Modafinil amd electrical coupling
Modafinil Increases Arousal Determined by P13 Potential Amplitude: An Effect Blocked by Gap Junction Antagonists Paige Beck; Angela Odle; Tiffany Wallace-Huitt, PhD; Robert D. Skinner, PhD; Edgar Garcia-Rill, PhD Center for Translational Neuroscience, Department of Neurobiology and Developmental Science, University of Arkansas for Medical Sciences, Little Rock, AR
Study Objectives: We recorded the effects of administration of the stimulant modafinil on the amplitude of the sleep state-dependent auditory P13 evoked potential in freely moving rats, a measure of arousal thought to be generated by the cholinergic arm of the reticular activating system, specifically the pedunculopontine nucleus (PPN). Design: Groups of rats were implanted for recording auditory evoked responses and the effects on P13 potential amplitude of intracranial injections into the PPN of neuroactive agents determined. Measurements and Results: The effects of intracranial injections into the PPN of modafinil showed that P13 potential amplitude increased in a dose-dependent manner at doses of 100, 200, and 300 µM. The effect was blocked by pretreatment with either of the gap junction an-
tagonists carbenoxolone (300 µM) or mefloquine (25 µM), which by themselves slightly decreased P13 potential amplitude. Conclusions: These results suggest that modafinil increases arousal levels as determined by the amplitude of the P13 potential, an effect blocked by gap junction antagonists, suggesting that one mechanism by which modafinil increases arousal may be by increasing electrical coupling. Keywords: Arousal, carbenoxolone, electrical coupling, gap junctions, mefloquine, Modafinil Citation: Beck P; Odle A; Wallace-Huitt T; Skinner RD; Garcia-Rill E. Modafinil Increases Arousal Determined by P13 Potential Amplitude: An Effect Blocked by Gap Junction Antagonists. SLEEP 2008;31(12):16471654.
WE RECENTLY DISCOVERED THAT CELL POPULATIONS WITHIN VARIOUS CELL GROUPS OF THE RETICULAR ACTIVATING SYSTEM (RAS) ARE ELECTRICALLY coupled.1,2 Slice-recorded neurons, probably GABAergic, including those in the pedunculopontine nucleus (PPN), the cholinergic arm of the RAS, were found to be electrically coupled.2 The stimulant modafinil is approved for the treatment of excessive sleepiness in narcolepsy, obstructive sleep apnea, and shift work disorder. A recent landmark study found that the mechanism of action of modafinil is to increase electrical coupling between cortical interneurons, thalamic reticular neurons, and inferior olivary neurons.3 We confirmed the fact that modafinil appears to increase electrical coupling in the RAS by showing that, in the absence of action potentials or fast synaptic transmission, modafinil decreased the input resistance of electrically coupled RAS cells, an effect blocked by the gap junction antagonists carbenoxolone or mefloquine.1,2 We hypothesize that increased electrical coupling of GABAergic RAS neurons by modafinil may decrease their input resistance and, consequently, GABA release, thus disinhibiting output cells, perhaps accounting for its stimulant properties. A measure of arousal in the whole animal is the click stimulus-induced P13 auditory evoked potential in the freely moving rat. The P13 potential has been shown to be sleep state dependent; it is present during waking and REM sleep but absent during slow wave sleep, in parallel with the activity pattern of PPN neurons.4 The P13 potential is also blocked by the cholin-
ergic antagonist scopolamine, suggesting that it is mediated by cholinergic projections,4 and is blocked by agents that decrease arousal such as anesthetics and alcohol.5,6 Significantly, the P13 potential is reduced or blocked by local injections of agents that have a net inhibitory effect on the PPN, demonstrating that the PPN mediates this vertex-recorded waveform.7,8 The present study was undertaken to determine if injections of modafinil into the PPN would affect the amplitude of the P13 potential in the awake, freely moving rat, and if any induced stimulant effect could be blocked by pretreatment locally at the level of the PPN with gap junction antagonists. METHODS Surgery The methods employed have been previously published.4-8 All animal procedures were approved by the Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (n = 16, 250 to 350 gm, Harlan) were anesthetized with ketamine HCl (60 mg/kg, i.m.) and xylazine (20 mg/kg, i.p.). Anesthetic levels were maintained such that the withdrawal reflex to paw pinch was absent during surgical procedures. The animal was placed in a stereotaxic frame using hollow ear bars, which protected the middle ear from injury. After reflection of the scalp, stainless steel screws for recording auditory evoked potentials were inserted epidurally at the vertex (Vx), 5.5 mm anterior to the interaural line, 1.0 mm lateral to the midline, bilaterally. A reference screw electrode was inserted into the frontal sinus. The screws were connected to a receptacle anchored to the skull. In addition, a pair of 25 gauge stainless steel hypodermic tubing guide cannulae (18 mm length) were implanted bilaterally at coordinates 1.0 mm anterior of the interaural line, 1.9 mm lateral of the midline, and 6.0 mm above the horizontal plane passing through the interaural line (1.0 mm dorsal to
Submitted for publication May, 2008 Accepted for publication May, 2008 Address correspondence to: E. Garcia-Rill, PhD, Professor and Director, Center for Translational Neuroscience, Dept. Neurobiology & Dev. Sci., Slot 847, University of Arkansas for Medical Sciences, Little Rock, AR 72205; Tel: (501) 686-5167; Fax: (501) 526-7928; E-mail:
[email protected] SLEEP, Vol. 31, No. 12, 2008
1647
Modafinil and Gap Junctions—Beck et al
