Activation of c-fos in GABAergic neurones in the preoptic area during sleep and in response to sleep deprivation

Abstract

Neurones in the median preoptic nucleus (MnPN) and the ventrolateral preoptic area (vlPOA) express immunoreactivity for c-Fos protein following sustained sleep, and display elevated discharge rates during both non-REM and REM sleep compared to waking. We evaluated the hypothesis that MnPN and vlPOA sleep-active neurones are GABAergic by combining staining for c-Fos protein with staining for glutamic acid decarboxylase (GAD). In a group of six rats exhibiting spontaneous total sleep times averaging 82.2 +/- 5.1% of the 2 h immediately prior to death, >75% of MnPN neurones that were Fos-immunoreactive (IR) were also GAD-IR. Similar results were obtained in the vlPOA. In a group of 11 rats exhibiting spontaneous sleep times ranging from 20 to 92%, the number of Fos + GAD-IR neurones in MnPN and vlPOA was positively correlated with total sleep time. Compared to control animals, Fos + GAD-IR cell counts in the MnPN were significantly elevated in rats that were sleep deprived for 24 h and permitted 2 h of recovery sleep. These findings demonstrate that a majority of MnPN and vlPOA neurones that express Fos-IR during sustained spontaneous sleep are GABAergic. They also demonstrate that sleep deprivation is associated with increased activation of GABAergic neurones in the MnPN and vlPOA.

Figure 1. Examples of Fos + GAD-IR double-labelled neurones (filled arrows) and GAD-IR single-labelled cells (open arrows) in the MnPN of one HSS (A) and one LSS (B) rat

Calibration = 20 μm.

Figure 2. Number and distribution of Fos-IR single-labelled, GAD-IR single-labelled and Fos + GAD-IR double-labelled neurones located in counting grids for the rostral (A) and caudal (B) MnPN from one HSS rat and one LSS rat

In the HSS rat, 81% of rostral MnPN neurones and 77% of caudal MnPN neurones that stained for Fos-IR were also positive for GAD-IR. •, Fos + GAD double labelled; ▵, GAD single labelled; □, Fos single labelled

Figure 3. Mean numbers of total Fos-IR neurones, Fos-IR single-labelled neurones, Fos + GAD-IR double-labelled neurones and the mean percentage of Fos-IR neurones that were double labelled for GAD-IR in the rostral MnPN, caudal MnPN and vlPOA for HSS and LSS rats

**P < 0.001, independent t test; *P < 0.01, independent t test.

Figure 4. Regression lines and scatter plots of the number of Fos + GAD-IR double-labelled neurones versus total sleep time in the 11 rats studied in the spontaneous sleep protocol

In each of the three POA subregions examined, there was a significant positive correlation between sleep time and Fos + GAD-IR cell counts.

Figure 5. Temporal distribution of sleep–waking state progression (lower panels) and continuous EEG delta power (upper panels) during 2 h of recovery sleep in a sleep deprived rat and a sleep deprived control rat

Abbreviations: W, waking; N, non-REM sleep; R, REM sleep.

Figure 6. Mean numbers of Fos + GAD-IR double-labelled neurones (A) and mean percentage of GAD-IR neurones that were double labelled for Fos-IR (B) in SD rats permitted 2 h of recovery sleep (n= 6), in SDC rats permitted 2 h of recovery sleep (n= 6) and in HSS rats (n= 6)

ANOVA indicated a significant treatment effect on Fos + GAD-IR cell counts in the rostral MnPN (F_2,15= 45.07, P < 0.001), the caudal MnPN (F_2,15) = 27.78, P < 0.001) and the vlPOA (F_2,15= 15.57, P < 0.001). Similarly, ANOVA indicated a significant treatment effect on the percentage of GAD-IR neurones that were double labelled for Fos-IR in the rostral MnPN (F_2,15= 49.65, P < 0.001) caudal MnPN (F_2,15= 14.29, P < 0.01) and vlPOA (F_2,15= 13.08, P < 0.01). **Significantly different from SD and HSS values, P < 0.01, Newman–Keuls. *Significantly different from HSS values, P < 0.05, Newman–Keuls.

Figure 7. Mean numbers of Fos + GAD-IR double-labelled neurones in SD and SDC rats that were not permitted recovery sleep

The cell counts were significantly higher in SD versus SDC rats in the rostral MnPN (F_1,10= 17.57, P < 0.01), the caudal MnPN (F_1,10= 16.91, P < 0.01) and the vlPOA (F_1,10= 15.57, P < 0.01).