William M. Roberts

Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: Capacitance measurements in saccular hair cells

Depolarization of isolated frog saccular hair cells caused Ca(2+)-dependent increases in membrane capacitance that we interpret as the fusion of synaptic vesicles with the plasma membrane. During a maintained depolarization to -10 mV, the capacitance increased at a rate corresponding to the fusion of approximately 500 vesicles per second at each active zone. Release continued at this high rate for up to 2 s, long enough to exhaust > 5 times the number of vesicles initially in close apposition to the plasma membrane at active zones. We therefore propose that hair cells are specialized for rapid replenishment of vesicles at release sites. Upon repolarization to -70 mV, the capacitance returned exponentially (time constant, approximately 14 s) to near the prestimulus level in perforated-patch recordings, but not in whole-cell recordings, suggesting that a mobile intracellular factor is required for membrane retrieval.

Depolarization Redistributes Synaptic Membrane and Creates a Gradient of Vesicles on the Synaptic Body at a Ribbon Synapse

We used electron tomography of frog saccular hair cells to reconstruct presynaptic ultrastructure at synapses specialized for sustained transmitter release. Synaptic vesicles at inhibited synapses were abundant in the cytoplasm and covered the synaptic body at high density. Continuous maximal stimulation depleted 73% of the vesicles within 800 nm of the synapse, with a concomitant increase in surface area of intracellular cisterns and plasmalemmal infoldings. Docked vesicles were depleted 60%-80% regardless of their distance from the active zone. Vesicles on the synaptic body were depleted primarily in the hemisphere facing the plasmalemma, creating a gradient of vesicles on its surface. We conclude that formation of new synaptic vesicles from cisterns is rate limiting in the vesicle cycle.

Calretinin modifies presynaptic calcium signaling in frog saccular haircells

To determine whether the concentrations of calcium-binding proteins present in some neurons and sensory cells are sufficient to influence presynaptic calcium signaling, we studied the predominant calcium-binding protein in a class of sensory hair cells in the frog ear. Based on antibody affinity and molecular weight, we identified this protein as calretinin. We measured its cytoplasmic concentration to be ∼1.2 mM, sufficient to bind ∼6 mM Ca2+. Calcium signaling was altered when the diffusible cytoplasmic components were replaced by an intracellular solution lacking any fast calcium buffer, and was restored by the addition of 1.2 mM exogenous calretinin to the intracellular solution. We conclude that calretinin, when present at millimolar concentration, can serve as a diffusionally mobile calcium buffer/transporter capable of regulating calcium signaling over nanometer distances at presynaptic sites.

An Image-Free Opto-Mechanical System for Creating Virtual Environments and Imaging Neuronal Activity in Freely Moving Caenorhabditis elegans

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.

A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans

This paper describes the fabrication and use of a microfluidic device for performing whole-animal chemical screens using non-invasive electrophysiological readouts of neuromuscular function in the nematode worm, C. elegans. The device consists of an array of microchannels to which electrodes are attached to form recording modules capable of detecting the electrical activity of the pharynx, a heart-like neuromuscular organ involved in feeding. The array is coupled to a tree-like arrangement of distribution channels that automatically delivers one nematode to each recording module. The same channels are then used to perfuse the recording modules with test solutions while recording the electropharyngeogram (EPG) from each worm with sufficient sensitivity to detect each pharyngeal contraction. The device accurately reported the acute effects of known anthelmintics (anti-nematode drugs) and also correctly distinguished a specific drug-resistant mutant strain of C. elegans from wild type. The approach described here is readily adaptable to parasitic species for the identification of novel anthelmintics. It is also applicable in toxicology and drug discovery programs for human metabolic and degenerative diseases for which C. elegans is used as a model.

Calcium signalling in hair cells: multiple roles in a compact cell

Ca2+ is critical for mechanosensory adaptation, frequency tuning, afferent synaptic transmission, and efferent modulation in hair cells. These four processes involve cytoplasmic Ca2+ in three independent signalling pathways. Recent work suggests that Ca2+ regulates a myosin adaptation motor, and that a mobile Ca2+ buffer is highly concentrated in hair cells. Focal Ca2+ entry and the cytoplasmic Ca2+ buffer help to separate these pathways by limiting the spread of Ca2+ signals.

Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta.

The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons.

