Ming-Fung Wu

Release of Hypocretin (Orexin) during Waking and Sleep States

Hypocretin (Hcrt or orexin) somas are located in the hypothalamus and project widely to forebrain and brainstem regions, densely innervating monoaminergic and cholinergic cells. Loss of Hcrt function results in the sleep disorder narcolepsy. However, the normal pattern of Hcrt release across the sleep–wake cycle is unknown. We monitored Hcrt-1 release in the basal forebrain, perifornical hypothalamus, and locus ceruleus (LC) across the sleep–wake cycle using microdialysis in freely moving cats and a sensitive solid phase radioimmunoassay. We found that the peptide concentration in dialysates from the hypothalamus was significantly higher during active waking (AW) than during slow-wave sleep (SWS). Moreover, Hcrt-1 release was significantly higher during rapid eye movement (REM) sleep than during SWS in the hypothalamus and basal forebrain. We did not detect a significant difference in release across sleep–waking states in the LC, perhaps because recovered levels of the peptide were lower at this site. Because there was a trend toward higher levels of Hcrt-1 release during AW compared with quiet waking (QW) in our 10 min dialysis samples, we compared Hcrt-1 levels in CSF in 2 hr AW and QW periods. Hcrt-1 release into CSF was 67% higher during AW than during QW. Elevated levels of Hcrt during REM sleep and AW are consistent with a role for Hcrt in the central programming of motor activity.

LOCUS COERULEUS NEURONS: CESSATION OF ACTIVITY DURING CATAPLEXY

Cataplexy, a symptom of narcolepsy, is a loss of muscle tone usually triggered by sudden, emotionally significant stimuli. We now report that locus coeruleus neurons cease discharge throughout cataplexy periods in canine narcoleptics. Locus coeruleus discharge rates during cataplexy were as low as or lower than those seen during rapid-eye-movement sleep. Prazosin, an alpha1 antagonist, and physostigmine, a cholinesterase inhibitor, both of which precipitate cataplexy, decreased locus coeruleus discharge rate. Our results are consistent with the hypothesis that locus coeruleus activity contributes to the maintenance of muscle tone in waking, and that reduction in locus coeruleus discharge plays a role in the loss of muscle tone in cataplexy and rapid-eye-movement sleep. Our results also show that the complete cessation of locus coeruleus activity is not sufficient to trigger rapid-eye-movement sleep in narcoleptics.

Activity of dorsal raphe cells across the sleep-waking cycle and during cataplexy in narcoleptic dogs

Cataplexy, a symptom associated with narcolepsy, represents a unique dissociation of behavioural states. During cataplectic attacks, awareness of the environment is maintained, as in waking, but muscle tone is lost, as in REM sleep. We have previously reported that, in the narcoleptic dog, noradrenergic cells of the locus coeruleus cease discharge during cataplexy. In the current study, we report on the activity of serotonergic cells of the dorsal raphe nucleus. The discharge patterns of serotonergic dorsal raphe cells across sleep–waking states did not differ from those of dorsal raphe and locus coeruleus cells recorded in normal rats, cats and monkeys, with tonic discharge in waking, reduced activity in non‐REM sleep and cessation of activity in REM sleep. However, in contrast with locus coeruleus cells, dorsal raphe REM sleep‐off neurones did not cease discharge during cataplexy. Instead, discharge continued at a level significantly higher than that seen in REM sleep and comparable to that seen in non‐REM sleep. We also identified several cells in the dorsal raphe whose pattern of activity was the opposite of that of the presumed serotonergic cells. These cells were maximally active in REM sleep and minimally active in waking and increased activity during cataplexy. The difference between noradrenergic and serotonergic cell discharge profiles in cataplexy suggests different roles for these cell groups in the normal regulation of environmental awareness and muscle tone and in the pathophysiology of narcolepsy.

