EFFECTS OF LATERAL HYPOTHALAMIC LESION WITH THE NEUROTOXIN HYPOCRETIN-2 SAPORIN ON SLEEP IN LONG EVANS RATS

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1 Neuroscience 116 (2003) EFFECTS OF LATERAL HYPOTHALAMIC LESION WITH THE NEUROTOXIN HYPOCRETIN-2 SAPORIN ON SLEEP IN LONG EVANS RATS D. GERASHCHENKO, a C. BLANCO-CENTURION, a M. A. GRECO a,b AND P. J. SHIROMANI a * a West Roxbury VA Medical Center and Harvard Medical School, 1400 VFW Parkway, West Roxbury, MA 02132, USA b SRI, International, 333 Ravenswood Avenue, Menlo Park, CA 94061, USA hypothalamus may explain the differences in severity of symptoms seen in human narcolepsy IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: hypothalamus, hypocretin saporin, lesion, NREM sleep, REM sleep, NeuN. Abstract Narcolepsy, a disabling neurological disorder characterized by excessive daytime sleepiness, sleep attacks, sleep fragmentation, cataplexy, sleep-onset rapid eye movement sleep periods and hypnagogic hallucinations was recently linked to a loss of neurons containing the neuropeptide hypocretin. There is considerable variability in the severity of symptoms between narcoleptic patients, which could be related to the extent of neuronal loss in the lateral hypothalamus. To investigate this possibility, we administered two concentrations (90 ng or 490 ng in a volume of 0.5 l) of the neurotoxin hypocretin-2 saporin, unconjugated saporin or saline directly to the lateral hypothalamus and monitored sleep, the entrained and free-running rhythm of core body temperature and activity. Neurons stained for hypocretin or for the neuronal specific marker were counted in the perifornical area, dorsomedial and ventromedial nucleus of the hypothalamus. More neuronal nuclei (NeuN) cells were destroyed by the higher concentration of hypocretin-2 saporin ( 55%) compared with the lower concentration ( 34%) in the perifornical area, although both concentrations lesioned the hypocretin neurons almost equally well (high concentration 91%; low concentration 88%). The high concentration of hypocretin-2 saporin also lesioned neurons in the dorsomedial nucleus of the hypothalamus and ventromedial nucleus of the hypothalamus. Narcoleptic-like sleep behavior was produced by both concentrations of the hypocretin-2- saporin. The high concentration produced a larger increase in non-rapid eye movement sleep amounts during the normally active night cycle than low concentration. Neither concentration of hypocretin-2 saporin disrupted the phase or period of the core temperature or activity rhythms. The low concentration of unconjugated saporin did not significantly lesion hypocretin or neurons and did not alter sleep. The high concentration of unconjugated saporin produced some loss of neuronal nuclei-immunoreactive (NeuN-ir) neurons and hypocretin immunoreactive neurons, but only a transient increase in non-rapid eye movement sleep. These results led us to conclude that the extent of hypocretin neuronal loss together with an accompanying loss of cells in the lateral *Corresponding author. Tel: ; fax: address: pshiromani@hms.harvard.edu (P. Shiromani). Abbreviations: CSF, cerebrospinal fluid; DMH, dorsomedial nucleus of the hypothalamus; EEG, electroencephalogram; EMG, electromyogram; HCRT, hypocretin; ir; immunoreactive; LH, lateral hypothalamus; MCH, melanin concentrating hormone; NeuN, neuronal nuclei; NREM, non-rem; REM, rapid eye movement; SAP, saporin; SCN, suprachiasmatic nucleus; SOREMPs, sleep-onset REM sleep periods; VMH, ventromedial nucleus of the hypothalamus /03$ IBRO. Published by Elsevier Science Ltd. All rights reserved. PII: S (02) The hypocretins, also known as orexins, are recently discovered peptides with a discrete localization in the lateral hypothalamus (LH) (De Lecea et al., 1998; Peyron et al., 1998; Sakurai et al., 1998). A single gene encodes hypocretin (HCRT), which is cleaved by proteolytic processing into two smaller peptides, HCRT1 (orexin A) and HCRT2 (orexin B) (De Lecea et al., 1998; Sakurai et al., 1998). HCRT-containing neurons project to the entire brain and spinal cord, providing especially heavy innervation to forebrain and brainstem neuronal populations implicated in wakefulness (Peyron et al., 1998; Trivedi et al., 1998; Greco and Shiromani, 2001). Recently, this peptide was implicated in the human sleep disorder narcolepsy based on the findings that canines with narcolepsy possess a mutation in the HCRT2 receptor (Lin et al., 1999). Transgenic mice with a deletion of the HCRT gene (Chemelli et al., 1999) or mice with a gene-specific ablation of the HCRT neurons (Hara et al., 2001) exhibit symptoms of narcolepsy. In human narcolepsy there is a massive loss of HCRT neurons (Peyron et al., 2000; Thannickal et al., 2000), and consistent with such a neuronal loss, levels of HCRT1 are undetectable in the cerebrospinal fluid (CSF) of human narcoleptic patients (Nishino et al., 2000b). A polymorphism in the HCRT gene associated with narcolepsy has been found (Gencik et al., 2001). The posterior hypothalamic region where the HCRT neurons are located was identified to be a wake-center by von Economo because he observed that patients suffering from the viral encephalitic epidemic of 1918 were excessively sleepy and postmortem analysis revealed damage to this region (von Economo, 1930). Since then a few studies have examined changes in sleep after electrolytic (McGinty, 1969; Nauta, 1946; Ranson, 1939; Shoham and Teitelbaum, 1982; Swett and Hobson, 1968) or excitotoxic (Denoyer et al., 1991; Sallanon et al., 1988) lesions of the posterior hypothalamus but the results have been inconsistent. In these lesion studies, no attempts were made to specifically identify the phenotype of the neurons that were lesioned, and the inconsistent effects on sleep might have occurred because the lesion methods did not destroy the appropriate neurons.

