We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

The goal of sequencing the entire human genome for $1,000 is almost in sight. However, the total costs including DNA sequencing, data management, and analysis to yield a clear data interpretation are unlikely to be lowered significantly any time soon to make studies on a population scale and daily clinical uses feasible. Alternatively, the targeted enrichment of specific groups of disease and biological pathway-focused genes and the capture of up to an entire human exome (~1% of the genome) allowing an unbiased investigation of the complete protein-coding regions in the genome are now routine. Targeted gene capture followed by sequencing with massively parallel next-generation sequencing (NGS) has the advantages of 1) significant cost saving, 2) higher sequencing accuracy because of deeper achievable coverage, 3) a significantly shorter turnaround time, and 4) a more feasible data set for a bioinformatic analysis outcome that is functionally interpretable. Gene capture combined with NGS has allowed a much greater number of samples to be examined than is currently practical with whole-genome sequencing. Such an approach promises to bring a paradigm shift to biomedical research of Mendelian disorders and their clinical diagnoses, ultimately enabling personalized medicine based on one’s genetic profile. In this review, we describe major methodologies currently used for gene capture and detection of genetic variations by NGS. We will highlight applications of this technology in studies of genetic disorders and discuss issues pertaining to applications of this powerful technology in genetic screening and the discovery of genes implicated in syndromic and non-syndromic hearing loss.

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The exquisite sensitivity of elasmobranch fishes to electric fields is thought to reside in electroreceptive organs called ampullae of Lorenzini. We measured the stimulus-response behavior of ampullary organs excised from skates. Under open-circuit conditions, the ampullary organ showed three distinct response states: spontaneous repetitive spikes, evoked spikes, and small, damped oscillatory responses. Under short-circuit conditions, the amplitude range for a linear current response to a sinusoidal (0.5 Hz) voltage clamp of an organ (assessed by spectral analysis of the harmonics generated) was 7-200 microV rms. Changes in the spike firing rate of the afferent nerve innervating the organ were evident for voltage clamps of the ampullary epithelium of 3 microV and the spike rate saturated for clamp steps exceeding 100 microV. Thus, the linear response range of the ampullary epithelium exceeded the range in spike firing rate of the afferent nerve. The steady-state transorgan electrical properties under voltage clamp conditions were obtained by analysis of complex admittance determinations in the frequency range 0.05-20 Hz for perturbations (< 100 microV rms) in the linear range. Admittance functions were distinctly related to the preparation states observed under open-circuit conditions. A negative real part in the organ admittance (i.e., a steady-state negative conductance generated by the preparation) was a common characteristic of the two (open-circuit) excitable states. The negative conductance was also confirmed by the direction of current flow through the ampullary epithelium in response to step voltage clamps. We conclude that the steady state-negative conductance is an essential property of the ampullary epithelium,and we suggest that the interplay of negative and positive conductances generated by ion channels in apical and basal membranes of receptor cells results in signal amplification that may contribute significantly to the electric field sensitivity of ampullary organs.

We used intracellular recording and single electrode voltage-clamp techniques to explore Ca2+ currents and their relation to graded and spike-mediated synaptic transmissions in leech heart interneurons. Low-threshold Ca2+ currents (activation begins below -50 mV) consist of a rapidly inactivating component (I(CaF)) and a slowly inactivating component (I(CaS)). The apparent inactivation kinetics of I(CaF) appears to be influenced by Ca2+; both the substitution of Ca2+ (5 mM) with Ba2+ (5 mM) in the saline and the intracellular injection of the rapid Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), from the recording microelectrode, significantly increase its apparent inactivation time constant. The use of saline with a high concentration of Ba2+ (37.5 mM) permitted exploration of divalent ion currents over a broader activation range, by acting as an effective charge carrier and significantly blocking outward currents. Ramp and pulse voltage-clamp protocols both reveal a rapidly activating and inactivating Ba2+ current (I(BaF)) and a less rapidly activating and slowly inactivating Ba2+ current with a broad activation range (I(BaS)). Low concentrations of Cd2+ (100-150 microM) selectively block I(BaS), without significantly diminishing I(BaF). The current that remains in Cd2+ lacks the characteristic delayed activation peak of I(BaS) and inactivates with two distinct time constants. I(BaF) appears to correspond to a combination of I(CaF) and I(CaS), i.e., to low-threshold Ca2+ currents, that can be described as T-like. I(BaS) appears to correspond to a Ca2+ current with a broad activation range, which can be described as L-like. Cd2+ (100 microM) selectively blocks spike-mediated synaptic transmission between heart interneurons without significantly interfering with low-threshold Ca2+ currents and plateau formation in or graded synaptic transmission between heart interneurons. Blockade of spike-mediated synaptic transmission between reciprocally inhibitory heart interneurons with Cd2+ (150 microM), in otherwise normal saline, prevents the expression of normal oscillations (during which activity in the two neurons consists of alternating bursts), so that the neurons fire tonically. We conclude that graded and spike-mediated synaptic transmission may be relatively independent processes in heart interneurons that are controlled predominantly by different Ca2+ currents. Moreover, spike-mediated synaptic inhibition appears to be required for normal oscillation in these neurons.