Rapidly inactivating and non‐inactivating calcium‐activated potassium currents in frog saccular hair cells

1 Using a semi‐intact epithelial preparation we examined the Ca2+‐activated K+ (KCa) currents of frog (Rana pipiens) saccular hair cells. After blocking voltage‐dependent K+ (KV) currents with 4‐aminopyridine (4‐AP) an outward current containing inactivating (Itransient) and non‐inactivating (Isteady) components remained. 2 The contribution of each varied greatly from cell to cell, with Itransient contributing from 14 to 90 % of the total outward current. Inactivation of Itransient was rapid (τ≈ 2–3 ms) and occurred within the physiological range of membrane potentials (V1/2=−63 mV). Recovery from inactivation was also rapid (τ≈ 10 ms). 3 Suppression of both Itransient and Isteady by depolarizations that approached the Ca2+ equilibrium potential and by treatments that blocked Ca2+ influx (application Ca2+‐free saline or Cd2+), suggest both are Ca2+ dependent. Both were blocked by iberiotoxin, a specific blocker of large‐conductance KCa channels (BK), but not by apamin, a specific blocker of small‐conductance KCa channels. 4 Ensemble‐variance analysis showed that Itransient and Isteady flow through two distinct populations of channels, both of which have a large single‐channel conductance (∼100 pS in non‐symmetrical conditions). Together, these data indicate that both Itransient and Isteady are carried through BK channels, one of which undergoes rapid inactivation while the other does not. 5 Inactivation of Itransient could be removed by extracellular papain and could later be restored by intracellular application of the ‘ball’ domain of the auxiliary subunit (β2) thought to mediate BK channel inactivation in rat chromaffin cells. We hypothesize that Itransient results from the association of a similar β subunit with some of the BK channels and that papain removes inactivation by cleaving extracellular sites required for this association.

Spikes and Membrane Potential Oscillations in Hair Cells Generate Periodic Afferent Activity in the Frog Sacculus

To look for membrane potential oscillations that may contribute to sensory coding or amplification in the ear, we made whole-cell and perforated-patch recordings from hair cells and postsynaptic afferent neurites in the explanted frog sacculus, with mechanoelectrical transduction (MET) blocked. Small depolarizing holding currents, which may serve to replace the in vivo resting MET current, evoked all-or-none calcium spikes (39–75 mV amplitude) in 37% of hair cells tested, and continuous membrane potential oscillations (14–28 mV; 15–130 Hz) in an additional 14% of cells. Spiking hair cells were on average taller and thinner than nonspiking hair cells, and had smaller outward currents through delayed rectifier channels (IKV) and noninactivating calcium-activated potassium channels (IBK,steady), and larger inward rectifier currents (IK1). Some spiking hair cells fired only a brief train at the onset of a current step, but others could sustain repetitive firing (3–70 Hz). Partial blockade of IBK changed the amplitude and frequency of oscillations and spikes, and converted some nonspiking cells into spiking cells. Oscillatory hair cells preferentially amplified sinusoidal stimuli at frequencies near their natural oscillation frequency. Postsynaptic recordings revealed regularly timed bursts of EPSPs in some afferent neurites. EPSP bursts were able to trigger afferent spikes, which may be initiated at the sodium channel cluster located adjacent to the afferent axons most peripheral myelin segment. These results show that some frog saccular hair cells can generate spontaneous rhythmic activity that may drive periodic background activity in afferent axons.

Patch voltage clamping with low-resistance seals : loose patch clamp

Publisher Summary This chapter discusses several loose-seal methods, and emphasizes the possible difficulties associated with each, describing how they can be avoided. The chapter also discusses applications in which the potential across the membrane is controlled. The basic loose-patch clamp, which employs a single loosely sealed extracellular pipette and no intracellular electrodes, is appropriate for studying rapidly activating currents, such as voltage-gated Na+ and K+ currents, in muscles and other large cells with stable resting potentials. This technique is not well suited to studying currents that are much smaller or slower, such as the voltage-gated Ca2+ current in skeletal muscles, because of artifacts associated with the low resistances of the loose seals. A combination of loose-seal patch recordings, tight-seal whole-cell recordings, and freeze-fracture electron micrographs indicate that the array of membrane particles seen at presynaptic active zones on hair cells are dusters of ion channels that contain a carefully regulated mixture of voltage-gated Ca2+ channels and Ca2+-activated K+ channels. Future uses of the loose-seal technique are likely to address a wider range of problems, such as local mechanisms of channel modulation that require preservation of the microscopic structure of the membrane or its spatial relationship to the cytoskeleton.