SEARCH

SEARCH BY CITATION

Keywords:

  • atonia;
  • GABAA receptors;
  • hippocampal activation;
  • hypoglossal motoneurons;
  • pons;
  • sleep

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Carbachol, a cholinergic agonist, and GABAA receptor antagonists injected into the pontine dorsomedial reticular formation can trigger rapid eye movement (REM) sleep-like state. Data suggest that GABAergic and cholinergic effects interact to produce this effect but the sites where this occurs have not been delineated. In urethane-anesthetized rats, in which carbachol effectively elicits REM sleep-like episodes (REMSLE), we tested the ability of 10 nL microinjections of carbachol (10 mm) and bicuculline (0.5 or 2 mm) to elicit REMSLE at 47 sites located within the dorsal pontine reticular formation at the levels -8.00 to -10.80 from bregma (B) (Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997). At rostral levels, most carbachol and some bicuculline injections elicited REMSLE with latencies that gradually decreased from 242 to 12 s for carbachol and from 908 to 38 s for bicuculline for more caudal injection sites. As the latencies decreased, the durations of bicuculline-elicited REMSLE increased from 104 s to over 38 min, and the effect was dose dependent, whereas the duration of carbachol-elicited REMSLE changed little (104–354 s). Plots of REMSLE latency versus the antero-posterior coordinates revealed that both drugs were maximally effective near B-8.80. At levels caudal to B-8.80, carbachol was effective at few sites, whereas bicuculline-elicited REMSLE to at least B-9.30 level. Thus, the bicuculline-sensitive sites extended further caudally than those for carbachol and antagonism of GABAA receptors both triggered REMSLE and controlled their duration, whereas carbachol effects on REMSLE duration were small or limited by its concurrent REMSLE-opposing actions.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Microinjections of cholinergic agonists, especially carbachol, into the dorsomedial pontine reticular formation have been extensively used to trigger rapid eye movement (REM) sleep-like state in behaving or decerebrate cats (reviewed by Baghdoyan, 1997; Datta and MacLean, 2007; Kubin, 2001). Pontine injections of other drugs, such as glutamate receptor agonists (e.g., kainic acid) (Onoe and Sakai, 1995) or GABAA receptor antagonists (e.g., bicuculline) (Xi et al., 1999a,b) also can effectively trigger REM sleep-like state. In cats, the most effective sites for carbachol have been localized to the peri-locus coeruleus alpha (peri-LCα) region (Vanni-Mercier et al., 1989; Yamamoto et al., 1990). Studies also suggested that triggering of REM sleep-like state involved an interaction between cholinergic and GABAergic mechanisms within the dorsal pontine reticular formation. For example, pontine cholinergic activation of REM sleep was found to be gated by stimulation of GABAA receptors, thus allowing REM sleep to occur in the absence of GABAergic inhibition but promoting wakefulness when the inhibition was present (Xi et al., 1999a,b, 2004). Supporting the concept of cholinergic–GABAergic interaction was also a microdialysis study in cats in which bicuculline increased acetylcholine release within the dorsomedial pontine reticular formation (Vazquez and Baghdoyan, 2004).

In rats, a site possibly homologous to the cat peri-LCα region has been localized to the sublaterodorsal nucleus (SLD) using iontophoretic application of bicuculline, gabazine or kainic acid (Boissard et al., 2002). The SLD (Swanson, 1998) corresponds to the dorsal subcoeruleus (SubCD) and subcoeruleus alpha (SubCA) nuclei in the atlas of Paxinos and Watson (1997) which we used in this study. However, carbachol injections into the dorsomedial pontine tegmentum in chronically instrumented, behaving rats were found to be less reliable and effective than in cats (Boissard et al., 2002; Deuveilher et al., 1997; Gnadt and Pegram, 1986; Okabe et al., 1998), whereas bicuculline injections proved to be very effective (Boissard et al., 2002; Pollock and Mistlberger, 2003; Sanford et al., 2003). This difference prompted suggestions that pontine REM sleep-generating mechanisms differ between cats and rats, with GABAergic mechanisms being more important in rats and cholinergic mechanisms in cats (Baghdoyan, 1997; Boissard et al., 2002; Luppi et al., 2007). Not supporting this concept, however, were the results with pontine carbachol injections in urethane-anesthetized rats in which the drug very effectively and repeatedly elicited REM sleep-like episodes (REMSLE) characterized by hippocampal and cortical activation, silencing of pontine noradrenergic neurons, depression of activity in XII motoneurons and slowing of the respiratory rate (Fenik et al., 2002, 2004, 2005b; Kubin, 2001; Kubin and Fenik, 2004; Lu et al., 2007; Rukhadze et al., 2008).

The concept that triggering of REM sleep by neurons in the dorsomedial pons involves an interaction between cholinergic and GABAergic mechanism implies that common, or closely anatomically apposed, neurons process the hypothesized convergence and interaction. One way to test this is to determine whether there is a close anatomical correspondence between the pontine sites at which cholinergic and GABAergic drugs control the generation of REM sleep. This, however, has not been done. Our goal was to map the distribution of the sites within the dorsomedial pontine reticular formation at which carbachol, or bicuculline, or both could elicit REMSLE. To achieve this, we tested the effects of microinjections of carbachol and bicuculline at multiple sites within the dorsomedial pons in an anesthetized rat, a preparation in which carbachol has a well-established ability to elicit REMSLE. Using small-volume injections (10 nL), we identified a region where both drugs elicited REMSLE most effectively and with shortest latencies, but we also found that bicuculline-sensitive sites were more widespread than those for carbachol, and that antagonism of GABAA receptors could both trigger REMSLE and control their duration, whereas carbachol effects on REMSLE duration were small. Preliminary results have been published (Fenik and Kubin, 2005).

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animal preparation

Experiments were performed on 21 adult male Sprague–Dawley rats (body weight: 395 ± 9.2 g [standard error, SE]) obtained from Charles River Laboratories (Wilmington, MA, USA). All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and followed the guidelines for the care and use of experimental animals established by the National Institutes of Health.

