Basal forebrain control of wakefulness and cortical rhythms

Abstract

Wakefulness, along with fast cortical rhythms and associated cognition, depend on the basal forebrain (BF). BF cholinergic cell loss in dementia and the sedative effect of anti-cholinergic drugs have long implicated these neurons as important for cognition and wakefulness. The BF also contains intermingled inhibitory GABAergic and excitatory glutamatergic cell groups whose exact neurobiological roles are unclear. Here we show that genetically targeted chemogenetic activation of BF cholinergic or glutamatergic neurons in behaving mice produced significant effects on state consolidation and/or the electroencephalogram but had no effect on total wake. Similar activation of BF GABAergic neurons produced sustained wakefulness and high-frequency cortical rhythms, whereas chemogenetic inhibition increased sleep. Our findings reveal a major contribution of BF GABAergic neurons to wakefulness and the fast cortical rhythms associated with cognition. These findings may be clinically applicable to manipulations aimed at increasing forebrain activation in dementia and the minimally conscious state.

Figure 1. Administration of CNO activates cholinergic BF hM3Dq⁺ neurons and produced a generalized decrease in the slow-wave sleep (SWS) EEG power density.

(a) Experimental design: bilateral injections of DIO-hM3Dq-AAV were placed into the BF of ChAT-IRES-Cre, Vglut2-IRES-Cre or Vgat-IRES-Cre mice, resulting in the expression of hM3Dq in BF cholinergic, glutamatergic or GABAergic neurons, respectively. (b) Coronal section from injected ChAT-IRES-Cre mouse showing hM3Dq-expressing BF cholinergic (mCherry⁺) neurons (scale bar, 1 mm). (c) Heat map generated from ten injection cases in the ChAT-IRES-Cre mouse; white colour=area of maximum overlap of hM3Dq-AAV transduction/expression across ten injection cases. (d) hM3Dq-expressing BF cholinergic neurons (left) visualized under IR-DIC (right) during whole-cell recordings (scale bar, 20 μm) showed a strong depolarizing and firing response to bath application of CNO (500 nM (e)). (f) Non-hM3Dq-expressing cholinergic BF neurons, recorded from ChAT-IRES-cre mice (scale bar, 20 μm) with hrGFP-AAV control injections did not respond to CNO (g). (h,i) Example compressed spectral array (CSA; 0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (h) or CNO (0.3 mg kg⁻¹, IP; ZT3 (i)) administration in a mouse with bilateral hM3Dq receptor expression in BF cholinergic neurons. The power of the slow EEG frequencies, in particular δ and θ (0.5–9 Hz), was markedly and uniquely decreased as compared with vehicle administration in the same mouse. (j–l) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg kg⁻¹, IP, ZT3=10 A.M., n=13 mice) or vehicle. (m–o) Power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the 3-h post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=10 A.M., n=9 mice) and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for SWS and REM sleep), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands following vehicle or CNO administrations. (p–r) EEG/EMG examples of wake (W), SWS and REM sleep (RS) from the first hour post injection of saline (top) or CNO (bottom) in a mouse with bilateral hM3Dq receptor expression in BF cholinergic neurons. The raw EEG and EMG traces following CNO injection in q provide unambiguous evidence of the existence of SWS when BF cholinergic neurons are activated. Note, however, that as compared with vehicle injection, SWS after CNO injection is characterized by decreased slow-wave activity (delta band, n) density. AC=anterior commissure; MPO=medial preoptic area; ox=optic chiasm. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS).

Figure 2. Administration of CNO activates glutamatergic BF hM3Dq⁺ neurons, but had little effect on sleep–wake or EEG power density.

