The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and subcellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show interanimal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in subcellular events such as organelle positions, we developed a microfluidic device to feed and immobilize Caenorhabditis elegans for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process in vivo . Subcellular imaging of an individual neuron in multiple animals, over 36 h in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an incre...

Listening to vocal music has been recently shown to improve language recovery in stroke survivors. The neuroplasticity mechanisms supporting this effect are, however, still unknown. Using data from a three-arm, single-blind, randomized controlled trial including acute stroke patients ( N  = 38) and a 3 month follow-up, we set out to compare the neuroplasticity effects of daily listening to self-selected vocal music, instrumental music, and audiobooks on both brain activity and structural connectivity of the language network. Using deterministic tractography, we show that the 3 month intervention induced an enhancement of the microstructural properties of the left frontal aslant tract (FAT) for the vocal music group compared with the audiobook group. Importantly, this increase in the strength of the structural connectivity of the left FAT correlated with improved language skills. Analyses of stimulus-specific activation changes showed that the vocal music group exhibited increased activations in the frontal...

Cells in the entorhinal cortex (EC) contain rich spatial information and project strongly to the hippocampus where a cognitive map is supposedly created. These cells range from cells with structured spatial selectivity, such as grid cells in the medial EC (MEC) that are selective to an array of spatial locations that form a hexagonal grid, to weakly spatial cells, such as non-grid cells in the MEC and lateral EC (LEC) that contain spatial information but have no structured spatial selectivity. However, in a small environment, place cells in the hippocampus are generally selective to a single location of the environment, while granule cells in the dentate gyrus of the hippocampus have multiple discrete firing locations but lack spatial periodicity. Given the anatomic connection from the EC to the hippocampus, how the hippocampus retrieves information from upstream EC remains unclear. Here, we propose a unified learning model that can describe the spatial tuning properties of both hippocampal place cells and...

Neuronal population activity in the hippocampal CA3 subfield is implicated in cognitive brain functions such as memory processing and spatial navigation. However, because of its deep location in the brain, the CA3 area has been difficult to target with modern calcium imaging approaches. Here, we achieved chronic two-photon calcium imaging of CA3 pyramidal neurons with the red fluorescent calcium indicator R-CaMP1.07 in anesthetized and awake mice. We characterize CA3 neuronal activity at both the single-cell and population level and assess its stability across multiple imaging days. During both anesthesia and wakefulness, nearly all CA3 pyramidal neurons displayed calcium transients. Most of the calcium transients were consistent with a high incidence of bursts of action potentials (APs), based on calibration measurements using simultaneous juxtacellular recordings and calcium imaging. In awake mice, we found state-dependent differences with striking large and prolonged calcium transients during locomotion...

The ability of neurons to produce behaviorally relevant activity in the absence of pathology relies on the fine balance of synaptic inhibition to excitation. In the hippocampal CA1 microcircuit, this balance is maintained by a diverse population of inhibitory interneurons that receive largely similar glutamatergic afferents as their target pyramidal cells, with EPSCs generated by both AMPA receptors (AMPARs) and NMDA receptors (NMDARs). In this study, we take advantage of a recently generated GluN2A-null rat model to assess the contribution of GluN2A subunits to glutamatergic synaptic currents in three subclasses of interneuron found in the CA1 region of the hippocampus. For both parvalbumin-positive and somatostatin-positive interneurons, the GluN2A subunit is expressed at glutamatergic synapses and contributes to the EPSC. In contrast, in cholecystokinin (CCK)-positive interneurons, the contribution of GluN2A to the EPSC is negligible. Furthermore, synaptic potentiation at glutamatergic synapses on CCK-p...

Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single-cell mRNA sequencing (scRNAseq) could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained fro...

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA level...

Astrocytes provide neurons with diffusible factors that promote synapse formation and maturation. In particular, glypican-4/GPC4 released from astrocytes promotes the maturation of excitatory synapses. Unlike other secreted factors, GPC4 contains the C-terminal GPI-anchorage signal. However, the mechanism by which membrane-tethered GPC4 is released from astrocytes is unknown. Using mouse primary astrocyte cultures and a quantitative luciferase-based release assay, we show that GPC4 is expressed on the astrocyte surface via a GPI-anchorage. Soluble GPC4 is robustly released from the astrocytes largely by proteolytic shedding and, to a lesser extent, by GPI-anchor cleavage, but not by vesicular release. Pharmacological, overexpression, and loss of function screens showed that ADAM9 in part mediates the release of GPC4 from astrocytes. The released GPC4 contains the heparan sulfate side chain, suggesting that these release mechanisms provide the active form that promotes synapse maturation and function. Overa...

Bombesin receptor subtype-3 (BRS3) is an orphan receptor that regulates energy homeostasis. We compared Brs3 driver mice with constitutive or inducible Cre recombinase activity. The constitutive BRS3-Cre mice show a reporter signal (Cre-dependent tdTomato) in the adult brain because of lineage tracing in the dentate gyrus, striatal patches, and indusium griseum, in addition to sites previously identified in the inducible BRS3-Cre mice (including hypothalamic and amygdala subregions, and parabrachial nucleus). We detected Brs3 reporter expression in the dentate gyrus at day 23 but not at postnatal day 1 or 5 months of age. Hypothalamic sites expressed reporter at all three time points, and striatal patches expressed Brs3 reporter at 1 day but not 5 months. Parabrachial nucleus Brs3 neurons project to the preoptic area, hypothalamus, amygdala, and thalamus. Both Cre recombinase insertions reduced Brs3 mRNA levels and BRS3 function, causing obesity phenotypes of different severity. These results demonstrate t...

Patch clamp electrophysiology is a common technique used in neuroscience to understand individual neuron behavior, allowing one to record current and voltage changes with superior spatiotemporal resolution compared with most electrophysiology methods. While patch clamp experiments produce high fidelity electrophysiology data, the technique is onerous and labor intensive. Despite the emergence of patch clamp systems that automate key stages in the typical patch clamp procedure, full automation remains elusive. Patch clamp pipettes can miss the target cell during automated experiments because of positioning errors in the robotic manipulators, which can easily exceed the diameter of a neuron. Further, when patching in acute brain slices, the inherent light scattering from non-uniform brain tissue can complicate pipette tip identification. We present a convolutional neural network (CNN), based on ResNet101, to identify and correct pipette positioning errors before each patch clamp attempt, thereby preventing t...