Orexin B saporin lesions in the lateral hypothalamus enhance photic masking of rapid eye movement sleep in the albino rat
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(2) 4. A. Ocampo-Garce´s et al.. lateral hypothalamus (LH). This method destroys neurones harbouring orexin ⁄ hypocretin receptors, including orexinergic neurones. LH-lesioned albino rats entrained to a 12 : 12 LD schedule display a nocturnal preference for REM sleep with a twofold increase in nocturnal REM sleep (Gerashchenko et al., 2001). The displacement of REM sleep preference to the active phase has also been observed in orexin ⁄ ataxin-3 heterozygote rats (Beuckmann et al., 2004; Zhang et al., 2007a), a putative animal model of human narcolepsy– cataplexy (Scammell et al., 2009). In the albino rat, photic input to the retina influences the activity of REM sleep generators, interfering with the circadian- and homeostatic-driven REM sleep propensity, a phenomenon known as photic masking. It has been established that darkness increases and light decreases the probability of NREM to REM sleep transitions in the albino rat (Alföldi et al., 1991; Borbély, 1976; Borbély et al., 1975). In this report, we explore the hypothesis that the REM sleep transposition to the active (nocturnal) phase is due to an exacerbation of photic masking of REM sleep as a consequence of damage to lateral hypothalamic neurones in the albino rat. We used a skeleton photoperiod (SP) protocol as a tool to reveal the impact of LH lesions on the photic masking of REM sleep (Moore-Ede et al., 1982). METHODS Lateral hypothalamic lesions Experiments complied with the policies of the American Physiological Society and were supervised by the Bioethics Committee of the Faculty of Medicine, University of Chile. Twenty-five male Sprague–Dawley rats weighing 250–300 g were kept in individual cages under a 12 : 12 LD schedule, at an ambient temperature of 21–24 C, with water and food ad libitum. Under ketamine (50 mg kg)1) ⁄ xylazine (10 mg kg)1) anaesthesia, rats were placed into a stereotaxic instrument and received bilateral microinjections of 0.5 lL containing 200 ng of orexin-B-saporin conjugate (Advanced Targeting System, San Diego, CA, USA) (n = 19) or saline solution (n = 6) in the lateral hypothalamus (LH), at the following coordinates (Swanson, 1998): 3.5 mm posterior to bregma ± 1.5 mm from the midline and 8.5 mm below the dura close to coordinates described by Gerashchenko et al. (2001). Microinjections were performed using a glass micropipette (20 lm at the tip). Analgesic and antibiotic treatment were administered at the end of this procedure.. Electrode implant At least 3 weeks after the microinjection procedure, rats were anaesthetized with ketamine ⁄ xylazine as above, and implanted with two cranial epidural electrode screws for electroencephalogram (EEG) and two stainless steel neck muscle electrodes for electromyogram (EMG). The electrode array was protected and affixed permanently to the skull by dental acrylic and. anchoring cranial screws. Analgesic (ketoprofen, 5 mg kg)1) and antibiotic (enrofloxacin 0.2 mg kg)1) treatment were administered at the end of this procedure and maintained for 3 days. Thereafter, rats were placed in cages inside a soundisolated cube under a 12 : 12 LD schedule. Illumination during the light phase was set near 500 lux. A dim red light (<10 lux) was placed inside the isolation cube for housekeeping. Sleep recordings After 1 week of recovery, the rats were connected to the sleep recording system by means of a counterbalanced cable attached to a slip-ring. Recordings began after at least 2 days of adaptation to recording conditions. An automated data acquisition system sampled, displayed and stored EEG and EMG. Sampling was performed at 250 Hz after analogue filtering and