Gerashchenko D, Murillo-Rodriguez E, Blanco-Centurion C, Lin L, Nishino S, Mignot E, Shiromani PJ (2005) Adenosine levels do not increase with 6 h waking in rats with lesions of the lateral hypothalamus. Neuroscience 2005 Abstracts 63.9. Society for Neuroscience, Washington, DC.

Summary: The hypocretin neurons in the lateral hypothalamus (LH) have been implicated in wakefulness, but it is not clear which projection is responsible for the arousal. One possibility is that the LH neurons induce wakefulness by driving the basal forebrain (BF) wake-active neurons (Gerashchenko and Shiromani, Cellular & Molec Neurosci, 29: 41, 2004). Here we measure adenosine (AD) levels in the BF as a marker of arousal and test the LH-BF circuit in Sprague-Dawley rats with lesions of the LH induced by hypocretin-2-saporin. 64 days after lesions the rats were kept awake (gentle handling) for six hours (ZT 3-9) and microdialysis samples (5ul) were collected hourly for 9 hours (24h after probe stabilization). AD levels were assessed using HPLC. Hypocretin-saporin ablated 95% of the hypocretin neurons and reduced CSF hypocretin levels (-75% versus control). AD levels increased with 6h waking in saline control rats (n=9), consistent with previous studies in cats (Strecker et al., Behav Brain Res 115: 183, 2000) and rats (Murillo-Rodriguez et al., Neuroscience 123: 361, 2004). However, in rats with LH lesions (n=5) such an increase with waking did not occur. Sleep drive was measured by conducting a rodent version of a multiple sleep latency test (MSLT). In this test, conducted over 10h (from ZT2-ZT12) the rats were kept awake for 20min and then allowed 20min to sleep. The lesioned rats had more sleep during the 20min sleep periods indicating a higher sleep drive. These results suggest that in narcolepsy when the HCRT LH neurons die, there is a loss of stimulation of the wake-active BF neurons and the decline in this pathway may be the cause of the increased sleep attacks. Supported by VA Medical Research and NIH

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Blanco-Centurion C, Gerashchenko D, Salin-Pascual RJ, Shiromani PJ (2004) Effects of hypocretin2-saporin and antidopamine-beta-hydroxylase-saporin neurotoxic lesions of the dorsolateral pons on sleep and muscle tone. Eur J Neurosci 19(10):2741-2752. doi: 10.1111/j.0953-816X.2004.03366.x

Summary: Narcolepsy is linked to the loss of orexin (or hypocretin)-containing neurons in the brain. These neurons are located in the perifornical region of the posterior hypothalamus and innervate the locus coeruleus (LC). To investigate the role of the LC in sleep the authors injected 0.3 µl of 192-Saporin (Cat. IT-01) or anti-DBH-SAP (Cat. #IT-03) at 1 µg/µl. They also used 0.3 µl of orexin-SAP (Cat. #IT-20) at either 90 ng/µl or 60 ng/µl in a separate group of animals. The results indicate that orexin innervation to the pons plays a role in arousal from sleep.

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Gerashchenko D, Murillo-Rodriguez E, Lin L, Xu M, Hallett L, Nishino S, Mignot E, Shiromani PJ (2003) Relationship between CSF hypocretin levels and hypocretin neuronal loss. Exp Neurol 184(2):1010-1016. doi: 10.1016/S0014-4886(03)00388-1

Summary: Narcolepsy has recently been shown to be a neurodegenerative disease. Data from several different sources indicate that narcoleptics have very low levels of hypocretin (HCRT)-containing neurons. The authors sought to verify a direct linkage between HCRT-containing neurons and HCRT levels in the CSF. Rats were lesioned with 45-90 ng of orexin-SAP (Cat. #IT-20) bilaterally into the lateral hypothalamus. Loss of HCRT neurons correlated with decreased levels of HCRT in the CSF.

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Gerashchenko D, Murillo-Rodriguez E, Lin L, Xu M, Hallett L, Nishino S, Mignot E, Shiromani PJ (2003) Relationship between CSF hypocretin levels and hypocretin neuronal loss. Neuroscience 2003 Abstracts 616.2. Society for Neuroscience, New Orleans, LA.

