The reticular arousal threshold during the transition from slow wave sleep to paradoxical sleep in the rat

Physiology& Behavior,Vol. 58, No. 1, pp. 199-202, 1995 Copyright© 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0031-9384/95 $9.50 + .00

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The Reticular Arousal Threshold During the Transition from Slow Wave Sleep to Paradoxical Sleep in the Rat BRIGITTE PIALLAT AND CLAUDE GOIq'ESMANN l Laboratoire de F'sychophysiologie, Facultd des Sciences, Universitd de Nice-Sophia Antipolis, Nice, France R e c e i v e d 1 F e b r u a r y 1994 PIALLAT, B. and C. GO'ITESMANN. The reticular arousal threshold during the transition from slow wave sleep to paradoxical sleep in the rat. PHYSIOL BEHAV 58 (1) 199-202, 1995.--The short-lasting intermediate stage of sleep occurring between slow wave sleep and paradoxical sleep showed frontal cortex spindles of higher amplitude and longer duration than during slow wave sleep, and hippocampal theta rhythm of lower frequency than during paradoxical sleep. During the intermediate stage, the mesencephalic reticular arousal threshold was slightly higher than during the slow wave sleep and much lower than in the paradoxical sleep. Thus, following this central reactivity test, the intermediate stage seems to be functionally nearer the slow wave sleep than the paradoxical sleep. Slow wave sleep Intermediate stage Theta rhythm Rat

Paradoxical sleep

PARADOXICAL SLEEP in humans is preceded and sometimes followed by a short-lasting electroencephalographic (EEG) stage associating spindles and K complexes, an index of slow wave sleep (SWS), and low voltage activity without rapid eye movements, an index of paradoxical sleep (PS) (19). Arousal during this "intermediate phase" is difficult to get and reveals " a feeling of indefinable disconffort, anxious perplexity and harrowing worry" (20; p. 280). This stage is extended at the expense of PS in oligophrenia (16), schizophrenia (17), and brief reactive psychosis (20). It is likely that this stage corresponds to the intermediate stage of sleep described in rats (5,6,11,25,35), cats (14), and mice (9). It lasts a few seconds and is characterized by cortical spindles of large amplitude, an index of advanced SWS, and low frequency theta rhythm in the dorsal hiplx)campus, an index of limbic activation because it occurs during active and/or attentive waking and PS. This intermediate stage (IS) is extended at the expense of PS by low doses of b~biturates (11), which also suppress the pontine cell activation of this sleep stage (12), and by benzodiazepines (8). Finally, the, thalamic transmission level of sensory inputs, controlled by brain-stem-activating influences (31) is the lowest of all sleep-waking stages (7). These data suggest that IS corresponds to a short-lasting period where brain-stem-ascending

Arousal threshold

Reticular formation

Spindles

influences of waking (3,21,24,27,31), which decrease during SWS, are at their lowest level or suppressed while the pontine influences of PS are still absent, or at low level (31) but inoperative. Thus, the forebrain would be shortly functionally disconnected from the brain stem. The acute intercollicular transected rat (15) and cat (14) show continuous association of cortical spindies and low-frequency theta rhythm. To know more about the brain state during this transitional period, we undertook a study of the arousal threshold by stimulating the midbrain reticular formation. ME'H-IOD Under penthiobarbital anesthesia (55 mg/kg IP), nine adult male Wistar rats (weighing 250-320 g) were implanted bilaterally with silver ball (1-mm diameter) electrodes to record the frontal corticogram from Krieg's area 10 (18). To record hippocampal theta rhythm, a bipolar electrode made up of coated (except at the tip) stainless steel wires (10/100 mm) was stereotaxically (26) placed in the CAm area (A: 5.4; L: 2.6; D: +7.4). A silver ball (I-nun diameter) was placed on each side of one orbit to record eye movements. Two twisted stainless steel wires (251100 nun) were inserted bilaterally in the dorsal neck muscles

i Requests for reprints should be addressed to C. Gottesmann, Laboratoire de Psychophysiologie, Facult¢ des Sciences, 06108 Nice cedex 2, France.

199

200

PIALLAT AND GOTFESMANN

TABLE 1 DURATIONAND AM~LITUDEOF CORTICALSPINDLES Slow WaveSleep

IntermediateStage

Rat

Duration(s)

Amplitude (/~V)

Duration(s)

Amplitude(#V)

1 2 3 4 5 6 7 8 9 Mean± SD

1.08 1.13 0.73 0.83 0.80 0.92 1.00 1.40 0.98 0.99 ± 0.07

341 366 362 440 316 320 341 383 250 369 ± 30

3.00 1.84 1.50 1.25 1.78 1.13 2.37 1.93 2.12 1.88 ±0.19"

572 503 716 558 391 562 499 950 312 559 ± 61t

*p < 0.001; Jp < 0.05. to record the electromyogram. A ground electrode was screwed in front of the olfactory bulb in the middle plane. The stimulation (Anapulse Stimulator, USA) was delivered by an identical bipolar electrode implanted in the midbrain reticular formation (A: 2.7; L: 1.8; D: +4.2). All the recording electrodes were soldered to a connector (Connectral, France) and secured to the skull of the animal with dental cement (Texton, UK). At the end of surgery, for prophylactic antibiotic therapy, each rat received an i.m. injection (50,000 u) of benzylpenicilline(Specia, France). Postoperative recovery with false cables for habituation lasted at least 1 week in natural day/night lighting, the rats being individually housed in the recording room with free access to commercial rat chow and water. The ambient temperature was maintained constant at 230(2.