conditioning of signals by means of an amplifier (Grass Model 15LT; Astromed, Inc., West Wartwick, RI, USA). Two independent scorers assigned 15-s epochs to wakefulness, NREM sleep or REM sleep by offline visual inspection of polygraphic recordings, as described elsewhere (Ocampo-Garcés and Vivaldi, 2002). Experimental protocol Rats were recorded uninterruptedly for at least 3 days under a 12 : 12 LD schedule (LD condition). Under LD, the light phase started at Zeitgeber time (ZT) 00 and ended at ZT 11:59. On the fourth experimental day the rats were transferred to an SP, consisting of continuous darkness interrupted by two 20-min light pulses (500 lux), timed at ZT 00 (morning pulse) and ZT 11:40 (evening pulse), so that light pulses of SP delimit the former light phase presented under the LD condition, as described by Borbély and Neuhaus (1978). We denominate the ZT 00–ZT 11:59 interval as a Ôrest phaseÕ, and the ZT 12–ZT 23:59 interval as an Ôactive phaseÕ for both LD and SP conditions. Reported data were obtained during the last 48 h under the LD condition and during 2 consecutive days under SP, excluding the first day after transference to SP. After completion of the experimental protocol, rat brains were processed for histology and immunohistochemistry. Histology and immunohistochemistry The rats were anaesthetized deeply with 7% chloral hydrate [350 mg kg)1 intraperitoneally) and perfused through the left ventricle with a saline flush (100 mL) followed by 500 mL of 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were postfixed in the same fixative for 2 h and transferred to 30% sucrose with 0.02% sodium azide in PBS until they sank. Brains were sliced in the coronal plane under dry ice, at 50 lm thickness, using a sliding microtome. We obtained three alternate series of sections from each brain. One series was stained with cresyl violet for Nissl substance and the other two were used for immunohistochemistry. Free-floating sections were incubated in 0.3% H2O2 in 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11.
(3) Lateral hypothalamus lesion and masking of REM sleep PBS for 30 min, rinsed in PBS and transferred to the blocking (0.4% Triton-X100, 0.02% sodium azide, 3% normal goat serum in PBS) solution for 1 h. The sections were transferred to the primary antibody incubation solution and left overnight at room temperature. This incubation solution contained either the anti-orexin-A (rabbit polyclonal, from Phoenix Pharmaceutical Inc., Burlingame, CA, USA) diluted 1 : 2 000 in the blocking solution or the melanin-concentrating hormone (MCH) antibody (1 : 20 000 rabbit polyclonal, from Phoenix Pharmaceutical Inc.). The sections were rinsed in PBS for 1 h before being incubated in the secondary antibody solution [biotin-SP-conjugated AffiniPure goat anti-rabbit immunoglobulin G (IgG) (H + L) from Jackson ImmunoResearch, West Grove, PA, USA; diluted 1 : 1 000 in 0.4% Triton-X100, 1.5% normal goat serum in PBS]. After rinsing for 40 min, the sections were incubated for 1 h in the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) diluted 1 : 500 in PBS, rinsed and incubated in a 0.05% 3-3¢ diaminobenzidine hydrochloride solution containing 0.003% H2O2. The specificity of the antibodies we used has been tested by preadsorption of the antisera with the respective antigen: orexin-A (Chen et al., 1999). We also checked and confirmed the published distribution of the antigens in brain tissues. We examined the Nissl-stained sections, and counted orexinA-immunoreactive (ir) or MCH-ir neurones in three sections from the hypothalamus in lesioned and intact rats. Examination of the Nissl-stained sections revealed neuronal injury adjacent to the site of orexin-B-saporin injections, which in many cases involved not only the lateral hypothalamic