Pontine reticular formation (PnO) administration of hypocretin-1 increases PnO GABA levels and wakefulness

Abstract

Study objectives: GABAergic transmission in the oral part of the pontine reticular formation (PnO) increases wakefulness. The hypothalamic peptide hypocretin-1 (orexin A) promotes wakefulness, and the PnO receives hypocretinergic input. The present study tested the hypothesis that PnO administration of hypocretin-1 increases PnO GABA levels and increases wakefulness. This study also tested the hypothesis that wakefulness is either increased or decreased, respectively, by PnO administration of drugs known to selectively increase or decrease GABA levels.

Design: Awithin-subjects design was used for microdialysis and microinjection experiments.

Setting: University of Michigan.

Patients or participants: Experiments were performed using adult male Crl:CD (SD)IGS BR (Sprague-Dawley) rats (n=46).

Interventions: PnO administration of hypocretin-1, nipecotic acid (a GABA uptake inhibitor that increases extracellular GABA levels), 3-mercaptopropionic acid (a GABA synthesis inhibitor that decreases extracellular GABA levels; 3-MPA), and Ringer solution (vehicle control).

Measurements and results: Dialysis administration of hypocretin-1 to the PnO caused a statistically significant, concentration-dependent increase in PnO GABA levels. PnO microinjection of hypocretin-1 or nipecotic acid caused a significant increase in wakefulness and a significant decrease in non-rapid eye movement (NREM) sleep and REM sleep. Microinjecting 3-MPA into the PnO caused a significant increase in NREM sleep and REM sleep and a significant decrease in wakefulness.

Conclusions: An increase or a decrease in PnO GABA levels causes an increase or decrease, respectively, in wakefulness. Hypocretin-1 may promote wakefulness, at least in part, by increasing GABAergic transmission in the PnO.

Figure 1

GABA levels in the pontine reticular nucleus, oral part (PnO) became stable by 42 minutes after dialysis probe insertion and remained stable for 2.8 hours. A. A representative chromatogram generated from a known quantity of GABA (in vitro standard, gray line, 0.913 pmol/10 μL) is shown superimposed on a typical chromatogram obtained by dialysis of the PnO (in vivo sample, black line; 0.107 pmol/10 μL), indicating the ability to identify GABA in brain samples. A sagittal diagram of the rat brain has been modified by adding a schematized microdialysis probe inserted in the PnO. The dialysis membrane at the tip of the probe is drawn to scale. Curved arrows indicate that Ringer solution was delivered to the probe through the inlet port and GABA from the PnO was collected at the outlet port. B. GABA levels in sequentially collected dialysis samples are plotted for the first 84 minutes after dialysis probe insertion into the PnO. Each bar indicates average GABA levels from 18 rats, plotted in 7 minute intervals. Thus, time 7 indicates minutes 1–7, and time 84 indicates minutes 78–84. C. GABA levels in the PnO remained stable for 2.8 hours of dialysis with Ringer solution. Each bar represents average PnO GABA levels in 18 dialysis samples (6 samples per rat x 3 rats).

Figure 2

Hypocretin-1 caused a concentration-dependent increase in pontine reticular nucleus, oral part (PnO) GABA levels. A-C. Each bar represents average GABA levels from 3 rats plotted in sequential 7 minute intervals during dialysis with Ringer solution (hatched bars), hypocretin-1 (solid bars), and Ringer solution (hatched bars). Dialysis samples 1–6 correspond in time to minutes 49–84 in Figure 1B. Asterisks indicate a significant increase compared to the average GABA level measured during dialysis with Ringer solution before delivery of hypocretin-1 (samples 1–6). D. Each concentration of hypocretin-1 was tested in 3 rats. The number of dialysis samples used to calculate mean ± SEM GABA levels was 16, 17, 17, 18, 18, and 18 for hypocretin-1 concentrations of 0, 1, 3, 10, 30, and 100 μM, respectively. Average GABA levels significantly (*) increased during dialysis with 10, 30, and 100 μM hypocretin-1 as compared to control dialysis with Ringer solution (0 μM hypocretin-1).

