Antisense oligonucleotide-mediated exon skipping of CHRNA1 pre-mRNA as potential therapy for Congenital Myasthenic Syndromes.

CHRNA1 encodes the α subunit of nicotinic acetylcholine receptors (nAChRs) and is expressed at the neuromuscular junction. Moreover, it is one of the causative genes of Congenital Myasthenic Syndromes (CMS). CHRNA1 undergoes alternative splicing to produce two splice variants: P3A(-), without exon P3A, and P3A(+), with the exon P3A. Only P3A(-) forms functional nAChR. Aberrant alternative splicing caused by intronic or exonic point mutations in patients leads to an extraordinary increase in P3A(+) and a concomitant decrease in P3A(-). Consequently this resulted in a shortage of functional receptors. Aiming to restore the imbalance between the two splice products, antisense oligonucleotides (AONs) were employed to induce exon P3A skipping. Three AON sequences were designed to sterically block the putative binding sequences for splicing factors necessary for exon recognition. Herein, we show that AON complementary to the 5' splice site of the exon was the most effective at exon skipping of the minigene with causative mutations, as well as endogenous wild-type CHRNA1. We conclude that single administration of the AON against the 5' splice site is a promising therapeutic approach for patients based on the dose-dependent effect of the AON and the additive effect of combined AONs. This conclusion is favorable to patients with inherited diseases of uncertain etiology that arise from aberrant splicing leading to a subsequent loss of functional translation products because our findings encourage the option of AON treatment as a therapeutic for these prospectively identified diseases.

Isatin, an endogenous MAO inhibitor, and a rat model of Parkinson's disease induced by the Japanese encephalitis virus.

A single dose of isatin (indole-2,3-dione)(i.p.), an endogenous MAO inhibitor, significantly increased norepinephrine and 5-hydroxytryptamine concentrations in the rat brain and also significantly increased acetylcholine and dopamine (DA) levels in the rat striatum. Urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. We have developed a rat model of Parkinson's disease induced by the Japanese encephalitis virus (JEV). The distribution of the pathological lesions of JEV-rats resemble those found in Parkinson's disease. Significant behavioral improvement was observed in JEV-rats after isatin, L-DOPA and selegiline administration using a pole test. Both isatin and selegiline prevented the decrease in striatum DA levels of JEV-rats. The increased turnover of DA (DOPAC/DA) induced by JEV was significantly inhibited by isatin, but not selegiline. These findings suggest that JEV-infected rats may serve as a model of Parkinson's disease and that exogenously administered isatin and selegiline can improve JEV-induced parkinsonism by increasing DA concentrations in the striatum.

Comparative study of the effects of isatin, an endogenous MAO-inhibitor, and selegiline on bradykinesia and dopamine levels in a rat model of Parkinson's disease induced by the Japanese encephalitis virus.

We previously reported that exogenously administered isatin, an endogenous monoamine oxidase (MAO) inhibitor, significantly increased acetylcholine (ACh) and dopamine (DA) levels in the rat striatum. Selegiline [(-)-deprenil] was developed as a MAO-B inhibitor more than 30 years ago and widely used in the treatment of Parkinson's disease. Effects of isatin or selegiline were investigated in Japanese encephalitis virus (JEV)-induced post-encephalitic parkinsonism rats by a pole test for detecting motor activity and by the determination of biogenic amine levels. Motor activity of JEV-induced rats receiving isatin (100 mg/kg per day for 1 week, i.p.) or selegiline (0.2 mg/kg per day for 1 week, i.p.) was significantly improved compared with that of untreated JEV-infected rats. Both isatin and selegiline prevented the decrease in striatal DA levels in JEV-rats. The increased turnover of DA (DOPAC/DA) induced by JEV was significantly inhibited by isatin, but not by selegiline. These results suggested that exogenously administered isatin and selegiline can improve JEV-induced parkinsonism by increasing DA concentrations in the striatum.

Roles of paraventricular catecholamines in feeding-associated corticosterone rhythm in rats.

