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Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline.

Choi S, Yu E, Rabello G, Merlo S, Zemmar A, Walton KD, Moreno H, Moreira JE, Sugimori M, Llinás RR - Front Synaptic Neurosci (2014)

Bottom Line: Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites.Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW.After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory Woods Hole, MA, USA ; Department of Neuroscience and Physiology, New York University School of Medicine New York, NY, USA.

ABSTRACT
Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa(++) amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5-10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

No MeSH data available.


Related in: MedlinePlus

Voltage clamp study indicating that RNS60 increases transmitter release without modifying calcium current or its relationship with transmitter release. (A) Set of traces recorded in Control ASW show the amplitude and time course of the presynaptic calcium current (black), the amplitude and time course of the postsynaptic response (green) elicited by the rapid voltage clamp step shown in the third trace (Pre Dep, black). (B) Set of traces recorded in RNS60 ASW with the same amplitude depolarizing pulses as in the control set; EPSPs are red. (C) Superposition of calcium currents (upper traces) and EPSPs (lower trace) from panel (A) for control (green) and panel (B) for RNS60 (red) ASW demonstrates that there was no change in the time course or amplitude of the presynaptic calcium current, but a clear increase in the EPSP amplitude in RNS60 compared to control ASW. (D) Plot of EPSP amplitude as a function of presynaptic depolarization step for the five synapses. (Set of recordings from each synapse use the same marker.) The oxygenated control is modified from Figure 3B in (Llinas et al., 1981b) and provides data from seven synapses superfused with control ASW oxygenated with a 99.5% 02 and 0.5% CO2 gas mixture or with 0.001% H2O2. (E) Mean EPSP and s.e.m. as a function of mean presynaptic depolarizations for synapses in panel (D). Oxygenated control is mean of data in Figure 3B in (Llinas et al., 1981b). [*T(4, 8) = 4.27, p < 0.01; **T(4, 8) = 5.1, p < 0.001; ***T(4, 8) = 3.54, p < 0.05, t-test].
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Figure 4: Voltage clamp study indicating that RNS60 increases transmitter release without modifying calcium current or its relationship with transmitter release. (A) Set of traces recorded in Control ASW show the amplitude and time course of the presynaptic calcium current (black), the amplitude and time course of the postsynaptic response (green) elicited by the rapid voltage clamp step shown in the third trace (Pre Dep, black). (B) Set of traces recorded in RNS60 ASW with the same amplitude depolarizing pulses as in the control set; EPSPs are red. (C) Superposition of calcium currents (upper traces) and EPSPs (lower trace) from panel (A) for control (green) and panel (B) for RNS60 (red) ASW demonstrates that there was no change in the time course or amplitude of the presynaptic calcium current, but a clear increase in the EPSP amplitude in RNS60 compared to control ASW. (D) Plot of EPSP amplitude as a function of presynaptic depolarization step for the five synapses. (Set of recordings from each synapse use the same marker.) The oxygenated control is modified from Figure 3B in (Llinas et al., 1981b) and provides data from seven synapses superfused with control ASW oxygenated with a 99.5% 02 and 0.5% CO2 gas mixture or with 0.001% H2O2. (E) Mean EPSP and s.e.m. as a function of mean presynaptic depolarizations for synapses in panel (D). Oxygenated control is mean of data in Figure 3B in (Llinas et al., 1981b). [*T(4, 8) = 4.27, p < 0.01; **T(4, 8) = 5.1, p < 0.001; ***T(4, 8) = 3.54, p < 0.05, t-test].