The animals were pre-anesthetized with isoflurane (2%) and then anesthetized with urethane (1 g kg−1, i.p., supplemented by 50 mg i.v. injections as needed). The trachea was intubated and a femoral artery and vein were catheterized for arterial blood pressure monitoring and fluid injections, respectively. The right XII nerve was cut and its central end placed inside a cuff electrode for recording (Fenik et al., 2001). The cervical vagi were cut to enhance XII nerve activity and eliminate its reflex modulation by pulmonary afferents. The head of the animal was placed in a stereotaxic holder, openings were made in the parietal bone on the right side, and the dura removed for inserting a drug-containing pipette into the pons and a recording electrode into the hippocampus. Two screws were attached to the skull (2 mm anterior and 2 mm to the right, and 3 mm posterior and 2 mm to the left, of the bregma [B]) to record the electroencephalogram (EEG). Hippocampal activity was recorded using an electrode made from two Teflon-insulated platinum wires (0.002″/0.004″ bare/coated diameter; A-M Systems, Carlsborg, WA, USA) with tips separated by 0.8 mm. The electrode was inserted into the hippocampus: 3.7 mm posterior to the bregma, 2.2 mm to the right from midline and 2.4 mm below the cortical surface.

The animals were paralyzed with pancuronium bromide (2 mg kg−1 i.v., supplemented with 1 mg kg−1 injections as needed) and artificially ventilated with a mixture of air and oxygen (30–60% O2) at a rate of 50–70 lung inflations min−1. After paralysis, the level of anesthesia was assessed by intermittently applying a pinch to the hind limb while recording the arterial blood pressure, EEG, hippocampal and XII nerve activity. Absence of pinch-induced changes in either the respiratory rate or XII nerve activity and only transient changes in blood pressure, EEG and hippocampal signals, similar to those before paralysis, indicated adequate level of anesthesia. Rectal temperature was maintained at 35.5–36.5 °C throughout the experiments. The end-expiratory CO2 (Columbus Instruments Capnograph, Columbus, OH, USA) was adjusted at the beginning of the experiment to maintain a steady respiratory modulation of XII nerve activity and then kept constant.

Electrophysiologic recordings

Electroencephalogram (bandwidth 0.5–100 Hz), hippocampal activity (2–20 Hz) and XII nerve activity (30–2500 Hz) were amplified with AC amplifiers (N101, Neurolog System; Digitimer, Hertfotdshire, UK). These signals were continuously monitored on an 8-channel chart recorder (TA-11; Gould Instruments, Valley View, OH, USA) and recorded using a 16-channel digital tape recorder (C-DAT; Cygnus Technology; Water Gap, PA, USA) together with tracheal pressure, end-expiratory CO2, arterial blood pressure and event markers. XII nerve activity was fed into a moving average circuit that had a time constant of 100 ms (MA-821 RSP; CWE, Inc., Ardmore, PA, USA).

Drug microinjections

Glass pipettes (A-M Systems) having tip diameters of 25–30 μm were filled with either carbamylcholine chloride (carbachol, 10 mm) or (−)-bicuculline methiodide (0.5 or 2.0 mm) in 0.9% saline (both obtained from Sigma, St. Louis, MO, USA). Pontamine sky blue dye (2%, ICN Biomedicals Inc., Aurora, OH, USA) was added to the carbachol solution to mark the injection sites. Ten nanoliters of either drug were injected over 10–20 s by applying pressure to the fluid in the pipette while movement of the meniscus was visually monitored with 2 nL resolution through a calibrated microscope.

Experimental protocol and data analysis

Once the animal was prepared for recording, first carbachol injection was made into the dorsomedial pontine reticular formation 1.1 mm lateral from the midline, 1.4 mm rostral from lambda and at depth 7.4 mm from the brain surface because we found this site to be reliably effective in our earlier studies (Fenik et al., 2002, 2004, 2005b; Kubin and Fenik, 2004; Lu et al., 2007). This injection was done to verify that the preparation was capable of generating REMSLE and was not analyzed. Then, at least one carbachol and one bicuculline injection were made at multiple pontine sites at 30 min intervals or at least 30 min after termination of any effects of the previous injection. To do this, a pipette containing one drug was removed and replaced with a pipette containing the other drug aiming at the same site. Since carbachol solution contained Pontamine sky blue, each site was marked with the dye. The distance between successively explored sites was 0.2–0.6 mm in rostro-caudal direction. The order in which the drugs were injected alternated from one site to the next, with up to four separate sites tested per experiment. The sites explored extended over nearly 3 mm rostro-caudally and less than a half of that distance medio-laterally or dorso-ventrally because, according to our earlier studies, the sites at which carbachol induces REMSLE occupy a longitudinal zone of the dorsomedial pontine tegmentum that has relatively limited medio-lateral and vertical extent (Fenik et al., 2005b,c; Lu et al., 2007).

The amplitude of XII nerve activity and central respiratory rate were measured from the moving average of XII nerve activity over 30–60 s periods centered around the peak of the evoked REMSLE and compared to the corresponding measurements obtained from a 30–60 s baseline period preceding the injection. The onset and offset of the effects were defined as the time when a 10% decrease from baseline XII nerve activity and a 50% recovery from the level of maximal depression occurred, respectively. REMSLE were defined as episodes of continuous depression of XII nerve activity and central respiratory rate that at least at their onset had distinct signs of cortical and hippocampal activation that we previously determined to be characteristic of REMSLE induced by carbachol in urethane-anesthetized rats (Fenik et al., 2002, 2005b; Kubin, 2001; Kubin and Fenik, 2004; Lu et al., 2007).

