm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.
RESUMEN
The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca2+ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca2+-dependent kinase regulates the clock gene transcription. However, the Ca2+ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca2+ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca2+ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca2+ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca2+ rhythms, whereas blocking of Ca2+ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors had a minor effect on either Ca2+ rhythms. We conclude that the in-phasic circadian Ca2+ rhythms in the cytosol and nucleus are mainly driven by Ca2+ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca2+ rhythms directly regulate transcription in situ.
RESUMEN
Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.
RESUMEN
Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 µm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 µm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.
Asunto(s)
Neuronas , Tacto , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/ultraestructura , Ratones , Microscopía Fluorescente/métodos , Neuronas/fisiología , FotonesRESUMEN
Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.
RESUMEN
Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of embryonic stem (ES) cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factor-responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.
Asunto(s)
Células Madre Embrionarias/citología , Neuronas/metabolismo , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Lentivirus/metabolismo , Ratones , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Medicina Regenerativa/métodos , Sinapsis/metabolismo , Factores de TiempoRESUMEN
Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200 µm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.
Asunto(s)
Calcio/metabolismo , Ritmo Circadiano , Proteínas Circadianas Period/metabolismo , Neuronas del Núcleo Supraquiasmático/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Análisis de la Célula Individual , Neuronas del Núcleo Supraquiasmático/citologíaRESUMEN
In mammals, the master circadian clock is located in the suprachiasmatic nucleus (SCN), where most neurons show circadian rhythms of intracellular Ca2+ levels. However, the origin of these Ca2+ rhythms remains largely unknown. In this study, we successfully monitored the intracellular circadian Ca2+ rhythms together with the circadian PER2 and firing rhythms in a single SCN slice ex vivo, which enabled us to explore the origins. The phase relation between the circadian PER2 and Ca2+ rhythms, but not between the circadian PER2 and firing rhythms, was significantly altered in Cry1/Cry2 double knockout mice, which display a loss of intercellular synchronization in the SCN. In addition, in Cry1/Cry2 double knockout mice, circadian Ca2+ rhythms were abolished in the dorsolateral SCN, but were maintained in the majority of the ventromedial SCN. These findings indicate that intracellular circadian Ca2+ rhythms are composed of an exogenous and endogenous component involving PER2 expression.
Asunto(s)
Calcio/metabolismo , Ritmo Circadiano , Núcleo Supraquiasmático/fisiología , Animales , Animales Recién Nacidos , Biomarcadores , Señalización del Calcio , Criptocromos/genética , Criptocromos/metabolismo , Espacio Intracelular/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismoRESUMEN
Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
Asunto(s)
Comunicación Celular , Transformación Celular Neoplásica/metabolismo , Metabolismo Energético , Células Epiteliales/metabolismo , Animales , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Técnicas de Cocultivo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Perros , Femenino , Genes ras , Glucosa/metabolismo , Glucólisis , Ácido Láctico/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
It was shown recently that the basal dendrites of cortical pyramidal neurons generate NMDA-spikes. In the present study, we made whole-cell recordings from hippocampal CA1 pyramidal neurons and examined whether NMDA receptor activation was involved in synaptic responses. At low input stimulus intensity, EPSPs with a fast decay time were induced. As the intensity of stimulation was increased in the presence of GABA receptor antagonists, a depolarizing after-potential (DAP) was generated in addition to a fast decaying potential. A DAP was never observed when the input was applied to the apical dendrites. The DAP was suppressed by hyperpolarization or by NMDA receptor antagonists, but not by Na+, K+, or Ca2+ channel blockers. One possible mechanism is that the morphology of the basal dendrites favors DAP generation. A compartmental model simulation showed that synaptic inputs to thinner shorter dendrites generated a potential that resembled a DAP. Our study shows that a synaptic input to the basal dendrites of a hippocampal pyramidal neuron can generate a NMDA receptor-mediated potential in the presence of GABA receptor blockade.
