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Theme : DreamsPontine Injections of Nitric Oxide Synthase Inhibitor L-NAME Consolidate Episodes of REM Sleep in the RatShinichi Okabe, Larry D. Sanford, Sigrid C. Veasey and Leszek Kubin Current Claim: REM sleep episodes are consolidated in rats following microinjections of the nitric oxide synthase inhibitor, L-NAME, into dorsal mesopontine tegmentum.
The microinjection system consisted of a 30 gauge cannula that extended 1.0 mm beyond the guide and was connected to a 1 µl motor-driven Hamilton microsyringe with PE10 tubing. Sterile saline was used as a vehicle for all drugs. The solutions were freshly prepared before each use under sterile conditions and filtered through a sterile 1.2 µm filter. After filling the injection system, a small air bubble was introduced into the tubing to monitor the movement of the fluid during the injection, and the cannula was inserted into the guide. The animal was allowed 15-40 min to adapt to these initial procedures and a microinjection (120-200 nl; mean: 160 nl ± 6 (SE); n = 22) was performed over a 30 s period. The cannula was gently withdrawn 3-5 min after the injection and the recording was continued for 330 min ± 7 (SE) (nominally: 6 h). In each rat, at the conclusion of the series of studies, a dye (Pontamine Sky Blue) was microinjected using the same technique (100-200 nl). The animal was then sacrificed, its brainstem removed and processed histologically to determine the location of the injection site. Using standard criteria, four behavioral states were distinguished based on the visual scoring of the chart records in successive 10 s epochs and direct observation of the animal through a video system: wakefulness (W), slow wave sleep (SWS), REMS, and drowsiness (D). The relationship of the levels and quality of the signals (cortical and hippocampal EEG, EOG and EMG) to animal's behavior was first assessed in each animal during the adaptation session by a simultaneous observation of the records and the animal. Scoring of the records from subsequent sessions was aided by the comparative data gathered from the same animal in the first session. All scoring was done by one person who was not blind to the treatments, but did not anticipate the main result of this study which became apparent only after all the scoring was completed. Each 10 s epoch was scored according to the predominant state during this period. The epochs scored as D corresponded mainly to the periods of transition between quiet wakefulness and the initial stages of SWS that could not be unequivocally classified as either W or SWS. In individual rats, D amount ranged from 2% ± 0.9 (SE) to 14% ± 8.3 (SE) of the total recording time. The scoring information was entered into a data base (Microsoft Excel) programmed to generate hypnograms for each session and calculate the numbers and durations of individual episodes of each behavioral state and the percentage of recording time spent in each state. Statistical comparisons were performed using a repeated measures analysis of variance (ANOVA) followed by a paired t-test and referred to the effects of saline microinjections as a control. Standard error (SE) is used throughout the Results to characterize the variability of mean values.
Figure 1 Figure 2 While the statistical analysis of individual parameters determined over the whole duration of the recording sessions revealed few significant effects of the treatments, there were two qualitative changes in the sleep-wake pattern which occurred to a variable extent in all five animals in a treatment-characteristic manner. First, following microinjections of 50 mM carbachol, the animals exhibited a profound suppression of sleep, with one animal remaining awake for the subsequent 4.5 h (whereupon the recording session was terminated). Second, following L-NAME, long REMS episodes (e.g., longer than 1.5 min, as described in the next section) occurred in all animals more frequently than after any other treatment, whereas injections of 10 mM carbachol (and 2 mM carbachol, additionally performed in two rats) produced many short REMS episodes (e.g., shorter than 1 min). These qualitative changes are illustrated in hypnograms from one rat in Fig. 1, and are analyzed quantitatively in the next section. Since sleep parameters are not stationary within a 6 h recording session in an experimental design similar to ours (e.g., Imeri et al., 1994; Bourgin et al., 1995; see Table 1), additional analysis was performed with the recording session divided into three 2 h long periods. Table 1 shows the amounts of different phases of the sleep-wake cycle for different treatments within the successive 2 h portions of the recording sessions. For the amount of REMS, application of a two-way repeated measures ANOVA revealed highly significant effect of both the treatment, i.e., saline, L-NAME and two doses of carbachol (F = 8.35, p = 0.0029, df = 4,3), and the period within the recording session, i.e., three successive 2-hour long segments (F = 66.74, p < 0.0001, df = 4,2). The interaction between the treatment and session segment was, however, insignificant (F = 1.85, p = 0.13, df = 4,6). Subsequent individual comparisons revealed that the percentage of REMS was increased during the fifth and sixth hours following injection of L-NAME, when compared to the corresponding periods of the control recording sessions. The second significant difference was that carbachol at the high concentration (50 mM) significantly reduced the amount of REMS during the first 2 (but not the subsequent 4) h following the injection (Table 1). The amount of W tended to be increased at the expense of SWS and REMS also during the remaining 4 h of the recording session, but the effect did not reach statistical significance. In contrast, 10 mM carbachol tended to increase the amount of REMS during the third through sixth hours of the