For spectral analysis of the EEG and hippocampal activity, the signals were digitized at 100 Hz sampling rate using Spike-2 software (Cambridge Electronics Design, Cambridge, UK). Spectral power in characteristic bands was then determined sequentially in 15 s intervals using a sleep scoring software (Somnologica; MedCare, Buffalo, NY, USA). The power of hippocampal signal was calculated in the theta range (3–5 Hz in urethane-anesthetized rats [Vertes et al., 1993; ]) and the EEG power in the 6–12 Hz range because carbachol-induced REMSLE have characteristic power increases in these bands (Fenik et al., 2005b; Lu et al., 2007). The peak values of the EEG and hippocampal powers during REMSLE were compared with baseline values. We previously reported that, in urethane-anesthetized rats, REMSLE may occasionally occur spontaneously (Rukhadze et al., 2008). Such spontaneous episodes have all features characteristic of the carbachol-induced REMSLE (typical pattern of hippocampal activation, depression of XII nerve activity and appropriate duration) but may occur without being preceded for at least 15 min by any intervention. It appears that multiple injections of carbachol at the same site increase the incidence of such spontaneous events and that some preparations may have a greater propensity for generation of spontaneous REMSLE than other, but otherwise it is not known how frequent such spontaneous episodes can be and what may facilitate their occurrence. In practical terms, episodes that start within seconds following an intervention leave little doubt that the intervention was the triggering factor, but when the effects occur with latencies of several minutes one needs to conduct additional tests to verify that the effect did not occur spontaneously. In this study, when REMSLE followed an injection with relatively long latencies (over 2 min), we repeated the injection of the same drug at the same site to confirm that the response was elicited again and had a similar latency. Therefore, the REMSLE described in this study represent only drug-elicited events. When more than one injection of the same drug was made at the same site and elicited a response, the properties of such response were averaged and it was reported as one. Thus, only one set of measurements is reported for the effects of carbachol and bicuculline at each injection site.

Histology

At the conclusion of the experiment, an additional dose of urethane was injected (1 g kg−1, i.v.) and the brain removed for histologic analysis. The brainstem was fixed in 10% phosphate-buffered formalin and cut into 50 μm sections in coronal plane. All sections containing Pontamine blue dye were serially mounted and stained with Neutral red to localize the injection sites. The number of slices multiplied by their thickness was used to determine the rostro-caudal coordinates of the injection sites. As an anatomical reference, we used the level where the ventral tegmental nucleus (VT) intersects with the medial longitudinal fasciculus. We chose this landmark because it is located adjacent to the investigated area (level B-8.72; Paxinos and Watson, 1997) and VT has a distinct appearance in Neutral red-stained sections.

Statistical analysis

After verification that the data were normally distributed, two-tailed Student’s t-test was used for statistical comparisons (SigmaStat; Jandel, Inc., San Rafael, CA, USA). Linear regression analysis was used to detect relationships between distinct parameters of the responses to carbachol and bicuculline, and its slope was considered significantly different from zero when P < 0.05. The variability of the means is characterized by the SE throughout the report. Differences between means were considered significant when P < 0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Extent of the explored area and general characteristics of bicuculline- and carbachol-elicited REMSLE

Bicuculline and carbachol were injected at 47 dorsomedial pontine sites located between the levels B-8.00 and B-10.80 (Paxinos and Watson, 1997). As described in our earlier publications and illustrated in Fig. 1a, the REMSLE triggered by carbachol were characterized by increased theta-like activity in the hippocampus, increased EEG power in the 6–12 Hz range, depression of XII nerve activity, decreased respiratory rate, and a small or no change of the arterial blood pressure (Fenik et al., 2005b; Kubin, 2001; Lu et al., 2007). For the 29 sites at which carbachol-elicited REMSLE, the latencies of the responses varied from 12 to 242 s (mean: 100 ± 15 s [SE]) and the episodes lasted 93–257 s (mean: 165 ± 8.5 s). Among the 32 sites at which bicuculline was effective, 2 mm bicuculline was injected at 25, and 0.5 mm bicuculline at the remaining seven sites. The latter concentration was used in selected experiments after we have determined the appropriate location of the most effective sites for bicuculline with the purpose to test whether a concentration and total dose much lower than those previously found effective in chronically instrumented, behaving rats (Pollock and Mistlberger, 2003; Sanford et al., 2003) can be effective in urethane-anesthetized rats. The latencies of the REMSLE elicited by bicuculline were generally longer than for carbachol and varied from 38 to 908 s (mean: 225 ± 35 s). Some of the bicuculline-induced episodes also lasted considerably longer than those elicited by carbachol, resulting in a much wider range of REMSLE durations, from 104 s to over 38 min. This wide range was due to the occurrence of two types of REMSLE elicited by bicuculline. At 10 of the 32 sites where bicuculline was effective, the bicuculline-induced REMSLE were nearly identical to those obtained with carbachol, they lasted 104–354 s (mean: 187 ± 24 s) and comprised an increase and then decline of the cortical and hippocampal activities that occurred parallel to the depression of XII nerve activity and decrease of the respiratory rate; we called such REMSLE mono-phasic. Fig. 1b shows a typical example of a mono-phasic REMSLE elicited by bicuculline from the same site from which carbachol elicited a similar REMSLE illustrated in Fig. 1a.

image

Figure 1.  Examples of short-lasting (mono-phasic) REM sleep-like episodes (REMSLE) elicited by carbachol (a) and 0.5 mm bicuculline (b) injected consecutively into the same pontine site located at the level B-8.72 from bregma. Both drugs elicited REMSLE characterized by similar hippocampal and cortical activations, depressions of XII nerve activity and reductions of the central respiratory rate. XII nerve activity is shown as the moving average (MA) of the signal, with each peak representing the magnitude of inspiratory activity in successive central respiratory cycles. The respiratory rate was calculated in successive 10 s intervals. The latency of the REMSLE elicited by bicuculline was characteristically longer than that elicited by carbachol (bicuculline was injected 240 s prior to the beginning of the trace in b), whereas the durations of the episodes were similar. The vertical lines show the onsets and offsets of both responses, as defined in the Methods. (c, d) Power spectra for cortical EEG determined under baseline conditions (thin line) and around the peak of the responses (thick line) for the carbachol- (c) and bicuculline-induced (d) REMSLE. The insets show the corresponding expanded power spectra (in logarithmic scale) for the 6–12 Hz range, where we observed characteristic increase during REMSLE.

