Fig. 1: Cholinergic interneurons are connected by a strong polysynaptic inhibitory pathway.

a Neurons expressing acetylcholine transferase (ChAT) are labeled in a ChAT-Cre mouse crossed to a tdTomato reporter and patched in whole-cell patch clamp configuration (n = 35 ChINs recorded in this configuration from 15 mice). Scale bar: 30 µm. b Schematic representation of a paired whole-cell recording from two neighboring ChINs expressing feedback and feed-forward polysynaptic inhibition. c ChINs are identified by their typical I–V response, including a pronounced sag, wide action potentials, depolarized membrane potential, and moderate inward rectification. d, e Example of a feed-forward inhibitory connection between ChINs as recorded in current- and voltage-clamp configurations. The synaptic currents scaled according to the voltage responses by 22.2 ± 2.9 pA mV⁻¹ (two-sided linear regression through the origin, R² = 0.86, n = 11 connections, p = 0.000016). f, g Polysynaptic inhibition was blocked by bath application of the nicotinic receptor antagonist DHβE (n = 14 connections, Z = −3.296, p = 0.00066) and the GABA_A receptor antagonist gabazine (n = 14 connections, Z = −3.296, p = 0.00098) but unaffected by AMPA and NMDA receptor antagonists NBQX and D-AP5 (n = 8 connections, Z = −0.14, p = 0.89, 2-tailed Wilcoxon signed-ranks test). h Example of a monosynaptic inhibitory connection from a fast-spiking interneuron (FSI) onto an MSN and a polysynaptic feed-forward inhibition between ChINs. Compared to the monosynaptic FSI-MSN connection, polysynaptic ChIN-ChIN connections exhibit longer onset latencies and trial to trial variability. i Onset latency histogram for monosynaptic connections between FSIs and MSNs (in red), compared to the polysynaptic connection between ChINs (in black). Mean latency for the polysynaptic connection (7.8 ms, n = 98 connections) is significantly larger (p = 3.0E−43, two-sided independent samples t-test) than the monosynaptic FSI onto MSN connection (1.8 ms, n = 20 connections).