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1.
Two-photon-excited fluorescence laser-scanning microscopy (2PLSM) has provided a wealth of information about the spatiotemporal properties of biological processes at the single cell and population level. Because such nonlinear optical methods allow for imaging deep within biological tissue, 2PLSM can be combined with patch-clamp techniques to obtain electrophysiological recordings from specific fluorescently labeled cells in vivo. Here a protocol referred to as two-photon targeted patching (TPTP) describes a method that may be used to record from cells in the intact animal labeled by virtually any type of fluorophore. We target neurons that have been optically and genetically identified using green fluorescent protein (GFP) expressed under the control of a specific promoter. TPTP when combined with genetic approaches therefore permits electrophysiological recordings from specified neurons and their compartments, including dendrites. This technique may be repeated in the same preparation many times over the course of several hours and is equally applicable to non-neuronal cell types. 相似文献
2.
Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. 相似文献
3.
在体膜片钳是指在整体动物上直接对其中枢神经元进行全细胞膜片钳记录的技术,在生理学和药理学研究中具有良好的应用前景.常规采用的是盲法记录,最近出现的可视法记录,采用双光子靶向膜片钳(two-photon targeted patching,TPTP)技术,通过基因操作在动物脑内目标神经元中构建特异表达的荧光标志,可以做到对特定神经元亚群的靶向研究.对这两种方法的原理和操作进行了简单的介绍. 相似文献
4.
《Cell calcium》2017
Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neural function and structure at the cellular and subcellular level in vivo. The applications range from imaging of subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysis of large neuronal or glial populations. However, the depth penetration is often limited to a few hundred micrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations originating from refractive index inhomogeneties of the tissue are the reasons for these limitations. The depth penetration of two-photon imaging can be enhanced through various approaches, such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cells with red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so far require the implementation of new instrumentation and/or time consuming staining protocols. Here we present a simple approach that can be readily implemented in combination with standard two-photon microscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. The approach allows in vivo functional imaging of neuronal populations with single cell resolution in all six layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are not restricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that the improved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates. 相似文献
5.
The crystalline-like structure of the optic lobes of the fruit fly Drosophila melanogaster has made them a model system for the study of neuronal cell-fate determination, axonal path finding, and target selection. For functional studies, however, the small size of the constituting visual interneurons has so far presented a formidable barrier. We have overcome this problem by establishing in vivo whole-cell recordings from genetically targeted visual interneurons of Drosophila. Here, we describe the response properties of six motion-sensitive large-field neurons in the lobula plate that form a network consisting of individually identifiable, directionally selective cells most sensitive to vertical image motion (VS cells). Individual VS cell responses to visual motion stimuli exhibit all the characteristics that are indicative of presynaptic input from elementary motion detectors of the correlation type. Different VS cells possess distinct receptive fields that are arranged sequentially along the eye's azimuth, corresponding to their characteristic cellular morphology and position within the retinotopically organized lobula plate. In addition, lateral connections between individual VS cells cause strongly overlapping receptive fields that are wider than expected from their dendritic input. Our results suggest that motion vision in different dipteran fly species is accomplished in similar circuitries and according to common algorithmic rules. The underlying neural mechanisms of population coding within the VS cell network and of elementary motion detection, respectively, can now be analyzed by the combination of electrophysiology and genetic intervention in Drosophila. 相似文献
6.
Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb 总被引:4,自引:0,他引:4
Inhibitory local circuits in the olfactory bulb play a critical role in determining the firing patterns of output neurons. However, little is known about the circuitry in the major plexiform layers of the olfactory bulb that regulate this output. Here we report the first electrophysiological recordings from Blanes cells, large stellate-shaped interneurons located in the granule cell layer. We find that Blanes cells are GABAergic and generate large I(CAN)-mediated afterdepolarizations following bursts of action potentials. Using paired two-photon guided intracellular recordings, we show that Blanes cells have a presumptive axon and monosynaptically inhibit granule cells. Sensory axon stimulation evokes barrages of EPSPs in Blanes cells that trigger long epochs of persistent spiking; this firing mode was reset by hyperpolarizing membrane potential steps. Persistent firing in Blanes cells may represent a novel mechanism for encoding short-term olfactory information through modulation of tonic inhibitory synaptic input onto bulbar neurons. 相似文献
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8.
Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models. 相似文献
9.
