The development of hypocretin (orexin) deficiency in hypocretin/ataxin-3 transgenic rats

Abstract

Narcolepsy is linked to a widespread loss of neurons containing the neuropeptide hypocretin (HCRT), also named orexin. A transgenic (TG) rat model has been developed to mimic the neuronal loss found in narcoleptic humans. In these rats, HCRT neurons gradually die as a result of the expression of a poly-glutamine repeat under the control of the HCRT promoter. To better characterize the changes in HCRT-1 levels in response to the gradual HCRT neuronal loss cerebrospinal fluid (CSF) HCRT-1 levels were measured in various age groups (2-82 weeks) of wild-type (WT) and TG Sprague-Dawley rats. TG rats showed a sharp decline in CSF HCRT-1 level at week 4 with levels remaining consistently low (26%+/-9%, mean+/-S.D.) thereafter compared with WT rats. In TG rats, HCRT-1 levels were dramatically lower in target regions such as the cortex and brainstem (100-fold), indicating decreased HCRT-1 levels at terminals. In TG rats, CSF HCRT-1 levels significantly increased in response to 6 h of prolonged waking, indicating that the remaining HCRT neurons can be stimulated to release more neuropeptide. Rapid eye movement (REM) sleep in TG rats (n=5) was consistent with a HCRT deficiency. In TG rats HCRT immunoreactive (HCRT-ir) neurons were present in the lateral hypothalamus (LH), even in old rats (24 months) but some HCRT-ir somata were in various stages of disintegration. The low output of these neurons is consistent with a widespread dysfunction of these neurons, and establishes this model as a tool to investigate the consequences of partial hypocretin deficiency.

Figure 1

HCRT-1 levels in wildtype (WT) and hypocretin-ataxin transgenic (TG) rats. HCRT-1 was measured in the CSF (top), cortex (middle) and brainstem (bottom) at various ages in WT and TG rats. CSF was extracted at two time points (ZT0 and ZT 8). Brain measurements were all made at ZT 3–5. TG rats have consistently low levels of HCRT-1, especially after the fourth week of age.

Figure 2

CSF HCRT-1 levels in response to prolonged waking. CSF was collected from male and female WT and TG rats at ZT8 without sleep deprivation (no TSD) and after 6h (ZT2–ZT8) of total sleep deprivation (TSD). Significance levels: * versus no TSD (P< 0.05); ** versus no TSD (P<0.01); ⇟=versus no TSD (P<0.001); # =WT versus TG (P< 0.001). The numbers within brackets represents the sample size of the group.

Figure 3

Percent (± SEM) wake, nonREM and REM sleep in old WT and TG rats. The line graphs summarize the average each hour across the 24h period whereas the bar graphs summarize the data during the 12h night and day cycles. The rats were implanted with sleep recording electrodes (isofluorane anesthesia) and two weeks later a 48 h sleep recording was made. CSF was not extracted from the TG rats but the diurnal distribution of REM sleep is indicative of a HCRT deficiency. Asterisks denote significant difference (P<0.05) versus WT.

Figure 4

Alternation between wakefulness (W), non-REM sleep (NR) and REM sleep in representative WT and TG rats. The figure represents a 40 minute segment of a sleep-wake recording during the night. Each panel (WT and TG) consists of a recording of the electroencephalogram (EEG), power of the EEG in the delta (0.3–4 hz; pink) and theta bands (4–12 hz; green), and integrated activity of the nuchal muscles (EMG). The sleep-wake state determination, based on the relationship of the EEG, power and EMG activity, is indicated at the bottom of each panel. In the WT rat there are normal transitions from W to non-REM to REM sleep. In TG rats, there are occasional occurrences of sleep-onset REM sleep period (SOREMP). The SOREMP is identified by the abrupt loss of EMG tone (near zero) during a wake bout. Note that the length of the SOREMP episode is quite similar to an average length of a REM sleep bout.

Figure 5

Hypocretin-immunoreactive neurons in the LH of representative WT and TG rats. The top four photos depict LH in WT rats and the lower four photos represent TG rats. HCRT-ir neurons are evident in 3 and 5 week old TG rats. These neurons are still visible in 16 week old rats, albeit they are much lighter.

Figure 6

Hypocretin-ir neurons in the LH of representative WT (63 weeks) (photo A and C) and TG (83 wks) (photos B, D and E) rats. In TG rats, HCRT-ir somata were evident in the LH (photo B) but the high density of HCRT processes seen in WT rats (photo A) was lacking. Photomicrographs C and D represent higher magnification (Nomarski optics) of the areas denoted by arrows in photos A and B. Note that in TG rats the somata are visible but they lack the axonal and dendritic processes emanating from it. Upon closer inspection some of the HCRT somata of TG rats were found to be in various stages of disintegration (photo E). Magnification: A=B, C=D.