Susanna Bianchi

Mutant Prion Protein Expression Causes Motor and Memory Deficits and Abnormal Sleep Patterns in a Transgenic Mouse Model

A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.

Interleukin-1β enhances non-rapid eye movement sleep when microinjected into the dorsal raphe nucleus and inhibits serotonergic neurons in vitro

Interleukin‐1 (IL‐1) and IL‐1 receptors are constitutively expressed in normal brain. IL‐1 increases non‐rapid eye movements (NREM) sleep in several animal species, an effect mediated in part by interactions with the serotonergic system. The site(s) in brain at which interactions between IL‐1 and the serotonergic system increase NREM sleep remain to be identified. The dorsal raphe (DRN) is the origin of the major ascending serotonergic pathways to the forebrain, and it contains IL‐1 receptors. This study examined the hypothesis that IL‐1 increases NREM sleep by acting at the level of the DRN. IL‐1β (0.25 and 0.5 ng) was microinjected into the DRN of freely behaving rats and subsequent effects on sleep–wake activity were determined. IL‐1β 0.5 ng increased NREM sleep during the first 2 h post‐injection from 33.5 ± 3.7% after vehicle microinjection to 42.9 ± 3.0% of recording time. To determine the effects of IL‐1β on electrophysiological properties of DRN serotonergic neurons, intracellular recordings were performed in a guinea‐pig brain stem slice preparation. In 26 of 32 physiologically and pharmacologically identified serotonergic neurons, IL‐1β superfusion (25 ng/mL) decreased spontaneous firing rates by 50%, from 1.6 ± 0.2 Hz (before IL‐1β superfusion) to 0.8 ± 0.2 Hz. This effect was reversible upon washout. These results show that IL‐1β increases NREM sleep when administered directly into the DRN. Serotonin enhances wakefulness and these novel data also suggest that IL‐1β‐induced enhancement of NREM sleep could be due in part to the inhibition of DRN serotonergic neurons.

Selective blockade of different brain stem muscarinic receptor subtypes: effects on the sleep-wake cycle

Changes induced in the sleep-wake cycle by pontine microinjections of muscarinic antagonists were studied in freely moving rats, instrumented for chronic polygraphic recordings. Pirenzepine (PIR), methoctramine (MET) and p-fluoro-hexahydro-siladifenidol (p-F-HHSiD), which are highly selective M1, M2 and M3 antagonists, respectively, were dissolved in 0.1 microliter of sterile isotonic saline (0.2 microliter of distilled water for p-F-HHSiD) and injected into the pontine reticular nucleus, where the administration of 0.5 microgram carbachol (a mixed muscarinic agonist) induced a 52% increase in the amount of desynchronized sleep (DS) over a 6 h recording period. The blockade of M2 receptors was shown to (i) antagonize DS, by increasing its latency and decreasing its percentage, (ii) decrease slow wave sleep, and (iii) enhance wakefulness. These effects were dose-dependent. No changes in the sleep-wake cycle were observed following microinjection of M1 or M3 antagonists. The results support the hypothesis that at the brain stem level only M2 receptors are involved in sleep mechanisms and, particularly, in the generation and maintenance of DS.

5-Hydroxytryptophan, but not L-tryptophan, alters sleep and brain temperature in rats.

The precise role of serotonin (5-hydroxytryptamine) in the regulation of sleep is not fully understood. To further clarify this role for 5-hydroxytryptamine, the 5-hydroxytryptamine precursors L-tryptophan (40 and 80 mg/kg) and L-5-hydroxytryptophan (25-, 50-, 75-, 100 mg/kg) were injected intraperitoneally into freely behaving rats 15 min prior to dark onset, and subsequent effects on sleep-wake activity and cortical brain temperature were determined. L-5-hydroxytryptophan, but not L-tryptophan, induced dose-dependent changes in sleep-wake activity. During the 12-h dark period, non-rapid eye movement sleep was inhibited in post-injection hours 1-2 by the two lowest L-5-hydroxytryptophan doses tested, while the two highest doses induced a delayed increase in non-rapid eye movement sleep in post-injection hours 3-12. These highest doses inhibited non-rapid eye movement sleep during the subsequent 12-h light period. The finding that L-5-hydroxytryptophan, but not L-tryptophan, induced a dose-dependent and long-lasting decrease in cortical brain temperature regardless of whether or not non-rapid eye movement sleep was suppressed or enhanced contributes to a growing list of conditions showing that sleep-wake activity and thermoregulation, although normally tightly coupled, may be dissociated. The initial non-rapid eye movement sleep inhibition observed following low doses of L-5-hydroxytryptophan may be attributable to increased serotonergic activity since 5-hydroxytryptamine may promote wakefulness per se, whereas the delayed non-rapid eye movement sleep enhancement after higher doses may be due to the induction by 5-hydroxytryptamine of sleep-inducing factor(s), as previously hypothesized. The period of non-rapid eye movement sleep inhibition beginning 12 h after administration of L-5-hydroxytryptophan doses that increase non-rapid eye movement sleep is characteristic of physiological manipulations in which non-rapid eye movement sleep is enhanced. The results of the present study suggest that the complex effects of 5-HT on sleep depend on the degree and time course of activation of the serotonergic system such that 5-HT may directly inhibit sleep, yet induce a cascade of physiological processes that enhance subsequent sleep.

