Synapse density regulates independence at unitary inhibitory synapses

Abstract

Neurotransmitter transporters may promote synapse specificity by limiting spillover between release sites. At GABAergic synapses, transport block prolongs synaptic responses when many inputs are activated, yet it is unclear whether transporters alter signaling by single axons. We found that unitary IPSCs generated by paired recordings between hippocampal interneurons and granule cells could be either prolonged or totally unaffected by block of GABA transporters. This variability was explained by the density of active release sites rather than the number of active sites. Prolongation by transport block required release from multiple sites and was enhanced by repetitive activation. Furthermore, transport-sensitive unitary IPSCs were accelerated when the release probability was reduced, indicating that cross talk prolonged the time course of IPSCs even when transport was intact. Our results suggest that the release site density regulates the degree of cross talk as well as the contribution of transporters to GABA clearance. Thus, interplay between release site density and transporter action determines the independence of unitary inhibitory synapses.

Fig. 1.

Prolongation of IPSCs by transport block depends on stimulus. a, Left, Block of GABA transporters with NO711 (20 μm) prolonged IPSCs evoked by focal stimulation of the granule cell layer. Calibration: 500 pA, 50 msec. a, Right, Spontaneous mIPSCs recorded in TTX were unaffected by transport block. Calibration: 20 pA, 20 msec. b, Unitary IPSCs (uIPSC) evoked by paired recordings of interneurons and granule cells were either prolonged or unaffected by transport block. Calibration:left, 120 pA, 20 msec, 50 mV; right, 62 pA, 20 msec, 50 mV. c, The relative prolongation of IPSCs by transport block varied according to stimulus type. The number of experiments is shown in parentheses. The effect of transport block was quantified by the weighted decay τ in NO711 normalized to the weighted decay τ in control (τ_NO711/τ_control).

Fig. 2.

Prolongation by transport block is not correlated with IPSC amplitude. a, uIPSCs were smaller than focally stimulated IPSCs (left), but there was no correlation between the uIPSC amplitude and the prolongation by NO711 (right). Data from all uIPSCs are shown with a linear fit (dotted line). Solid line indicates no effect of transport block (τ_NO711/τ_control = 1).b, Left, At NO711-sensitive synapses, the second uIPSC in a paired-pulse protocol was slightly less prolonged by NO711 compared with the first. Right, There was no correlation between the paired-pulse ratio and the prolongation by NO711 in individual paired recordings.

Fig. 3.

Prolongation of uIPSCs requires release from multiple sites. Top, An individual experiment showing changes in the uIPSC amplitude and half-width in NO711 and NO711 + Cd²⁺ (10 μm). Failures are included for illustration purposes. Bottom, Averaged uIPSCs from the time points indicated (failures excluded). NO711 prolonged the uIPSC. Cd²⁺ reduced the uIPSC amplitude to that of an mIPSC and blocked the prolongation (n = 4).

Fig. 4.

Transport block promotes spillover.a, The low-affinity competitive antagonist SR95103 (5 μm) reduced the uIPSC amplitude to a similar extent in the absence and presence of NO711. Averaged uIPSCs from the time points indicated show that the antagonist accelerated the uIPSC decay only in NO711 (n = 5), suggesting that transport block promotes delayed activation of receptors by a low concentration of GABA. b, Using a similar protocol with IPSCs evoked by focal stimulation, SR95103 accelerated the decay of the multi-fiber IPSC when transport was blocked (n = 6). The stimulus artifacts are blanked for clarity. c, Comparison of the change in half-width for single-fiber unitary and multi-fiber evoked responses. In the absence of transport block, SR95103 had no effect on the uIPSC decay.

Fig. 5.

Repetitive stimulation increases functional synaptic density. a, Left, Transport block prolonged the decay of uIPSCs after repetitive stimulation (10 action potentials at 100 Hz), even in cases in which there was no effect on the single uIPSC. Right, Increasing the number of stimuli enhanced the prolongation by NO711. b, A second uIPSC evoked at 4 msec after the first unmasked greater prolongation by transport block. Middle, The second uIPSC is normalized and aligned with the peak of the single uIPSC. In control, the second uIPSC had the same time course as a single uIPSC.Right, Transport block produced a greater effect on the second response compared with the single. c, With transport intact, the decay of many uIPSCs was prolonged after a train of stimuli (mean prolongation to 148 ± 16%;n = 8).

Fig. 6.

Correlation between synaptic cross talk and sensitivity to uptake block. a, An example of the reduction in uIPSC amplitude (top) and half-width (bottom) when release probability was reduced by Cd²⁺. When Cd²⁺ was washed from the slice, this uIPSC was robustly prolonged by transport block.Insets illustrate averaged responses (failures excluded) from the indicated time points. b, Cd²⁺ reduced the weighted decay of most uIPSCs. Individual experiments are connected by lines, andsolid symbols indicate the mean ± SEM. Cd²⁺ also reduced the uIPSC rise time from 341 ± 50 to 256 ± 23 μsec (n = 6;p = 0.04; one-tailed t test), consistent with a reduction in release asynchrony (Kraushaar and Jonas, 2000). uIPSCs recorded in 0 Cd²⁺ reflect averaged responses from control and wash periods. c, There was a strong correlation (r = 0.93; p= 0.024) between the acceleration in the uIPSC decay in Cd²⁺ and the prolongation of the decay produced by subsequent transport block.

Fig. 7.

uIPSCs evoked by basket cells and axo-axonic cells have different sensitivity to transport block. a1, Representative morphology of a basket cell in the dentate gyrus visualized with Cy5 fluorescence. The dotted lineindicates the boundary of the granule cell layer. The location of the granule cell shown in a2 is indicated with anasterisk. Scale bar, 110 μm. ml, Molecular layer; gcl, granule cell layer.a2, Basket cell axon varicosities (red) overlap the soma and dendrites of the postsynaptic granule cell (green), confirming the identity of the basket cell. The outline of the granule cell was traced and filled for clarity. Scale bar, 8 μm. a3, The uIPSC from this basket cell–granule cell pair was slightly prolonged by NO711. b1, This presynaptic interneuron had characteristics of an axo-axonic cell. The postsynaptic granule cell was also filled with biocytin (asterisk). Scale bar, 110 μm. b2, Putative synaptic contacts formed on the granule cell axon (arrowhead). The image was located just to the hilar side of the asterisk inb1. Scale bar, 8 μm. b3, The uIPSC from this pair was robustly prolonged by NO711.