Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 1998 Jul 15;18(14):5160-9.
doi: 10.1523/JNEUROSCI.18-14-05160.1998.

Neuromodulators enhance transmitter release by two separate mechanisms at the inhibitor of crayfish opener muscle

Affiliations
Comparative Study

Neuromodulators enhance transmitter release by two separate mechanisms at the inhibitor of crayfish opener muscle

A Vyshedskiy et al. J Neurosci. .

Abstract

A presynaptic voltage control method has been used to investigate the modulatory effects of serotonin (5-HT) and okadaic acid (OA) on the inhibitory junction of the crayfish opener muscle. Instead of using action potentials, we used 20 msec pulses depolarized to 0 mV to activate transmitter release. This approach allowed us to monitor two separate physiological parameters related to the release process. The first parameter, transmitter release kinetics, is characterized as the delay when inhibitory postsynaptic conductance reaches its half-maximum (IPSG50). The second parameter, the total area of IPSG (IPSGarea), estimates total transmitter output. We have reported previously that the F2 component of synaptic facilitation is associated with a decrease in IPSG50 but without a change in IPSGarea. These results raised the possibility that IPSG50 and IPSGarea could be mediated by separate mechanisms that were modulated independently. To explore this possibility, we investigated the effects of 5-HT (100-200 nM) and OA (2.5 microM) on the two parameters. 5-HT and OA enhanced IPSG neither by changing the sensitivity of postsynaptic receptors, as tested by iontophoretically ejected GABA, nor by elevating resting and action potential-activated presynaptic free calcium, as monitored by fura-2 imaging. 5-HT and OA decreased IPSG50 by 3.0 +/- 1.4 and 3.6 +/- 1.1 msec, respectively, and increased IPSGarea by 50 +/- 21 and 37 +/- 6%, respectively. The ability of F2 facilitation to accelerate release kinetics was reduced in the presence of the modulators, suggesting that the mechanism underlying the accelerated release kinetics was shared by the two modes of synaptic enhancement. This report demonstrates that the acceleration in release kinetics and the increase in total release are two separate mechanisms for enhancing transmitter output and that these two mechanisms can be activated without changes in presynaptic calcium dynamics.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
5-HT and OA enhance action potential-mediated transmitter release of the inhibitor.A, Time courses of peak IPSGs enhanced by 100 nm 5-HT (A1) and 2.5 μm OA (A2). Peak IPSGs were normalized to the average IPSG measured 5 min before the modulators were applied. Insets in A1 andA2, Control (1 and dashed line) and enhanced (2 and solid line) IPSPs that represent the averages of 60 and 75 trials, respectively. ΔECl andRm measured before and after 5-HT was applied were 9 mV, 0.45 MΩ and 8.3 mV, 0.4 MΩ, respectively. ΔECl and Rmmeasured before and after OA was applied were 10 mV, 0.6 MΩ and 10 mV, 0.7 MΩ, respectively. B, Averaged time courses showing the effects of 5-HT (n = 12) and OA (n = 5) on IPSG amplitude. Open triangles in B2 represent averaged results (n = 5) from control experiments in which perfusion was stopped but in which no OA was added. All experiments were performed at 23°C.
Fig. 2.
Fig. 2.
5-HT and OA have no effect on GABA receptors of muscle fibers. A, 5-HT (A1) and OA (A2) do not changegGABA. gGABAamplitudes were calculated from membrane potential changes in a muscle fiber activated by iontophoretically applied GABA.Insets, GABA-activated membrane potential changes before (1 and dashed line) and after (2 and solid line) modulator application. The traces represent the averages of 38 trials. B, IPSGs calculated from action potential-activated IPSPs in the same muscle fibers. Both 5-HT (B1) and OA (B2) increase the IPSG amplitude. ΔECl and Rmmeasured before and after 5-HT application were 7.5 mV, 0.21 MΩ and 7.5 mV, 0.21 MΩ, respectively. The same parameters measured before and after OA application were 8.8 mV, 0.29 MΩ and 9 mV, 0.3 MΩ, respectively. All experiments were performed at 23°C.
Fig. 3.
Fig. 3.
Effects of 5-HT and OA on [Ca2+]i level. Resting [Ca2+]i levels are not changed by 5-HT (A) or OA (B). Activity-dependent calcium influx was activated by a train of 10 Hz action potentials for 30 sec. 5-HT (200 nm) did not create any detectable change in the [Ca2+]iincrease activated by the action potential train (A). OA (2.5 μm) application caused a small but significant decrease in the [Ca2+]i increase activated by the same stimulation as in A (B).
Fig. 4.
Fig. 4.
