Kun-Ruey Shieh

Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and rCLOCK, in the rat brain.

High densities of mRNAs for three rhythm-related genes, rPeriod1 (rPer1), rPer2, and rClock, which share high homology in Drosophila and mice, were found in the hypothalamic suprachiasmatic nucleus (SCN). The SCN, however, is not the only brain region that expresses these genes. To understand the distributions and possible physiological roles of these rhythm-related genes, we examined the gene expressions of rPer1, rPer2, and rClock in different brain regions by serial coronal, sagittal, and horizontal brain sections in Sprague-Dawley male rats. Animals were housed in a light-controlled room (lights on from 0600 to 1800 h) and killed at 1000 or 1200 h, which corresponds to Zeitgeber time 4 or 6. Semi-quantitative in situ hybridization with (35)S-riboprobes was used to evaluate mRNA levels. The mRNAs of rPer1, rPer2, and rClock were widely distributed in the rat CNS, including the olfactory bulb, cortex, piriform cortex, SCN, ventromedial hypothalamus, arcuate nucleus, hippocampus, mammillary nucleus, pontine nucleus, superior and inferior colliculus, cerebellum, median eminence/pars tuberalis, pineal gland, and pituitary. The expression patterns of mRNAs for rPer1 and rPer2 were almost identical. In contrast, different expression patterns were observed between rClock and rPer1 or rPer2 in several brain regions, including the hypothalamic supraoptic and suprachiasmatic nuclei, the paraventricular zone of the caudate putamen, the superior olivary nucleus, and anterior and intermediate lobes of the pituitary. These findings suggest that the different expression patterns observed for rPer1, rPer2, and rClock might be due to their different physiological role(s) in those brain regions.

Cocaine- and amphetamine-regulated transcript in the nucleus accumbens participates in the regulation of feeding behavior in rats

The present studies aimed to determine whether cocaine- and amphetamine-regulated transcript (CART) peptide in the nucleus of accumbens shell (AcbSh) is implicated in the regulation of food intake. Bilateral intranuclear injections of CART peptide (55-102, 1 microg/microl/side) into the AcbSh decreased food intake with no change in locomotion activity and attenuated the orexigenic effect of the GABA(A) agonist muscimol (100 ng/microl/side) in male Sprague-Dawley rats. Decreased food intake after bilateral intranuclear injections of CART was more sustained in freely fed rats than in food-deprived rats, suggesting fuel availability is an important factor in modulating the function of CART in the regulation of feeding. Our anatomical findings indicate that in addition to the perifornical region and the arcuate nucleus, some neurons within the AcbSh also project within the AcbSh. Moreover, many of these efferent cells contain CART immunoreactivity, including those which reside within the AcbSh, suggesting that accumbal CART circuitry is involved in the central function of the nucleus accumbens. Furthermore, fasting suppressed CART mRNA levels in the AcbSh, paraventricular nucleus of the hypothalamus, arcuate nucleus, and the perifornical region, indicating that the Acb is sensitive to fuel availability to an extent similar to those regions in the hypothalamus. Our findings are the first to demonstrate that CART mRNA in the AcbSh is sensitive to metabolic challenges and that injection of CART peptide into the AcbSh has an inhibitory effect on food intake.

Effects of the cocaine- and amphetamine-regulated transcript peptide on the turnover of central dopaminergic neurons

The effects of the cocaine- and amphetamine-regulated transcript (CART) peptide on central dopaminergic (DA) neurons were examined in ovariectomized, estrogen-primed Sprague-Dawley rats in both the morning and afternoon. Intracerebroventricular administration of 1 microg, but not lower doses of the CART peptide (55-102), either in the morning or afternoon produced a prolonged increase in the 3,4-dihydroxyphenylacetic acid (DOPAC) level in the median eminence (ME) and a corresponding decrease of serum prolactin (PRL) levels, which resulted from stimulation of tuberoinfundibular dopaminergic neurons. The CART peptide stimulated DOPAC levels in the striatum (ST), nucleus accumbens (NA), hypothalamic paraventricular nucleus (PVN), and periventricular (A14), but had no effect in the medial prefrontal cortex (MPFC) or suprachiasmatic nucleus (SCN). These effects of the CART peptide on stimulation of central DA systems and inhibition of PRL levels are specific because the inactive form of the CART peptide (0.1 and 1 microg) could not induce a similar response. Stimulatory effects of the CART peptide on different central DA systems displayed differential time-response profiles in the NA and ST, ME, and PVN and A14. These findings indicate that the CART peptide may selectively regulate certain central DA neuronal activities.

Depression- and anxiety-like behaviors of a rat model with absence epileptic discharges.

