Subcortical Circuits Among Pedunculopontine Nucleus, Thalamus and Basal Ganglia Play Important Roles in Paroxysmal Arousal in Genetic Rat Models of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

Abstract

A part of autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is caused by mutant CHRNA4. The pathomechanisms underlying motor seizures followingly brief/sudden awakening (paroxysmal arousal) in ADSHE seizures remain to be clarified. This study determined extracellular levels of ACh and L-glutamate in the pedunculopontine nucleus (PPN) and its projection regions, including the thalamus and basal ganglia, during wakefulness, slow-wave sleep (SWS) and paroxysmal arousal of transgenic rats bearing rat S286L-mutant Chrna4 (S286L-TG), corresponding to human S284L-mutant CHRNA4, using microdialysis. The expression of connexin43 and pannexin1 in the plasma membrane of the PPN was determined using capillary immunoblotting. The expressions of connexin43 and pannexin1 in the PPN plasma membrane of S286L-TG were larger than the wild type. The extracellular L-glutamate levels in the PPN and projection regions of S286L-TG consistently increased during both wakefulness and SWS compared to the wild type. The extracellular levels of ACh and L-glutamate in the PPN and projection regions decreased accompaning SWS in the wild type. In S286L-TG, this decreasing extracellular ACh level was observed, whereas decreasing L-glutamate level was impaired. Both extracellular levels of ACh and L-glutamate in the PPN and projection regions drastically increased during paroxysmal arousal. Hemichannel inhibitors suppressed the increasing releases of ACh and L-glutamate induced by paroxysmal arousal but decreased and did not affect extracellular levels of L-glutamate and ACh during wakefulness and SWS, respectively. In particular, under hemichannels inhibition, decreasing L-glutamate release accompanying SWS was observed in S286L-TG. This study elucidated that enhanced hemichannels are predominantly involved in the dysfunction of glutamatergic transmission compared to AChergic transmission during the interictal stage in S286L-TG, whereas the hyperactivation of hemichannels contributes to the generation of paroxysmal arousal. Therefore, the hyperactivated excitatory tripartite synaptic transmission associated with hemichannels in the PPN and projection regions plays important roles in epileptogenesis/ictogenesis in S286L-TG.

Keywords: ACh; ADSHE; glutamate; hemichannel; tripartite synaptic transmission.

Figure 1

Based on the established networks, the present study was designed to determine the transmission abnormalities in the AChergic and glutamatergic projections from the pedunculopontine nucleus (PPN) to its projection regions, such as the thalamus (RTN and MoTN) and subthalamic nucleus (STN) [21,22,23,24,25,26], and afferents from the substantia nigra reticulata (SNr) to the PPN [27,28,29,30,31].

