Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB260

Poster Communications

Foundations of neocortical high-fidelity synaptic transmission

G. Bornschein1, J. Eilers1, H. Schmidt1

1. 2, Carl-Ludwig Institute for Physiology, Leipzig, Germany.

Highly reliable synaptic transmission between layer 5 pyramidal neurons (L5PNs) is thought to be crucial for neocortical feedforward excitation and ensemble formation (Frick et al., 2008; Markram et al., 1997). However, the synaptic foundations of this high reliability are largely unclear. We analyzed these synapses in 3 - 4 weeks old C57Bl6 mice that were decapitated under deep Isoflurane inhalation anesthesia before excising the brain and cutting coronar slices of neocortical S1 region. In paired recordings from connected L5PNs we found strong depression of EPSC amplitudes (A) at 50 Hz (median A2/A1 = 0.78, 0.69 - 0.90 IQR, n = 86 pairs) and an exceptionally low initial synaptic failure rate (Fsyn = 0, 0 - 0.04) that does not significantly increase after 5 stimulations (Fsyn = 0.07, 0.05 - 0.14, n = 24, P = 0.061, ANOVA on ranks). Using mean-variance analysis of EPSC amplitudes at different extracellular Ca2+ concentrations ([Ca2+]e) we show that L5PN synapses operate at high vesicular release probability of 0.66 (0.55 - 0.71, n = 5) and 8 (3-19) release sites. Dual-dye two-photon Ca2+ imaging with the low-affinity Ca2+ indicator Fluo-5F and the Ca2+-insensitive Alexa-594 revealed that Ca2+ influx into individual presynaptic terminals was small (129 nM, 79 - 219 nM, n = 41 cells) but highly reliable with no failures in single AP-induced Ca2+ influx and low trial-to-trial signal variability. The majority of the Ca2+ influx occurred through ω-agatoxin-IVA (AgTx)-sensitive P/Q-type Ca2+ channels (64%, 50 - 66%, n = 11). Transmitter release was almost exclusively mediated by P/Q-type channels, since AgTx blocked EPSC amplitudes by 85% (82 - 88%, n = 7 pairs). Numerical simulation of influx-release coupling predicts a coupling distance of 32 nm. Indeed, transmitter release was insensitive to 10 mM EGTA since median EPSC amplitudes remained stable at 94% (71 - 102%, n = 5) of the baseline amplitude (P = 0.418 vs. 100%, t-test) after 40 min of presynaptic buffer application. This finding corroborates that the Ca2+ channels are tightly coupled to the Ca2+ sensor. We further investigated sustained synaptic transmission and vesicle replenishment during trains of stimuli at different frequencies. Cumulative EPSC amplitudes were plotted to estimate frequency-dependent reloading rates (at 50 Hz: 0.74 pA/ms, 0.62 - 0.84, n = 9) and the initial size of the ready-releasible pool (92 pA, 69 - 131 pA, n = 9; Schneggenburger et al., 1999). From our data we conclude that the high reliability of sustained synaptic transmission between L5PNs is ensured at several stages translating electrical activity into transmitter release, ranging from highly reliable AP-mediated Ca2+ influx and high pr of tightly coupled synaptic vesicles to rapid vesicle replenishment. These features allow L5PNs to reliably generate and spread neocortical oscillations (Sanchez-Vives and McCormick, 2000; Silva et al., 1991).

Where applicable, experiments conform with Society ethical requirements