Theme : Dreams

Injection of 6-Hydroxydopamine into the Ventral Tegmental Area Suppresses the Increase in Arterial Pressure during REM Sleep in the Rat

Hiroyoshi Sei1, Keiko Ikemoto2, Ryohachi Arai2 and Yusuke Morita1
1Department of Physiology, School of Medicine, University of Tokushima, Tokushima, 770-8503, Japan and 2Department of Anatomy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Abstract
We have examined the effect of injection of 6-hydroxydopamine (6-OHDA) into the ventral tegmental area (VTA) on the changes in arterial blood pressure (AP) and heart rate (HR) during the transition from non-rapid eye movement (NREM) sleep to REM sleep. The 6-OHDA-treated rats showed suppression of the increase of AP and HR during REM sleep and of theta frequency in the cortical electroencephalogram (EEG) during wakefulness (W) and REM sleep. It is suggested that midbrain dopaminergic neurons are involved in the control of AP and HR during REM sleep and in the EEG theta activity.

Current Claim: The injection of 6-OHDA into the rat VTA suppresses the increase of AP and HR during REM sleep.

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Arterial blood pressure (AP) increases upon going from non-rapid-eye movement (NREM) sleep to REM sleep in the rat (Del Bo et al., 1982; Lacombe et al., 1988; Obal et al., 1994; Sei and Morita, 1996a, 1996b), rabbit (Lenzi et al., 1987), cat (Kanamori et al., 1995; Kanamori et al., 1994; Sei et al., 1989; Sei et al., 1994) and human (Mancia and Zanchetti, 1980). Observations in decerebrated cats indicate that forebrain structure is necessary for this to occur (Kanamori et al., 1995; Kanamori et al., 1994). It is, however, still unknown what mechanism in the brain plays the main role on the changes of AP and HR during REM sleep.
During REM sleep, several neural groups, such as noradrenergic, histaminergic and serotonergic neurons, show virtual cessation of firing (Hobson et al., 1998; Jones, 1994; Sakai et al., 1990). It is reasonably imagined that these groups are not directly involved in the AP increase during REM sleep, even if their disinhibitory effects play an important role on the AP change. In contrast, dopaminergic neurons are reported to maintain their firing rate throughout the sleep-wake cycle (Jacobs, 1985) and to be involved in AP control. Electrical and chemical stimulation of the ventral tegmental area (VTA), the mesolimbic dopaminergic system, causes an increase in AP (Cornish and van den Buuse, 1994, 1995). The aim of this study was to investigate the effect of injection into the VTA of the dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), on the change of AP and HR during REM sleep. As we have previously shown the existence of a correlation between cortical electroencephalographic (EEG) theta frequency and the change in AP during REM sleep (Sei and Morita, 1996a), the effect of 6-OHDA injection on the EEG theta frequency was also studied.

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Male Wistar rats (280-320g) were used and were kept under a 12-h light:dark cycle (light on, 06:00-18:00 h) and at a controlled ambient temperature (23±1°C). Food and water were available ad libitum.

Surgery

Under sodium pentobarbital anesthesia (50-60mg/kg. ip), the rats received bilateral injections of 6-OHDA (Sigma, 10 µg in 2 µl of saline containing 0.02% ascorbic acid, n=6) or vehicle (n=6) in the VTA, using the following coordinates with respect to the bregma: -5.3 mm AP, 0.8 mm ML, -8.2 mm DV (Paxinos and Watson, 1986). The solution was injected over 12 min at a rate of 0.17 µl/min, using a Harvard pump (model 1140-001), through a 26-gauge steel cannula connected to a Gastight syringe. The cannula was left in place for 5 min before slowly retracting it. The rats were then implanted with electrodes to record the cortical EEG (two round-tip miniature screws over the frontal and occipital cortex) and electromyogram (EMG, two stainless wire electrodes in the neck muscle). After a 2-week recovery period from brain lesion surgery, the rats were implanted with a telemetry device in order to record the AP. A thin catheter (0.7 mm outer diameter, 8 cm long) of a telemetric system (TA11PA-C4; Data Sciences Int. [DSI]) for AP recording was inserted into the descending aorta through its wall, and the transmitter was fixed in the peritoneal cavity.

Recordings

Following surgery, each rat was placed in a square plastic cage (length and width 30 cm, depth 35 cm) in a sound-attenuated and air-conditioned (23±1°C) room under the same light:dark cycle as before surgery. The rats were connected by a cable with a slip-ring to a polygraph to record the EEG and EMG, and a receiver for telemetric AP recording was placed under the cage. The signal from the AP transmitter was automatically calibrated and the ambient pressure was subtracted using a dual ambient pressure monitor (C11PR and UA10, DSI). Polygraphic recordings over 12h (6:00-18:00h) were made following a minimum 10-day period of recovery and adaptation from the second operation. The polygraphic signals were passed through an anti-aliasing (i.e., high cut 30 Hz) filter and digitized using a CED 1401 data processor (Cambridge Electronic Design Ltd. [CED]) at a rate of 100 Hz for all channels, then stored on hard disk.