area (Figs 1 and 2), but also neighbouring regions. Injury to neighbouring regions consisted of a moderate to high reduction in neuronal density, significant glial infiltration and sometimes retraction of the tissue. Statistics Statistical analysis was carried out by means of Intercooled Stata 9.2 (Stata Corporation, College Station, TX, USA) for Windows. The values of relevant results presented throughout the present manuscript correspond to arithmetic averages (± standard errors). The values of several variables were calculated, as stated below, for 2-h blocks, half-day blocks (rest or active phase) and whole day. All rats had at least two consecutive days recorded under the LD condition and 2 days under SP. The 2 days of each condition were averaged for each rat at the corresponding time resolution to obtain a single value for LD and for SP. The achrophase values of sleep–wake states of control rats were estimated by a half-hour moving average and averaged for each rat to obtain a single value for each photoperiod condition and expressed as ZT time. The magnitude of the LH lesion among orexin-B-saporinlesioned rats is expressed as the percentage of the average number of orexin-A-ir neurones observed in control rats. Rats were segregated into three groups according to lesion size: (i) control rats (n = 9), including sham-lesioned rats (n = 6), and animals with failed neurotoxic injection (n = 3), where no 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11. 5. Figure 1. Photomicrographs of a lesion (left column) aimed at the lateral hypothalamic area (LHA), evaluated in adjacent Nissl-stained sections (upper row), orexin-A-immunoreactive (ir) (middle row) and MCH-ir (lower row). The right column shows corresponding sections from an intact rat. Note that at this antero-posterior level, the lesion (indicated by discontinuous lines) was unilateral and involved several hypothalamic nuclei and the caudal part of the anteromedial (AM) thalamic nucleus. AHN: anterior hypothalamic nucleus; ARH: arcuate hypothalamic nucleus; fx: fornix; mtt; mammillothalamic tract; V3: third ventricle; VMH: ventromedial hypothalamic nucleus. Scale bar = 0.5 mm.. cell loss could be detected at the LH after Nissl staining and immunostaining for MCH or orexin-A; (ii) rats with small lesions that killed <49% of orexin-A-ir neurones (n = 4); and (iii) rats with large lesions, >49% of orexinA-ir neurones killed (n = 6). As explained below (see Results), results for the small-lesioned group did not differ from those of control rats, according to a one-way analysis of variance (anova) and post hoc Bonferroni multiple comparison test for factor Ônumber of orexin-A-ir remaining neuronesÕ. A two-way repeated-measures anova for factors ÔphotoperiodÕ (LD versus SP) and Ôphase of dayÕ (rest and active), and the between-factor ÔlesionÕ (control versus large lesion), was performed on the sleep data. A post hoc t-test was performed whenever a significant effect was obtained on factors or interactions: within-subject comparisons for factor LD cycle were performed with a paired StudentÕs t-test and betweensubject comparisons using an unpaired StudentÕs t-test. Nonparametric statistics for matched (WilcoxonÕs signed-rank test) and unmatched data (Mann–Whitney U-test) were applied for percentages. SpearmanÕs non-parametric correlation was used to establish relationships between the size of the lesion and the.
