Endogenous Melatonin is Not Obligatory for the Regulation of the Rat ...

ENDOGENOUS MELATONIN AND SLEEP-WAKE REGULATION

Endogenous Melatonin is Not Obligatory for the Regulation of the Rat SleepWake Cycle Simon P. Fisher, PhDa; David Sugden, PhD Division of Reproduction and Endocrinology, School of Biomedical and Health Sciences, King’s College London, London, UK

Study Objectives: Though melatonin and melatonin receptor agonists are in clinical use and under development for treating insomnia, the role of endogenous melatonin in the regulation of the sleep-wake cycle remains uncertain. Some clinical case reports suggest that reduced nocturnal melatonin secretion is linked to sleep disruption, but pineal-gland removal in experimental animals has given variable results. Design: The present study examined the effects of pinealectomy on the diurnal sleep-wake cycle of rats implanted with a radiotransmitter to allow continuous measurement of cortical electroencephalogram, electromyogram, and core temperature (Tc) without restraint in their home cages. Measurements and Results: Tc was slightly (0.2oC) but significantly lower after pineal removal. The total amount and diurnal distribution of locomotor activity, wake, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep were unaltered in pinealectomized rats compared to sham-operated controls. Sleep consolidation measured by determining wake, NREM sleep, and REM sleep bout length and frequency was also unchanged. The EEG power spectrum during NREM sleep was unchanged, but a significant decrease in theta power (5-8 Hz) during REM sleep episodes was found. Conclusions: Our data provide no evidence that endogenous circulating melatonin plays a role in regulating the sleep-wake cycle in rats. However, because cortical theta oscillations are generated in the CA1-3 layer of the hippocampus, neurons known to express melatonin receptors, this suggests that a lack of melatonin following pineal removal influences the function of these neurons and is consistent with previous work suggesting that endogenous melatonin is an important regulator of hippocampal physiology. Keywords: Pinealectomy, sleep, rat, radiotelemetry, melatonin Citation: Fisher SP; Sugden D. Endogenous melatonin is not obligatory for the regulation of the rat sleep-wake cycle. SLEEP 2010;33(6):833-840.

A SLEEP-PROMOTING �� ACTION OF �� MELATONIN �� FOL� �� LOWING ADMINISTRATION TO MAN AND EXPERI� MENTAL ANIMALS HAS OFTEN BEEN REPORTED, though the effectiveness of melatonin has been questioned.1,2 Nevertheless, a novel melatonin receptor agonist was approved by the US Food and Drug Administration for the treatment of insomnia in 2005,3 and additional melatonin-related com� pounds or�� melatonin formulations are in various stages of clini� cal development.4-8 However, the role of endogenous melatonin in the regulation of the sleep-wake cycle remains uncertain. There are several published clinical case reports that link sleep disruption to impaired pineal melatonin secretion as a result of surgery to remove a pineal tumor.9-11 In many of these stud� ies, administration of exogenous melatonin did improve sleep consolidation; however, it is difficult to know if impaired me� latonin secretion, the initial tumor, or trauma associated with its surgical removal was responsible for the sleep disturbances reported. In addition, patients with Smith-Magenis syndrome, a rare neurodevelopmental condition, exhibit an inverted pat� tern of melatonin secretion and commonly suffer from daytime sleepiness and disturbed nocturnal sleep.12 This would sug� gest that melatonin secretion at inappropriate times may have a significant impact on sleep-wake regulation. Again, it’s not clear if the reversed melatonin rhythm and sleep disturbances arise from a common neurologic problem, though other circa�

