Glycogen Synthase Kinase 3ß Is a Key Regulator in the Inhibitory Effects of Accumbal Cocaine- and Amphetamine-Regulated Transcript Peptide 55-102 on Amphetamine-Induced Locomotor Activity.

Microinjection of cocaine- and amphetamine-regulated transcript (CART) peptide 55-102 into the nucleus accumbens (NAcc) core significantly attenuates psychostimulant-induced locomotor activity. However, the molecular mechanism remains poorly understood. We examined the phosphorylation levels of Akt, glycogen synthase kinase 3ß (GSK3ß), and glutamate receptor 1 (GluA1) in NAcc core tissues obtained 60 min after microinjection of CART peptide 55-102 into this site, followed by systemic injection of amphetamine (AMPH). Phosphorylation levels of Akt at Thr308 and GSK3ß at Ser9 were decreased, while those of GluA1 at Ser845 were increased, by AMPH treatment. These effects returned to basal levels following treatment with CART peptide 55-102. Furthermore, the negative regulatory effects of the CART peptide on AMPH-induced changes in phosphorylation levels and locomotor activity were all abolished by pretreatment with the S9 peptide, an artificially synthesized indirect GSK3ß activator. These results suggest that the CART peptide 55-102 in the NAcc core plays a negative regulatory role in AMPH-induced locomotor activity by normalizing the changes in phosphorylation levels of Akt-GSK3ß, and subsequently GluA1 modified by AMPH at this site. The present findings are the first to reveal GSK3ß as a key regulator of the inhibitory role of the CART peptide in psychomotor stimulant-induced locomotor activity.

Ezrin-radixin-moesin proteins are regulated by Akt-GSK3ß signaling in the rat nucleus accumbens core.

The ezrin-radixin-moesin (ERM) proteins are a family of membrane-associated proteins known to play roles in cell-shape determination as well as in signaling pathways. We have previously shown that amphetamine decreases phosphorylation levels of these proteins in the nucleus accumbens (NAcc), an important neuronal substrate mediating rewarding effects of drugs of abuse. In the present study, we further examined what molecular pathways may be involved in this process. By direct microinjection of LY294002, a PI3 kinase inhibitor, or of S9 peptide, a proposed GSK3ß activator, into the NAcc core, we found that phosphorylation levels of ERM as well as of GSK3ß in this site are simultaneously decreased. These results indicate that ERM proteins are under the regulation of Akt-GSK3ß signaling pathway in the NAcc core. The present findings have a significant implication to a novel signal pathway possibly leading to structural plasticity in relation with drug addiction.

Locomotor sensitization is expressed by ghrelin and D1 dopamine receptor agonist in the nucleus accumbens core in amphetamine pre-exposed rat.

Ghrelin modulates mesolimbic dopaminergic pathways in the brain in addition to its role in feeding. We investigated what roles ghrelin in the nucleus accumbens (NAcc) core may play in mediating locomotor activating effects of amphetamine (AMPH). First, when rats were administered with AMPH (1 mg/kg, i.p.) following a bilateral microinjection of ghrelin (0.1 or 0.5 µg/side) into the NAcc core, their locomotor activity was significantly enhanced, while these effects were blocked by co-microinjection of ghrelin receptor antagonist (0.5 µg/side) into this site. Second, we pre-exposed rats to saline or amphetamine (1 mg/kg, i.p.) every 2 to 3 days for a total of four times. After 2 weeks of drug-free withdrawal period, we examined the effect of saline, ghrelin (0.5 µg/side), D1 dopamine receptor agonist, SKF81297 (0.5 µg/side) or ghrelin (0.5 µg/side) + SKF81297 (0.5 µg/side) directly microinjected into the NAcc core on locomotor activity. When we measured rats' locomotor activity for 1 hour immediately following microinjections, only ghrelin + SKF81297 produces sensitized locomotor activity, while all others have no effects. These results suggest that ghrelin may have a distinct role in the NAcc core to provoke the sensitized locomotor activity induced by psychomotor stimulants, and further, it may produce these effects by interaction with D1 dopamine receptors.

Incidence of three roots and/or four root canals in the permanent mandibular first molars in a Korean sub-population.