Depression and/or anxiety are major comorbidities of epilepsy. However, the contribution of absence epileptic discharges in psychiatric syndromes is inconclusive. This study aimed to clarify the influence of absence seizure in anxiety- and depression-like behaviors using normal Wistar rats and Long-Evans rats with spontaneous spike-wave discharges (SWDs). Anxiety-like behaviors were evaluated by the open field (OF) and elevated plus maze (EPM) tests, and depression-like behaviors by the forced swimming (FS) and sucrose consumption (SC) tests. Long-Evans rats displayed significantly higher frequency and longer duration in the open arms of the EPM and in the center zone of the OF than did Wistar rats. Normalized behavioral indexes by movement also were significantly higher in Long-Evans rats. An excess of SWD numbers was associated with lower indexes and worse movement in the two behavioral tests. Ethosuximide eliminated the seizure frequency-dependent relationship and also significantly increased all indexes of the EPM test. Additionally, Long-Evans rats revealed significantly longer immobility in the FS test and lower consumption of sucrose solution in the SC test than did Wistar rats. Meanwhile, no relationship was found between immobility of the FS test and SWD number. Ethosuximide ameliorated depression-like behavior of Long-Evans rats that was equal to that of Wistar rats. Thus, Long-Evans rats showed seizure frequency-related exacerbation in anxiety-like behavior; and they displayed a depressive propensity. Our data suggest that generalized SWDs may have distinct influences in anxious and depressive behaviors.

Effects of estradiol on the stimulation of dopamine turnover in mesolimbic and nigrostriatal systems by cocaine- and amphetamine-regulated transcript peptide in female rats.

The present studies aimed to determine whether estradiol (E(2)) modulates the stimulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the mesolimbic and nigrostriatal dopaminergic systems. I.c.v. administration of the CART peptide (55-102, 1 microg/3 microl) increased dopamine turnover (3,4-dihydroxyphenylacetic acid, DOPAC) in the nucleus accumbens (NA) and striatum (ST) in ovariectomized (OVX) female Sprague-Dawley rats with E(2)-priming. This stimulation of NA and ST DOPAC contents by CART peptide was found in OVX+E(2) female rats, but not in OVX only female rats, suggesting E(2) is an important factor in modulating the stimulatory effect of CART in the regulation of NA and ST DOPAC contents. This stimulation by CART peptide was also restored by treatment with the water-soluble form of E(2), but not by treatment with the membrane-impermeable form of E(2) in OVX female rats, suggesting that E(2) acts through intracellular rather than extracellular mechanisms to modulate the effects of CART peptide. Furthermore, the effects of water-soluble form of E(2) were blocked by E(2) antagonist, tamoxifen, but not by testosterone antagonist, flutamide. Our findings are the first to demonstrate that that E(2) plays a regulatory role in stimulation of CART peptide in mesolimbic and nigrostriatal dopaminergic systems in female rats, and E(2) acts through its own receptor(s) and intracellular mechanisms.

Differential effects of melanin concentrating hormone on the central dopaminergic neurons induced by the cocaine‐ and amphetamine‐regulated transcript peptide

Stimulatory effects of cocaine‐ and amphetamine‐regulated transcript (CART) peptide on central mesolimbic, nigrostriatal and mesocortical dopaminergic (DA) neurons were examined in female Sprague–Dawley rats. We also determined the different blocking effects of melanin concentrating hormone (MCH) on the stimulation by CART peptide in central DA systems. Intracerebroventricular administration of 1 µg CART peptide (55–102) produced increases in 3,4‐dihydroxyphenylacetic acid (DOPAC) levels in the nucleus accumbens (NA) at 15 and 45 min, and in the striatum (ST) at 15 min, but not in the medial prefrontal cortex (MPFC). We found that the agonist of α‐melanocyte stimulating hormone (α‐MSH), MT II, at 10 µg had a stimulatory effect on the NA and ST DOPAC levels similar to the CART peptide. In contrast, 1 µg MCH and the antagonist of α‐MSH, HS014, significantly decreased NA and ST DOPAC levels. However, only MCH prevented the stimulatory effect of CART peptide on DOPAC levels in the NA, but not in the ST. These results indicate that the stimulation of CART peptide on central DA neurons is region‐specific, and that this effect can be blocked by MCH but not by the antagonist of α‐MSH.

Gonadal hormones-mediated effects on the stimulation of dopamine turnover in mesolimbic and nigrostriatal systems by cocaine- and amphetamine-regulated transcript (CART) peptide in male rats

Estradiol and testosterone modulated behavioral and neurochemical activities in the mesolimbic and nigrostriatal dopaminergic systems have been reported. We examined whether estradiol and testosterone affect stimulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the mesolimbic and nigrostriatal dopaminergic systems in this study. Intracerebroventricular administration of CART peptide increased dopamine turnover in the nucleus accumbens and striatum in male rats. Stimulation of dopamine turnover in nucleus accumbens and striatum by CART peptide were found in intact male rats, but not in castrated male rats. This stimulation was restored in castrated male rats by testosterone or estradiol priming, or by treatment with the water-soluble form of estradiol, but not by treatment with the membrane-impermeable form of estradiol. Estradiol and testosterone antagonists blocked testosterones effects, but only estradiol antagonist blocked estradiols effects. Moreover, treatment of dihydrotestosterone also restored the stimulation in castrated male rats. This dihydrotestosterones effect was blocked by a testosterone antagonist, but not by an estradiol antagonist. All of these findings indicate that gonadal hormones play a regulatory role in stimulation of CART peptide in mesolimbic and nigrostriatal dopaminergic systems, and suggest that acts through intracellular rather than extracellular mechanisms.

