Ventral hippocampal CA1 (vCA1) projections to the amygdala are necessary for contextual fear memory. Here we used in vivo Ca 2+ imaging in mice to assess the temporal dynamics by which ensembles of vCA1 neurons mediate encoding and retrieval of contextual fear memories. We found that a subset of vCA1 neurons were responsive to the aversive shock during context conditioning, their activity was necessary for memory encoding, and these shockresponsive neurons were enriched in the vCA1 projection to the amygdala. During memory retrieval, a population of vCA1 neurons became correlated with shock-encoding neurons, and the magnitude of synchronized activity within this population was proportional to memory strength. The emergence of these correlated networks was disrupted by inhibiting vCA1 shock responses during memory encoding. Thus, our findings suggest that networks of cells that become correlated with shock-responsive neurons in vCA1 are essential components of contextual fear memory ensembles.

Sleep is an essential behavioral state of rest that is regulated by homeostatic drives to ensure a balance of sleep and activity, as well as independent arousal mechanisms in the central brain. Dopamine has been identified as a critical regulator of both sleep behavior and arousal. Here, we present results of a genetic screen that selectively restored the Dopamine Receptor (DopR/DopR1/dumb) to specific neuroanatomical regions of the adult Drosophila brain to assess requirements for DopR in sleep behavior. We have identified subsets of the mushroom body that utilizes DopR in daytime sleep regulation. These data are supported by multiple examples of spatially restricted genetic rescue data in discrete circuits of the mushroom body, as well as immunohistochemistry that corroborates the localization of DopR protein within mushroom body circuits. Independent loss of function data using an inducible RNAi construct in the same specific circuits also supports a requirement for DopR in daytime sleep. Additional circuit activation of discrete DopR+ mushroom body neurons also suggests roles for these subpopulations in sleep behavior. These conclusions support a new separable function for DopR in daytime sleep regulation within the mushroom body. This daytime regulation is independent of the known role of DopR in nighttime sleep, which is regulated within the Fan-Shaped Body (FSB). This study provides new neuroanatomical loci for exploration of dopaminergic sleep functions in Drosophila, and expands our understanding of sleep regulation during the day vs. night.

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Background: Stem cell transplantation (Tx) has emerged as a promising new experimental treatment for stroke; understanding its mechanism of action will facilitate the translation of stem cell therapy to the clinic. The ultimate change in brain plasticity is manifested at the synaptic level, however, the synaptic remodeling after stem cell therapy remains unknown. Here we evaluate the effect of transplanted human neural progenitor cells (hNPCs) on the peri-infarct synaptic remodeling in the post-ischemic brain. Materials and Methods: We use array tomography, a high-resolution proteomic imaging method, to determine how hNPCs affect the number and subtype of glutamate and GABA synapses after stroke. Vehicle or hNPCs were transplanted into the ischemic cortex of Nude rats 7 days after distal middle cerebral artery occlusion. Neurological recovery was assessed weekly using a battery of behavioral tests. The arrays of serial ultrathin sections (70 nm), removed from the peri-infarct cortex at 1 and 4 weeks post-Tx, were stained using multiple synaptic markers and imaged in cortical layer 2/3 and 5. Computational analysis of the resultant staining pattern was used to identify and quantify subtypes of glutamate and GABA synapses. Results: Tx of hNPCs significantly improved behavioral recovery after stroke compared to vehicle-treated rats (4 weeks post-transplantation; p<0.01) without altering the infarct size. hNPC-treated rats had a higher density of VGluT1-containing glutamate synapses (0.223 vs 0.185 synapses/μm3, p<0.05), and GluA2-containing glutamate synapses (0.091 vs 0.069 synapses/μm3, p<0.05) in layer 5 at 4 weeks post-Tx, compared to vehicle-treated rats. However, hNPCs had did not alter total number of glutamate synapses. This synaptic increase was cortical layer-specific observed in layer 5 but not .in layer 2/3. hNPCs had no detectable effect on the density of GABA synapses in either layer 5 or 2/3 at 1 week or 4 weeks post-Tx. Conclusions: These results provide novel new information about the organization of synaptic circuitry and its plasticity after stem cell therapy. These data suggest that stem cells alter the subunit composition of glutamate synapses after stroke and this is coincident with stem cell-induced functional recovery.

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Introduction: Neural stem cell (NSC) transplantation is being explored as a potential therapy for ischemic stroke (IS). Although research has shown the efficacy of NSCs in IS, little is known about the gene expression profile of the NSCs after transplantation. To date this has been challenging due to the issue of trying to distinguish factors expressed by the transplanted cells from that of surrounding host cells. We have overcome this critical barrier by adapting the novel TRAP (Translating Ribosome Affinity Purification) approach to selectively purify mRNA from transplanted NSCs. Hypothesis: Application of TRAP technology can enrich isolation of human NSC transcripts from IS rats and enabling identification of the in vivo gene expression profile of transplanted NSCs. Methods: Nude rats were subjected to permanent distal cerebral artery occlusion and after 7 days fetal human NSCs expressing GFP under RPL10a promoter were transplanted into the motor cortex of IS and non-ischemic (NI) rats. Seven days post-transplantation, the graft site was dissected and processed using the TRAP methodology. Seven housekeeping genes (HKG) were analyzed by qPCR in TRAP and non-TRAP samples to determine the level of enrichment of human transcripts. The RNA isolated was converted to cDNA libraries and analyzed using RNAseq technology. Results: The expression level of HKG was consistent between samples and groups when analyzed by the Pearson correlation (r ≥ 8.7), indicating that TRAP samples are compatible between them for sequencing analysis. Furthermore, the average enrichment of human HKG was 22.7-fold (range: 3.37-22.76) and 58.8-fold (range: 1.31-154.94) in IS and NI rats, respectively. Preliminary data from MiSeq analysis detected 317 genes differentially expressed between IS and NI rats. Gene ontology analysis of these genes showed enrichment of genes involved in the intracellular process, stem cell biology, extracellular region, and signaling and secreted proteins. Conclusion: Our data demonstrate that the application of TRAP is feasible to enrich human NSC transcripts from rat cortex, making analysis of their expression profile in the stroke brain possible. This sets the stage to probe the molecular mechanism of action of transplanted stem cells.