The Delayed Neuropathological Consequences of Traumatic Brain Injury in a Community-Based Sample.

The late neuropathological effects of traumatic brain injury have yet to be fully elucidated, particularly with respect to community-based cohorts. To contribute to this critical gap in knowledge, we designed a multimodal neuropathological study, integrating traditional and quantitative approaches to detect pathologic changes in 532 consecutive brain autopsies from participants in the Adult Changes in Thought (ACT) study. Diagnostic evaluation including assessment for chronic traumatic encephalopathy (CTE) and quantitative immunoassay-based methods were deployed to examine levels of pathological (hyperphosphorylated) tau (pTau) and amyloid (A) ß in brains from ACT participants with (n = 107) and without (n = 425) history of remote TBI with loss of consciousness (w/LOC). Further neuropathological assessments included immunohistochemistry for α-synuclein and phospho-TDP-43 pathology and astro- (GFAP) and micro- (Iba1) gliosis, mass spectrometry analysis of free radical injury, and gene expression evaluation (RNA sequencing) in a smaller sub-cohort of matched samples (49 cases with TBI and 49 non-exposed matched controls). Out of 532 cases, only 3 (0.6%-none with TBI w/LOC history) showed evidence of the neuropathologic signature of chronic traumatic encephalopathy (CTE). Across the entire cohort, the levels of pTau and Aß showed expected differences for brain region (higher levels in temporal cortex), neuropathological diagnosis (higher in participants with Alzheimer's disease), and APOE genotype (higher in participants with one or more APOE ε4 allele). However, no differences in PHF-tau or Aß1-42 were identified by Histelide with respect to the history of TBI w/LOC. In a subset of TBI cases with more carefully matched control samples and more extensive analysis, those with TBI w/LOC history had higher levels of hippocampal pTau but no significant differences in Aß, α-synuclein, pTDP-43, GFAP, Iba1, or free radical injury. RNA-sequencing also did not reveal significant gene expression associated with any measure of TBI exposure. Combined, these findings suggest long term neuropathological changes associated with TBI w/LOC may be subtle, involve non-traditional pathways of neurotoxicity and neurodegeneration, and/or differ from those in autopsy cohorts specifically selected for neurotrauma exposure.

Visual tuning properties of genetically identified layer 2/3 neuronal types in the primary visual cortex of cre-transgenic mice.

The putative excitatory and inhibitory cell classes within the mouse primary visual cortex V1 have different functional properties as studied using recording microelectrode. Excitatory neurons show high selectivity for the orientation angle of moving gratings while the putative inhibitory neurons show poor selectivity. However, the study of selectivity of the genetically identified interneurons and their subtypes remain controversial. Here we use novel Cre-driver and reporter mice to identify genetic subpopulations in vivo for two-photon calcium dye imaging: Wfs1(+)/Gad1(-) mice that labels layer 2/3 excitatory cell population and Pvalb(+)/Gad1(+) mice that labels a genetic subpopulation of inhibitory neurons. The cells in both mice were identically labeled with a tdTomato protein, visible in vivo, using a Cre-reporter line. We found that the Wfs1(+) cells exhibited visual tuning properties comparable to the excitatory population, i.e., high selectivity and tuning to the angle, direction, and spatial frequency of oriented moving gratings. The functional tuning of Pvalb(+) neurons was consistent with previously reported narrow-spiking interneurons in microelectrode studies, exhibiting poorer selectivity than the excitatory neurons. This study demonstrates the utility of Cre-transgenic mouse technology in selective targeting of subpopulations of neurons and makes them amenable to structural, functional, and connectivity studies.

Molecular and anatomical signatures of sleep deprivation in the mouse brain.