Figure 2

Panels (A–C) indicate fluctuations of extracellular levels of L-glutamate, ACh and GABA of the wild type (grey columns) and S286L-TG (red, green and blue columns) during wakefulness (wake), SWS and paroxysmal arousal (PA) in the PPN (A1–C1), MoTN (A2–C2), STN (A3–B3), SNr (A4) and RTN (B4), respectively. Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM), L-glutamate (μM) and GABA (μM). Panel (D) indicates the expression of connexin43 (Cx43) and pannexin1 (PANX1) in the plasma membrane fraction of the PPN between wild type (grey columns) and S286L-TG (black columns). Ordinates indicate the mean ± SD (n = 6) of the relative expression of connexin43 and pannexin1 per GAPDH. Right-side panels indicate the pseudo-gel images of connexin43, pannexin1 and GAPDH, obtained using capillary immunoblotting. * p < 0.05, ** p < 0.01 relative to wakefulness, @ p < 0.05, @@ p < 0.01 relative to wild type ## p < 0.01 relative to SWS using MANOVA with Scheffe’s post hoc test or Student’s T-test. F-values of L-glutamate between wakefulness and SWS were in the PPN [F_genotype(1,10) = 7.0 (p < 0.05), F_event(1,10) = 7.2 (p < 0.05), F_{genotype*event}(1,10) = 2.6 (p > 0.1)], MoTN [F_genotype(1,10) = 17.4 (p < 0.01), F_event(1,10) = 27.1 (p < 0.01), F_{genotype*event}(1,10) = 25.3 (p < 0.01)], STN [F_genotype(1,10) = 5.8 (p < 0.05), F_event(1,10) = 5.7 (p < 0.05), F_{genotype*event}(1,10) = 0.5 (p > 0.1)] and SNr [F_genotype(1,10) = 13.8 (p < 0.05), F_event(1,10) = 1.9 (p > 0.1), F_{genotype*event}(1,10) = 5.4 (p < 0.05)]. F-values of ACh between wakefulness and SWS were in the PPN [F_genotype(1,10) = 0.1 (p > 0.1), F_event(1,10) = 9.2 (p < 0.05), F_{genotype*event}(1,10) = 0.1 (p > 0.1)], MoTN [F_genotype(1,10) = 0.3 (p > 0.1), F_event(1,10) = 13.2 (p < 0.01), F_{genotype*event}(1,10) = 0.4 (p > 0.1)], STN [F_genotype(1,10) = 0.7 (p > 0.1), F_event(1,10) = 23.0 (p < 0.01), F_{genotype*event}(1,10) = 0.5 (p > 0.1)] and RTN [F_genotype(1,10) = 0.1 (p > 0.1), F_event(1,10) = 21.9 (p < 0.01), F_{genotype*event}(1,10) = 0.1 (p > 0.1)]. F-values of GABA between wakefulness and SWS were in the PPN [F_genotype(1,10) = 0.1 (p > 0.1), F_event(1,10) = 0.1 (p > 0.1), F_{genotype*event}(1,10) = 0.2 (p > 0.1)] and MoTN [F_genotype(1,10) = 0.3 (p > 0.1), F_event(1,10) = 31.4 (p < 0.01), F_{genotype*event}(1,10) = 13.0 (p < 0.01)]. F-values of L-glutamate among wakefulness, SWS and IID of S286L-TG were in the PPN [F(2,10) = 67.4 (p < 0.01)], MoTN [F(1.0,5.2) = 83.9 (p < 0.01)], STN [F(1.1,5.3) = 45.9 (p < 0.01)] and SNr [F(2,10) = 76.5 (p < 0.01)]. F-values of ACh among wakefulness, SWS and interictal discharge of S286L-TG were in the PPN [F(2,10) = 43.7 (p < 0.01)], MoTN [F(1.1,5.5) = 39.4 (p < 0.01)], STN [F(1.0,5.1) = 14.8 (p < 0.01)] and RTN [F(2,10) = 51.2 (p < 0.01]. F-values of GABA among wakefulness, SWS and IID of S286L-TG were in the PPN [F(2,10) = 34.7 (p < 0.01)] and MoTN [F(2,10) = 53.4 (p < 0.01)].

Figure 3

Fluctuations of extracellular ACh levels during wakefulness (wake), SWS and paroxysmal arousal in the PPN, MoTN, STN and RTN. Fluctuations of extracellular ACh levels of the wild type (grey columns) and S286L-TG (green columns) during wakefulness, SWS and paroxysmal arousal (PA) in the PPN (A), MoTN (B), STN (C) and RTN (D) under the perfusion of MRS containing 1 μM neostigmine. Ordinates indicate the mean ± SD (n = 6) of extracellular ACh level (nM). * p < 0.05, ** p < 0.01 relative to wakefulness ## p < 0.01 relative to SWS using MANOVA with Scheffe’s post hoc test. F-values between wakefulness and SWS were in the PPN [F_genotype(1,10) = 0.6 (p > 0.1), F_event(1,10) = 69.2 (p < 0.01), F_{genotype*event}(1,10) = 0.1 (p > 0.1)], MoTN [F_genotype(1,10) = 0.7 (p > 0.1), F_event(1,10) = 44.4 (p < 0.01), F_{genotype*event}(1,10) = 0.3 (p > 0.1)], STN [F_genotype(1,10) = 0.2 (p > 0.1), F_event(1,10) = 7.7 (p < 0.05), F_{genotype*event}(1,10) = 0.2 (p > 0.1)] and RTN [F_genotype(1,10) = 0.8 (p > 0.1), F_event(1,10) = 20.7 (p < 0.01), F_{genotype*event}(1,10) = 0.1 (p > 0.1)]. F-values of ACh among wakefulness, SWS and interictal discharge of S286L-TG were in the PPN [F(1.1,5.5) = 41.2 (p < 0.01)], MoTN [F(1.1,5.5) = 40.8 (p < 0.01)], STN [F(1.0,5.1) = 50.2 (p < 0.01)] and RTN [F(2,10) = 67.9 (p < 0.01].

