Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1+ progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a+ progenitors generated Purkinje cells. The olig2+ eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a+ progenitors in the VZ but some originated from the atoh1+ progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.

The Drosophila mushroom body (MB) is a key associative memory center that has also been implicated in the control of sleep. However, the identity of MB neurons underlying homeostatic sleep regulation, as well as the types of sleep signals generated by specific classes of MB neurons, has remained poorly understood. We recently identified two MB output neuron (MBON) classes whose axons convey sleep control signals from the MB to converge in the same downstream target region: a cholinergic sleep-promoting MBON class and a glutamatergic wake-promoting MBON class. Here, we deploy a combination of neurogenetic, behavioral, and physiological approaches to identify and mechanistically dissect sleep-controlling circuits of the MB. Our studies reveal the existence of two segregated excitatory synaptic microcircuits that propagate homeostatic sleep information from different populations of intrinsic MB "Kenyon cells" (KCs) to specific sleep-regulating MBONs: sleep-promoting KCs increase sleep by preferentially activating the cholinergic MBONs, while wake-promoting KCs decrease sleep by preferentially activating the glutamatergic MBONs. Importantly, activity of the sleep-promoting MB microcircuit is increased by sleep deprivation and is necessary for homeostatic rebound sleep (i.e., the increased sleep that occurs after, and in compensation for, sleep lost during deprivation). These studies reveal for the first time specific functional connections between subsets of KCs and particular MBONs and establish the identity of synaptic microcircuits underlying transmission of homeostatic sleep signals in the MB.

The formation of functional neuronal circuits relies on accurate migration and proper axonal outgrowth of neuronal precursors. On the route to their targets migrating cells and growing axons depend on both, directional information from neurotropic cues and adhesive interactions mediated via extracellular matrix molecules or neighbouring cells. The inactivation of guidance cues or the interference with cell adhesion can cause severe defects in neuronal migration and axon guidance. In this study we have analyzed the function of the MAM domain containing glycosylphosphatidylinositol anchor 2A (MDGA2A) protein in zebrafish cranial motoneuron development. MDGA2A is prominently expressed in distinct clusters of cranial motoneurons, especially in the ones of the trigeminal and facial nerves. Analyses of MDGA2A knockdown embryos by light sheet and confocal microscopy revealed impaired migration and aberrant axonal outgrowth of these neurons; suggesting that adhesive interactions mediated by MDGA2A are required for the proper arrangement and outgrowth of cranial motoneuron subtypes.

Vocal learning in songbirds requires a basal ganglia circuit termed the anterior forebrain pathway (AFP). The AFP is not required for song production, and its role in song learning is not well understood. Like the mammalian striatum, the striatal component of the AFP, Area X, receives dense dopaminergic innervation from the midbrain. Since dopamine (DA) clearly plays a crucial role in basal ganglia-mediated motor control and learning in mammals, it seems likely that DA signaling contributes importantly to the functions of Area X as well. In this study, we used voltammetric methods to detect subsecond changes in extracellular DA concentration to gain better understanding of the properties and regulation of DA release and uptake in Area X. We electrically stimulated Ca(2+)- and action potential-dependent release of an electroactive substance in Area X brain slices and identified the substance as DA by the voltammetric waveform, electrode selectivity, and neurochemical and pharmacological evidence. As in the mammalian striatum, DA release in Area X is depressed by autoinhibition, and the lifetime of extracellular DA is strongly constrained by monoamine transporters. These results add to the known physiological similarities of the mammalian and songbird striatum and support further use of voltammetry in songbirds to investigate the role of basal ganglia DA in motor learning.

Sodium channels in the somata and dendrites of hippocampal CA1 pyramidal neurons undergo a form of long-lasting, cumulative inactivation that is involved in regulating back-propagating action potential amplitude and can influence dendritic excitation. Using cell-attached patch-pipette recordings in the somata and apical dendrites of CA1 pyramidal neurons, we determined the properties of slow inactivation on response to trains of brief depolarizations. We find that the amount of slow inactivation gradually increases as a function of distance from the soma. Slow inactivation is also frequency and voltage dependent. Higher frequency depolarizations increase both the amount of slow inactivation and its rate of recovery. Hyperpolarized resting potentials and larger command potentials accelerate recovery from slow inactivation. We compare this form of slow inactivation to that reported in other cell types, using longer depolarizations, and construct a simplified biophysical model to examine the possible gating mechanisms underlying slow inactivation. Our results suggest that sodium channels can enter slow inactivation rapidly from the open state during brief depolarizations or slowly from a fast inactivation state during longer depolarizations. Because of these properties of slow inactivation, sodium channels will modulate neuronal excitability in a way that depends in a complicated manner on the resting potential and previous history of action potential firing.

We can resolve multiple discrete features within a focal region of m spatial dimensions by first isolating each on the basis of n >/= 1 unique optical characteristics and then measuring their relative spatial coordinates. The minimum acceptable separation between features depends on the point-spread function in the (m + n)d-dimensional space formed by the spatial coordinates and the optical parameters, whereas the absolute spatial resolution is determined by the accuracy to which the coordinates can be measured. Estimates of each suggest that near-field fluorescence excitation microscopy/spectroscopy with molecular sensitivity and spatial resolution is possible.

Commentary: Inspired by my earlier work (see below) in single molecule imaging and the isolation of multiple exciton recombination sites within a single probe volume, here I proposed the principle which would eventually lead to PALM. Indeed, all methods of localization microscopy, including PALM, fPALM, PALMIRA, STORM, dSTORM, PAINT, GSDIM, etc. are specific embodiments of the general principle of single molecule isolation and localization I introduced here.

Locomotion requires coordinated motor activity throughout an animal's body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

This paper identifies the prospects of using aphid species as ideal genetic model systems for the study of evolutionary developmental biology and genetic control of polyphenisms. The advantages and disadvantages of using aphids as genetic model organisms are discussed.