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. 2017 Feb 1;37(5):1352-1366.
doi: 10.1523/JNEUROSCI.1405-16.2016. Epub 2016 Dec 30.

Cholinergic, Glutamatergic, and GABAergic Neurons of the Pedunculopontine Tegmental Nucleus Have Distinct Effects on Sleep/Wake Behavior in Mice

Daniel Kroeger  1 , Loris L Ferrari  1 , Gaetan Petit  1   2 , Carrie E Mahoney  1 , Patrick M Fuller  1 , Elda Arrigoni  1 , Thomas E Scammell  3
Affiliations

Cholinergic, Glutamatergic, and GABAergic Neurons of the Pedunculopontine Tegmental Nucleus Have Distinct Effects on Sleep/Wake Behavior in Mice

Daniel Kroeger et al. J Neurosci. .

Abstract

The pedunculopontine tegmental (PPT) nucleus has long been implicated in the regulation of cortical activity and behavioral states, including rapid eye-movement (REM) sleep. For example, electrical stimulation of the PPT region during sleep leads to rapid awakening, whereas lesions of the PPT in cats reduce REM sleep. Though these effects have been linked with the activity of cholinergic PPT neurons, the PPT also includes intermingled glutamatergic and GABAergic cell populations, and the precise roles of cholinergic, glutamatergic, and GABAergic PPT cell groups in regulating cortical activity and behavioral state remain unknown. Using a chemogenetic approach in three Cre-driver mouse lines, we found that selective activation of glutamatergic PPT neurons induced prolonged cortical activation and behavioral wakefulness, whereas inhibition reduced wakefulness and increased non-REM (NREM) sleep. Activation of cholinergic PPT neurons suppressed lower-frequency electroencephalogram rhythms during NREM sleep. Last, activation of GABAergic PPT neurons slightly reduced REM sleep. These findings reveal that glutamatergic, cholinergic, and GABAergic PPT neurons differentially influence cortical activity and sleep/wake states.

Significance statement: More than 40 million Americans suffer from chronic sleep disruption, and the development of effective treatments requires a more detailed understanding of the neuronal mechanisms controlling sleep and arousal. The pedunculopontine tegmental (PPT) nucleus has long been considered a key site for regulating wakefulness and REM sleep. This is mainly because of the cholinergic neurons contained in the PPT nucleus. However, the PPT nucleus also contains glutamatergic and GABAergic neurons that likely contribute to the regulation of cortical activity and sleep-wake states. The chemogenetic experiments in the present study reveal that cholinergic, glutamatergic, and GABAergic PPT neurons each have distinct effects on sleep/wake behavior, improving our understanding of how the PPT nucleus regulates cortical activity and behavioral states.

Keywords: PPT; chemogenetic; mouse; sleep.