Summary: In the sleep disorder narcolepsy there is a massive reduction in the number of neurons containing the neuropeptide, hypocretin (HCRT). Most narcoleptic patients also have low to negligible levels of HCRT in the cerebrospinal fluid (CSF). However, the relationship between HCRT neurons and HCRT levels is not known, making it difficult for investigators to estimate how many HCRT-containing neurons might be present based on measurements of CSF HCRT levels. A relationship between neuronal loss and CSF levels of the ligand is known in other degenerative diseases, such as Parkinson’s, but not in narcolepsy. To identify this relationship, hypocretin-2-saporin, the neurotoxin that kills hypocretin neurons, or saline were administered to the lateral hypothalamus and CSF was extracted at zeitgeber times (ZT) 0 (time of lights-on) or ZT 8 at various intervals (2, 4, 6, 12, 21, 36, 60 days) after the neurotoxin administration. Compared to saline animals (n=8), rats with an average loss of 73% of HCRT neurons (n=9) had a 50% decline in CSF HCRT levels on day 60. The decline in HCRT levels was evident by day 6 and there was no recovery or further decrease. The decline in HCRT was correlated with increased REM sleep. Rats with an average loss of 14.4% of HCRT neurons (n=4) showed no significant decline in CSF HCRT levels compared to saline rats. In rats with 73% loss of HCRT neurons, the HCRT levels were not substantially increased by 6h prolonged wakefulness indicating that surviving neurons were not able to increase the output of HCRT to compensate for the HCRT neuronal loss. From these data we conclude that since most narcoleptics have more than 80% reduction of CSF HCRT that in these patients most HCRT neurons are lost.

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Gerashchenko K, Blanco-Centurion C, Greco MA, Shiromani PJ (2003) Effects of lateral hypothalamic lesion with the neurotoxin hypocretin-2-saporin on sleep in Long-Evans rats. Neuroscience 116:223-235. doi: 10.1016/s0306-4522(02)00575-4

Summary: Recent data has linked narcolepsy to the loss of neurons containing the neuropeptide hypocretin, also known as orexin. The authors wished to investigate whether the variance in severity of narcolepsy could be explained by the extent of loss of these neurons. After injection of 90 or 490 ng of orexin-SAP (Cat. #IT-20) into the lateral hypothalamus, the measurement of several parameters demonstrated the severity of narcolepsy may be linked to the degree of loss of neurons expressing the orexin receptor.

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Gerashchenko D, Blanco-Centurion C, Shiromani PJ (2002) Hypocretin2-saporin (HCRT2-SAP) lesions of the lateral hypothalamus does not affect the entrained or free-running rhythm of core body temperature. Neuroscience 2002 Abstracts 776.2. Society for Neuroscience, Orlando, FL.

Summary: Hypocretin (HCRT)neurons are present only in the lateral hypothalamus (LH) from where they project heavily to major arousal centers. HCRT neurons are lost in the sleep disorder narcolepsy, an illness characterized by an increased tendency to fall asleep during the normal active period. As such, it is hypothesized that HCRT neurons are responsible for “waking-up” the brain. To test this hypothesis we monitored the rhythm of core body temperature during entrained & free-run conditions after lesions of the HCRT neurons. 23 male Long-Evans rats implanted with sleep recording electrodes and a temperature transmitter were given one of two concentrations (90 ng/0.5 ìl vs 490 ng/0.5 ìl) of the neurotoxin hypocretin2-saporin (HCRT2-SAP) or unconjugated saporin to the LH. Control rats received saline (n=5). After surgery, sleep and temperature were continuously recorded for 21d in entrained conditions followed by 21d in continuous darkness. Both concentrations of the HCRT2-SAP lesioned HCRT neurons (88% vs 91% HCRT loss). However, HCRT lesions did not disrupt the entrained rhythm of core temperature by either advancing or delaying the phase position of the temperature rhythm. In the saline rats, the free-run period of temperature rhythm (tau) was 24.16 (±0.07) and this was not significantly different in the HCRT2-SAP or SAP rats. These results indicate that in the absence of HCRT, the animal wakes up at the correct time of day but then is not able to stay awake.

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Gerashchenko D, Kohls MD, Greco MA, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-saporin (Hcrt-sap) as a tool to examine hypocretin function. Neuroscience 2001 Abstracts 410.7. Society for Neuroscience, San Diego, CA.