Etienne Lonchamp Jean-Luc Dupont Laetitia Wioland Rapha?l Courjaret Corinne Mbebi-Liegeois Emmanuel Jover Frédéric Doussau Michel R. Popoff Jean-Louis Bossu Jean de Barry Bernard Poulain 《PloS one》2010,5(9)
Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons. 相似文献
10.
Recent recordings from spinal neurons in hatchling frog tadpoles allow their type-specific properties to be defined. Seven
main types of neuron involved in the control of swimming have been characterized. To investigate the significance of type-specific
properties, we build models of each neuron type and assemble them into a network using known connectivity between: sensory
neurons, sensory pathway interneurons, central pattern generator (CPG) interneurons and motoneurons. A single stimulus to
a sensory neuron initiates swimming where modelled neuronal and network activity parallels physiological activity. Substitution
of firing properties between neuron types shows that those of excitatory CPG interneurons are critical for stable swimming.
We suggest that type-specific neuronal properties can reflect the requirements for involvement in one particular network response
(like swimming), but may also reflect the need to participate in more than one response (like swimming and slower struggling).
Action Editor: Eberhard E. Fetz 相似文献
11.
The connectivity diagram of neocortical circuits is still unknown, and there are conflicting data as to whether cortical neurons are wired specifically or not. To investigate the basic structure of cortical microcircuits, we use a two-photon photostimulation technique that enables the systematic mapping of synaptic connections with single-cell resolution. We map the inhibitory connectivity between upper layers somatostatin-positive GABAergic interneurons and pyramidal cells in mouse frontal cortex. Most, and sometimes all, inhibitory neurons are locally connected to every sampled pyramidal cell. This dense inhibitory connectivity is found at both young and mature developmental ages. Inhibitory innervation of neighboring pyramidal cells is similar, regardless of whether they are connected among themselves or not. We conclude that local inhibitory connectivity is promiscuous, does not form subnetworks, and can approach the theoretical limit of a completely connected synaptic matrix. 相似文献
12.
Active dendritic conductances dynamically regulate GABA release from thalamic interneurons 总被引:1,自引:0,他引:1
Inhibitory interneurons in the dorsal lateral geniculate nucleus (dLGN) process visual information by precisely controlling spike timing and by refining the receptive fields of thalamocortical (TC) neurons. Previous studies indicate that dLGN interneurons inhibit TC neurons by releasing GABA from both axons and dendrites. However, the mechanisms controlling GABA release are poorly understood. Here, using simultaneous whole-cell recordings from interneurons and TC neurons and two-photon calcium imaging, we find that synchronous activation of multiple retinal ganglion cells (RGCs) triggers sodium spikes that propagate throughout interneuron axons and dendrites, and calcium spikes that invade dendrites but not axons. These distinct modes of interneuron firing can trigger both a rapid and a sustained component of inhibition onto TC neurons. Our studies suggest that active conductances make LGN interneurons flexible circuit-elements that can shift their spatial and temporal properties of GABA release in response to coincident activation of functionally related subsets of RGCs. 相似文献
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A major unanswered question concerning "pain" circuitry is the extent to which different populations of primary afferent nociceptor engage the same or different ascending pathways. In the present study, we followed the transneuronal transport of a genetically expressed lectin tracer, wheat germ agglutinin, in Na(V)1.8-expressing nociceptors of the nonpeptide class. We found that interneurons of lamina II are at the origin of the major ascending circuits targeted by the nonpeptide nociceptors. These interneurons contact lamina V projection neurons, which in turn predominantly target fourth-order neurons in the amygdala, hypothalamus, bed nucleus of the stria terminalis, and to a remarkable extent, the globus pallidus. These circuits differ greatly from the lamina I-based projection that is targeted by the peptide class of nociceptors. Our results indicate that parallel, perhaps independent pain pathways arise from different nociceptor classes and that motor as well as limbic targets predominate in the circuits that originate from the nonpeptide population. 相似文献
16.
High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions. 相似文献
17.
Striatal dopamine plays key roles in our normal and pathological goal-directed actions. To understand dopamine function, much attention has focused on how midbrain dopamine neurons modulate their firing patterns. However, we identify a presynaptic mechanism that triggers dopamine release directly, bypassing activity in dopamine neurons. We paired electrophysiological recordings of striatal channelrhodopsin2-expressing cholinergic interneurons with simultaneous detection of dopamine release at carbon-fiber microelectrodes in striatal slices. We reveal that activation of cholinergic interneurons by light flashes that cause only single action potentials in neurons from a small population triggers dopamine release via activation of nicotinic receptors on dopamine axons. This event overrides ascending activity from dopamine neurons and, furthermore, is reproduced by activating ChR2-expressing thalamostriatal inputs, which synchronize cholinergic interneurons in vivo. These findings indicate that synchronized activity in cholinergic interneurons directly generates striatal dopamine signals whose functions will extend beyond those encoded by dopamine neuron activity. 相似文献
18.
