Conducting these experiments under anesthesia allowed us to make microinjections of sufficiently small volume to obtain the first to date map of the dorsomedial pontine sites that may play a key role in GABAergic and cholinergic triggering of REM sleep in the rat. We identified a region where both carbachol and bicuculline effectively triggered REMSLE with shortest latencies and a caudal extension of this region where only bicuculline was effective. We also determined that, at least in under urethane anesthesia, bicuculline more powerfully affected the duration of REMSLE than carbachol.
The animal model of REM sleep used in our study, urethane-anesthetized, paralyzed, vagotomized and artificially ventilated rat, allows one to repeatedly elicit REMSLE with small doses and volumes of carbachol from the same pontine region where carbachol injections in chronically instrumented, behaving cats were previously shown to produce a long-lasting state that was often indistinguishable from natural REM sleep (Baghdoyan et al., 1987; Hernández-Peón et al., 1963; Vanni-Mercier et al., 1989; see Baghdoyan, 1997; Kubin, 2001 for reviews). In urethane-anesthetized rats, dorsomedial pontine carbachol injections reliably elicit 2–4 min REMSLE that have four characteristics features: appearance of theta-like rhythm (3–5 Hz), cortical activation in the 6–12 Hz range, suppression of XII nerve activity, and reduction in the respiratory rate (Fenik et al., 2002, 2004, 2005b; Kubin, 2001; Rukhadze et al., 2008). Importantly, similar to natural REM sleep, noradrenergic locus coeruleus (LC) and ventrolateral pontine A5 cells cease firing during REMSLE (Fenik et al., 2002; Kubin, 2001). The hippocampal theta-like rhythm has a lower frequency range (3–5 Hz) in urethane-anesthetized rats than the theta rhythm (6–8 Hz) recorded in unanesthetized, behaving rats, but it has been shown that both originate from activation of a similar network and can be triggered by the similar stimuli under both conditions (Dringenberg and Vanderwolf, 1998; Vertes, 1984). Other rhythmic activities also can be expected to be slower under anesthesia than in behaving animals. Thus, the increased activation of the cortical EEG in the 6–12 Hz range that we determined to be characteristic of REMSLE is likely to correspond to a faster rhythm in behaving rats, such as cortical activation in the 20–30 Hz range that occurs during natural REM sleep in behaving rats (Campell and Feinberg, 1993). It is noteworthy that, in urethane-anesthetized rats, carbachol can elicit more than one type of cortical activation from the pontine reticular formation, including some effects that are not compatible with REM sleep. For example, our earlier study revealed that, under urethane anesthesia, carbachol elicits activation of cortical EEG and hippocampal theta-like activity from ventralmost regions of the nucleus pontis oralis, but the responses were different from the REMSLE, as defined in this study, because XII nerve activity, central respiratory rate and LC cell activity were increased, and cortical activation did not include increased power in the 6–12 Hz band (Fenik et al., 2005c). Importantly, the ability of pontine carbachol to elicit REMSLE is suppressed in urethane-anesthetized rats by activation of cells in the same posterior hypothalamic region the activation of which inhibits generation of natural REM sleep (Lu et al., 2007). Thus, the REMSLE activated from the dorsomedial pontine tegmentum by carbachol in urethane-anesthetized rats have many features in common with natural REM sleep and are likely to be generated by at least a subset of the same neurons that are responsible for the generation of natural REM sleep.
In experiments conducted under anesthesia, one can make relatively small-volume (10 nL in this study) microinjections when compared to experiments in behaving rats in which, for technical reasons, one needs to use considerably larger volumes (50–250 nL) (Bourgin et al., 1995; Deuveilher et al., 1997; Gnadt and Pegram, 1986; Marks and Birabil, 2001; Okabe et al., 1998; Pollock and Mistlberger, 2003; Sanford et al., 2003). Smaller injection volumes result in a smaller spread of the drugs, thereby improving the spatial resolution and minimizing any potentially confounding effects elicited from regions adjacent to the site of interest.