Sleep deprivation (SD) leads to a suite of cognitive and behavioral impairments, and yet the molecular consequences of SD in the brain are poorly understood. Using a systematic immediate-early gene (IEG) mapping to detect neuronal activation, the consequences of SD were mapped primarily to forebrain regions. SD was found to both induce and suppress IEG expression (and thus neuronal activity) in subregions of neocortex, striatum, and other brain regions. Laser microdissection and cDNA microarrays were used to identify the molecular consequences of SD in seven brain regions. In situ hybridization (ISH) for 222 genes selected from the microarray data and other sources confirmed that robust molecular changes were largely restricted to the forebrain. Analysis of the ISH data for 222 genes (publicly accessible at http://sleep.alleninstitute.org) provided a molecular and anatomic signature of the effects of SD on the brain. The suprachiasmatic nucleus (SCN) and the neocortex exhibited differential regulation of the same genes, such that in the SCN genes exhibited time-of-day effects while in the neocortex, genes exhibited only SD and waking (W) effects. In the neocortex, SD activated gene expression in areal-, layer-, and cell type-specific manner. In the forebrain, SD preferentially activated excitatory neurons, as demonstrated by double-labeling, except for striatum which consists primarily of inhibitory neurons. These data provide a characterization of the anatomical and cell type-specific signatures of SD on neuronal activity and gene expression that may account for the associated cognitive and behavioral effects.

Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor.

Genetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.

Wnt signalling regulates adult hippocampal neurogenesis.

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/beta-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.

Redefining the boundaries of the hippocampal CA2 subfield in the mouse using gene expression and 3-dimensional reconstruction.

The morphology of neurons in the main divisions of the hippocampal complex allow the easy identification of granule cells in the dentate gyrus and pyramidal cells in the CA1 and CA3 regions of Ammon's horn. However, neurons in the CA2 subfield have been much more difficult to reliably identify. We have recently identified a set of genes whose expression is restricted to either the dentate gyrus, CA1, CA2, or CA3. Here we show that these genes have an essentially nonoverlapping distribution throughout the entire septotemporal extent of the hippocampus. 3-Dimensional reconstruction of serial sections processed for in situ hybridization of mannosidase 1, alpha (CA1), bcl-2-related ovarian killer protein (CA3), and Purkinje cell protein 4 (dentate gyrus+CA2) was used to define the boundaries of each subregion throughout the entire hippocampus. The boundaries observed for these three genes are recapitulated across a much larger set of genes similarly enriched in specific hippocampal subregions. The extent of CA2 defined on the basis of gene expression is somewhat larger than that previously described on the basis of structural anatomical criteria, particularly at the rostral pole of the hippocampus. These results indicate that, at least at the molecular level, there are robust, consistent genetic boundaries between hippocampal subregions CA1, CA2, CA3, and the dentate gyrus, allowing a redefinition of their boundaries in order to facilitate functional studies of different neuronal subtypes in the hippocampus.

Mice lacking methyl-CpG binding protein 1 have deficits in adult neurogenesis and hippocampal function.

DNA methylation-mediated epigenetic regulation plays critical roles in regulating mammalian gene expression, but its role in normal brain function is not clear. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, has been shown to bind methylated gene promoters and facilitate transcriptional repression in vitro. Here we report the generation and analysis of MBD1-/- mice. MBD1-/- mice had no detectable developmental defects and appeared healthy throughout life. However, we found that MBD1-/- neural stem cells exhibited reduced neuronal differentiation and increased genomic instability. Furthermore, adult MBD1-/- mice had decreased neurogenesis, impaired spatial learning, and a significant reduction in long-term potentiation in the dentate gyrus of the hippocampus. Our findings indicate that DNA methylation is important in maintaining cellular genomic stability and is crucial for normal neural stem cell and brain functions.

A genetic method for selective and quickly reversible silencing of Mammalian neurons.

Genetic methods for neuronal silencing have great promise for allowing selective inactivation of specific cell types within complex neural systems. Present methods, however, are limited in their reversibility by the slow time scale (days) of transcriptional regulation. We report the rapid and reversible inactivation of mammalian cortical neurons expressing the insect G-protein-coupled receptor AlstR (Drosophila allatostatin receptor) [corrected] after application of its peptide ligand allatostatin (AL). The onset and reversal of inactivation could be achieved rapidly, within minutes. Moreover, the effects of AL were selective for AlstR-transfected neurons. The AlstR/AL system is therefore a promising genetic method for selective and quickly reversible silencing of neuronal activity.