Heritability of dark pulse triggering of paradoxical sleep in rats

Physiology&Behavior,Vol. 52, pp. 127-131, 1992

0031-9384/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

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Heritability of Dark Pulse Triggering of Paradoxical Sleep in Rats CYNTHIA

LEUNG, BERNARD

M. B E R G M A N N ,

ALLAN RECHTSCHAFFEN

AND RUTH

M. B E N C A l

Sleep Research Laboratory, University of Chicago, Chicago, IL 60637 Received 2 A u g u s t 1991 LEUNG, C., B. M. BERGMANN, A. RECHTSCHAFFEN AND R. M. BENCA. Heritabilityof darkpulse triggeringofparadoxical sleep in rats. PHYSIOL BEHAV 52(1) 127-131, 1992.--A previous study showed that albino Lewis (L) rats could be triggered into paradoxical sleep (PS) by dark pulse stimulation, i.e., turning off cage lights, whereas brown Norway (BN) rats showed no evidence of PS triggering by dark pulses (2). The transmission of the PS triggering behavior was studied in L X [L X BN]F~ hybrid backcross (BC) animals. Albino BC rats increased PS% during 5-minute dark pulses to three times the average PS% for the preceding 5 minutes of lights-on. In contrast, no significant PS triggering was observed in pigmented BC rats. These data support the hypothesis that PS triggering by dark pulse stimulation is related to albinism in these rat strains. The absence of a connection between PS triggering and total daily amounts of PS suggests independent genetic transmission of these two parameters. Genetics

REM sleep

Paradoxical sleep

Rats

Light stimulation

STRAIN-SPECIFIC differences in daily total sleep (TS) amount, paradoxical sleep (PS), and diurnal variations of sleep have been documented in rats and mice (7,22,26). [The term paradoxical sleep is used instead of rapid-eye-movement (REM) sleep because rapid eye movements are not so prominent in rats as in other mammals and were not recorded.] These and other studies have suggested that different sleep parameters may be inherited independently and through different modes of transmission, although specific genes that influence sleep have not been identified. Lisk and Sawyer showed that outbred Sprague-Dawley rats could be triggered into PS by brief dark pulses during the normal light period (15). This finding was confirmed by Rechtschaffen et al., who also demonstrated a negative rebound of PS, i.e., the rats had significantly less PS in the light period immediately following the dark pulses such that daily total PS amounts were conserved (19). This result suggested that regulation of PS induction and PS amount may be under independent control. Increased PS amounts during dark periods have also been demonstrated using short light:dark paradigms (5,19). In addition, when rats were placed on light:dark cycles ranging from 2.5:2.5 to 15:15 minutes, both conservation (19) and increases (5) in total daily PS amounts were observed. Recent studies from this laboratory showed that Lewis (L) and Brown Norway (BN) rats were significantly different in two PS parameters (2,22). Brown Norway rats had significantly more daily PS than L rats; [L X BN]F~ rats, the offspring of L and BN matings, showed intermediate amounts. Lewis rats were triggered into PS by dark pulse stimulation, whereas BN rats showed no

evidence of PS triggering, even when the dark pulses interrupted high background light levels. Of particular interest was the fact that L and Sprague-Dawley rats, which are both albino strains, showed PS triggering, but the pigmented BN rats did not. Pigmented [L x BN]Ft rats also failed to show PS triggering, as described below. To determine if dark pulse triggering in these strains of rats is related to albinism, we undertook a comparison of PS triggering in albino vs. pigmented rats using backcross (BC) animals, produced by mating [L x BN]FI hybrid rats with inbred L rats. This particular mating strategy was used because resulting offspring would be either homozygous or heterozygous for albinism; our hypothesis was that PS triggering would occur only in homozygous albino BC rats. METHOD Subjects were male rats between 3 and 6 months of age and weighing between 300 and 500 g at the time of surgery. Inbred L (albino) and hybrid [L X BN]F~ (brown) rats obtained from Harlan Sprague Dawley (Indianapolis, IN) were mated using reciprocal crosses (i.e., L females X [L X BN]FI males and [L X BN]F~ females X L males) to produce three phenotypic classes of BC progeny: albino, brown, and hooded. Rats were anesthetized by intraperitoneal injection of ketamine (87 mg/kg) and xylazine (l 3 mggkg). They were chronically implanted with screw electrodes in the skull to record EEG and with silver plate electrodes under the temporalis muscles to record EMG, according to standard procedures for this laboratory

Requests for reprints should be addressed to Ruth Benca, M.D., Ph.D., Sleep Research Laboratory, 5743 S. Drexel Ave., Chicago, IL 60637.

