Theta oscillations are believed to play an important role in the coordination of neuronal firing in the entorhinal (EC)-hippocampal system but the underlying mechanisms are not known. We simultaneously recorded from neurons in multiple regions of the EC-hippocampal loop and examined their temporal relationships. Theta-coordinated synchronous spiking of EC neuronal populations predicted the timing of current sinks in target layers in the hippocampus. However, the temporal delays between population activities in successive anatomical stages were longer (typically by a half theta cycle) than expected from axon conduction velocities and passive synaptic integration of feed-forward excitatory inputs. We hypothesize that the temporal windows set by the theta cycles allow for local circuit interactions and thus a considerable degree of computational independence in subdivisions of the EC-hippocampal loop.

Circadian rhythms in animals are regulated at the level of individual cells and by systemic signaling to coordinate the activities of multiple tissues. The circadian pacemakers have several physiological outputs, including daily locomotor rhythms. Several redox-active compounds have been found to function in regulation of circadian rhythms in cells, however, how particular compounds might be involved in regulating specific animal behaviors remains largely unknown. Here the effects of hydrogen peroxide on Drosophila movement were analyzed using a recently developed three-dimensional real-time multiple fly tracking assay. Both hydrogen peroxide feeding and direct injection of hydrogen peroxide caused increased adult fly locomotor activity. Continuous treatment with hydrogen peroxide also suppressed daily locomotor rhythms. Conditional over-expression of the hydrogen peroxide-producing enzyme superoxide dismutase (SOD) also increased fly activity and altered the patterns of locomotor activity across days and weeks. The real-time fly tracking system allowed for detailed analysis of the effects of these manipulations on behavior. For example, both hydrogen peroxide feeding and SOD over-expression increased all fly motion parameters, however, hydrogen peroxide feeding caused relatively more erratic movement, whereas SOD over-expression produced relatively faster-moving flies. Taken together, the data demonstrate that hydrogen peroxide has dramatic effects on fly movement and daily locomotor rhythms, and implicate hydrogen peroxide in the normal control of these processes.

Animals use information from multiple sensory organs to generate appropriate behavior. Exactly how these different sensory inputs are fused at the motor system is not well understood. Here we study how fly neck motor neurons integrate information from two well characterized sensory systems: visual information from the compound eye and gyroscopic information from the mechanosensory halteres. Extracellular recordings reveal that a subpopulation of neck motor neurons display "gating-like" behavior: they do not fire action potentials in response to visual stimuli alone but will do so if the halteres are coactivated. Intracellular recordings show that these motor neurons receive small, sustained subthreshold visual inputs in addition to larger inputs that are phase locked to haltere movements. Our results suggest that the nonlinear gating-like effect results from summation of these two inputs with the action potential threshold providing the nonlinearity. As a result of this summation, the sustained visual depolarization is transformed into a temporally structured train of action potentials synchronized to the haltere beating movements. This simple mechanism efficiently fuses two different sensory signals and may also explain the context-dependent effects of visual inputs on fly behavior.

The sense of taste is a specialized chemosensory system dedicated to the evaluation of food and drink. Despite the fact that vertebrates and insects have independently evolved distinct anatomic and molecular pathways for taste sensation, there are clear parallels in the organization and coding logic between the two systems. There is now persuasive evidence that tastant quality is mediated by labeled lines, whereby distinct and strictly segregated populations of taste receptor cells encode each of the taste qualities.

Cortical information processing is under state-dependent control of subcortical neuromodulatory systems. Although this modulatory effect is thought to be mediated mainly by slow nonsynaptic metabotropic receptors, other mechanisms, such as direct synaptic transmission, are possible. Yet, it is currently unknown if any such form of subcortical control exists. Here, we present direct evidence of a strong, spatiotemporally precise excitatory input from an ascending neuromodulatory center. Selective stimulation of serotonergic median raphe neurons produced a rapid activation of hippocampal interneurons. At the network level, this subcortical drive was manifested as a pattern of effective disynaptic GABAergic inhibition that spread throughout the circuit. This form of subcortical network regulation should be incorporated into current concepts of normal and pathological cortical function.