Effects of local destruction of the brain catecholaminergic neurons were examined on the light- and feeding-associated circadian rhythms in plasma corticosterone in rats. 6-Hydroxydopamine (6-OHDA), a selective and long-lasting neurotoxin of the catecholaminergic neurons, was microinjected into the following discrete areas of the brain: the paraventricular nucleus (PVN), median eminence (ME), suprachiasmatic nucleus (SCN), ventromedial hypothalamic nucleus (VMH), lateral hypothalamic nucleus (LH), and the ascending bundle of noradrenergic neurons (NAB). And the feeding-associated as well as the light-associated circadian rhythms in plasma corticosterone were determined. The light-associated circadian rhythm was assayed under a 24-h light-dark cycle with free access to food, whereas the feeding-associated circadian rhythm was measured under restricted daily feeding in which rats had free access to food at a fixed time of day. 6-OHDA reduced the norepinephrine concentrations in respective regions to 10-30% of the control value, except for the LH. The light-associated circadian rhythm was not affected by 6-OHDA into the SCN or PVN. By contrast, 6-OHDA into the PVN or ventral NAB suppressed the feeding-associated circadian peak. 6-OHDA into the VMH and LH showed some effects on plasma corticosterone level but not on the feeding-associated circadian rhythm. 6-OHDA had no systematic effect on plasma corticosterone level when injected into the SCN, ME, and dorsal NAB. These findings indicate that the catecholaminergic neurons projecting to the PVN are involved in the feeding-associated but not in the light-associated circadian rhythms.

SCN-independent circadian oscillators in the rat.

In this short overview, features of the two suprachiasmatic nucleus (SCN)-independent oscillators are described. One is restricted daily feeding (RF)-associated oscillator and the other is methamphetamine-dependent oscillator. We found that both oscillators have free-running period of circadian range, and express themselves in the special state of energy supply or energy deficiency. The expression of the RF-associated oscillator depends heavily on the catecholaminergic input from the medulla, whereas the methamphetamine-dependent oscillator appears to depend on the sympathomimetic effects of the drug. It is plausible that overt rhythms of both oscillators are complex, mainly because of the interaction of hourglass principle with circadian clock mechanism.

Methamphetamine effects on rat circadian clock depend on actograph.

Methamphetamine effects on the rest-activity rhythm were examined in 12 blinded rats using two different actographs, an Animex and a running-wheel. D-Methamphetamine was administered chronically by dissolving it in drinking water. During methamphetamine treatment, the rest-activity rhythm measured by an Animex showed a clear sign of relative coordination in addition to the general enhancement of activity level. Analyses of pre- and posttreatment activity rhythms revealed that neither the phase nor the period was affected by methamphetamine treatment. On the other hand, the circadian period was lengthened by methamphetamine treatment when locomotor activity was measured by a running-wheel. These results confirmed our previous findings that the chronic treatment of methamphetamine modified the expression of the circadian rhythms but did not affect the underlying oscillation when measured by an Animex, and further indicated that methamphetamine could affect the underlying oscillation when rats had free access to a running-wheel. It is concluded that the effects of methamphetamine on the circadian clock depend on actograph.

Methamphetamine induced locomotor rhythm entrains to restricted daily feeding in SCN lesioned rats.

Rats were lesioned in the SCN and treated with methamphetamine dissolved in drinking water. A robust rhythm appeared in spontaneous locomotor activity which was not affected by blinding. Periodic food restriction (RF) of a 24 hr period was imposed on SCN lesioned rats with free-access to food for 4 or 6 hr per day, while water was given ad lib. The locomotor rhythm induced by methamphetamine treatment was phase-set by RF immediately in most cases but with transients in some. The phase-angle difference (psi) between food presentation and the activity onset became more negative by increasing the dose of methamphetamine. Because there was a positive correlation between methamphetamine dose and the period of locomotor rhythm, the change in psi was most likely due to lengthening of the period. After the termination of the RF schedule, the locomotor rhythm started to free-run from the prior phase set by RF. These results indicate that the methamphetamine dependent locomotor rhythm entrains to RF.

Rhythms in behaviors, body temperature and plasma corticosterone in SCN lesioned rats given methamphetamine.

In aperiodic rats with lesions in the suprachiasmatic nuclei (SCN), rhythms with a circadian period in spontaneous locomotion, wheel-running, feeding, drinking, body temperature and plasma corticosterone were restored by chronic administration of methamphetamine. These rhythms were not entrained by a light-dark cycle. Wheel-running, feeding and drinking rhythms in individual rats were in phase in terms of ultradian bout as well as circadian fluctuation. Rhythms of the intraperitoneal temperature appeared accompanying the spontaneous locomotor rhythm. The phase relation between the two rhythms was similar to that of SCN dependent rhythms. Plasma corticosterone also fluctuated in a circadian fashion. The corticosterone peak preceded the activity onset of locomotor rhythm by a few hours, which was similar to the phase relation observed in the SCN intact animals. It is concluded that the oscillatory mechanism underlying the spontaneous locomotor rhythm in SCN lesioned and methamphetamine treated rats drives also other physiological rhythms. The phase-relations among them were similar to those of rhythms driven by the circadian pacemaker in the SCN.