dian output pathways seem to function normally (e.g., cortisol, growth hormone, and temperature rhythms).13 Evidence against an important role comes from another study that reported an absence of detectable plasma melatonin in 3 tetraplegic patients because disruption of the neural pathway to the pineal gland prevented melatonin synthesis, yet normal sleep-wake patterns were reported.14 In animal studies, Huber et al.15 examined sleep regulation in 3 strains of mice, 1 of which (C57BL/6J) does not synthe� size endogenous melatonin because of a mutation in the Aanat gene, resulting in a severely truncated, nonfunctional AANAT protein.16 C57BL/6J mice do not show any major differences in their sleep-wake cycle, compared with mice that do produce melatonin.15 There is also little consensus in the studies that have examined the effect of surgical pinealectomy on sleep in the rat. Rechtschaffen et al.17 failed to identify any difference in the amount of non-rapid eye movement (NREM) or rapid eye movement (REM) sleep in pinealectomized rats housed under a 12:12 light:dark cycle. Mouret et al.18 also found no change over 24 hours but reported an increase in REM sleep during the dark period and a decrease during the light period. In con� trast, Mendelson and Bergmann19 reported a modest increase in NREM sleep but no change in REM sleep. Finally, Kawakami et al.20 examined pinealectomized rats housed in constant dark� ness and reported a decrease in the amplitude of the circadian rhythm of NREM and REM sleep. Human studies in which melatonin has been administered have typically given doses that raise blood melatonin levels well beyond their usual nocturnal physiologic range (~100 pg/mL, reviewed by Sack et al.21). Few studies have utilized doses that result in blood levels similar to nocturnal levels. However, the work published by Dollins et al.22 and Zhdanova et al.23 gave low doses (0.1 - 0.3 mg) of melatonin, which elevated blood mela�

Submitted for publication July, 2009 Submitted in final revised form November, 2009 Accepted for publication December, 2009 Address correspondence to: Dr. David Sugden, Division of Reproduction and Endocrinology, Room 2.12N Hodgkin Building, King’s College London, London SE1 1UL, UK; E-mail: [email protected] SLEEP, Vol. 33, No. 6, 2010

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Pineal Removal and Sleep—Fisher and Sugden

tonin concentration to typical nighttime levels, and did produce a reduction in sleep latency but no change in sleep time, efficiency, or consolidation. The aim of the present study was to perform an in-depth analysis of the effect of pinealectomy on the sleep-wake param� eters in the rat to examine the physiologic role of endogenous melatonin. The study utilized radiotelemetry to enable longterm continuous recording of electroencephalogram (EEG) and electromyogram (EMG) to allow discrimination of wake, NREM sleep, and REM sleep in freely behaving, unrestrained, and unstressed rats in their home environment.

end of the dark period (i.e., 19:00-07:00). Urine samples were stored at -20oC until assay and then diluted 1:50 or 1:250 as appropriate with assay buffer. Urinary aMT6s was measured in duplicate using a sensitive radioimmunoassay previously vali� dated for rat urine, which uses iodinated aMT6s26 purchased from Stockgrand Ltd (University of Surrey, UK). Creatinine Assay Because 12-hour urine volume could not be accurately meas� ured (because of evaporation), aMT6s excretion was stand� ardized per unit of creatinine excreted. There is little tubular reabsorption of creatinine; therefore, it can be used as an indica� tor of the glomerular filtration rate of the kidneys.27 Creatinine concentration was determined colorimetrically with the Jaffé reaction using a creatinine assay kit (CR510, Randox Laborato� ries Ltd, UK) on duplicate aliquots of each urine sample.28

METHODS Animals Male Sprague-Dawley rats (250-300 g, Harlan, UK) were housed individually in a humidity-controlled, quiet room with food and water provided ad libitum. Room temperature was main� tained between 20oC and 22oC, and rats were maintained under a 12-hour light/12-hour dark cycle with lights on from 07:00 to 19:00, with light intensity during the day approximately 80 µW/ cm2 at cage level. An environmental climate monitor (SwiftBase International, Ltd., UK) was installed to continuously record room temperature and light level and ensure no unexpected changes in room temperature or L:D schedule. All �� procedures were in accor� dance with the UK Animal Scientific Procedures Act (1986).