2 224 D. Gerashchenko et al. / Neuroscience 116 (2003) The HCRT-containing neurons possess an autoreceptor (Horvath et al., 1999), and to lesion such hypocretin receptor bearing neurons, we have created a neurotoxin by conjugating the ribosomal inactivating protein saporin (SAP) (Gerashchenko et al., 2001) to the HCRT/orexin receptor-binding ligand HCRT2/orexin-B. The HCRT saporin (HCRT2 SAP) specifically binds to HCRT receptors and lesions HCRT neurons (Gerashchenko et al., 2001). Microinjection of HCRT2 SAP into the LH produces narcoleptic behavior that is directly correlated with the loss of HCRT neurons (Gerashchenko et al., 2001). The present study was done to further characterize the sleep abnormality following LH lesions produced by the HCRT2 SAP. Using two concentrations of the HCRT2 SAP versus unconjugated SAP and saline, we found a persistent hypersomnia in response to a greater loss of HCRT and adjacent LH neurons. We also found that animals with loss of HCRT and adjacent LH neurons continued to wake up at the same time of day, indicating that the circadian system was intact in these animals. This suggests that HCRT and adjacent LH neurons are not responsible for arousal at a specific time of day, and that the variability in the severity of symptoms seen in human narcolepsy may depend on the extent of posterior hypothalamic damage. EXPERIMENTAL PROCEDURES Animals and surgical preparation The studies were conducted in accordance with the principles and procedures described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Twenty-three male Long Evans rats ( g) were housed singly in Plexiglas cages with wood shavings, and with food and water available ad libitum. The rats were housed in a room where the temperature (21 C) and the lights were controlled (7:00 a.m. to 7:00 p.m. lights on; 100 lux). The rats were implanted under anesthesia (i.m. injection of a cocktail of acepromazine, [0.75 mg/kg], xylazine [20.5 mg/kg] and ketamine [22 mg/kg]) with electrodes to record the electroencephalogram (EEG) and electromyogram (EMG). Four miniature stainless steel screw electrodes were positioned in the skull to sit on the surface of the cortex and were used to record the EEG. Two miniature screws were inserted 2 mm on either side of the midline and 3 mm anterior to bregma (frontal cortex). The other two screws were located 2 mm on either side of the midline and 6 mm behind bregma (occipital cortex). The cortical EEG was recorded from two contralateral screws (frontal-occipital). To record muscle activity (EMG), two flexible multistranded wires were inserted in the nuchal muscles. The electrodes were placed in a plastic plug and secured onto the skull by using dental cement. At the time the sleep recording electrodes were implanted, bilateral microinjections of HCRT2 SAP, unconjugated SAP or pyrogen-free saline were made to the LH. After the surgery, the rats were returned to their home cages and EEG and EMG recordings were collected continuously for at least three weeks. The rats were then placed in total darkness for an additional three weeks and the rhythm of core body temperature during free-run conditions was recorded. After the experiment, the rats were perfused (after overdose of Nembutal) and formalinfixed brains were used for histological analysis. All rats were weighed on the day of the surgery and two months after the surgery. Drug groups and microinjection method The following five groups were used: (1) saline (n 5); (2) unconjugated SAP (90 ng/0.5 l, n 3); (3) unconjugated SAP (490 ng/0.5 l, n 4); (4) HCRT2 SAP (90 ng/0.5 l, n 5); (5) HCRT2 SAP (490 ng/0.5 l, n 6). The HCRT2 SAP conjugate (Advanced Targeting Systems, San Diego, CA, USA), SAP (Sigma, Saint Louis, MO, USA), or pyrogen-free saline was delivered via a glass micropipette with a tip diameter of 20 m by using a Picospritzer. All test substances were injected bilaterally in a volume of 0.5 l. After injection, the pipette was left in place for 5 min and then withdrawn slowly. All injections were made in the LH at the following coordinates relative to bregma: A 3.3 mm; L 1.6 to 1.8 mm; V 8.2 mm below the dura (Paxinos and Watson, 1986). The concentration of HCRT2 SAP used was based on our study (unpublished observations) on the effects of various concentrations of the HCRT2 SAP (45, 90, 245, 490 ng/0.5 l) in lesioning the HCRT receptor containing histaminergic cells in the tuberomammillary nucleus. We found that clearly discernible cell loss was first evident with 90 ng/0.5 l. The 490-ng/0.5- l concentration represents the full strength of the neurotoxin. Analysis of sleep wake states EEG and EMG signals were recorded on a Grass polygraph and onto a Jaz disk using an analog digital board (National Instruments, Austin, TX, USA). The EEG data were filtered at 70 Hz and 0.3 Hz by using a Grass electroencephalograph and continuously sampled at 128 Hz. The 24-h EEG and EMG recordings obtained on the 2nd, 6th, 14th and 21st day postinjection were scored manually on a computer (Icelus software, Mark Opp, Ann Arbor, MI, USA) in 12-s epochs for awake, rapid eye movement (REM) sleep and non-rem (NREM) sleep by staff blind to the type of drug administered to the rats. Wakefulness was identified by the presence of desynchronized EEG and high EMG activity. NREM sleep consisted of high-amplitude slow waves together with a low EMG tone relative to waking. REM sleep was identified by the presence of desynchronized EEG and/or theta activity coupled with absence of EMG activity. The amount of time spent in wakefulness, NREM and REM sleep was determined for each hour. After the EEG data were scored, the code was