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Figures

Figure 1.
Figure 1.
AAV injection sites in vGluT2-Cre, ChAT-Cre, and vGAT-Cre mice. Injections were mapped on coronal atlas drawings at three brainstem levels containing the PPT. Injection sites (gray shading) are plotted to encompass 90% of mCherry-expressing neurons, and darker shades of gray represent overlapping injection sites across mice. The red boundary highlights the PPT as shown in the Franklin and Paxinos (2008) mouse brain atlas, but the cholinergic PPT neurons actually extend across a larger region, as shown in VanderHorst and Ulfhake (2006). N = 8 mice per group. SC, Superior colliculus; IC, inferior colliculus; PAG, periaqueductal gray; DR, dorsal raphe nucleus; PBG, parabigeminal nucleus; xscp, decussation of the superior cerebellar peduncle; 4N, trochlear nucleus; 5N trigeminal motor nucleus.
Figure 2.
Figure 2.
hM3-mCherry is expressed eutopically in vGluT2-Cre, ChAT-Cre, and vGAT-Cre mice. In vGluT2-cre mice, FISH for vGluT2 mRNA colocalizes with mCherry immunostaining. In ChAT-Cre mice, immunostaining for ChAT colocalizes with mCherry. In vGAT-Cre mice, FISH for vGAT mRNA colocalizes with mCherry. Scale bar, 100 μm.
Figure 3.
Figure 3.
Major axonal projections of glutamatergic, cholinergic and GABAergic PPT neurons. A–L, Projections of each cell type were mapped and selected levels are shown on coronal slices: Injection site (A, E, and I), basal forebrain area (B, F, and J), midthalamic area (C, G, and K), and mammillary region (D, H, and L). Glutamatergic PPT neurons project densely to basal forebrain targets such as the SI (B), several thalamic nuclei (C), and especially to midbrain dopaminergic cell groups (D). Cholinergic PPT neurons project to some of the same targets that glutamatergic neurons project to, but with less dense projections (E–H). GABAergic neurons do not send long projections to these regions and most axons remain within ∼200 μm of the injection site (I–L). N = 2 mice per group. Scale bar in L = 500 μm. ac, Anterior commissure; aq, aqueduct; CL, centrolateral thalamic nucleus; OC, optic chiasm; MMN, medial mammillary nucleus; PF, parafasicular thalamic nucleus; SUM, supramammillary nucleus; VMT, ventromedial thalamic nucleus; VPM, ventral posteromedial thalamic nucleus.
Figure 4.
Figure 4.
Functional validation of hM3 in each mouse line. A, CNO (0.3 mg/kg) strongly increases fos in hM3-mCherry-expressing neurons in all three lines of mice compared with saline (n = 3 mice of each line per condition). B, Sample traces of in vitro, whole-cell, current-clamp recordings of PPT neurons in vGluT2-Cre, ChAT-Cre, and vGAT-Cre mice expressing hM3-mCherry or hM4-Cherry (vGluT2-Cre only). Application of CNO (5 μm) for 1–5 min (black bars above traces) activates glutamatergic and cholinergic PPT neurons but has no effect on GABAergic neurons. Dashed horizontal line indicates 0 mV. **p < 0.01; ***p < 0.001: ****p < 0.0001 versus saline.
Figure 5.
Figure 5.
Chemogenetic activation of glutamatergic PPT neurons increases wake. CNO substantially increases wake and reduces NREM and REM sleep for ∼7 h compared with saline (n = 8 mice). CNO (0.3 mg/kg) and saline were injected intraperitoneally at 8:00 A.M. **p < 0.01; ***p < 0.001 versus saline.
Figure 6.
Figure 6.
Activation of glutamatergic PPT neurons produces quiet wake and mild anxiety. A, After injection of CNO at 8:00 A.M., mice are mainly in quiet wake with modest amounts of time spent exploring, grooming, or nesting from 8:30 A.M. until 10:30 A.M. After injection of saline, mice are mainly asleep with a small amount of time in quiet wake during the same time interval. For comparison to injections in the light period, injections of saline in the dark period at 8:00 P.M. are associated with active wake behaviors, including exploring, grooming, and feeding from 8:30 P.M. until 10:30 P.M. (n = 4 mice). B, With open-field testing, CNO increased time in the corners compared with saline or no injection (n = 4 mice). *p < 0.05.
Figure 7.
Figure 7.
Activation of glutamatergic PPT neurons increases wheel running. CNO administered at 9:00 A.M. increases wheel running for 2 h to a level similar to that seen during the dark period (n = 4 mice). *p < 0.05 versus saline.
Figure 8.
Figure 8.
Chemogenetic inhibition of glutamatergic PPT neurons mildly decreases wake and increases NREM sleep. In mice expressing the inhibitory hM4 receptor in vGluT2 PPT neurons, CNO slightly decreased the average amount of wake and increased NREM sleep during the 6 h after injections (n = 8 mice). CNO (1.0 mg/kg) and saline were injected intraperitoneally at 8:00 P.M. *p < 0.05.
Figure 9.
Figure 9.
Activation of cholinergic PPT neurons increases light NREM sleep. In ChAT-Cre mice, CNO (0.3 mg/kg) increases light NREM sleep and reduces deep NREM sleep for ∼3 h. The amounts of wake and REM sleep are essentially unchanged (n = 8 mice). *p < 0.05; **p < 0.01; ***p < 0.001 versus saline.
Figure 10.
Figure 10.
Activation of cholinergic PPT neurons reduces EEG power in the delta range and other slow frequencies during NREM sleep. Administration of CNO (0.3 mg/kg) substantially reduces power of low EEG frequencies during NREM sleep, with large reductions in the delta and theta ranges. CNO does not alter the REM sleep EEG spectrum (n = 4 mice). *p < 0.05 versus saline.
Figure 11.
Figure 11.
Activation of cholinergic PPT neurons alters state-space characteristics of NREM sleep. Each dot represents EEG spectral characteristics of a 4 s epoch (n = 8 mice; see Materials and Methods). CNO reduces the amount of deep NREM sleep (gray dots) and increases the amount of light NREM sleep (black dots). CNO also reduces EEG total power. Histograms on the axes are overlapping.
Figure 12.
Figure 12.
Activation of GABAergic PPT neurons has little effect on sleep/wake behavior. Administration of CNO (0.3 mg/kg) in vGAT-Cre mice during the day does not alter the amounts of wake and NREM sleep, but slightly reduces REM sleep (n = 8 mice). *p < 0.05; **p < 0.01 versus saline.
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