Genetic Activation, Inactivation, and Deletion Reveal a Limited And Nuanced Role for Somatostatin-Containing Basal Forebrain Neurons in Behavioral State Control

Abstract

Recent studies have identified an especially important role for basal forebrain GABAergic (BF^VGAT) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BF^VGAT neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BF^VGAT neurons and somatostatin-containing BF^VGAT neurons (BF^SOM neurons), and it was recently reported that optogenetic activation of BF^SOM neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the rest period of the animal. This finding was unexpected given that most BF^SOM neurons are not NREM sleep active and that central administration of the synthetic somatostatin analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we used a combination of genetically driven chemogenetic and optogenetic activation, chemogenetic inhibition, and ablation approaches to further explore the in vivo role of BF^SOM neurons in arousal control. Our findings indicate that acute activation or inhibition of BF^SOM neurons is neither wakefulness nor NREM sleep promoting and is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24 h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BF^SOM neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BF^SOM neurons are electrically heterogeneous and are not NREM sleep or wake promoting per se, but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry.SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide range of higher-level neurobiological processes, including regulating and maintaining normal levels of electrocortical and behavioral arousal. The respective in vivo roles of BF cell populations and their neurotransmitter systems in the regulation of electrocortical and behavioral arousal remains incompletely understood. Here we seek to define the neurobiological contribution of GABAergic somatostatin-containing BF neurons to arousal control. Understanding the respective contribution of BF cell populations to arousal control may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia, and the cognitive impairments of normal aging.

Keywords: AAV; DREADD; EEG; arousal; diphtheria; optogenetic.

Figure 1.

Evidence that BF^SOM hM3Dq⁺ and hM4Di⁺ neurons are excited and inhibited, respectively, by CNO in in vitro brain slices, experimental design, and BF^SOM cell distribution. Injections of DIO-hM3Dq-mCherry-AAV, DIO-hM4Di-mCherry-AAV, or DIO-mCherry-AAV were placed into the BF of SOM-ires-Cre mice, resulting respectively in the expression of hM3Dq-mCherry, hM4Di-mCherry, or mCherry in BF^SOM neurons. Whole-cell recordings in brain slices were conducted 5–6 weeks after the AAV injections. A, Microphotographs showing the distribution of the recorded neurons labeled with biocytin. After incubation in fluorescent streptavidin, 13 of 29 recorded neurons were recovered, mapped, and represented (red dots, indicating BF^SOM neurons with LTS, n = 7; green dots representing non-LTS BF^SOM neurons, n = 6) over the images of two recorded slices (rostral level, top image; caudal level, lower image). Scale bar, 1 mm. Ac, Anterior commissure; 3V, third ventricle; Ox, optical chiasm; VP, ventral pallidum; SI, substantia innominata; MCPO, magnocellular preoptic nucleus; HDB, horizontal diagonal band). B, C, Firing properties of two distinct types of BF^SOM neurons: one group of BF^SOM neurons responds to depolarizing (left) and hyperpolarizing (right) current pulse protocols with LTS (B), and the other group has no LTS (C). The vast majority of BF^SOM neurons have an I_h. D, E, hM3Dq-mCherry-expressing BF^SOM neurons (top) visualized under IR-DIC during whole-cell recordings (bottom; scale bars, 20 μm) showed an increase in firing frequency in response to the bath application of CNO (1 μm; dotted lines, 0 mV). F, Averaged response on the firing frequency of hM3Dq-expressing BF^SOM neurons (n = 6) to 10 min application of CNO (0.5–1 μm). G, Confocal images of a recorded BF^SOM neuron (filled with biocytin and labeled in blue; top) that expresses hM3Dq-mCherry (native fluorescence; bottom; scale bars, 20 μm). H, I, hM4Di-mCherry expressing BF^SOM neurons (top) visualized under IR-DIC (bottom; scale bars, 20 μm) reduced their firing frequency in response to CNO (1 μm; dotted lines, 0 mV). J, Averaged response of hM4Di-expressing BF^SOM neurons to CNO (0.5–1 μm; n = 6). K, Confocal image of a recorded BF^SOM neuron (top, biocytin labeled in blue; bottom, mCherry native fluorescence; scale bars, 20 μm). L–N, Non-hM3Dq or non-hM4Di expressing BF^SOM neurons, recorded from SOM-Cre mice injected with DIO-mCherry-AAV, control injections (top: mCherry; bottom: IR-DIC; scale bars: 20 μm) did not respond to CNO (1 μm; dotted lines, 0 mV; n = 6). O, Confocal images of a recorded BF^SOM neuron (top, biocytin labeled in blue; bottom, mCherry native fluorescence; scale bars, 20 μm). P, Experimental design for the chemogenetic-based in vivo experiments. Q, Coronal section from a Som-ires-cre, lox-GFPL10 reporter cross (box shows targeted region of BF; scale bar, 1 mm).