Download figure to PowerPoint

At the remaining 22 effective sites, the bicuculline-induced REMSLE started with distinct peaks in the powers of cortical and hippocampal activity that were similar to those characterizing the mono-phasic episodes but then the episodes continued for a considerable period with a moderately elevated cortical power in the 6–12 Hz range that declined slowly until a response termination point. In 13 of such prolonged REMSLE, the power of hippocampal theta-like activity also remained elevated throughout the duration of the episode. As with the mono-phasic REMSLE, XII nerve activity was depressed and the respiratory rate was reduced throughout the duration of these prolonged episodes. We termed such episodes bi-phasic because they always started with a pattern similar to mono-phasic REMSLE and were then followed by a second phase of elevated cortical power in 6–12 Hz and depressed XII nerve activity. Fig. 2 shows a typical bi-phasic REMSLE. The durations of the bi-phasic REMSLE were from 325 s to 38 min (mean: 966 ± 100 s). Indeed, all REMSLE lasting more than 354 s started with an initial peak of cortical activation typical of the mono-phasic episodes followed by prolonged cortical activation of moderate magnitude. The depression of XII nerve activity and respiratory rate decrease were most prominent during the initial peak of the bi-phasic REMSLE (Fig. 2). During three of the 22 bi-phasic episodes, a second transient increase of cortical and hippocampal powers reminiscent of the mono-phasic REMSLE occurred superimposed on the partial cortical and hippocampal activations characterizing the second phase of the bi-phasic episodes. These additional peaks also were associated with a transient strengthening of depression of XII nerve activity and reduction of the respiratory rate. Together, these observations suggested that the first and second phase of the bi-phasic REMSLE represented related but distinct phenomena, and that an initial activation of a mono-phasic REMSLE was necessary for the occurrence of a bi-phasic episode.

image

Figure 2.  Example of a typical bi-phasic REM sleep-like episode (REMSLE) elicited by 2 mm bicuculline injection made at the level B-8.80 from bregma. Note the characteristic bout of cortical and hippocampal activation at the beginning of the episode followed by a prolonged period of moderately increased cortical and hippocampal powers and continuing depression of XII nerve activity and reduced respiratory rate. The respiratory rate was calculated in successive 15 s intervals. The two vertical lines show the onset and offset of the episode. (b, c) Power spectra for cortical EEG measured under baseline conditions (thin lines) and at two times during the episode (thick lines). The insets show corresponding expanded power spectra (in logarithmic scale) for the 6–12 Hz range in which power of cortical EEG is characteristically increased during REMSLE. The spectrum obtained during the initial phase (b) also shows prominently reduced power in the low frequency range and a prominent peak in the theta-like range (3–5 Hz). The last two changes are in this example relatively larger than in most REMSLE, whereas the increase in the 6–12 Hz range is typical. The power spectrum during the later stage of this REMSLE (c) shows the same features but they are of considerably lower magnitude. The periods from which these spectra were derived are marked by horizontal lines under the EEG traces.

Download figure to PowerPoint

Among the 22 bi-phasic REMSLE, 21 were elicited with 2 mm bicuculline and one with 0.5 mm bicuculline. The latter had a relatively short duration (354 s), but included a prolonged cortical and hippocampal activation beyond the initial peak similar to that in all other bi-phasic episodes and the site from which it was elicited was surrounded by many other sites that yielded clearly bi-phasic REMSLE. Of the 10 mono-phasic bicuculline-induced REMSLE, five were elicited with 2 mm bicuculline and the other five with 0.5 mm bicuculline. Further analysis of the distribution of the injection sites, latencies and durations of the episodes, presented in the subsequent sections, revealed that both the location of the injection site and bicuculline concentration determined the episode type.

In addition to REMSLE defined by their characteristic patterns of cortical and hippocampal activation, reduced XII nerve activity and decreased respiratory rate, carbachol, but not bicuculline, occasionally elicited transient cortical and hippocampal activations that were not accompanied by any XII nerve or respiratory rate changes. Four responses of this type were observed. They occurred with a very short latency (<10 s), lasted less than 60 s, and all were elicited from sites located within the caudal part of the investigated region. The low number of such responses in our present data set precluded a more detailed analysis of their properties and distribution of their sites of origin.

Site-dependence of the effects elicited by carbachol and bicuculline

Fig. 3 shows the distribution of all injection sites plotted on the closest standard cross-sections from a rat brain atlas (Paxinos and Watson, 1997). The effects produced by carbachol are shown on the left-hand side and those elicited from the corresponding sites by bicuculline on the right-hand side of each cross-section. At the most rostral level (B-8.00), all but one of the seven injections of carbachol-elicited REMSLE, whereas only one of the seven bicuculline injections was effective. At the next level (B-8.30), eight sites were tested and of those seven yielded REMSLE in response to carbachol and four in response to bicuculline. The trend towards improved effectiveness of bicuculline continued with more caudally placed injections, so that all but two bicuculline injections placed between the levels B-8.72 and B-9.30 were effective. In contrast to the increasing effectiveness of bicuculline, the effectiveness of carbachol declined at, and caudal to, the level B-8.80. Whereas at the level B-8.72, all but one of the 11 carbachol injections were effective, this proportion declined to five of nine at B-8.80 and to just one of five at B-9.16. Further caudally, none of the seven carbachol injections elicited REMSLE, but two produced transient activations described in the preceding section. Thus, based on the distribution of the effective sites, the rostro-caudal extent of the region from which carbachol-elicited REMSLE tended to be shifted rostral relative to the region where bicuculline was effective.

