Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

Dendritic spike induction of postsynaptic cerebellar LTP

The architecture of parallel fiber (PF) axons contacting cerebellar Purkinje neurons (PNs) retains spatial information over long distances. PF synapses can trigger local dendritic calcium spikes, but whether and how this calcium signal leads to plastic changes that decode the PF input organization is unknown. By combining voltage and calcium imaging, we show that PF-elicited calcium signals, mediated by voltage-gated calcium channels, increase non-linearly during high-frequency bursts of electrically constant calcium spikes because they locally and transiently saturate the endogenous buffer. We demonstrate that these non-linear calcium signals, independently of NMDA or metabotropic glutamate receptor activation, can induce PF long-term potentiation (LTP). Two-photon imaging in coronal slices revealed that calcium signals inducing LTP can be observed by stimulating either the PF or the ascending fiber pathway. We propose that local dendritic calcium spikes, evoked by synaptic potentials, provide a unique mechanism to spatially decode PF signals into cerebellar circuitry changes

Automatic Neuron Detection in Calcium Imaging Data Using Convolutional Networks

Calcium imaging is an important technique for monitoring the activity of thousands of neurons simultaneously. As calcium imaging datasets grow in size, automated detection of individual neurons is becoming important. Here we apply a supervised learning approach to this problem and show that convolutional networks can achieve near-human accuracy and superhuman speed. Accuracy is superior to the popular PCA/ICA method based on precision and recall relative to ground truth annotation by a human expert. These results suggest that convolutional networks are an efficient and flexible tool for the analysis of large-scale calcium imaging data.Comment: 9 pages, 5 figures, 2 ancillary files; minor changes for camera-ready version. appears in Advances in Neural Information Processing Systems 29 (NIPS 2016

Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data

The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network-based approach based on calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer activity propagation between the activities of different neurons. The resulting functional networks were then modeled as directed graphs and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions

Continuous volumetric imaging via an optical phase-locked ultrasound lens

In vivo imaging at high spatiotemporal resolution is key to the understanding of complex biological systems. We integrated an optical phase-locked ultrasound lens into a two-photon fluorescence microscope and achieved microsecond-scale axial scanning, thus enabling volumetric imaging at tens of hertz. We applied this system to multicolor volumetric imaging of processes sensitive to motion artifacts, including calcium dynamics in behaving mouse brain and transient morphology changes and trafficking of immune cells