recording sessions (p < 0.15 and p < 0.08 for the two successive epochs, respectively; Table 1) and had no effect on the amount of SWS. A separate analysis of the changes in the mean duration of individual REMS episodes performed within the successive 2 hour long segments of recording revealed a significant increase following L-NAME during the third through sixth hours of the recording session. For example, during the last 2 h, the REMS episode duration increased from 56 s ± 13 in control to 98 s ± 12 (p < 0.006). In contrast, following 50 mM carbachol, the durations of both REMS and SWS episodes were reduced during the first 2 h of the recording session. The average REMS episode duration decreased from 71 s ± 13 in control to 36 s ± 11 (p < 0.012), and the average duration of SWS episode from 122 s ± 37 to 74 s ± 28 (p < 0.04). There was no effect of 10 mM carbachol on SWS amount or the duration of SWS episodes in any portion of the recording session. Changes in the duration of REMS episodes Since the total counts of REMS episodes were different following different treatments, we converted the rough distributions of episode durations to the distributions of the percentage amounts of REMS obtained through REMS episodes of different durations (Fig. 2, main histograms). With this transformation, the rightward shift towards long REMS episodes following L-NAME and the leftward shift following 10 mM carbachol became even more apparent. We performed two analyses of the significance of the effects of different treatments on the amount of REMS obtained through episodes shorter than 1.5 min (i.e., 90 s in the histograms of Fig. 2), and longer than, or equal to, 1.5 min. In the first analysis, we compared, on bin-to-bin basis, the distributions of REMS amount separately with the range of short and long episodes. For the bins within the 0-90 s range, there were significantly lesser amounts of REMS after L-NAME than after saline (p < 0.0061, df = 7, paired t-test). In contrast, within the 90-250 s range, the bin-to-bin amount of REMS was larger after L-NAME than after saline (p < 0.05, df = 16). Following 10 mM carbachol, the bins with short REMS episodes contained more REMS than following saline (p < 0.03, df = 7). In contrast, within the 90-250 s range, the amount of REMS was larger after L-NAME than after saline (p < 0.05, df = 16). Following 10 mM carbachol, the bins with short REMS episodes contained more REMS than following saline (p < 0.03, df = 7). Finally, there was a significant difference between L-NAME and 10 mM carbachol for the range covering short-episodes (p < 0.03, df = 7), and between L-NAME and 50 mM carbachol for the long episode range (p < 0.006, df = 16). The latter, however, was probably due to the overall reduced amount of REMS following 50 mM carbachol. On the other hand, the differences among saline, L-NAME and 10 mM carbachol did not result from the differences in the overall amount of REMS following these treatments because the amounts of REMS after these treatments across the whole recording session were very similar, 11.3%, 14% and 15.2%, respectively, and not significantly different (see the first section of the Results). Furthermore, similar differences between treatments were obtained in the analysis described here when either 1 min or 2.5 min was used as the cut-off line between the short and the long episodes. We chose to describe the data using a 1.5 min cut-off as it was an even number close to the median for the distribution of the rough numbers of REMS episodes after L-NAME. In the second analysis, we used a two-way repeated measures ANOVA to compare the total amounts of REMS obtained through short (< 90 s) and long (> 90 s) REMS episodes following different treatments. There was a significant effect of the treatment (F = 4.84, p = 0.0196, df = 4,3) and of the treatment x REMS episode length interaction (F = 4.84, p = 0.0277, df = 3,1). Subsequent individual comparisons revealed that, within the short-episode range, the rats obtained less REMS following L-NAME (3.0% ± 0.4) than after saline (4.9% ± 0.8; p < 0.03). The amount of REMS obtained in short episodes after L-NAME also was less than that after 10 mM carbachol (6.7% ± 1.0; p < 0.02). Within the long episode range, the amount of REMS obtained following L-NAME (11.0% ± 1.9) was significantly more than after saline (6.4% ± 1.8; p < 0.02). Importantly, when we performed these comparisons after having substituted the treatments, i.e., saline and L-NAME or L-NAME and 10 mM carbachol with the order in which these treatments were applied, which involved reversing the order in two animals, the first two comparisons yielded insignificant differences, and the significance of the third dropped to p < 0.042. Thus, the effects were not due to a gradually developing adaptation of the animals to the recording conditions. Finally, as with the bin-to-bin analysis, similar results were obtained when 1 min or 2.5 min were used as cut-off line between short and long REMS episodes. Thus, the REMS episode-prolonging effect of L-NAME could be detected for a relatively wide range of REMS episodes of intermediate durations. The same analysis applied to the distribution of durations of SWS episodes did not reveal any consistent effects of either L-NAME or 10 mM carbachol, as expected from the lack of any effects of these treatments on the total amount of SWS (Table 1). Owing to the greatly reduced total amount of SWS following 50 mM carbachol, the distribution of the durations of SWS episodes was not analyzed. Thus, the most prominent effect of L-NAME was to shift the distribution of REMS episode durations rightwards, towards longer episodes. This could occur secondarily to the reported general wakefulness-reducing (Bagetta et al., 1993; Dzoljic and de Vries, 1994; Dzoljic et al., 1996) and anxiolytic (Guimarães et al., 1994) effect of inhibition of NO synthesis, whose one manifestation could be a reduced probability of occurrence of spontaneous arousals terminating some REMS episodes. With this in mind, we have determined the fraction of REMS episodes terminated with a transition to W following saline and L-NAME injections. These ratios were 0.55 ± 0.11 and 0.54 ± 0.05, respectively, and not significantly different. Therefore, these data did not support the possibility that the prolongation of REMS episodes following L-NAME microinjections was the result of a reduced vulnerability of the animals to spontaneous, REMS-terminating arousals. Localization of the sites of microinjections