the PPN).9 This placement was used in order to minimize cell damage in the PPN. The end of each cannula was fitted with a plastic cap and stylette to close off the open end of the cannula and prevent internal blockage of the cannula. Averages of P13 potentials routinely were collected from both Vx electrodes, but only the left side was used for analysis. None of the electrodes became unusable, thus there was no need to use the back-up Vx electrodes. This channel is frequently used to monitor EEG; however, previous studies have determined that bilateral injections induce consistent responses in both left and right Vx electrodes, and unilateral injections can induce asymmetrical responses, so these were not used.4-8 Pairs of hooked (insulated stainless steel except the last 2 mm) wires (100 μm diameter) were inserted into the dorsal nuchal muscles (belly of the splenius m.) bilaterally (2-3 mm intertip distance) and anchored by stitches to the fascia to monitor electromyographic (EMG) signals (routine monitoring of startle response). Penicillin G was given i.m. and the animals placed in a warm environment during recovery. Rats were housed individually in a vivarium with 12:12 light/dark schedule (lights on at 06:00) and food and water ad libitum. Recordings began after a 1-week recovery period. Before surgery, rats were acclimated to the chamber for 2 days for increasing periods of time up to 30 min. During the recovery week, animals were placed in the recording chamber daily for 30 min to habituate them to the chamber. Tethering was used for increasing periods of time until the rats accepted the tether for at least 30 min. Great care was taken to ensure that stress was not evident during the recording or injection procedures; previous studies using immobilization stress revealed that the P13 potential is invariably and markedly reduced in amplitude immediately after the beginning of the stressor and during the entire period of stress.10 The present results show no trend towards any effects of stress during recordings, particularly after injections, since injections of saline, various concentrations of modafinil, and modafinil after pretreatment with gap junction antagonists showed no decrements in P13 potential amplitude (see below).
was lit by a 12 V DC bulb and a peephole viewer allowed undetected observation of the animal. Wires from the swivel commutator were connected to a plug that mated with the receptacle implanted on the animal. Following a 15-min acclimation period in the chamber, auditory evoked responses were recorded during quiet waking, judged from constant observation of the animal’s behavior, EEG, and EMG. All subjects were tested between 09:00 and 13:00 to control for possible time of day effects. Evoked potentials from the Vx were amplified (10,000x) and filtered at 3 Hz-1 kHz.4-8 The EMG signal was amplified (10,000x) and filtered at 30 Hz-3 kHz. All recordings were amplified using Grass Instrument (Quincy, MA) model P511 amplifiers and digitized at a rate of 10 kHz, averaged, stored on computer hard drive, and analyzed using SuperScope software (GW Instruments, Somerville, MA). Measurement of the amplitude of the P13 auditory evoked potential, a positive waveform starting at a latency of 7-9 msec and peaking at 12-14 msec,4-8 was made from the beginning of the wave to its peak. Each stimulus consisted of two 103 dB rarefaction clicks, 0.1 msec duration, each at an interclick interval of 1 msec (total duration of each 2-click stimulus 200% amplitude. Such deviations extended beyond the window for each channel, triggering deletion prior to averaging. Injections After 3 pre-injection control recordings were made, animals received bilateral injections using a 32G stainless steel tube (19 mm length) to extend 1.0 mm beyond the lower end of the guide cannulae (dorsal boundary of PPN). Vehicle for all injected substances was 0.05% DMSO in saline, but we also used control injections of 0.9% physiological saline. The injectors were connected to a 5 µL syringe (Hamilton Co. Reno, NV) by a 10 cm long flexible polyethylene tube (0.38 mm ID). Concentrations of injected solutions were adjusted so that each injection was 0.1 µL. Injections were delivered in sequence over 10-20 sec while the animals were gently held. Care was taken to determine if backflow occurred after each injection, which was detected in one case, so that the recordings were not used in the analysis. The animal was returned to the recording chamber within 2 min of the beginning of the injections, and recordings began immediately. After injection, recordings were carried out every 10 min for 1 h. One group of 8 animals was used for the dose-response study. Note that, in all cases, 3 pre-injection recordings were made and these were averaged to obtain the pre-injection control for every agent injected. The dose-response animals received, in random order, saline, vehicle, 100 µM modafinil (MOD), 200 µM MOD, or 300 µM MOD. The other group of 8 animals was used for the antagonist study and received in
Recordings Auditory evoked potentials were recorded from the Vx of unrestrained, alert rats placed in a sound-attenuating chamber (2 × 2 × 2 ft). Earphones were mounted on opposite sides of the recording chamber 4.25 inches above the floor to create a diffuse sound field within the chamber. A diffuse sound field is desired because the rats are free to move within the chamber. Input to the earphones consisting of 100 µsec rectangular pulses was provided by a Grass Instrument Click-Tone Stimulator (Model S10CTCM). Measurements were made in the chamber using a Radio Shack sound level meter set for a 32-10,000 Hz range and in averaging mode. The click stimulus was produced at 99.9 Hz (fastest setting on the S10CTCM) in the recording chamber with the door open. Measurements were: (1) 103 dB sound pressure level (SPL) at the speaker on one side and 102 dB SPL on the other, (2) 84 dB SPL at the center of the chamber, and 82 dB SPL at the back corners, all at 4.25 in. above the floor of the recording chamber. All stimulus levels were well above the threshold of the P13 potential that was measured as ~70 dB SPL at the center of the chamber.4-8 A constant white noise background (50 dB SPL measured at center of chamber) was provided by a 4 in diameter fan. The chamber SLEEP, Vol. 31, No. 12, 2008
