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.
Highlights
Determining the biological variables that control both electrical and chemical synaptic transmission between nerve cells, or between nerve terminals and muscular or glandular systems, has been a very significant area of physiological exploration over the decades
We address the effect of artificial seawater (ASW) based on RNS60 (Khasnavis et al, 2012; Mondal et al, 2012), a physically modified isotonic saline that has been altered to include charge-stabilized nanostructures with an oxygen nanobubble core
ELECTROPHYSIOLOGICAL STUDIES The initial experiments tested the ability of presynaptic activation to generate a postsynaptic response (Hagiwara and Tasaki, 1958; Takeuchi and Takeuchi, 1962; Kusano, 1968) in the presence of RNS60 ASW
Summary
Determining the biological variables that control both electrical and chemical synaptic transmission between nerve cells, or between nerve terminals and muscular or glandular systems, has been a very significant area of physiological exploration over the decades. RNS60 is a physically modified normal saline solution generated by using a rotor/stator device that incorporates controlled turbulence and Taylor-Couette-Poiseuille (TCP) flow under high oxygen pressure. Sodium chloride (0.9% NaCl), USP pH 5.6 (4.5–7.0, Hospira), is processed at 4◦C with a flow rate of 32 mL/s under 1 atm of oxygen backpressure (7.8 mL/s gas flow rate) while maintaining a rotor speed of 3450 rpm. These conditions generate a strong shear layer at the interface between the vapor and liquid phases near the rotor cavities, which correlates with the generation of small bubbles from cavitation. When tested after 24 h, the oxygen content was 55 ± 5 ppm
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