broken to reveal the identity of each rat. Sleep-onset REM sleep periods (SOREMPs) were identified as REM sleep episodes in the day or night that occurred after 2 min or more of wakefulness with less than 2 min of an intervening episode of NREM sleep. Each SOREMP was identified by a computer program and then substantiated by visual inspection of the corresponding EEG and EMG recordings. Details of the criteria used to identify SOREMPs in rats have been described previously (Gerashchenko et al., 2001). Core body temperature recording At the time the sleep recording electrodes were implanted, a transmitter to record core temperature (model XM-FH, Mini Mitter, Bend, OR, USA) was also implanted in the abdominal cavity. This transmitter also recorded gross motor activity. The signal from the transmitter was collected every 10 min by using telemetry equipment (Mini Mitter, Bend, OR, USA) over a six-week period. The temperature and activity signals were recorded during entrained (12-:12-h light on and light off) conditions. Three weeks after the injections, the lights were turned off and temperature recordings were made during constant darkness conditions (free-run) for three weeks. The free-run periods (Tau) of temperature and activity rhythms were determined for the final week in the free-run conditions.

3 D. Gerashchenko et al. / Neuroscience 116 (2003) Immunohistochemistry Two months after the injections of test substances, the animals were deeply anesthetized with pentobarbital (150 mg/kg i.p.) and perfused transcardially with 0.9% saline (50 ml) followed by 500 ml of phosphate-buffered 4% paraformaldehyde (ph 7.0). The brains were postfixed overnight, equilibrated in 30% sucrose, and stored at 4 C. One-in-five series of coronal sections were cut at 30 m on a sliding microtome. Each set of coronal brain sections was incubated overnight at room temperature in the primary antibody. After washing, the sections were incubated with the secondary antibody for 1 h (Chemicon, Temecula, CA, USA; 1:250 dilution) and then reacted with avidin biotin complex for 1 h (Vector Laboratories, Burlingame, CA, USA). The 3,3 -diaminobenzidine (DAB) method was used to visualize the reaction product. Omission of the primary antiserum resulted in no specific staining. Antibodies Rabbit anti HCRT1 (1:70,000, Peninsula Laboratories, Inc., San Carlos, CA, USA) and mouse anti-neun monoclonal (1:1000; Chemicon) antibodies were used. The secondary antibodies, biotinylated anti-rabbit immunoglobulin G (IgG) and biotinylated F(ab') 2 fragments of anti-mouse IgG (both from donkey), were obtained from Chemicon. In situ hybridization In situ hybridization was performed as previously described (Greco and Shiromani, 2001). The HCRT-receptor 1 probe was generously provided by Drs. Nahid Waleh and Thomas S. Kilduff (Stanford Research Institute, Palo Alto, CA, USA) The chromosomal RNA was transcribed from a linearized plasmid using T7 (antisense) or T3 (sense) RNA polymerase and S-UTP with a riboprobe kit (Promega, Madison, WI, USA). Acetylated tissue was incubated overnight at 55 C in hybridization buffer containing probe (10 c.p.m./ml); washed successively in 2 (standard saline citrate; SSC) 1-mM (dithiothreitol; DTT) (50 C, 1 h), 0.2 SSC 1-mM DTT (55 C, 1 h), 0.2 SSC 1-mM DTT (60 C, 1 h); dehydrated; exposed to film; and developed after 48 h. Cell counts A person who did not know the lesion status of the rats counted all clearly stained HCRT-immunoreactive (ir) somata in 11 sections (one in five series) that encompassed the full extent of HCRT distribution (between 10.6 and 30.8 mm from bregma [Paxinos and Watson, 1986]). To identify whether other neurons were destroyed, we counted in 11 sections NeuN-positive neurons within a 0.2-mm rectangular grid positioned above the fornix (see box in Fig. 2). In a previous report (Gerashchenko et al., 2001), we had counted neurons containing melanin concentrating hormone (MCH) and adenosine deaminase, and found that HCRT2-SAP (450 ng) lesioned these neurons. In this study, we used NeuN, a protein specific to neurons, to provide another index of neuronal loss. To identify neuronal loss in adjacent neuronal populations, NeuN-ir nuclei were counted bilaterally within a 0.2-mm rectangular grid in five sections in the dorsomedial nucleus of the hypothalamus (DMH), and seven sections in the ventromedial nucleus of the hypothalamus (VMH). All NeuN-ir cells were identified and counted using a computer program similar to the NIH-Image program (3D-DOCTOR, Version 3.0.5, Able Software Corporation, ). Camera lucida drawings (Nikon microscope, Eclipse E400 Model, Melville, NY, USA) were made to identify the lesioned area. Statistical analysis Analysis of variance and t-tests with Bonferroni correction (where appropriate) were used to compare changes in sleep parameters (SYSTAT, Version 8.0, SPSS Inc., 1998). The same statistical methods were also used to compare counts of HCRT-ir and NeuN-ir cells in each anatomic region. Statistical significance was evaluated at the P 0.05 level. Neuronal loss RESULTS Fig. 1 summarizes the lesioned area in rats treated with HCRT2 SAP (450 ng), HCRT2 SAP (90 ng) and SAP (450 ng). Little or no neuronal loss was apparent with the low concentration of unconjugated SAP. The higher concentration of unconjugated SAP produced a lesion but this was much smaller compared with the conjugated SAP (see Fig. 1). Loss of HCRT neurons and NeuN-positive neurons in the DMH, VMH, and perifornical area was assessed quantitatively. The HCRT neurons located laterally were primarily lesioned (Fig. 2A, D), whereas a few HCRT-ir neurons located medially remained. Compared