1648
Modafinil and Gap Junctions—Beck et al
random order saline, 300 µM carbenoxolone (CBX), 25 µM mefloquine (MEF), 300 µM MOD, 300 µM CBX + 300 µM, or 25 µM MEF + 300 µM MOD. Note that, in the case of double injections in which the antagonist pre-treatment preceded MOD, no recordings were carried out between the 2 injections, so that responses could be directly compared following MOD and following antagonist + MOD. This protocol allowed us to test the effects of (a) any agent vs its preinjection control, (b) any agent vs vehicle control, and (c) any agent vs saline control over time. The pre-injection average P13 potential amplitude was designated as 100%, and subsequent post-injection amplitudes were calculated as percent amplitude of this control recording. Injection of vehicle or saline did not change P13 potential amplitude throughout the recording period. No animal was injected more than twice per week or more than 5 times overall. It should be noted that injections required that the animal be removed from the chamber, and that the absolute amplitude of the initial (preinjection control) recordings be similar across trials, implying no lasting effects of the preceding surgery or injection days earlier. Moreover, there was no detectable difference in the pre-injection control amplitudes in each of the 3 trials using different concentrations of MOD. That is, there seemed to be no lasting effects of surgery or of prior injections that could account for the effects described below. Previous studies have tested the effects of double injections of saline or saline plus agonist or antagonist, and found no significant effects of the double injection protocol.7 Moreover, the effects described in the present manuscript are quite specific, showing that double injections of CBX+MOD or MEF+MOD do not change P13 potential amplitude significantly, while MEF alone, CBX alone, and MOD alone all changed P13 potential amplitude significantly and in different directions (see Results). This argues against any unspecific effects of double injections. The concentrations of MOD tested were 100, 200, and 300 µM, and of the gap junction antagonists CBX (300 µM) and MEF (25 µM) (pretreatment or alone), were in the same range as those used in slice recording studies.1-3 These agents are water insoluble and were made up in the same vehicle used during in vitro studies.1-3 All agents were purchased from Sigma, except MEF, which was supplied by NCI (see Acknowledgments). The protocol used for single and double injections was as follows:
At the conclusion of the microinjection experiments, rats were deeply anesthetized, 0.2 µL of Fast Green dye was injected at the same sites at which neuroactive compounds had been injected and the brains processed histochemically for NADPH diaphorase.11 Only data from animals in which the injection site overlapped with NADPH diaphorase-positive PPN neurons were included in the analysis. The injections typically spread in a teardrop fashion over a region 1 mm in diameter, as in previous studies.4-8 Previous studies have determined that only injections within the PPN exhibit significant effects on the P13 potential, while injections dorsal or medial to the PPN show no significant effects. In the current series, all implanted cannulae were located within or at the edge of NADPH diaphorase-positive regions, so that negative controls were not available. Previous studies have determined that similar injections outside NADPH diaphorase-positive regions do not produce significant changes in P13 potential amplitude.7,12,13 However, lack of negative control injections may represent a limitation to our conclusions. Statistical Analyses The first analysis carried out was a repeated measures one-way analysis of variance (ANOVA) to test the pre-injection control recording for each agent vs each of the subsequent post-injection recordings and main effects were followed with post hoc contrasts using the Newman-Keuls test. This provided a measure of the post injection effect of an individual agent over time. Results were also evaluated using 2-factor, 2-way repeated measures analysis of variance (ANOVA) and main effects were followed with post hoc contrasts using the Newman-Keuls test. A 2-factor repeated measures model with repeats on both factors was fit to the data, such that responses from the animals were recorded at all combinations of injection concentration and time. An advantage of such an experimental design is that it is possible to take advantage of the correlation of the observations within rats, which leads to fewer experimental subjects to achieve a given level of statistical power. In our study, the within-animal correlation of the repeated observations was approximately 0.31 which, after calculation, implies that 16-18 animals per concentration would have been required to achieve the same statistical power as 6-8 animals measured at all levels in our study. This provided a measure, for example, of the post-injection effects of a particular concentration of MOD vs post injection of saline or of vehicle control, over time. Additionally, the data did not suggest that normal distribution assumption was violated, namely the test for non-normality of the residuals. The Kolmogorov-Smirnov test for normality on the residuals was not significant (P = 0.33), leading to the conclusion that the data did not suggest a departure from normal distribution. Finally, a 3-factor, 2-way repeated measures ANOVA was run to compare the effects of antagonist alone, modafinil alone, and pre-treatment with antagonist plus modafinil across every time point, along with the same post hoc test.
