Jen-Wei Lin

Modulation of available vesicles and release kinetics at the inhibitor of the crayfish neuromuscular junction

We have investigated the effect of serotonin (5-HT) and okadaic acid (OA) on presynaptic processes at the crayfish inhibitory neuromuscular junction. Two different physiological parameters of transmitter release were examined: release kinetics and the size of the readily releasable pool of vesicles (RRP). Using a paired pulse stimulus and high frequency trains, we established that a single broad action potential, recorded in 20 mM tetraethylammonium and 1 mM 4-amino-pyridine, released the RRP in its entirety. Thus, by measuring the amplitude of inhibitory postsynaptic potential (IPSC) we were able to directly assess the effects of 5-HT and OA on the RRP. Serotonin at 200 nM and OA at 2.5 microM each significantly increased IPSC above control levels and the effects of these two modulators were comparable. Both modulators also induced a leftward shift in the rising phase of IPSC, i.e. an apparent acceleration in release kinetics. The shift caused by OA was significantly more pronounced than that induced by 5-HT. This apparent acceleration in release was not associated with a corresponding change in the presynaptic Ca2+ transient measured at a 2 kHz resolution, suggesting that modulation was not due to an acceleration in Ca2+ channel kinetics. In view of the comparable increase in the size of the RRP by the modulators, the differential modulation of release kinetics suggests that these two parameters may be modulated by separate biochemical processes.

A whole-cell clamp study of dendrodendritic synaptic activities in mitral cells of turtle olfactory bulb slices.

A procedure for preparing slices from the turtle olfactory bulb is described in this report. Individual layers of the bulb could be identified in the slices which enabled visual identification of cell types. Mitral cells retained extensive dendritic arborizations in slices of typical thickness, 300-400 microm. The presence of extensive dendritic processes was consistent with the difficulties we encountered in our attempt to achieve adequate space clamp. On the few occasions where an adequate space clamp of a mitral cell was achieved, calcium current exhibited a threshold of - 50 mV and reached its maximal level at - 10 mV. In all cases where calcium current was analysed (n=46), the current exhibited little inactivation. Depolarizing steps in 50% of the mitral cells triggered a burst of feedback synaptic activity after termination of the step. The intensity of feedback activity correlated closely with the amplitude of the depolarizing step, reaching its maximal level at - 10 mV and declining with further depolarization. The bell-shaped relationship between the feedback activity and mitral cell depolarization is consistent with the hypothesis that the feedback activity is mediated by reciprocal synapses on the mitral cell dendrite. This hypothesis is further supported by the inhibitory nature of the feedback synaptic activity: (i) the polarity of the feedback synaptic current could be inverted at the predicted chloride equilibrium potential, (ii) the feedback activity could be completely blocked by 10 microM bicuculline. The analysis of spontaneous synaptic activity showed that it was mostly inhibitory because its polarity could be reversed at the predicted chloride equilibrium potential. In some mitral cells, the frequency of spontaneous activity was noticeably increased when the holding potential was depolarized. This correlation could be attributed to the activation of dendrodendritic synapses. Results shown in this report demonstrate that dendrodendritic synapses are viable in turtle olfactory bulb slices. In addition, the suppression of feedback inhibition by large depolarizing steps of mitral cells suggests that the control of mitral cell dendritic potential is adequate to suppress calcium influx during large depolarizing steps.

Electrophysiological events recorded at presynaptic terminals of the crayfish neuromuscular junction with a voltage indicator

The water‐soluble voltage indicator JPW1114 was used to stain thin axons and terminal varicosities of the crayfish neuromuscular junction. A slow, overnight injection protocol was developed to brightly stain fine structures without cytotoxicity. Fluorescence transients filtered at 2 kHz showed that the duration of terminal action potentials was shorter than that of those recorded in the main trunk of the axons. In addition, the repolarization phases of the terminal and axonal action potentials overlapped in time, suggesting that the entire axonal arborization repolarizes simultaneously. Manipulating resting membrane potential, ±15–20 mV, did not alter the peak level or duration of action potentials if they fired in isolation. A prolongation of action potential, by 23%, could be induced if a 10‐spike burst at 100 Hz was fired from depolarized membrane potential. No such change was observed when the high frequency train was fired from resting or hyperpolarized levels. Microelectrodes in the main trunk of axons typically recorded a depolarizing after‐potential (DAP) following an action potential initiated from resting membrane potential. The DAP could be inverted and enlarged by depolarization and hyperpolarization, respectively. Fluorescence transients recorded from terminals exhibited similar DAP characteristics. The ratio of DAP to action potential amplitude recorded from terminals was similar to that recorded from the main axon. Thus, the entire axonal arborization returned to resting level in a spatially uniform manner during the DAP. The functional significance of DAP is discussed in the light of these observations.