with the saline-treated rats (Fig. 2A and Fig. 3B), both concentrations of the HCRT2 SAP produced a significant reduction in the numbers of HCRT-positive cells in the LH (88% neuronal loss for the concentration of 90 ng (t , P 0.001) and 91% for the concentration of 490 ng (t , P 0.001); between groups F (4, 22) , P 0.001). Consistent with the neuronal loss, expression of HCRT receptor mrna was also reduced (Fig. 2B, E). The higher concentration of HCRT2 SAP destroyed more NeuN-positive cells in the perifornical area (34% neuronal loss for the concentration of 90 ng (t 3.060, P 0.028) and 55% for the concentration of 490 ng, t 5.190, P 0.001); between groups F (4, 21) 7.878, P 0.001; Fig. 2C, F and Fig. 3A). As shown in Fig. 1, the extent of the lesion produced by the higher concentration of HCRT2 SAP was also greater, with the lesion extending dorsally into the midline thalamus, medially into the DMH and ventrally into the VMH. In the DMH and VMH, the higher concentration of HCRT2 SAP produced a significant reduction of NeuN-ir neurons (DMH, t 3.384, P 0.014, between groups F (4, 21) , P 0.009; VMH, t 2.816, P 0.048, between groups F (4, 21) 2.970, P 0.009; Fig. 4A and B). The lower concentration of the HCRT2 SAP (90 ng) or SAP (90 and 490 ng) did not cause a significant loss of NeuN neurons in the DMH or VMH (Fig. 4A and B). Analysis of sleep data In the present study, sleep recordings obtained on days 2, 6, 14, and 21 after HCRT2 SAP injection were analyzed. In the saline-treated rats, there were no significant differences in sleep between days 6, 14 and 21. Therefore, the data from these days were combined (Fig. 5). The diurnal distribution of sleep wake states across 24 h in the saline- and HCRT2 SAP treated rats is shown in Fig. 5. The percentage of each sleep wake state during 12 h of day and night is presented in Table 1. Salinetreated rats (n 5) were awake more at night and asleep during the day, as is typical of nocturnal rodents (Fig. 5 and Table 1). On postinjection night 6, the rats treated with HCRT2 SAP (90 or 490 ng) exhibited more sleep time

4 226 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 1. Camera lucida drawings of lesion in rats treated with 90 ng of hypocretin-2 saporin (A), 450 ng of hypocretin-2 saporin (B), and 450 ng of saporin (C). Microinjection of these agents was made to the lateral hypothalamus and loss of NeuN labeled neurons was used to demarcate the lesion area. Three schematic sections from the rat atlas of Paxinos and Watson (Paxinos and Watson, 1986) are shown at the top of the figure to identify the location of the drawings relative to bregma. B, bregma.

5 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 2. Effects of hypocretin-2 saporin on hypocretin-containing neurons and expression of hypocretin mrna receptor in the lateral hypothalamus. A, B and C represent the lateral hypothalamic sections of saline-treated rats. D, E and F represent the sections of the rats treated with 90 ng of hypocretin-2 saporin. Hypocretin-containing neurons were identified by immunohistochemistry (A, D), and expression of hypocretin mrna receptor was identified by in situ hybridization (B, E). NeuN immunostaining and the rectangular boundaries used to count stained cells are shown in C and F. DMH, dorsomedial nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus; mt, mammillothalamic tract; f, fornix; opt, optic tract; 3V, 3rd ventricle. The scale bar in F applies also to photomicrographs A, C and D. The scale bar in E applies to photomicrograph B. than saline-treated rats (Table 1). These rats had significant increases in both NREM and REM sleep at night (Table 1). The significant increase in NREM and REM sleep was also present on night 14. On night 21 postinjection, rats treated with 90 ng of HCRT2 SAP continued to have significantly elevated amounts of REM sleep during the night (Table 1), whereas the rats given the higher concentration of HCRT2 SAP (490 ng) continued to have significantly higher amounts of both NREM and REM sleep at night compared with saline-treated rats (Fig. 5, Table 1). The increase in sleep at night in rats lesioned with HCRT2 SAP occurred because of an increase in the duration of bouts of both REM sleep and NREM sleep (Table 2). During the night, these rats had significantly more fragmented sleep, as indicated by more transitions between wake NREM sleep (Table 2). During the day cycle of the 6th day postinjection, NREM sleep levels were significantly higher in rats given HCRT2 SAP (90 or 490 ng) but then on succeeding day cycles they returned to normal levels. During the day, the HCRT2 SAP (90 or 490 ng) treated rats had a significant decline in REM sleep, with the higher concentration producing a greater reduction in REM sleep (Fig. 5, Table 1). Previously, we found that albino rats also had a similar increase in REM sleep at night and a decrease during the day cycle (Gerashchenko et al., 2001). Unconjugated SAP (490 ng) increased NREM sleep during both lights-on and -off periods on the 6th postinjection day (Table 1), but by day 14 and 21, NREM sleep amounts returned to control levels (Table 1). Low concentration of SAP (90 ng) produced only transient changes in the amounts of REM sleep but no change in NREM sleep. All tested parameters of sleep/wakefulness (Fig. 5, Tables 1, 2, 3) were not different from control values in rats treated with 90 ng of SAP on day 21 postinjection. Sleep-onset REM sleep periods Both concentrations of HCRT2 SAP induced SOREMPs. As in the previous study (Gerashchenko et al., 2001), these were evident primarily during the night cycle (Fig. 6). The higher concentration of HCRT2 