Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo

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.     

Two-photon compatibility and single-voxel, single-trial detection of subthreshold neuronal activity by a two-component optical voltage sensor

Minimally invasive measurements of neuronal activity are essential for understanding how signal processing is performed by neuronal networks. While optical strategies for making such measurements hold great promise, optical sensors generally lack the speed and sensitivity necessary to record neuronal activity on a single-trial, single-neuron basis. Here we present additional biophysical characterization and practical improvements of a two-component optical voltage sensor (2cVoS), comprised of the neuronal tracer dye, DiO, and dipicrylamine (DiO/DPA). Using laser spot illumination we demonstrate that membrane potential-dependent fluorescence changes can be obtained in a wide variety of cell types within brain slices. We show a correlation between membrane labeling and the sensitivity of the magnitude of fluorescence signal, such that neurons with the brightest membrane labeling yield the largest ΔF/F values per action potential (AP; ∼40%). By substituting a blue-shifted donor for DiO we confirm that DiO/DPA works, at least in part, via a Förster resonance energy transfer (FRET) mechanism. We also describe a straightforward iontophoretic method for labeling multiple neurons with DiO and show that DiO/DPA is compatible with two-photon (2P) imaging. Finally, exploiting the high sensitivity of DiO/DPA, we demonstrate AP-induced fluorescence transients (fAPs) recorded from single spines of hippocampal pyramidal neurons and single-trial measurements of subthreshold synaptic inputs to granule cell dendrites. Our findings suggest that the 2cVoS, DiO/DPA, enables optical measurements of trial-to-trial voltage fluctuations with very high spatial and temporal resolution, properties well suited for monitoring electrical signals from multiple neurons within intact neuronal networks.     

A microprobe for parallel optical and electrical recordings from single neurons in vivo

Recording electrical activity from identified neurons in intact tissue is key to understanding their role in information processing. Recent fluorescence labeling techniques have opened new possibilities to combine electrophysiological recording with optical detection of individual neurons deep in brain tissue. For this purpose we developed dual-core fiberoptics-based microprobes, with an optical core to locally excite and collect fluorescence, and an electrolyte-filled hollow core for extracellular single unit electrophysiology. This design provides microprobes with tips < 10 μm, enabling analyses with single-cell optical resolution. We demonstrate combined electrical and optical detection of single fluorescent neurons in rats and mice. We combined electrical recordings and optical Ca2(+) measurements from single thalamic relay neurons in rats, and achieved detection and activation of single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP transgenic mice. The microprobe expands possibilities for in vivo electrophysiological recording, providing parallel access to single-cell optical monitoring and control.     

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.     

96-well electroporation method for transfection of mammalian central neurons

Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.     

Single-cell electroporation for gene transfer in vivo

We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted in GFP expression in single neurons and glia. In vivo imaging showed morphologies, dendritic arbor dynamics, and growth rates characteristic of healthy cells. Coelectroporation of two plasmids resulted in expression of both proteins, while electroporation of fluorescent dextrans allowed direct visualization of transfer of molecules into cells. This technique will allow unprecedented spatial and temporal control over gene delivery and protein expression.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Imaging of respiratory-related population activity with single-cell resolution

The pre-B?tzinger complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been analyzed predominantly by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network, voltage-sensitive dyes have been used mainly, so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now have succeeded in using more sensitive Ca(2+) imaging to study respiratory neurons in rhythmically active brain stem slices of neonatal rats. For the visualization of neuronal activity, fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment (100 mM mannitol) during dye loading. Rhythmic population activity was imaged with single-cell resolution using a sensitive charge-coupled device camera and a x20 objective, and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Rhythmic neurons located deeper became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory and 26% as expiratory neurons. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a noninvasive approach with single-cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.     

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.     

Labeling neurons in vivo for morphological and functional studies

Increasingly sophisticated strategies for labeling cells in vivo are providing unprecedented opportunities to study neurons in living animals. Transgenic expression of genetically encoded reporters enables us to monitor changes in neuronal activity in response to sensory stimuli, and the labeling of single neurons with fluorescent proteins allows the dynamics of neuronal connectivity to be observed in transgenic animals over periods ranging from minutes to months. Advances in transient labeling techniques such as viral infection and electroporation provide a rapid means by which to analyze neuronal gene function in vivo. These new approaches to labeling, manipulating and imaging neurons in intact organisms are transforming the way in which the nervous system is studied.     

Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain

Electroporation is becoming more popular as a technique for transfecting neurons within intact tissues. One of the advantages of electroporation over other transfection techniques is the ability to precisely target an area for transfection. Here we highlight this advantage by describing methods to restrict transfection to either a single cell, clusters of cells, or to include large portions of the brain of the intact Xenopus tadpole. Electroporation is also an effective means of gene delivery in the retina. We have developed these techniques to examine the effects of regulated gene expression on various neuronal properties, including structural plasticity and synaptic transmission. Restriction of transfection to individual cells aids in imaging of neuronal morphology, while bulk cell transfection allows examination of the affects of gene expression on populations of cells by biochemical assays, imaging, and electrophysiological recording.     

15N-labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using (15)N-labeled brain tissue as an internal standard and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits.     

Hepatocyte growth factor/c-MET axis-mediated tropism of cord blood-derived unrestricted somatic stem cells for neuronal injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.     

Simultaneous imaging of morphological plasticity and calcium dynamics in dendrites

The structure and function of the nervous system are intricately connected. To investigate their relationship it is essential to image neuronal structure and function simultaneously with high spatio-temporal resolution. For this purpose, we describe here a two-step strategy. First, to visualize neurons and their entire dendritic arborization in neuronal tissue, we use ballistic delivery or single-cell electroporation of a fluorescent calcium indicator and a red fluorescent dye. Second, dual wavelength wide-field fluorescence microscopy or confocal microscopy enables imaging structural plasticity of dendrites (including filopodia and spines) and calcium dynamics together. We routinely apply this strategy to developing neurons in live tissue, but mature neurons can also be loaded and imaged as described. For labeling cells and setting up imaging equipment, approximately 2 h are required.     

Use of herpes virus amplicon vectors to study brain disorders

There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting the natural ability of viruses to insert genetic material into cells. When delivered to brain cells, these vectors cause infected cells to increase the expression of the genes of interest. The ability to deliver genes into neurons in vitro and in vivo with herpes simplex virus (HSV) amplicon vectors has made it possible to carry out exactly these sorts of experiments. This technology has the potential to offer new insights into the etiology of a wide variety of neuropsychiatric disorders. We describe the use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression, and discuss the considerations that enter into the use of these vectors both in vitro and in vivo. The HSV amplicon virus is a user-friendly vector for the delivery of genes into neurons that has come of age for the study of brain function.     

Genetic labeling of neuronal subsets through enhancer trapping in mice

The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain. Enhancer trapping has proved extremely useful for the genetic manipulation of selective cell types in Drosophila. We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase. This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions. Enhancer detection by lentiviral transgenesis could thus provide a complementary method for generating transgenic mouse libraries for the genetic labeling and manipulation of neuronal subsets.     

Immunohistochemical localization of P-glycoprotein in rat brain and detection of its increased expression by seizures are sensitive to fixation and staining variables.

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood-brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons.     

An Adenoviral Vector-Based System to Study Neuronal Gene Expression: Analysis of the Rat Tyrosine Hydroxylase Promoter in Cultured Neurons

Abstract: We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.     

抑郁症易感基因和抗抑郁药物靶点相关[]神经细胞类型及相互作用网络分析

基于抑郁症的全基因组关联分析研究(GWAS),对于获得的单核苷酸多态性位点(SNP)使用Haploreg软件进行基因注释,得到SNP注释的102个易感基因.。使用MAGMA软件对GWAS的汇总统计数据做基因水平的分析,获得了270个校正之后显著的基因,两者合并共得到320个抑郁症易感基因。通过药物数据库Drugbank获取133个抗抑郁药物靶点基因。使用EWCE包对抑郁症易感基因和抗抑郁药物靶点在三套脑组织单细胞测序数据中,分别进行神经细胞类型富集分析。结果发现大脑皮质的GABA神经元(抑制性神经元)和谷氨酸能神经元(兴奋性神经元)是抑郁症易感基因和抗抑郁药物靶点共同的神经元。这两种类型的神经细胞可能是抗抑郁药物与抑郁症易感基因相互作用的神经细胞,另外少突胶质前体细胞可能是抑郁症特有的易感神经细胞。使用Network Calculator软件构建网络并进行进行网络拓扑学参数分析。结果表明抑郁症易感基因与抗抑郁药物靶点组成了一个具有显著的相互连接的网络。本研究从单细胞层面揭示抑郁症的遗传机制,在网络层面为寻找新的抗抑郁药物靶点提供了一定的启示。