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TABLE 1 DARK PULSE TRIGGERING OF PS IN BC RATS Preceding 5 Minutes Lights-on

5 Minutes Dark Pulse

Following 5 Minutes Lights-on

8.60 ± 2.75 10.88 ± 2.05

24.03 _+ 8.47* 8.70 + 2.99~

4.92 _+ 1.70"t 12.85 ± 2.96

10.15 ± 2.84 13.40 _+ 2.51

28.74 _+ 11.23" 10.91 _+ 3.44~

6.05 ± 2.00t 15.83 ± 3.80

Number of animals in brackets. Values expressed as means +_ SD. All data were obtained at 150 lux background light intensity. * Differs significantly from preceding and following lights-on periods with p < 0.01. f Differs significantly from preceding lights-on periods with p < 0.01. Differs significantly from following lights-on periods with p < 0.05.

(3,4). Electrodes were attached to a nine-pin miniature connector cemented to the skull. On recovery, rats were attached to a recording cable suspended from a c o m m u t a t o r allowing free m o v e m e n t and were placed in individual sound-attenuated recording chambers on a 12-hour light:12-hour dark cycle. Cage temperature was maintained at 26°C. Food and water were available ad lib. Rats were run three at a time with phenotype-to-cage assignment alternated in successive trials to control for differences in environment. After adaptation to the recording cages, 4 days of baseline sleep data followed by 4 days of dark pulse stimulation data were obtained and used for the reported analyses. Cage lights were automatically turned on and off by a computer-directed timer. Beginning 40 minutes into the normal 12hour lights-on period, five-minute dark pulses occurred every

40-

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RESUt.IS The effects of dark pulse stimulation on albino and pigmented BC rats are shown in Table 1 and Fig. 1. Albino rats were trig-

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half hour (i.e., five minutes lights-off lbllowed by 25 minutes lights-on) tbr a total of 17 trials per day. The dark pulse trials were followed by 2 hours, 50 minutes of lights-on to maintain the overall 12:12 light:dark cycle. Light intensity at the cage floor, as measured by an LX-101 Lux Meter (Lutron), was approximately 150 lux during the lights-on period and less than I lux when lights were turned off. Because prior studies had shown that dark pulse triggering of PS increased with more intense background illumination (2,19), all rats that did not show PS triggering at 150 lux were subjected to an additional 2 days of dark pulse stimulation at a light intensity of 3600 lux. Beginning at least 3 days after surgery, lateral EEG, midline EEG containing hippocampal theta, and temporalis E M G activities were recorded continuously on a Grass polygraph, converted to digital signals, and stored in a computerized sleep staging system (4). Each 24 hours of recording was scored in 30second epochs as waking (W), PS, or one of three NREM sleep stages: low EEG voltage (LS), medium EEG voltage (HSI), or high EEG voltage (HS2). Computer scoring was regularly validated against manual scoring of polygraphic output. Diurnal rhythm amplitude and phase of wake and PS were calculated as the amplitude and acrophase of a cosine curve fitted to the mean distribution of waking or PS fractions (in 5minute epochs) of the baseline recording days, as described by Halberg et al. (11). Mean values for various sleep parameters were evaluated using analysis of variance (ANOVA) and Bonferroni-corrected paired comparison t-tests. Triggering was evaluated by the Wilcoxon signed rank test: a rat was considered to trigger if the Wilcoxon test on PS time for the 5 minutes preceding the dark pulse vs. the 5 minutes of the dark pulse produced an increase at p less than 0.05 over all trials for that rat.