SAP induced more SOREMPs at night compared with the values of the saline group (90 ng, day 6: average S.E.M , t 2.571, df 8, P 0.033; 90 ng, day 21: average S.E.M , t 2.500, df 8, P 0.037; 490 ng, day 6: average S.E.M , t 2.631, df 9, P 0.027; 490 ng, day 21: average S.E.M ; t 2.261, df 9, P 0.050). SOREMPs were rarely seen in rats injected with saline or unconjugated SAP (Fig. 6). Core temperature Fig. 7 summarizes the entrained diurnal rhythm of core temperature in saline versus HCRT2 SAP treated rats 21 days after injection. In all three groups, the temperature peak occurred soon after lights off and the temperature nadir occurred soon after lights on. This indicates that the HCRT2 SAP lesions did not disrupt the entrained rhythm of core temperature by either advancing or delaying the phase position of the temperature rhythm. Saline-treated rats demonstrated a robust diurnal rhythm in core temperature, with average core temperature at night being significantly greater than during the day (average day S.E.M ; average night S.E.M ; t 3.771, df 4, P 0.02). This diurnal distribution of core temperature is consistent with the

6 228 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 3. Number of NeuN-immunoreactive (A) neurons in the perifornical area and HCRT-immunoreactive (B) neurons in the lateral hypothalamus of rats treated with saline, saporin (SAP), or hypocretin-2 saporin (HCRT2 SAP). Both concentrations of HCRT2 SAP produced a significant decline in neurons, with the higher concentration of HCRT2 SAP producing a greater loss of NeuN-immunoreactive neurons. The total number of HCRT- or NeuN-immunoreactive cells was determined in 30- m-thick sections, as described in the Experimental Procedures. Asterisks denote P 0.05 compared with saline. higher amount of wakefulness at night. The mean temperature did not differ between day and night in the HCRT2 SAP (490 ng) treated rats (average day S.E.M ; average night S.E.M ; t 0.48; df 4). This is also consistent with the reduction of a diurnal difference in NREM and REM sleep in these rats (see Fig. 5). During the day, the core temperature in the HCRT2 SAP (490 ng) was not significantly different from that of saline rats. However, during the night the core temperature of these rats was significantly lower (t 2.416; df 8; P 0.04) compared with that of saline rats (Fig. 7). Core temperature in rats administered the low concentration of HCRT2 SAP (90 ng) was not different compared with that of saline-treated rats. The temperature in SAP-treated rats also was not significantly different from that of salinetreated rats. Because endogenous rhythms such as temperature and activity are very strongly influenced by light, we placed the rats in constant darkness (free-run condition) and continued to observe the rhythm of core body temperature and activity for three weeks. In rats, in the absence of any external cues, the period of the temperature and activity Fig. 4. Number of NeuN-immunoreactive neurons in the dorsomedial nucleus (A) and the ventromedial nucleus of the hypothalamus (B). Data are presented as mean S.E.M. in each group of rats. *P 0.05 versus saline group. rhythms lengthens gradually and is generally about 24.2 h. In the present study, in the saline-treated rats, the free-run period of temperature and activity rhythm (tau) was ( 0.07) and this was not significantly different in the HCRT2 SAP ( ) or SAP-treated rats ( ) (Fig. 8). This indicates that the lesions of the LH and HCRT neurons did not affect the phase or period of the endogenous rhythm of core temperature or activity rhythm. Body weight Two months postinjection the weight of rats given 90 ng of unconjugated SAP was not significantly different from that of saline-treated rats (saline % increase in weight; 90 ng of SAP % increase in weight; t ; df 6). The high concentration of SAP (490 ng) induced a little reduction in body weight ( % reduction in weight; t ; df 7; P vs. saline group). Rats given both concentrations of HCRT2 SAP had a significant reduction in weight compared with salinetreated rats ( % for the 90-ng concentration (t 2.312; df 8; P 0.050); % for the 490-ng concentration (t 4.872; df 9; P 0.001)). DISCUSSION Human narcolepsy has been linked to a loss of HCRT neurons (Peyron et al., 2000; Thannickal et al., 2000) but

7 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 5. Mean ( S.E.M.) percentage of wakefulness, non rapid eye movement (NREM) and rapid eye movement (REM) sleep during 24 h in rats administered hypocretin-2 saporin (HCRT2 SAP) or saline into the lateral hypothalamus. The 24 h are represented in 2-h blocks. The dark bar represents the 12-h lights-off period. On day 6 postinjection, the rats treated with either concentration of HCRT2 SAP (90 or 490 ng) experienced significantly more NREM and REM sleep at night compared with saline-treated rats. On day 21 postinjection, nighttime amounts of NREM and REM sleep were elevated in the rats treated with 490 ng of HCRT2 SAP, and REM sleep levels were increased in rats treated with 90 ng of HCRT2 SAP. Table 1. Average percent ( S.E.M.) of wakefulness, NREM, or REM sleep, in rats administered hypocretin2-saporin (HCRT2-SAP), saporin (SAP), or saline into the lateral hypothalamus Group Day postinjection Light-off period Light-on period Wakefulness NREM sleep REM sleep Wakefulness NREM sleep REM sleep Saline Saporin ng * * * Saporin * * * ng * * * * HCRT2-SAP * * 90 ng * * * * * * * * * * * HCRT2-SAP ng * * * * * * * * * * * * * * * The values were calculated during 12 h lights-off or 12 h lights-on period. * P 0.05, significant difference compared with the values of the saline group.

