Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Nov;18(11):1641-7.
doi: 10.1038/nn.4143. Epub 2015 Oct 12.

Basal Forebrain Circuit for Sleep-Wake Control

Affiliations
Free PMC article

Basal Forebrain Circuit for Sleep-Wake Control

Min Xu et al. Nat Neurosci. .
Free PMC article

Abstract

The mammalian basal forebrain (BF) has important roles in controlling sleep and wakefulness, but the underlying neural circuit remains poorly understood. We examined the BF circuit by recording and optogenetically perturbing the activity of four genetically defined cell types across sleep-wake cycles and by comprehensively mapping their synaptic connections. Recordings from channelrhodopsin-2 (ChR2)-tagged neurons revealed that three BF cell types, cholinergic, glutamatergic and parvalbumin-positive (PV+) GABAergic neurons, were more active during wakefulness and rapid eye movement (REM) sleep (wake/REM active) than during non-REM (NREM) sleep, and activation of each cell type rapidly induced wakefulness. By contrast, activation of somatostatin-positive (SOM+) GABAergic neurons promoted NREM sleep, although only some of them were NREM active. Synaptically, the wake-promoting neurons were organized hierarchically by glutamatergic→cholinergic→PV+ neuron excitatory connections, and they all received inhibition from SOM+ neurons. Together, these findings reveal the basic organization of the BF circuit for sleep-wake control.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Genetically defined BF cell types. (a) Fluorescence image of BF (box in coronal diagram) showing tdTomato-expressing cholinergic neurons in ChAT-Cre × Ai14 mouse. (b) Fluorescence images of tdTomato (red) and immunohistochemical (IHC) staining or in situ hybridization for ChAT, VGLUT2, PV, and SOM (green) in ChAT-Cre × Ai14 mouse. (c–e) Similar to b, but for VGLUT2-Cre (c), PV-Cre (d) and SOM-Cre (e) mice crossed with Ai14 mouse. Red, green, and yellow arrows indicate cells that are tdTomato+, immunostaining/in situ hybridization+, and both, respectively. Scale bar, 20 μm.
Figure 2
Figure 2
Identification of BF cell types using ChR2 tagging and optrode recording. (a) Schematic of optrode recording in freely moving mice. (b) Example recording of laser-evoked spiking in ChAT-ChR2 mice. Blue lines, laser pulses (5 ms, 16 and 33Hz). Scale bars, 50 μV, 200 ms. (c) Raster plot and peri-stimulus time histogram (PSTH, bin size, 1 ms) for laser-evoked spikes of an example ChR2-tagged neuron. Dashed line, laser onset time. (d) Distribution of P values of Kolmogorov-Smirnov (K-S) test for all 85 optogenetically identified BF neurons. Colored circle, K-S test of spike timing distribution before and after laser onset for each neuron (cell types are color coded). Gray circles, K-S test after random shuffling of laser onset time. Dashed line, P = 0.0005. (e) Distribution of latency of laser-evoked spiking for the 85 neurons. The latency was measured by change point analysis (see Methods). (f) Waveform comparison between laser-evoked and spontaneous spikes. Upper panel, averaged waveforms of laser-evoked spikes (blue) and spontaneous spikes (gray) of an example neuron. Lower panel, distribution of correlation coefficient between laser-evoked and spontaneous spike waveforms for all 85 optogenetically identified BF neurons.
Figure 3
Figure 3
Firing rates of identified BF cell types across natural sleep-wake cycles. (a) Firing rates of an example ChAT+ neuron over 108 min. Top panel, EEG power spectrogram (0–25 Hz). Middle panel, EMG trace. Bottom panel, firing rate of the ChAT+ neuron. Scale bars, 3 spikes/s, 500 s. Brain states are color coded (wake, gray; REM, orange; NREM, white). (b) Summary of firing rate modulation of 12 ChAT+ neurons (from 6 mice). Gray shading, <2 fold firing rate change between brain states. Note that in this 2D plot, firing rates are compared explicitly between wake and NREM and between REM and NREM, but not between wake and REM states. Neurons that are maximally active during REM sleep are located in the upper left quadrant and part of the upper right quadrant (closer to the vertical than horizontal axis). (c, d), Similar to a & b, for VGLUT2+ neurons (6 mice). (e, f), PV+ neurons (5 mice). (g, h), SOM+ neurons (5 mice). Scale bars (c, e, g), 10 spikes/s, 500 s.
