Mapping of Nb-labeled (Orx− and) MCH− and VGAT+ sleep-max-active neurons in the LH. Nb-labeled sleep-max neurons were distributed in the region of Orx+ and MCH+ neurons, where Orx+ W-max neurons (Lee et al., 2005b) and MCH+ P-max neurons (Hassani et al., 2009b), were previously recorded. The Nb+/MCH− sleep-max neurons comprised S-max neurons (blue, n = 9) and P-max neurons (green, n = 39). Localization of Nb-labeled varicosities in adjacent sections for some sleep-max neurons allowed identification of (2/2) S-max MCH− neurons as VGAT+ (blue filled triangles, including the cell shown in Fig. 1A–C, depicted here as the largest blue triangle in A5.8). Similarly, localization of Nb-labeled varicosities allowed identification of (8/10) P-max MCH− neurons as VGAT+ (green filled triangles, including the cell shown in Fig. 1D–F, depicted here as the largest green filled triangle in A6.2). The remaining (2/10) P-max MCH− cells were VGAT− (green x’s). Cells were mapped according to the appropriate levels (A ~5.4, 5.8, and 6.2 mm from interaural zero) through the LH where Orx+ cells are distributed (Henny & Jones, 2006a). Abbreviations: Arc, arcuate nucleus; cp, cerebral peduncle; DMH, dorsomedial hypothalamic nucleus; f, fornix; ic, internal capsule; LH, lateral hypothalamus; mt, mammillo-thalamic tract; ot, optic tract; PH, posterior hypothalamus; STh, subthalamic nucleus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta. Scale bar, 1 mm.

Discharge of P-max-active MCH−/VGAT+ cell across sleep-wake states. (A) Data from Nb-labeled unit (c102u04, also shown in Fig. 1D–F) showing the sleep-wake recording scored per 10 sec epochs for sleep and wake stages along with simultaneous EEG frequency and amplitude (μV/Hz with frequency on y axis and amplitude differentially scaled according to color from blue to red, over the low frequency, 0–30 Hz from ~0–100 μV, and the high frequency, 30–60 Hz from ~0–25 μV), EMG amplitude (arbitrary units) and unit spike rate (Hz) over the recording session. Note that the unit showed its lowest firing rates during aW (periods indicated by red dashed vertical lines) and high EMG activity and its highest rates during PS (periods indicated by solid green vertical lines) and low EMG activity. Across stages, its discharge rate was significantly, negatively correlated with EMG amplitude (r = −0.71). Horizontal lines (marked as 1, 2 and 3) indicate 10 sec recording epochs of aW, SWS and PS respectively shown in C. (B) Bar graph showing mean spike rate (Hz) of the unit across sleep-wake stages. Note that the unit fired at its lowest rate during aW (1.08 Hz), progressively increased its rate through qW, tSWS and SWS (1.97 Hz) and more markedly increased its rate during tPS to reach its maximal rate during PS (6.22 Hz). (C) Polygraphic records from 10 sec epochs (indicated in A) of the unit together with EEG and EMG activity during aW (1), SWS (2) and PS (3). Note that during waking, the unit fired only minimally and ceased firing altogether during active periods when EMG activity was prominent (C1). It fired periodically in one or two spikes during SWS (C2). It discharged almost continuously and at its highest rates during PS (C3) with an instantaneous firing frequency of 15.38 Hz and tendency toward irregular phasic grouping of spikes. Calibrations: horizontal, 1 s; vertical, 1 mV (EEG, EMG), 2 mV (unit). Abbreviations: OB, olfactory bulb; PF, prefrontal cortex; RS, retrosplenial cortex.