Figure 2.

Absence of sleep–wake changes following chemogenetic activation of BF^SOM neurons. A, B, Photomicrographs showing transfection of BF^SOM neurons (A) and their robust activation (B) following CNO in vivo (green arrows indicate hM3Dq+ cells expressing c-Fos). C1–F3, Sleep–wake phenotypes following injection of vehicle and CNO (0.3 mg/kg and 0.9 mg/kg) in BF^SOM-hM3Dq mice. C, Hourly amount (±SEM) of the vigilance stages and sleep latencies in BF^SOM-hM3Dq mouse group (n = 8 mice). D, Power spectrum changes (±SEM) over baseline during the 3 h postinjection period for vehicle injection compared with the 3 h postinjection period for CNO (0.3 and 0.9 mg/kg) administration and the quantitative changes (±SEM) in power for the delta (δ. 0.5–5 Hz), theta (θ, 5–9 Hz), sigma (σ, 9–15 Hz), beta (β, 15–30 Hz), low-gamma (lγ, 30–60 Hz), and high-gamma (hγ, 60–120 Hz) frequency bands (n = 8 mice). E, F, Number of episodes (±SEM) of wakefulness (W), NREM sleep, or REM sleep in each bout length (E) and time-weighted frequency histograms (F) showing the proportion (±SEM) of wakefulness, NREM sleep, or REM sleep amounts in each bout length to the total amount of wakefulness, NREM sleep, or REM sleep during the 3 h postinjection period for vehicle injection compared with the 3 h postinjection period for CNO (0.3 and 0.9 mg/kg) administration (n = 8). Light green star p < 0.05 between CNO 0.3 mg/kg and control injection; dark green star p < 0.05 between CNO 0.9 mg/kg and control injection, two-way ANOVA followed by a post hoc Bonferroni test. Scale bars: A, 400 μm; A inset, 100 μm; B, 70 μm.

Figure 3.

Absence of sleep–wake changes following chemogenetic inhibition of BF^SOM neurons. Sleep–wake phenotypes following the injection of vehicle and CNO (0.3 and 0.9 mg/kg) in BF^SOM-hM4Di mice. A, Hourly amount of wakefulness (W; A1), NREM sleep (NREMS; A2) and REM sleep (REMS; A3) and sleep latencies in BF^SOM-hM4Di mouse group (n = 5 mice). B, Power spectrum changes (±SEM) over baseline during the 3 h postinjection period for vehicle injection compared with the 3 h postinjection period for CNO (0.3 and 0.9 mg/kg) administration and the quantitative changes (±SEM) in power for the delta (δ, 0.5–5 Hz), theta (θ, 5–9 Hz), sigma (σ, 9–15 Hz), beta (β, 15–30 Hz), low-gamma (lγ, 30–60 Hz), and high-gamma (hγ, 60–120 Hz) frequency bands (n = 4 mice). C1–C3, D1–D3, Number of episodes (±SEM) of W, NREMS, or REMS in each bout length (C) and in time-weighted frequency histograms (D) showing the proportion (±SEM) of W, NREMS, or REMS amounts in each bout length to the total amount of wakefulness, NREM sleep, or REM sleep during the 3 h postinjection period for vehicle injection compared with the 3 h postinjection period for CNO (0.3 and 0.9 mg/kg) administration (n = 5). *p < 0.05 between CNO 0.9 mg/kg and control injection, two-way ANOVA followed by a post hoc Bonferroni test.

Figure 4.

Absence of sleep–wake changes following optogenetic activation of BF^SOM neurons. A, Schematic showing experimental setup. B1, B2, Histological verification of ChR2-mCherry-expressing neurons within the BF (red), together with optical fiber placement (*) in both a BF^SOM-ChR2 (B1) and BF^VGAT-ChR2 (B2) mouse. Scale bar, 200 μm. C1, C2, Arousal state probability plots from BF^SOM-ChR2 mice showing wakefulness (blue), NREM sleep (purple), and REM sleep (red) over time as a percentage of the total number of trials over all mice (n = 7) before, during, and after blue light stimulation (light blue box) at either 2.5 Hz (C1) or 10 Hz (C2). D1, D2, Arousal state probability plots from BF^VGAT-ChR2 mice showing wakefulness (blue), NREM sleep (purple), and REM sleep (red) over time as a percentage of the total number of trials over all mice (n = 2) before, during, and after blue light stimulation (light blue box) at either 2.5 Hz (D1) or 10 Hz (D2).

