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.

Early milestones in the understanding of echolocation in bats.

Almost 80 years ago, Griffin and Galambos discovered the phenomenon of echolocation in bats. Since then, the field has grown exponentially as new generations of investigators have joined the chase and technological advances have revolutionized working with ultrasound in the laboratory and in the field. Today our understanding of the diversity of behavioral and neural adaptations for echolocation constitutes one of the paramount triumphs of neuroethology. At the invitation of the editor in chief, I here review some of the important milestones in the discovery and early understanding of echolocation in bats through about the mid-1980s.

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.

Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves muscle function in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin-glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (<3.5 weeks) ages and demonstrated upregulation of the utrophin-glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (>6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting.

Depolarization-induced Ca2+ entry preferentially evokes release of large quanta in the developing Xenopus neuromuscular junction.

The amplitude histogram of spontaneously occurring miniature synaptic currents (mSCs) is skewed positively at developing Xenopus neuromuscular synapses formed in culture. To test whether the quantal size of nerve-evoked quanta (eSCs) distributes similarly, we compared the amplitude histogram of single quantum eSCs in low external Ca(2+) with that of mSCs and found that nerve stimulation preferentially released large quanta. Depolarization of presynaptic terminals by elevating [K(+)] in the external solution or by direct injection of current through a patch pipette increased the mSC frequency and preferentially, but not exclusively, evoked the release of large quanta, resulting in a second broad peak in the amplitude histogram. Formation of the second peak under these conditions was blocked by the N-type Ca(2+) channel blocker, ω-conotoxin GVIA. In contrast, when the mSC frequency was elevated by thapsigargin- or caffeine-induced mobilization of internal Ca(2+), formation of the second peak did not occur. We conclude that the second peak in the amplitude histogram is generated by Ca(2+) influx through N-type Ca(2+) channels, causing a local elevation of internal Ca(2+). The mSC amplitude in the positively skewed portion of the histogram varied over a wide range. A competitive blocker of acetylcholine (ACh) receptors, d-tubocurarine, reduced the amplitude of smaller mSCs in this range relatively more than that of larger mSCs, suggesting that this variation in the mSC amplitude is due to variable amounts of ACh released from synaptic vesicles. We suggest that Ca(2+) influx through N-type Ca(2+) channels preferentially induces release of vesicles with large ACh content.

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.

The role of integrins in the modulation of neurotransmitter release from motor nerve terminals by stretch and hypertonicity.

Integrins are found at most or all synapses and play a variety of roles. At frog neuromuscular junctions, mechanical tension on integrins due to muscle stretch or hypertonicity causes a powerful modulation of release efficacy. Understanding the mechanism(s) of integrin-mediated modulation will likely further our understanding of mechanisms of neurotransmitter release. The modulation of evoked release with stretch occurs with no detectable delay, does not adapt, and bypasses the Ca(2+) triggering step in vesicle fusion. It depends primarily on integrin bonds to native ligands and requires that one or more proteins in the link between integrins and vesicle fusion be dephosphorylated. Hypertonicity, studied in both frog and Drosophila terminals, causes a larger but slower phasic-tonic change in spontaneous release, which is also Ca(2+)-independent and mostly dependent on integrins, but not dependent on the phosphorylation state of molecules in its pathway of action. In Drosophila, the integrin-dependent component involves the cAMP/PKA pathway, and is absent in mutants lacking PKA. Both stretch and hypertonicity responses in frog terminals are enhanced by agents that elevate PKA levels, suggesting that, in frogs, the cAMP/PKA cascade primarily determines the size of the pool of vesicles available for release by the integrin-mediated mechanism and is not a direct intermediary in the modulation. Evoked release is affected little or even inhibited by hypertonicity. In Drosophila, the inhibition can be explained by a decrease in Ca(2+) influx. The effect of hypertonicity on evoked release in frogs may similarly be a balance between mechanisms that enhance spontaneous release and those that suppress I (Ca).

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A.

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.