(4) 6. A. Ocampo-Garce´s et al.. sleep parameters, using the whole pool of orexin-B-saporinlesioned and control rats. The number of MCH-ir cells was obtained from a subset of rats (seven control and six lesioned animals). A Pearson correlation was applied to correlate the number of orexin-A-ir and MCH-ir cells.. RESULTS Cell count in control and lesioned rats The control group included six rats that received 0.5 lL saline injections in the LH. Three control rats had failed neurotoxic injections, where no cell loss could be detected at the LH after Nissl staining and immunostaining for MCH or orexin-A. The cell count of orexin-A-ir neurones in the LH of control rats was, on average, 665.5 (±37.8). Sixteen other rats were subjected to bilateral microinjections of 200 ng of orexin-B-saporin conjugate. Five were affected severely during the 3-week postlesion interval and were excluded from the electrode implant. The remaining rats were chronically implanted for polysomnography. One of them was discarded because of the poor quality of EEG recordings. Of the remaining 10 rats, nine completed the recording sessions successfully and one completed the 3-day LD recordings. The 10 lesioned and recorded rats were ranked according to the number of remaining orexin-A-ir cells in the LH. Taking the average control rat cell count as a reference, six rats (Fig. 2) had lesions that affected more than 49% of orexin-A-ir neurones (large-lesioned group). On average, this group presented with 200.6 (±43.7) orexin-A-ir neurones, equivalent to 30.1% of those in the control group. Four rats had lesions that affected less than 49% of orexin-A-ir neurones (smalllesioned group), with an average of 520.5 (±32.8) orexin-A-ir neurones, equivalent to 78.2% of control values. A one-way anova for the three-level factor Ônumber of orexin-A-ir remaining neuronesÕ was significant (F(2,16) = 32.4, P < 0.001). Bonferroni multiple comparisons demonstrated a significant difference between the large-lesioned group and both other groups (P < 0.001).. The orexin-B-saporin lesion affected MCH-ir cells The number of MCH-ir cells was studied in seven control and six lesioned animals. The scatterplot presented in Fig. 3 displays the relationship between the number of the remaining orexin-A-ir neurones and MCH-ir neurones. The associated PearsonÕs correlation coefficient is 0.63 (n = 13, P = 0.02). Thus, orexin-B-saporin lesions affected different neuronal types in the hypothalamus. As a consequence, the loss of orexin-A-ir should be taken as a measure of LH damage, rather than as a cell-specific orexinergic neurotoxic lesion. Sleep states under 12 : 12 LD and SP conditions The main effect of the large LH lesion in rats was the transposition of REM sleep preference to the active phase. Figure 2. Schematic drawings of transverse sections through the lateral hypothalamic area (LHA), showing the six largest lesions, depicted at three levels posterior to bregma. The level in mm is indicated at the lower left of each diagram (Swanson, 1998).. under LD. Large-lesioned rats exhibited on average 50% less REM sleep during the rest phase (P = 0.002) and 82% more REM sleep during the active phase (P = 0.002) than controls (Table 1). No significant differences in the total time spent in each state under the LD schedule were observed. Wakefulness during the active phase of large-lesioned rats was reduced by 11.2%, compared to that of controls (P = 0.043). After being transferred to SP, control and large-lesioned rats displayed different patterns of response (Table 1). 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11.
(5) Lateral hypothalamus lesion and masking of REM sleep Table 1 State amount [minutes ± standard error of the mean (SEM)] and fraction occurring during rest phase (% of whole day ± SEM) in control and large lesioned rats under 12 : 12 light ⁄ dark (LD) and skeleton photoperiod (SP). 2000. MCH-ir cells. 7. 1600. 1200. 800 200. 400. 600. 