Asunto(s)
Dendritas/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Galopamilo/análogos & derivados , Hipocampo/citología , Lidocaína/análogos & derivados , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Valina/análogos & derivados , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Dendritas/efectos de la radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Antagonistas del GABA/farmacología , Galopamilo/farmacología , Lidocaína/farmacología , Masculino , Modelos Neurológicos , Técnicas de Placa-Clamp/métodos , Células Piramidales/efectos de los fármacos , Células Piramidales/efectos de la radiación , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Bloqueadores de los Canales de Sodio/farmacología , Estadísticas no Paramétricas , Valina/farmacologíaRESUMEN
Although it has been reported that dendritic neurotransmitter releases from amacrine cells are regulated by the intracellular Ca(2+) concentration ([Ca(2+)](i)), their spatiotemporal patterns are not well explained. Fast Ca(2+) imagings of amacrine cells in the horizontal slice preparation of goldfish retinas under whole-cell patch-clamp recordings were undertaken to better investigate the spatiotemporal patterns of dendritic [Ca(2+)](i). We found that amacrine cell dendrites showed inhomogeneous [Ca(2+)](i) increases in both Na(+) spiking cells and cells without Na(+) spikes. The spatiotemporal properties of inhomogeneous [Ca(2+)](i) increases were classified into three patterns: local, regional and global. Local [Ca(2+)](i) increases were observed in very discrete regions and appeared as discontinuous patches, presumably evoked by local excitatory postsynaptic potentials. Regional [Ca(2+)](i) increases were observed in either a single or a small number of dendrites, presumably reflecting the result of dendritic action potentials. Global [Ca(2+)](i) increases were observed in the entire dendrites of a cell and were mediated by Na(+) action potentials or multiple Na(+) action potentials riding on slow depolarization. Ca(2+)-mediated potentials also evoked global [Ca(2+)](i) increase in cells without Na(+) spikes. These spatiotemporal dynamics of dendritic Ca(2+) signals may reflect multiple modes of synaptic integration on the dendrites of amacrine cells.
Asunto(s)
Potenciales de Acción/fisiología , Células Amacrinas/fisiología , Señalización del Calcio/fisiología , Dendritas/fisiología , Carpa Dorada/fisiología , Animales , Técnicas In Vitro , Retina/fisiología , Factores de TiempoRESUMEN
Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute to the information processing in the retinal circuits. However, many of previous electrophysiological and immunocytochemical studies employed slice preparations cut by vertical direction in which most of these cells and their neurites were severely damaged and removed. This might lead to the underestimation of active and passive conductance in horizontally expanding neurites, and also missing of morphological information of horizontal structures. Here, we describe an alternative slicing method of horizontally cut preparation of the retina. The slice is made horizontally at the inner layer of the retina using a vibratome slicer after the retina is embedded in the low-temperature melting agarose gel. This horizontal slice preparation enables us to directly access cells in the inner retina by patch-clamp recording, calcium imaging, single RT-PCR, and immunocytochemistry. The method described here would offer an alternative strategy for studying the functions of neurons and neural circuits in the retina.
Asunto(s)
Microtomía/métodos , Retina/citología , Neuronas Retinianas/citología , Animales , Diseño de Equipo , Geles/química , Humanos , Inmunohistoquímica , Microtomía/instrumentación , Técnicas de Placa-Clamp , Retina/ultraestructura , Neuronas Retinianas/ultraestructura , Sefarosa/química , Adhesión del Tejido/métodosRESUMEN
Single-point laser scanning confocal imaging produces signals with high spatial resolution in living organisms. However, photo-induced toxicity, bleaching, and focus drift remain challenges, especially when recording over several days for monitoring circadian rhythms. Bioluminescence imaging is a tool widely used for this purpose, and does not cause photo-induced difficulties. However, bioluminescence signals are dimmer than fluorescence signals, and are potentially affected by levels of cofactors, including ATP, O(2), and the substrate, luciferin. Here we describe a novel time-lapse confocal imaging technique to monitor circadian rhythms in living tissues. The imaging system comprises a multipoint scanning Nipkow spinning disk confocal unit and a high-sensitivity EM-CCD camera mounted on an inverted microscope with auto-focusing function. Brain slices of the suprachiasmatic nucleus (SCN), the central circadian clock, were prepared from transgenic mice expressing a clock gene, Period 1 (Per1), and fluorescence reporter protein (Per1::d2EGFP). The SCN slices were cut out together with membrane, flipped over, and transferred to the collagen-coated glass dishes to obtain signals with a high signal-to-noise ratio and to minimize focus drift. The imaging technique and improved culture method enabled us to monitor the circadian rhythm of Per1::d2EGFP from optically confirmed single SCN neurons without noticeable photo-induced effects or focus drift. Using recombinant adeno-associated virus carrying a genetically encoded calcium indicator, we also monitored calcium circadian rhythms at a single-cell level in a large population of SCN neurons. Thus, the Nipkow spinning disk confocal imaging system developed here facilitates long-term visualization of circadian rhythms in living cells.