5-HT and OA accelerate release kinetics and increase IPSGarea. A, Pre- (lower) and postsynaptic (upper) potentials recorded 5 min before (dashed line) and ∼50 min after (solid line) 100 nm 5-HT (A1) and 2.5 μm OA (A2) were applied. Presynaptic steps were 20 msec in duration and depolarized to 0 mV. A1 andA2 share the same time scale. B, IPSGs converted from IPSPs shown in A. 5-HT (B1) and OA (B2) increase IPSGarea significantly. The kinetics of IPSG is quantified by measuring the interval between the beginning of the pulse and the half-maximum point of IPSG (○). B1 andB2 share the same time scale. C, Changes of IPSG50 over time after 5-HT (C1) and OA (C2) application. Y-scales are identical on both graphs. Averaged results, with SD, are shown at the end (see Results).D, Time course of change in IPSGarea in response to 5-HT (D1) and OA (D2) application. Y-scales are identical on both graphs. Averaged results, with SD, are shown at the end (see Results). ΔECl,Rm, and τm of the muscle fiber measured before and after 5-HT application were 10.1 mV, 0.47 MΩ, 9.2 msec and 10.3 mV, 0.47 MΩ, 8.9 msec, respectively. The membrane characteristics of the muscle fiber used in the OA experiment were 5.6 mV, 0.66 MΩ, 13.8 msec and 6.2 mV, 0.75 MΩ, 17.0 msec, respectively.
Fig. 5.
Fig. 5.
Interaction between synaptic enhancement mediated by F2 facilitation and modulators. A, IPSG50measured from both control (○) and facilitated (•) IPSGs is decreased by 100 nm 5-HT (A1) and 2.5 μm OA (A2). The dotted lines are used to highlight the fact that 5-HT did not decrease IPSG50 beyond the level achieved by F2 facilitation, whereas OA was able to reduce IPSG50 beyond that level.B, The effect of F2 facilitation is quantified by calculating the difference between control and facilitated IPSG50 (ΔIPSG50). Both 5-HT (B1) and OA (B2) decrease ΔIPSG50. The averaged percentage change in ΔIPSG50 is shown also, with SD (see Results). Membrane characteristics of the muscle fiber used in the 5-HT experiment were 10.4 mV, 0.89 MΩ, 13.5 msec and 10.4 mV, 0.88 MΩ, 13.3 msec. The same parameters measured from the OA experiment were 6.6 mV, 0.38 MΩ, 9.0 msec and 6.8 mV, 0.39 MΩ, 9.3 msec.
Fig. 6.
Fig. 6.
Multiple mechanisms for enhancement transmitter release. A, A schematic drawing to illustrate the two mechanisms that can enhance transmitter release. The solid curve in the upper panel represents the time course of vesicular release activated by a 20 msec presynaptic pulse (lower trace, solid line). Transmitter release can be increased by accelerating release kinetics (wide-spaced dotted line and α↑) and/or by increasing the total number of vesicles released without altering release kinetics (dashed line and V↑). A brief presynaptic depolarization with a duration of 2.5 msec (lower trace, dashed line) allows one to observe only the beginning of transmitter release and therefore does not provide sufficient resolution to observe the mechanisms underlying release (double arrows). Inset, The transmitter release process is modeled according to a simple reaction in whichV represents the concentration of available vesicles andR represents release; the secretion step has a forward reaction rate of α, which is modulated by calcium ions.B, Block diagram illustrating different mechanisms that could increase transmitter output and the relationship between activity-dependent and modulator-mediated synaptic enhancement. 5-HT and OA are both able to increase protein phosphorylation. Physiological effects of protein phosphorylation include an acceleration in release kinetics and an increase in total release. Both mechanisms result in an increase in action potential-activated release. Facilitation processes have access only to the kinetic branch of the flow chart. It remains unclear whether the increase in [Ca+2]i associated with F2 facilitation accelerates release kinetics by way of protein phosphorylation (dotted arrow) or processes further downstream (dashed arrow).

Similar articles

Cited by

References

    1. Atwood HL, Dixon D, Wojtowicz JM. Rapid introduction of long-term synaptic changes at crustacean neuromuscular junction. J Neurobiol. 1989;20:373–385. - PubMed
    1. Bertram R, Sherman A, Stanley EF. Single-domain/bound calcium hypothesis of transmitter release and facilitation. J Neurophysiol. 1996;75:1919–1931. - PubMed
    1. Bialojan C, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphorylation. Biochem J. 1988;256:283–290. - PMC - PubMed
    1. Bittner GD. Synaptic plasticity at the crayfish opener neuromuscular preparation. J Neurobiol. 1989;20:386–406. - PubMed
    1. Byers D, Davis RL, Kiger JA. Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster. Nature. 1981;289:79–81. - PubMed

Publication types

MeSH terms

LinkOut - more resources