Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors

Abstract

Photoreceptors have depolarized resting potentials that stimulate calcium-dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca2+ dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole cell recordings from light-adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post-synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca2+ influx by lowering extracellular Ca2+, by application of Cd2+ and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca2+. We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd2+ or by increased intracellular Ca2+ buffering in rods, but not in cones, that were voltage-clamped at −70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked EPSCs suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca2+-independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran-conjugated pHrodo. Spontaneous release in rods occurred more frequently at non-ribbon sites than evoked release events. The function of Ca2+-independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.

Distinct Actions of Voltage-Activated Ca2+Channel Block on Spontaneous Release at Excitatory and Inhibitory Central Synapses

“…To further test the action of VACC activity on spontaneous release, we used a structurally different type of VACC blocker, MVIIC, which is a peptide toxin specific for block of N-and P/Q-type VACCs. N-, P/Q-, and R-type VACCs are expressed in neocortical nerve terminals and have been shown to contribute to spontaneous and evoked release at these synapses (Cao and Tsien, 2005;Bucurenciu et al, 2010). Application of a saturating concentration of MVIIC (5 M) had no effect on mEPSC or mIPSC rise time, decay phase, or amplitude ( Fig.…”

“…All animal procedures were approved by the VA Portland Health Care System (VAPORHCS) and Oregon Health and Science University Institutional Animal Care and Use Committees. Mouse pups of either sex were used to prepare acute slices from the neocortex or the medial nucleus of the trapezoid body (MNTB) at postnatal day 12 (P12)-P16 and P10 -P12, respectively, as described previously (Forsythe, 1994;Borst and Sakmann, 1996;Taschenberger and von Gersdorff, 2000). Animals were anesthetized using isoflurane and decapitated.…”

“…5). One advantage of this preparation is that principal cells usually receive one large glutamatergic calyx-type synapse on their soma and multiple small bouton-type inhibitory synapses that release glycine or GABA (von Gersdorff and Borst, 2002). Thus, in voltage-clamp mode, we acquired and resolved mEPSCs and mIPSCs simultaneously from the same postsynaptic cell by using bath and pipette solutions that widely separated the reversal potentials for excitatory and inhibitory transmissions (see Materials and Methods; Fig.…”

Calcium dependence of spontaneous neurotransmitter release

“…However, even within sensory cells the role of VACCs can be more complex. In photoreceptors there are both VACC‐dependent and [Ca 2+ ] i ‐independent components of spontaneous release, with the former being more concentrated around ribbons (Cork, Van Hook, & Thoreson, ).…”

“…However, even within sensory cells the role of VACCs can be more complex. In photoreceptors there are both VACCdependent and [Ca 21 ] i -independent components of spontaneous release, with the former being more concentrated around ribbons (Cork, Van Hook, & Thoreson, 2016 (a) Fluorescent micrograph labeled for synaptophysin (green) and DAPI (blue) illustrates that a large number of nerve terminals may synapse onto a single neocortical neuron. Spontaneous release will increase with the number of synaptic contacts, along with other factors including temperature.…”