Phase setting of circadian locomotor rhythm of infant rats.

Postnatal phase setting of pup circadian rhythm by nursing mother was examined in rats by mother-pup exchange experiments. To know the effect of phase reversal of the nursing mother, pups were exchanged after birth so that a mother rat raised pups, half of which were born to her and the other half which came from a reversed mother, and activity onset and offset of pup locomotor rhythm were measured at weaning. Effects of litter size were also examined. Direct entrainment to light-dark cycles was excluded by blinding pups. In the unexchanged group, a strong positive correlation was detected between the phase of the circadian rhythm at weaning and the free-running period measured after weaning. The area covered by the 99% confidence interval of the regression line was used for the phase reference zone, and the circadian rhythm of an exchanged pup whose phase at weaning was located outside the reference zone was regarded as having been influenced by the nursing mother. The complete phase reversal occurred in two out of seven exchanged pups when litter size was relatively large (n = 5-7) and in 9 out of 11 when litter size was small (n = 2). It is concluded that the nursing mother rat is capable of phase setting the circadian locomotor rhythm of blinded pups. This maternal effect seems to be related to litter size.

Restricted daily feeding during nursing period resets circadian locomotor rhythm of infant rats.

Phase setting of the circadian rhythm by restricted daily feeding (RF) was examined in rat pups. Feeding of virgin, pregnant, and nursing rats together with pups was restricted to 4 h at a fixed time of the day. Phases of the circadian rhythm developed in neonatally blinded pups were measured on the day of weaning to evaluate the effect of RF on phase setting. The activity onset and offset of the locomotor rhythm were used for the phase reference. In blinded pups born and raised under ad libitum feeding, the phase at weaning was a function of the free-running period measured after weaning and located in a narrow range (control zone). When RF was imposed from 2 wk prior to the conception (C) until weaning (3w), the pups phases were located outside the control zone and related to the feeding time. To identify the period in which the phase setting is effective, RF was imposed during 5 different periods (C-0w, C-1w, C-2w, 2-6w, and 3-6w). The circadian rhythm of all pups in the C-2w group and some in the C-1w was phase set, but those in other groups were not affected. It is concluded that RF imposed during the early nursing period is capable of phase setting the circadian system of blinded infant rats.

The ventromedial hypothalamic nucleus is not essential for the prefeeding corticosterone peak in rats under restricted daily feeding.

The role of the ventromedial hypothalamic nucleus (VMH) in the generation of the prefeeding corticosterone peak was examined in rats under restricted daily feeding, under which daily meal supply was restricted to a fixed time in the early light period. Rats were lesioned in the VMH bilaterally and subjected to restricted daily feeding during two different post-operative phases and with two different durations of food presentation. A restricted feeding with free access to meal for 4 hr was imposed on the VMH-lesioned rats from 2 to 4 weeks after the lesion, when the daily food intake increased significantly (dynamic phase). The restricted feeding induced the prefeeding hormone peak in sham operated rats, but failed to develop it in the VMH-lesioned rats. On the other hand, the hormone peak appeared in the VMH-lesioned rats subjected to the feeding schedule from 8 to 10 weeks after the lesion, when the daily food intake was not different from the control (static phase). Moreover, the VMH-lesioned rats showed the hormone peak even in the dynamic phase when the access to meal was shortened to 1 hr. These results indicate that the VMH is not essential for the generation of the prefeeding corticosterone peak under restricted daily feeding, and suggest that a special metabolic state observed during the dynamic phase of VMH lesion prevents the development of the feeding-associated oscillation or its expression upon plasma corticosterone level.

Activity rhythms in the circadian domain appear in suprachiasmatic nuclei lesioned rats given methamphetamine.

Female rats were lesioned in the suprachiasmatic nuclei (SCN) electrolytically and treated with methamphetamine. The SCN lesions abolished the circadian locomotor rhythm completely. When methamphetamine was administered in the drinking water, robust rhythmicities in locomotor activity appeared in the SCN lesioned rats, which did not entrain to the 24 hr light cycle. The period of the activity rhythm was dose-dependent; the lower the concentration of methamphetamine was, the shorter the period of the rhythm became. When rats were treated with 0.005% methamphetamine, the mean period was 26.4 hours. In addition, activity time (alpha) became shorter, rest time (rho) longer and alpha/rho ratio lower, when methamphetamine concentration was decreased. After methamphetamine withdrawal, the rhythmicity disappeared and locomotor activity became aperiodic again. When methamphetamine was administered continuously by means of an osmotic minipump, similar rhythmicities appeared in locomotor activity of the SCN lesioned rats. It is concluded that methamphetamine manifests an activity rhythm whose period is in the circadian range. The rhythmicity is independent of the SCN and is not entrained by the light-dark cycle.