Data analysis

Off-line data analyses were performed with a Spike 2 analyzing program (CED). The mean AP (MAP) was calculated as the average of the consecutive 100 digitized AP points, that is, in the 1-s epochs, including 5-6 AP beats. As we focused on alteration of the AP variability accompanying the change of vigilance stage, we did not analyze the systolic and diastolic AP values in this study. Heart rate (HR) was determined from the AP signal. Systolic peak points of the AP signal were detected and its interbeat-interval was measured by the Spike 2. HR was then calculated as a reciprocal of the mean interbeat-interval in the consecutive 1-s epochs. In this report, we paid particular attention to the changes in the MAP and HR during successive periods of W, NREM and REM sleep. The MAP and HR were averaged for each sleep stage in each rat. Furthermore, in order to observe the changes in MAP and HR during the transition from NREM to REM sleep and from REM sleep to wake, the MAP and HR were averaged for the period from 1 min before to 1 min after the onset and end of REM sleep respectively. For this time series analysis, as the sample interval of 1 sec made a too large data size to test the significance by analysis of variance (ANOVA), MAP and HR were averaged over every 5 sec.

The mean frequency of the theta rhythm in the cortical EEG during W and REM sleep was obtained by frequency analysis of 1026-point fast Fourier transformation and was calculated as follows:

mean frequency of theta rhythm = (p f)/ p

p: power spectral density (PSD)

f: frequency (4.0 f 12.0)

In order to exclude any contribution from incomplete REM sleep, data for REM sleep lasting less than one min and interrupted by short episodes of NREM sleep or W were excluded from the analyses.

Histology

Under deep sodium pentobarbital anesthesia (100-120 mg/kg, ip), each rat was perfused through the left cardiac ventricle with saline, followed by 0.1 M phosphate buffer containing 4% paraformaldehyde, then the brains were immediately removed and postfixed with the same fixative overnight. They were then rinsed in 0.1 M phosphate buffer containing 15% sucrose and 0.1% sodium azide at 4°C for 3-4 days. Thirty µm thick brain sections at the level of midbrain were cut using a cryostat in the coronal plane, and tyrosine hydroxylase (TH) immunohistochemistry performed on the sections; the details are provided elsewhere (Ikemoto et al., 1997).

Statistical analysis

The Mann-Whitney U-test was used to compare average values of MAP, HR and EEG theta frequency during each sleep stage between 6-OHDA- and vehicle-treated rats. The time course changes in the MAP and HR were assessed by ANOVA. Fisher's PLSD test was used for post-hoc comparison between the two groups. A p-value of less than 0.05 was considered to indicate statistical significance.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 1 shows examples of the histological results. In the 6-OHDA-treated rats, VTA TH immunoreactive neurons were almost absent in comparison with vehicle-treated rats. Neuron loss in the 6-OHDA-treated rats appeared to extend to the antero-medial part of substantia nigra. However, on visual inspection, during recording sessions, the behavior of the 6-OHDA-treated rats seemed not to be impaired.
Examples of polygraphic recordings from 6-OHDA- and vehicle-treated rats are shown in Fig. 2. In vehicle-treated rats, the AP showed a tonic increase with several superimposed phasic surges during REM sleep, while in 6-OHDA-treated rats, it showed a tonic slight decrease and no phasic events. In vehicle-treated rats, the HR showed a great variation during REM sleep, while in 6-OHDA-treated rats, it showed no large phasic events.

Averaged values for MAP, HR and mean frequency of the cortical EEG theta rhythm during each sleep stage in the vehicle-treated (n=6) and 6-OHDA-treated (n=6) rats are summarized in Fig. 3. Table 1 shows the number of episodes and the averaged duration of each sleep stage used for the analyses of the MAP, HR and EEG theta rhythm. REM duration in the 6-OHDA-treated rats was significantly longer than that in the vehicle-treated rats. As shown in Fig. 3, in the vehicle-treated rats, the MAP during REM sleep was significantly higher than in NREM sleep, while the converse was the case in 6-OHDA-treated rats (p<0.05 by Wilcoxon rank test, not marked in the figure). A significant difference in MAP between the two groups was only seen during REM sleep. Average HR during REM sleep also tended to be lower in 6-OHDA-treated rats than vehicle-treated rats, although it did not reach the significance level (p=0.23). EEG theta frequency in the 6-OHDA-treated rats was significantly lower than in the vehicle-treated rats during both W and REM sleep.