8 230 D. Gerashchenko et al. / Neuroscience 116 (2003) Table 2. Average number of transitions to NREM, REM sleep, or wakefulness and duration of wakefulness, NREM, or REM sleep in rats administered hypocretin2-saporin (HCRT2-SAP), saporin (SAP), or saline into the lateral hypothalamus during lights-off period Group Day postinjection Average number of transitions Average duration of bouts (min) Wake-NREM NREM-REM NREM-Wake Wake NREM REM Saline Saporin ng Saporin * 490 ng * HCRT2-SAP * 90 ng HCRT2-SAP ng * * * * * * * * P 0.05, significant difference compared with the values of the saline group. it is not known to what extent loss of other neurons in the LH and surrounding posterior hypothalamus contribute to the disease and the variation in symptoms. The currently available models of narcolepsy cannot adequately answer this question, since in the murine model (Chemelli et al., 1999; Hara et al., 2001), only the HCRT neurons are affected, whereas in the canine model, the disease is due to a mutation in the HCRT2 receptor (Lin et al., 1999). On the other hand, the HCRT2 SAP (Gerashchenko et al., 2001) could be used, since the number of neurons lost would depend on the concentration of the neurotoxin. Using two concentrations of HCRT2 SAP, we find that when HCRT neurons and a greater number of adjacent LH neurons are lost, there is a corresponding increase in total sleep time, in addition to SOREMPs and increased REM sleep. The extent of HCRT neuronal loss together with an accompanying loss of cells in the LH may explain the differences in severity of symptoms seen in human narcolepsy. HCRT2 SAP induced neuronal loss in the LH In a previous study, we demonstrated specificity of the HCRT2 SAP (Gerashchenko et al., 2001). Using fluorescent-activated cell sorting analysis, we found that the conjugate binds with a high affinity to HCRT receptor bearing Chinese hamster ovary cells, but not to substance-p receptor bearing cells (Gerashchenko et al., 2001). We also demonstrated that HCRT2 SAP binds to the HCRT2 receptor and, to a lesser degree, to the HCRT1 receptor Table 3. Average number of transitions to NREM, REM sleep, or wakefulness and duration of wakefulness, NREM, or REM sleep in rats administered hypocretin2-saporin (HCRT2-SAP), saporin (SAP), or saline into the lateral hypothalamus during lights-on period Group Day postinjection Average number of transitions Wake-NREM NREM-REM NREM- Wake Average duration of bouts (min) Wake NREM REM Saline Saporin * * ng * * Saporin * * ng HCRT2-SAP * * * * * 90 ng * * HCRT2-SAP * * * * ng * * * * * * * P 0.05, significant difference compared with the values of the saline group.

9 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 6. Number of sleep-onset rapid eye movement sleep periods (SOREMPs) during nighttime in rats 2, 6 and 21 days following injection of hypocretin-2-saporin, unconjugated saporin, or saline. *P 0.05 versus saline group. (Gerashchenko et al., 2001), which is consistent with the properties of the ligand alone (Sakurai et al., 1998). Cytotoxic effects in the brain were demonstrated by applying the HCRT2 SAP in the LH, where it destroyed some immunohistochemically identified cells but not others (Gerashchenko et al., 2001). HCRT neurons are known to contain the HCRT receptor (Horvath et al., 1999), but to what extent these receptors are present on non-hcrt neurons in the LH is still unclear. In the present study, the two concentrations destroyed an equal number of HCRT neurons but the higher concentration destroyed more NeuN-labeled neurons. Sparing of some cells in this and our previous study (Gerashchenko et al., 2001) might be related to presence and subtype of HCRT receptor on the neurons. In the present study, unconjugated SAP did not lesion as many neurons as the HCRT2-conjugated SAP (Figs. 3 and 4), a finding that is consistent with previous reports that intracellular entry of the SAP is facilitated by conjugating SAP with a ligand (Wiley, 1992). Unconjugated SAP also did not produce as severe or long-lasting sleep changes as HCRT2 SAP. The two concentrations of HCRT2 SAP produced quite similar loss of HCRT neurons, and both concentrations also produced a decrease in NeuN-labeled neurons. However, there were more NeuN neurons lost with the higher concentration of HCRT2 SAP. The higher concentration also produced neuronal loss that extended into the thalamus and to adjacent nuclei such as the DMH and VMH. Thalamic lesions cause insomnia (Lugaresi et al., 1986; Marini et al., 1988), but in our study the rats had more sleep, including REM sleep. On the other hand, damage to the DMH may have caused the weight loss in rats administered 490 ng of HCRT2 SAP. DMH lesions have been shown previously to reduce body weight (Bernardis and Bellinger, 1998). Rats given 490 ng of unconjugated SAP or 90 ng of HCRT2 SAP also lost weight ( 9.4% and 13.5%, respectively). Since these rats did not have a significant reduction in the number of NeuN-positive neurons (Fig. 4) in the DMH, the weight loss may be due to the loss of some LH neurons in these animals. Postoperative aphagia and adipsia associated with dramatic loss of body weight is typically seen in rats with electrolytic lesions of the LH (Bernardis and Bellinger, 1993; Bernardis et al., 1999). It is possible that loss of MCH neurons also contributes to body weight loss following the LH lesion. MCH-deficient mice weigh less because of a significant decrease in food intake and increased metabolic rate (Shimada et al., 1998) and previously (Gerashchenko et al., 2001) we found that HCRT2 SAP (490 ng) destroyed some MCH neurons. Effects of HCRT neuronal loss on core temperature rhythm The rhythm of core body temperature during entrained and free-run conditions was monitored to determine whether in the HCRT2 SAP lesioned rats the biological clock, the suprachiasmatic nucleus (SCN), is functioning normally. The rats given the high concentration of HCRT2 SAP (490 ng) slept more at night when they should have been awake, and this could have occurred because these rats