Figure 4

Effects of perfusion with tetrodotoxin on extracellular ACh levels in the PPN, MoTN, STN and RTN. Under the perfusion of MRS containing without (A) or with (B) 1 μM neostigmine, the effects of perfusion with 1 μM TTX (green columns) into the PPN, MoTN, STN and RTN on extracellular ACh level in the PPN, MoTN, STN and RTN, respectively. Ordinates indicate the mean ± SD (n = 6) of extracellular ACh level (nM). * p < 0.05, ** p < 0.01 relative to control (TTX free) using Student’s T-test.

Figure 5

Effects of perfusion with hemichannel inhibitors into the PPN on extracellular levels of L-glutamate and ACh during wakefulness (wake) and SWS in the MoTN, STN, SNr and RTN of the wild type. Panels (A,B) and (C,D) indicate the effects of perfusion with 300 μM probenecid (pannexin1-hemichannel inhibitor) and 100 μM carbenoxolone (non-selective connexin43-hemichannel inhibitor) on extracellular levels of L-glutamate (panels A and C) and ACh (panels B and D) in the MoTN (panels A1–D1), STN (panels A2–D2), SNr (panels A3 and C3) and RTN (panels B3 and D3) of the wild type during wakefulness and SWS. Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM) and L-glutamate (μM). Grey columns indicate the levels of control (perfusion with MRS alone). Red and green columns indicate the levels of L-glutamate and ACh during the perfusion with MRS containing probenecid or carbenoxolone, respectively. * p < 0.05, ** p < 0.01 relative to wakefulness using MANOVA with Scheffe’s post hoc test. F-values of effects of probenecid on L-glutamate level in the MoTN [F_PBN(1,5) = 1.9 (p > 0.1), F_event(1,5) = 85.2 (p < 0.01), F_PBN*event(1,5) = 0.4 (p > 0.1)], STN [F_PBN(1,5) = 0.3 (p > 0.1), F_event(1,5) = 9.1 (p < 0.01), F_PBN*event(1,5) = 0.2 (p > 0.1)] and SNr [F_PBN(1,5) = 0.3 (p > 0.1), F_event(1,5) = 37.5 (p < 0.01), F_PBN*event(1,5) = 0.1 (p > 0.1)]. F-values of effects of carbenoxolone on L-glutamate level in the MoTN [F_CBX(1,5) = 0.5 (p > 0.1), F_event(1,5) = 30.1 (p < 0.01), F_CBX*event(1,5) = 0.1 (p > 0.1)], STN [F_CBX(1,5) = 0.1 (p > 0.1), F_event(1,5) = 6.7 (p < 0.05), F_CBX*event(1,5) = 0.1 (p > 0.1)] and SNr [F_CBX(1,5) = 3.5 (p > 0.1), F_event(1,5) = 21.3 (p < 0.01), F_CBX*event(1,5) = 0.1 (p > 0.1)]. F-values of effects of probenecid on ACh level in the MoTN [F_PBN(1,5) = 1.6 (p > 0.1), F_event(1,5) = 16.1 (p < 0.01), F_PBN*event(1,5) = 0.1 (p > 0.1)], STN [F_PBN(1,5) = 0.3 (p > 0.1), F_event(1,5) = 12.0 (p < 0.01), F_PBN*event(1,5) = 0.1 (p > 0.1)] and SNr [F_PBN(1,5) = 1.0 (p > 0.1), F_event(1,5) = 17.1 (p < 0.01), F_PBN*event(1,5) = 0.2 (p > 0.1)]. F-values of effects of carbenoxolone on ACh level in the MoTN [F_CBX(1,5) = 0.1 (p > 0.1), F_event(1,5) = 30.9 (p < 0.01), F_CBX*event(1,5) = 3.6 (p > 0.1)], STN [F_CBX(1,5) = 0.2 (p > 0.1), F_event(1,5) = 17.4 (p < 0.01), F_CBX*event(1,5) = 1.2 (p > 0.1)] and SNr [F_CBX(1,5) = 0.1 (p > 0.1), F_event(1,5) = 9.8 (p < 0.01), F_CBX*event(1,5) = 0.6 (p > 0.1)].