Spatial variation in membrane excitability modulated by 4-AP-sensitive K+ channels in the axons of the crayfish neuromuscular junction

Current-clamp recordings were made from the primary (1°) and secondary (2°) branching points (BPs) of axons at the crayfish neuromuscular junction. Action potential (AP) firing initiated by current injected at the 2° BP showed strong adaptation or high-frequency firing at threshold current, whereas AP firing frequency at the 1° BP exhibited a gradual rise with increasing current amplitude. The voltage threshold for AP (V(TH)) was higher at the 2° BP than the 1° BP. 4-Aminopyridine (4-AP) at 200 μM increased AP amplitude and duration at both BPs but reduced threshold current at the 2° BP more than at the 1° BP. This blocker lowered V(TH) at both BPs, but the difference between the BPs remained. Firing patterns evoked at the 2° BP became similar to those evoked at the 1° BP in 4-AP. Thus 4-AP-sensitive channels may be more concentrated in the distal axon and control AP initiation and firing patterns there. Orthodromic APs between the two BPs were also compared. There was no difference in AP amplitude between the two BPs, but AP half-width recorded at the 2° BP was longer than that at the 1° BP. AP duration at both BPs increased gradually, by ∼17%, during a 100-Hz, 500-ms train (in-train rise). Normalized AP half-widths revealed a smaller fractional in-train rise at the 2° BP. Thus, although distal APs were broader, AP duration there was under more stringent control than that of the proximal axon. 4-AP increased AP amplitude and duration of the entire orthodromic train and reduced the magnitude of the in-train rise in AP half-width at both BPs. However, this blocker did not uncover a clear difference between the two BPs. Thus 4-AP-sensitive channels concentrated in distal axon may be essential in preventing unintended firing and modulating AP waveform without interfering with orthodromic AP propagation.

Spatial gradient in TTX sensitivity of axons at the crayfish opener neuromuscular junction

At the crayfish opener neuromuscular junction, axons branch repeatedly before synapsing onto muscle fibers as varicosities. Excitability of these axons was examined with two-electrode current clamp before and after partial block of Na(+) channels with 1 nM tetrodotoxin. 4-Aminopyridine (200 μM) was added to homogenize nonuniformity in K(+) channel density. The impact of tetrodotoxin was evaluated in terms of action potential (AP) amplitude, rate of rise, and threshold. All three parameters were more severely affected at the secondary than the primary branching point (BP). Both BPs fired continuously during 1-s current steps before tetrodotoxin. After tetrodotoxin, the secondary BP fired only in brief bursts, whereas the primary BP still fired continuously. Despite this diminished excitability at the secondary BP, no failure in orthodromic AP conduction was observed. AP waveform at terminals (AP(f)) was examined with voltage indicators. For orthodromic APs, reduction in AP amplitude and prolongation of AP rise time in tetrodotoxin were more pronounced in terminals than at the secondary BP. For APs initiated at the secondary BP, AP(f) sometimes failed to show a spikelike waveform in tetrodotoxin. This degraded AP(f) was not due to averaging variable AP invasion into terminals, because the variance of AP(f) traces did not increase in tetrodotoxin. Tetrodotoxin applied in the absence of 4-aminopyridine showed an impact on the distal axon similar but less distinct than that recorded with 4-aminopyridine. In conclusion, the distal axon is more sensitive to tetrodotoxin than the proximal axon, such that AP waveform degrades as it propagates toward terminals in tetrodotoxin.

Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction.

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca(2+) channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](o), did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.