Supplementary Figure 8: Nicotinic modulation of VTA neurons.

(a) Effect of carbachol puffing on VTA DA neurons (n = 9 neurons) that were labeled and identified as TH+. (b) Example of VTA neurons showing classical dopaminergic features with low spontaneous firing and/or presence of Ih current. (c) Carbachol puffs (200 µm) provoke excitatory postsynaptic potentials (EPSPs) that are blocked by mecamylamine (n = 9 neurons; 5 µM) but not by DhβE (1 µM) or methylcaconitine (500 nM). (d) Carbachol-induced EPSPs were not blocked by bicuculline (10 µM), CNQX (10 µM) or APV (10 µM) (n = 4 neurons). (e) The mecamylamine effect on EPSP size was significantly different to the control condition (t = 7.281, two-tailed t-test, P = 0.000342). (f) Effect of carbachol puffing on a VTA non-DA neuron, confirmed as TH- by immunofluorescence, and showing classical non-DA neuron features such as higher firing rate and lack of Ih current. (g) Carbachol puffing provokes robust EPSPs that are blocked by bath application of MLA (500 nM) but not by DHβE (1 µM) or mecamylamine (5 µM). Scale bar in fluorescent images: 20 µm. Acetylcholine has been reported to induce strong somatic excitation of dopamine neurons in the VTA (Eddine, R., et al. (2015) Sci Rep 5, 8184) and its effects consist of a presynaptic component, modulating glutamate release (Schilstrom, B., et al. (1998) Neuroscience 82, 781–789), and a postsynaptic component, involving nicotinic and muscarinic mechanisms (Gronier, B., et al. (2000) Psychopharmacology 147, 347-355; Mameli-Engvall, M., et al. (2006) Neuron 50, 911-921; Mansvelder, H.D., et al. (2002) Neuron 33, 905-919). Furthermore, muscarinic agonists increase the frequency of spontaneous action potentials in DA neurons (Lacey, M.G., et al. (1990) Pharmacol Exp Ther 253, 395-400; Scroggs, R.S., et al. (2001) J Neurophysiol 86, 2966-2972) and induce glutamate release concomitantly with a decrease in GABA release in the VTA (Grillner, P., et al. (2000) Neuroscience 96, 299-307; Mansvelder, H.D., et al. (2000) Neuron 27, 349-357). Our results are in agreement with other studies showing that mecamylamine was able to abolish the effects mediated by cholinergic agonists in DA neurons (Zhang, L., et al. (2005) J Physiol 586, 469-481) or following place preference associated with carbachol injections in the VTA (Ikemoto, S. & Wise R.A., et al. (2002) J Neurosci 22, 9895-9904). While our experiments seem to indicate an effect predominantly mediated by nicotinic receptors, muscarinic receptors are highly expressed in the VTA and are also likely to contribute to the modulation of DA neurons, as shown previously (Forster, G.L. and Blaha C.D. (2000) Eur J Neurosci 12, 3596–3604).