Experimentally monitored calcium dynamics at synaptic active zones during neurotransmitter release in neuron-muscle cell cultures.

Ca-dependent K (BK) channels at varicosities in Xenopus nerve-muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca] resulting from different rates and durations of Ca entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca tail currents produce mean peak [Ca] ~ 30 μM; with continued influx, [Ca] reaches ~45-60 μM at different rates depending on Ca driving force and duration of influx. Both I and release are dependent on Ca microdomains composed of both N- and L-type Ca channels.

Patch clamp recordings of hair cells isolated from zebrafish auditory and vestibular end organs.

The senses of hearing and balance in vertebrates are transduced by hair cells in the inner ear. Hair cells from a wide variety of organisms have been described electrophysiologically but this is the first report of the application of these techniques to the genetically tractable zebrafish model system. Auditory and vestibular hair cells isolated from zebrafish lagenae and utricles were patch clamped and both inward and outward currents under voltage clamp, and changes in membrane potential under current clamp were recorded.

Simultaneous pre- and post-synaptic electrophysiological recording from Xenopus nerve-muscle co-cultures.

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs).

Patch clamp recordings in inner ear hair cells isolated from zebrafish.

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously(1-4) but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained.

Monitoring transient Ca2+ dynamics with large-conductance Ca2+-dependent K+ channels at active zones in frog saccular hair cells.

Neurotransmitter release from the basolateral surface of auditory and vestibular hair cells is mediated by Ca(2+) influx through voltage-gated Ca(2+) channels. Co-localization of large-conductance Ca(2+)-activated K(+) (BK) channels at the active zones of these cells affords them with an optimal location to act as reporters of the Ca(2+) concentration changes at active zones of transmitter release. In this report we use BK channels in frog (Rana pipiens) hair cells to monitor dynamic changes in intracellular Ca(2+) concentration during transient influxes of Ca(2+), showing that BK current magnitude and delay to onset are correlated with the rate and duration of Ca(2+) entry through Ca(2+) channels.