Manipulation of neuronal activity through genetically targeted actuator molecules is a powerful approach for studying information flow in the brain. In these approaches the genetically targeted component, a receptor or a channel, is activated either by a small molecule (chemical genetics) or by light from a physical source (optogenetics). We developed a hybrid technology that allows control of the same neurons by both optogenetic and chemical genetic means. The approach is based on engineered chimeric fusions of a light-generating protein (luciferase) to a light-activated ion channel (channelrhodopsin). Ionic currents then can be activated by bioluminescence upon activation of luciferase by its substrate, coelenterazine (CTZ), as well as by external light. In cell lines, expression of the fusion of Gaussia luciferase to Channelrhodopsin-2 yielded photocurrents in response to CTZ. Larger photocurrents were produced by fusing the luciferase to Volvox Channelrhodopsin-1. This version allowed chemical modulation of neuronal activity when expressed in cultured neurons: CTZ treatment shifted neuronal responses to injected currents and sensitized neurons to fire action potentials in response to subthreshold synaptic inputs. These luminescent channelrhodopsins - or luminopsins – preserve the advantages of light-activated ion channels, while extending their capabilities. Our proof-of-principle results suggest that this novel class of tools can be improved and extended in numerous ways. 相似文献
19.
The gonadotropin-releasing hormone (GnRH) neurons are the key output cells of a complex neuronal network controlling fertility in mammals. To examine calcium homeostasis in postnatal GnRH neurons, we generated a transgenic mouse line in which the genetically encodable calcium indicator ratiometric Pericam (rPericam) was targeted to the GnRH neurons. This mouse model enabled real-time imaging of calcium concentrations in GnRH neurons in the acute brain slice preparation. Investigations in GnRH-rPericam mice revealed that GnRH neurons exhibited spontaneous, long-duration (~8s) calcium transients. Dual electrical-calcium recordings revealed that the calcium transients were correlated perfectly with burst firing in GnRH neurons and that calcium transients in GnRH neurons regulated two calcium-activated potassium channels that, in turn, determined burst firing dynamics in these cells. Curiously, the occurrence of calcium transients in GnRH neurons across puberty or through the estrous cycle did not correlate well with the assumption that GnRH neuron burst firing was contributory to changing patterns of pulsatile GnRH release at these times. The GnRH-rPericam mouse was also valuable in determining differential mechanisms of GABA and glutamate control of calcium levels in GnRH neurons as well as effects of G-protein-coupled receptors for GnRH and kisspeptin. The simultaneous measurement of calcium levels in multiple GnRH neurons was hampered by variable rPericam fluorescence in different GnRH neurons. Nevertheless, in the multiple recordings that were achieved no evidence was found for synchronous calcium transients. Together, these observations show the great utility of transgenic targeting strategies for investigating the roles of calcium with specified neuronal cell types. 相似文献
20.
Extensive tangential cell migrations have been described in the developing mammalian, avian, and reptilian forebrain, and they are viewed as a powerful developmental mechanism to increase neuronal complexity in a given brain structure. Here, we report for the first time anatomical and cell tracking evidence for the presence of important migratory processes in the developing forebrain of the anamniote Xenopus laevis. Combining developmental gene expression patterns (Pax6, Nkx2.1, Isl1, Lhx5, Lhx9, and Dll3), neurotransmitter identity (GABA, NOS, ChAT), and connectivity information, several types of putative migratory cell populations and migration routes originating in the ventral pallium and the subpallium are proposed. By means of in vivo cell tracking experiments, pallio-subpallial and subpallio-pallial migrating neurons are visualized. Among them, populations of Nkx2.1(+) striatal interneurons and pallial GABAergic interneurons, which also express the migratory marker doublecortin, are identified. Finally, we find that these tangentially migrating pallial interneurons travel through an "isl1-free channel" that may guide their course through the subpallium. Our findings strongly suggest that the developing Xenopus telencephalon shares many similarities with amniotes in terms of neuronal specification and migrations. However, some differences are discussed, particularly with regard to the evolution of the pallium. 相似文献