image

Figure 3.  Distribution of all 47 injection sites plotted on the closest standard brainstem cross-sections representing the indicated antero-posterior levels from bregma according to Paxinos and Watson (1997). The effects produced by carbachol are shown on the left-hand side and those elicited from the same sites by bicuculline by the symmetrically located symbols on the opposite side of each cross-section. Filled symbols represent injections that elicited mono-phasic episodes and dotted symbols show bi-phasic REMSLE as indicated (see text for the characteristics of the two types of episodes). Based on the distribution of the effective sites, the rostro-caudal extent of the region where carbachol elicited REMSLE was located rostral relative to the region where bicuculline was effective. The sites at which the long-lasting, bi-phasic REMSLE were elicited by bicuculline were located only caudal to B-8.30 and extended to the level B-9.30, whereas carbachol effectiveness decreased caudal to the level B-8.72. 7, facial motor nucleus; Gi, gigantocellular reticular region; IRt, intermediate reticular region; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LDTV, laterodorsal tegmental nucleus, ventral part; Mo5, trigeminal motor nucleus; PC, nucleus pontis caudalis; PO, nucleus pontis oralis; scp, superior cerebellar peduncle; SubCA, nucleus subcoeruleus, alpha; VT, ventral tegmental nucleus; VTx, the site of intersection between the VT and the medial longitudinal fasciculus.

Download figure to PowerPoint

Whereas all effective bicuculline injections placed at the levels B-8.00 and B-8.30 were mono-phasic, all injections of 2 mm bicuculline and one of 0.5 mm bicuculline made at the levels from B-8.72 to B-9.30 elicited bi-phasic REMSLE. Thus, the tendency for increased effectiveness of bicuculline at more caudal levels was reflected in both the increased proportion of effective injections and considerably longer duration of the episodes.

At the two most caudal levels explored (B-9.68 and B-10.80), the effectiveness of bicuculline was greatly diminished; only one out of the four injections made at these levels was effective, and it produced only a mono-phasic REMSLE even though the concentration used was 2 mm.

Dependence of the latency and duration of REMSLE on the rostro-caudal coordinates of the injection site

Fig. 4 shows the distributions of REMSLE latencies elicited by carbachol and bicuculline in relation to the antero-posterior coordinates of the injection site. To relate the anatomical representation of the injection sites shown in Fig. 3 to the plots shown in Fig. 4, the same symbols are used in both figures to designate different effects of carbachol and bicuculline, and the plots in Fig. 4 have sectors that correspond to the standard cross-sections in Fig. 3. For completeness, the sites from which no REMSLE were elicited are plotted at one latency value that is higher than any of the actually observed REMSLE latencies, 5 min for carbachol and 16 min for bicuculline (open symbols).

image

Figure 4.  Relationship between the latencies of the REMSLE elicited by carbachol (a) or bicuculline (b) and the antero-posterior coordinates of the injection sites. The plots are vertically divided into the levels corresponding to the standard sections shown in Fig. 3, and the symbols used to designate different effects are the same as in Fig. 3. Injections that did not elicit any effects are shown by open symbols at latencies 5 and 16 min for carbachol and bicuculline, respectively. Both carbachol and bicuculline elicited REMSLE with shortest latencies at the levels B-8.72 and B-8.80; at more caudal levels, carbachol became ineffective whereas the latencies of the REMSLE elicited by bicuculline were still short through the level B-9.30. The site of intersection between the ventral tegmental nucleus and the medial longitudinal fasciculus (VTx) was used as the anatomical reference point.

Download figure to PowerPoint

One feature apparent in both plots in Fig. 4 is that, at the rostral end of the investigated region (right-hand side), REMSLE latencies steadily decrease with more caudally placed injections. The distance–latency plots for both drugs tended point to a level around B-8.80 at which the latencies were shortest. At this level, the mean latency for the carbachol-elicited REMSLE was 30.0 ± 9.4 s (n = 5), and for the REMSLE induced by 2 mm bicuculline (all bi-phasic) it was 96.4 ± 14 s (n = 5). Even at this level where both drugs elicited REMSLE with shortest latencies, the latencies of the episodes elicited with 2 mm bicuculline were significantly longer than those for the REMSLE elicited by carbachol (P < 0.01).

To better define the antero-posterior coordinates of the region where both carbachol and bicuculline were most effective, as determined by the response latency, we applied linear regression analysis to the latency-injection site coordinate data for the REMSLE elicited by carbachol, 0.5 and 2 mm bicuculline from the sites located between the levels B-8.00 and B-8.80 (Fig. 5). This analysis revealed a statistically significant correlation between the latencies and the rostro-caudal location of the injection site for both drugs. Importantly, the separate regression lines for the REMSLE elicited with bicuculline at the two concentrations and carbachol converged and intersected around 0.3 mm caudal to the intersection point between the VT and the medial longitudinal fasciculus (VTx), corresponding to the level B-8.80 and suggesting that the injections placed at this level were particularly close to neurons responsible for the generation of REMSLE.

image

Figure 5.  Relationships between REMSLE latencies and the antero-posterior coordinates of the injection sites could be approximated with separate linear regressions for the episodes elicited by 0.5 and 2 mm bicuculline and carbachol. The plot shows data for effective injections made at, or rostral to, B-8.80. The vertical lines delineate the range of antero-posterior coordinates corresponding to the standard sections shown in Fig. 3, and the symbols used to designate different effects are the same as in Fig. 3. All three regression lines point to level B-8.80 as the region of highest sensitivity for both bicuculline and carbachol, and the differences in the slopes of the regressions are consistent with the diffusion rate being faster for carbachol than bicuculline. The site of intersection between the ventral tegmental nucleus and the medial longitudinal fasciculus (VTx) was used as the anatomical reference point. R, linear regression coefficient.