PS% of total time

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FIG. 1. Average values of PS percent of recording time (±SE) for successive 30-second epochs of the dark pulses and the preceding and following 5-minute lights-on periods for the 9 albino and 11 pigmented BC rats. For this figure only, data obtained during higher intensity background light level (3600 lux) were included for the single albino animal that failed to show significant triggering at the lower light level.

HERITABILITY OF PS TRIGGERING IN RATS

129

TABLE 2 SLEEP STAGEPERCENTAGESFOR BASELINEVS. TRIGGERINGDAYS

Baseline Albino [9] Pigmented [11] Triggering Albino [9] Pigmented [1 l]

W%

PS%

LS%

HS1%

HS2%

33.49 _+ 1.64 33.48 + 2.01

7.10 + 1.44 7.61 +_0.94

2.88 -+ 0.63 2.99 _+0.75

27.89 _ 1.15 27.40 + 1.00

28.49 + 2.23 27.91 + 1.09

36.59 _+ 3.34 35.51 _+2.40

6.80 _+ 1.17 7.75 _+ 1.19

2.92 _+0.95 2.85 -+ 0.74

25.45 -+ 1.41* 26.13+-- 1.36

28.03 _+2.49 27.56 +_ 1.37

Number of animals in brackets. Wake and sleep stage percentages of total time are expressed as means +_ SD. W, wake; PS, paradoxical sleep; LS low-voltage NREM sleep; HS1 and HS2, medium- and highvoltage NREM sleep, respectively. * Differs significantly from HSI% during baseline at p < 0.02.

gered into PS and showed an average three-fold increase in PS during dark pulses over the preceding 5-minute lights-on periods whether PS was calculated either as a percentage of total time or of total sleep (Table I ). Maximal PS triggering occurred within 1-2 minutes following the initiation of the dark pulses (Fig. 1). A significant negative rebound in PS% was observed for the following 5-minute lights-on periods, i.e., PS amounts during lightson were less following dark pulses than preceding dark pulses. Amounts of TS during the dark pulses and the preceding and following lights-on periods were not significantly different and averaged approximately 80% of total time. Individually, eight out of nine albino rats tested showed PS triggering, as defined by an increase in PS during the 5-minute dark pulse trials as compared to the preceding 5-minute lights-on periods, which was significant by the Wilcoxon signed ranks test. The weakest triggering response was 32 PS increases vs. 18 declines (p = 0.04). The strongest positive response in a nontriggering rat was 20 increases vs. 17 declines (p = 0.5). The single albino rat which did not trigger at 150 lux triggered at a higher background light intensity (3600 lux), with an average twofold increase in PS% during the dark pulses and a negative rebound of PS in the first 5 minutes of the following lights-on periods. None of the 11 individual pigmented BC rats showed PS triggering as defined by the criterion above. Moreover, two pigmented BC rats showed a significant suppression of PS during dark pulses by the same criterion. As a group, pigmented BC rats showed a decrease of 20% in PS percent of time during the dark pulses compared to the preceding 5 minute lights-on periods. This decrease was not significant (p < 0.15) after Bonferroni correction; however, in contrast to the albino BC animals, PS amount in the pigmented BC group was significantly increased in the 5-minute lights-on periods following dark pulses as compared to the dark pulses (Table 1). In addition, three pigmented [L 5< BN]Ft rats tested showed no evidence of PS triggering (results not shown). There were also no significant differences in TS parameters for the 5-minute periods before, during, and after the dark pulse trials. No pigmented rats showed PS triggering when exposed to the higher background light level of 3600 lux (results not shown). Comparisons of brown vs. hooded animals showed no significant differences for any sleep parameters on baseline or dark pulse stimulation days, so data from these two phenotypic groups were combined. A Yatescorrected chi-square test of triggering at 150 lux in pigmented vs. albino BC rats gave a significant (p < 0.001) difference between groups.