Sleep studies Two weeks after transmitter implantation, baseline EEG, EMG, Tc, and locomotor activity were recorded for a 48-hour period in 6 sham-operated and 6 pinealectomized rats. EEG, EMG, Tc, and locomotor activity data were transmitted to a Data Sciences radio receiver (RPC-1) placed underneath each rat cage. Signals were then routed via a data exchange matrix to a PC running Dataquest A.R.T. software (version 3.01). The EEG and EMG data were continuously sampled using DSI Dataquest Gold acquisition software at 500 Hz, with a 100-Hz filter cutoff. EEG and EMG signals were band-pass filtered (0.5-35 Hz for EEG and 80-120 Hz for EMG) and then used to identify vigilance states. Sleep-wake stages were scored as wake, NREM sleep, and REM sleep in 10-second epochs using a semiautomated approach, as has been described previously.25 The sleep-scoring procedure consisted an automated initial step using SleepSign® software (Kissei Comtec, Nagano, Japan), fol� lowed by a review of all epochs by an experienced sleep scorer to eliminate errors in the assignment of wake, NREM sleep, and REM sleep stages using the EEG/EMG criteria defined pre� viously.29 The total percentage of each vigilance state for the 12-hour light, 12-hour dark, and total 24-hour period was de� termined. Mean bout duration and frequency for wake, NREM sleep, and REM sleep were compared for pinealectomized and sham-operated rats. Mean bout duration was defined as the mean duration of each sleep-wake stage (in seconds) calculated per hour. Frequency was defined as the number of occurrences of a particular sleep-wake stage per hour. To obtain power spectra, the EEG of all NREM and REM epochs for each individual rat were analyzed offline using fast Fourier transformation (512-point Hanning window, 0.5-30 Hz with 1-Hz resolution) using the built-in function within Sleep� Sign.® For each rat, power for each 1-Hz frequency bin was expressed as a percentage of total power (0.5-30 Hz). Mean percentage total power was then calculated for sham-operated and pinealectomized groups. Urinary aMT6s concentration in the dark and light periods were compared using a Student’s t test (Figure 1), whereas the analysis of the effect of pinealectomy on locomotor activity, Tc, and vigilance states utilized an unpaired Student t test (Figure 2 and Table 1). Time-course and EEG power spectra comparisons between sham-operated and pinealectomized groups used a

Pinealectomy and Surgical Implantation of the Radiotelemetry System Rats were anesthetized with ketamine (75 mg/kg intraperito� neal; Fort Dodge Animal Health Ltd, Southampton, UK) com� bined with medetomidine (0.5 mg/kg ip; Pfizer, Sandwich, UK). The pineal gland was removed using the method described by Hoffman and Reiter.24 Sham operations were performed in an identical fashion, with the exception that the bone disc above the pineal was not fully removed to prevent damage to the gland or its nerve supply. Two weeks after surgery, 12-hour urine collections were made for measurement of 6-hydroxymelatonin sulfate (aMT6s) in urine to confirm complete removal of the gland. Pinealectomized rats exhibiting a significant increase in urinary aMT6s during the 12hour dark period were excluded from further experimentation. Pinealectomized and sham-operated rats were implanted with a radiotelemetry transmitter (Model TL11M2-F40-EET, Data Sciences International, St. Paul, MN) into the peritoneal cav� ity under ketamine/medetomidine anesthesia.25 EEG leads were connected to stainless-steel screws placed on the skull (2 mm an� terior to lambda on the right hand side, 2 mm anterior to bregma on the left hand side) with the screw tip resting on the dura. EMG leads were inserted bilaterally into either side of the musculus cervicoauricularis and sutured in place. Core body temperature (Tc) and locomotor activity data were obtained from the body of the implant in the abdomen. All implanted rats remained healthy and gave clear EEG/EMG recordings allowing discrimination of sleep-wake stages for longer than 6 months. Radioimmunoassay of aMT6s Twelve-hour urine samples were collected from individual rats at the end of the light period (i.e., 07:00-19:00) and the SLEEP, Vol. 33, No. 6, 2010

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2-way analysis of variance. Interactions between treatment and time of day or EEG frequency were also assessed. Posthoc mul� tiple comparisons were made using the Bonferroni test (Figures 3 to 6). Any differences were considered statistically significant when P was less than 0.05. RESULTS Urinary aMT6s Excretion The radioimmunoassay for aMT6s was sensitive (2 pg/tube could be measured) and linear to 100 pg/tube (R2 = 0.995). The intraassay coefficient of variation was 14% at 1.25 ng/mL (n = 6) and 9.5% at 16.25 ng/mL (n = 5). Serial dilution of a urine sample collected during the night from a sham-operated rat gave a line that was linear (R2 = 0.989) and parallel to the aMT6s standard curve. In sham-operated rats, urinary aMT6s concentration was ap� proximately 7-fold higher during the dark period (11.9 ± 2.5 ng/mg creatinine) than during the light period (1.6 ± 0.2 ng/mg creatinine, mean ± SEM, n = 6; P < 0.01, Figure 1). In pinealec� tomized rats, both daytime and nighttime urinary aMT6s was significantly lower than excretion in sham-operated rats during daytime (P < 0.0001) and showed no significant diurnal differ� ence (light period, 0.39 ± 0.08 ng/mg creatinine; dark period 0.33 ± 0.03 ng/mg creatinine, mean ± SEM, n = 6).

