CAFFEINE-MEDIATED MODULATION OF CONDUCTION VELOCITY IN HIPPOCAMPAL MOSSY FIBER AXONS

Axonal excitability is essential for reliable action potential propagation, yet the functional contribution of intracellular organelles such as the axonal endoplasmic reticulum (ER) remains poorly understood. Recent ultrastructural studies have revealed a continuous network of tubular ER extending along axons and expanding at presynaptic varicosities, raising the possibility that axonal ER contributes to neuronal signaling. Hippocampal mossy fiber axons selectively express type 2 ryanodine receptors (RyR2), suggesting a role for ER-mediated Ca²⁺ signaling in regulating axonal function. We examined the effect of caffeine, a RyR activator, on mossy fiber excitability using experimental and computational approaches. Overall, caffeine suppressed axonal spike amplitude and delayed propagation. In extracellular field-potential recordings, fiber volleys isolated under Ca²⁺-free conditions showed reversible amplitude reduction and prolonged latency during bath application of 10 mM caffeine. Crucially, to dissociate axonal propagation from changes at the stimulation site or slice-wide effects, we performed loose-patch recordings from individual mossy fiber boutons with focal caffeine perfusion restricted to the recording region. Under these conditions, local caffeine application similarly reduced spike amplitude and increased latency, directly demonstrating slowed action potential propagation, independent of changes in threshold at the stimulation site. In parallel, computational simulations showed that a modest reduction in voltage-gated Na⁺ conductance (90–95% of control) reproduced the experimental changes in spike timing and waveform, including alterations in the second derivative of membrane potential. These results support the notion that RyR2-mediated Ca²⁺ release from axonal ER transiently suppresses mossy fiber excitability by modulating sodium channel–dependent action potential propagation.