Neuronal Wiring: Linking Dendrite Placement to Synapse Formation

SummaryUnderstanding the processes that drive the formation of synapses between specific neurons within a circuit is critical to understanding how neural networks develop. A new study of synapse formation between motor neurons and pre-synaptic partners highlights the importance of dendrite placement

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

Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature

Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSFcNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine

ZebraZoom: an automated program for high-throughput behavioral analysis and categorization

International audienceThe zebrafish larva stands out as an emergent model organism for translational studies involving gene or drug screening thanks to its size, genetics, and permeability. At the larval stage, locomotion occurs in short episodes punctuated by periods of rest. Although phenotyping behavior is a key component of large-scale screens, it has not yet been automated in this model system. We developed ZebraZoom, a program to automatically track larvae and identify maneuvers for many animals performing discrete movements. Our program detects each episodic movement and extracts large-scale statistics on motor patterns to produce a quantification of the locomotor repertoire. We used ZebraZoom to identify motor defects induced by a glycinergic receptor antagonist. The analysis of the blind mutant atoh7 revealed small locomotor defects associated with the mutation. Using multiclass supervised machine learning, ZebraZoom categorized all episodes of movement for each larva into one of three possible maneuvers: slow forward swim, routine turn, and escape. ZebraZoom reached 91% accuracy for categorization of stereotypical maneuvers that four independent experimenters unanimously identified. For all maneuvers in the data set, ZebraZoom agreed with four experimenters in 73.2–82.5% of cases. We modeled the series of maneuvers performed by larvae as Markov chains and observed that larvae often repeated the same maneuvers within a group. When analyzing subsequent maneuvers performed by different larvae, we found that larva–larva interactions occurred as series of escapes. Overall, ZebraZoom reached the level of precision found in manual analysis but accomplished tasks in a high-throughput format necessary for large screens

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

International audienceImaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo micros-copy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo micros-copy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes

Fast calcium imaging at 100 fps with HiLo microscopy reveals different dynamics in soma and initial segment.

<p>(<b>A</b>) HiLo image of motor neurons expressing GCaMP5G with ROIs. Scale bar 5 μm. (<b>B</b>) Time course of calcium signals in soma, initial segment and axon, recorded at 100 fps. In the axon (ROI 1, gray) and the initial segments (ROI 4, light blue; ROI 5, magenta) a much faster rise is observed than in the somata (ROI 2, orange; ROI 3, dark blue). Somatic signals show a linear rise and a mono-exponential decay (Inset: blue somatic signal with red dotted fit, decay time 1.7 s, R² = 0.998). (<b>C</b>) The kinetics of calcium transients in different compartments were stable over time as shown with a recording performed 2.5 min later.</p

Optical section in embryonic spinal cord of zebrafish obtained with HiLo microscopy.

<p>(<b>A</b>) Lateral view of the spinal cord of transgenic zebrafish embryo expressing the fluorescent protein GCaMP5 pan-neuronally in <i>Tg(elavl3</i>:<i>GCaMP5G)</i> obtained with HiLo microscopy. <b>(B)</b> Same image obtained in UWF mode. The spatial distribution of GCaMP5G remains hidden under a haze of background fluorescence due to missing optical sectioning.Scale bars in both panels are 20 μm. <b>(C)</b> Corresponding z-stack in 3D visualization, imaged in HiLo mode. The pools of neurons on each side of the spinal cord are visible in two separate planes. <b>(D)</b> Same reconstruction in WF mode (grid step 10 μm/line). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143681#pone.0143681.s002" target="_blank">S1 Movie</a>.</p

Suppression of non-specific signals.

<p>Nonspecific signals appeared in UWF mode (A, C) and were suppressed in HiLo mode (B, D) (ROIs numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143681#pone.0143681.g003" target="_blank">Fig 3A</a>). The benefit is consistent in the somatic signal (A, B) and particularly important for the axonal signals (C, D) which are smaller and therefore more difficult to distinguish from the unspecific signals. Furthermore HiLo mode allows observing that calcium spikes occasionally did not occur in a single axon (black trace, arrow), despite occurrence in the neighboring axon (magenta trace) (D), while UWF mode hindered the observation of this phenomenon (C). Note that some bleaching is seen over the 80 s recording time. Traces were offset vertically for visualization. Raw data are shown in all panels. Acquisition rate was 25 fps.</p

Implementation of HiLo Microscopy to enable optical sectioning at high frame rate in thick biological tissue.

<p>(<b>A</b>) Setup: A 473nm laser beam is expanded with lens L1 to a divergent beam to illuminate the diffuser D. The diffuser is imaged by lens L2 onto the galvanometric mirror GM. The galvanometric mirror is then imaged by lens L3 into a conjugated plane of the back focal plane of the objective O within the microscope body. Fluorescence images are acquired either with a CCD camera or a CMOS camera. (<b>Inset</b>) Fluorescent Rhodamine layer imaged with speckled (SWF) and uniform (UWF) widefield illumination at 200 frames per second (fps), confirming successful uniform/speckled illumination. (<b>B–C</b>) Illustration of the optical Sectioning: (<b>B</b>) A single fluorescent layer appears as a sharp intensity peak (width 2.1 μm) in a z-stack in HiLo mode (solid green line), but with almost constant intensity in uniform wide-field (UWF) mode (blue dotted line). (<b>C</b>) Two Rhodamine layers can be separated in HiLo mode (solid green line), but in UWF mode only broad peaks (due to spherical aberration) whose positions do not correspond to the layers are visible. Inset: Experimental arrangement: red: Rhodamine layers; gray: supporting coverslips.</p

HiLo microscopy enhances calcium signals.

<p>(<b>A</b>) <i>In vivo</i> HiLo image of motor neurons of a zebrafish embryo expressing GCaMP5G in soma (ROI1), axons (ROIs 2–4), or background (ROIs 5–7). Scale bar 5 μm. (<b>B</b>) ΔF/F time series in the soma (ROI 1) shows that HiLo mode leads to a larger signal than UWF mode. (<b>C</b>) For axons (ROI 4) the gain in HiLo mode is even larger. Acquisition rate was 25fps.</p