An Integrated Platform for In Vivo Electrophysiology in Spatial Cognition Experiments.

Spatial cognition research requires behavioral paradigms that can distinguish between different navigational elements, such as allocentric (map-like) navigation and egocentric (e.g., body centered) navigation. To fill this need, we developed a flexible experimental platform that can be quickly modified without the need for significant changes to software and hardware. In this paper, we present this inexpensive and flexible behavioral platform paired with software which we are making freely available. Our behavioral platform serves as the foundation for a range of experiments, and although developed for assessing spatial cognition, it also has applications in the nonspatial domain of behavioral testing. There are two components of the software platform, "Maze" and "Stim Trigger." While intended as a general platform, presently both programs can work in conjunction with Neuralynx and Open Ephys electrophysiology acquisition systems, allowing for precise time stamping of neural events. The Maze program includes functionality for automatic reward delivery based on user defined zones. "Stim Trigger" permits control of brain stimulation via any equipment that can be paired with an Arduino board. We seek to share our software and leverage the potential by expanding functionality in the future to meet the needs of a larger community of researchers.

A thalamo-parietal cortex circuit is critical for place-action coordination.

The anterior and lateral thalamus (ALT) contains head direction cells that signal the directional orientation of an individual within the environment. ALT has direct and indirect connections with the parietal cortex (PC), an area hypothesized to play a role in coordinating viewer-dependent and viewer-independent spatial reference frames. This coordination between reference frames would allow an individual to translate movements toward a desired location from memory. Thus, ALT-PC functional connectivity would be critical for moving toward remembered allocentric locations. This hypothesis was tested in rats with a place-action task that requires associating an appropriate action (left or right turn) with a spatial location. There are four arms, each offset by 90°, positioned around a central starting point. A trial begins in the central starting point. After exiting a pseudorandomly selected arm, the rat had to displace the correct object covering one of two (left versus right) feeding stations to receive a reward. For a pair of arms facing opposite directions, the reward was located on the left, and for the other pair, the reward was located on the right. Thus, each reward location had a different combination of allocentric location and egocentric action. Removal of an object was scored as correct or incorrect. Trials in which the rat did not displace any objects were scored as "no selection" trials. After an object was removed, the rat returned to the center starting position and the maze was reset for the next trial. To investigate the role of the ALT-PC network, muscimol inactivation infusions targeted bilateral PC, bilateral ALT, or the ALT-PC network. Muscimol sessions were counterbalanced and compared to saline sessions within the same animal. All inactivations resulted in decreased accuracy, but only bilateral PC inactivations resulted in increased non selecting, increased errors, and longer latency responses on the remaining trials. Thus, the ALT-PC circuit is critical for linking an action with a spatial location for successful navigation.

Tau Pathology Profile Across a Parietal-Hippocampal Brain Network Is Associated With Spatial Reorientation Learning and Memory Performance in the 3xTg-AD Mouse.

In early Alzheimer's disease (AD) spatial navigation is one of the first impairments to emerge; however, the precise cause of this impairment is unclear. Previously, we showed that, in a mouse model of tau and amyloid beta (Aß) aggregation, getting lost represents, at least in part, a failure to use distal cues to get oriented in space and that impaired parietal-hippocampal network level plasticity during sleep may underlie this spatial disorientation. However, the relationship between tau and amyloid beta aggregation in this brain network and impaired spatial orientation has not been assessed. Therefore, we used several approaches, including canonical correlation analysis and independent components analysis tools, to examine the relationship between pathology profile across the parietal-hippocampal brain network and spatial reorientation learning and memory performance. We found that consistent with the exclusive impairment in 3xTg-AD 6-month female mice, only 6-month female mice had an ICA identified pattern of tau pathology across the parietal-hippocampal network that were positively correlated with behavior. Specifically, a higher density of pTau positive cells predicted worse spatial learning and memory. Surprisingly, despite a lack of impairment relative to controls, 3-month female, as well as 6- and 12- month male mice all had patterns of tau pathology across the parietal-hippocampal brain network that are predictive of spatial learning and memory performance. However, the direction of the effect was opposite, a negative correlation, meaning that a higher density of pTau positive cells predicted better performance. Finally, there were not significant group or region differences in M78 density at any of the ages examined and ICA analyses were not able to identify any patterns of 6E10 staining across brain regions that were significant predictors of behavioral performance. Thus, the pattern of pTau staining across the parietal-hippocampal network is a strong predictor of spatial learning and memory performance, even for mice with low levels of tau accumulation and intact spatial re-orientation learning and memory. This suggests that AD may cause spatial disorientation as a result of early tau accumulation in the parietal-hippocampal network.

Mesenchymal stem cell-derived extracellular vesicles ameliorate Alzheimer's disease-like phenotypes in a preclinical mouse model.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs. Methods: To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aß) and glial fibrillary acidic protein (GFAP) levels. Results: The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aß plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aß plaques was found in the brain of EV-treated mice compared to saline. Conclusions: Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis.

Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior.

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers in fru P1 neurons differ across development and between the sexes. We validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.

Impaired Hippocampal-Cortical Interactions during Sleep in a Mouse Model of Alzheimer's Disease.