Figure 1—Excretion of 6-hydroxymelatonin sulfate (aMT6s) in urine collected at the end of the light and dark periods in pinealectomized (Pinex) and sham-operated (Sham) rats, expressed as nanograms of aMT6s per mg creatinine. Data are mean ± SEM (n = 6) for each group. **P < 0.01 significant difference between light and dark samples in shamoperated rats.

Effect of pinealectomy on locomotor activity and Tc There were no significant differences in either the mean lo� comotor activity during the entire 24-hour period or the light and dark periods of sham-operated and pinealectomized rats (Figure 2A), though, as expected, higher activity levels were exhibited during the dark period. Continuous recording of over 48 hours revealed a clear diurnal rhythm in Tc in both shamoperated and pinealectomized rats (data not shown). Mean Tc Table 1—Amounts of the 3 vigilance states (wake, NREM sleep, and REM sleep) during an entire 24-hour period, 12-h hour light and 12-h hour dark periods, and the light-dark difference in sham-operated and pinealectomized rats Surgery Sham-operated Period Wake, h 24-h 12.61 ± 0.11 12-h L 3.91 ±0.19 12-h D 8.69 ± 0.26 LD difference 4.78 ± 0.47

NREM, h 8.89 ± 0.05 6.10 ± 0.20 2.79 ± 0.19 3.31 ± 0.38

REM, h 2.50 ± 0.09 1.99 ± 0.03 0.52 ± 0.09 1.47 ± 0.11

NREM, h 9.15 ± 0.15 5.99 ± 0.10 3.16 ± 0.10 2.83 ± 0.14

REM, h 2.49 ± 0.08 1.88 ± 0.05 0.61 ± 0.04 1.27 ± 0.05

Pinealectomized Period Wake, h 24-h 12.36 ± 0.12 12-h L 4.14 ± 0.08 12-h D 8.23 ± 0.10 LD difference 4.09 ± 0.16

Figure 2—The effect of pinealectomy on locomotor activity (A) and mean core body temperature (Tc; B) measured over 24-h, or the 12-h light and 12-h dark periods. Values shown are mean ± SEM for 6 pinealectomized (Pinex) and 6 sham-operated (Sham) rats. *P < 0.05 significant difference between Sham and Pinex rats.

Data are averaged for 2 separate diurnal cycles and is the mean ± SEM of 6 rats. NREM refers to non-rapid eye movement; REM, rapid eye movement; L, light period; D, dark period. SLEEP, Vol. 33, No. 6, 2010

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and pinealectomized rats (Figure 6). The NREM EEG spectra gave a peak power at 1.5 to 3.5 Hz and showed no significant differences between sham-operated and pinealectomized rats at any frequency band. However, for REM sleep episodes, pine� alectomized rats, compared with sham-operated rats, exhibited a highly significant decrease (P < 0.001) in power in the theta range (5-8 Hz), which was evident in both the light and dark periods (Figure 6). DISCUSSION In sham-operated rats housed under a 12:12 light:dark cycle, a robust diurnal rhythm in urinary aMT6s was clearly evident. However, in pinealectomized rats, there was no significant dif� ference in the concentration of aMT6s in urine collected dur� ing the day (light period) and night (dark period) periods, and aMT6s in pinealectomized rats was only approximately 3% of the value seen in the urine of sham-operated rats at night. This confirms that surgery to remove the gland was successful. In addition, the finding that both daytime and nighttime levels in pinealectomized rats were significantly lower (only 22% of sham levels; P < 0.0001) than daytime urinary aMT6s in shamoperated rats indicates that the entire gland was removed. Uri� nary excretion of aMT6s, the major metabolite of melatonin, has been previously shown to be a reliable surrogate of blood melatonin levels, reflecting the nocturnal synthesis of melaton� in by the pineal gland.30 Pineal gland removal did not change the total locomotor activity of rats, nor did it alter the diurnal rhythm of activity. This finding agrees with an earlier report that failed to detect any change in locomotor activity or rhythm.31 In contrast, a small, but significant, decrease in mean Tc over 24 hours (Tc ~0.2oC) was observed after pineal gland removal. A small fall in mean Tc during the light and dark periods was also appar� ent but failed to reach statistical significance (P = 0.054 in the light period; P = 0.10 in the dark period). A previous report also found a small decrease (~0.3oC) in mean Tc after pinealec� tomy,32 which the authors suggested was related to a change in setpoint. It’s interesting to speculate that the small change in Tc may reflect a change in heat-loss mechanisms mediated by circulating melatonin. Previous in vitro work33,34 has shown that physiologic concentrations of melatonin can activate mela� tonin receptor-mediated changes in the tone of the rat tail artery, a major mechanism for regulating heat loss in this species. In pinealectomized rats, loss of circulating melatonin may impair nocturnal vasoconstriction in the tail, leading to enhanced heat loss and the small decrease in Tc observed. Regulation of pe� ripheral heat loss may not be the only mechanism involved, as the small change in Tc after pineal gland removal was apparent also during the light period. In the present study, sham-operated rats spent approximately 52% of each 24-hour period in wake, 38% in NREM sleep, and 10% in REM sleep. These proportions are comparable to sleepwake data previously reported by other investigators (reviewed by 35), indicating that the semiautomated sleep-wake scoring method used accurately measured the sleep-wake cycle. In ad� dition, as expected, a clear and stable diurnal rhythm in wake, NREM sleep, and REM sleep was apparent in all rats recorded. Because telemetry implants were used to record EEG and EMG for determination of sleep, each rat was recorded in its normal