800. Orexin-A-ir cells Figure 3. Orexin-B-saporin injections in the lateral hypothalamic area (LHA) damaged both MCH-immunoreactive (ir) and orexin-A-ir neurones. Relationship between the number of orexin-A-ir neurons and MCH-ir neurones in the LHA of control (n = 7; open symbols) and orexin-B-saporin lesioned rats (n = 6; closed symbols).. Large-lesioned rats showed a major increase (88.4%) in REM sleep occurring during the rest phase under SP, compared to LD (P = 0.004). Thus, total REM sleep of large-lesioned rats during the rest phase under SP now almost equalled that of controls, whereas the time spent in REM sleep during the active phase remained greater than in control rats (P = 0.017). As a result, the rest phase ⁄ total ratio increased from 44.4 to 63.0% (P = 0.018), a value close to, but still different from, the 73.6% observed among control rats (P = 0.007). Control rats exhibited minor changes in sleep state amount and distribution. In this sense, NREM sleep increased 18% (P = 0.022) and wakefulness dropped 12% (P = 0.011) during the active phase under SP with respect to LD. REM sleep of control rats was largely unaffected after exposure to SP. Large-lesioned rats suffered an 18% reduction in total wakefulness (P = 0.009) and a 14% increase of whole day NREM sleep (P = 0.024). Sleep state mean curves under LD and SP conditions The increase in REM sleep observed among large-lesioned rats under SP was more evident in the first two-thirds of the rest phase (Fig. 4, upper row). In contrast, the amount of REM sleep observed during the active phase was unaffected by the LD cycle condition in this group. These rats also showed transient increases in NREM sleep and reductions of wakefulness during the rest phase. The control group exhibited minor changes after being exposed to SP compared to the LD condition. NREM sleep increased transiently in the early rest phase and wakefulness decreased, mainly during the middle of the active phase. Amount of REM sleep was almost unaffected by the change in the LD schedule in the control group (Fig. 4, lower row). The sleep–wake states acrophases were almost unaffected by photoperiod. The acrophases estimated for REM sleep under LD and SP were ZT 8.8 (±0.5) and 8.3 (±0.8), respectively (P > 0.05). 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11. REM sleep Whole day LD SP Rest phase LD SP Active phase LD SP Rest ⁄ total LD SP NREM sleep Whole day LD SP Rest phase LD SP Active phase LD SP Rest ⁄ total LD SP Wakefulness Whole day LD SP Rest phase LD SP Active phase LD SP Rest ⁄ total LD SP. Control. Large lesion. 106.0 ± 8.3 111.0 ± 7.3. 89.5 ± 8.7 118.0 ± 14.5. 79.1 ± 7.2 79.2 ± 5.5. 39.7 ± 6.1 74.8 ± 10.5*. 27.3 ± 2.6 28.4 ± 2.7. 49.8 ± 6.1 43.3 ± 5.3. 73.9 ± 1.9 73.6 ± 1.8. 44.4 ± 5.7§ 63.0 ± 3.1à,§. 553.0 ± 18.9 595.0 ± 21. 562.0 ± 31.5 644.0 ± 24.4*. 374.0 ± 11.9 367.0 ± 9.9. 346.0 ± 32 371.0 ± 16.4. 179.0 ± 11.3 212.0 ± 12.5*. 216.0 ± 19.9 273.0 ± 15.9. 67.8 ± 1.4 63.6 ± 1.2. 61.2 ± 4.1 57.6 ± 1.6§. 779.0 ± 24.3 731.0 ± 37.7*. 789.0 ± 38.3 678.0 ± 35*. 267.0 ± 16.2 258.0 ± 13.7. 334.0 ± 37.5 274.0 ± 22.7. 512.0 ± 13.1 453.0 ± 25.5*. 454.0 ± 25.2 404.0 ± 20.1. 34.1 ± 1.3 36.4 ± 1.0. 42.0 ± 3.1§ 40.3 ± 1.8. *P < 0.05, paired StudentÕs t-test versus LD. P < 0.05, unpaired StudentÕs t-test versus control. à P < 0.05, WilcoxonÕs signed-rank test versus LD. § P < 0.05, Mann–Whitney U-test versus control. REM, rapid eye movement; NREM, non-rapid eye movement.. Masking of REM sleep as a function of LH lesion magnitude As described, the most striking modification produced by transference to SP was the enhancement of REM sleep during the rest phase of the large-lesion group. The upper graph of Fig. 5 presents scatterplots of REM sleep amount during the rest phase, expressed as percentage of total REM sleep under LD and SP conditions, for the whole pool of lesioned animals (n = 10), including small-lesioned rats (n = 4). SpearmanÕs non-parametric correlation coefficient (rho) for LD condition values was 0.697 (n = 10, P < 0.05) and for the SP condition.