OBJECTIVES: The purpose of this study was to evaluate the prevalence of three-rooted permanent mandibular first molars (PMFMs) with four canals and their morphological characteristics among a Korean population from using cone-beam computed tomography scans (CBCTs). MATERIALS AND METHODS: Among the 705 CBCTs screened, 472 patient cases possessing at least one PMFM were identified. A total of 780 PMFMs were evaluated in axial section series to determine the number of roots and canals. The incidences of three-rooted PMFMs were compared with regard to gender and location. For distal root(s) with two canals, inter-orifice distances (IOD) between distobuccal and distolingual canals were measured at pulpal floor and furcation levels. The difference of IOD between males and females was also analyzed using chi-square tests. RESULTS: Among the 472 CBCTs of 225 females and 247 males, 84 females and 107 males were found to have at least one three-rooted PMFM. Among the 780 PMFMs, 191 PMFMs (24.5%, 89 of 397 left and 102 of 383 right) were found to have three roots. The prevalence of distal root(s) with two canals was 34.2% (267 of 780). From the molars with two distal canals, the mean IOD between distobuccal and distolingual canals at the pulpal floor level was 3.1 mm in males and 2.9 mm in females (p = 0.0428). CONCLUSIONS: The occurrence of three-rooted PMFMs among a Korean population was 24.5% and was higher than other countries and ethnicities. Understanding the prevalence of PMFMs with a distolingual root and/or canal in a Korean population and the IOD between distobuccal and distolingual canals may be useful for successful endodontic treatments. CLINICAL RELEVANCE: Acknowledgment of potential incidence of three-rooted permanent mandibular first molars with four canals and the distance between two distal canals may increase the success rate of root canal treatment by reducing the missing canal untreated.

JBPOS0101 regulates amyloid beta, tau, and glial cells in an Alzheimer's disease model.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease characterized by cognitive dysfunction and memory loss as the main symptoms. The deposition of amyloid beta (Aß) and tau hyperphosphorylation are hallmarks of AD and are major therapeutic targets. However, the exact etiology has not yet been fully elucidated; thus, no drug that cures the disease has been approved. JBPOS0101 is a phenyl carbamate compound that has been tested as a drug for epileptic diseases. In our previous study, we showed that JBPOS0101 attenuated the accumulation of Aß as well as the deficits in learning and memory in the 5xFAD mouse model. Here, we tested the dose effect (70 or 35 mg/kg) of JBPOS0101 on the memory defect and pathological markers and further investigated the underlying mechanisms in 5xFAD mice. In the behavior tests, JBPOS0101 treatment ameliorated deficits in learning and memory. Moreover, JBPOS0101 attenuated Aß accumulation and tau phosphorylation. The elevated phosphorylation levels of the active GSK3ß form (GSK3ß-y216) in 5xFAD, which are responsible for tau phosphorylation, decreased in the JBPOS0101-treated groups. Furthermore, the elevation of reactive astrocytes and microglia in 5xFAD mice was attenuated in JBPOS0101-treated groups. These data suggest that JBPOS0101 may be a new drug candidate to lessen amyloid- and tau-related pathology by regulating glial cells.

JBPOS0101 attenuates amyloid-ß accumulation and memory loss in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a major neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) in the brain. Defects in Aß clearance or the interference of Aß homeostasis could result in Aß aggregation. JBPOS0101 has been studied for its antiepileptic activity. It showed a neuroprotective effect and prevented memory deficits in lithium-pilocarpine-induced status epilepticus rats. In this study, we tested the effect of JBPOS0101 in an AD model. We showed that JBPOS0101 attenuated the accumulation of Aß in 5XFAD mouse brains. Moreover, the treatment of JBPOS0101 rescued the deficits in learning and memory in 5XFAD mice. These data suggest that JBPOS0101 could be a potential therapeutic drug candidate for AD.

Blockade of group II metabotropic glutamate receptors produces hyper-locomotion in cocaine pre-exposed rats by interactions with dopamine receptors.

It was previously reported that blockade of group II metabotropic glutamate receptors (mGluRs) produces hyper-locomotion in rats previously exposed to amphetamine, indicating that group II mGluRs are well positioned to modulate the expression of behavioral sensitization by amphetamine. The present study further examined the locomotor activating effects of specific blockade of these receptors after cocaine pre-exposures. First, rats were pre-exposed to seven daily injections of cocaine (15mg/kg, IP). When challenged the next day with an injection of either saline or the group II mGluR antagonist LY341495 (0.5, 1.0 or 2.5mg/kg, IP), they produced hyper-locomotor activity, measured by infrared beam interruptions, to LY341495 compared to saline in a dose-dependent manner. Second, rats were pre-exposed to either saline or seven daily injections of cocaine (15mg/kg, IP). Three weeks later, when they were challenged with an injection of either saline or LY341495 (1.0mg/kg, IP), only rats pre-exposed to cocaine produced hyper-locomotor activity to LY341495 compared to saline. These effects, however, were not present when dopamine D1 (SCH23390; 5 or 10microg/kg), but not D2 (eticlopride; 10 or 50microg/kg), receptor antagonist was pre-injected, indicating that this cocaine-induced hyper-locomotor activity to LY341495 may be mediated in dopamine D1 receptor-dependent manner. These results suggest that group II mGluRs may be adapted to interact with dopaminergic neuronal signaling in mediating the sensitized locomotor activity produced by repeated cocaine pre-exposures.