Our previous study showed that L rats, with low daily PS%, were triggered into PS by dark pulses and that BN rats, with high daily PS%, were not (2). However, in the present study there were no significant differences in total daily PS% between triggering and nontriggering rats. In the albino BC rats, the correlation between PS% and average magnitude of the triggering response (i.e., PS% during dark pulses/PS% during previous 5minute lights-on periods) was not significant (r = 0.43). Apart from the issue of the significance, the size of the correlation indicated that only a small portion of the triggering effect could be accounted for by baseline PS amount. Thus, PS induction was regulated independently from daily PS amounts in the albino BC rats. The effects of triggering on sleep stage parameters are shown in Table 2. Daily amounts of PS were unchanged by the triggering paradigm. This result agrees with earlier studies which demonstrated that widely spaced dark pulses could trigger PS in albino rats without changing the total daily amount of PS (2,19). There was a small but statistically significant (p < 0.02) decrease in medium voltage NREM (HSI) sleep for baseline vs. triggering days in albino BC rats. However, there were no significant differences in overall wake or sleep stage percentages between albino and pigmented BC rats. Means of amplitudes and phases of circadian wake and PS rhythms are shown in Table 3. Both groups of animals showed significant circadian patterns of wake and PS, although pigmented BC rats had significantly weaker wake rhythms than albino BC rats (p < 0.01). Acrophases of wake and PS rhythms were not significantly different between groups. DISCUSSION All albino BC rats showed PS triggering in response to dark pulses, with all but one albino rat triggering at the standard light level. No pigmented BC rats showed PS triggering. The triggering mechanism is unknown, but deafly the triggering effect was unrelated to differences or changes in daily total sleep, daily PS, or in amounts of TS during the dark pulse trials and the preceding and following lights-on periods. The intensity of the triggering effect is known to vary with light intensity (2,19). Previous studies showed that L rats triggered at light levels as low as 50 lux, although the triggering response was greater at higher light levels. However, to document that light intensity was not a significant factor in the failure of the

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l_titJN(i EI ,\1. TABLE 3 CIRCADIAN RHYTHMS Waking Rhythm

Albino [9] Pigmented [l 1]

PS Rhythm

Amplitude

Acrophase

Amplitude

Acrophase

0.2118 _+0.0357* 0.1414 _+0.0475

95.37 _+ 13.63 103.49 _+ 38.05

0.0432 _+0.0182 0.0378 _+0.0170

310.86 .+_22.70 269.46 +_ 51.22

Number of animals in brackets. Values expressed as means _+ SD. Amplitude is that of the fitted cosine for wake or PS fraction of time. Acrophase is the phase angle of the fitted cosine measured in degrees (0 ° to 180° represents the 12-hour dark phase and 180 ° to 360 ° represents the 12-hour light phase). * Albino waking rhythm amplitude differs significantly from pigmented waking rhythm amplitude with p < 0.01.

pigmented BC rats to show the PS triggering effect, all nontriggering animals were subjected to 2 days of dark pulse trials at a light intensity of 3600 lux. None of the pigmented rats triggered at the higher light level, although the single albino rat that failed to show PS triggering at the standard background light level did trigger at 3600 lux. Furthermore, the standard light level of 150 lux was clearly adequate to maintain circadian rhythms in pigmented as well as albino rats. No pigmented rats triggered in response to dark pulse stimulation, suggesting that ability to trigger PS in rats may be related to albinism. Albino mammals, including rats, exhibit anomalies of the central visual pathways (8-10). In the normal rat brain, the lateral geniculate nucleus contains clearly defined layers of nerve cells, but in albino rats these layers are fused and/or irregular. Also, the optic nerve fibers in albino species show increased crossover to the contralateral hemisphere. Therefore, it is possible that albino rats could have abnormalities in any of several neural pathways leading from the retina to the pontine areas where PS is generated. Previous studies have shown that the tendency for PS varies with circadian phase (12,16). Any abnormalities in the visual pathways associated with albinism could also have consequences for entrainment of circadian rhythms by light and therefore for PS regulation. However, a recent study revealed no significant differences between albino and pigmented rats in pathways from the retina to the SCN (13). Furthermore, it is unlikely that major differences in circadian parameters account for differences in PS triggering since albino and pigmented BC rats showed no significant differences in diurnal rhythms of PS (Table 3). Borbrly and colleagues have shown circadian differences in the susceptibility to trigger PS in albino rats (5,6), with greatest PS triggering occurring during the inactive phase of the circadian cycle. Although our studies did not specifically examine the effects of circadian phase on PS triggering, the data indicate that pigmented BC rats did not show any PS triggering, even though they were tested during their inactive phases. Guillery found that it was not the albino gene itself (the c gene) but the lack of pigmentation which accompanies it that was associated with abnormalities of the visual pathways (8). An association between albinism and sleep was described by Valatx (26), who found that in mice, coat color genes, particularly the albino gene, appeared to correlate with sleep parameters, including PS amounts. But, as in the case of the abnormal visual pathways, the albino gene per se did not appear to be critical, because mice carrying a spontaneous mutation for albinism slept like the pigmented parental strain. Either the c gene responsible for albinism or one or more other genes in close proximity to it may be involved in PS triggering or induction. Examination of

