Ethanol selectively inhibits enhanced vesicular release at excitatory synapses: real-time visualization in intact hippocampal slices.
BACKGROUND
Conflicting information exists concerning the actions of ethanol on vesicular release at excitatory synapses. Because long-term alterations in synaptic transmission are thought to underlie neuroadaptive responses to ethanol, we have directly measured the actions of ethanol on release dynamics at an intact central synapse.


METHODS
Here we investigated the effects of ethanol on release dynamics in hippocampal slices using confocal microscopy with the lipophilic dye, FM1-43, complemented by a patch clamp analysis of AMPA miniature excitatory postsynaptic currents (mEPSCs). After a pretreatment/loading paradigm with sulforhodamine (S-Rhd) and FM1-43, stable, dense punctate FM1-43 staining in the CA1 stratum radiatum was evident.


RESULTS
FM1-43 fluorescence destaining was dose-dependently induced by perfusion with elevated K+ (20-60 mM). Cadmium inhibited K+-induced destaining, whereas nifedipine had no significant effect. Ethanol (25-75 mM) inhibited K+-induced destaining with high efficacy and had no effect on basal destaining. Both omega-Conotoxin GVIA and omega-Agatoxin IVA inhibited K+-induced destaining with high efficacy. The combination of omega-Conotoxin GVIA and omega-Agatoxin IVA occluded the inhibitory effect of ethanol, indicating that ethanol inhibition of release was dependent on inhibition of N/P/Q-voltage-gated calcium channels (VGCCs). Patch clamp studies of AMPA mEPSCs revealed similar findings in that vesicular release was enhanced with K+ depolarization in an ethanol-sensitive manner.


CONCLUSIONS
These findings indicate that the FM1-43/S-Rhd method is a stable and powerful approach for direct real-time measurement of vesicular release kinetics in intact brain slice preparations and that ethanol inhibits vesicular release induced by depolarization via inhibition of N/P/Q-VGCCs.
Maldve
Regina E
RE
Division of Pharmacology and Toxicology, the College of Pharmacy, University of Texas at Austin, Austin, Texas, USA.
Chen
Xiaochun
X
Zhang
Tao A
TA
Morrisett
Richard A
RA
D000818:Animals; D000431:Ethanol / Q000494:pharmacology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects* / Q000502:physiology; D005260:Female; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D018408:Patch-Clamp Techniques / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000187:drug effects / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors
D000431:Ethanol