Figure 3—Diurnal distribution of non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in pinealectomized (Pinex) and sham-operated (Sham) rats. Shaded area indicates the dark period of the Light:Dark cycle. Data are presented as the percentage of time in each hour and are mean values ± SEM from 6 rats per group.

(Figure 2B) was slightly, but significantly, lower in pinealec� tomized rats (37.4oC ± 0.05oC, mean ± SEM, n = 6; P = 0.04) compared with sham-operated animals (37.6oC ± 0.04oC, mean ± SEM, n = 6). When mean Tc was compared separately for the light and dark periods, there was a tendency for the mean Tc to decrease in pinealectomized rats, although this just failed to reach statistical significance for both periods (P = 0.054, light period; P = 0.10, dark period). Effect of Pinealectomy on the Sleep-Wake Cycle Pinealectomy had no effect on the percentage of time rats spent in wake, NREM sleep, and REM sleep over the whole 24hour period or on the percentage of time spent in each vigilance state during the 12-hour light period and 12-hour dark period (Table 1). An hour-by-hour analysis of NREM and REM sleep in sham and pinealectomized rats over 48 hours also showed no significant differences in the diurnal distribution of time spent in NREM and REM sleep (Figure 3). An hour-by-hour analysis of the mean duration of each bout of wake, NREM sleep, and REM sleep throughout the 48-hour recording period showed that wake-bout duration was consist� ently low during daytime and was increased and somewhat more variable during the night. NREM sleep-bout duration clearly in� creased following light onset but then declined gradually during the day (Figure 4). REM sleep-bout duration showed a prominent day-night difference clearly linked to the light-dark transition, with episodes approximately 4-fold longer during the daytime than at night. Pinealectomy did not significantly alter mean wake, NREM, or REM bout duration or its diurnal distribution. An hourly analysis of the mean frequency of wake, NREM, and REM bouts showed a clear diurnal pattern with increased bout frequency for all 3 vigilance states during daytime (Fig� ure 5). Thus, during the daytime, NREM and REM episodes are more frequent and longer than at night, whereas rats have more but shorter wake episodes during daytime than at night. No significant differences in bout frequency were seen between sham-operated and pinealectomized rats. The EEG power spectra during NREM sleep epochs during both the dark and light period were compared in sham-operated SLEEP, Vol. 33, No. 6, 2010

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Figure 4—Bout duration analysis for Wake (A), non-rapid eye movement (NREM) sleep (B), and rapid eye movement (REM) sleep (C) in pinealectomized (Pinex) and sham-operated (Sham) rats. Shaded area indicates the dark period of the light:dark cycle. Data points represent mean hourly values ± SEM for 6 rats per group.

