Faculty Bibliography

Inhibitory neurons are critical for regulating effective transfer of sensory information and network stability. The precision of inhibitory function likely derives from the existence of a variety of interneuron subtypes. Their specification is largely dependent on the locale of origin of interneuron progenitors. Neocortical and hippocampal inhibitory neurons originate the subpallium, namely in the medial and caudal ganglionic eminences (MGE and CGE), and in the preoptic area (POA). In the hippocampus, neuronal nitric oxide synthase (nNOS)-expressing cells constitute a numerically large GABAergic interneuron population. On the contrary, nNOS-expressing inhibitory neurons constitute the smallest of the known neocortical GABAergic neuronal subtypes. The origins of most neocortical GABAergic neuron subtypes have been thoroughly investigated, however, very little is known about the origin of, or the genetic programs underlying the development of nNOS neurons. Here, we show that the vast majority of neocortical nNOS-expressing neurons arise from the MGE rather than the CGE. Regarding their molecular signature, virtually all neocortical nNOS neurons co-express the neuropeptides somatostatin (SST) and neuropeptide Y (NPY), and about half of them express the calcium-binding protein calretinin (CR). nNOS neurons thus constitute a small cohort of the MGE-derived SST-expressing population of cortical inhibitory neurons. Finally, we show that conditional removal of the transcription factor Sox6 in MGE-derived GABAergic cortical neurons results in an absence of SST and CR expression, as well as reduced expression of nNOS in neocortical nNOS neurons. Based on their respective abundance, origin and molecular signature, our results suggest that neocortical and hippocampal nNOS GABAergic neurons likely subserve different functions and have very different physiological relevance in these two cortical structures.

Exchange mechanisms across the blood-cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood-CSF interface.

The development of two-component expression systems in Drosophila melanogaster, one of the most powerful genetic models, has allowed the precise manipulation of gene function in specific cell populations. These expression systems, in combination with site-specific recombination approaches, have also led to the development of new methods for clonal lineage analysis. We present a hands-on user guide to the techniques and approaches that have greatly increased resolution of genetic analysis in the fly, with a special focus on their application for lineage analysis. Our intention is to provide guidance and suggestions regarding which genetic tools are most suitable for addressing different developmental questions.

The current publishing system with its merits and pitfalls is a mending topic for debate among scientists of various disciplines. Editors and reviewers alike, both face difficult decisions about the judgment of new scientific findings. Increasing interdisciplinary themes and rapidly changing dynamics in method development of each field make it difficult to be an "expert" with regard to all issues of a certain paper. Although unintended, it is likely that misunderstandings, human biases, and even outright mistakes can play an unfortunate role in final verdicts. We propose a new community-driven publication process that is based on network statistics to make the review, publication, and scientific evaluation process more transparent.

In a forward genetic screen in Drosophila, we have isolated insomniac, a mutant that severely reduces the duration and consolidation of sleep. Anatomically restricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. We show that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals.

BACKGROUND: Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. METHODS AND RESULTS: In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor gamma 1 (PPARgamma1) via the cardiac alpha-myosin heavy-chain promoter. The PPARgamma transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARgamma agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARgamma transgenic mice. CONCLUSIONS: Our findings support an important link between PPARgamma activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.

Holographic speckle is a major impediment for the emerging applications of multiphoton holographic projection in biomedical imaging, photo-stimulation and micromachining. Time averaging of multiple shifted versions of a single hologram ("shift-averaging") is a computationally-efficient method that was recently shown to deterministically eliminate holographic speckle in single-photon applications. Here, we extend these results and show, computationally and experimentally, that in two-photon holographic excitation shift-averaging also reduces holographic speckle better than "random" averaging of multiple calculated holograms.

Recurrent excitatory circuits face extreme challenges in balancing efficacy and stability. We recorded from CA3 pyramidal neuron pairs in rat hippocampal slice cultures to characterize synaptic and circuit-level changes in recurrent synapses resulting from long-term inactivity. Chronic tetrodotoxin treatment greatly reduced the percentage of connected CA3-CA3 neurons, but enhanced the strength of the remaining connections; presynaptic release probability sharply increased, whereas quantal size was unaltered. Connectivity was decreased in activity-deprived circuits by functional silencing of synapses, whereas three-dimensional anatomical analysis revealed no change in spine or bouton density or aggregate dendrite length. The silencing arose from enhanced Cdk5 activity and could be reverted by acute Cdk5 inhibition with roscovitine. Our results suggest that recurrent circuits adapt to chronic inactivity by reallocating presynaptic weights heterogeneously, strengthening certain connections while silencing others. This restricts synaptic output and input, preserving signaling efficacy among a subset of neuronal ensembles while protecting network stability.

Transmembrane receptors allow a cell to communicate with its environment in response to a variety of input signals. These can be changes in the concentration of ligands (e.g. hormones or neurotransmitters), temperature, pressure (e.g. acoustic waves or touch), transmembrane potential, or light intensity. Many important receptors have now been characterized in atomic detail and our understanding of their functional properties has markedly increased in recent years. As a consequence, these sophisticated molecular machines can be reprogrammed to respond to unnatural input signals. In this Review, we show how voltage-gated and ligand-gated ion channels can be endowed with synthetic photoswitches, and how the resulting artificial photoreceptors can be used to optically control neurons with exceptional temporal and spatial precision. They work well in animals and might find applications in the restoration of vision and the optical control of other sensations. The combination of synthetic photoswitches and receptor proteins contributes to the field of optogenetics and adds a new functional dimension to chemical genetics. As such, we propose to call it "optochemical genetics".