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Different priming states of synaptic vesicles underlie distinct release probabilities at hippocampal excitatory synapses

Summary A stunning example of synaptic diversity is the postsynaptic target cell-type-dependent difference in synaptic efficacy in cortical networks. Here, we show that CA1 pyramidal cell (PC) to fast spiking interneuron (FSIN) connections have 10-fold larger release probability (Pv) than those on oriens lacunosum-moleculare (O-LM) interneurons. Freeze-fracture immunolabeling revealed that different nano-topologies and coupling distances between Ca2+ channels and release sites (RSs) are not responsible for the distinct Pv. Although [Ca2+] transients are 40% larger in FSINs innervating boutons, when [Ca2+] entry is matched in the two bouton populations, EPSCs in O-LM cells are still 7-fold smaller. However, application of a phorbol ester analog resulted in a ∼2.5-fold larger augmentation at PC – O-LM compared to PC – FSIN synapses, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct RS occupancies and demonstrate that incompletely primed, but docked, vesicles limit the output of PC – O-LM synapses.

: Comparison of active zone (AZ) areas determined by visual inspection or DBSCAN based outlining, Related to figure 2.
(A) Raw images of three axon terminal P-faces labeled for Munc13-1 and Cav2.1 with gold particles.
(B) AZ areas (blue) are outlined manually based on visual inspection of loose clusters of IMPs.
(C) AZ areas (yellow) are outlined based on DBSCAN clustering of all IMPs on the axonal Pface. Yellow dots label IMPs within the cluster, purple dots label IMPs outside the cluster.
(D) AZ areas determined by manual (blue) or DBSCAN based (yellow) outlining in 10 axon terminals.
(F) Density of IMPs within DBSCAN determined clusters (2203.6 ± 489 IMP / µm 2 ) is on average 2.5 ± 1 times higher than outside the cluster (= 948.9 ± 244 IMP / µm 2 ). (A) Reduction of the amplitude of uEPSCs recorded in O-LM cells when the presynaptic PCs are recorded in whole-cell configuration. Individual (thin traces) and averaged (thick traces) uEPSCs evoked by three action potential at 40 Hz are shown from the beginning (black) and end (purple) of the recording period from the same pair (left). Superimposed averaged traces of the 1st uEPSCs at the beginning (black) and end (purple) of the recording (top right). The amplitude of the 1 st uEPSCs is significantly reduced (n = 28, p = 0.0001, Wilcoxon signed-rank test) from the beginning (0 -10 minutes) to the end (20 -30 minutes) of a 30-minute-long recording period without any drug application (bottom right).
(B) Scatter plot between the uEPSCs peak amplitude ratios (last 10 min / first 10 min) and the success rate ratios (last 10 min / first 10 min) demonstrates that a larger rundown is accompanied by a larger increase in failure rate.
(C) Perforated patch-clamp recording of a PC. Differential interference contrast (DIC) image of a PC with an attached gramicidin-containing pipette after sealing (top, left). An epifluorescent image of the Alexa Fluor 594 filled pipette (top, right). After perforation of the membrane, the fluorophore in the pipette does not diffuse into the PC (bottom, left). When the plasma membrane ruptures, the Alexa 594 fluorophore diffuses into the PC (bottom, center). Maximum intensity projection image of the same PC after developing the biocytin with Cy3-coupled streptavidin.
(D) uEPSCs does not show a significant rundown (n = 6, p = 0.83, Wilcoxon signed-rank test) when the presynaptic PC is recorded in the perforated patch-clamp configuration. Panels are the same as in A.
(E) The effect of 1 µM PDBU on the uEPSCs when the presynaptic PCs were recorded in the whole-cell patch-clamp configuration. Panels are the same as in A. PDBU does not have a significant effect when not-corrected for the rundown (n = 18, p = 0.117, Wilcoxon signed-rank test). Box plots represent interquartile range, squares and whiskers indicate mean and SD in all panels. (C) Immunofluorescent labeling for Elfn1, gephyrin and mGluR1α+ in CA1 so. (C right) High magnification view a representative mGluR1α+ dendrite.