Single Injection ↓ + 5 min ↓ + 5 min ↓ │+ 5, 10, 15, 25, 35, 45, and 55 min post injection recordings Double injection ↓ + 5 min ↓ + 5 min ↓ │ + 20 min ║ + 5, 10, 15, 25, 35, 45, and 55 min post inj. recordings
RESULTS
Legend
Saline and Vehicle Controls
↓ = 3 CTL pre-inj. recordings │ = Inj. of saline, vehicle, MOD (3 conc'ns), CBX or MEF ║ = Inj. of MOD 20 min after CBX or MEF SLEEP, Vol. 31, No. 12, 2008
Figure 1A shows representative averages of the P13 potential in the same rat after control (CTL) recordings, after 100 1649
Modafinil and Gap Junctions—Beck et al
Figure 1—Effects of MOD on P13 potential amplitude. A. Representative averages from a single rat showing a control average (top record), one 25 min after intracranial MOD at 100 µM (middle record), and one 25 min after MOD at 300 µM (bottom record). The stimuli were applied at the arrow labeled S, and the amplitude of the P13 potential was measured between the second and third arrows, at the peak of the potential, approximately 13 msec after the stimulus. Calibration bars as shown. B. Percent change in P13 potential amplitude in a group of 8 rats recorded from 5-55 min after injection of saline or vehicle (X), after MOD at 100 µM (open circles), after 200 µM (open squares), and after 300 µM (filled circles). Note that the CTL value is 100% for every injection, i.e., saline, 100, 200, or 300 µM MOD, and post injection comparisons were made against these controls. The asterisks denote significant (P < 0.01) increases compared to pre-injection control using a repeated measures one-way ANOVA (2-way results provided in the text), showing significance at 35 min after 100 µM, at 25 and 35 min after 200 µM, and at 10-55 min after 300 µM MOD.
µM MOD and after 300 µM MOD, demonstrating the increase in amplitude evident 25 min following intracranial injection into the PPN. Figure 1B is a graph of the average P13 potential amplitudes normalized to the average control amplitude in 3 pre-injection recordings in a group of 8 rats. The average P13 potential amplitudes following saline injection are denoted by X, and show no significant change in recordings at 5-55 min (pre-injection control vs each post injection recording, one-way repeated measures ANOVA, df = 7, F = 0.8, P > 0.05, NS), nor was there a difference between vehicle and saline injection (pre- and post-injection values at each time point after saline vs vehicle, 2-way repeated measures ANOVA saline vs vehicle, df = 15, F = 1.02, P > 0.05, NS).
numerical increase that reached statistical significance at 25 and 35 min post-injection, then remained numerically elevated at levels similar to 100 µM injections. Following injections of MOD at 300 µM (filled circles), the average P13 potential amplitude became statistically significant at 10 min, and remained significantly increased at 15, 25, 35, 45, and 55 min. That is, the highest concentration had not returned to nonsignificant levels by the end of the recordings. Repeated measures one-way ANOVA showed that there was a significant increase in P13 potential amplitude compared to saline following the 100 µM injection (post-injection MOD vs post-injection saline at each time point, df = 7, F = 14.18, P < 0.0001), with post hoc significance at the 35 min time point only (P < 0.001). After the 200 µM injection, there was also a statistically significant increase (post-injection MOD vs post-injection saline at each time point, one-way repeated measures ANOVA df = 7, F = 3.81, P < 0.0009), with post hoc significance at the 25 and 35 min time points (P < 0.001). Following injection of 300 µM MOD, P13 potential amplitude increased significantly compared to preinjection controls (post-injection MOD vs post-injection saline at each time point, one-way repeated measures ANOVA df = 7, F = 33.23, P < 0.0001), with post hoc significance of indi-
MOD vs Pre-Injection Control Following injections of MOD at 100 µM (open circles), there was a variable response in average amplitude, but no significant increase was evident until 35 min post-injection (> 55% increase), and subsequent recordings failed to show significant increases. After injections of MOD at 200 µM (open squares), the average amplitude of the P13 potential showed a gradual SLEEP, Vol. 31, No. 12, 2008
1650
Modafinil and Gap Junctions—Beck et al
Figure 2—Effects of gap junction antagonists on MOD-induced changes on P13 potential amplitude. A. Percent change in average P13 potential amplitude after saline (X), and after MOD at 300 µM (filled circles, see significance in Figure 1B), as well as after CBX alone at 300 µM (filled triangles) and CBX at 300 µM followed 20 min later by MOD at 300 µM (open triangles) compared to pre-injection control for each agent. CBX alone (filled triangles) induced transient decreases compared to saline alone at 10, 25, and 55 min (P < 0.01, asterisks not shown for clarity), while pretreatment with CBX blocked the effects of MOD at all time points (open triangles) and was not different from saline (X). When a 3-factor, 2-way analysis between MOD at 300 µM (filled circles), CBX at 300 µM (filled triangles) and CBX+MOD (open triangles) was done (pre- and post injection values at all time points after MOD vs CBX vs CBX+MOD), significant increases were present for MOD vs CBX+MOD at 25, 35, and 55 min (**P < 0.01), as well as 45 min (*P < 0.05) (upper asterisks in Fig. 2A), suggesting the presence of CBX suppressed the increases seen with MOD alone (shown in Fig. 1B). Comparison between CBX and CBX+MOD showed only significance at 10 min (*P < 0.05, asterisk at bottom of Fig. 2A). Significant differences between MOD alone and CBX alone were present at 10-55 min (P < 0.01, asterisks not shown for clarity). B. Percent change in average P13 potential amplitude after saline (X), and after MOD at 300 µM (filled circles), as well as after MEF alone at 25 µM (filled squares) and MEF at 25 µM followed 20 min later by MOD at 300 µM (open squares) compared to pre-injection control for each agent. One-way ANOVA comparison between MEF alone and saline induced significant decreases at 5, 10, 15, 25, 35 ( P < 0.01), and 45 min (P < 0.05) (asterisks not shown for clarity), but pretreatment with MEF blocked the effects of MOD at all time points (open squares) and was not different from saline (X). When a 3-factor, 2-way analysis (pre- and post injection values at all time points after MOD vs MEF vs MEF+MOD between MOD at 300 µM (filled circles) and MEF+MOD (open squares) was done, significant increases were present comparing MOD vs MEF+MOD at 10 and 15 min (*P < 0.05) and 25-55 min (**P < 0.01) (upper asterisks in Fig. 2B), suggesting the presence of MEF suppressed the increases seen with MOD alone (as shown in Fig. 1B). When MEF vs MEF+MOD were compared, significant decreases were present only at 10 and 35 min (**P < 0.01) (lower asterisks in Fig. 2B). Significant differences between MOD alone and MEF alone were present at 10-55 min (P < 0.01, asterisks not shown for clarity).
