Are nicotinic receptors involved in regulating REM sleep architecture of the rat?
Nathaniel C. Charles, A.A., Rafael de Jesus Cabeza, PhD.
Department of Biological Sciences
The University of Texas at El Paso
El Paso, TX 79968
Correspondence: Nathaniel C. Charles
Dr. Rafael Cabeza
Department of Biological Sciences
The University of Texas at El Paso
El Paso, TX 79968
Using the rat as a model, we studied the effects of nicotine and chlorisondamine, a nicotinic receptor antagonist, on REM sleep. The purpose behind these experiments was to try and understand if nicotinic receptors play a physiological role in regulating REM sleep architecture. Nicotine showed a bi-modal dose response effect (dose range from 0.03 to 1 mg/kg) with 0.1 mg/kg resulting in a higher REM sleep percentage and 1 mg/kg resulting in a decrease. Nicotine primarily affected the average REM sleep bout duration, though a small effect was noted on REM sleep bout frequency.
The experiments are not yet completed and no data is available on the effects of chlorisondamine on baseline levels of REM sleep or the ability of chlorisondamine to block the effects of 0.1 or 1.0 mg/kg nicotine. The effects of chlorisondamine on REM sleep deprivation recovery or immobilization recovery are also not yet available for analysis.
Rapid eye movement sleep (REM sleep) is a recurrent state that occurs during the sleeping phase of most mammals, with both REM sleep bout duration and the frequency of REM sleep bouts being specific to the species (13). The architecture of REM sleep in the rat has been modeled by a stochastic model and shows a complex structure even under baseline sleeping conditions (10). The architecture of this state can easily be affected by drugs and stressful conditions, such as REM sleep deprivation or through immobilization stress (in the rat) (1, 16, 24). It is not yet clear how the centers regulating REM sleep are altered to affect these observed changes.
Three pontine nuclei believed to regulate and establish REM sleep have been identified (12). These centers, the dorsal raphe (DR), the locus coeruleus (LC), and the laterodorsal tegmentum (LDT) appear to function in a reciprocal fashion to establish the normal REM sleep rhythm and architecture (12). According to the Hobson-McCarley model, activity by the cholinergic LDT neurons leads to the expression of REM sleep but the serotonergic DR and the noradrenergic LC neurons inhibit neuronal firing by the LDT. Normally, the DR and LC suppress the expression of REM sleep but these centers are purported to send collaterals that inhibit their own activity. When these two groups of neurons become silent, those of the LDT are able to gain activity and initiate and maintain REM sleep. The LDT neurons are believed to send excitatory projections to the DR and LC, such that, as the LDT neurons remain active they re-activate those of the DR and LC, thus leading to a renewal of their inhibition and the termination of REM sleep (see figure 1 for a schematic diagram of these connections). Though several aspects of this model have been confirmed and some of the receptors affecting the activity of these three groups of neurons have been studied, much remains to be elucidated in this model. In particular, the role of nicotinic receptors present in these nuclei has not been thoroughly studied and their role in regulating the REM sleep structure is not well understood (6).
Studies of the effects of nicotine on sleep have not helped to clarify this issue. It is known that smoking of tobacco or the use of the nicotine patch has an alerting effect that suppresses sleep in general and that when smokers attempt to quit smoking they have an altered REM sleep pattern (4, 9, 19, 20,-22, 25). Furthermore, work by Velazquez-Moctezuma and colleagues have shown that injection of nicotine into the pedunculopontine tegmentum increases REM sleep, though not to as large an extent as carbachol, a general cholinergic agonist (26). Chlorisondamine is a non-competitive nicotinic channel blocker with very prolonged blocking capabilities in the central nervous system (over 80 days following a single dose)(2, 3, 5-8, 14-15, 17-18, 23, 27). This compound has been found to block both behavioral and neurochemical effects of nicotine on the brain (3, 5, 7, 17-18, 27). However, work in this laboratory, using chlorisondamine has found that this antagonist has no effect on the architecture of baseline REM sleep (unpublished results). These findings have failed to clarify the role of this nicotinic receptor (most likely the a4b2 subtype) in regulating REM sleep. It is possible that these receptors play a highly limited role in normal REM sleep or that their role is more important in REM sleep responses to stress. It is even possible that these receptors are only of pharmacological importance and that they play no role in the regulation of REM sleep architecture. Therefore, we propose investigating the role of nicotinic receptors in the response of REM sleep to stress.