Download figure to PowerPoint

In contrast to a robust dependence of REMSLE latency on the antero-posterior level of the injection for the sites located at the levels from B-8.00 to B-8.80, we did not find an expected latency increase for carbachol injections placed at, and more caudal to, the level B-9.16 (note in Fig. 4a many ineffective carbachol injections at these levels and not a single site for which the latency would increase above the lowest levels observed at B-8.80). Thus, it appeared that, at these caudal levels, carbachol abruptly lost its effectiveness to elicit REMSLE. This was different from the effects of caudally placed bicuculline injections, for which many REMSLE with latencies as short as those obtained from the level B-8.80 were also elicited from sites located at the levels B-9.16 and B-9.30. At the more caudal levels, the drug was either ineffective or produced a REMSLE with a considerably longer latency (nearly 8 min).

Similar to the AP coordinates–latency relationships shown in Fig. 4, the plots of REMSLE duration versus AP coordinates shown in Fig. 6 indicated that the injections placed at the levels B-8.72 and B-8.80 were most effective for both carbachol and bicuculline because the durations of REMSLE elicited from these levels were longer than for the injections placed at either more rostral or more caudal levels. The carbachol-induced REMSLE elicited from the levels B-8.72 and B-8.8 lasted 194 ± 11 s (n = 15) and were significantly longer than those elicited from the levels B-8.00 and B-8.30 (mean: 132 ± 7 s, n = 13; P < 0.001) (Fig. 6a). For bicuculline (Fig. 6b), the corresponding AP coordinates–duration relationship was similar to that for carbachol. The mean duration of the REMSLE elicited by 2 mm bicuculline from the levels B-8.72 and B-8.80 was 1040 ± 130 s (n = 15), but it was only 135 ± 19 s (n = 4) for the effective injections at the levels B-8.00 and B-8.30 (P < 0.01). The latter was not different from the duration of the REMSLE elicited by carbachol from the same levels, which reflected the fact that bicuculline did not elicit any bi-phasic REMSLE from these rostral levels. It is also noteworthy that, at the levels B-8.72 and B-8.80, bicuculline effects on the duration and type of REMSLE were dose-dependent because all but one injection of 0.5 mm bicuculline elicited the shorter, mono-phasic REMSLE, whereas with 2 mm bicuculline all the REMSLE elicited from these levels were of the bi-phasic type.

image

Figure 6.  Relationship between the durations of the REMSLE elicited by carbachol (a) or bicuculline (b) and the antero-posterior coordinates of the injection sites plotted in the same format as in Fig. 4. The injections that did not elicit REMSLE are shown as yielding effects of zero duration (open symbols). Note that the REMSLE elicited by carbachol were 5–10 times shorter than many of those elicited by bicuculline, and that the longest-lasting REMSLE were elicited by either drug from the levels B-8.72 and B-8.80. The site of intersection between the ventral tegmental nucleus and the medial longitudinal fasciculus (VTx) was used as the anatomical reference point.

Download figure to PowerPoint

Since REMSLE latencies are expected to be lowest and the durations of the episodes longest near the most effective region, we tested whether REMSLE latencies and durations were inversely correlated. Fig. 7 shows linear regressions separately applied to the data points for the REMSLE elicited by carbachol and the two concentrations of bicuculline. The latencies and durations of the REMSLE elicited by carbachol and 0.5 mm bicuculline were weakly correlated, with the regression being statistically significant for carbachol only (P < 0.01). The regression line for 2 mm bicuculline was significant (P < 0.01) and much steeper than the other two lines owing to the fact that all the REMSLE elicited with latencies shorter than 6 min (thus from the sites located close to the most effective region) were of the bi-phasic type.

image

Figure 7.  Relationship between the latencies and durations of REMSLE elicited by carbachol and bicuculline. Linear regression analysis was separately applied to the data for mono-phasic REMSLE elicited by carbachol, REMSLE evoked by 0.5 mm bicuculline (all but one, mono-phasic), and REMSLE elicited by 2 mm bicuculline (all but five, bi-phasic). The symbols used to designate different effects are the same as in Fig. 3. The slopes of the regression lines for 0.5 mm bicuculline and carbachol were similar (the latter was statistically significant). These slopes were nearly flat compared to the regression line for 2 mm bicuculline. The reason for this was that all the REMSLE elicited with latencies shorter than 6 min (thus from the sites located close to the most effective region) were of the much longer-lasting, bi-phasic type. R, linear regression coefficient.

Download figure to PowerPoint

Magnitudes of the changes in XII nerve activity, respiratory rate, and cortical and hippocampal powers during carbachol- and bicuculline-elicited REMSLE

All these four distinct measures of REMSLE magnitude were changed similarly for carbachol and bicuculline during the episodes. During the 29 carbachol-induced REMSLE, XII nerve activity was depressed to 22.0 ± 2.2% of pre-carbachol level (P < 0.001), the respiratory rate decreased from 46.9 ± 1.1 to 34.4 ± 2.5 min−1 (P < 0.001), the power of hippocampal activity in the 3–5 Hz range increased by 565 ± 78% (P < 0.001) and EEG power in the 6–12 Hz range increased by 275 ± 25% (P < 0.001). For the 32 REMSLE elicited by either 0.5 or 2 mm bicuculline, XII nerve activity was depressed to 25.8 ± 2.4% of the baseline (P < 0.001), the respiratory rate decreased from 45.9 ± 1.2 to 34.5 ± 2.0 min−1 (P < 0.001), the power of hippocampal activity in the 3–5 Hz range increased by 663 ± 148% (P < 0.001) and EEG power in the 6–12 Hz range increased by 263 ± 28% (P < 0.001). Using Student’s t-test, we found no significant effect of the order of the injections (carbachol first or bicuculline first) on REMSLE parameters for either the magnitude or durations of the responses elicited by 10 mm carbachol or 2 mm bicuculline.