Experimental Procedure Four recording channels were used to characterize SWS, IS, and PS stages: (a) interhemispheric bipolar recording of the frontal cortex; (b) a bipolar recording of the left dorsal hippocampus (CA1 area) for theta rhythm; (c) a bipolar electrooculogram from the right eye orbit; and (d) a bipolar electromyogram from the dorsal neck muscles. Each morning the rats were connected to recording cables and were recorded on the EEG apparatus (Alvar, France) at a speed of 10 or 15 mm/s during 6 h in the daylight period (from 10:00 a.m. to 16:00 p.m.). The stimulations (100 Hz, rectangular pulses of 100 #S during 2 s) of increasing intensity were delivered in a given behavioral stage at irregular time intervals. After spontaneously induced sleep, the animals were stimulated during well-established SWS and PS outside the rapid eye movement periods, up to behavioral arousal, and during IS (characterized by the association of cortical spindles and hippocampal theta rhythm), IS being chosen following SWS and prior to PS. Each rat was stimulated during several days to obtain at least ten values for each stage. At the end of the experiments, the animals were sacrified by barbiturate overdose given in two steps and the position of deep electrodes was verified histologically. Statistical tests were carried out by means of the two-tailed Student's t-test to compare the EEG parameters during the sleep stages and the arousal threshold, preceded by the Snedecor Ftest for the arousal threshold. RESULTS The intermediate stage was characterized in nine rats by cortical spindles of higher amplitude than during SWS (559 ± 61

vV vs. 369 --- 30, mean ___ SD, p < 0.05). Their duration was larger during IS (1.89 ___0.19 s vs. 0.99 ± 0.07,p < 0.001) (Table 1). The theta rhythm was also of lower frequency than during PS outside the eye movement periods (7.2 ± 0.2 c/s vs. 7.8 ± 0.1, p < 0.01) (Table 2). As shown in Table 3, the arousal threshold quantified in eight rats was slightly higher during IS as compared to SWS (77 ± 22 #A vs. 61 ± 22, mean ± SD, p < 0.03; F(2, 14) = 21.09, p < 0.01), and much higher during PS than during IS (132 ± 45 #A vs. 77 ± 22, p < 0.001). DISCUSSION The intermediate stage in rats, cats, and mice is characterized by simultaneous EEG activities of SWS (cortical spindles) and PS (hippocampal theta rhythm). However, these pattern properties differ from those in SWS and PS. The duration and amplitude of spindles is higher than during SWS and the theta rhythm intrinsic frequency is lower than during the basic activity of PS, i.e., outside the eye movement bursts hypersynchronization. Thus, IS is a sleep stage with EEG-specific characteristics. The high amplitude spindles presumably occur because the thalamic reticular nucleus, which is the generator of spindles (30), shows highly active neurons during the transition from SWS to PS (22), and the theta rhythm seems to appear because the median rapbe TABLE 2 FREQUENCYOF HIPPOCAMPALTHETARHYTHM(c/s) Rat 1 2

3 4

5 6 7 8 9 Mean ± SD *p < 0.01.

Intermediate Stage

Paradoxical Sleep

7.6

8.2

7.0 6.7 7.3 7.2 7.5 6.3 8.0 7.1 7.2 ± 0.2

8.0 7.8 8.2 7.5 7.9 7.2 7.5 8.2 7.8 + 0.1"

AROUSAL THRESHOLD

201

TABLE 3 AROUSAL THRESHOLD BY STIMULATIONOF THE MIDBRAIN RETICULAR FORMATION (pA) Rat

Slow Wave l~leep IntermediateStage ParadoxicalSleep

1

40 45 70 35 90 150 35 40 75 75 85 125 75 90 210 65 85 150 75 85 125 90 95 150 61 ± 212 77 ± 22 132 ± 45 = ..... ~, < 0.03 ...... ~ ~ ..... - p < 0.001 .....

2 3 4 5 6 7 8 Mean± SD Student's ~test

nucleus, whose lesion induces continuous theta rhythm during waking (23), becomes silent prior to PS (27). The high amount of low-frequency theta rhythm (associated with cortical spindles) of the acute intercollicular transected rat and cat, without brainstem-ascending activating influences, is suppressed after midhypothalamic transection (10). Consequently, the theta rhythm of IS might occur through the disinhibition of a posterior hypothalamic trigger (2,28,34). The slightly higher frequency during PS

without eye movements is certainly related to brain-stem-ascending influences, which are probably issued from the nucleus reticularis pontis oralis (33). This theta rhythm is suppressed by atropine (13). Thus, it is "atropine sensitive," following Vanderwolf's (32) terminology, like the theta rhythm occurring during PS outside the rapid eye movement bursts. It is already well established in cats (4) and rats (12,29) that the midbrain reticular arousal threshold is significantly higher during PS than during SWS. Our results show that during IS this threshold is intermediate, but much nearer to that of SWS than of PS. This result suggests that the brain stem activation, which is important during waking and decreases during SWS, further decreases during IS, while the pontine influences inducing the high arousal threshold, and the masseteric reflex's strong inhibition during PS (36) are still absent or only begin to appear. The result showing a greater proximity of IS to SWS is in opposition to neurochemical data, because atropine delays both IS and PS, which implies that these two stages have in common a muscarinic support (1). Further studies are necessary to know the precise functional relationship between IS, SWS, and PS. A recent study showed in rats that the acoustical stimulus arousal threshold is higher during well-established SWS and IS than during PS (25). However, as mentioned by the authors, peripheral stimulation is "particularly dependent of the experimental design (novelty of stimulus, degree of meaningfulness of stimulus, increase in stimulus strength, criteria for response)" (p. 476), which is less the case with central stimulation.

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