Figure 5.

Sleep–wake quantitative changes following selective ablation of BF^SOM neurons. A, Schematic showing experimental design. B, Cartoon of construct expressing the cellular toxin DTA in a cre-dependent configuration (NB mCherry is expressed in transfected cre-negative cells). C, SOM⁺ neurons of the BF of the SOM-ires-cre,lox-L10GFP mouse. D, Injection of DTA-AAV into BF of SOM-ires-cre, lox-L10GFP mouse; red neurons show extent of cellular transfection and label surviving cells. E, A corresponding section from the same mouse shown in D showing a nearly complete loss of SOM (green) cells following DTA-driven ablation. F, the dorsally situated lateral septum, which contains a large number of SOM⁺ cells, was unaffected by the DTA-AAV targeting the BF (same mouse as E; scale bar C–E, 200 μm; F, 400 μm). G, Hourly amount (±SEM) of wakefulness (W; G1), NREM sleep (NREMS; G2) and REM sleep (REMS; G3) in BF^SOM-DTA and control mouse groups (n = 11 and 5 mice, respectively). H, Amount (±SEM) of the vigilance stages during the first 4 h of the dark period (19–23 stages), during the light, dark, and 24 h periods in BF^SOM-DTA and control mouse groups (n = 11 and 5 mice, respectively). *p < 0.05, **p < 0.01, two-way ANOVA followed by a post hoc Bonferroni test (hourly amounts and light/dark analysis) or paired t test (19–23 and 24 h).

Figure 6.

Sleep–wake qualitative changes following selective ablation of BF^SOM neurons. A, B, Number of episodes (±SEM) of wakefulness (W; A1, B1), NREM sleep (NREMS, A2, B2), or REM sleep (REMS, A3, B3) in each bout length (A) and time-weighted frequency histograms (B) showing the proportion (±SEM) of W, NREMS, or REMS amounts in each bout length to the total amount of W, NREMS, or REMS during the first 4 h of the dark period (19–23), during the light, dark, and 24 h periods in BF^SOM-DTA and control mouse groups (n = 11 and 5 mice, respectively). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA followed by a post hoc Bonferroni test. C, D, Power spectrum (±SEM) over total power during the dark period (19–01; C) and the light period (10–13; D) and the power band (±SEM) for the delta (δ, 0.5–5 Hz), theta (θ, 5–9 Hz), sigma (σ, 9–15 Hz), beta (β, 15–30 Hz), low-gamma (lγ, 30–60 Hz), and high-gamma (hγ, 60–120 Hz) frequency bands in BF^SOM-DTA and control mouse groups (n = 8 and 5 mice, respectively). **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA followed by a post hoc Bonferroni test.

Figure 7.

SOM-IRES-cre: a hypomorphic allele? A, Comparison of hourly amounts (±SEM) of baseline wakefulness (W; A1), NREM sleep (NREMS; A2) and REM sleep (REMS; A3) in BF^SOMcre/cre (homozygous for the Cre allele, n = 11 mice) and BF^SOMcre/wt (heterozygous for the Cre allele, n = 10 mice) mouse groups. B1, C1, D1, Amount (±SEM) of the vigilance stages during the light, dark, and 24 h baseline periods in BF^SOMcre/cre compared with BF^SOMcre/wt mouse groups (n = 11 and 10 mice, respectively). *p < 0.05, **p < 0.01, two-way ANOVA followed by a post hoc Bonferroni test (hourly amounts and light/dark analysis) or paired t test (24 h). B2, C2, D2, Number of episode (±SEM) of wakefulness (W), NREM sleep (NREMS), or REM sleep (REMS) in each bout length during the light and 24 h periods in BF^SOMcre/cre compared with BF^SOMcre/wt mouse groups (n = 11 and 10 mice, respectively). **p < 0.01, ****p < 0.0001, two-way ANOVA followed by a post hoc Bonferroni test. E1, E2 and E3, Power spectrum (±SEM) over total power during the dark period (19–01) and power band (±SEM) for the delta (δ, 0.5–5 Hz), theta (θ, 5–9 Hz), sigma (σ, 9–15 Hz), beta (β, 15–30 Hz), low-gamma (lγ, 30–60 Hz), and high-gamma (hγ, 60–120 Hz) frequency bands, in BF^SOMcre/cre compared with BF^SOMcre/wt mouse groups (n = 8 and 9 mice, respectively). *p < 0.05, ****p < 0.0001, two-way ANOVA followed by a post hoc Bonferroni test.