In contrast to a clear dependence of REMSLE latencies and durations on the coordinates of the injection site, the maximal decrements of XII nerve activity and respiratory rate, and the maximal increases of cortical and hippocampal powers in the characteristic bands were weakly related to the coordinates of the injection site (Fig. 8). Although all REMSLE-associated changes tended to be most prominent at the same levels, B-8.8 and B-8.72, where both carbachol and bicuculline were found to be most effective based on their response latencies and durations, the relationship between the coordinates of the injection site and the magnitude of the effect was not significant for any of the four outputs when tested separately for each drug. Similarly, the magnitude of depression of XII nerve activity, decrease of the respiratory rate, and the maximal increases of cortical or hippocampal powers were not different for carbachol and bicuculline at either concentration. Thus, based on the magnitudes of the changes during REMSLE, the episodes occurred in a nearly all-or-none fashion regardless of the injection site or the triggering drug.

image

Figure 8.  Magnitudes of the changes in XII nerve activity (a), respiratory rate (b), EEG power in the 6–12 Hz range (c) and hippocampal power in the 3–5 Hz range (d) during REMSLE elicited by carbachol and bicuculline shown in relation to the antero-posterior coordinates of the injection sites. Each graph shows combined data for all REMSLE elicited by either carbachol, 0.5 or 2 mm bicuculline. The symbols used to designate different drugs and effects are the same as in Fig. 3. There were only weak trends for the magnitudes of the changes to be larger at the levels B-8.72 and B-8.80, where the latencies were the shortest (see Figs 4 and 5), and for 2 mm bicuculline to produce larger effects than 0.5 mm bicuculline. The site of intersection between the ventral tegmental nucleus and the medial longitudinal fasciculus (VTx) was used as the anatomical reference point.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Technical considerations