(6) 8. A. Ocampo-Garce´s et al. NREM sleep. REM sleep. *. 70. ** * *. 15 60 50. 10. Wakefulness 80. *. * *. 60. Minutes in state. 40 40. 5 30 SP LD. SP LD. SP LD. *. **. 70 15. 80. 10. 60. *. 60 50. *. 40 5. 40. 30 SP LD. 0. 6 12 18. 0. SP LD. 0. 6 12 18. 0. SP LD. 0. 6 12 18. 0. Masking of REM sleep observed at morning and evening light pulses The 10-min moving average of REM sleep obtained on pooled state-by-epoch arrays of control and large-lesioned rats is presented in Fig. 6. Under LD, lesioned and control rats exhibited striking differences at ZT 0. The sustained light phase starting at ZT 0 was associated with a long-lasting suppression of REM sleep among large-lesioned rats, whereas the control group showed an increasing trend stabilizing REM sleep at 5–10% of recording time. In contrast, largelesioned and control rats exhibited similar time profiles associated with the morning light pulse under SP. The 20-min light pulse starting at ZT 0 produced a marked suppression of REM sleep, followed by REM sleep induction after lights-off, which lasted for at least 1.5 h in both groups. The evening light pulse determined a strong light suppression of REM sleep among control rats. After the evening light pulse, control rats exhibited a pronounced increase of REM sleep compared to the LD condition. The large-lesion group exhibited a transient decrease in REM sleep during the evening light pulse. The LD transition at ZT 12 was followed. 80 60 40 20. 40. SP-LD difference. was 0.38 (n = 9, P > 0.05). Hence, the magnitude of the LH lesion is correlated negatively with the amount of rest phase REM sleep under LD schedule. The absence of correlation observed under SP suggests that the response of REM sleep to darkness during the rest phase was almost unaffected by LH lesion size. The ÔSP minus LDÕ paired difference for that variable directly evaluates the photic masking on REM sleep (Fig. 5, lower graph). It is correlated negatively with the number of remaining orexin-A-ir neurones (SpearmanÕs rho = )0.72, n = 9, P < 0.05). In other words, the larger the lesion, the more masking of REM sleep during the rest phase.. REM sleep during rest phase (%). Zeitgeber time. Figure 4. Mean curves of states of the sleep– wake cycle under 12 : 12 light ⁄ dark (LD; open symbols) and skeleton photoperiod (SP; closed symbols). Symbols correspond to the mean (± standard error of the mean) of 2 h of recording time in large orexin-B-saporin lesioned rats (upper row) and control animals (lower row). Data of late active phase and early rest phase are double-plotted. LD schedule is depicted at the abscissa: white bars correspond to light phase under 12 : 12 LD condition and 20-min light pulses under SP, black bars correspond to darkness intervals, and grey bar identifies darkness hours of rest phase under skeleton photoperiod. *P < 0.05, paired StudentÕs t-test comparisons among animals, for factor LD schedule.. 20. 0. –20 25. 50. 75. 100. Orexin-A-ir neurons (% of controls) Figure 5. Relationship between the number of orexin-A-immunoreactive (ir) neurones and the percentage of total rapid eye movement (REM) sleep occurring during the rest phase among orexin-B saporin lesioned rats. The number of orexin-A-ir neurones is expressed as a percentage of the mean of control rats. Upper scatterplot: open circles correspond to values obtained under 12 : 12 light ⁄ dark (LD) cycle (n = 10) and closed circles to skeleton photoperiod (SP) values (n = 9). Lower scatterplot: triangles represent the arithmetic difference of SP minus LD condition obtained for each animal (n = 9). See text for statistical analysis.. by an irregular pattern of REM sleep in lesioned rats under both LD and SP. 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11.
(7) Lateral hypothalamus lesion and masking of REM sleep Light–dark transitions Morning. Evening. 25. REM sleep (% of recording time). 20 15 10 5 0 25 20 15 10 5 0. SP LD. 23. 0. 1. 11. 12. 