Highly Specific Role of Hypocretin (Orexin) Neurons: Differential Activation as a Function of Diurnal Phase, Operant Reinforcement versus Operant Avoidance and Light Level

Hypocretin (Hcrt) cell loss is responsible for narcolepsy, but Hcrts role in normal behavior is unclear. We found that Hcrt knock-out mice were unable to work for food or water reward during the light phase. However, they were unimpaired relative to wild-type (WT) mice when working for reward during the dark phase or when working to avoid shock in the light or dark phase. In WT mice, expression of Fos in Hcrt neurons occurs only in the light phase when working for positive reinforcement. Expression was seen throughout the mediolateral extent of the Hcrt field. Fos was not expressed when expected or unexpected unearned rewards were presented, when working to avoid negative reinforcement, or when given or expecting shock, even though these conditions elicit maximal electroencephalogram (EEG) arousal. Fos was not expressed in the light phase when light was removed. This may explain the lack of light-induced arousal in narcoleptics and its presence in normal individuals. This is the first demonstration of such specificity of arousal system function and has implications for understanding the motivational and circadian consequences of arousal system dysfunction. The current results also indicate that comparable and complementary specificities must exist in other arousal systems.

Lateral geniculate spikes, muscle atonia and startle response elicited by auditory stimuli as a function of stimulus parameters and arousal state

We have investigated the motor and ponto-geniculo-occipital (PGO) wave response to startle eliciting stimuli in the unanesthetized cat. We found that the amplitude of the PGO spike recorded in the lateral geniculate nucleus (LGN) increases monotonically with increasing intensities of auditory stimuli. In contrast, the motor response to low intensity (less than 75 dB) stimuli is characterized by electromyographic (EMG) suppression, while at higher intensities an EMG excitation is superimposed on this suppression. Thus PGO elicitation is accompanied by EMG suppression at low intensities and by a net EMG excitation at high intensities. While the amplitude of the auditory elicited PGO response is a graded function of stimulus intensity, somatic stimuli tend to elicit the PGO response in all-or-none fashion. Both the motor and PGO responses to sensory stimulation change with behavioral state. The EMG suppression by auditory stimulation increases in duration during the transition to rapid eye movement (REM) sleep. Elicited PGO amplitude is highest in transitional sleep, lower in quiet waking and REM sleep and lowest in active waking. Prepulse inhibition of PGO spikes is greatly attenuated during transitional and REM sleep. We hypothesize the existence of 3 phasic response systems, a motor suppression system, a motor excitation (startle) system and a PGO elicitation system. While these systems are triggered concurrently by intense phasic stimuli in waking, they are modulated independently by stimulus intensity and behavioral state, and have different rates of habituation. These systems act in concert to produce behavioral responses to sudden onset stimuli.

Cholinergic mechanisms in startle and prepulse inhibition: effects of the false cholinergic precursor N-aminodeanol.

We examined the effects of cholinergic deficiency on prepulse inhibition (PPI) of the acoustic startle. Rats treated with a choline-free diet that contained the false cholinergic precursor N-aminodeanol showed great deficit in PPI. This deficit does not appear to be secondary to an increase of stereotyped behaviors. Startle threshold was also greatly reduced, as these rats startled to the 70-dB prepulse and the baseline startle amplitude was increased by 60% over the control rats. Arecoline (4 mg/kg) partially reversed the deficit in PPI. This improvement persisted beyond the period of drug treatment. On the other hand, scopolamine (1 mg/kg) reduced PPI in the control rats. These results suggest that cholinergic systems play a major role in both the elicitation and prepulse inhibition of startle.

Intravenously administered hypocretin‐1 alters brain amino acid release: an in vivo microdialysis study in rats

We have reported that intravenous administration of hypocretin (Hcrt or orexin) reverses the symptoms of narcolepsy in genetically narcoleptic dogs. We have also reported that the onset of symptoms in canine genetic narcolepsy is accompanied by degenerative changes in forebrain regions, particularly the septal nucleus and amygdala. In the present in vivo microdialysis study we have investigated the effect of intravenous administration of Hcrt‐1 (orexin‐A) to anaesthetized rats on glutamate and GABA release in the amygdala, a region with moderate Hcrt innervation, and in the cerebellar cortex, a region with sparse or no Hcrt innervation. We found that intravenous Hcrt administration caused a marked (> 60 %) and sustained (> 50 min) increase in glutamate release within the amygdala, but no change in release in the cerebellar cortex. We did not detect a significant change in GABA release. When calcium‐free artificial cerebrospinal fluid was used as the microdialysis perfusate, Hcrt‐1 no longer produced an increase in glutamate release. Hcrt may act via the calcium‐dependent regulation of glutamate release in certain nuclei of the central nervous system.