Muramyl dipeptide and IL-1 effects on sleep and brain temperature after inhibition of serotonin synthesis

The role of the interactions between serotonin (5-HT) and muramyl dipeptide (MDP) and interleukin-1 (IL-1) in sleep control and thermoregulation was evaluated. To this purpose, MDP and IL-1 were injected intracerebroventricularly at dark onset into freely moving rats pretreated twice intraperitoneally with para-chlorophenylalanine (PCPA) (300 mg/kg), which depletes brain 5-HT and causes insomnia. Fever and slow-wave sleep (SWS) enhancement induced by 150 pmol MDP were completely blocked in PCPA-pretreated rats. Only the first phase of the biphasic increase in SWS induced by 2.5 ng IL-1 was suppressed by PCPA pretreatment, whereas fever remained unaffected. These results suggest that 1) MDP effects on both sleep-wake activity and brain cortical temperature are mediated by the serotonergic system; 2) the mechanisms mediating the first and the second phases of IL-1-induced SWS excess are different: 5-HT could be involved in the first phase, but not in the second one; and 3) the 5-HT system does not appear to be involved in IL-1-induced fever.

Differential effects of M2 and M3 muscarinic antagonists on the sleep-wake cycle.

To study the role of muscarinic receptor subtypes in sleep control, methoctramine (25, 50, 75 micrograms), a highly selective M2 antagonist, was injected intra-cerebroventricularly into freely moving rats. Methoctramine induced a dose-dependent increase in desynchronized sleep (DS) latency (from 62.7 +/- 10 min following saline to 122.4 +/- 13.8 min with the lowest dose) and a 75% decrease in the amount of DS in 6 h recordings. 4DAMP (a M3/M1 selective antagonist) did not significantly change DS latency and percentage time, but it reduced wakefulness (from 38 +/- 2.8% following saline to 25.3 +/- 3.7% with a dose of 2.5), and increased slow wave sleep. The results suggest that M2 muscarinic receptors play a selective role in DS physiology.

M1 and M3 muscarinic receptors: specific roles in sleep regulation.

P-FLUORO-HEXAHYDRO-SILA-DIFENIDOL hydrochloride (p-F-HHSiD) (15, 30 μg) and pirenzepine (7.5, 15, 30 μg), which are highly selective M, and M1 muscarinic antagonists, respectively, were injected intracerebroventricularly into freely moving rats. p-F-HHSiD (30 μg) reduced wakefulness (W) (from 34.7 ± 3.1 to 24.9 ± 1.3 min) and increased slow wave sleep (SWS) (from 56.7 ± 2.4 to 67.2 ± 1.5 min); however, it did not modify desynchronized sleep (DS) latency and percentage in 6 h recordings. W and SWS were not affected by pirenzepine (7.5, 15, 30 μg) which decreased significantly DS amount but left unaffected DS latency. The results suggest that each muscarinic receptor subtype may induce different and specific changes in sleep phases and cortical desynchronization processes.

Muscarinic receptor subtypes in the medial preoptic area and sleep-wake cycles.

To clarify which muscarinic receptor subtype(s) mediate changes in sleep and cortical temperature (Tcort) induced by carbachol microinjections into the medial preoptic area (MPA), pirenzepine, tripitramine and ± p <>-fluoro-hexahydro-sila-difenidol (p-F-HHSiD), which are highly selective muscarinic M1, M2 and M3 antagonists, respectively, were microinjected into the MPA of rats. Whereas pirenzepine (3.45 and 7.08 nmol) and p-F-HHSiD (3.90 and 7.80 nmol) were without effect, tripitramine (0.67 and 3.37 nmol) enhanced wakefulness, decreased slow wave and desynchronized sleep, and raised Tcort with the higher dose. The data suggest that in the MPA only M2 muscarinic subtypes may be functionally important in mediating the cholinergic effects on sleep and thermoregulation.