The averaged values for the time course changes in the MAP and HR during the transition from NREM to REM sleep and from REM sleep to W are shown in Fig. 4. Table 2 exhibits the results of ANOVA for the data in Fig. 4. As indicated by the ANOVA results (f values between groups), the difference in both MAP and HR between the two groups was greater in the later part of REM sleep than in the earlier part. In the 6-OHDA-treated rats, the increase in MAP was suppressed throughout almost the whole of REM sleep, while the HR was only suppressed significantly around the end of REM sleep. MAP and HR in the vehicle-treated rats gradually increased during REM sleep, while little change was seen in the 6-OHDA-treated rats.

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The present study shows that injection of 6-OHDA into the VTA suppressed the increase of AP and HR during REM sleep and the frequency of EEG theta rhythm during both W and REM sleep. The average in both MAP and HR during W and NREM sleep was not affected.
Although 6-OHDA is also thought to destroy other catecholaminergic neurons and fibers (Stein and Wise, 1971), the dopaminergic neurons are the most likely candidates for involvement in the AP change during REM sleep, as, of the catecholaminergic neural groups, only dopaminergic neurons are reported to function during REM sleep (Jacobs, 1985). For example, noradrenergic neurons in the locus coeruleus stop their firing during REM sleep. The present findings, therefore, indicate that the midbrain dopaminergic system is involved in the changes of AP and HR during REM sleep in the rat.

The duration of REM sleep in the 6-OHDA-treated rats was significantly increased. It has been reported that dopamine is also involved in the regulation of REM sleep duration. Dopamine D1 receptor antagonists increase the amount and duration of REM sleep, whereas D1 agonists suppress REM sleep (Trampus et al., 1991; Trampus and Ongini, 1990). Therefore, the increase of REM sleep duration in the 6-OHDA-treated rats is thought to be caused by the midbrain dopamine depletion.

We have previously reported that acceleration of the cortical EEG theta wave precedes the phasic surge in AP during REM sleep (Sei and Morita, 1996a). As for hippocampal theta wave, it has been reported that the central noradrenergic system is not essentially involved in the determination of the theta frequency (Kolb and Whishaw, 1977; Robinson et al., 1977). As, in this study, VTA 6-OHDA injection also causes a decrease in EEG theta frequency, and the cortical EEG theta rhythm during REM sleep in the rat has been reported to show synchrony with that from the hippocampus, it is suggested that midbrain dopaminergic neurons are involved in the hippocampal theta activity. However, at present, it is completely unknown whether the suppression of the changes in the AP and HR during REM sleep are caused by the suppression of the limbic (including hippocampus) activity, indicated by the lowered frequency of theta rhythm, or by the loss of direct or indirect descending signals from, or via, the VTA dopaminergic system.

VTA dopaminergic neurons (A10) are implicated in motivated behavior and/or initiation of action (Le Moal and Simon, 1991). It has been reported that the lesions of the midbrain dopaminergic system severely impair behavioral arousal (Jones et al., 1973). Furthermore, in Parkinson's disease, there is a marked impairment of psychomotor function. Oneiric behavior (Sastre and Jouvet, 1979; Soh et al., 1992) during REM sleep, observed in the cat with a pontine tegmental lesion, suggests the possibility that the central motor system actively functions during REM sleep, although the actual behavioral action is inhibited by muscle atonia. It has been reported that AP and HR increase during exercise or voluntary movement, and that the cardiovascular sympathetic outflow increases immediately before, or concomitantly with, the onset of exercise and voluntary movement (Matsukawa et al., 1991; Matsukawa and Ninomiya, 1987; Vissing and Hjortso, 1996). These findings indicate that, during behavior, descending autonomic activation originates from the higher nervous system (central command), in order to adjust the blood supply for exercise or movement. Thus, it can be hypothesized that, during REM sleep, AP is increased by central command in parallel with the activated motor system without any actual action, and that the midbrain dopaminergic system is involved in this central command to the AP. Observation of the autonomic function during oneiric behavior and/or the temporal relationship in the phasic events between AP and muscle twitches during REM sleep may provide us with additional information on the relationship between AP control and motor function during REM sleep.

As shown by the EEG theta frequency, the loss of VTA catecholaminergic neurons and fibers affects hippocampal function during both W and REM sleep. However, the average MAP and HR during W are not affected, suggesting that other neural groups, such as catecholaminergic neurons outside the VTA, histaminergic or serotonergic neurons, may compensate for the loss in VTA catecholaminergic function, thus maintaining the AP at the same level as in intact rats. Midbrain dopaminergic neurons are suggested to be involved in the control of AP and HR, their role being particularly evident during REM sleep.

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The authors do not wish to include any acknowledgments at this time.

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