Mentions: Presynaptic calcium currents were elicited by graded depolarizing step pulses after pharmacological block of the voltage-gated sodium and potassium conductances (Llinas et al., 1976, 1981a; Augustine and Charlton, 1986). Figure 4A illustrates the presynaptic calcium current (Pre ICa), postsynaptic EPSP, and presynaptic voltage pulse (Pre Dep) at three levels of presynaptic depolarization in control (top traces, green) and RNS60 (bottom traces, red) ASW. The calcium current and EPSP traces are superimposed in Figure 4B. It is immediately apparent that the posstsynaptic response amplitude was larger in RNS60 (red) than in Control (green) ASW and that presynaptic inward calcium current was not significantly modified by RNS60. Note that the difference between the control and RNS60 EPSPs for the largest presynaptic depolarization is less than that for the middle depolarization. This is because the presynaptic membrane is close to the equilibrium potential for calcium, reducing ICa++ and the EPSP amplitude (Llinas et al., 1981a). The EPSP amplitude is plotted in Figure 4C for five synapses as a function of presynaptic voltage clamp depolarization. Each synapse has a different marker and the EPSPs recorded in Control ASW (green) RNS60 ASW (red) may be compared for each synapse. Note that the increase in transmitter release varied among synapses, but in every case was larger in the RNS60 ASW and reached a maximum value. Once this value was attained, we did not observe any further increase with protracted superfusion, suggesting that conditions for optimal transmitter release had been reached. When the mean amplitude of the postsynaptic response in control and RNS60 ASW were compared, significant differences were seen at three levels of depolarization. As may be seen in Figure 4D, depolarizing pulses were not exactly the same amplitude across synapses. To calculate the mean EPSP amplitude, the responses were assigned to one of four groups according to the presynaptic depolarization (two depolarization values, 16.5 and 25 mV, were not included a group). There was a significant difference in EPSP recorded in control and RSN60 ASW in three presynaptic depolarization groups: 38 mV, [T(1, 8) = 4.27, p < 0.01]; 43 mV, [T(1, 8) = 5.1, p < 0.001], 48 mV, [T(1, 8) = 3.54, p < 0.01]. RNS60 did not change the decay constant of the EPSPs. This suggests that there was not a significant change in the passive properties (resistance or capacitance) of the postsynaptic membrane (τ, control 2.99 ± 0.7 ms; RNS60 2.36 ± 0.3 ms, n = 9).


Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline.

Choi S, Yu E, Rabello G, Merlo S, Zemmar A, Walton KD, Moreno H, Moreira JE, Sugimori M, Llinás RR - Front Synaptic Neurosci (2014)