vidual time points at 10-55 min (P < 0.001) (shown in Figure 1B). When the one-way repeated measures ANOVA compared P13 potential amplitude after injections of 300 µM MOD to the control (pre-injection) recordings, significant differences were evident between groups (pre-injection control vs post injection recordings, df = 7, F = 5.13, P < 0.001) with post hoc differences at 25 min (P < 0.05), 35 min (P < 0.01) and 55 min (P < 0.01) (not shown).
15, 25, 35, 45, and 55 min), the results showed that there was a significant difference across concentration (pre- and post injection values at each time point after saline vs each concentration of MOD, df = 3, F = 40.13, P < 0.0001), and across time (df = 7, F = 13.33, P < 0.0001), as well as an interaction between concentration and time (df = 21, F = 4.33, P < 0.0001). Post hoc comparisons showed significant differences compared to vehicle or saline at 35 min for 100 µM, at 25 min for 200 µM, and at 25, 35, 45, and 55 min for 300 µM (not shown).
MOD vs Saline Controls
CBX vs Pre-Injection Control and vs CBX+MOD
When the data were analyzed using a repeated measures 2-way ANOVA comparing the effects of MOD at each concentration vs injection of saline (saline, 100, 200, and 300 µM MOD vs 5, 10, SLEEP, Vol. 31, No. 12, 2008
Figure 2A is a graph of the P13 potential average amplitudes after saline (X) and 300 µM MOD (filled circles). In this group 1651
Modafinil and Gap Junctions—Beck et al
time (df = 7, F = 3.46, P < 0.01), between time and drug (df = 14, F = 5.58, P < 0.01), and between different agents (df = 2, F = 13.04, P < 0.01). Post hoc testing revealed that MOD differed significantly from MEF+MOD at 10 and 15 min (P < 0.05), and 25-55 min (P < 0.01) (asterisks at top of Figure 2B), while MEF differed significantly from MEF+MOD only at the 10 and 35 min (P < 0.01) time points (asterisks at bottom of Figure 2B). Post hoc results comparing MOD vs MEF showed significant differences at 10-55 min (P < 0.01, asterisks omitted for clarity).
of 8 animals, we injected CBX at 300 µM (filled triangles) and recorded for 1 h, to determine the effects of this putative gap junction antagonist alone. We also pretreated with CBX at 300 µM and waited 20 min before injecting MOD at 300 µM (open triangles), to determine the effects of this agent on the effect observed after MOD at the highest concentration tested in the dose-response curve. The graph shows that one-way repeated measures ANOVA revealed that CBX by itself led to a significant decrease in P13 potential amplitude (pre-injection control vs post-injection recordings at each time point, df = 7, F = 12.11, P < 0.0001), specifically at 10, 25, and 55 min (post hoc P < 0.001, asterisks not shown for clarity), while MOD at 300 µM showed increases at 10-55 min (P < 0.001, shown in top asterisks in Figure 1A). However, when we injected CBX at 300 µM followed 20 min later by MOD at 300 µM (CBX+MOD), both the increases in amplitude induced by MOD alone, and the decreases in amplitude induced by CBX alone were completely blocked, and were identical to saline. We then compared the effects of CBX vs MOD vs CBX+MOD in a 3-factor, 2-way repeated measures ANOVA testing 8 time points vs 3 agents. The results showed a significant effect over time (df = 7, F = 3.46, P < 0.01), between time and agents (df = 14, F = 4.84, P < 0.01), and between different agents (df = 2, F = 12.64, P < 0.01). Post hoc testing revealed that MOD differed significantly from CBX+MOD at 25, 35, and 55 min (P < 0.01), and 45 min (P < 0.05) (asterisks at top of Figure 2A), while CBX did not differ significantly from CBX+MOD at any time point except 10 min (P < 0.05) (asterisks at bottom of Figure 2A). Post hoc results comparing MOD alone vs CBX alone showed significant differences at 10-55 min (P < 0.01, asterisks omitted for clarity).