Figure 6

Effects of perfusion with hemichannel inhibitors into the PPN on extracellular levels of L-glutamate and ACh during wakefulness (wake), SWS and paroxysmal arousal in the MoTN, STN, SNr and RTN of S286L-TG. Panels (A,B) and (C,D) indicate the effects of perfusion with 300 μM probenecid (pannexin1-hemichannel inhibitor) and 100 μM carbenoxolone (non-selective connexin43-hemichannel inhibitor) on extracellular levels of L-glutamate (panels A and C) and ACh (panels B and D) in the MoTN (panels A1–D1), STN (panels A2–D2), SNr (panels A3 and C3) and RTN (panels B3 and D3) of S286L-TG during wakefulness, SWS and paroxysmal arousal (PA). Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM) and L-glutamate (μM). Grey columns indicate the levels of control (perfusion with MRS alone). Red and green columns indicate the levels of L-glutamate and ACh during the perfusion with MRS containing probenecid or carbenoxolone, respectively. * p < 0.05, ** p < 0.01 relative to wakefulness, @ p < 0.05, @@ p < 0.01 relative to control ## p < 0.01 relative to SWS using MANOVA with Scheffe’s post hoc test. F-values of effects of probenecid on L-glutamate level in the MoTN [F_PBN(1,5) = 40.3 (p < 0.01), F_event(2,10) = 249.9 (p < 0.01), F_PBN*event(2,10) = 9.1 (p < 0.01)], STN [F_PBN(1,5) = 15.8 (p < 0.01), F_event(2,10) = 100.2 (p < 0.01), F_PBN*event(2,10) = 12.4 (p < 0.01)] and SNr [F_PBN(1,5) = 4.6 (p > 0.05), F_event(2,10) = 20.4 (p < 0.01), F_PBN*event(2,10) = 31.4 (p < 0.01)]. F-values of effects of carbenoxolone on L-glutamate level in the MoTN [F_CBX(1,5) = 122.3 (p < 0.01), F_event(2,20) = 121.2 (p < 0.01), F_CBX*event(2,20) = 24.9 (p < 0.1)], STN [F_CBX(1,5) = 43.9 (p < 0.01), F_event(2,20) = 50.9 (p < 0.01), F_CBX*event(2,20) = 3.3 (p > 0.05)] and SNr [F_CBX(1,5) = 10.5 (p < 0.01), F_event(2,20) = 25.6(p < 0.01), F_CBX*event(2,20) = 9.5 (p < 0.01)]. F-values of effects of probenecid on ACh level in the MoTN [F_PBN(1,5) = 6.8 (p < 0.05), F_event(2,10) = 116.8 (p < 0.01), F_PBN*event(2,10) = 4.4 (p < 0.05)], STN [F_PBN(1,5) = 23.4 (p < 0.01), F_event(2,10) = 24.3(p < 0.01), F_PBN*event(2,10) = 8.7 (p < 0.01)] and RTN [F_PBN(1,5) = 16.3 (p < 0.01), F_event(2,10) = 67.7 (p < 0.01), F_PBN*event(2,10) = 9.8 (p < 0.01)]. F-values of effects of carbenoxolone on ACh level in the MoTN [F_CBX(1,5) = 47.9 (p < 0.01), F_event(2,20) = 32.6 (p < 0.01), F_CBX*event(2,20) = 11.5 (p < 0.1)], STN [F_CBX(1,5) = 15.6 (p < 0.05), F_event(2,20) = 41.8 (p < 0.01), F_CBX*event(2,20) = 7.4 (p < 0.05)] and RTN [F_CBX(1,5) = 21.3 (p < 0.01), F_event(2,20) = 39.0 (p < 0.01), F_CBX*event(2,20) = 16.8 (p < 0.01)].