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was supported by grant HL-47600. We thank Ms Tyana M. Singletary for assistance with histology.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Baghdoyan, H. A. Cholinergic mechanisms regulating REM sleep. In: W. J.Schwartz (Ed.) Sleep Science: Integrating Basic Research and Clinical Practice. Karger, Basel, 1997: 88116.
  • Baghdoyan, H. A., Rodrigo-Angulo, M. L., McCarley, R. W. and Hobson, J. A. A neuroanatomical gradient in the pontine tegmentum for the cholinoceptive induction of desynchronized sleep signs. Brain Res., 1987, 414: 245261.
  • Boissard, R., Gervasoni, D., Schmidt, M. H., Barbagli, B., Fort, P. and Luppi, P. H. The rat ponto-medullary network responsible for paradoxical sleep onset and maintenance: a combined microinjection and functional neuroanatomical study. Eur. J. Neurosci., 2002, 16: 19591973.
  • Boissard, R., Fort, P., Gervasoni, D., Barbagli, B. and Luppi, P. H. Localization of the GABAergic and non-GABAergic neurons projecting to the sublaterodorsal nucleus and potentially gating paradoxical sleep onset. Eur. J. Neurosci., 2003, 18: 16271639.
  • Bourgin, P., Escourrou, P., Gaultier, C. and Adrien, J. Induction of rapid eye movement sleep by carbachol infusion into the pontine reticular formation in the rat. Neuroreport, 1995, 6: 532536.
  • Campell, I. G. and Feinberg, I. A cortical EEG frequency with a REM-specific increase in amplitude. J. Neurophysiol., 1993, 69: 13681371.
  • Datta, S. and MacLean, R. R. Neurobiological mechanisms for the regulation of mammalian sleep–wake behavior: reinterpretation of historical evidence and inclusion of contemporary cellular and molecular evidence. Neurosci. Biobehav. Rev., 2007, 31: 775824.
  • Datta, S., Siwek, D. F., Patterson, E. H. and Cipolloni, P. B. Localization of pontine PGO wave generator sites and their anatomical projections in the rat. Synapse, 1998, 30: 409423.
  • Deuveilher, S., Hars, B. and Hennevin, E. Pontine microinjection of carbachol does not reliably enhance paradoxical sleep in rats. Sleep, 1997, 20: 593607.
  • Dringenberg, H. C. and Vanderwolf, C. H. Involvement of direct and indirect pathways in electrocorticographic activation. Neurosci. Biobehav. Rev., 1998, 22: 243257.
  • Fenik, V. and Kubin, L. Dorsomedial pontine injections of bicuculline and carbachol elicit similar REM sleep-like effects in urethane-anesthetized rats. Sleep, 2005, 28(Suppl.): A9.
  • Fenik, V., Fenik, P. and Kubin, L. A simple cuff electrode for nerve recording and stimulation in acute experiments on small animals. J. Neurosci. Methods, 2001, 116: 147150.
  • Fenik, V., Marchenko, V., Janssen, P., Davies, R. O. and Kubin, L. A5 cells are silenced when REM sleep-like signs are elicited by pontine carbachol. J. Appl. Physiol., 2002, 93: 14481456.
  • Fenik, V., Davies, R. O. and Kubin, L. Combined antagonism of aminergic excitatory and amino acid inhibitory receptors in the XII nucleus abolishes REM sleep-like depression of hypoglossal motoneuronal activity. Arch. Ital. Biol., 2004, 142: 237249.
  • Fenik, V. B., Davies, R. O. and Kubin, L. Noradrenergic, serotonergic and GABAergic antagonists injected together into the XII nucleus abolish the REM sleep-like depression of hypoglossal motoneuronal activity. J. Sleep Res., 2005a, 14: 419429.
  • Fenik, V. B., Davies, R. O. and Kubin, L. REM sleep-like atonia of hypoglossal (XII) motoneurons is caused by loss of noradrenergic and serotonergic inputs. Am. J. Respir. Crit. Care Med., 2005b, 172: 13221330.
  • Fenik, V. B., Ogawa, H., Davies, R. O. and Kubin, L. Carbachol injections into the ventral pontine reticular formation activate locus coeruleus cells in urethane-anesthetized rats. Sleep, 2005c, 28: 551559.
  • Gnadt, J. W. and Pegram, G. V. Cholinergic brainstem mechanisms of REM sleep in the rat. Brain Res., 1986, 384: 2941.
  • Goutagny, R., Comte, J. C., Salvert, D., Gomeza, J., Yamada, M., Wess, J., Luppi, P. H. and Fort, P. Paradoxical sleep in mice lacking M3 and M2/M4 muscarinic receptors. Neuropsychobiology, 2005, 52: 140146.
  • Hernández-Peón, R., Chávez-Ibarra, G., Morgane, P. J. and Timo-Iaria, C. Limbic cholinergic pathways involved in sleep and emotional behavior. Exp. Neurol., 1963, 8: 93111.
  • Khan, I. M., Yaksh, T. L. and Taylor, P. Ligand specificity of nicotinic acetylcholine receptors in rat spinal cord: studies with nicotine and cytisine. J. Pharmacol. Exp. Ther., 1994, 270: 159166.
  • Kimura, H., Kubin, L., Davies, R. O. and Pack, A. I. Cholinergic stimulation of the pons depresses respiration in decerebrate cats. J. Appl. Physiol., 1990, 69: 22802289.
  • Kubin, L. Carbachol models of REM sleep: recent developments and new directions. Arch. Ital. Biol., 2001, 139: 147168.
  • Kubin, L. and Fenik, V. Pontine cholinergic mechanisms and their impact on respiratory regulation. Respir. Physiol. Neurobiol., 2004, 143: 235249.
  • Lu, J., Sherman, D., Devor, M. and Saper, C. B. A putative flip-flop switch for control of REM sleep. Nature, 2006, 441: 589594.
  • Lu, J. W., Fenik, V. B., Branconi, J. L., Mann, G. L., Rukhadze, I. and Kubin, L. Disinhibition of perifornical hypothalamic neurones activates noradrenergic neurones and blocks pontine carbachol-induced REM sleep-like episodes in rats. J. Physiol. (Lond.), 2007, 582: 553567.
  • Luppi, P. H., Gervasoni, D., Verret, L., Goutagny, R., Peyron, C., Salvert, D., Leger, L. and Fort, P. Paradoxical (REM) sleep genesis: the switch from an aminergic–cholinergic to a GABAergic–glutamatergic hypothesis. J. Physiol. (Paris), 2007, 100: 271283.
  • Maloney, K. J., Mainville, L. and Jones, B. E. c-Fos expression in GABAergic, serotonergic, and other neurons of the pontomedullary reticular formation and raphe after paradoxical sleep deprivation and recovery. J. Neurosci., 2000, 20: 46694679.
  • Marks, G. A. and Birabil, C. G. Comparison of three muscarinic agonists injected into the medial pontine reticular formation of rats to enhance REM sleep. Sleep Res. Online, 2001, 4: 1724.
  • Okabe, S., Sanford, L. D., Veasey, S. C. and Kubin, L. Pontine injections of nitric oxide synthase inhibitor, L-NAME consolidate episodes of REM sleep in the rat. Sleep Res. Online, 1998, 1: 4148.
  • Onoe, H. and Sakai, K. Kainate receptors: a novel mechanism in paradoxical (REM) sleep generation. Neuroreport, 1995, 6: 353356.
  • Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordinates, Compact 3rd edn. Academic Press, San Diego, 1997.
  • Pollock, M. S. and Mistlberger, R. E. Rapid eye movement sleep induction by microinjection of the GABAA antagonist bicuculline into the dorsal subcoeruleus area of the rat. Brain Res., 2003, 962: 6877.
  • Rukhadze, I., Fenik, V. B., Branconi, J. L. and Kubin, L. Fos expression in pontomedullary catecholaminergic cells following REM sleep-like episodes elicited by pontine carbachol in urethane-anesthetized rats. Neuroscience, 2008, 152: 208222.
  • Sakai, K. and Onoe, H. Critical role for M3 muscarinic receptors in paradoxical sleep generation in the cat. Eur. J. Neurosci., 1997, 9: 415423.
  • Sanford, L. D., Tang, X., Xiao, J., Ross, R. J. and Morrison, A. R. GABAergic regulation of REM sleep in reticularis pontis oralis and caudalis in rats. J. Neurophysiol., 2003, 90: 938945.
  • Swanson, L. W. Brain Maps: Structure of the Rat Brain, 2nd edn. Elsevier, Amsterdam, 1998.
  • Taguchi, O., Kubin, L. and Pack, A. I. Evocation of postural atonia and respiratory depression by pontine carbachol in the decerebrate rat. Brain Res., 1992, 595: 107115.
  • Vanni-Mercier, G., Sakai, K., Lin, J. S. and Jouvet, M. Mapping of cholinoceptive brainstem structures responsible for the generation of paradoxical sleep in the cat. Arch. Ital. Biol., 1989, 127: 133164.
  • Vazquez, J. and Baghdoyan, H. A. GABAA receptors inhibit acetylcholine release in cat pontine reticular formation: implications for REM sleep regulation. J. Neurophysiol., 2004, 92: 21982206.
  • Vertes, R. P. Brainstem control of the events of REM sleep. Prog. Neurobiol., 1984, 22: 241288.
  • Vertes, R. P., Colom, L. V., Fortin, W. J. and Bland, B. H. Brainstem sites for the carbachol elicitation of the hippocampal theta rhythm in the rat. Exp. Brain Res., 1993, 96: 419429.
  • Xi, M. C., Morales, F. R. and Chase, M. H. A GABAergic pontine reticular system is involved in the control of wakefulness and sleep. Sleep Res. Online, 1999a, 2: 4348.
  • Xi, M. C., Morales, F. R. and Chase, M. H. Evidence that wakefulness and REM sleep are controlled by a GABAergic pontine mechanism. J. Neurophysiol., 1999b, 82: 20152019.
  • Xi, M. C., Morales, F. R. and Chase, M. H. Interactions between GABAergic and cholinergic processes in the nucleus pontis oralis: neuronal mechanisms controlling active (rapid eye movement) sleep and wakefulness. J. Neurosci., 2004, 24: 1067010678.
  • Yamamoto, K., Mamelak, A. N., Quattrochi, J. J. and Hobson, J. A. A cholinoceptive desynchronized sleep induction zone in the anterodorsal pontine tegmentum: locus of the sensitive region. Neuroscience, 1990, 39: 279293.