CADENCE - Neuroinformatics Tool for Supervised Calcium Events Detection.

CADENCE is an open Python 3-written neuroinformatics tool with Qt6 graphic user interface for supervised calcium events detection. In neuronal ensembles recording during calcium imaging experiments, the output of instruments such as Celena X, Zeiss LSM 5 Live confocal microscope and Miniscope is a movie showing flashing cells somata. There are few pipelines to convert video to relative fluorescence ΔF/F, from simplest ImageJ plugins to sophisticated tools like MiniAn (Dong et al. in Elife 11, https://doi.org/10.7554/eLife.70661 , 2022). Minian, an open-source miniscope analysis pipeline. Elife, 11.). While in some areas of study relative fluorescence ΔF/F may be the desired result in itself, researchers of neuronal ensembles are typically interested in a more detailed analysis of calcium events as indirect proxy of neuronal electrical activity. For such analyses, researchers need a tool to infer calcium events from the continuous ΔF/F curve in order to create a raster representation of calcium events for later use in analysis software, such as Elephant (Denker, M., Yegenoglu, A., & Grün, S. (2018). Collaborative HPC-enabled workflows on the HBP Collaboratory using the Elephant framework. Neuroinformatics, 19.). Here we present such an open tool with supervised calcium events detection.

The role of the parafascicular thalamic nucleus in action initiation and steering.

The parafascicular (Pf) nucleus of the thalamus has been implicated in arousal and attention, but its contributions to behavior remain poorly characterized. Here, using in vivo and in vitro electrophysiology, optogenetics, and 3D motion capture, we studied the role of the Pf nucleus in behavior using a continuous reward-tracking task in freely moving mice. We found that many Pf neurons precisely represent vector components of velocity, with a strong preference for ipsiversive movements. Their activity usually leads velocity, suggesting that Pf output is critical for self-initiated orienting behavior. To test this hypothesis, we expressed excitatory or inhibitory opsins in VGlut2+ Pf neurons to manipulate neural activity bidirectionally. We found that selective optogenetic stimulation of these neurons consistently produced ipsiversive head turning, whereas inhibition stopped turning and produced downward movements. Taken together, our results suggest that the Pf nucleus can send continuous top-down commands that specify detailed action parameters (e.g., direction and speed of the head), thus providing guidance for orienting and steering during behavior.

Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory.

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.