Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.     

Imaging of molecular dynamics regulated by electrical activities in neural circuits and in synapses

One of the major challenges in brain research is to unravel a network of molecules, neurons, circuits and systems that are responsible for dynamic and hierarchical brain functions. To understand molecular events that occur in synapses could be an important key to exploring the mechanism of information processing. A spatiotemporal recording method is required to observe neuronal activities in a particular local circuit and to resolve single synaptic potential with high resolution. As alternative methods, real-time imaging using fluorescent probes and optical recording methods are also a powerful approach for investigating the molecular dynamics of biological events in neurons in vitro and in vivo. Recently, optical imaging techniques have become of great importance to visualize the molecular dynamics in a micron-sized compartment of a single neuron such as neuronal synapse. In general, the presynaptic axon forms synapses at the postsynaptic site on the dendritic spines in the mammalian central nervous system. Subsets of the synapses undergo a series of enduring changes in spine shape and density as well as alterations in electrophysiological functions. Here we describe recent optical imaging studies conducted by elaborate methods and techniques that provide evidence for the link between neural activity and molecular dynamics.     

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.     

Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.     

Three-Dimensional Distribution of Sensory Stimulation-Evoked Neuronal Activity of Spinal Dorsal Horn Neurons Analyzed by In Vivo Calcium Imaging

The spinal dorsal horn comprises heterogeneous populations of interneurons and projection neurons, which form neuronal circuits crucial for processing of primary sensory information. Although electrophysiological analyses have uncovered sensory stimulation-evoked neuronal activity of various spinal dorsal horn neurons, monitoring these activities from large ensembles of neurons is needed to obtain a comprehensive view of the spinal dorsal horn circuitry. In the present study, we established in vivo calcium imaging of multiple spinal dorsal horn neurons by using a two-photon microscope and extracted three-dimensional neuronal activity maps of these neurons in response to cutaneous sensory stimulation. For calcium imaging, a fluorescence resonance energy transfer (FRET)-based calcium indicator protein, Yellow Cameleon, which is insensitive to motion artifacts of living animals was introduced into spinal dorsal horn neurons by in utero electroporation. In vivo calcium imaging following pinch, brush, and heat stimulation suggests that laminar distribution of sensory stimulation-evoked neuronal activity in the spinal dorsal horn largely corresponds to that of primary afferent inputs. In addition, cutaneous pinch stimulation elicited activities of neurons in the spinal cord at least until 2 spinal segments away from the central projection field of primary sensory neurons responsible for the stimulated skin point. These results provide a clue to understand neuronal processing of sensory information in the spinal dorsal horn.     

Neuronal population activity and functional imaging.

Human functional brain imaging detects blood flow changes that are thought to reflect the activity of neuronal populations and, thus, the responses of neurons that carry behaviourally relevant information. Since this relationship is poorly understood, we explored the link between the activity of single neurons and their neuronal population. The functional imaging results were in good agreement with levels of population activation predicted from the known effects of sensory stimulation, learning and attention on single cortical neurons. However, the nature of the relationship between population activation and single neuron firing was very surprising. Population activation was strongly influenced by those neurons firing at low rates and so was very sensitive to the baseline or 'spontaneous' firing rate. When neural representations were sparse and neurons were tuned to several stimulus dimensions, population activation was hardly influenced by the few neurons whose firing was most strongly modulated by the task or stimulus. Measures of population activation could miss changes in information processing given simultaneous changes in neurons' baseline firing, response modulation or tuning width. Factors that can modulate baseline firing, such as attention, may have a particularly large influence on population activation. The results have implications for the interpretation of functional imaging signals and for cross-calibration between different methods for measuring neuronal activity.     

Targeted whole-cell recordings in the mammalian brain in vivo

While electrophysiological recordings from visually identified cell bodies or dendrites are routinely performed in cell culture and acute brain slice preparations, targeted recordings from the mammalian nervous system are currently not possible in vivo. The "blind" approach that is used instead is somewhat random and largely limited to common neuronal cell types. This approach prohibits recordings from, for example, molecularly defined and/or disrupted populations of neurons. Here we describe a method, which we call TPTP (two-photon targeted patching), that uses two-photon imaging to guide in vivo whole-cell recordings to individual, genetically labeled cortical neurons. We apply this technique to obtain recordings from genetically manipulated, parvalbumin-EGFP-positive interneurons in the somatosensory cortex. We find that both spontaneous and sensory-evoked activity patterns involve the synchronized discharge of electrically coupled interneurons. TPTP applied in vivo will therefore provide new insights into the molecular control of neuronal function at the systems level.     

