Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators

Genetically encoded Ca2+ indicators (GECIs) based on fluorescent proteins (XFPs) and Ca2+-binding proteins [like calmodulin (CaM)] have great potential for the study of subcellular Ca2+ signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca2+ concentration ([Ca2+]) is complicated by the nonlinear interactions between Ca2+ binding and GECI fluorescence. We have characterized GECIs in pyramidal neurons in cultured hippocampal brain slices, focusing on indicators based on circularly permuted XFPs [GCaMP (Nakai et al., 2001), Camgaroo2 (Griesbeck et al., 2001), and Inverse Pericam (Nagai et al., 2001)]. Measurements of fluorescence changes evoked by trains of action potentials revealed that GECIs have little sensitivity at low action potential frequencies compared with synthetic [Ca2+] indicators with similar affinities for Ca2+. The sensitivity of GECIs improved for high-frequency trains of action potentials, indicating that GECIs are supralinear indicators of neural activity. Simultaneous measurement of GECI fluorescence and [Ca2+] revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively

Stereotyped odor-evoked activity in the mushroom body of Drosophila revealed by green fluorescent protein-based Ca2+ imaging

To study the representation of olfactory information in higher brain centers, we expressed a green fluorescent protein-based Ca2+ sensor, G-CaMP, in the Drosophila mushroom body (MB). Using two-photon microscopy, we imaged odor-evoked G-CaMP fluorescence transients in MB neurons [Kenyon cells (KCs)] with single-cell resolution. Odors produced large fluorescence transients in a subset of KC somata and in restricted regions of the calyx, the neuropil of the MB. In different KCs, odor-evoked fluorescence transients showed diverse changes with odor concentration: in some KCs, fluorescence transients were evoked by an odor at concentrations spanning several orders of magnitude, whereas in others only at a narrow concentration range. Different odors produced fluorescence transients in different subsets of KCs. The spatial distributions of KCs showing fluorescence transients evoked by a given odor were similar across individuals. For some odors, individual KCs with fluorescence transients evoked by a particular odor could be found in similar locations in different flies with spatial precisions on the order of the size of KC somata. These results indicate that odor-evoked activity can have remarkable spatial specificity in the MB

Deep and fast live imaging with two-photon scanned light-sheet microscopy

We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology

Glutamate Induces the Elongation of Early Dendritic Protrusions via mGluRs in Wild Type Mice, but Not in Fragile X Mice

Fragile X syndrome (FXS), the most common inherited from of autism and mental impairment, is caused by transcriptional silencing of the Fmr1 gene, resulting in the loss of the RNA-binding protein FMRP. Dendritic spines of cortical pyramidal neurons in affected individuals are abnormally immature and in Fmr1 knockout (KO) mice they are also abnormally unstable. This could result in defects in synaptogenesis, because spine dynamics are critical for synapse formation. We have previously shown that the earliest dendritic protrusions, which are highly dynamic and might serve an exploratory role to reach out for axons, elongate in response to glutamate. Here, we tested the hypothesis that this process is mediated by metabotropic glutamate receptors (mGluRs) and that it is defective in Fmr1 KO mice. Using time-lapse imaging with two-photon microscopy in acute brain slices from early postnatal mice, we find that early dendritic protrusions in layer 2/3 neurons become longer in response to application of glutamate or DHPG, a Group 1 mGluR agonist. Blockade of mGluR5 signaling, which reverses some adult phenotypes of KO mice, prevented the glutamate-mediated elongation of early protrusions. In contrast, dendritic protrusions from KO mice failed to respond to glutamate. Thus, absence of FMRP may impair the ability of cortical pyramidal neurons to respond to glutamate released from nearby pre-synaptic terminals, which may be a critical step to initiate synaptogenesis and stabilize spines

Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy

ANNINE-6 and ANNINE-6plus are voltage-sensitive dyes that when combined with two-photon microscopy are ideal for recording of neuronal voltages in vivo, in both bulk loaded tissue and the dendrites of single neurons. Here, we describe in detail but for a broad audience the voltage sensing mechanism of fast voltage-sensitive dyes, with a focus on ANNINE dyes, and how voltage imaging can be optimized with one-photon and two-photon excitation. Under optimized imaging conditions the key strengths of ANNINE dyes are their high sensitivity (0.5%/mV), neglectable bleaching and phototoxicity, a linear response to membrane potential, and a temporal resolution which is faster than the optical imaging devices currently used in neurobiology (order of nanoseconds). ANNINE dyes in combination with two-photon microscopy allow depth-resolved voltage imaging in bulk loaded tissue to study average membrane voltage oscillations and sensory responses. Alternatively, if ANNINE-6plus is applied internally, supra and sub threshold voltage changes can be recorded from dendrites of single neurons in awake animals. Interestingly, in our experience ANNINE-6plus labeling is impressively stable in vivo, such that voltage imaging from single Purkinje neuron dendrites can be performed for 2 weeks after a single electroporation of the neuron. Finally, to maximize their potential for neuroscience studies, voltage imaging with ANNINE dyes and two-photon microscopy can be combined with electrophysiological recording, calcium imaging, and/or pharmacology, even in awake animals

A Synaptic Mechanism for Temporal Filtering of Visual Signals

The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties

Monitoring neural activity with bioluminescence during natural behavior

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca^(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior

Two-way communication with neural networks in vivo using focused light

Neuronal networks process information in a distributed, spatially heterogeneous manner that transcends the layout of electrodes. In contrast, directed and steerable light offers the potential to engage specific cells on demand. We present a unified framework for adapting microscopes to use light for simultaneous in vivo stimulation and recording of cells at fine spatiotemporal resolutions. We use straightforward optics to lock onto networks in vivo, to steer light to activate circuit elements and to simultaneously record from other cells. We then actualize this 'free' augmentation on both an 'open' two-photon microscope and a leading commercial one. By following this protocol, setup of the system takes a few days, and the result is a noninvasive interface to brain dynamics based on directed light, at a network resolution that was not previously possible and which will further improve with the rapid advance in development of optical reporters and effectors. This protocol is for physiologists who are competent with computers and wish to extend hardware and software to interface more fluidly with neuronal networks.National Institutes of Health (U.S.) (Postdoctoral Fellowship)Simons Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Predoctoral Fellowship)National Institutes of Health (U.S.)Simons Foundatio

Neural Representations of Airflow in Drosophila Mushroom Body

The Drosophila mushroom body (MB) is a higher olfactory center where olfactory and other sensory information are thought to be associated. However, how MB neurons of Drosophila respond to sensory stimuli other than odor is not known. Here, we characterized the responses of MB neurons to a change in airflow, a stimulus associated with odor perception. In vivo calcium imaging from MB neurons revealed surprisingly strong and dynamic responses to an airflow stimulus. This response was dependent on the movement of the 3rd antennal segment, suggesting that Johnston's organ may be detecting the airflow. The calyx, the input region of the MB, responded homogeneously to airflow on. However, in the output lobes of the MB, different types of MB neurons responded with different patterns of activity to airflow on and off. Furthermore, detailed spatial analysis of the responses revealed that even within a lobe that is composed of a single type of MB neuron, there are subdivisions that respond differently to airflow on and off. These subdivisions within a single lobe were organized in a stereotypic manner across flies. For the first time, we show that changes in airflow affect MB neurons significantly and these effects are spatially organized into divisions smaller than previously defined MB neuron types