Julie A. Harris

A mesoscale connectome of the mouse brain

Comprehensive knowledge of the brain’s wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.

Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio).

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Reversing EphB2 depletion rescues cognitive functions in Alzheimer model

Amyloid-β oligomers may cause cognitive deficits in Alzheimer’s disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer’s disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-β-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer’s disease.

Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institutes Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.

Many Neuronal and Behavioral Impairments in Transgenic Mouse Models of Alzheimer’s Disease Are Independent of Caspase Cleavage of the Amyloid Precursor Protein

Previous studies suggested that cleavage of the amyloid precursor protein (APP) at aspartate residue 664 by caspases may play a key role in the pathogenesis of Alzheimers disease. Mutation of this site (D664A) prevents caspase cleavage and the generation of the C-terminal APP fragments C31 and Jcasp, which have been proposed to mediate amyloid-β (Aβ) neurotoxicity. Here we compared human APP transgenic mice with (B254) and without (J20) the D664A mutation in a battery of tests. Before Aβ deposition, hAPP–B254 and hAPP–J20 mice had comparable hippocampal levels of Aβ1-42. At 2–3 or 5–7 months of age, hAPP–B254 and hAPP–J20 mice had similar abnormalities relative to nontransgenic mice in spatial and nonspatial learning and memory, elevated plus maze performance, electrophysiological measures of synaptic transmission and plasticity, and levels of synaptic activity-related proteins. Thus, caspase cleavage of APP at position D664 and generation of C31 do not play a critical role in the development of these abnormalities.

Human P301L-mutant tau expression in mouse entorhinal-hippocampal network causes tau aggregation and presynaptic pathology but no cognitive deficits.

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.

Afferent regulation of neuron number in the cochlear nucleus: cellular and molecular analyses of a critical period.

The neurons of the cochlear nucleus are dependent on input from the auditory nerve for survival during a critical period of development in a variety of vertebrate species. The molecules that underlie this age-dependent vulnerability to deafferentation are for the most part unknown, although recent studies have begun to yield interesting candidate genes. Here, we review the studies that originally described the presence of afferent dependent neuron survival in the cochlear nucleus and the age-dependency of this effect, as well as more recent work that seeks to understand the mechanisms underlying the neuron loss that occurs and the basis of this critical period. While much of the past work on cochlear nucleus neuronal susceptibility has been conducted looking at one or two genes at a time, recent advances in genomics make it possible to screen tens of thousands of genes while looking for candidate genes that are determinants of the critical period response to afferent deprivation.

Efferent pathways of the mouse lateral habenula

The lateral habenula (LHb) is part of the habenula complex of the dorsal thalamus. Recent studies of the LHb have focused on its projections to the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg), which contain γ‐aminobutyric acid (GABA)ergic neurons that mediate reward prediction error via inhibition of dopaminergic activity. However, older studies in the rat have also identified LHb outputs to the lateral and posterior hypothalamus, median raphe, dorsal raphe, and dorsal tegmentum. Although these studies have shown that the medial and lateral divisions of the LHb have somewhat distinct projections, the topographic specificity of LHb efferents is not completely understood, and the relative extent of these projections to brainstem targets is unknown. Here we have used anterograde tracing with adeno‐associated virus–mediated expression of green fluorescent protein, combined with serial two‐photon tomography, to map the efferents of the LHb on a standard coordinate system for the entire mouse brain, and reconstruct the efferent pathways of the LHb in three dimensions. Using automated quantitation of fiber density, we show that in addition to the RMTg, the median raphe, caudal dorsal raphe, and pontine central gray are major recipients of LHb efferents. By using retrograde tract tracing with cholera toxin subunit B, we show that LHb neurons projecting to the hypothalamus, VTA, median raphe, caudal dorsal raphe, and pontine central gray reside in characteristic, but sometimes overlapping regions of the LHb. Together these results provide the anatomical basis for systematic studies of LHb function in neural circuits and behavior in mice. J. Comp. Neurol. 523:32–60, 2015.

Pharmacogenomic testing to prevent aminoglycoside-induced hearing loss in cystic fibrosis patients: potential impact on clinical, patient, and economic outcomes

Background: Aminoglycosides are commonly used in cystic fibrosis patients to treat Pseudomonas aeruginosa respiratory infections. Aminoglycoside-induced hearing loss may occur in 1%–15% of patients with cystic fibrosis, ranging from mild to severe. Recently, a genetic test to identify patients with a mitochondrial mutation (A1555G) that may predispose patients to this adverse event has become available. Although the A1555G variant is very rare, it seems to confer a high risk of severe hearing loss in patients exposed to aminoglycosides.Objective: The objective was to evaluate the potential clinical, patient, and economic outcomes associated with the use of A1555G testing in a cystic fibrosis population, and explore data gaps and uncertainty in its clinical implementation.Methods: We developed a decision-analytic model to evaluate a hypothetical cohort of patients with cystic fibrosis from a societal perspective. Clinical and economic data were derived primarily from a critical literature review. The incidence of aminoglycoside-induced severe hearing loss, quality-adjusted life-years, and total health care costs were evaluated. Sensitivity analyses were conducted to evaluate uncertainty in our results.Results: In the base-case analysis, A1555G testing decreased the risk of severe aminoglycoside-induced hearing loss by 0.12% in the cystic fibrosis population. The discounted incremental cost per quality-adjusted life-years gained was

Family Communication During the Cancer Experience

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