Fig. 5.

Co-transmission of GABA and acetylcholine is cross-regulated by autoreceptors and mediated by distinct calcium channels. a Optical stimulations of cholinergic fibres trigger composite membrane potential responses (average of 50 traces) of an inhibitory neuron recorded in str. lacunosum-moleculare (green). Cells were held at approximately −50 mV to record hyperpolarising and depolarising components concurrently. Early hyperpolarization was confirmed to be of GABAergic origin by bath application of the GABA_AR blocker gabazine (magenta, 10 µM, which abolished quick hyperpolarization in 9 out of 9 tested cells), while the late depolarisation was confirmed to be cholinergic by the application of the alpha4-type nicotinic acetylcholine receptor blocker DHβE (black, 1 µM, which abolished depolarisation in 5 out of 5 tested cells). Cells with identifiable nicotinic responses were used to further investigate the properties of GABA and acetylcholine release from cholinergic fibres. b Increase in GABAergic response after AChR block (Fig. 3d) suggested that presynaptic muscarinic receptors cross-modulated GABA (and possibly ACh) release from cholinergic fibres. Therefore, putative presynaptic muscarinic receptors were selectively inhibited via bath application of atropine (n = 6), which led to a significant increase in amplitude of both the GABAergic hyperpolarization and nicotinic depolarisation (green, see Supplementary Note 8). c Bath application of the selective M2 antagonist AFDX-116 (10 µM, n = 6) resulted in changes similar to that of atropine application (for data see Supplementary Note 8). d Blocking GABA_BRs with CGP-55845 (1 µM, n = 7) also resulted in increased GABAergic and cholinergic responses suggesting a dual modulatory role (for data see Supplementary Note 8). e, f Testing roles of distinct voltage-dependent calcium channels (VDCC) in release of GABA and ACh . e Blockade of P/Q-type VDCC, using selective antagonist ω-agatoxin (1 µM, puff application), reliably decreased GABAergic response component but did not alter cholinergic component. At control conditions, 5 short (1 ms) light pulses at 10 Hz evoked a composite event of GABAergic hyperpolarization and cholinergic depolarisation (green, average of 20 traces). In response to ω-agatoxin application (magenta, n = 9), the GABAergic component (measured as IPSP amplitude) decreased robustly (magenta arrowhead) and the cholinergic component (measured as EPSP area) showed no change (for details see Supplementary Note 8). f Blockade of N-type VDCC, using selective blocker ω-conotoxin (1 µM), reliably reduced cholinergic response component (n = 6) but left GABAergic component unchanged (for details see Supplementary Note 4)