10 232 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 7. Core temperature rhythm in entrained conditions (12-h lights on/lights off) 21 days after administration of saline or hypocretin-2 saporin (HCRT2 SAP) into the lateral hypothalamus. Values represent average core temperature ( C) at 10-min intervals for each group of rats. Average temperature during the lights-off period in rats given the high concentration of HCRT2 SAP was significantly lower compared with that of saline-treated rats (P 0.04). Nevertheless, these rats showed a temperature peak and nadir at the same phase position as saline-treated rats. When these rats were placed in constant dark conditions for three weeks, the period of the temperature rhythm was not different from that of saline-treated rats. were not receiving an appropriate awakening signal from the SCN. Nevertheless, these rats continued to display a temperature peak and nadir at the same time of day as saline-treated rats, indicating that the SCN is sending out a signal at the appropriate time of day. The rats given the low concentration of HCRT2 SAP also displayed intact entrained and free-run temperature and activity rhythms (Fig. 8), indicating that the SCN is functioning properly in rats with a massive loss of HCRT neurons. SCN function has not been assessed in the murine model of narcolepsy. In human narcoleptics there is a profound loss of HCRT neurons, but the circadian clock functions normally (Dantz et al., 1994), a finding that is consistent with the present results in HCRT2 SAP-treated rats. Animal models of narcolepsy versus human narcolepsy At present, narcoleptic-like behavior associated with the dysfunction of the HCRT system has been described in humans (Nishino et al., 2000b; Peyron et al., 2000; Thannickal et al., 2000; Overeem et al., 2001; Kales et al., 1982), dogs (Lin et al., 1999), mice (Chemelli et al., 1999; Hara et al., 2001), and rats (Gerashchenko et al., 2001). The existence of several animal models of narcolepsy allows us to compare symptoms between animal and human narcolepsy. An important symptom in human narcolepsy is excessive daytime sleepiness, which is defined as a continuous subjective feeling of sleepiness and the presence of irresistible sleep attacks (Overeem et al., 2001). As measured by the multiple sleep latency test, human narcoleptics have excessive daytime sleepiness but generally do not have an increased amount of sleep over a 24-h period. Sleep polysomnography conducted either at home or in the laboratory has found only few differences in total sleep measures between patients with narcolepsy and normal humans. Among the differences are higher daytime amounts of REM sleep and stage 1 sleep (drowsiness) in narcoleptic patients, whereas amounts of NREM sleep (stages 3 and 4 of sleep) usually do not differ significantly (Broughton et al., 1988; Nobili et al., 1995). Interestingly, when the amount of sleep in untreated patients with narcolepsy is compared with that of normal habitual nappers, slow-wave sleep amounts are significantly higher in patients with narcolepsy (Broughton et al., 1998). As in human narcolepsy, narcoleptic dogs display fragmentation of vigilance states characterized by significantly shorter mean duration of wake, drowsy, and deep sleep episodes (Nishino et al., 2000a; Kaitin et al., 1986). Day-

11 D. Gerashchenko et al. / Neuroscience 116 (2003) Fig. 8. Activity rhythms of representative rats administered saline (A) or high concentration of hypocretin-2 saporin (HCRT2-SAP) (B) into the lateral hypothalamus. Each panel depicts activity patterns during a 12-h:12-h light dark entrainment cycle and during dark dark (free-run) conditions. Activity was recorded every 10 min from a transmitter (Mini Mitter, Bend, OR) implanted in the abdominal cavity. The time after administration of saline or HCRT2 SAP is identified to the left of each panel (week 2, week 4, etc). Each line represents a 48-h record of activity pattern, with the second 24-h activity record being repeated in the first half of the subsequent line. The 12-h:12-h light dark entrained condition is represented by the dark (lights-off) and white (lights-on) bars at the top of each chart. Midway in each chart, the asterisk depicts the point where the lights were turned off and the animals were maintained in a dark environment devoid of any external lighting cues. Under such conditions, the rat s intrinsic clock dictates periods of rest and activity. Saline-treated rats (representative rat depicted in panel A) were more active and awake in the lights-off period and this pattern was maintained during the free-run condition. Rats administered HCRT2 SAP (representative rat depicted in panel B) were most active when the lights were turned off (during entrained condition) but then activity levels declined during the rest of the time because the animals had hypersomnia. Most important, during the free-run condition