The concentration of carbachol used in this study (10 mm) was the same as in our earlier studies (e.g., Fenik et al., 2002, 2005b; Kimura et al., 1990; Lu et al., 2007; Rukhadze et al., 2008; Taguchi et al., 1992) because our present goal was to relate the location of bicuculline-sensitive sites to the location of carbachol-sensitive sites for which our earlier experiments indicated that 10 nL injections of 10 mm carbachol offer a satisfactory spatial resolution. At this concentration, carbachol may act as agonist for both muscarinic and nicotinic cholinergic receptors (Khan et al., 1994) and both actions could contribute to triggering and maintaining the REMSLE in this study.
We used two concentrations of bicuculline because little is known about effective concentrations of this drug in studies of REM sleep. The lower dose was used in selected experiments to test whether the drug could be effective in much lower doses than those used in earlier studies in chronically instrumented, behaving rats (Pollock and Mistlberger, 2003; Sanford et al., 2003) and at a concentration that is nearly certain to act specifically through GABAA receptors, as we discussed elsewhere (Fenik et al., 2005a). We found that 0.5 mm bicuculline could elicit mono-phasic and at least one bi-phasic REMSLE, thus strengthening our confidence about pharmacologic specificity of these effects. Bicuculline at the higher dose could both spread farther and produce effects with shorter latencies. We found this to be the case, but the latencies of the effects of carbachol were still consistently shorter than for bicuculline. This was likely due to an over twice larger molecular weight of bicuculline than carbachol. Since both compounds are positively charged, carbachol is expected to diffuse faster and be effective at longer distances than bicuculline. Our results with injections in the rostral part of the investigated region were consistent with the expected diffusion rate difference between bicuculline and carbachol. Based on microinjection experiments in vivo, one cannot unequivocally verify whether the enhanced effectiveness and ability of 2 mm bicuculline to elicit bi-phasic REMSLE were due to its wider diffusion. However, the findings that continuous iontophoretic administration of gabazine, a more selective than bicuculline antagonist of GABAA receptors, also produced long-lasting (up to 90 min) episodes of REM sleep-like state in rats (Boissard et al., 2002) suggests that the effects that we obtained with 2 mm bicuculline reflected its specific action on pontine GABAA receptors involved in the control of REM sleep.
We explored the dorsomedial pontine reticular region over a length of nearly 3 mm, whereas dorso-ventrally our microinjections covered 1.0–1.5 mm. The reasons for a limited dorso-ventral extent of our exploration were twofold. First, most of the available evidence points to the dorsal aspects of the medial pontine reticular formation as playing a key role in triggering of REM sleep. Second, as mentioned earlier, we previously determined that, at least within the rostral part of the explored region, carbachol injections into the ventral pontine reticular formation elicit in urethane-anesthetized rats an effect that includes generation of theta-like activity but otherwise significantly differs from REM sleep (Fenik et al., 2005c). Since our aim was to explore the sites where carbachol-elicited REM sleep-like effects and test the same sites with bicuculline, exploration of the ventral pontine reticular formation would not be compatible with the goal of our study.
REM sleep-like effects of carbachol in anesthetized versus chronically instrumented, behaving rats
Carbachol is relatively ineffective in enhancing REM sleep-like state in behaving rats (Boissard et al., 2002; Deuveilher et al., 1997; Gnadt and Pegram, 1986; Marks and Birabil, 2001; Okabe et al., 1998; Pollock and Mistlberger, 2003). Based on the earlier data demonstrating that carbachol is extremely effective in inducing REM sleep-like state in cats but not in rats, it has been suggested that there are fundamental differences between the pontine REM sleep-generating network in rats and cats (Baghdoyan, 1997; Boissard et al., 2002; Luppi et al., 2007). However, our experience from urethane-anesthetized rats has been that carbachol can effectively and repeatedly elicits REMSLE in this preparation. Our present data reveal that the region where carbachol is most effective is relatively small. This, combined with the evidence from behaving rats that pontine carbachol also has a wakefulness-promoting action (Bourgin et al., 1995; Marks and Birabil, 2001; Okabe et al., 1998), suggests that it may be difficult to selectively administer carbachol into an optimal site in chronically instrumented, behaving rats without producing additional REM sleep-opposing effects from adjacent regions. For example, a spread of carbachol following a 100 nL injection was estimated to be 1.0–1.5 mm (Gnadt and Pegram, 1986), whereas our present data show that the antero-posterior extent of the region from which carbachol elicits REMSLE is of the order of 0.5 mm. Anesthesia also may limit wake-promoting effects of carbachol, thereby facilitating its REMSLE-triggering action. Thus, as discussed elsewhere (Kubin, 2001), the reported differences in carbachol effectiveness between cats and rats do not necessarily point to fundamental species differences and instead may be of technical or quantitative nature. To date, only one specific neurochemical species difference in the role of pontine cholinergic mechanisms of REM sleep has emerged; whereas muscarinic type 3 receptors promote REM sleep in cats and mice (Goutagny et al., 2005; Sakai and Onoe, 1997), their action in rats opposes generation of REM sleep (Marks and Birabil, 2001). In our study, while we found an expected increase of REMSLE latency when carbachol microinjections were placed rostral to the most effective region (around B-8.80), the effectiveness of carbachol dropped abruptly with injections placed slightly caudal to that region. Such a sudden decrease of carbachol effectiveness was difficult to explain on the basis of increased diffusion distance (as discussed earlier) and suggested an REMSLE-opposing action of carbachol in the caudal half of the explored region.