Maternal phase control of fetal circadian oscillation during pregnancy.

Mother-pups interaction is essential for the normal development of individuals during ontogeny. In this review, the emphasis was on a special function in a special stage of development, i.e., fetal entrainment to pregnant mother of the circadian rhythm which is one of the fundamental autonomic functions in the organism. To this end, the basic assumptions and problems were introduced and the validity of the assumption was defended. Based on this, the various factors of postnatal life which might affect the phase angle of overt hormone rhythm were experimentally evaluated. Finally, by introducing experimental designs which were to exclude disturbing effects of the nursing mother, we found that the fetal oscillation was possibly entrainable by pregnant mother as early as in the middle of gestation. Possible mechanism of the entrainment and adaptive implications of the findings were discussed.

Characterization of the prandial plasma corticosterone peak in the freely moving rat.

To characterize the prandial plasma corticosterone peak, three experiments were done on adult male Wistar rats: (1) the prandial hormone peak was followed at three different times of day, 0800 h, 1900 h, and 0100 h, in starved rats; (2) a 20% glucose solution was infused into the jugular vein or was given as a drinking solution after a 24-h starvation; (3) in order to determine the role of vagal proprioceptive inputs, the hormone peak was examined at two times of day, 0800 h and 0100 h, in starved vagotomized rats. The prandial hormone peak was more conspicuous at midnight than in the daytime. Exposure to food pellets without feeding failed to induce a hormone rise. The peak appeared after oral ingestion, but not after intravenous infusion, of glucose solution. Ingestion of water alone was without effect. The peak was not affected by bilateral vagotomy. Thus, as far as glucose as a metabolite is concerned, the possibility that glucose per se works directly on the brain or indirectly through glucoreceptors in the gut was excluded. Possible modes of action of glucose ingestion are discussed.

Disorganization of the rat activity rhythm by chronic treatment with methamphetamine.

Remarkable changes in the circadian activity rhythm of rats were observed when they were chronically treated with methamphetamine dissolved in drinking water. The circadian rhythm was phase-delayed with respect to the light-dark (LD) cycle, and showed signs of relative coordination. In some rats, the circadian organization was disturbed and two activity components appeared, with one component free-running and the other entrained by the LD cycle. After methamphetamine withdrawal, these changes disappeared rapidly but there were transient periods of 2-3 days before establishment of a stable entrainment to the LD cycle. The changes in the circadian rhythm persisted even in constant darkness (DD). The period around 24 hr in DD was significantly shorter during methamphetamine treatment than after the drug withdrawal. These results indicate that neither alteration of the sensitivity to light nor lengthening of the intrinsic period is involved in the methamphetamine induced disorganization of the circadian rhythm. Possible mechanisms are discussed in terms of a multi-oscillatory system.

Food ingestion is more important to plasma corticosterone dynamics than water intake in rats under restricted daily feeding.

The roles of food and/or water ingestion in the regulation of plasma corticosterone level were examined in rats under restricted daily feeding. When the time of food-pellets and water supply was restricted to 2 hours in the early light period (meal feeding) for 2 weeks, the corticosterone level increased prior to meal (prefeeding peak). A similar prefeeding hormone peak was observed when supply of food-pellets was restricted to 2 hours with free-access to water (food restriction). In contrast, when water supply was restricted to 2 hours with free-access to food-pellets (water restriction), the hormone level before water supply did not increase as much as that under meal feeding or food restriction. Shortening of an available time for water under water restriction or prolongation of the restriction schedule failed to elevate the hormone level furthermore. On the other hand, the high prefeeding corticosterone level before meal decreased subsequently to meal feeding (prandial fall), which was not observed when rats were kept fasting during the meal time. This prandial fall of the hormone level was not observed by water intake alone, and closely related to food-pellets ingestion. It is concluded that food ingestion is more important than water intake to the formation of the prefeeding corticosterone peak and to the prandial fall of the hormone level under restricted daily feeding.

Response curve, free-running period, and activity time in circadian locomotor rhythm of rats.