Human immunodeficiency virus type-1 vulnerates nascent neuronal cells

Macrophages or microglial cells are the major target cells for HIV-1 infection in the brain. The infected cells release neurotoxic factors that may cause severe neuronal cell damage, especially in the basal ganglia and hippocampus. In this study, we used rat OHC to examine the region-specific neuronal cell damage caused by HIV-1-infected macrophages. When OHC was cocultured with HIV-1-infected MDM, we found that neuronal cells at the GCL of the DG were preferentially killed via apoptosis, and that projection of MF from GCL to PCL of the CA3 region was severely disturbed. We marked precursor cells around the DG region by using an EGFP-expressing retrovirus vector and found that these cells lost the ability to differentiate into neurons when exposed to HIV-1-infected MDM. In the DG, new neurons are normally incorporated into GCL or PCL, while in the presence of HIV-1-infected MDM, mature neurons failed to be incorporated into those layers. These data indicate that the neurotoxic factor(s) released from HIV-1-infected macrophages impede(s) neuronal cell repair in brain tissue. This suggests that DG is the region of the hippocampus most vulnerable to neuronal damage caused by HIV-1 infection, and that its selective vulnerability is most likely due to the highly active neurogenesis that takes place in this region.     

Analysis of the neuronal selectivity underlying low fMRI signals

BACKGROUND: A prevailing assumption in neuroimaging studies is that relatively low fMRI signals are due to weak neuronal activation, and, therefore, they are commonly ignored. However, lower fMRI signals may also result from intense activation by highly selective, albeit small, subsets of neurons in the imaged voxel. We report on an approach that could form a basis for resolving this ambiguity imposed by the low (mm range) spatial resolution of fMRI. Our approach employs fMR-adaptation as an indicator for highly active neuronal populations even when the measured fMRI signal is low.RESULTS: In this study, we first showed that fMRI-adaptation is diminished when overall neuronal activity is lowered substantially by reducing image contrast. We then applied the same adaptation paradigm, but this time we lowered the fMRI signal by changing object shape. While the overall fMRI signal in category-related regions such as the face-related pFs was drastically reduced for non-face stimuli, the adaptation level obtained for these stimuli remained high. We hypothesize that the relatively greater adaptation level following exposure to "nonoptimal" object shapes is indicative of small subsets of neurons responding vigorously to these "nonoptimal" objects even when the overall fMRI activity is low.CONCLUSIONS: Our results show that fMR-adaptation can be used to differentiate between neuronal activation patterns that appear similar in the overall fMRI signal. The results suggest that it may be possible to employ fMR-adaptation to reveal functionally heterogeneous islands of activity, which are too small to image using conventional imaging methods.     

Threshold-free population analysis identifies larger DRG neurons to respond stronger to NGF stimulation

Sensory neurons in dorsal root ganglia (DRG) are highly heterogeneous in terms of cell size, protein expression, and signaling activity. To analyze their heterogeneity, threshold-based methods are commonly used, which often yield highly variable results due to the subjectivity of the individual investigator. In this work, we introduce a threshold-free analysis approach for sparse and highly heterogeneous datasets obtained from cultures of sensory neurons. This approach is based on population estimates and completely free of investigator-set parameters. With a quantitative automated microscope we measured the signaling state of single DRG neurons by immunofluorescently labeling phosphorylated, i.e., activated Erk1/2. The population density of sensory neurons with and without pain-sensitizing nerve growth factor (NGF) treatment was estimated using a kernel density estimator (KDE). By subtraction of both densities and integration of the positive part, a robust estimate for the size of the responsive subpopulations was obtained. To assure sufficiently large datasets, we determined the number of cells required for reliable estimates using a bootstrapping approach. The proposed methods were employed to analyze response kinetics and response amplitude of DRG neurons after NGF stimulation. We thereby determined the portion of NGF responsive cells on a true population basis. The analysis of the dose dependent NGF response unraveled a biphasic behavior, while the study of its time dependence showed a rapid response, which approached a steady state after less than five minutes. Analyzing two parameter correlations, we found that not only the number of responsive small-sized neurons exceeds the number of responsive large-sized neurons--which is commonly reported and could be explained by the excess of small-sized cells--but also the probability that small-sized cells respond to NGF is higher. In contrast, medium-sized and large-sized neurons showed a larger response amplitude in their mean Erk1/2 activity.     

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.     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Detecting action potentials in neuronal populations with calcium imaging.