Effects of the cocaine- and amphetamine-regulated transcript peptide on the turnover of dopamine in tuberoinfundibular neurons and serum prolactin levels: studies using estrogen, melanin concentrating hormone, and melanocortin

Effects of the cocaine- and amphetamine-regulated transcript (CART) peptide on tuberoinfundibular dopaminergic (TIDA) neurons were examined in female and male Sprague-Dawley rats in the morning and afternoon. We also examined the blocking effects of melanin concentrating hormone (MCH) and the antagonists of alpha-melanocyte stimulating hormone (alpha-MSH), SHU9119 and HS014, on stimulation induced by the CART peptide in TIDA systems. Intracerebroventricular administration of 1 mug CART peptide (55-102) at 45 min, either in the morning or afternoon, produced an increase in the median eminence (ME) DOPAC (3,4-dihydroxyphenylacetic acid) level and a corresponding decrease in serum prolactin (PRL) levels. This resulted from stimulation of TIDA neurons regardless of castration, and whether or not male and female rats were estrogen-primed. The stimulatory effects of the CART peptide on ME DOPAC levels were similar in the morning and afternoon in both male and female rats. Central treatment with 1 microg SHU9119, HS014, or MCH significantly decreased the ME DOPAC levels and elevated serum PRL levels in female rats. However, only MCH prevented the stimulatory effect of the CART peptide on TIDA neurons. These results indicate that stimulation by the CART peptide on TIDA neurons is gender-independent; and this stimulatory effect can be blocked by MCH, but not the antagonists of alpha-MSH.

Hepatic circadian-clock system altered by insulin resistance, diabetes and insulin sensitizer in mice.

Circadian rhythms are intrinsic rhythms that are coordinated with the rotation of the Earth and are also generated by a set of circadian-clock genes at the intracellular level. Growing evidence suggests a strong link between circadian rhythms and energy metabolism; however, the fundamental mechanisms remain unclear. In the present study, neonatal streptozotocin (STZ)-treated mice were used to model the molecular and physiological progress from insulin resistance to diabetes. Two-day-old male C57BL/6 mice received a single injection of STZ and were tested for non-obese, hyperglycemic and hyperinsulinemic conditions in the early stage, insulin resistance in the middle stage, and diabetes in the late stage. Gene expression levels of the hepatic circadian-clock system were examined by real-time quantitative PCR. Most of the components of the hepatic circadian-clock gene expression system, such as the mRNAs of Bmal1 (brain and muscle Arnt-like protein-1), Per2 (period 2) and Cry1 (cryptochrome 1), were elevated, and circadian patterns were retained in the early and middle stages of insulin-resistant conditions. The insulin sensitizer, rosiglitazone, returns the physiological and molecular changes associated with the diabetic phenotype to normal levels through peroxisome proliferator-activated receptor γ (PPARγ) rather than PPARα. Early and chronic treatment with rosiglitazone has been shown to be effective to counter the diabetic condition. Over time, this effect acts to attenuate the increased gene expression levels of the hepatic circadian-clock system and delay the severity of diabetic conditions. Together, these results support an essential role for the hepatic circadian-clock system in the coordinated regulation and/or response of metabolic pathways.

Circadian-clock system in mouse liver affected by insulin resistance

Circadian rhythms are exhibited in the physiological and behavioral processes of all mammals; they are generated by intracellular levels of circadian oscillators, which are named as a set of circadian-clock genes. These genes compose the transcriptional/translational feedback loops to regulate not only circadian rhythmicity, but also energy metabolism. Previous studies have shown that obesity and diabetes cause the dysregulation of the circadian-clock system, and vice versa. However, some diabetes subjects are lean with insulin resistance and the mechanisms of insulin resistance without obesity are much less well known. Therefore, whether insulin resistance alone is enough to influence the expression of circadian-clock genes is uncertain. This study employs a neonatal streptozotocin (STZ)-treated paradigm in mice to model the molecular and physiological progress of nonobese insulin resistance. A single injection of STZ into 2-d-old male C57BL/6 mice induces nonobese, hyperglycemic and hyperinsulinemic conditions, and the levels of gene expression in the liver by a real-time quantitative polymerase chain reaction are then measured. Although the levels of Bmal1 (brain and muscle Arnt-like protein-1), Per2 (period 2), and Cry1 (cryptochrome 1) mRNA expression in the liver change during the progress of insulin resistance conditions, the gene expression patterns still show circadian rhythmicity. This study suggests that changes in the hepatic circadian-clock gene expression mark an early event in the metabolic disruption associated with insulin resistance. Furthermore, 2 wks of treatment with the thiazolidinedione, pioglitazone, fully resolve the dysfunction in metabolic parameters and the changes in circadian-clock gene expression from early insulin resistance conditions. These results indicate that the circadian-clock system is sensitive to insulin resistance, and that treatment with thiazolidinediones can resolve changes in the circadian-clock system in a timely manner. Thus, strengthening the peripheral circadian-clock system may counteract the adverse physiological consequences in the metabolic syndrome.