Summary: The hypocretin (Hcrt) peptides are linked to narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither lesions of this nor any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. To facilitate lesioning the Hcrt neurons, the ribosome inactivating protein, saporin (SAP), was conjugated to Hcrt-2/orexin B. In vitro binding studies indicated specificity of Hcrt-SAP since it preferentially bound to CHO-cells containing the HcrtR2/OX2 receptor compared to the HcrtR1/OX1 receptor, but not to KNRK cells stably transfected with the NK1 receptor. In vivo specificity was confirmed since administration of the toxin to the LH eliminated some neurons (Hcrt, MCH, and histamine) but not others (alpha-MSH). When the toxin was administered to the LH, rats (n=19) had increased slow wave sleep, REM sleep, and sleep-onset REM sleep periods at night. These were negatively correlated with the loss of Hcrt-containing neurons (r=-0.74; p<0.01). Toxin applied to hypothalamic neurons that are not Hcrt positive but contain the Hcrt receptor lesioned the neurons but did not produce narcoleptic-like sleep. These findings indicate the utility of Hcrt-SAP as a tool and also demonstrate that damage to the LH that also includes a substantial loss of Hcrt neurons is likely to produce the sleep disturbances that occur in narcolepsy.

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Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat. J Neurosci 21(18):7273-7283. doi: 10.1523/JNEUROSCI.21-18-07273.2001 PMID: 11549737

Summary: Orexin (also knows as hypocretin) peptides are produced exclusively by neurons in the lateral hypothalamus, however non-specific lesioning in this region has not produced narcoleptic-like sleep. Gerashchenko et al. use orexin-SAP (490 ng/0.5 µl; Cat. #IT-20) to specifically eliminate orexin neurons in rats. The treated rats displayed several sleep disturbances found in narcolepsy, including increased slow-wave sleep, and sleep-onset REM sleep periods. The data suggest that orexin-SAP can be used to create a model for narcolepsy in rats.

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Gerashchenko D, Salin-Pascual R, Shiromani PJ (2001) Effects of hypocretin-saporin injections into the medial septum on sleep and hippocampal theta. Brain Res 913:106-115. doi: 10.1016/s0006-8993(01)02792-5

Summary: Hypocretin, also known as orexin, neurons are located only in the lateral hypothalamus. Recently, the loss of these neurons was shown to be associated with narcolepsy. The authors used orexin-SAP (100 ng/0.5 µl; Cat. #IT-20) to eliminate parvalbumin and cholinergic neurons (orexin B receptor-expressing) in the rat medial septum. They used 192-Saporin (1 µg/ 1 µl; Cat. #IT-01) to contrast the effect and eliminate only cholinergic neurons (NGF/p75 receptor-expressing). Hippocampal theta activity was completely eliminated in orexin-SAP treated rats by day 12, suggesting that orexin neurons influence cognitive processes critical for survival.

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Gerashchenko D, Greco MA, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2000) Orexin-B conjugated to saporin lesions LH and TMN neurons and produces narcoleptic-like sleep in rats. Neuroscience 2000 Abstracts 566.27. Society for Neuroscience, New Orleans, LA.

Summary: A dysfunction of the hypocretin/orexin (Hcrt/Ox) system was recently linked with the sleep disorder, narcolepsy. To provide an experimental method that could be used to inactivate Hcrt/Ox receptor bearing neurons, we linked the toxin, saporin, to the orexin receptor binding ligand, orexin-B. Eighteen male Sprague-Dawley rats (400-450 g) were administered orexin-saporin (0.5 ul; 490 ng) to the lateral hypothalamus (LH) (where Hcrt/Ox containing neurons are located) or tuberomammillary nucleus (TMN) (where Hcrt/Ox receptor containing neurons are present) and sleep was recorded for 3 weeks. A significant reduction in the numbers of TMN and Hcrt/Ox neurons in the LH was detected 3 to 5 days after toxin administration and complete loss occurred by 2 weeks. Rats with extensive cell loss exhibited more REM sleep, nonREM sleep, and multiple sleep onset REM periods during the night. In the only two available animal models of human narcolepsy, the dysfunction in the orexin system is inherited and in the entire animal which makes it difficult to localize specific brain regions or circuits underlying narcolepsy. Orexin-saporin provides a method of determining the contribution of a specific Hcrt/Ox innervation in the regulation of behavior.

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