The study of neural circuits requires methods for simultaneously recording the activity of populations of neurons. Here, using calcium imaging of neocortical brain slices we take advantage of the ubiquitous distribution of calcium channels in neurons to develop a method to reconstruct the action potentials occurring in a population of neurons. Combining calcium imaging with whole-cell or perforated patch recordings from neurons loaded with acetoxymethyl ester or potassium salt forms of calcium indicators, we demonstrate that each action potential produces a stereotyped calcium transient in the somata of pyramidal neurons. These signals are detectable without averaging, and the signal-to-noise is sufficient to carry out a reconstruction of the spiking pattern of hundreds of neurons, up to relatively high firing frequencies. This technique could in principle be applied systematically to follow the activity of neuronal populations in vitro and in vivo.     

Dissecting cascade computational components in spiking neural networks

Finding out the physical structure of neuronal circuits that governs neuronal responses is an important goal for brain research. With fast advances for large-scale recording techniques, identification of a neuronal circuit with multiple neurons and stages or layers becomes possible and highly demanding. Although methods for mapping the connection structure of circuits have been greatly developed in recent years, they are mostly limited to simple scenarios of a few neurons in a pairwise fashion; and dissecting dynamical circuits, particularly mapping out a complete functional circuit that converges to a single neuron, is still a challenging question. Here, we show that a recent method, termed spike-triggered non-negative matrix factorization (STNMF), can address these issues. By simulating different scenarios of spiking neural networks with various connections between neurons and stages, we demonstrate that STNMF is a persuasive method to dissect functional connections within a circuit. Using spiking activities recorded at neurons of the output layer, STNMF can obtain a complete circuit consisting of all cascade computational components of presynaptic neurons, as well as their spiking activities. For simulated simple and complex cells of the primary visual cortex, STNMF allows us to dissect the pathway of visual computation. Taken together, these results suggest that STNMF could provide a useful approach for investigating neuronal systems leveraging recorded functional neuronal activity.     

Scale-Free Topology of the CA3 Hippocampal Network: A Novel Method to Analyze Functional Neuronal Assemblies

Cognitive mapping functions of the hippocampus critically depend on the recurrent network of the CA3 pyramidal cells. However, it is still not known in detail how network activity patterns emerge, or how they encode information. By using functional multineuron calcium imaging, we simultaneously recorded the activity of >100 neurons in the CA3 region of hippocampal slice cultures. We utilized a novel computational method to analyze the multichannel spike trains and to depict functional neuronal assemblies. By means of event synchronization and the correlation matrix analysis method, we found that: 1), the average functional neuronal cluster consists of 23 neurons, and neurons could be part of multiple assemblies; 2), the clustering strength, size, and mean distance among cells in neuronal assemblies follow a power-law-like distribution; 3), the clustering strength and size of neuronal assemblies are not correlated with the total number of neurons and their physical distance; and 4), the clustering distance of neuronal assemblies is weakly correlated with the total number of neurons and their physical distance. These findings suggest that the functional organization of the spontaneously firing CA3 hippocampal network is a scale-free structure in slice culture.     

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP¹, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons^2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding⁶ (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER⁷ with a calcium imaging technique⁸ using GCaMP3.0^{1, 9}, I have established an experimental system, where we can monitor activity of neurons in the feeding center – the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca²⁺ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.     

Optical calcium imaging in the nervous system of Drosophila melanogaster

BACKGROUND: Drosophila melanogaster is one of the best-studied model organisms in biology, mainly because of the versatility of methods by which heredity and specific expression of genes can be traced and manipulated. Sophisticated genetic tools have been developed to express transgenes in selected cell types, and these techniques can be utilized to target DNA-encoded fluorescence probes to genetically defined subsets of neurons. Neuroscientists make use of this approach to monitor the activity of restricted types or subsets of neurons in the brain and the peripheral nervous system. Since membrane depolarization is typically accompanied by an increase in intracellular calcium ions, calcium-sensitive fluorescence proteins provide favorable tools to monitor the spatio-temporal activity across groups of neurons. SCOPE OF REVIEW: Here we describe approaches to perform optical calcium imaging in Drosophila in consideration of various calcium sensors and expression systems. In addition, we outline by way of examples for which particular neuronal systems in Drosophila optical calcium imaging have been used. Finally, we exemplify briefly how optical calcium imaging in the brain of Drosophila can be carried out in practice. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Drosophila provides an excellent model organism to combine genetic expression systems with optical calcium imaging in order to investigate principles of sensory coding, neuronal plasticity, and processing of neuronal information underlying behavior. This article is part of a Special Issue entitled Biochemical, Biophysical and Genetic Approaches to Intracellular Calcium Signaling.     

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.