Faculty Bibliography

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures. A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

The apical dendrites of many neurons contain proximal and distal compartments that receive synaptic inputs from different brain regions. These compartments also contain distinct complements of ion channels that enable the differential processing of their respective synaptic inputs, making them functionally distinct. At present, the molecular mechanisms that specify dendritic compartments are not well understood. Here, we report that the extracellular matrix protein Reelin, acting through its downstream, intracellular Dab1 and Src family tyrosine kinase signaling cascade, is essential for establishing and maintaining the molecular identity of the distal dendritic compartment of cortical pyramidal neurons. We find that Reelin signaling is required for the striking enrichment of HCN1 and GIRK1 channels in the distal tuft dendrites of both hippocampal CA1 and neocortical layer 5 pyramidal neurons, where the channels actively filter inputs targeted to these dendritic domains.

The proper regulation of translation is required for the expression of long-lasting synaptic plasticity. A major site of translational control involves the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) by PKR-like endoplasmic reticulum (ER) kinase (PERK). To determine the role of PERK in hippocampal synaptic plasticity, we used the Cre-lox expression system to selectively disrupt PERK expression in the adult mouse forebrain. Here, we demonstrate that in hippocampal area CA1, metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) is associated with increased eIF2alpha phosphorylation, whereas stimulation of early- and late-phase long-term potentiation (E-LTP and L-LTP, respectively) is associated with decreased eIF2alpha phosphorylation. Interesting, although PERK-deficient mice exhibit exaggerated mGluR-LTD, both E-LTP and L-LTP remained intact. We also found that mGluR-LTD is associated with a PERK-dependent increase in eIF2alpha phosphorylation. Our findings are consistent with the notion that eIF2alpha phosphorylation is a key site for the bidirectional control of persistent forms of synaptic LTP and LTD and suggest a distinct role for PERK in mGluR-LTD.

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.

Concentration of the neuronal marker, N-acetylaspartate (NAA), a quantitative metric for the health and density of neurons, is currently obtained by integration of the manually defined peak in whole-head proton (1 H)-MRS. Our goal was to develop a full spectral modeling approach for the automatic estimation of the whole-brain NAA concentration (WBNAA) and to compare the performance of this approach with a manual frequency-range peak integration approach previously employed. MRI and whole-head 1 H-MRS from 18 healthy young adults were examined. Non-localized, whole-head 1 H-MRS obtained at 3 T yielded the NAA peak area through both manually defined frequency-range integration and the new, full spectral simulation. The NAA peak area was converted into an absolute amount with phantom replacement and normalized for brain volume (segmented from T1 -weighted MRI) to yield WBNAA. A paired-sample t test was used to compare the means of the WBNAA paradigms and a likelihood ratio test used to compare their coefficients of variation. While the between-subject WBNAA means were nearly identical (12.8 +/- 2.5 mm for integration, 12.8 +/- 1.4 mm for spectral modeling), the latter's standard deviation was significantly smaller (by ~50%, p = 0.026). The within-subject variability was 11.7% (+/-1.3 mm) for integration versus 7.0% (+/-0.8 mm) for spectral modeling, i.e., a 40% improvement. The (quantifiable) quality of the modeling approach was high, as reflected by Cramer-Rao lower bounds below 0.1% and vanishingly small (experimental - fitted) residuals. Modeling of the whole-head 1 H-MRS increases WBNAA quantification reliability by reducing its variability, its susceptibility to operator bias and baseline roll, and by providing quality-control feedback. Together, these enhance the usefulness of the technique for monitoring the diffuse progression and treatment response of neurological disorders

The hedgehog (HH) pathway is well known for its mitogenic and morphogenic functions during development, and HH signaling continues in discrete populations of cells within many adult mammalian tissues. Growing evidence indicates that HH regulates diverse quiescent stem cell populations, but the exact roles that HH signaling plays in adult organ homeostasis and regeneration remain poorly understood. Here, we review recently identified functions of HH in modulating the behavior of tissue-specific adult stem and progenitor cells during homeostasis, regeneration and disease. We conclude that HH signaling is a key factor in the regulation of adult tissue homeostasis and repair, acting via multiple different routes to regulate distinct cellular outcomes, including maintenance of plasticity, in a context-dependent manner.

Theta oscillations in the limbic system depend on the integrity of the medial septum. The different populations of medial septal neurons (cholinergic and GABAergic) are assumed to affect different aspects of theta oscillations. Using optogenetic stimulation of cholinergic neurons in ChAT-Cre mice, we investigated their effects on hippocampal local field potentials in both anesthetized and behaving mice. Cholinergic stimulation completely blocked sharp wave ripples and strongly suppressed the power of both slow oscillations (0.5-2 Hz in anesthetized, 0.5-4 Hz in behaving animals) and supratheta (6-10 Hz in anesthetized, 10-25 Hz in behaving animals) bands. The same stimulation robustly increased both the power and coherence of theta oscillations (2-6 Hz) in urethane-anesthetized mice. In behaving mice, cholinergic stimulation was less effective in the theta (4-10 Hz) band yet it also increased the ratio of theta/slow oscillation and theta coherence. The effects on gamma oscillations largely mirrored those of theta. These findings show that medial septal cholinergic activation can both enhance theta rhythm and suppress peri-theta frequency bands, allowing theta oscillations to dominate.

Alzheimer's disease is a neurodegenerative disorder that is the most common cause of dementia in the elderly today. One of the earliest reported signs of Alzheimer's disease is olfactory dysfunction, which may manifest in a variety of ways. The present study sought to address this issue by investigating odor coding in the anterior piriform cortex, the primary cortical region involved in higher order olfactory function, and how it relates to performance on olfactory behavioral tasks. An olfactory habituation task was performed on cohorts of transgenic and age-matched wild-type mice at 3, 6 and 12 months of age. These animals were then anesthetized and acute, single-unit electrophysiology was performed in the anterior piriform cortex. In addition, in a separate group of animals, a longitudinal odor discrimination task was conducted from 3-12 months of age. Results showed that while odor habituation was impaired at all ages, Tg2576 performed comparably to age-matched wild-type mice on the olfactory discrimination task. The behavioral data mirrored intact anterior piriform cortex single-unit odor responses and receptive fields in Tg2576, which were comparable to wild-type at all age groups. The present results suggest that odor processing in the olfactory cortex and basic odor discrimination is especially robust in the face of amyloid beta precursor protein (AbetaPP) over-expression and advancing amyloid beta (Abeta) pathology. Odor identification deficits known to emerge early in Alzheimer's disease progression, therefore, may reflect impairments in linking the odor percept to associated labels in cortical regions upstream of the primary olfactory pathway, rather than in the basic odor processing itself.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex(1). Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene(2,3), to drive the expression of specific genes through in utero electroporation.