Figure 7

Effects of perfusion with inhibitors of AMPAR and NMDAR into the PPN on extracellular levels of L-glutamate and ACh during wakefulness (wake) and SWS in the MoTN, STN, SNr and RTN of the wild type. Panels (A,B) and (C,D) indicate the effects of perfusion with 3 μM perampanel (selective AMPAR inhibitor) and 3 μM MK801 (selective NMDAR inhibitor) on extracellular levels of L-glutamate (panels A and C) and ACh (panels B and D) in the MoTN (panels A1–D1), STN (panels A2–D2), SNr (panels A3 and C3) and RTN (panels B3 and D3) of the wild type during wakefulness and SWS. Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM) and L-glutamate (μM). Grey columns indicate the levels of control (perfusion with MRS alone). Red and green columns indicate the levels of L-glutamate and ACh during the perfusion with MRS containing probenecid or carbenoxolone, respectively. * p < 0.05, ** p < 0.01 relative to wakefulness, @ p < 0.05, @@ p < 0.01 relative to control (inhibitor free) using MANOVA with Scheffe’s post hoc test. F-values of effects of perampanel on L-glutamate level in the MoTN [F_PER(1,5) = 54.5 (p < 0.01), F_event(1,5) = 157.4 (p < 0.01), F_PER*event(1,5) = 1.3 (p > 0.1)], STN [F_PER(1,5) = 18.8 (p < 0.01), F_event(1,5) = 19.7 (p < 0.01), F_PER*event(1,5) = 0.3 (p > 0.1)] and SNr [F_PER(1,5) = 11.6 (p < 0.01), F_event(1,5) = 36.0 (p < 0.01), F_PER*event(1,5) = 0.1 (p > 0.1)]. F-values of effects of MK801 on L-glutamate level in the MoTN [F_MK801(1,5) = 17.1 (p < 0.01), F_event(1,5) = 37.3 (p < 0.01), F_MK801*event(1,5) = 2.4 (p > 0.1)], STN [F_MK801(1,5) = 3.5 (p > 0.1), F_event(1,5) = 32.3 (p < 0.01), F_MK801*event(1,5) = 2.4 (p > 0.1)] and SNr [F_MK801(1,5) = 1.9 (p > 0.1), F_event(1,5) = 14.5 (p < 0.01), F_MK801*event(1,5) = 0.6 (p > 0.1)]. F-values of effects of perampanel on ACh level in the MoTN [F_PER(1,5) = 3.9 (p > 0.1), F_event(1,5) = 7.1 (p < 0.05), F_PER*event(1,5) = 0.1 (p > 0.1)], STN [F_PER(1,5) = 2.1 (p > 0.1), F_event(1,5) = 21.1 (p < 0.01), F_PER*event(1,5) = 0.2 (p > 0.1)] and RTN [F_PER(1,5) = 0.6 (p > 0.1), F_event(1,5) = 8.0 (p < 0.01), F_PER*event(1,5) = 0.4 (p > 0.1)]. F-values of effects of MK801 on ACh level in the MoTN [F_MK801(1,5) = 6.7 (p < 0.05), F_event(1,5) = 9.1 (p < 0.05), F_MK801*event(1,5) = 9.2 (p < 0.05)], STN [F_MK801(1,5) = 17.9 (p < 0.01), F_event(1,5) = 13.4 (p < 0.01), F_MK801*event(1,5) = 0.4 (p > 0.1)] and RTN [F_MK801(1,5) = 8.2 (p < 0.05), F_event(1,5) = 6.9 (p < 0.05), F_MK801*event(1,5) = 0.3 (p > 0.1)].

Figure 8

Effects of perfusion with inhibitors of AMPAR and NMDAR into the PPN on extracellular levels of L-glutamate and ACh during wakefulness, SWS and paroxysmal arousal in the MoTN, STN, SNr and RTN of S286L-TG. Panels (A,B) and (C,D) indicate the effects of perfusion with 3 μM perampanel (AMPAR inhibitor) and 3 μM MK801 (NMDAR inhibitor) on extracellular levels of L-glutamate (panels A and C) and ACh (panels B and D) in the MoTN (panels A1–D1), STN (panels A2–D2), SNr (panels A3 and C3) and RTN (panels B3 and D3) of S286L-TG during wakefulness, SWS and paroxysmal arousal (PA). Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM) and L-glutamate (μM). Grey columns indicate the levels of control (perfusion with MRS alone). Red and green columns indicate the levels of L-glutamate and ACh during the perfusion with MRS containing perampanel or MK801, respectively. * p < 0.05, ** p < 0.01 relative to wakefulness, @ p < 0.05, @@ p < 0.01 relative to control and # p < 0.05, ## p < 0.01 relative to SWS using MANOVA with Scheffe’s post hoc test. F-values of effects of perampanel on L-glutamate level in the MoTN [F_PER(1,5) = 59.5 (p < 0.01), F_event(2,10) = 65.3 (p < 0.01), F_PER*event(2,10) = 14.2 (p < 0.01)], STN [F_PER(1,5) = 115.0 (p < 0.01), F_event(2,10) = 54.0 (p < 0.01), F_PER*event(2,10) = 6.8 (p < 0.05)] and SNr [F_PER(1,5) = 38.7 (p < 0.01), F_event(2,10) = 58.1 (p < 0.01), F_PER*event(2,10) = 23.0 (p < 0.01)]. F-values of effects of MK801 on L-glutamate level in the MoTN [F_MK801(1,5) = 18.3 (p < 0.01), F_event(2,20) = 135.9 (p < 0.01), F_MK801*event(2,20) = 2.5 (p > 0.1)], STN [F_MK801(1,5) = 52.8 (p < 0.01), F_event(2,20) = 220.0 (p < 0.01), F_MK801*event(2,20) = 23.7 (p < 0.01)] and SNr [F_MK801(1,5) = 9.6 (p < 0.05), F_event(2,20) = 17.0 (p < 0.01), F_MK801*event(2,20) = 3.6 (p > 0.1)]. F-values of effects of perampanel on ACh level in the MoTN [F_PER(1,5) = 8.3 (p < 0.05), F_event(2,10) = 60.5 (p < 0.01), F_PER*event(2,10) = 4.3 (p < 0.05)], STUN [F_PER(1,5) = 19.6 (p < 0.01), F_event(2,10) = 57.4 (p < 0.01), F_PER*event(2,10) = 20.2 (p < 0.01)] and RTN [F_PER(1,5) = 19.5 (p < 0.01), F_event(2,10) = 64.5 (p < 0.01), F_PER*event(2,10) = 9.8 (p < 0.01)]. F-values of effects of MK801 on ACh level in the MoTN [F_MK801(1,5) = 50.1 (p < 0.01), F_event(2,20) = 57.8 (p < 0.01), F_MK801*event(2,20) = 17.2 (p < 0.01)], STN [F_MK801(1,5) = 6.8 (p < 0.05), F_event(2,20) = 45.5 (p < 0.01), F_MK801*event(2,20) = 1.9 (p > 0.1)] and RTN [F_MK801(1,5) = 61.0 (p < 0.01), F_event(2,20) = 57.3 (p < 0.01), F_MK801*event(2,20) = 30.1(p < 0.01)].