Immunostaining of Nb-labeled sleep-active neurons. (A) As shown in merged image (A1), an Nb-labeled cell (#c103u01) that discharged maximally during SWS, as S-max (green, arrowhead) was found intermingled with Orx+ (blue) and MCH+ (red) neurons in the LH. As also shown at higher magnification (A2, A3 and A4), the Nb+ neuron was clearly immunonegative for both Orx and MCH. (B) Found in an adjacent section, the Nb-labeled axon varicosities of the same neuron (B1, green, arrowheads, magnified in B2 and B5) were positively immunostained for VGAT (B3 and B6, red and B4 and B7, yellow in merged images). (C) Another Nb-labeled varicosity of the same neuron (C1, green, arrowhead, magnified in C2), was immunonegative for VGluT2 (C3, red and C4, in merged image). (D) As shown in merged image (D1), an Nb-labeled cell (#c102u04) that discharged maximally during PS, as P-max (green, arrowhead) was intermingled with Orx+ (blue) and MCH+ (red) neurons in the LH. As also shown at higher magnification (D2, D3 and D4), the Nb+ neuron was clearly immunonegative for both Orx and MCH. (E) Found in an adjacent section, the Nb-labeled axon varicosities of the same neuron (E1, green, arrowheads, magnified in E2 and E5) were positively immunostained for VGAT (E3 and E6, red and E4 and E7, yellow in merged images). (F) Another Nb-labeled axon varicosity of the same neuron (F1, green, arrowhead, magnified in F2) was immunonegative for VGluT2 (F3, red and F4, green in merged image). Scale bars in D1, for A1 and D1 = 40 μm; in D4 for insets A2–A4 and D2–D4 = 20 μm; in F1, for B1, C1, E1 and F1 = 5 μm; and in F4 for insets B2–B7, C2–C4, E2–E7 and F2–F4 = 2 μm.

Discharge of S-max-active MCH−/VGAT+ cell across sleep-wake states. (A) Data from Nb-labeled unit (c103u01, also shown in Fig. 1A–C) showing the sleep-wake recording scored per 10 sec epochs for sleep and wake stages along with simultaneous EEG frequency and amplitude (μV/Hz with frequency on y axis and amplitude differentially scaled according to color from blue to red, over the low frequency, 0–30 Hz from ~0–100 μV, and the high frequency, 30–60 Hz from ~0–25 μV), EMG amplitude (arbitrary units) and unit spike rate (Hz) over the recording session. Note that the unit discharged at moderately low rates during both aW (showing high EMG activity; periods indicated by red dashed vertical lines) and PS (showing low EMG activity; periods indicated by solid green vertical lines), and at its highest rates during SWS. Across stages, its discharge rate was significantly, positively correlated with delta EEG (r = 0.34). Horizontal lines (marked as 1, 2 and 3) indicate 10 sec recording epochs of aW, SWS and PS respectively shown in C. (B) Bar graph showing mean spike rate (Hz) of the unit across sleep-wake stages. Note that the unit fired at a relatively low rate during aW (7.27 Hz), increased its rate through qW, tSWS to reach its maximal rate during SWS (13.35 Hz) and decreased its rate slightly during tPS to reach a lower rate during PS (9.95 Hz). C, Polygraphic records from 10 sec epochs (indicated in A) of the unit together with EEG and EMG activity during aW (1), SWS (2) and PS (3). Note that during waking, despite an overall moderate discharge rate, the unit notably slows down its firing during active periods when EMG activity was prominent (C1). It fired continuously and tonically in single spikes during SWS (C2), with an instantaneous firing frequency of 14.9 Hz. It discharged also at moderate rates during PS (C3). Calibrations: horizontal, 1 s; vertical, 1 mV (EEG, EMG), 2 mV (unit). Abbreviations: OB, olfactory bulb; PF, prefrontal cortex; RS, retrosplenial cortex.

Discharge of sleep-max Nb+/MCH− and VGAT+ cells across sleep-wake stages. (A) As a group, all S-max Nb+/MCH− cells, including VGAT+ cells (n = 9) discharged maximally during SWS (top row). S-max VGAT+ neurons (n = 2) discharged with a similar profile (middle row). In all S-max MCH− cells, the discharge rate changed progressively in positive association with delta EEG activity across sleep-wake stages (bottom row, normalized averages from all sleep-max MCH− units). (B) As a group, the P-max Nb+/MCH− cells, including VGAT+ cells (n = 39) discharged maximally during PS (top row). P-max VGAT+ neurons (n = 8) discharged with a similar profile across sleep-wake stages (middle row). In all P-max MCH− cells, the discharge rate changed progressively across sleep-wake stages in negative association with EMG amplitude (bottom row, normalized averages from all sleep-max MCH− units).