the HCRT2 SAP rats continued to be most active at the same time of day despite of a loss of HCRT and neighboring LH neurons, indicating that the HCRT and surrounding LH neurons are not the primary recipients of a signal to awaken from the circadian oscillator. time amounts of drowsy state, light sleep, deep sleep, and REM sleep are not significantly different between narcoleptic and normal dogs (Nishino et al., 2000a; Kaitin et al., 1986). A behavioral phenotype that resembles narcolepsy is also displayed by HCRT knockout mice (Chemelli et al., 1999) and mice with an acquired loss of HCRT neurons (HCRT/ataxin-3 mice) (Hara et al., 2001). These mice have fragmented sleep, little diurnal variation in the amount of REM sleep, and behavioral arrests. HCRT knockout mice have increased NREM sleep and REM sleep time during the normally active lights-off period. Nighttime amounts of NREM sleep in HCRT/ataxin-3 mice, however, are not different from that of wild-type mice (Hara et al., 2001). Similar to that in the HCRT knockout and HCRT/ ataxin-3 mice, lesions of the LH with HCRT2 SAP (90 ng) increase REM sleep during the night, reduce REM sleep during the day, and produce SOREMPs and sleep fragmentation. When a higher concentration of HCRT2 SAP (490 ng) is used, other LH neurons in addition to the HCRT neurons are destroyed and these rats have an increase in NREM sleep during the night. On the other hand, nighttime amounts of NREM sleep were not different from control values in narcoleptic dogs and ataxin-3 mice (Lin et al., 1999; Hara et al., 2001). Taken together, these data suggest that the HCRT neuronal loss produces the REM sleep abnormalities and when adjacent LH neurons are also lost, there is also a hypersomnia during the night in rats. Interestingly, in human narcolepsy, during the early stages of the illness, there is a profound hypersomnia, which then resolves gradually to sleep attacks and SOREMPs as the illness progresses (Kales et al., 1982). In the present study, both 90 and 490 ng of HCRT2 SAP produced a significant increase in the total sleep amounts calculated over 24 h on day 6 postinjection (25% and 34% increase, respectively). In the 90-ng HCRT2 SAP rats, the hypersomnia returned to normal levels by day 21, but the REM sleep abnormalities persisted. The transient hypersomnia in the SAP-treated animals might be due to loss of some HCRT and LH neurons. In these rats there was no long-term hypersomnia or SOREMPs, perhaps because the unconjugated SAP did not destroy a sufficient number of the LH and HCRT neurons. Convincing episodes of cataplexy alone, without an overlying SOREMP, were not present in the HCRT2-SAP treated rats. In fact, clear, unambiguous incidences of cataplexy have not been observed in the HCRT/orexin null mutant mice, either. In canine narcolepsy, specific incidences of cataplexy are

12 234 D. Gerashchenko et al. / Neuroscience 116 (2003) triggered in response to food or play and are short, lasting on the average 23 s (Wu et al., 1999). It is quite possible that stimuli that trigger cataplexy need to be identified in rodents. Alternatively, in order for cataplexy to occur, one would have to lesion the brainstem effector neurons implicated in triggering cataplexy. Nevertheless, both human narcoleptics and the high concentration of HCRT2 SAP treated rats are excessively sleepy during the normal wake active period. We suggest that the hypersomnia results from the loss of HCRT- and adjacent non HCRT-containing LH neurons. We reach this conclusion based on the sleep changes in the low concentration of HCRT2 SAP treated rats. These rats, like the HCRT/ataxin knockout mice, have REM sleep abnormalities at night but without a corresponding hypersomnia. In these rats, the diurnal rhythm of temperature is similar to that of saline-treated rats. On the other hand, the rats given the higher concentration of HCRT2 SAP have a greater loss of adjacent non HCRTcontaining neurons. These rats also have a hypersomnia at night. Loss of non-hcrt wake-related neurons produced by high concentrations of HCRT2 SAP may have accounted for the hypersomnia seen in the present study. HCRT neurons are not the only population of wake active neurons in the perifornical lateral hypothalamic area. Based on the electrophysiological characteristics of the neurons in this area, 53% of recorded neurons were classified as wake-/rem-related and 38% as wake-related (Alam et al., 2002). These results suggest that other neurons in the LH are also responsible for wakefulness, compared with the HCRT neurons. Moreover, recent findings suggest that in some narcoleptics, CSF levels of HCRT are normal (Ripley et al., 2001), raising the possibility that in these patients not all of the HCRT neurons have been lost and/or there is additional loss of other adjacent LH neurons. One can rule out neurons containing MCH as being responsible for narcolepsy, since they are not lost in human narcoleptics (Thannickal et al., 2000) and MCH knockout mice do not display narcoleptic behavior (Shimada et al., 1998). Thus, there appears to be an additional cell type not measured in human narcoleptics and which is destroyed in our studies. Destruction of these neurons in addition to the HCRT neurons might be causing hypersomnia. Taken together, the results show that when HCRT neurons are lost, REM sleep is increased, including inadvertent triggering of REM sleep episodes during purposeful behavior. 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