Figure 9

Fluctuations of extracellular levels L-glutamate and ACh in the MoTN and STN before and after paroxysmal arousal and nocturnal paroxysmal dystonia. Panels A and B indicate the extracellular levels of L-glutamate and ACh in the MoTN (panels A1 and B1) and STN (panels A2 and B2) of S286L-TG before and after paroxysmal arousal (opened circles) and nocturnal paroxysmal dystonia (red and blue circles). Ordinates indicate the mean ± SD (n = 6) of extracellular levels of ACh (nM) and L-glutamate (μM) associated with paroxysmal arousal (n = 6) and those of individuals associated with nocturnal paroxysmal dystonia. Abscissas indicate before (pre) and after the paroxysmal arousal or nocturnal paroxysmal dystonia (min). Panel (C) indicates the typical ECoG during wakefulness (black), SWS (blue), paroxysmal arousal (red) and nocturnal paroxysmal dystonia (green). Red and green arrows indicate the onsets of paroxysmal arousal and nocturnal paroxysmal dystonia, respectively.

Figure 10

Proposed hypothesis for functional abnormalities among the PPN and its projection regions in S286L-TG. The AChergic neurons in the PPN project to the thalamus (RTN and MoTN) and STN [21,22], and glutamatergic neurons project mainly to the MoTN and STN [23,24]. GABAergic neurons in the RTN, which receives AChergic terminals via α4β2-nAChRs [10,11,16], project to various glutamatergic neurons in the thalamus [26]. SNr receives mainly glutamatergic terminals from the STN [31] and projects both GABAergic and glutamatergic terminals to the PPN [27,28,29]. In the PPN, glutamatergic neurons receive glutamatergic terminals via both AMPAR and NMDAR, whereas AChergic neurons receive mainly NMDAR. During wakefulness, glutamatergic transmissions from the PPN to its projection regions of S286L-TG are enhanced compared to the wild type, whereas AChergic transmission between S286L-TG and the wild type is almost equal. During SWS, in the wild type, both AChergic and glutamatergic transmissions from the PPN to its projection regions decrease compared to wakefulness. In S286L-TG, AChergic transmission from the PPN to its projection regions also decreases compared to wakefulness, whereas that of glutamate does not change between wakefulness and SWS. During PA (sudden/brief awakening with polyspikes), both AChergic and glutamatergic transmissions drastically increase compared to wakefulness and SWS, which are suppressed by inhibition of hemichannels, NMDAR and AMPAR in the PPN. Glutamatergic transmission from the PPN to its projection regions is suppressed by inhibition of both AMPAR and NMDAR in the PPN, whereas AChergic transmission is predominantly suppressed by inhibition of NMDAR in the PPN. Hemichannel inhibitor decreases both AChergic and glutamatergic transmissions in S286L-TG but does not affect those in the wild type. Hemichannel-dependent releases of ACh and L-glutamate during SWS are larger than those during wakefulness in S286L-TG.