Materials and Methods
Eleven male Sprague-Dawley rats weighing between 400-600 grams (approximately 7 months old) and raised in the local vivarium were used for this study. The animals were housed individually with a 12L: 12D lighting schedule (lights on at 06:30) with food and water available ad libidum. Animals were handled on a daily basis to reduce stress and they were given a sweet cereal as a treat at the end of each handling period.
In order to implant electroencephalogram (EEG) and electromyogram (EMG) electrodes, the animals were first anesthetized with an intramuscular injection of 2 ml/kg of a cocktail containing, 6.3 mg/ml xylazine, 2.5 mg/ml acepromazine maleate, and 25 mg/ml ketamine HCL. Four screw EEG electrodes (1 frontal, 2 parietal) (Plastics One, Roanoke, VA), one screw reference electrode (1 occipital), and two EMG electrodes (Plastics One, Roanoke, VA) (threaded into the nuccal muscles) were permanently implanted. An intracerebro ventricular (I.C.V.) 22 gauge guide cannula was stereotaxicaly placed into the right lateral ventricle (AP: -.4, ML: -1.4, DV: -3.6 from Bregma). The electrode ends were secured to a six-electrode pedestal (Plastics One, Roanoke, VA) and the whole assembly was secured to the calvarium using crainioplastic cement (Plastics One, Roanoke, VA). Animals requiring additional anesthetic were given 0.1 ml increments of the cocktail until no foot pinch response was elicited. Surgical wounds were treated with 2% lidocaine and a topical antibiotic (containing neomycin, polymixin B, and bacitracin); animals were monitored until they regained consciousness. Wounds were cleaned daily and received antibiotic ointment daily until healed.
Animals were given two weeks to recover from surgery. During this time the rats were introduced to the recording chamber in the presence of the recording cable (Plastics One, Roanoke, VA). Following the recovery period we conducted a 6-hour baseline recording of sleep and wakefulness starting 4 hours after lights on +/- 30 minutes using Grass model 8 recorders connected to the Grass PolyVIEW Pro software. Computerized data management was used for acquisition, analysis, and storage of data. Records were visually scored and quantified for sleep and wakefulness in six-second epochs. This was done by evaluating the EEG waves and EMG muscle tone.
Nicotine at doses of 0.03, 0.1, 0.3, and 1 mg/kg (in a buffered physiologic saline solution containing 50 mM HEPES, pH=7) or vehicle was administered subcutaneous via a 25-gauge needle.
Chlorisondamine, dissolved in a physiological saline solution, was administered at a dose of 10 mg/5 ml (total volume of 5 ml) I.C.V. using a 28-gauge injection cannula over a 5-minute period using a syringe pump (kd Scientific).
As previously mentioned, surgical placement of EEG, EMG electrodes and stereotaxic placement of an I.C.V. cannula using aseptic techniques in the anesthetized rat was first conducted. The rats recovered for a 2 week period, then had
baseline EEG and EMG recordings conducted (6-hour recording, start @ 4 hrs. into light cycle). Next, the animals were placed into one of two study groups; a chlorisondamine group, 6 rats, and a vehicle control group, 5 rats.
Nicotine dose response
The nicotine dose response study allowed us to determine the effects of several doses of nicotine, 0, 0.03, 0.1, 0.3, and 1.0 mg/kg, on sleep and wakefulness. The dose treatment schedule (all animals receiving all doses) in this manipulation was randomized using a random number generator function in Microsoft Excel. The interval of time between each of the 5 administrations was 5 days.