Discussion The results of intracranial injections into the PPN described demonstrate that (a) modafinil increased P13 potential amplitude in a dose-dependent fashion, peaking at 25-45 min after injection, (b) the increase in P13 potential amplitude was blocked by pretreatment with the gap junction antagonists carbenoxolone or mefloquine, and (c) these gap junction blockers slightly reduced P13 potential amplitude when administered on their own. These findings suggest that (a) modafinil may increase arousal at least in part by acting at the level of the PPN, (b) since its effects are blocked by two different gap junction antagonists, it may act by increasing electrical coupling, and (c) gap junction antagonists on their own may tend to decrease arousal as determined using P13 potential amplitude. Some methodological considerations and limitations of this study should be considered. For example, the P13 potential is a volume-conducted potential that, although obviously modulated by the PPN, its precise localization is unknown since similar waveforms can be recorded in deep structures such as the PPN and the intralaminar thalamus.11 Moreover, intracranial injections can spread over a 1 mm region centered immediately dorsal to the posterior PPN, which may have included the medial midbrain reticular formation and the anterior pole of the locus coeruleus. Previous studies have determined that the effective injection sites must include the PPN.7,8 In the present study, all cannulae were found to impinge on the PPN so that no off-site injection controls were available. Since injections of modafinil and gap junction blockers did not impinge on the anterior pole of locus coeruleus, which has gap junctions, we are certain that this region was not affected. While there are no other nuclei in the region known to have gap junctions, the absence of negative injection controls suggest the results must be regarded with some caution. The P13 potential can be occluded by excessive EMG activity, especially in neck muscles. The monitoring of EMGs is critical to acquiring uncontaminated waveforms, so that an online selection process is made (see Methods). Despite the excitatory effects of MOD on the P13 potential, this did not result in excessive EMG activation, requiring that only 1-2 trials per average be discarded. The monitoring of EEG was likewise essential to monitor shifting into slow wave sleep, but this was not evident during control and MOD recordings, although some instances of slow wave activity were evident after CBX or MEF alone. This required discarding 2-3 trials and opening the cage door in 2 cases in order to shift the animal to quiet waking. These limitations have not been prohibitive to securing accurate and reproducible findings on the P13 potential over the last 10 years.4-8,10,12,13
MEF vs Pre-Injection Control and vs MEF+MOD We also tested a second gap junction antagonist, MEF, on its ability to affect the P13 potential by itself and when used as pretreatment before MOD. Figure 2B is a graph of the P13 potential average amplitudes after saline or vehicle (X) and 300 µM MOD (filled circles). In this group of 8 animals, we injected MEF at 25 µM (filled squares) and recorded for 1 hr, to determine the effects of this putative gap junction antagonist alone. We also pretreated with MEF at 25 µM and waited 20 min before injecting MOD at 300 µM (open squares), to determine the effects of this agent on the effect observed after MOD at the highest concentration tested in the dose-response curve. The graph shows that One-way repeated measures ANOVA showed that MEF by itself led to a significant decrease in P13 potential amplitude (pre-injection control vs post injection recordings at each time point, df = 7, F = 23.10, P < 0.0001), specifically at 5, 10, 15, 25, 35, and 45 min (post hoc P < 0.001 for all but 45 min, P < 0.05) (Figure 2B), while MOD at 300 µM showed increases at 10-55 min (P < 0.001, shown in figure 1B). However, when we injected MEF at 25 µM followed 20 min later by MOD at 300 µM (MEF+MOD), both the increases in amplitude induced by MOD alone and the decreases in amplitude induced by MEF alone were completely blocked, and were similar to saline. We then compared the effects of MEF vs MOD vs MEF+MOD in a 3-factor, 2-way repeated measures ANOVA testing 8 time points vs 3 agents. The results showed a significant effect over SLEEP, Vol. 31, No. 12, 2008
1652
Modafinil and Gap Junctions—Beck et al
A number of studies have convincingly demonstrated that the P13 potential is sleep state dependent (active during waking and REM sleep but not slow wave sleep), blocked by the muscarinic cholinergic antagonist scopolamine and rapidly habituating, all suggestive of its origins at the level of the RAS.4-6 Moreover, intracranial injections into the region of the PPN have shown that this vertex-recorded waveform is modulated by the PPN.7,8 In addition, systemic interventions that decrease arousal (alcohol, halothane, head trauma)7 and anesthetics like propofol8 reduce the amplitude of the P13 potential. The P13 potential thus appears to be a measure of ascending RAS output.14 Cells in the PPN were found to exhibit spikelets, thought to reflect electrical coupling, that could in theory also be from sodium-mediated dendritic spikes, but these remained despite the presence of QX314 in the recording electrode. This intracellular sodium channel antagonist would have blocked all dendritic spikes, but the spikelets always remained.2,15 The discovery of electrical coupling in the RAS, including the PPN, provides a novel mechanism of sleep-wake control based on the interactions, some reciprocal, between known transmitter systems and the ensemble activity mediated by electrical coupling.2,15 The finding that a mechanism of action of the stimulant modafinil may be through increasing electrical coupling,3 especially in the RAS (modafinil decreased input resistance without changing membrane potential in the presence of fast synaptic blockers or sodium channel blockers, an effect blocked in vitro by gap junction blockers),1,2 helps explain some of the effects ascribed to this agent, such as increasing glutamatergic, adrenergic, and histaminergic