Blockade of group II metabotropic glutamate receptors in the nucleus accumbens produces hyperlocomotion in rats previously exposed to amphetamine.

The neurotransmitter glutamate is known to participate in both the induction and expression of locomotor sensitization by psychostimulant drugs like amphetamine. Previously, it was reported that subtype nonselective blockade of metabotropic glutamate receptors (mGluRs) in the nucleus accumbens (NAcc) produces hyperlocomotion in rats previously exposed to amphetamine. The present experiments examined whether group II mGluRs may contribute to this effect. Rats in different groups were administered five injections of either saline or amphetamine (1.0 mg/kg, i.p.), one injection given every third day. Two weeks later, they were tested for 2 h following an injection of either saline or the group II mGluR antagonist LY341495. In one experiment, test injections were administered systemically (saline or LY341495, 1.0 mg/kg, i.p.). Rats previously exposed to amphetamine showed a greater locomotor response to LY341495 on the test compared to controls previously exposed to saline. This hyperlocomotor response was absent in rats tested with a combination of LY341495 and the group II mGluR agonist LY379268 (1.0 mg/kg, i.p.). In a second experiment, different rats were tested following microinjections into the NAcc (saline or LY341495, 0.1, 10 or 100 microg/0.5 microl/side). Again, rats previously exposed to amphetamine showed a greater dose-dependent locomotor response to LY341495 on the test relative to saline-exposed controls. Locomotor activity in saline-exposed rats challenged with LY341495 did not differ from that observed in rats previously exposed and tested with saline in either experiment. These results indicate that group II mGluRs, particularly those found in the NAcc, are well positioned to modulate the expression of locomotor sensitization by amphetamine.

Inhibition of PKMζ in the nucleus accumbens core blocks the expression of locomotor sensitization induced by amphetamine.

Behavioral sensitization is a form of drug-induced, long-lasting, non-associative memory. Protein kinase M zeta (PKMζ) has been shown to play a role in maintaining associative long-term memory in various brain regions. In the present study, rats were pre-exposed to either saline or amphetamine (AMPH) (1 mg/kg, i.p.). After 1 week of drug-free withdrawal period, they were challenged with AMPH (1 mg/kg, i.p.) following a bilateral microinjection of either saline or zeta inhibitory peptide (ZIP) (5 nmole/side) into the NAcc core. In the AMPH pre-exposed group, the ZIP microinjection blocked the enhanced increase of locomotion by AMPH challenge. These results suggest that PKMζ in the NAcc core plays a role in the expression of AMPH-induced locomotor sensitization, and further extend the role of PKMζ in long-term memory to non-associative form of drug-related memory.

Amphetamine dephosphorylates ERM proteins in the nucleus accumbens core and lithium attenuates its effects.

The ezrin-radixin-moesin (ERM) proteins have been implicated not only in cell-shape determination but also in cellular signaling pathway. We have previously shown that cocaine decreases phosphorylation levels of these proteins in the nucleus accumbens (NAcc), an important brain area mediating addictive behaviors. Here we further revealed that the phosphorylation levels of ERM were decreased in the NAcc core, but not in the shell, by a single injection of amphetamine (AMPH) (2 mg/kg, i.p.). When lithium (100 mg/kg, i.p.) was co-administered with AMPH, the decreases of phosphorylation levels for ERM by AMPH were recovered back to basal levels in the NAcc core. Together, these results suggest that psychomotor stimulants like AMPH regulate phosphorylation levels of ERM in the NAcc core and lithium-involved signaling pathway has a regulatory role in the opposite direction in this site.

Microinjection of ghrelin in the nucleus accumbens core enhances locomotor activity induced by cocaine.

Ghrelin has a regulatory role not only in the rewarding aspect for feeding but also in drug addiction by interaction with mesolimbic dopaminergic pathways in the brain. Previously it has been shown that systemic ghrelin enhances cocaine-induced hyper-locomotor activity. However, it has not been determined yet what effects ghrelin may produce on cocaine-induced locomotor behavior when microinjected into the nucleus accumbens core, where cocaine actually produces its locomotor activating effects. In the present study, when rats were administered cocaine (15 mg/kg, i.p.) following a bilateral microinjection of ghrelin (0.1 or 0.5 µg/side) into the NAcc core, their locomotor activity was significantly enhanced, while these effects were inhibited by co-microinjection of ghrelin receptor antagonist (0.5 µg/side) into this site. When ghrelin alone microinjected, basal locomotor activity was unchanged. These results expand previous similar findings obtained by systemic ghrelin to more specific neuronal site, the nucleus accumbens core, and further suggest that it may work in this site to positively contribute to the expression of locomotor sensitization.