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

Ca2+-dependent K+ (BK) channels at varicosities in Xenopus nerve-muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca2+]AZ resulting from different rates and durations of Ca2+ entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca2+ tail currents produce mean peak [Ca2+]AZ ~ 30 µM; with continued influx, [Ca2+]AZ reaches ~45-60 µM at different rates depending on Ca2+ driving force and duration of influx. Both IBK and release are dependent on Ca2+ microdomains composed of both N- and L-type Ca channels. Domains collapse with a time constant of ~0.6 ms. We have constructed an active zone (AZ) model that approximately fits this data, and depends on incorporation of the high-capacity, low-affinity fixed buffer represented by phospholipid charges in the plasma membrane. Our observations suggest that in this preparation, (1) some BK channels, but few if any of the Ca2+ sensors that trigger release, are located within Ca2+ nanodomains while a large fraction of both are located far enough from Ca channels to be blockable by EGTA, (2) the IBK is more sensitive than the excitatory postsynaptic current (EPSC) to [Ca2+]AZ (K1/2-26 µM vs. ~36 µM [Ca2+]AZ); (3) with increasing [Ca2+]AZ, the IBK grows with a Hill coefficient of 2.5, the EPSC with a coefficient of 3.9; (4) release is dependent on the highest [Ca2+] achieved, independent of the time to reach it; (5) the varicosity synapses differ from mature frog nmjs in significant ways; and (6) BK channels are useful reporters of local [Ca2+]AZ.

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). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis, molecular mechanisms of neurotransmitter release, the role of diffusible messengers in neuromodulation, and in vitro synaptic plasticity.

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. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

Electrophysiological properties of BK channels in Xenopus motor nerve terminals.

Single channel properties of Ca(2+)-activated K(+) (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone-muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca(2+) affinity of the channel (K(d)= 7.7 microM at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca(2+)](i) from 1 microM to 1 mM. The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at > or =+20 mV. The activation rate increased approximately 10-fold in response to a [Ca(2+)](i) increase from 1 to 100 microM, but increased only approximately 2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca(2+)](i) of 100 microM or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca(2+) affinity and rapid Ca(2+) binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca(2+)] at the inner membrane surface.