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Comparative Study
. 2006 Nov 29;26(48):12487-96.
doi: 10.1523/JNEUROSCI.3106-06.2006.

Release probability-dependent scaling of the postsynaptic responses at single hippocampal GABAergic synapses

Agota A Biró  1 , Noémi B Holderith, Zoltan Nusser
Affiliations
Comparative Study

Release probability-dependent scaling of the postsynaptic responses at single hippocampal GABAergic synapses

Agota A Biró et al. J Neurosci. .

Abstract

The amount of neurotransmitter released after the arrival of an action potential affects the strength and the trial-to-trial variability of postsynaptic responses. Most studies examining the dependence of synaptic neurotransmitter concentration on the release probability (P(r)) have focused on glutamatergic synapses. Here we asked whether univesicular or multivesicular release characterizes transmission at hippocampal GABAergic synapses. We used multiple probability functional analysis to derive quantal parameters at inhibitory connections between cannabinoid receptor- and cholecystokinin (CCK)-expressing interneurons and CA3 pyramidal cells. After the recordings, the cells were visualized and reconstructed at the light-microscopic level, and the number of boutons mediating the IPSCs was determined using electron microscopy (EM). The number of active zones (AZs) per CCK-immunopositive bouton was determined from three-dimensional EM reconstructions, thus allowing the calculation of the total number of AZs for each pair. Our results reveal an approximate fivefold discrepancy between the numbers of functionally determined release sites (17.4 +/- 3.2) and structurally identified AZs (3.7 +/- 0.9). Channel modeling predicts that a fivefold to sevenfold increase in the peak synaptic GABA concentration is required for the fivefold enhancement of the postsynaptic responses. Kinetic analysis of the unitary IPSCs indicates that the increase in synaptic GABA concentration is most likely attributable to multivesicular release. This change in the synaptic GABA concentration transient together with extremely low postsynaptic receptor occupancy permits a P(r)-dependent scaling of the postsynaptic response generated at a single hippocampal GABAergic synaptic contact.

