Fig. 5: Optogenetic and pharmacological dissection of PPN and LDT effects.

a Schematic of the in vivo optogenetic and acetylcholine antagonists experiment design. b, c pSPNs decreased their activity during ChR2-PPN activation but not in the presence of cholinergic blockers (n = 7 neurons, n = 7 rats; one-way ANOVA F(2,20) = 5.24, P = 0.0161; Bonferroni post hoc analyses: before/during P = 0.014, before/after P = 0.611, during/after P = 0.221). d In vivo optogenetic experiment design where ChR2 was transduced in the midbrain and halorhodopsin (NpHR; AAV-DIO-NpHR3.0-mCherry) in the striatum (n = 7). e pCINs (n = 8) and pSPNs (n = 33) firing rate changes during stimulation of ChR2, NpHR, or both. pCINs activity was significantly higher during ChR2 activation than during NpHR activation or both (ChR2: +83.10 ± 32.09%; NpHR: −71.19 ± 5.61%; ChR2/NpHR: −6.97 ± 22.22%; one-way ANOVA F(2,23) = 11.59, P = 0.0004). pSPNs activity was significantly lower during ChR2 compared to NpHR and ChR2/NpHR (ChR2: −85.07 ± 1.38%; ChR2/NpHR: −61.40 ± 15.46%; paired t-test, t(42) = −9.744, P = 2.48 × 10⁻¹²). f pSPNs normalized instantaneous firing rate using the same stimulation protocols (color lines represent significant differences; cluster-based permutation test, P < 0.05). pSPNs inhibition by midbrain axons seems to partly depend on CINs activity. f Circuit mechanisms underlying the excitatory effects of PPN on CINs (n = 8 rats). ChR2 was activated in either the striatum or the parafascicular thalamic nucleus (PF), and glutamate antagonists (CNQX + AP5) were locally delivered. h Optogenetically inhibited pCINs increased their firing following ChR2-stimulation in the striatum but not the PF, thus ruling out antidromic activation (%-change in firing rate: NpHR, −64.6 ± 7.18, n = 13; ChR2-STR, 84.49 ± 9.35, n = 11; ChR2-PF, −22.9 ± 4.88, n = 7; univariate ANOVA F_(2,28) = 70.102, P < 0.001; Tukey HSD post hoc: ChR2-STR vs. ChR2-PF P < 0.001). i Neurons activated by PPN axons were unable to respond to electrical pulses in the PF in the presence of glutamate blockers (%-change in firing rate pre-drug: 107.71 ± 12.98, n = 8; post-drug: −6.39 ± 8.27, n = 6; paired two-tailed t-test t(5) = 5.198 P = 0.003), confirming glutamate blockers efficiency. j Activated striatal neurons were still responding to PPN stimulation in the presence of glutamate blockers (n = 3), suggesting that the excitatory responses are independent of glutamate transmission (paired two-tailed t-test; pre-drug: t(2) = −3.828 P = 0.031; CNQX + AP5: t(2) = −2.904 P = 0.051). Individual data points and mean ± SEM are shown. *P < 0.05. All experiments have been replicated at least three times.