Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures

Abstract

Paired pulse depression (PPD) is a common form of short-term synaptic plasticity. The aim of this study was to characterise PPD at the level of a single inhibitory bouton. Low-density collicular cultures were loaded with the Ca²⁺ indicator Oregon Green-1, active boutons were stained with RH414, and action potentials were blocked with TTX. Evoked IPSCs (eIPSCs) and presynaptic Ca²⁺ transients were recorded in response to direct presynaptic depolarisation of an individual bouton. The single bouton eIPSCs had a low failure rate (< 0.1), large average quantal content (3-6) and slow decay (τ₁ = 15 ms, τ₂ = 81 ms). The PPD of eIPSCs had two distinct components: PPD_fast and PPD_slow (τ = 86 ms and 2 s). PPD_slow showed no dependence on extracellular Ca²⁺ concentration, or on the first eIPSC's failure rate or amplitude. Most probably, it reflects a release-independent inhibition of exocytosis. PPD_fast was only observed in normal or elevated Ca²⁺. It decreased with the failure rate and increased with the amplitude of the first eIPSC. It coincided with paired pulse depression of the presynaptic Ca²⁺ transients (τ = 120 ms). The decay of the latter was accelerated by EGTA, which also reduced PPD_fast. Therefore, a suppressive effect of residual presynaptic Ca²⁺ on subsequent Ca²⁺ influx is considered the most likely cause of PPD_fast. PPD_fast may also have a postsynaptic component, because exposure to a low-affinity GABA_A receptor antagonist (TPMPA; 300 μM) counteracted PPD_fast, and asynchronous IPSC amplitudes were depressed for a short interval following an eIPSC. Thus, at these synapses, PPD is produced by at least two release-independent presynaptic mechanisms and one release-dependent postsynaptic mechanism.