Voltage clamp study indicating that RNS60 increases transmitter release without modifying calcium current or its relationship with transmitter release. (A) Set of traces recorded in Control ASW show the amplitude and time course of the presynaptic calcium current (black), the amplitude and time course of the postsynaptic response (green) elicited by the rapid voltage clamp step shown in the third trace (Pre Dep, black). (B) Set of traces recorded in RNS60 ASW with the same amplitude depolarizing pulses as in the control set; EPSPs are red. (C) Superposition of calcium currents (upper traces) and EPSPs (lower trace) from panel (A) for control (green) and panel (B) for RNS60 (red) ASW demonstrates that there was no change in the time course or amplitude of the presynaptic calcium current, but a clear increase in the EPSP amplitude in RNS60 compared to control ASW. (D) Plot of EPSP amplitude as a function of presynaptic depolarization step for the five synapses. (Set of recordings from each synapse use the same marker.) The oxygenated control is modified from Figure 3B in (Llinas et al., 1981b) and provides data from seven synapses superfused with control ASW oxygenated with a 99.5% 02 and 0.5% CO2 gas mixture or with 0.001% H2O2. (E) Mean EPSP and s.e.m. as a function of mean presynaptic depolarizations for synapses in panel (D). Oxygenated control is mean of data in Figure 3B in (Llinas et al., 1981b). [*T(4, 8) = 4.27, p < 0.01; **T(4, 8) = 5.1, p < 0.001; ***T(4, 8) = 3.54, p < 0.05, t-test].
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Figure 4: Voltage clamp study indicating that RNS60 increases transmitter release without modifying calcium current or its relationship with transmitter release. (A) Set of traces recorded in Control ASW show the amplitude and time course of the presynaptic calcium current (black), the amplitude and time course of the postsynaptic response (green) elicited by the rapid voltage clamp step shown in the third trace (Pre Dep, black). (B) Set of traces recorded in RNS60 ASW with the same amplitude depolarizing pulses as in the control set; EPSPs are red. (C) Superposition of calcium currents (upper traces) and EPSPs (lower trace) from panel (A) for control (green) and panel (B) for RNS60 (red) ASW demonstrates that there was no change in the time course or amplitude of the presynaptic calcium current, but a clear increase in the EPSP amplitude in RNS60 compared to control ASW. (D) Plot of EPSP amplitude as a function of presynaptic depolarization step for the five synapses. (Set of recordings from each synapse use the same marker.) The oxygenated control is modified from Figure 3B in (Llinas et al., 1981b) and provides data from seven synapses superfused with control ASW oxygenated with a 99.5% 02 and 0.5% CO2 gas mixture or with 0.001% H2O2. (E) Mean EPSP and s.e.m. as a function of mean presynaptic depolarizations for synapses in panel (D). Oxygenated control is mean of data in Figure 3B in (Llinas et al., 1981b). [*T(4, 8) = 4.27, p < 0.01; **T(4, 8) = 5.1, p < 0.001; ***T(4, 8) = 3.54, p < 0.05, t-test].
Mentions: Presynaptic calcium currents were elicited by graded depolarizing step pulses after pharmacological block of the voltage-gated sodium and potassium conductances (Llinas et al., 1976, 1981a; Augustine and Charlton, 1986). Figure 4A illustrates the presynaptic calcium current (Pre ICa), postsynaptic EPSP, and presynaptic voltage pulse (Pre Dep) at three levels of presynaptic depolarization in control (top traces, green) and RNS60 (bottom traces, red) ASW. The calcium current and EPSP traces are superimposed in Figure 4B. It is immediately apparent that the posstsynaptic response amplitude was larger in RNS60 (red) than in Control (green) ASW and that presynaptic inward calcium current was not significantly modified by RNS60. Note that the difference between the control and RNS60 EPSPs for the largest presynaptic depolarization is less than that for the middle depolarization. This is because the presynaptic membrane is close to the equilibrium potential for calcium, reducing ICa++ and the EPSP amplitude (Llinas et al., 1981a). The EPSP amplitude is plotted in Figure 4C for five synapses as a function of presynaptic voltage clamp depolarization. Each synapse has a different marker and the EPSPs recorded in Control ASW (green) RNS60 ASW (red) may be compared for each synapse. Note that the increase in transmitter release varied among synapses, but in every case was larger in the RNS60 ASW and reached a maximum value. Once this value was attained, we did not observe any further increase with protracted superfusion, suggesting that conditions for optimal transmitter release had been reached. When the mean amplitude of the postsynaptic response in control and RNS60 ASW were compared, significant differences were seen at three levels of depolarization. As may be seen in Figure 4D, depolarizing pulses were not exactly the same amplitude across synapses. To calculate the mean EPSP amplitude, the responses were assigned to one of four groups according to the presynaptic depolarization (two depolarization values, 16.5 and 25 mV, were not included a group). There was a significant difference in EPSP recorded in control and RSN60 ASW in three presynaptic depolarization groups: 38 mV, [T(1, 8) = 4.27, p < 0.01]; 43 mV, [T(1, 8) = 5.1, p < 0.001], 48 mV, [T(1, 8) = 3.54, p < 0.01]. RNS60 did not change the decay constant of the EPSPs. This suggests that there was not a significant change in the passive properties (resistance or capacitance) of the postsynaptic membrane (τ, control 2.99 ± 0.7 ms; RNS60 2.36 ± 0.3 ms, n = 9).

Bottom Line: Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites.Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW.After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory Woods Hole, MA, USA ; Department of Neuroscience and Physiology, New York University School of Medicine New York, NY, USA.

ABSTRACT
Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa(++) amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5-10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

No MeSH data available.


Related in: MedlinePlus