13. Zeitgeber time Figure 6. Rapid eye movement (REM) sleep expression at morning and evening light ⁄ dark (LD) transitions of large orexin-B-saporin lesion (upper row) and control rats (lower row). Each data point is centred on a 10-min moving average, with a 15-s time resolution, of the percentage of REM sleep obtained from pooled data of each rat category. Thin lines correspond to the 12 : 12 LD condition and bold lines to the skeleton photoperiod (SP). Bars at lower abscissa indicate the timing of light (white) dark (black) intervals. Grey bars correspond to the dark period at rest phase and vertical dotted lines delimit morning and evening 20-min light pulses under skeleton photoperiod.. DISCUSSION Orexin-B-saporin lesions of the LH and the sleep–wake cycle in rats Gerashchenko et al. (2001) first described the effect on sleep states of orexin-B-saporin lesions in the lateral hypothalamus of albino rats, recorded under LD. They described a noctural (active phase) transposition of REM sleep preference and a loss of NREM sleep and wakefulness modulation during the 24-h period. Our large-lesion group consistently exhibited a 50% reduction in REM sleep during the light phase and an 81.6% excess during dark with respect to control rats under LD (Table 1). The fraction of REM sleep observed during rest phase is correlated positively with the percentage of surviving orexin-ir neurones. The 24-h modulation of wakefulness was mildly affected by the neurotoxic lesion. NREM sleep modulation was refractory to the lesion procedure in terms of the total amount of time in each state and phase distribution, a fact that could be explained by more surviving LH neurones in our large-lesioned group. Body temperature, locomotor activity and drinking circadian rhythms survive orexin-B-saporin LH lesions, without 2010 European Sleep Research Society, J. Sleep Res., 20, 3–11. 9. affecting the phase of the circadian output (Gerashchenko et al., 2003; Mistlberger et al., 2003). This suggests that the mechanism underlying the nocturnal phase preference for REM sleep among LH lesioned rats is not a phase adjustment of REM sleep circadian modulation. The fact that simultaneous lesions induced by means of saporin-conjugated neurotoxins in orexin-recipient nuclei of the ascending activating system involved in state control do not shift REM sleep phase preference (Blanco-Centurion et al., 2007) suggests that the displacement of REM sleep to the active phase may be a consequence of direct damage to the lateral hypothalamus. Orexin-B-saporin neurotoxic lesions affect neurones harbouring orexin receptors, including orexinergic and MCH-ir neurones, that are intermingled around the perifornical region of the LH (Broberger et al., 1998). Substantial evidence has been amassed that relates orexinergic activity and sleep–wake state regulation (Sakurai, 2007). Growing evidence supports a REM sleep-promoting role for MCH-ir in rats (Lagos et al., 2009; Verret et al., 2003). Previous reports described the concurrent loss of orexinergic and MCH-ir after orexin-Bsaporin lesions in the LH (Gerashchenko et al., 2001; Mistlberger et al., 2003). Furlong and Carrive (2007) found that the lesion areas were more extensive than suggested by orexin or MCH staining. As depicted in Fig. 3, we found a linear correlation between the loss of orexin-A-ir and MCH-ir neurones. These results argue against ascribing the observed sleep–wake state changes to specific neuronal damage. However, it should be noted that specific damage to orexinergic neurones, such as that observed in orexin ⁄ ataxin-3 transgenic rats (Beuckmann et al., 2004; Zhang et al., 2007a) and mice (Zhang et al., 2007b), induced a marked nocturnal (active) phase preference of REM sleep under a 12 : 12 LD schedule. It is possible that the nocturnal phase preference for REM sleep observed in orexin-B-saporin lesion and in orexin ⁄ ataxin-3 models shares a common neurobiological substrate in the damaged orexinergic system. It should be noted that the genetic substrates of the orexin ⁄ ataxin-3 rat models are the albino Wistar (Beuckmann et al., 2004) and Sprague– Dawley (Zhang et al., 2007a) strains, and that of mice is the pigmented C57BL ⁄ 6 strain (Zhang et al., 2007b), all of which exhibit strong masking of REM sleep by the LD cycle. Sleep–wake cycle under SP and masking of the sleep–wake cycle Consistent with the work of Borbély and Neuhaus (1978), which reported no change in the phase preference of sleep states, our control group presented no significant changes in sleep–wake states acrophases after being transferred to SP. A significant reduction in NREM sleep modulation due to an increase in NREM sleep and a corresponding reduction in wakefulness during the active phase, without affecting REM sleep modulation when recorded under SP, was also observed. In sharp contrast, large-lesioned rats exhibited a huge increase in (88.4%) REM sleep after being transferred to SP during the rest phase, and recovered the typical rest phase preference of REM sleep (63% of total REM sleep occurring during rest.