Figure 5—Bout frequency analysis for Wake (A), non-rapid eye movement (NREM) sleep (B), and rapid eye movement (REM) sleep (C) in pinealectomized (Pinex) and sham-operated (Sham) rats. Shaded area indicates the dark period of the light:dark cycle. Data points represent mean hourly values ± SEM for 6 rats per group.

home-cage environment, avoiding the need for any handling or restraint during data collection, preventing potential restriction of movement, mild stress, or anxiety associated with tethered systems for monitoring sleep. Because the transmitter implant� ed in the abdomen is small (~2% of the body weight of the rat), it had no acute or long-term effect on behavior, growth, or diurnal rhythms. Pineal removal did not significantly alter the proportion of time spent in wake, NREM sleep, or REM sleep measured over an entire 24-h light:dark cycle. Nor was there any change when wake, NREM, and REM data were calculated separately for the 12-hour light and 12-hour dark periods (Table 1). Further, an hour-by-hour analysis of mean wake, NREM sleep, and REM sleep for sham-operated and pinealectomized rats failed to reveal any significant differences at any single time point (Figure 3). A more detailed analysis of wake, NREM, and REM bout duration and frequency was undertaken to investigate the pos� sibility that consolidation of vigilance states might be altered by the absence of circulating melatonin after pineal removal. Bout

frequency and duration for each vigilance state after pinealec� tomy have not been examined in previous studies.17-20 In shamoperated rats, wake bout frequency showed a distinct increase at light onset and a sharp fall soon after dark onset (Figure 5). Wake-bout duration (Figure 4) was consistently low during the light period but was generally longer, but more variable, during the dark period. Thus, during the daytime, rats have frequent but short periods of awakening, whereas, at night, wake bouts are fewer but longer. For NREM and REM sleep, a clear diur� nal rhythm in both bout duration and frequency was apparent; a sharp increase in bout frequency and, in particular, bout dura� tion was observed on both recording days immediately follow� ing light onset. The clear diurnal pattern in wake, NREM sleep, and REM sleep bout frequency and duration were not altered by pinealectomy, suggesting that circulating melatonin has no apparent influence on the mechanisms that regulate sleep-wake consolidation in the rat. Taken together, these findings indicate that a physiologic level of endogenous circulating melatonin is not obligatory for the regulation of the diurnal sleep-wake cycle

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Figure 6—Effect of pinealectomy on non-rapid eye movement (NREM) and rapid eye movement (REM) electroencephalographic (EEG) power spectra. Mean power spectrum for NREM sleep during the light (A) and dark (B) period and mean power spectrum for REM sleep during the light (C) and dark (D) period. Each point shown represents the mean percentage total power ± SEM in each 1-Hz frequency band for 6 pinealectomized (Pinex) and sham-operated (Sham) rats. ***P < 0.001 significant difference between Sham and Pinex rats at 7.5 Hz.

in rats housed under a typical alternating light-dark photoperiod. The diurnal sleep-wake cycle under these conditions probably reflects both circadian and homeostatic regulation; thus, it’s un� likely that physiologic levels of circulating melatonin influences either of these mechanisms. �� However, it’s important to recog� nize that the current study used a 12-hour:12-hour light-dark cycle with abrupt light and dark transitions (as is common in animal housing facilities), which is not an environmental light� ing regime that rats would typically encounter in nature. Clearly, in the present experiments, pineal removal had little effect on the sleep-wake cycle, but we cannot rule out a role of endogenous melatonin in rats housed in a more naturalistic environment. This would include exposure to gradual changes in the intensity and the spectral composition of light at dawn and dusk and giv� ing these nocturnal animals the opportunity to escape light and self-select the photoperiod to which they are exposed. Some previous studies have also failed to detect any change in the daily amount of NREM or REM sleep.17,18,20 In contrast, Mendelson and Bergmann19 found a significant increase in SLEEP, Vol. 33, No. 6, 2010

NREM sleep and, in addition, reported that pinealectomized rats did not show the expected increase in NREM sleep dur� ing rebound from 24-hour total sleep deprivation. A change in the amplitude of the circadian20 or diurnal18 rhythm in REM sleep has been previously observed, though only the former study reported a decrease in the diurnal rhythm in NREM sleep. However, none of these previous studies reported measurement of plasma melatonin or its major metabolite, aMT6s, in urine in either sham-operated or pinealectomized rats to check the success of pineal gland removal. Furthermore, previous stud� ies employed a tethered recording setup, which may have per� turbed natural sleep-wake behavior, perhaps accounting for the sleep changes reported in earlier work,17-20 whereas our studies employed telemetry to allow natural sleep recording without perturbation of the animals in their home cage. A comparison of the EEG power spectrum during NREM sleep in sham-operated and pinealectomized rats did not show any significant change in EEG power in the delta band (0.53.5 Hz; often referred to as slow-wave activity). Hypnotic 838


