The Small and Dynamic Pre-primed Pool at the Release Site; A Useful Concept to Understand Release Probability and Short-Term Synaptic Plasticity?

Abstract

Advanced imaging techniques have revealed that synapses contain nanomodules in which pre- and post-synaptic molecules are brought together to form an integrated subsynaptic component for vesicle release and transmitter reception. Based on data from an electrophysiological study of ours in which release from synapses containing a single nanomodule was induced by brief 50 Hz trains using minimal stimulation, and on data from such imaging studies, we present a possible modus operandi of such a nanomodule. We will describe the techniques and tools used to obtain and analyze the electrophysiological data from single CA3-CA1 hippocampal synapses from the neonatal rat brain. This analysis leads to the proposal that a nanomodule, despite containing a number of release locations, operates as a single release site, releasing at most a single vesicle at a time. In this nanomodule there appears to be two separate sets of release locations, one set that is responsible for release in response to the first few action potentials and another set that produces the release thereafter. The data also suggest that vesicles at the first set of release locations are primed by synaptic inactivity lasting seconds, this synaptic inactivity also resulting in a large heterogeneity in the values for vesicle release probability among the synapses. The number of vesicles being primed at this set of release locations prior to the arrival of an action potential is small (0-3) and varies from train to train. Following the first action potential, this heterogeneity in vesicle release probability largely vanishes in a release-independent manner, shaping a variation in paired-pulse plasticity among the synapses. After the first few action potentials release is produced from the second set of release locations, and is given by vesicles that have been recruited after the onset of synaptic activity. This release depends on the number of such release locations and the recruitment to such a location. The initial heterogeneity in vesicle release probability, its disappearance after a single action potential, and variation in the recruitment to the second set of release locations are instrumental in producing the heterogeneity in short-term synaptic plasticity among these synapses, and can be seen as means to create differential dynamics within a synapse population.

Keywords: glutamate; hippocampus; nanomodule; plasticity; release probability; release site; synapse; vesicle.

Figure 1

Schematic drawing of a functional release site (nanomodule) at rest. The schematic release site contains five release locations. The three red release locations constitute the pre-primed source pool, responsible for phasic release. Here one of these release locations has a docked and pre-primed vesicle. The two green release locations are for recruited vesicles, responsible for tonic release. Voltage-gated calcium channels are indicated in the presynaptic membrane and a nanocluster of AMPA receptors are indicated in the postsynaptic membrane.

Figure 2

Minimal train stimulation of a unitary synaptic input. (A) Five consecutive example sweeps from one synaptic input in response to minimal train stimulation, 10 impulses 50 Hz. (B) Release pattern for the synaptic input shown in (A). Release is indicated with a black bar and failure is indicated by a white bar. (C) Average train response for the synaptic input shown in (A,B). Adapted from Hanse and Gustafsson (2001a).

Figure 3

Quantal size is independent of release probability. (A) Release probability for one synaptic input repeatedly activated with a train consisting of 10 impulses at 50 Hz. (B1) All EPSCs (n = 44) from the 1st position in the train where the release probability was 0.61. (B2) All EPSCs (n = 22) from the 2nd to the 10th position in the train where the release probability was 0.11. To minimize the risk for potential influence of desensitization only EPSCs that were not immediately preceded by another EPSC were included. (B3) Average of the EPSCs included in (B1,B2) superimposed. (C) Summary plot (n = 19 synaptic inputs) shows both the relative amplitude of the EPSCs (open black circles) and the average release probability (filled green circles) as a function of stimulus position in the train. Adapted from Hanse and Gustafsson (2001c).

Figure 4

Minimal train stimulation of a synapse lacking pre-primed pool. (A) Release pattern for one synaptic input repeatedly activated with a train consisting of 10 impulses at 50 Hz. Release is indicated with a black bar and failure is indicated by a white bar. Note the absence of release in the 1st position of the train. (B) Release probability plotted against the position in the stimulus train. The release probability curve is fitted with exponential function indicating a time constant of increased release probability of 56 ms. (C) Release probability in the 2nd position of the train as a function of train length (increasing from 2 to 10). Only trials that up to the train length had contained one release event were selected for the calculation of the release probability in the 2nd position. Note that this curve decays to zero within five stimuli showing that no 1-release trials remain after the 5th stimulus. Adapted from Hanse and Gustafsson (2001d).

Figure 5

Determination of the pre-primed pool and P_ves1. (A) Release pattern for one synaptic input repeatedly activated with a train consisting of 10 impulses at 50 Hz. Release is indicated with a black bar and failure is indicated by a white bar. (B) Release probability in 1st position of the train as a function of train length (increasing from 1 to 10). Red squares represent trials that up to the train length had only contained one release event, and those trials were selected for the calculation of the release probability in the 1st position. In other words, for each train-length x, all trials that contained a single release event in the first × positions were selected out from all trials in a given experiment, and P₁ was calculated. Note that this curve decays to a plateau within five stimuli indicating that no 1-release trials remain after the 5th stimulus. Blue squares represent trials that up to the train length had contained two release events, the second release event in the last position of the examined train length. (C) Summary graph of single release trials from 43 synaptic inputs. Adapted from Hanse and Gustafsson (2001d).

Figure 6

Activity-dependent normalization of P_ves. Vesicle release probability (P_ves) as a function of stimulus position in a 50 Hz train. The synaptic inputs were divided into five groups according their P_ves in the 1st stimulus position. P_ves was calculated using the equation P_ves(n) = 1 – (1 − P_(n))^1/pool(n), where P_(n) is the release probability at stimulus position n and pool_(n) is the size of the pre-primed pool at the nth stimulation. The pre-primed pool was estimated after subtraction of the average release probability curve for synapses lacking pre-primed pool (“Zero P₁” in Figure 8C). Adapted from Hanse and Gustafsson (2001a).

Figure 7

Schematic drawing of a functional release site (nanomodule) during activity. The schematic release site contains five release locations. The three red release locations constitute the pre-primed source pool, responsible for phasic release. The two green release locations are for recruited vesicles, responsible for tonic release. During activity, priming and release occur at the release locations for recruited vesicles and the release locations for pre-primed vesicles do not contribute as indicated by black crosses. The red and white release location indicates recent exocytosis of a pre-primed vesicle. Voltage-gated calcium channels are indicated in the presynaptic membrane and a nanocluster of AMPA receptors are indicated in the postsynaptic membrane.

Figure 8

Heterogeneity in frequency facilitation/depression among the synapses. (A) Relationship between facilitation/depression (P_8–10/P₁) and the size of the pre-primed pool for 43 synaptic inputs. (B) Relationship between facilitation/depression (P_8–10/P₁) and P_ves1 for 43 synaptic inputs. (C) Release probability at each stimulus position in a 50 Hz train for three groups of synaptic inputs; High P₁ (n = 21, blue circles), moderate P₁ (n = 22, black open circles) and synaptic inputs with zero P₁ (n = 9, red squares). Adapted from Hanse and Gustafsson (2001a).