FACILITATION OF THE ACOUSTIC STARTLE REFLEX BY PONTO-GENICULO-OCCIPITAL WAVES : EFFECTS OF PCPA

The relationship between ponto-geniculo-occipital (PGO) waves and motor activity during waking and non-rapid eye movement (non-REM) sleep stages was studied in cats treated with the serotonin synthesis inhibitor p-chlorophenylalanine (PCPA). PGO waves appeared in waking after daily treatment with PCPA. The magnitude of the acoustic startle elicited in the absence of prior PGO waves was increased (by a mean of 555%) by the PCPA treatment as compared to that of the pre-drug level. When startle-eliciting stimuli were presented shortly after the occurrence of the PGO wave, the response amplitude was further enhanced as compared to that of the baseline startle. The effect was maximal 50 ms following the peak of the PGO wave (average 192% of the baseline level), with return to the baseline startle level within 200 ms. A similar effect could also be seen with waking eye-movement potentials (EMPs) in drug-naive animals. Over half of the spontaneous PGO waves were found to be preceded or followed by discrete head-body movements. After PCPA, the amplitude of auditory-evoked LGN PGO waves increased during quiet waking (QW) while those in non-REM and REM sleep states did not change. It was concluded that serotonergic systems produce a tonic suppression of startle response and PGO amplitude in waking. PGO spikes in waking are associated with a phasic facilitation of the sensorimotor mechanisms involved in startle.

REM sleep deprivation reduces auditory evoked inhibition of dorsolateral pontine neurons

In many dorsolateral pontine neurons, auditory stimulation produces an initial excitation followed by a sustained inhibition. We now report that rapid eye movement (REM) sleep deprivation, for periods of from 22-48 h, reduced this auditory evoked inhibition of unit discharge. Inhibition returned to baseline levels after recovery REM sleep. Prior work indicates that the auditory evoked inhibition seen in noradrenergic cells in this region is partially mediated by norepinephrine. We hypothesize that the reduction in inhibition that we see is a consequence of either downregulation/desensitization of norepinephrine receptors or reduced norepinephrine release resulting from REM sleep deprivation.

Treatment with immunosuppressive and anti-inflammatory agents delays onset of canine genetic narcolepsy and reduces symptom severity.

All Doberman pinschers and Labrador retrievers homozygous for a mutation of the hypocretin (orexin) receptor-2 (hcrtr2) gene develop narcolepsy under normal conditions. Degenerative changes and increased display of major histocompatibility complex class II antigens have been linked to symptom onset in genetically narcoleptic Doberman pinschers. This suggests that the immune system may contribute to neurodegenerative changes and narcoleptic symptomatology in these dogs. We therefore attempted to alter the course of canine genetic narcolepsy, as an initial test of principle, by administering a combination of three immunosuppressive and anti-inflammatory drugs chosen to suppress the immune response globally. Experimental dogs were treated with a combination of methylprednisolone, methotrexate and azathioprine orally starting within 3 weeks after birth, and raised in an environment that minimized pathogen exposure. Symptoms in treated and untreated animals were quantified using the food elicited cataplexy test (FECT), modified FECT and actigraphy. With drug treatment, time to cataplexy onset more than doubled, time spent in cataplexy during tests was reduced by more than 90% and nighttime sleep periods were consolidated. Short-term drug administration to control dogs did not reduce cataplexy symptoms, demonstrating that the drug regimen did not directly affect symptoms. Treatment was stopped at 6 months, after which experimental animals remained less symptomatic than controls until at least 2 years of age. This treatment is the first shown to affect symptom development in animal or human genetic narcolepsy. Our findings show that hcrtr2 mutation is not sufficient for the full symptomatic development of canine genetic narcolepsy and suggest that the immune system may play a role in the development of this disorder.