Site-dependence of the effects of carbachol and bicuculline
We found that the sites from which bicuculline-elicited REMSLE spanned from the level B-8.72 to B-9.30, whereas carbachol could trigger REMSLE only from a rostral part of this region. Our analysis of REMSLE latencies relative to the antero-posterior location of the injection sites revealed that both drugs had a common region of highest sensitivity located near the levels B-8.72 and B-8.80. This most effective region encompasses the ventral part of the laterodorsal tegmental nucleus (LDTV) and the rostral portion of the SubCD of Paxinos and Watson (1997), or the SLD and adjacent reticular formation of Swanson (1998). The site also may be homologous to the peri-LCα region, a cholinoceptive REM sleep triggering region in cats (Vanni-Mercier et al., 1989). At the more caudal sites (B-9.16 and B-9.30), only bicuculline could elicit REMSLE, whereas carbachol was not effective. This bicuculline only-sensitive region includes the SubCA and the caudal portion of the SubCD (Paxinos and Watson, 1997).
Data from cats suggest that the REM sleep-promoting actions of carbachol and bicuculline converge at the level of some common neurons located within the dorsomedial pontine tegmentum, with GABAergic action being exerted downstream from the cholinergic actions (Xi et al., 2004). Injections of muscimol, a GABAA receptor agonist, at the same sites as carbachol abolished the carbachol-induced REM sleep when they were made prior to, or right after, carbachol injection. In contrast, bicuculline injections could induce REM sleep-like state even when preceded by injection of scopolamine, a muscarinic antagonist, which blocked the effects of carbachol (Xi et al., 2004). This result could be explained by assuming that bicuculline and carbachol acted on the same dorsomedial pontine neurons, but it could also be produced by bicuculline acting on separate, non-cholinoceptive pontine neurons capable of triggering of REM sleep-like state; such neurons could be additionally responsible for the maintenance of this state. Our data provide support for both possibilities because we found that neurons located near the LDTV and rostral part of SubCD had similarly high sensitivity to both drugs, but near the caudal part of SubCD and SubCA, bicuculline effectively produced long-lasting REMSLE but carbachol was ineffective. Our finding of bicuculline-sensitive but carbachol-insensitive sites may also explain why two studies in chronically instrumented, behaving rats in which carbachol was administered at relatively caudal levels reported ineffectiveness of carbachol and strong REM sleep-activating effects of bicuculline (Boissard et al., 2002; Pollock and Mistlberger, 2003).
REM sleep is characterized and identified by synchronized occurrence of postural atonia, eye movements, ponto-geniculo-occipital (PGO) waves, cortical activation and hippocampal theta rhythm, but lesion and microinjection experiments indicate that different parts of the dorsomedial pontine tegmentum may control individual aspects of this state (reviewed by Datta and MacLean, 2007). In our experiments, we regarded simultaneous occurrence of depression of XII nerve activity and activation of the cortical EEG as the key phenomenon indicating that an injection elicited a REMSLE. The use of different outputs or criteria may lead to different delineations of putative REM sleep-related regions. For example, pontine sites where carbachol elicits theta-like rhythm in anesthetized rats are distributed more widely than those identified as REM sleep-related in the present study (Fenik et al., 2005c; Vertes et al., 1993). In another recent lesion study in behaving rats (Lu et al., 2006), a site important for the generation of REM sleep-related theta rhythm in the cortical EEG was reported to be small and located in a dorsal part of the caudal pontine region, the pre-locus coeruleus area, that we have not explored in the present study. In the same study, lesions of a more ventral region which we found responsive to bicuculline, but not carbachol, resulted in REM sleep without atonia. In another study, pontine sites for the generation of PGO-like activity in anesthetized rats in response to 50 nL carbachol injections were also localized to a similar area (Datta et al., 1998). The region that we found to be sensitive to carbachol is located rostral to the regions that others have explored and identified as controlling individual phenomena of REM sleep. Conversely, we found that bicuculline could trigger multiple REM sleep-like events (atonia, cortical activation, theta-like rhythm) from at least a part of the caudal region that was previously implicated in generation of selected distinct phenomena of REM sleep.