Chlorisondamine or saline was microinjected I.C.V., as described above, and 4 days after the injection a 24-hour baseline recording of sleep and wakefulness was conducted.
The following manipulations have not yet been conducted:
Animals in each treatment group then received both a high dose (1 mg/kg) and a low dose (0.1 mg/kg) of nicotine, subcutaneously, in a randomized fashion with 5 days between treatments and their sleep was recorded for 6 hours as before to investigate if the chlorisondamine blocked the effects of nicotine.
Platform sleep deprivation:
Five days after the last nicotine treatment animals will be deprived of REM sleep using the platform technique (24). Rats from both groups were placed on both a secure, elevated, 3 and 6.5 inch diameter platform with approximately 2 inches of water in the bottom of the cage in a randomized fashion for 24 hours with water and food available ad libidum. The large platform allows animals to sleep normally but controls for the stress effects of having limited mobility and being suspended above water. The small platform allows animals to have non-REM sleep but whenever they enter REM sleep they fall into the water because of the atonia that occurs during REM sleep, thus greatly decreasing the amount of REM sleep they get. Immediately after the platform manipulation, rats were returned to their home cages and recovery sleep was recorded for 24 hours. Five days were allowed to transpire between treatments to insure no lingering effects of the previous treatment on the next.
Five days after the platform manipulation, rats received an immobilization stress manipulation. This manipulation was chosen because rats respond to this stress by increasing REM sleep bout durations and thus the amount of REM sleep they get (1,16). In this manipulation, the animals were placed, depending on weight, into 2.5 or 3.5 inch diameter pipes (with ventilation and cooling slits cut every 0.5 inches) that restricted their movement for a period of 2 hours just prior to lights off. Following this, rats were returned to their home cages and their sleep was recorded for 24 hours.
Nicotine response (High dose /Low Dose)
In order to insure that the responses to nicotine were still blocked by the chlorisondamine, the high and low dose treatments of nicotine were repeated in the fashion previously described and sleep monitored for 6 hours. At the end of the study rats were euthanized in accordance with IACUC protocol.
The first part of our study involved determining if nicotine had an effect on the amount of REM sleep in rats. Rats treated subcutaneously with nicotine showed changes in the amount of REM sleep they had within a 6 hour period following the nicotine injections. As can be seen in figure 2, the effect of nicotine on REM sleep was bimodal with a maximal increase occurring at 0.1 mg/kg and a maximal decrease occurring at 1 mg/kg, as compared to the saline treatment. Analysis of Variance with a Tukey post hoc test showed a significant difference between the 0.1 and the 1.0 mg/kg doses but not between saline and any of the doses. Though not significant, we feel that once all of the data is analyzed that these two dose points will differ from saline. This bi-modal response is not atypical, with the treatment of many drugs showing similar bi-modality effects.
A change in the amount of time spent in REM sleep may be due to several architectural changes. It is possible to change the percent time spent in REM sleep by changing the frequency of REM sleep bouts or by changing the mean REM sleep bout duration or both. Analysis of the frequency of REM sleep bouts, as demonstrated by the absolute number of bouts occurring in the six hour recording, shows that there was a small, but non-significant, increase in this parameter at the 0.1 and 0.3 mg/kg doses (Figure 3). Thus, the primary effect of nicotine on percent of REM sleep appears not to involve major changes in the frequency of REM sleep bouts. This would suggest that the major effect of nicotine should be to change the average REM sleep bout duration. When we analyzed this parameter (Figure 4) we found that only at the 1.0 mg/kg dose was there a substantial decrease in average duration. The increase in REM sleep percentage seen at the 0.1 mg/kg dose is therefore due to both an increase in frequency and duration, though neither change is statistically significant. However, the decrease in the percent of REM sleep seen at the 1.0 mg/kg dose is due solely to a decrease in the average REM sleep bout duration.