We found that the primary effect of L-NAME within the rat dorsal mesopontine tegmentum is REMS enhancing, and manifested as a prolongation of individual REMS episodes (consolidation of REMS), rather than an increase in overall REMS amount. It is particularly noteworthy that the effects of L-NAME on REMS were more robust than those of the low concentrations of carbachol. This appears to contrast with many earlier studies showing extremely powerful REMS-promoting effects of carbachol (see Jones, 1991 for review). However, most of these data have been obtained from studies in cats. In the fewer studies performed in rats, the effects of carbachol are less impressive (e.g., Gnadt and Pegram, 1986; Shiromani and Fishbein, 1986; Bourgin et al., 1995). The relatively weaker effect of carbachol in rats may be related to the recent finding that, in this species, only very low doses of carbachol effectively promote REMS, whereas higher doses induce wakefulness (Bourgin et al., 1995; see also Mastrangelo et al., 1994). In our study, the lower doses of carbachol used were in the upper regions of the range which had a significant REMS-promoting effect, whereas the higher doses used were similar to those promoting W at the expense of both SWS and REMS in the study of Bourgin et al. (1995). As in the latter study, we found significant sleep-reducing effects of the higher dose and a tendency towards an increase in REMS following the lower doses, including a higher frequency of occurrence of short REMS episodes following 10 mM carbachol than after control injections. Thus, our results with carbachol are consistent with those of Bourgin et al. (1995), and suggest that similar regions of the reticular formation were involved in the response to carbachol in both studies. NO release, presumably from cell bodies and terminals of the pontine cholinergic and NO synthase-containing neurons, can have complex pre- and postsynaptic effects (Zhuo et al., 1994; Garthwaite and Boulton, 1995). One well-documented effect of local administrations of NO synthase inhibitors in the CNS is a reduction in acetylcholine release (Prast et al., 1995; Leonard and Lydic, 1997). It appears that such a functionally anticholinergic effect in the pontine tegmentum should lead to a reduction in REMS. However, pontine microdialysis with an NO synthase inhibitor reduced the baseline acetylcholine level, rather than attenuate the state-dependent changes in acetylcholine release (Leonard and Lydic, 1997). Thus, it is possible that, in our rat experiments, L-NAME lowered the mean level of pontine acetylcholine to a point where its REMS-promoting effects prevailed over those promoting wakefulness (discussed above). This alone cannot explain the significant prolongation of REMS episodes observed in our study, and suggests a more specific contribution of NO to the mechanism of termination of REMS episodes. Such a contribution awaits further studies. One may hypothesize, however, that by controlling the baseline amount of acetylcholine released in the pons, NO may be capable of enhancing either REMS or wakefulness. Such a mode of action could be suitable for long-term, homeostatic regulation of sleep-wake cycle. Effects of NO other than its modulatory action on acetylcholine release may also be relevant for its ability to regulate REMS at the pontine level. For example, inhibition of NO synthesis may decrease the release of glutamate (see Prast et al., 1995 for references), dopamine and serotonin (e.g., Lorrain and Hull, 1993). A decreased release of serotonin could indeed lead to enhancement of REMS (Portas et al., 1996; Horner et al., 1997). Whether any of those modes of L-NAME action contributed to our result needs to be addressed in future experiments. The possibility that L-NAME acts as a muscarinic antagonist was also considered in other systems (Buxton et al., 1993). However, muscarinic antagonists such as atropine reduce REMS, including REMS episode duration (Shiromani and Fishbein, 1986; Arnaud et al., 1994; Imeri et al., 1994; Benington and Heller, 1995). In contrast, L-NAME in our study tended to increase, rather than reduce the amount of REMS. Thus, a direct antimuscarinic action of L-NAME seems unlikely. Regardless of the specific neurotransmitter systems involved, our results suggest that endogenous NO release in the pontine tegmentum contributes to the shortening of REMS episodes and, therefore, may be seen as a wakefulness (or arousal)-promoting mechanism. Such a function of NO in the pons would be consistent with the recently discussed arousal-promoting effects of NO in the thalamus (Pape, 1995). Moreover, the potential role played by NO in synaptic plasticity and memory consolidation (see Garthwaite and Boulton, 1995 for a review) may be functionally related to our finding suggesting that NO contributes to the regulation of REMS episode durations. The link here is the hypothesis that consolidated REMS episodes have a beneficial effect on learning and memory (Giuditta et al., 1995). Thus, the pontine acetylcholine and NO-containing neurons, through their diverging projections, may play an important role in these processes not only in thalamo-cortical and hippocampal pathways, but also at the pontine level. Conclusion
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