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Figures

Figure 1.
Figure 1.
Determination of the number of functional release sites and EM identification of the number of boutons for a basket cell– pyramidal cell pair (AB624). A, Membrane potential responses of the interneuron to ±400 pA current injections. B, Trains of presynaptic APs (red) evoke uIPSCs (light blue, individual traces; dark blue, averaged trace) in the postsynaptic pyramidal cell. Main panel, LM reconstruction of the biocytin-labeled presynaptic basket (soma and dendrites are black, axon is red) and postsynaptic pyramidal cell (soma and dendrites are blue, axon is gray). Inset, The location of EM-identified boutons is shown at a higher magnification. EM images illustrate two synaptic contacts between the filled presynaptic axon terminals and the soma of the pyramidal cell. The arrowheads point to synaptic junctions. Scale bar, 0.2 μm. C, Plot of peak amplitude of uIPSCs versus time. The horizontal bars indicate the epochs used for MPFA in E. Arrows indicate the start of washin of different extracellular solutions ([Ca2+] and [Mg2+] are in millimolars). D, Representative traces are shown from epochs indicated by the gray bars in panel C. Individual uIPSCs are light blue; the averaged current traces are dark blue. E, Quantal parameters were derived from the parabola fitted to the variance versus mean current plot. Error bars indicate the theoretical error (σvar) in the variance. The fit was weighted by 1/σvar 2.
Figure 2.
Figure 2.
Functional and structural determination of the number of release sites (AB651). A, LM reconstruction of the presynaptic MFA IN (soma and dendrites are black, axonal arbor is red) and the postsynaptic pyramidal cell (soma and dendrites are blue, partial axonal arbor is light gray, arrowhead indicates the Schaffer collateral heading toward the CA1). Inset, The location of the five contact sites at a higher magnification. str. lac. mol., Stratum lacunosum moleculare; str. rad., stratum radiatum; str. luc., stratum lucidum; str. pyr., stratum pyramidale; str. oriens, stratum oriens. B, Responses of the presynaptic interneuron to depolarizing and hyperpolarizing current injections (±400 pA). C1–C5, Electron micrographs illustrate the synaptic junctions (arrowheads) between presynaptic boutons (b) and postsynaptic dendrites (d; numbers 1–3, 5) or soma (s; number 4). Scale bars, 0.2 μm D, Trains of presynaptic APs (red) in the GABAergic interneuron evoked uIPSCs (blue) in a pyramidal cell. In this pair, release only occurred after ∼20 APs. The CB1 receptor antagonist/inverse agonist AM251 (10 μm; shown in inset) effectively increased the initial Pr such that small-amplitude IPSCs are already evoked by the first APs. E, The peak amplitude of single AP-evoked uIPSCs (0.33 Hz in 10 μm AM251) is plotted as a function of time. Horizontal black and gray bars indicate epochs of postsynaptic responses at different Pr conditions used for determination of quantal parameters. Arrows indicate the start of washin of different extracellular solutions; [Ca2+] and [Mg2+] are in millimolars. Bottom, Representative uIPSCs under low- and high-Pr conditions. The gray bars in the top panel indicate the epochs from where 25 individual traces (light blue) and the averaged current traces (dark blue) are shown. F, Parabola fitted to the variance versus mean current plot yielded an NF of 41 and a q of 26.5 pA. Error bars indicate the theoretical error (σvar) in the variance. The fit was weighted by 1/σvar 2. Because of the large discrepancy between NF(mean) (41) and NF(0.025) (6), this cell was not included in our final analysis.
Figure 3.
Figure 3.
Morphological characterization of CCK-immunopositive axon terminals. A, Three-dimensional reconstruction of a CCK-positive bouton (corresponds to bouton 2 in Table 2) that establishes four AZs (orange, indicated by numbers) on a pyramidal cell soma. Inset, The bouton from a different angle. A1–A6, Electron micrographs of the CCK-immunopositive (immunogold particles) bouton shown in A. AZs are recognized from the rigid apposition of presynaptic and postsynaptic membranes and from the clusters of vesicles. Arrowheads mark the edges of the AZs, and the numbers correspond to those in A. Numbers in the top right corner represent the number of the sections from the series. B, A representative dendritic bouton (bouton 7 in Table 2) with two release sites. Inset, The bouton from a different angle. B1–B4, Two pairs of consecutive EM images illustrate the two AZs of the bouton shown in B. Arrowheads mark the edges of the AZs. Numbers in the top right corner correspond to the number of sections from the series. C, One of the largest dendritic boutons (bouton 9 in Table 2) contains only a single release site. D, The number of AZs has a positive correlation with the bouton volume for CCK+ axon terminals making somatic synapses (filled triangle), but no correlation was found for boutons synapsing on dendrites (filled circle). Open symbols above the plot represent boutons of the recorded INs synapsing on somata (open triangle) and dendrites (open circle). In LM reconstructions, each side of the cubes is 0.5 μm; EM images are all at the same magnification. Scale bar, 0.2 μm.
Figure 4.
Figure 4.
Estimating changes in peak GABA concentration underlying a fivefold increase in peak open probability of postsynaptic GABAA receptors. The peak Po of GABAA receptors are plotted against the peak cleft GABA concentration using three kinetic models. Synaptic GABA concentration transients were modeled with an instantaneous rise and single-exponential decay time of 0.1 ms (gray) or 0.3 ms (black). Vertical lines (solid lines for τGABA = 0.3 ms, dashed lines for τGABA = 0.1 ms) delineate the changes in peak [GABA] for which a fivefold increase in the peak Po occurs in the steepest part of the curve. Kinetic parameters of the GABAA receptor models were adopted from Jones and Westbrook (1995; J &W model), Mozrzymas et al. (1999; M & C model), and Haas and Macdonald, (1999; H & M model).
Figure 5.
Figure 5.
Kinetic analysis of uIPSCs. A, The 10–90% rise time of uIPSCs does not change from low- to high-Pr conditions. B, The weighted decay time constant of uIPSCs was larger at high-Pr conditions (open symbols, individual uIPSCs; filled symbols, mean ± SEM). C, Distribution of the ratio of IPSC decay time constants at high- and low-Pr conditions. Only 1 of 13 cells showed acceleration, whereas in the rest of the cells there was a slowing of the decay under the high-Pr condition. D, Representative uIPSCs under low-Pr (gray) and high-Pr (black) conditions (AB607) are normalized to their peak amplitudes. Exponential fits to the decay are superimposed. Inset, Non-peak scaled uIPSCs and the presynaptic AP.
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