Phase response curves (PRC) for the spontaneous locomotor rhythm were constructed by applying short light pulses to rats in constant darkness (DD). The offset of locomotor activity as well as the onset was taken as a phase reference (offset PRC vs. onset PRC). The amount of phase shift yielded by light pulses was evaluated on the next day of pulse treatment (immediate PRC) and also after completion of a new steady state (steady state PRC). Significant differences in shape were observed between the onset and offset PRCs as well as between the immediate and steady state PRCs. In the immediate onset PRC, an area under the phase advance part (A) was absent, while it was present in the immediate offset PRC. In contrast, the steady state PRCs for activity onset and offset were essentially the same. The shape of steady state PRC depended on the free-running period in DD (tau). In the PRCs of long tau rhythms, the range covered by the phase delay area (D) was lengthened without changing its amplitude, resulting in a larger D/A ratio. A strong positive correlation was detected between tau and activity time (alpha). The steady state PRC shapes also depended on alpha; the D/A ratio was larger in a long alpha rhythm than in a short alpha. These results are in good agreement with the hypothesis that the circadian locomotor rhythm of nocturnal rodents is regulated by two coupled oscillators.

Ethosecretogram: relation of behavior to plasma corticosterone in freely moving rats.

An ethosecretogram, which illustrates the phase relation between animal behavior and plasma hormone levels, was constructed in conscious rats. A catheter was placed into the right atrium in male rats 2 days before blood sampling. Plasma corticosterone levels were monitored individually through the catheter every 5 min over a period of several hours under freely moving conditions on an Animex, which records spontaneous locomotor activity. The results showed that the bursts of locomotor activity were temporally correlated with those of plasma corticosterone level, the peak of the former preceding the hormone peak by 15 min. Regarding the type of locomotor activity, feeding and grooming were significantly correlated with the subsequent hormone elevation, whereas drinking was not. In a separate series of experiments, effects of feeding and drinking on plasma corticosterone levels were examined in conscious rats that had been fasted or water deprived for 24 or 48 h. In both cases, feeding induced a more pronounced elevation than drinking, although locomotor activity counts per se increased more sharply in the latter. It is concluded that in the ethosecretogram specific types of locomotor activity such as feeding and grooming induce a phase-locked elevation of plasma corticosterone under freely moving conditions.

Dissociation of circadian rhythms in rats with a hypothalamic island.

Complete isolation of the medial basal hypothalamus, including the suprachiasmatic nuclei in the isolated island, from the rest of the central nervous system was performed in rats. The circadian rhythm of plasma corticosterone level remained essentially intact in 5 of 16 rats with complete islands, whereas that of spontaneous locomotor activity was decomposed into ultradian bursts in 15 rats, resulting in a clear dissociation of the two rhythms in four rats. One rat, whose circadian rhythm of both variables persisted after the hypothalamic isolation, showed a diurnal activity. The 24-h patterns of plasma corticosterone of the other rats could be characterized as either episodic or continuously low throughout the day. A prefeeding corticosterone peak was detected under restricted feeding in rats with episodic fluctuations but not in those with continuously low hormone levels. It is concluded that the hypothalamic island includes the fundamental structures necessary for the manifestation of the entrained circadian rhythm of plasma corticosterone in rats under ad libitum feeding, whereas it contains only a part of that for spontaneous locomotor activity. The coupling pathways from the circadian oscillator(s) to these overt rhythms seem to be separate in the central nervous system, and the circadian rhythm of plasma corticosterone is not a direct consequence of that of locomotor activity.

Hormonal rhythm and feeding behavior.

In this review, attempts were made to clarify the role of feeding behavior in modifying the basal 24-h pattern of plasma corticosterone in freely moving rats. An ethosecretogram, consisting of locomotor activity and plasma corticosterone levels in conscious rats over a period of several hours, was constructed. By analyzing the ethosecretogram, it was found that the feeding effectively induced an elevation of plasma corticosterone. Conscious rats were then exposed to various kinds of feeding regimens, emphasis being laid on the effect of restricted daily feeding on the circadian skeleton of plasma hormone rhythm. We found that feeding has a bidirectional effect on hormone level along with an initiation of an interval timer mechanism which in turn uniquely modify the basal pattern of circadian hormone rhythm, especially when a pre-feeding hormone peak is formed under restricted daily feeding schedules. Furthermore, it was shown that the pre-feeding hormone peak persisted after the termination of restricted feeding and free-ran in parallel with the circadian hormone peak. It is concluded that the pre-feeding hormone peak is driven by a damped oscillator which is entrainable to restricted feeding. It is most likely that this damped oscillator is loosely driven by the circadian oscillator which is not entrainable to restricted feeding.