Figure 3

Microdialysis sites were localized to the pontine reticular formation, oral part (PnO). The vertical cascade of coronal sections shows the location of each microdialysis probe. Probe membranes are denoted by shaded cylinders and are drawn to scale. The digitized image of a cresyl-violet-stained section shows a typical microdialysis probe tract. The arrow points to the most ventral portion of the dialysis site. The coronal diagrams were modified from a rat brain atlas, and numbers at the right of each diagram indicate mm from bregma.

Figure 4

The temporal organization of sleep and wakefulness was normal following pontine reticular nucleus (PnO) microinjection of Ringer solution (A), hypocretin-1 (B), nipecotic acid (C), and 3-mercaptopropionic acid (D). Each graph plots the time course of sleep and wakefulness for 2 hours following a single microinjection, which was made during wakefulness. Bar height indicates states of wakefulness (lowest bars), non-rapid eye movement (NREM) sleep (intermediate height bars) and rapid eye movement (REM) sleep (highest bars). Sleep architecture was normal following drug administration, in that REM sleep was always preceded by NREM sleep and REM sleep was most frequently followed by an episode of wakefulness.

Figure 5

Pontine reticular nucleus, oral part (PnO) microinjection of hypocretin-1 caused a reversible increase in wakefulness and decrease in sleep. In the first hour after microinjection, the percentage of wakefulness (A) increased by 30%, and the percentage of NREM sleep (B) and REM sleep (C) decreased by 36% and 68%, respectively. Hypocretin-1 increased REM latency (D) by 79% but did not affect NREM latency (D). Microinjection of hypocretin-1 decreased the number of episodes of wakefulness (E, 38%), NREM sleep (F, 41%), and REM sleep (G, 71%). All dependent measures of sleep and wakefulness returned to control (Ringer solution) levels during the second hour of recording. Hypocretin-1 microinjection sites (H) were localized to the PnO and are depicted by filled circles on 2 coronal diagrams modified from a rat brain atlas. Numbers at the right of each diagram indicate mm posterior to bregma. Hypocretin-1 microinjection sites (n = 10) spanned from −7.80 to −8.26 mm from bregma.

Figure 6

Nipecotic acid significantly (*) increased wakefulness and decreased sleep in the first hour after microinjection into the pontine reticular nucleus, oral part (PnO). Nipecotic acid caused a 20% increase in wakefulness (A), a 39% decrease in non-rapid eye movement (NREM) sleep (B), and a 70% decrease in rapid eye movement (REM) sleep (C). Nipecotic acid also increased latency to onset of REM sleep by 49% (D). Nipecotic acid decreased the number of episodes of wakefulness (E, 39%), NREM sleep (F, 46%), and REM sleep (G, 68%). Nipecotic acid microinjection sites (H) were localized to the PnO and are represented by filled circles on 3 coronal diagrams modified from a rat brain atlas. Numbers at the right of each diagram indicate mm posterior to bregma. Sites where nipecotic acid was microinjected ranged from −7.82 to −8.73 mm from bregma.

Figure 7

Pontine reticular nucleus, oral part (PnO) microinjection of 3-mercaptopropionic acid (3-MPA) significantly (*) and reversibly increased sleep and decreased wakefulness. Microinjection of 3-MPA decreased wakefulness by 29% (A), increased non-rapid eye movement (NREM) sleep by 68% (B), and increased rapid eye movement (REM) sleep by 193% (C) during the first hour after PnO microinjection. Although the effects did not reach statistical significance, 3-MPA showed a trend toward decreasing the latency to onset of NREM sleep and REM sleep (D) by 42% and 29%, respectively. During the first hour, an increase in the number of episodes of wakefulness (E, 28%), NREM sleep (F, 31%), and REM sleep (G, 190%) was also observed after 3-MPA microinjection, but only the increase in the number of NREM sleep and REM sleep episodes achieved statistical significance. 3-MPA microinjection sites (H) were localized to the PnO and are indicated by filled circles on 3 coronal diagrams modified from a rat brain atlas. Numbers at the right of each diagram indicate mm posterior to bregma. The PnO sites where 3-MPA was microinjected extended from −7.97 to −8.51 mm from bregma.