(8) 10. A. Ocampo-Garce´s et al.. phase). Under SP the amount of REM sleep in the largelesioned group did not differ from that of the control group in the rest phase. As depicted in Fig. 5, the REM sleep increase under SP (SP minus LD) is correlated negatively with the number of surviving orexin-A-IR neurones. This result suggests that the photic masking of REM sleep is a function of the size of the lesion in the LH. To confirm the existence of an exacerbated masking process in the lesioned rat, it would be expected that after a reverse transition from SP to LD, REM sleep of lesioned rats should recover the nocturnal predominance. This prediction was not tested in our experiment. The photic masking of REM sleep consists in a change in REM sleep propensity due to the photic stimulation of the retina. An immediate 10% increase in REM sleep has been described after transferring from LD to constant light (LL) or constant darkness (Borbély and Neuhaus, 1978). It has been also described that REM sleep tends to increase under SP (Borbély and Neuhaus, 1978; Trachsel et al., 1986). Dark pulses applied during rest or active phase of albino rats enhance NREM to REM sleep transitions (Alföldi et al., 1991; Baracchi et al., 2008; Benca et al., 1993). Although the description of REM sleep masking has been centred mainly on REM sleep enhancement by darkness, there are consistent reports of REM sleep suppression by light. Benca et al. (1996) proposed that ÔREM sleep induction following lights-off may in part represent a release from REM sleep suppression by lightÕ. Because the amount of rest phase REM sleep in largelesioned rats under SP does not differ from that of control rats, we propose that the surge in REM sleep seen in the large-lesioned group under the SP versus LD condition is not attributable to an REM sleep triggering effect of darkness, but rather the release of an exaggerated suppression of REM sleep exerted by light under the LD schedule. The largelesioned rats show 54% and 81% more active phase (nocturnal) REM sleep than do control rats under SP and LD, respectively, suggesting that the REM sleep triggering effect of darkness may also be augmented in the large-lesioned group. REM sleep masking among different rat strains The photic masking of sleep states is not circumscribed to albino rats, as it has been also described among pigmented rats (Benca et al., 1993, 1998) and C57BL ⁄ 6 mice (Deboer et al., 2007). The albino rat is an optimal animal model to elucidate the neural connectivity that underlies the sleep–wake state masking phenomenon, because the REM sleep triggering effect has not been observed among pigmented rats. Instead, Benca et al. (1993, 1998) reported suppression of REM sleep by dark pulses among pigmented strains, such as hooded Long Evans, Dark Agouti and Brown Norway rats. In a report on orexin-Bsaporin lesions induced in the LH of pigmented Long Evans rats under LD, the disruption of REM sleep temporal organization became apparent as a loss of the 24-h modulation, without the prominent nocturnal REM sleep predominance described in the albino strain (Gerashchenko et al.,. 2003), suggesting that the nocturnal preference of REM sleep reported among neurotoxic lesioned rats is a specific process of albino strains. We propose that the expression of sleep–wake states could suffer the enhancement of photic masking processes when interfering with the modulation conveyed by LH pathways on sleep-related structures, as occurs among neurotoxic-lesioned rats and rodent models of narcolepsy–cataplexy. DECLARATIONS OF INTEREST None. ACKNOWLEDGEMENTS This work was supported by Grant FONDECYT 1061089. We thank Christian López for his excellent work in animal care. REFERENCES Alföldi, P., Franken, P., Tobler, I. and Borbély, A. A. Short light–dark cycles influence sleep stages and EEG power spectra in the rat. Behav. Brain Res., 1991, 43: 125–131. Baracchi, F., Zamboni, G., Cerri, M. et al. Cold exposure impairs dark-pulse capacity to induce REM sleep in the albino rat. J. Sleep Res., 2008, 17: 166–179. Benca, R. M., Obermeyer, W. H., Bergmann, B. M., Lendvai, N. and Gilliland, M. A. Failure to induce rapid eye movement sleep by dark pulses in pigmented inbred rat strains. Physiol. 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Figure
![Table 1 State amount [minutes ± standard error of the mean (SEM)] and fraction occurring during rest phase (% of whole day ± SEM) in control and large lesioned rats under 12 : 12 light ⁄ dark (LD) and skeleton photoperiod (SP)](https://thumb-us.123doks.com/thumbv2/123dok_es/7275313.441594/5.892.456.813.135.812/minutes-standard-fraction-occurring-control-lesioned-skeleton-photoperiod.webp)
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