ACKNOWLEDGMENTS This work was supported by a project grant from the Well� come Trust (ref. GR065816) and a Medical Research Council CASE studentship to Simon Fisher. We are grateful to Dr Alex Kulla (Eli Lilly, UK) for his expert assistance in setting up the SleepSign® scoring software and to Rachel Tanner (Eli Lilly, UK) for her advice on telemetry surgery. a Dr. Fisher’s current affiliation is with the Circadian and Vi� sual Neuroscience Group, Nuffield Laboratory of Ophthalmol� ogy, The John Radcliffe Hospital, University of Oxford, UK.

drugs such as zolpidem and increased sleep pressure follow� ing sleep deprivation increase EEG delta power during NREM sleep,36,37 which is thought to represent a quantitative estimate of the homeostatic sleep process. In contrast to NREM sleep, a highly significant (P < 0.001) decrease in theta power at 7.5 Hz occurred during REM sleep. This was apparent in REM sleep episodes recorded during both the light period and the dark period (Figure 6). Because the power spectrum was al� tered by pinealectomy only for REM and not NREM sleep, it seems unlikely that the decrease observed is an artifact caused by differences in electrode placement. Furthermore, a simi� lar decrease occurred in REM sleep recorded during the light period (when there is little circulating melatonin even in in� tact rats) and the dark period (when melatonin is elevated in sham-operated rats but essentially absent from the circulation in pinealectomized rats). This suggests that the change in EEG power during REM sleep is not simply due to an acute lack of melatonin. Theta oscillations (5-8 Hz) are one of the hallmark electro� physiologic characteristics of the mammalian hippocampus.38 In lower mammals such as rodents, theta activity is associated with specific behavior states, namely REM sleep, and loco� motor activity, including exploratory behaviors.39 These theta oscillations are generated in CA1 and CA3 pyramidal neurons in the hippocampus.38 High-affinity melatonin-binding sites have been reported in the hippocampal region,40 as well as MT1 and MT2 receptor mRNA transcripts,41 and both receptor sub� type proteins, identified by immunohistochemistry, have been localized in pyramidal neurons of the hippocampal CA1-4 region.42,43 Furthermore, application of melatonin alters excit� ability and synaptic transmission in the hippocampus41,44 and in� hibits hippocampal long-term potentiation via an MT2-receptor subtype,45 and endogenous melatonin suppresses memory con� solidation after nighttime acquisition.46 �� Interestingly, pinealec� tomy was recently reported to reduce hippocampal CA1 and CA3 cell number, which was reversed by melatonin administra� tion.47 The change in theta power during REM sleep observed in the present study after pineal removal and the loss of circulat� ing melatonin may reflect the loss of CA1-3 neurons or changes in their intrinsic excitability or an alteration of synaptic plastic� ity in the hippocampus. Clearly, the absence of a nocturnal peak in circulating mela� tonin after pinealectomy does not have a dramatic effect on rat sleep, nor on the diurnal organization of the sleep-wake cycle, nor on sleep consolidation judged by the lack of effect on wake, NREM sleep, and REM sleep bout frequency or duration. The findings suggest that endogenous circulating melatonin has no role in the generation, organization, or diurnal timing of sleep. Because administration of melatonin itself has been reported to increase sleep and melatonin agonists are used as a treatment for insomnia, this raises important questions about the mechanism or mechanisms by which the sleep-promoting effect is gener� ated. Although the affinity of melatonin for its receptors is very high (typical Ki < 1 nM), relatively large doses of melatonin are usually administered to give a sleep-promoting effect, and this may lead to concentrations much greater than necessary for selective melatonin-receptor activation. Further studies are needed to clarify the role of melatonin receptors in the sleeppromoting action of administered melatonin. SLEEP, Vol. 33, No. 6, 2010

DISCLOSURE STATEMENT This was not an industry supported study. The authors have indicated no financial conflicts of interest. REFERENCES

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