Carbonated beverages are commonly available and immensely popular, but little is known about the cellular and molecular mechanisms underlying the perception of carbonation in the mouth. In mammals, carbonation elicits both somatosensory and chemosensory responses, including activation of taste neurons. We have identified the cellular and molecular substrates for the taste of carbonation. By targeted genetic ablation and the silencing of synapses in defined populations of taste receptor cells, we demonstrated that the sour-sensing cells act as the taste sensors for carbonation, and showed that carbonic anhydrase 4, a glycosylphosphatidylinositol-anchored enzyme, functions as the principal CO2 taste sensor. Together, these studies reveal the basis of the taste of carbonation as well as the contribution of taste cells in the orosensory response to CO2.

Behaviour is governed by activity in highly structured neural circuits. Genetically targeted sensors and switches facilitate measurement and manipulation of activity in vivo, linking activity in defined nodes of neural circuits to behaviour. Because of access to specific cell types, these molecular tools will have the largest impact in genetic model systems such as the mouse. Emerging assays of mouse behaviour are beginning to rival those of behaving monkeys in terms of stimulus and behavioural control. We predict that the confluence of new behavioural and molecular tools in the mouse will reveal the logic of complex mammalian circuits.

Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme-substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse-chase experiments, high-content screening, and numerous other protocols.

Many theoretical advances have been made in applying probabilistic inference methods to improve the power of sequence homology searches, yet the BLAST suite of programs is still the workhorse for most of the field. The main reason for this is practical: BLAST’s programs are about 100-fold faster than the fastest competing implementations of probabilistic inference methods. I describe recent work on the HMMER software suite for protein sequence analysis, which implements probabilistic inference using profile hidden Markov models. Our aim in HMMER3 is to achieve BLAST’s speed while further improving the power of probabilistic inference based methods. HMMER3 implements a new probabilistic model of local sequence alignment and a new heuristic acceleration algorithm. Combined with efficient vector-parallel implementations on modern processors, these improvements synergize. HMMER3 uses more powerful log-odds likelihood scores (scores summed over alignment uncertainty, rather than scoring a single optimal alignment); it calculates accurate expectation values (E-values) for those scores without simulation using a generalization of Karlin/Altschul theory; it computes posterior distributions over the ensemble of possible alignments and returns posterior probabilities (confidences) in each aligned residue; and it does all this at an overall speed comparable to BLAST. The HMMER project aims to usher in a new generation of more powerful homology search tools based on probabilistic inference methods.

Administration of aminoglycoside antibiotics can precipitate sudden, profound bouts of weakness that have been attributed to block of presynaptic voltage-activated calcium channels (VACCs) and failure of neuromuscular transmission. This serious adverse drug reaction is more likely in neuromuscular diseases such as myasthenia gravis. The relatively low affinity of VACC for aminoglycosides prompted us to explore alternative mechanisms. We hypothesized that the presynaptic Ca(2+)-sensing receptor (CaSR) may contribute to aminoglycoside-induced weakness due to its role in modulating synaptic transmission and its sensitivity to aminoglycosides in heterologous expression systems. We have previously shown that presynaptic CaSR controls a non-selective cation channel (NSCC) that regulates nerve terminal excitability and transmitter release. Using direct, electrophysiological recording, we report that neuronal VACCs are inhibited by neomycin (IC(50) 830 +/- 110 microM) at a much lower affinity than CaSR-modulated NSCC currents recorded from acutely isolated presynaptic terminals (synaptosomes; IC(50) 20 +/- 1 microM). Thus, at clinically relevant concentrations, aminoglycoside-induced weakness is likely precipitated by enhanced CaSR activation and subsequent decrease in terminal excitability rather than through direct inhibition of VACCs themselves.