Spatial learning is impaired in humans with preclinical Alzheimer's disease (AD). We reported similar impairments in 3xTg-AD mice learning a spatial reorientation task. Memory reactivation during sleep is critical for learning-related plasticity, and memory consolidation is correlated with hippocampal sharp wave ripple (SWR) density, cortical delta waves (DWs), cortical spindles, and the temporal coupling of these events-postulated as physiological substrates for memory consolidation. Further, hippocampal-cortical discoordination is prevalent in individuals with AD. Thus, we hypothesized that impaired memory consolidation mechanisms in hippocampal-cortical networks could account for spatial memory deficits. We assessed sleep architecture, SWR-DW dynamics, and memory reactivation in a mouse model of tauopathy and amyloidosis implanted with a recording array targeting isocortex and hippocampus. Mice underwent daily recording sessions of rest-task-rest while learning the spatial reorientation task. We assessed memory reactivation by matching activity patterns from the approach to the unmarked reward zone to patterns during slow-wave sleep (SWS). AD mice had more SWS, but reduced SWR density. The increased SWS compensated for reduced SWR density so there was no reduction in SWR number. In control mice, spindles were phase-coupled with DWs, and hippocampal SWR-cortical DW coupling was strengthened in post-task sleep and was correlated with performance on the spatial reorientation task the following day. However, in AD mice, SWR-DW and spindle-DW coupling were impaired. Thus, reduced SWR-DW coupling may cause impaired learning in AD, and spindle-DW coupling during short rest-task-rest sessions may serve as a biomarker for early AD-related changes in these brain dynamics.

Impaired Spatial Reorientation in the 3xTg-AD Mouse Model of Alzheimer's Disease.

In early Alzheimer's disease (AD) spatial navigation is impaired; however, the precise cause of this impairment is unclear. Recent evidence suggests that getting lost is one of the first impairments to emerge in AD. It is possible that getting lost represents a failure to use distal cues to get oriented in space. Therefore, we set out to look for impaired use of distal cues for spatial orientation in a mouse model of amyloidosis (3xTg-AD). To do this, we trained mice to shuttle to the end of a track and back to an enclosed start box to receive a water reward. Then, mice were trained to stop in an unmarked reward zone to receive a brain stimulation reward. The time required to remain in the zone for a reward was increased across training, and the track was positioned in a random start location for each trial. We found that 6-month female, but not 3-month female, 6-month male, or 12-month male, 3xTg-AD mice were impaired. 6-month male and female mice had only intracellular pathology and male mice had less pathology, particularly in the dorsal hippocampus. Thus, AD may cause spatial disorientation as a result of impaired use of landmarks.

A region of euchromatin coincides with an extensive tandem repeat on the mouse (Mus musculus) inactive X chromosome.

Euchromatic features are largely absent from the human inactive X chromosome (Xi), with the exception of several large tandem repeats that can be detected as euchromatin bands at metaphase. Despite residing megabases apart, these tandem repeats make frequent inactive X-specific interactions. The mouse homologue has been reported for at least one of the tandem repeats, but whether the mouse Xi is also characterized by distinct bands of euchromatin remains unknown. We examined the mouse Xi for the presence of euchromatin bands by examining the pattern of histone H3 dimethylated at lysine 4 and detected two major signals. The first band resides in the subtelomeric region of band XF5 and may correspond to the pseudoautosomal region. The second band localizes to XE3 and coincides with an extensive complex repeat composed of a large tandem and inverted repeat segment as well as several large short interspersed nuclear element (SINE)-rich tandem repeats. Fluorescence in situ hybridization reveals that sequences with homology to the repeat region are scattered along the length of the Y chromosome. Immunofluorescence analysis of histone H3 trimethylated at lysine 9 on metaphase chromosomes indicates that the repeat region corresponds to a band of constitutive heterochromatin on the male X and female active X chromosomes, whereas the euchromatin signal appears to be female specific. These data suggest that the band of euchromatin observed at XE3 is unique to the mouse Xi, comparable to the chromatin arrangement of several large tandem repeats located on the human X chromosome.

The macrosatellite DXZ4 mediates CTCF-dependent long-range intrachromosomal interactions on the human inactive X chromosome.

The human X-linked macrosatellite DXZ4 is a large tandem repeat located at Xq23 that is packaged into heterochromatin on the male X chromosome and female active X chromosome and, in response to X chromosome, inactivation is organized into euchromatin bound by the insulator protein CCCTC-binding factor (CTCF) on the inactive X chromosome (Xi). The purpose served by this unusual epigenetic regulation is unclear, but suggests a Xi-specific gain of function for DXZ4. Other less extensive bands of euchromatin can be observed on the Xi, but the identity of the underlying DNA sequences is unknown. Here, we report the identification of two novel human X-linked tandem repeats, located 58 Mb proximal and 16 Mb distal to the macrosatellite DXZ4. Both tandem repeats are entirely contained within the transcriptional unit of novel spliced transcripts. Like DXZ4, the tandem repeats are packaged into Xi-specific CTCF-bound euchromatin. These sequences undergo frequent CTCF-dependent interactions with DXZ4 on the Xi, implicating DXZ4 as an epigenetically regulated Xi-specific structural element and providing the first putative functional attribute of a macrosatellite in the human genome.

YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas.

DXZ4 is an X-linked macrosatellite composed of 12-100 tandemly arranged 3-kb repeat units. In females, it adopts opposite chromatin arrangements at the two alleles in response to X-chromosome inactivation. In males and on the active X chromosome, it is packaged into heterochromatin, but on the inactive X chromosome (Xi), it adopts a euchromatic conformation bound by CTCF. Here we report that the ubiquitous transcription factor YY1 associates with the euchromatic form of DXZ4 on the Xi. The binding of YY1 close to CTCF is reminiscent of that at other epigenetically regulated sequences, including sites of genomic imprinting, and at the X-inactivation centre, suggesting a common mode of action in this arrangement. As with CTCF, binding of YY1 to DXZ4 in vitro is not blocked by CpG methylation, yet in vivo both proteins are restricted to the hypomethylated form. In several male carcinoma cell lines, DXZ4 can adopt a Xi-like conformation in response to cellular transformation, characterized by CpG hypomethylation and binding of YY1 and CTCF. Analysis of a male melanoma cell line and normal skin cells from the same individual confirmed that a transition in chromatin state occurred in response to transformation.