transmission and decreasing GABAergic transmission.16 The increase in electrical coupling is thought to shunt the current and thereby decrease input resistance of GABAergic neurons, decreasing their output, thus disinhibiting other systems.3 The present results showed that, in the freely moving whole animal, modafinil injected into the PPN induced a significant but transient increase in P13 amplitude at 100 µM, an increase at more than one time point at 200 µM, and a lasting increase at 300 µM (Figure 1B), suggesting a dose-dependent effect. Conversely, gap junctions can be blocked through membrane fluidization such as that induced by the anesthetics halothane and propofol,17,18 the same agents that reduce P13 potential amplitude.5,6 One possibility is that a mechanism by which these agents may induce sleep and/or anesthesia is through blockade of electrical coupling in the RAS. The effects of modafinil on RAS cells were evident in the absence of changes in resting membrane potential or of changes in the amplitude of induced EPSCs, and its effects were blocked by carbenoxolone or low concentrations of the gap junction blocker mefloquine, also in the absence of changes in resting membrane potential or in the amplitude of induced EPSCs.1-3 This suggests that these compounds do not act indirectly by affecting voltage-sensitive channels such as potassium channels, but rather modulate electrical coupling in the RAS via gap junctions. In order to block the effects of modafinil on the P13 potential, we used two different gap junction antagonists (Figure 2). Carbenoxolone is a putative gap junction blocker that decreases the synchronicity of gamma oscillations19 and seizure activity.20 Quinine and a related compound, mefloquine, also block gap junctions.21,22 Mefloquine is particularly useful because it blocks Cx 36 (specific to neurons) and Cx 50 gap junctions at low concentrations, but not Cx 43, SLEEP, Vol. 31, No. 12, 2008
Cx 32, or Cx 26 gap junctions.23 Each of these agents also has a number of side effects; however, given their disparate structure, they may do so through different mechanisms. Previous studies have shown that both carbenoxolone and mefloquine affect various neuronal processes independently of each other and of their antagonism of gap junction conductance, some of which may be due to effects on astrocytic gap junctions or 5-HT3 receptors.23-25 The present study used both carbenoxolone and mefloquine in an attempt to minimize the possible confounding effects of both drugs so that, by showing common effects, they may be ascribed to their antagonism of gap junctions. Mefloquine may also block potassium channels, but its effects in the RAS do not appear to include these, since such blockade would lead to an inward current and increase spikelets, not decrease them, as we recently reported.1-2 Each agent was found to significantly reduce P13 potential amplitude when administered by itself, suggesting that they may mildly decrease arousal levels as measured by P13 potential amplitude. Moreover, mefloquine appeared to induce greater inhibition of P13 potential amplitude than carbenoxolone (two vs one time point, compare asterisks at bottom of Figure 2A vs 2B), indicating that this may be a more effective gap junction blocker in the PPN. Direct comparisons between the two drugs need to be carried out in order to confirm this potential effect. An interesting observation is the time course of the effects of modafinil, carbenoxolone and mefloquine, peaking only after 25-35 min after intracranial injections. A similar delay is present after superfusion of these agents in slices.1-3 These agents are thought to require entry into the cell in order to affect gap junctions from the inside, probably because the close apposition of gap junctions may not permit direct extracellular effects. Although the mechanisms by which these agents affect gap junctions intracellularly are not well understood, some evidence suggests that they may act via phosphorylation of gap junctions through protein kinase and similar pathways.3 We assume that their lipophilic nature allows them to enter the cell to affect these enzymes, but it requires time to induce an effect. At present, it is not known how modafinil increases Cx-36 electrical coupling, whether it mobilizes endoplasmic pools and inserts hemi-channels into the membrane, or promotes migration of hemi-channels to formation plaques, or promotes alignment of hemi-channels on both cells, or simply helps open closed channels. Similarly, the exact site of action of carbenoxolone and mefloquine are unknown, so that the effects observed in these studies represent an algebraic summation of effects and not necessarily a competitive blockade at the same site of action. Much more work is called for to understand the actions of these agents. Importantly, each agent counteracted the effects of modafinil, canceling its excitatory effects on P13 potential amplitude, suggesting that indeed modafinil may act via gap junctions, and its impact may be reduced or blocked by gap junction antagonists. Nevertheless, additional studies using congeners of these agents that do not affect gap junctions need to be performed in order to determine if their parallel effects are specific to gap junctions. In summary, our results suggest that the stimulant modafinil increases the amplitude of the sleep state-dependent P13 potential, an effect blocked by the gap junction antagonists carbenoxolone and mefloquine. These results suggest that modafinil may 1653
Modafinil and Gap Junctions—Beck et al
act via gap junctions to increase vigilance. More generally, the finding that gap junctions modulate arousal states introduces an added mechanism by which sleep and waking, and perhaps anesthesia, may be controlled.
9. 10.
Abbreviations CBX DMSO MEF MOD NADPH PPN RAS
11.
carbenoxolone dimethyl sulfoxide mefloquine modafinil nicotinamide adenine dinucleotide phosphate, reduced form pedunculopontine nucleus reticular activating system
12. 13. 14.
Acknowledgments Supported by USPHS grants NS20246 and RR20146. We would like to thank the Drug Synthesis and Chemistry Branch, National Cancer Institute, for the supply of mefloquine.