(a) Coronal section from injected Vglut2-IRES-Cre mouse showing hM3Dq-expressing BF glutamatergic (mCherry⁺) neurons (scale bar, 1 mm). (b) Heat map generated from ten injection cases in the Vglut2-IRES-Cre mouse; white colour=area of maximum overlap of hM3Dq-AAV transduction/expression across ten injection cases. (c) Region of BF containing the recorded hM3Dq-expressing glutamatergic neurons (left panel, native mCherry-hM3Dq signal, scale bar, 300 μm) and a representative hM3Dq-expressing BF glutamatergic neuron (top right panel) visualized under IR-DIC (bottom right panel) during whole-cell recordings (scale bar, 10 μm). (d) Whole-cell current clamp recordings of BF glutamatergic neurons expressing hM3Dq showed the expected depolarization and increased firing response to CNO (500 nM) when applied in vitro. (e,f) Non-hM3Dq-expressing glutamatergic BF neurons, recorded from Vglut2-IRES-Cre mice injected with hrGFP-AAV (scale bar, 30 μm) did not respond to CNO. (i–k) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg kg⁻¹, IP, ZT3=10 A.M., n=11 mice) or vehicle. (l–n) Power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the 3-h post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=10 A.M., n=9 mice) and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for slow-wave sleep (SWS) and REM sleep), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands following vehicle or CNO administrations. (o,p) Example CSA (0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (o) or CNO (0.3 mg kg⁻¹, IP; ZT3; (p)) administration in a mouse with bilateral hM3Dq receptor expression in BF glutamatergic neurons. A black bar (4–6 bins=1–1.5 Hz large) was inserted to mask the 60 Hz contamination of the EEG recordings. (q–s) EEG/EMG examples of wake (W), SWS and REM sleep (RS) from the first hour post injection of saline (top) or CNO (bottom) in a mouse with bilateral hM3Dq receptor expression in BF glutamatergic neurons. The raw EEG and EMG traces following CNO injection provide unambiguous evidence of the existence of the three sleep–wake stages while BF glutamatergic neurons are activated. AC, anterior commissure; CPu, caudate putamen; HDB, diagonal band of Broca; MCPO, magnocellular pre-optic nucleus; MPO, medial preoptic area; SI, substantia innominata; ox, optic chiasm. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS).

Figure 3. Administration of CNO activates GABAergic BF hM3Dq⁺ neurons, and strongly promoted wake and an increase in fast EEG power density.

(a) Coronal section from injected Vgat-IRES-Cre mouse showing hM3Dq-expressing BF GABAergic (mCherry⁺) neurons (scale bar, 1 mm). (b) Heat map generated from ten injection cases in the Vgat-IRES-Cre mouse; white colour=area of maximum overlap of hM3Dq-AAV transduction/expression across ten injection cases. (c) hM3Dq-expressing BF GABAergic neurons (left) visualized under IR-DIC (right) during whole-cell recordings (scale bar, 20 μm) showed a strong depolarizing and firing response to bath application of CNO (500 nM; (d)). (e) Non-hM3Dq-expressing GABAergic BF neurons, recorded from Vgat-IRES-Cre, lox-GFP mice (scale bar, 20 μm) and (f) Vgat-IRES-Cre mice injected with hrGFP-AAV (scale bar, 20 μm) did not respond to CNO (e–h). (i–k) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg kg⁻¹, IP, ZT3=1000 hours, n=14 mice) or vehicle. Note that activation of BF GABAergic neurons induced a large and sustained increase in wakefulness (W) at the expense of both slow-wave sleep (SWS) and REM sleep (RS). (l,m) power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the first hour post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=1000 hours, n=12 mice; (l)) or the first hour containing SWS (n=9, (m)) and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for SWS), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands following vehicle or CNO administrations. (n) EEG/EMG examples from the first hour post-injection show a normal waking state following CNO injection as compared with vehicle injection. (o,p) Example CSA (0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (o) or CNO (0.3 mg kg⁻¹, IP; ZT3; (p)) administration in a mouse with bilateral hM3Dq receptor expression in BF GABAergic neurons. The power of the fast EEG frequencies, in particular high γ (60–150 Hz) was markedly increased as compared with vehicle administration in the same mouse. A black bar (4–6 bins=1–1.5 Hz large) was inserted to patch over the 60 Hz contaminations of the EEG recordings. AC, anterior commissure; BF, basal forebrain; MPO, medial preoptic area; ox,optic chiasm. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS).

Figure 4. Administration of CNO activates GABAergic BF hM4Di⁺ neurons, and promoted slow-wave sleep (SWS) and an increase in slow EEG power density.

