Faculty Bibliography

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".

Dopamine (DA) is a key transmitter in the basal ganglia, yet DA transmission does not conform to several aspects of the classic synaptic doctrine. Axonal DA release occurs through vesicular exocytosis and is action potential- and Ca(2+) -dependent. However, in addition to axonal release, DA neurons in midbrain exhibit somatodendritic release by an incompletely understood, but apparently exocytotic, mechanism. Even in striatum, axonal release sites are controversial, with evidence for DA varicosities that lack postsynaptic specialization, and largely extrasynaptic DA receptors and transporters. Moreover, DA release is often assumed to reflect a global response to a population of activities in midbrain DA neurons, whether tonic or phasic, with precise timing and specificity of action governed by other basal ganglia circuits. This view has been reinforced by anatomical evidence showing dense axonal DA arbors throughout striatum, and a lattice network formed by DA axons and glutamatergic input from cortex and thalamus. Nonetheless, localized DA transients are seen in vivo using voltammetric methods with high spatial and temporal resolution. Mechanistic studies using similar methods in vitro have revealed local regulation of DA release by other transmitters and modulators, as well as by proteins known to be disrupted in Parkinson's disease and other movement disorders. Notably, the actions of most other striatal transmitters on DA release also do not conform to the synaptic doctrine, with the absence of direct synaptic contacts for glutamate, GABA, and acetylcholine (ACh) on striatal DA axons. Overall, the findings reviewed here indicate that DA signaling in the basal ganglia is sculpted by cooperation between the timing and pattern of DA input and those of local regulatory factors. This article is part of a Special Issue entitled: Function and Dysfunction of the Basal Ganglia

Skeletal muscle development involves the differentiation of myoblasts into myotubes; likely involving connexins (Cx) and the gap junctional exchange of secondary messengers and metabolites. Some patients with oculodentodigital dysplasia (ODDD), a rare primarily autosomal dominant disease caused by mutations in the gene encoding Cx43, become less ambulatory with aging and exhibit symptoms that may reflect defects in skeletal muscle development, maintenance, and repair. In this study, the role of Cx43 in skeletal muscle development was examined in differentiation-competent L6 myoblasts and two ODDD-linked Cx43-mutant mouse lines. Undifferentiated L6 myoblasts exhibited high levels of Cx43-based gap junctions which were inhibited by the co-expression of I130T and G60S mutants. Upon myotube formation, Cx43 was rapidly down-regulated and gap junctions were lost but myotubes continued to enlarge. Cx43-mutant mice heterozygous for the I130T mutation exhibited similar body weight, tibial length and muscle fiber size as littermate controls. While mutant mice heterozygous for the G60S mutation also had similar tibial length their overall size and muscle fibre diameter were significantly smaller. Overall, Cx43 regulation may be important for initial myoblast differentiation but not linked to myotube growth and while our mutant mouse studies suggest that some ODDD patients are predicted to have smaller muscle fibres, this appears to be dependent on the site of the Cx43 mutation or other unknown etiologies

Neostriatal cholinergic interneurons are believed to be important for reinforcement-mediated learning and response selection by signaling the occurrence and motivational value of behaviorally relevant stimuli through precisely timed multiphasic population responses. An important problem is to understand how these signals regulate the functioning of the neostriatum. Here we describe the synaptic organization of a previously unknown circuit that involves direct nicotinic excitation of several classes of GABAergic interneurons, including neuroptide Y-expressing neurogilaform neurons, and enables cholinergic interneurons to exert rapid inhibitory control of the activity of projection neurons. We also found that, in vivo, the dominant effect of an optogenetically reproduced pause-excitation population response of cholinergic interneurons was powerful and rapid inhibition of the firing of projection neurons that is coincident with synchronous cholinergic activation. These results reveal a previously unknown circuit mechanism that transmits reinforcement-related information of ChAT interneurons in the mouse neostriatal network