15. 16.
Disclosure Statement 17.
This was not an industry supported study. Dr. Garcia-Rill has received research support and has consulted for Sepracor. The other authors have indicated no financial conflicts of interest.
18.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
19.
Heister DS, Hayar A, Charlesworth A, Yates C, Zhou Y, GarciaRill E. Evidence for electrical coupling in the SubCoeruleus (SubC) nucleus. J Neurophysiol 2007;97:3142-7. Garcia-Rill E, Heister DS, Ye M, Charlesworth A, Hayar A. Electrical coupling: novel mechanism for sleep-wake control. Sleep 2007;30:1405-14. Urbano FJ, Leznik E, Llinas R. Modafinil enhances thalamocortical activity by increasing neuronal electrotonic coupling. Proc Natl Acad Sci U S A 2007;104:12554-9. Miyazato H, Skinner RD, Reese NB, Boop FA, Garcia-Rill E. A middle-latency auditory-evoked potential in the rat. Brain Res Bull 1995;37:247-55. Miyazato H, Skinner RD, Cobb M, Andersen B, Garcia-Rill E. Midlatency auditory evoked potentials in the rat - effects of interventions which modulate arousal. Brain Res Bull 1999;48:545-53. Homma Y, Teneud L, Skinner RD, Garcia-Rill E. Effects of propofol on the sleep state-dependent P13 midlatency auditory evoked potential in the rat. Brain Res Bull 2003;61:189-96. Miyazato H, Skinner RD, Garcia-Rill E. Neurochemical modulation of the P13 midlatency auditory evoked potential in the rat. Neurosci 1999;92:911-20. Mamiya N, Buchanan R, Wallace T, Skinner RD, Garcia-Rill E. Nicotine suppresses the P13 auditory evoked potential by acting on the pedunculopontine nucleus in the rat. Exp Brain Res 2005;164:109-19.
SLEEP, Vol. 31, No. 12, 2008
20. 21. 22. 23. 24.
25.
1654
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 2nd ed. Orlando, FL: Academic Press; 1986. Miyazato H, Skinner RD, Garcia-Rill E. Locus coeruleus involvement in the effects of immobilization stress on the P13 midlatency auditory evoked potential in the rat. Prog Neuropsychopharmacol Biol Psychiatry 2000;24:1177-201. Vincent SR, Satoh K, Armstrong DM, Fibiger HC. NADPHdiaphorase: A selective histochemical marker for the cholinergic neurons in the pontine reticular formation. Neurosci Lett 1983;43:31-6. Miyazato H, Skinner RD, Crews T, Williams K, Garcia-Rill E. Serotonergic modulation of the P13 midlatency auditory evoked potential in the rat. Brain Res Bull 2000;51:387-91. Teneud L, Miyazato H, Skinner RD, Garcia-Rill E. Cholinergic modulation of the sleep state-dependent P13 midlatency auditory evoked potential in the rat. Brain Res 2000;884:196-200. Garcia-Rill E, Skinner RD. The sleep state-dependent P50 midlatency auditory evoked potential. In: Lee-Chiong TL, Carskadon MA, Sateia MJ, eds. Sleep medicine. Philadelphia: Hanley & Belfus; 2001:697-704. Garcia-Rill E, Charlesworth A, Heister D, Ye M, Hayar A. The developmental decrease in REM sleep: The role of transmitters and electrical coupling. Sleep 2008:31;673-90. Ballon JS, Feifel D. A systematic review of Modafinil: Potential clinical uses and mechanisms of action. J Clin Psychiatry 2006;67:554-66. Evans WH, Boitano S. Connexin mimetic peptides: specific inhibitors of gap-junctional intercellular communication. Biochem Soc Trans 2001;29:606-12. He DS, Burt JM. Mechanism and selectivity of the effects of halothane on gap junction channel function. Circ Res 2000;86:1-10. Gigout S, Louvel J, Kawasaki H, et al. Effects of gap junction blockers on human neocortical synchronization. Neurobiol Dis 2006;22:496-508. Gareri P, Condorelli D, Belluardo N, et al. Anticonvulsant effects of carbenoxolone in genetically epilepsy prone rats (GEPRs). Neuropharmacology 2004;47:1205-16. Srinivas M, Hopperstad MG, Spray DC. Quinine blocks specific gap junction channel subtypes. Proc Natl Acad Sci U S A 2001;98:10942-7. Gajda Z, Szupera Z, Blaszo G, Szente M. Quinine, a blocker of neuronal Cx36 suppresses seizure activity in rat neocortex in vivo. Epilepsia 2005;46:1581-91. Cruikshank SJ, Hopperstad M, Younger M, Connors BW, Spray DC. Potent block of Cx36 and Cx50 gap junction channels by mefloquine. Proc Natl Acad Sci U S A 2004;101:12364-9. Rouach N, Segal M, Koulakoff A, Giaume C, Avignone E. Carbenoxolone blockade of neuronal network activity in culture is not mediated by an action on gap junctions. J Physiol 2003;553:729-45. Thompson AJ, Lochner M, Lummis S. The antimalarial drug quinine, chloroquinine and Mefloquine are antagonists at 5-HT3 receptors. Br J Pharmacol 2007;151:666-77.
Modafinil and Gap Junctions—Beck et al
