Figure 1.
Fos+ and GAD67 mRNA+ neurons in the vlPAG/DpMe region and the SLD after paradoxical sleep deprivation and hypersomnia.
A,C: Low power photomicrographs showing frontal sections double-labeled for Fos and GAD67 at the level of the vlPAG/dDpMe region in PSD (A) and PSR (C) animals. B, D: Enlargements showing several Fos+/GAD+ neurons (arrows) characterized by a blue diffuse cytoplasmic staining and a brown nuclear staining in the lateral and the medial parts of the vlPAG/dDpMe region in PSD (B) and PSR (D) animals, respectively. E: Low power photomicrograph showing a frontal section double-labeled for Fos and GAD67 at the pontine level in a PSR animal. F: Enlargement of E showing the presence in the SLD of a large number of Fos+ and GAD negative labeled cells characterized by a brown nuclear staining. Scale bars: 500 µm for A, C and E; 25 µm for B, D and F. Abbreviations: 3, oculomotor nucleus; Aq, Sylvius aqueduct; dDpMe, dorsal part of the deep mesencephalic nucleus; DpMe, deep mesencephalic nucleus; DRN, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; LDTg, laterodorsal tegmental nucleus; me5, mesencephalic trigeminal tract; mlf, medial longitudinal fasciculus; PnC, pontine reticular nucleus, caudal part; SLD, sublaterodorsal nucleus; vlPAG, ventrolateral periaqueductal gray.
Figure 2.
Activated GABAergic neurons in the vlPAG/DpMe region after paradoxical sleep deprivation and hypersomnia (Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization).
Schematic distribution of Fos+ (small black dots) and Fos-GAD (large red dots) neurons in the vlPAG/dDpMe region in PSD (A) and PSR (B) animals (sections −7.40 from Bregma). Double-labeled neurons in the vlPAG are more abundant in the lateral and ventral portions of the vlPAG in the PSD animal and in its medial part in the PSR animal.
Table 1.
Number of GABAergic activated neurons in Control, Paradoxical sleep deprived or hypersomniac rats.
Figure 3.
Activated GABAergic neurons at medullary level after paradoxical sleep hypersomnia.
Photomicrographs of Fos (brown nuclear staining) and GAD67 mRNA (blue diffuse cytoplasmic staining) double-labeled sections from PSR rats at the medullary level. Low (A) and high (B) power photomicrographs showing the rDPGi. Numerous Fos+ and GAD-negative neurons are observed in this structure at high magnification (B). (C–F) Photomicrographs showing low (C) and high magnifications (D–F) of the medullary reticular nuclei in a PSR animal. Note the presence of numerous Fos-GAD cells (black arrows) in the DPGi (D), the GiV (E) and the LPGi (F). Abbreviations: 4 V, 4th ventricle; Amb, ambiguus nucleus; Cb, cerebellum; g7, genu of the facial nerve; mlf, medial longitudinal fasciculus; cDPGi, caudal part of the dorsal paragigantocellular nucleus; GiV, gigantocellular reticular nucleus, ventral part; IO, inferior olive; LPGi, lateral paragigantocellular nucleus; MVe, medial vestibular nucleus; Pr, prepositus nucleus; rDPGi, rostral part of the dorsal paragigantocellular nucleus. Scale bars, 500 µm for A and C; 50 µm for B, E and F and 25 µm for D.
Figure 4.
Muscimol injection sites inducing paradoxical sleep hypersomnia.
A: Localization on rostro-caudal frontal sections (ß, Bregma) of the Mus injections sites inducing either a PS hypersomnia (black circles), a strong excitation of the animals (grey circles) or no effects on the sleep-wake cycle (empty circles). One circle can represent one or more ejections. B: Percentages of the three vigilance states before and after Mus and NaCl iontophoretic ejections (n = 5) in the vlPAG/dDpMe region. Significance values indicated are: *P<0.05, **P<0.01 and ***P<0.001 vs NaCl.
Figure 5.
Paradoxical sleep characteristics after muscimol injection in the vlPAG/dDpMe region.
A: EEG spectrogram analysis of the 3000 s following one Mus ejection in the vlPAG/dDpMe region. Rectified EMG amplitude, EEG trace and spectrogram analysis are presented for 200 s corresponding to one PS episode induced by the Mus ejection. B: PS episodes induced by Mus ejections share the same properties as the control one, an active EOG, a flat EMG and a dominant theta band frequency activity. C: Average power spectrum analysis for Mus and NaCl ejections in the vlPAG/dDpMe region.
Figure 6.
Model of the network responsible for PS onset and maintenance.
The SLD contains glutamatergic neurons responsible for the onset and maintenance of PS. They induce muscle atonia and sensory inhibition via direct excitatory projections to the glycinergic/GABAergic neurons localized in the medullary ventral reticular nuclei (GiA, GiV, RMg) and EEG activation via direct intralaminar thalamic projections. During W and SWS, the SLD neurons are tonically inhibited by GABAergic PS-off neurons localized in the vlPAG/dDpMe region. At the onset and during PS these PS-off neurons are tonically inhibited by the co-localized GABAergic PS-on neurons as well as those of the DPGi and LPGi. These GABAergic PS-on neurons are also responsible for the inhibition of locus coeruleus (LC) noradrenergic and dorsal raphe (DRN) serotonergic neurons during PS. In normal condition, the onset of PS is not possible directly from W because the wake-active hypothalamic hypocretinergic neurons and brainstem monoaminergic neurons tonically excite the vlPAG/dDpMe PS-off GABAergic neurons. The decrease or cessation of activity of these wake-active neurons during SWS weaken the activity of vlPAG/dDpMe PS-off GABAergic neurons inducing a desinhibition of the co-localized GABAergic PS-on neurons and by this way PS.