differences between L and BN rats at these other loci may help to localize the position of the genes involved in PS triggering. A previous study had shown PS enhancement in pigmented rats during dark periods using a 10 minute: 10 minute light:dark cycle paradigm (24). The nonalbino DA strain had an increased frequency of PS onsets during dark periods, suggesting that some pigmented strains might show PS triggering by dark pulses. However, the overall increase in PS percentage during dark periods compared to light periods was much lower in the DA rats than in the albino Sprague-Dawley rats; these results are consistent with our findings of differences in PS triggering by clark pulses in albino vs. pigmented strains. Alternatively, it is possible that BN rats have a defective PS triggering response to dark pulses as compared to other nonalbino strains of rats. Discrepancies between our findings and those of Tobler and Borbrly (24) may also be related to differences in the experimental paradigms used, In addition to triggering PS immediately after the switch to lights-off, short light:dark cycles may also cause shifts in PS patterns without specifically triggering PS. In studies using 60:60 minute light:dark cycles (1,6), rats showed increased overall amounts of PS during the dark periods, but the rise in PS began during the late portion of the light phase, before lights were turned off. This phenomenon is different from the triggering of PS as a specific event following the initiation of the dark phase (see Fig. 1). Since data pertaining to the kinetics of the PS induction in DA rats were not shown (24), we cannot determine the extent to which DA rats show dark pulse triggering of PS in this specific sense vs. a synchronization of PS rhythms to short light:dark cycles with phase delays and/or advances, as described for albino rats on short light:dark cycles by Borb~ly (5). In another study of the effects of short light:dark cycles on the sleep of pigmented rodents, it was found that PS% was increased during short light periods compared to dark periods in the golden hamster (25). Similarly, the pigmented BC rats in the present study showed a significantly increased PS% during the lights-on periods immediately after the dark pulses. The magnitude of the difference in PS% between the dark pulses and the following 5-minute lights-on periods was comparable to that seen in the golden hamster (25). It is not clear whether the identification of genes involved in PS regulation and induction might have relevance for clinical disorders marked by abnormal triggering or reduced latency of REM sleep, such as narcolepsy, a heritable disorder characterized by abnormal PS induction. Depressed patients and their close relatives are known to have shortened REM latencies (i.e., the amount of time from sleep onset to REM in these individuals

HERITABILITY OF PS T R I G G E R I N G IN RATS

131

is much shorter than normal) and are hypersensitive to REM induction with cholinergic drugs (21,23). The data from this study imply that strong PS triggering by dark pulses is associated with the albino gene in rats. However, it is not known whether the p h e n o m e n o n is specific to rats, since data on albinos of other species have not been reported. The differential heritability of PS triggering and PS amount, and the independence of both from N R E M parameters in pigmented and albino rats, suggest that different aspects of each sleep stage

(e.g., induction, distribution, and duration) can be inherited through separate groups of genes. ACKNOWLEDGEMENTS This study was supported by a grant from the American Narcolepsy Association, NIH grant NS 27730, and NIMH grant MH 19051 to Ruth M. Benca, and NIMH grants MH 4151 and MH 18428 to Allan Rechtschaffen. We would like to thank William Obermeyer for assistance with statistical analyses.

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