(a) Experimental design: bilateral injections of DIO-hM4Di-AAV were placed into the BF of Vgat-IRES-Cre mice, resulting in expression of hM4Di on BF GABAergic neurons. (b,c) hM4Di-expressing BF GABAergic neurons (left) visualized under IR-DIC (right) during whole-cell recordings (scale bar, 20 μm) showed a strong membrane hyperpolarization and reduction in firing to bath application of CNO (500 nM; c). (d) coronal section from injected Vgat-IRES-Cre mouse showing hM4Di-expressing BF GABAergic (mCherry⁺) neurons (scale bar, 1 mm). (e) Heat map generated from seven injection cases in the Vgat-IRES-Cre mouse; white colour=area of maximum overlap of hM4Dq-AAV transduction/expression across seven injection cases. (f–h) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg kg⁻¹, IP, ZT12=1900 hours, n=7 mice) or vehicle; inset in f shows ∼30% decrease in wake during the 3-h post CNO injection period. (i–k) Power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the 3-h post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=1900 hours, n=5 mice) administration and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for SWS and REM sleep), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands following vehicle or CNO administrations. (l–n) EEG/EMG examples of wake (W), SWS and REM sleep (RS) from the first hour post injection of saline (top) or CNO (bottom) in a mouse with bilateral hM4Di receptor expression in BF GABAergic neurons. The raw EEG and EMG traces following CNO injection provide unambiguous evidence of the existence of the three sleep–wake stages while BF GABAergic neurons are inhibited. (o,p) Example compressed spectral array (CSA; 0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (o) or CNO (0.3 mg kg⁻¹, IP; ZT12; (p)) administration in a mouse with bilateral hM4Di receptor expression in BF GABAergic neurons. A black bar (4–6 bins=1–1.5 Hz large) was inserted to mask the 60 Hz contamination of the EEG recordings. AC, anterior commissure; CPu, caudate putamen; MPO, medial preoptic area; SI, substantia innominata; ox, optic chiasm. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS).

Figure 5. Acute activation of glutamatergic thalamocortical (TC) hM3Dq⁺ neurons did not promote wake, but did increase high-frequency EEG activity during wake, slow-wave sleep (SWS) and REM sleep.

Large bilateral injections of hM3Dq-AAV were placed into the thalamus of Vglut2-IRES-Cre mice (n=8). Rostral and caudal coronal sections from a representative hM3Dq-AAV-injected mouse show widespread expression of hM3Dq in midline and intralaminar TC neurons (a–c). CNO administration produced robust thalamic c-Fos expression in mice with hM3Dq-AAV injections ((d,e) corresponding coronal section showing hM3Dq expression in same mouse as panel a3). High-power photomicrograph (e) of thalamic region in box of d shows the robust expression of c-Fos following CNO administration (scale bar, 250 μm). (f–h) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg/kg, IP, ZT3=1000 hours, n=8 mice) or vehicle. (i–k) Power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the 3-h post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=1000 hours, n=8 mice) and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for SWS and REM sleep), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) bands following vehicle or CNO administrations. (l,m) Example CSA (0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (l) or CNO (0.3 mg kg⁻¹, IP; ZT3; m) administration in a mouse with bilateral hM3Dq receptor expression in thalamus glutamatergic neurons. A black bar (4–6 bins=1–1.5 Hz large) was inserted to mask the 60 Hz contamination of the EEG recordings. (n–p) EEG/EMG examples of wake (W), SWS and REM sleep (RS) from the first hour post injection of saline (top) or CNO (bottom) in a mouse with bilateral hM3Dq receptor expression in thalamus glutamatergic neurons. The raw EEG and EMG traces following CNO injection provide unambiguous evidence of the existence of the three sleep–wake stages while thalamus glutamatergic neurons are activated. AM, anteriormedial thalamus; CM, centromedial thalamus; Hi, hippocampus; IMD, intermediodorsal thalamus; LPMR, lateral posterior thalamus, mediorostral; PVA, paraventricular thalamus, anterior; RTn, reticular thalamus; VPM, ventral thalamus, posteromedial. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS). scale bar (a–d), 2 mm.