The remainder of the experiment is still in progress and no data is presently available for discussion.
REM sleep architecture appears to be affected by the subcutaneous administration of nicotine at the aforementioned varying doses. It is possible that this is in part, due to nicotine’s high affinity for nicotinic acetylcholine receptors of the subtype, ? 4?2. The binding of nicotine to these receptors is most likely responsible for the changes in REM sleep architecture that are described in the results. At the high dose (1 mg/kg of nicotine), changes seen in the REM sleep architecture could be due to desensitization of these receptors, such a conclusion being consistent with other bi-modal drug dose responces. Conversely, lower doses of nicotine, specifically 0.1 mg/kg, seem to increase REM sleep percentage. The mechanism of this increase is not well understood but appears to involve small changes in both the frequency of REM sleep bouts and an increase in the mean REM sleep bout duration. What is of interest is that both 0.1 and 1 mg/kg doses affected the average duration of the REM sleep bouts. Now, according to the Hobson-McCarley model of REM sleep regulation (12), the cholinergic inputs to the DR and the LC account for the duration of any given REM sleep bout. Our data indicate that nicotinic receptors are not directly responsible for the cholinergic stimulation of these pontine centers since the model would predict that such stimulation should shorten the bout duration and desensitization of such receptors should prolong it. Indeed, our results show just the opposite effect, given that all identified nicotinic receptors in mammals are stimulatory. It is possible that the nicotinic receptors are presynaptic in nature and, we would propose, are located on inhibitory terminals such as GABAergic terminals. At this point it is difficult to explain the effect of nicotine on REM sleep based on this model and further work is necessary to locate which nicotinic receptors are involved in this effect. It is possible that the nicotinic receptors found in the DR and LC are not the ones accounting for the changes seen in REM sleep with nicotine. Velazquez Moctezuma et al. (26) have reported that nicotine injections into the pedunculopontine tegmentum can increase REM sleep in the cat. Thus, it is possible that the effect of nicotine on REM sleep is via such receptors. What is of interest for the purpose of our work is that nicotine appears to have a bi-modal effect on REM sleep, allowing us to test the notion that chlorisondamine will block these effects. As yet these results are not available and further discussion will be necessary when the rest of the experiment is completed and analyzed.
We gratefully acknowledge Mr. Cesar Moreno for his extensive technical assistance and Ms. Meghan Townsend and Mr. Jesus Nunez for their assistance with manipulations and data entry. This study was made possible through the financial support of NIH Grant #: 2R25GM49011 and NIH Grant # G12RR08124.
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Figure 1. The reciprocal interaction model of sleep. Three pontine nuclei, the dorsal raphe (DR), the locus coeruleus (LC), and the laterodorsal tegmentum (LDT) are believed to regulate and establish REM sleep. Negative symbols (-), represent inhibitory connections, and positive symbols (+), represent excitatory connections. The model is a representation of the Hobson-McCarley reciprocal interaction model of REM sleep regulation (***).
Figure 2. The effect of nicotine on percentage of REM sleep. Percentage of REM sleep for each dose of nicotine is plotted with solid squares (mean +/- sem for between 4 to 7 animals). The horizontal line on graph represents the mean +/- sem for the saline group (n=7). The data are plotted on a log 10 scale for the nicotine dose.
Figure 3. The effect of nicotine on REM sleep bout duration. REM sleep bout durations for each dose of nicotine are plotted with solid squares (mean +/- sem for between 4 to 7 animals). The horizontal line on graph represents the mean +/- sem for the saline group (n=7). The data are plotted on a log 10 scale for the nicotine dose.
Figure 4. The effect of nicotine on REM sleep bout number. The number of REM sleep bouts for each dose of nicotine are plotted with solid squares (mean +/- sem for between 4 to 7 animals). The horizontal line on graph represents the mean +/- sem for the saline group (n=7). The data are plotted on a log 10 scale for the nicotine dose.