Differential effects of carbachol and bicuculline on REMSLE duration
Based on the temporal pattern of cortical and hippocampal activation and duration of the effects of bicuculline, we identified REMSLE of two types, mono- and bi-phasic. The bi-phasic episodes always started with what appeared to be a mono-phasic episode that was then considerably extended from a typical duration of 2–4 min to as much as 38 min. The two successive phases of cortical and hippocampal activation during bi-phasic REMSLE suggested that bicuculline had two actions, one to trigger REMSLE similar to those triggered by carbachol and the other to maintain REMSLE once they were initiated. The durations of REMSLE elicited by 2 mm bicuculline at the levels from B-8.72 to B-9.30 were at least 5 times longer than those of the REMSLE elicited by either carbachol or bicuculline from more rostral or more caudal sites. Bicuculline 0.5 mm was also effective in this region, but the REMSLE it elicited were shorter than those obtained with 2 mm bicuculline. Thus, the effects of bicuculline on REMSLE duration were strongly dose dependent.
In contrast to bicuculline, all carbachol-induced REMSLE observed in the present study were short-lasting and of mono-phasic type. Even the longest episodes elicited by carbachol rarely exceeded 4 min and their duration minimally increased when the injections were placed at the sites yielding REMSLE with shortest latencies (Fig. 7). The contrast between the pattern and range of durations for the REMSLE elicited by carbachol and bicuculline suggests that pontine cholinergic activation triggers REMSLE, whereas antagonism of GABAA receptors can both trigger these episodes and control their duration. This view is supported by the findings that pontine injections of carbachol in behaving rats increased REM sleep by increasing the frequency of the REM sleep episodes but not their durations (Bourgin et al., 1995; Marks and Birabil, 2001; Okabe et al., 1998), whereas bicuculline effectively increased the duration of REM sleep episodes in chronically instrumented, behaving rats (Boissard et al., 2002; Sanford et al., 2003). What is less certain is whether carbachol’s inability to produce long-lasting REMSLE, as we found in this study reflects a unique feature of REM sleep control by pontine cholinergic activation. In a study with carbachol injections in unanesthetized, decerebrate rats, in which REMSLE were identified on the basis of a parallel suppression of activity in postural and cranial muscles and changes in respiratory rate, the average episode duration was 14.5 min (Taguchi et al., 1992). In another study in urethane-anesthetized, paralyzed and artificially ventilated rats, we occasionally observed REMSLE lasting up to 13 min when carbachol was injected multiple times into the same dorsomedial pontine site (Rukhadze et al., 2008). Thus, while the data with GABAA receptor antagonists (Boissard et al., 2002; Sanford et al., 2003, and this study) point to a major role of the removal of pontine GABAergic inhibition in the maintenance of REM sleep, the contribution of cholinergic activation to this aspect of the control of REM sleep in rats may vary with experimental and behavioral conditions.
In conclusion, we found that, in urethane-anesthetized rats, the pontine cholinergic/GABAergic REMSLE-generating region consists of two parts. The rostral part, LDTV and rostral SubCD, is sensitive to both carbachol and bicuculline, whereas the caudal part that includes the SubCA and caudal SubCD regions is sensitive to bicuculline only. We suggest that the rostral part is responsible for triggering of REM sleep whereas the caudal part is additionally involved in the maintenance of the state. During wake and slow wave sleep, neurons located in both parts are under GABAergic inhibition which may originate from both local (Maloney et al., 2000) and remotely located (Boissard et al., 2003) GABAergic neurons.