Figure 4
Figure 4
Effects of BF neuron activation on sleep-wake states. (a) Schematic of optogenetic stimulation experiment. (b) An example trial of ChAT+ neuron activation. Shown are EEG power spectrum, EEG traces during selected periods (indicated by boxes) and EMG trace during the whole trial. Blue bar, period of laser stimulation (10 ms pulses, 10 Hz, 60 s). Scale bar, 10 s. (c) Probability of wake, NREM or REM states before, during, and after laser stimulation of ChAT+ neurons (n = 5 mice). Error bar, ± s.e.m. Blue shading, period of laser stimulation. (d) Laser-induced change in the probability of each state (difference between the 60s periods before and during laser stimulation) in ChAT-ChR2 (filled bar) and ChAT-eYFP (open bar, n = 3) mice. (e, f) Similar to (c, d), for VGLUT2-ChR2 (n = 6) and VGLUT2-eYFP (n = 5) mice. (g, h) PV-ChR2 (n = 6) and PV-eYFP (n = 4) mice. (i, j) SOM-ChR2 (n = 7) and SOM-eYFP (n = 4) mice. The number of trials per mouse was 24–36. *P ≤ 0.05, ***P ≤ 0.001 (difference between ChR2 and eYFP mice, two-way ANOVA followed by Bonferroni post-hoc test).
Figure 5
Figure 5
Local connectivity of BF cell types. (a) Schematic of slice experiment using two strategies. The first strategy (upper right) is to use double transgenic mice. Shown are fluorescence image of a small BF area (red box in coronal diagram) showing ChR2-eYFP-expressing ChAT+ neurons (green) and tdTomato-expressing PV+ neurons (red) in an example experiment. Scale bar, 30 μm. Blue light was used to activate ChR2-expressing (presynaptic) neurons and whole-cell recordings were made from fluorescently labeled postsynaptic neurons. The second strategy (lower right) requires single-cell gene-expression analysis. Recordings were made from unlabeled neurons and the cell type is identified using RT-PCR. (b–e) Synaptic interactions between multiple pairs of pre- and postsynaptic cell types. (b) VGLUT2+ to ChAT+, PV+ and SOM+ neuron connections. Top, example light-evoked excitatory responses (red) recorded under current clamp, blocked by AMPA receptor antagonist CNQX (10 μM, black traces). Short blue bar, light pulse (5 ms). Scale bars, 1 mV, 200 ms. Bottom, population summary of input strength (measured by voltage area, integral of EPSP), each circle (using double transgenic mice) or triangle (based on single-cell gene-expression analysis, performed in the presence of mAChR, nAChR antagonists) represents one cell. Red, significant excitatory response (P < 0.05, t-test); Gray, no significant response. Error bar, ± s.e.m. (c) Similar to b, for ChAT+ to VGLUT2+, PV+ and SOM+ connections (recorded under current clamp). Blue, significant inhibitory response (P < 0.05); black, after application of AChR antagonists. ChAT→VGLUT2 excitatory response was blocked by nAChR antagonists MLA (methyllycaconitine, α7-containing nAChR antagonist, 5 nM) and DhβE (dihydro-β-erythroidine, non-α7 nAChR antagonist, 500 nM), inhibitory response blocked by mAChR antagonist scopolamine (20 μM). ChAT→PV response was blocked by MLA + DhβE. ChAT→SOM excitatory responses were blocked by MLA, DhβE and scopolamine, and inhibitory responses were blocked by scopolamine. Scale bars, 1 mV, 200 ms. All experiments indicated by triangle were performed in the presence of glutamate and GABA receptor antagonists. (d) PV+ to VGLUT2+, ChAT+ and SOM+ connections (voltage clamp). Among all recorded neurons (VGLUT2+, n = 11; ChAT+, n = 17; SOM+, n = 8), inhibitory responses were detected only in two VGLUT2+ neurons, which were blocked by GABAA receptor antagonist bicuculline (‘bic’, 20 μM, black trace). Scale bars, 10 pA, 50 ms. (e) SOM+ to VGLUT2+, ChAT+ and PV+ connections (voltage clamp). All inhibitory responses (blue) were blocked by bicuculline (black). Scale bars, 10 pA, 50 ms. (f) Diagram of BF local circuit. Light circles, wake-promoting neurons. Dark circle, sleep-promoting SOM+ neurons (containing both wake/REM-active and NREM-active neurons). Excitatory and inhibitory connections are indicated by red and blue lines, respectively. Gray line with cross indicates tested connection with no detectable response. Connection strength is represented qualitatively by line thickness.

Similar articles

See all similar articles

Cited by 95 articles

See all "Cited by" articles
Feedback