Visualization of the existence of growth hormone secretagogue receptor in the rat nucleus accumbens.

The potential role of the ghrelin receptor, also known as the growth hormone secretagogue receptor (GHSR), within the nucleus accumbens (NAcc) in regulating drug addiction and feeding has been documented; however, the pattern of its expression in this site remains elusive. In this study, we characterized the expression patterns of GHSR1a and 1b, two subtypes of GHSRs, within the NAcc of the rat brain by immunohistochemistry. We visually detected GHSR signals, for the first time, at the protein level in the NAcc in which they were mostly expressed in neurons including both medium spiny neurons (MSNs) and non-MSNs. Furthermore, GHSR1a was found expressed as localized near the cellular membrane or some in the cytoplasm, whereas GHSR1b expressed solely throughout the large cytoplasmic area. The existence and subcellular expression pattern of GHSRs in the NAcc identified in this study will contribute to improving our understanding about the role of GHSR-mediated neurosignaling in feeding and drug addiction.

Repeated exposure to methiopropamine increases dendritic spine density in the rat nucleus accumbens core.

Repeated exposure to classical psychomotor stimulants, like amphetamine (AMPH), produces locomotor sensitization and accompanied structural plasticity of dendritic spines in the nucleus accumbens (NAcc). Following our previous report that repeated administration of methiopropamine (MPA), a structural analog to meth-AMPH, produces locomotor sensitization, it was examined in the present study whether this behavioral change also accompanies with structural plasticity in the NAcc in a similar way to AMPH. A week after adeno-associated viral vectors containing enhanced green fluorescent protein (eGFP) were microinjected into the NAcc core, rats were repeatedly injected with saline, AMPH (1 mg/kg, IP), or MPA (5 mg/kg, IP) once every 2-3 days for a total of 4 times. Two weeks after last injection, all rats were perfused and their brains were processed for immunohistochemical staining. The image stacks for dendrite segments of medium spiny neuronal cells in the NAcc core were obtained and dendritic spines were quantitatively analyzed. Interestingly, it was found that the number of total spine density, with thin spine as a major contributor, was significantly increased in MPA compared to saline pre-exposed group, in a similar way to AMPH. These results indicate that MPA, a novel psychoactive substance, has similar characteristics with AMPH in that they both produce structural as well as behavioral changes, further supporting MPA's dependence and abuse potential.

A novel synthetic cathinone, α-pyrrolidinopentiothiophenone (PVT), produces locomotor sensitization in rat: Implications for GSK3ß connections in the nucleus accumbens core.

A novel psychoactive substance, α-pyrrolidinopentiothiophenone (α-PVT), is a structural analog to amphetamine. Recently, it has been shown that α-PVT has an abuse potential similar to psychomotor stimulants like cocaine or amphetamine. However, it has not been performed yet to determine whether α-PVT develops behavioral sensitization, a well-known phenomenon for psychomotor stimulants. In the present study, rats were first pre-exposed to either saline or α-PVT (20 mg/kg, IP) with a total of four injections in every 2-3 days of interval. Then, 2-weeks after withdrawal, locomotor activity was measured with a challenge dose (10 mg/kg, IP) of α-PVT and the nucleus accumbens core region was taken out. Similar to psychomotor stimulants, repeated administration of α-PVT produced locomotor sensitization. Further, the phosphorylation levels of GSK3ß in the nucleus accumbens core were found to be decreased only in rats with sensitization developed, but not in those with acute or non-sensitized. Correlation analysis revealed that the phosphorylation levels of GSK3ß have a strong negative correlation with locomotor activity only in rats with α-PVT pre-exposed, but not in those with its acute injection. These results suggest that a certain level of change in the phosphorylation levels of GSK3ß in the nucleus accumbens core may involve in mediating the expression of locomotor sensitization by repeated injection of α-PVT in rats.

Low cocaine- and amphetamine-regulated transcript (CART) peptide levels in human cerebrospinal fluid of major depressive disorder (MDD) patients.

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART) peptide is a candidate neuropeptide as a biomarker for major depressive disorder (MDD) because of its effects on emotion and distribution covering brain areas involved in the pathophysiology of MDD symptoms. However, it is unknown whether CART peptide levels are altered in the cerebrospinal fluid (CSF) of patients with MDD patients and are correlated with MDD symptoms. METHODS: Subjects were 24 patients with MDD and 25 healthy controls matched for age, gender and ethnicity (Japanese). We measured CSF CART levels by a commercially available immunoassay kit and analyzed the relationships of the levels with antidepressant dose and symptoms assessed with the 21 item Hamilton Depression Rating Scale (HAMD-21). RESULTS: CSF CART levels were significantly decreased in the patients than in the controls (p < 0.05). In MDD patient group, the CART levels had a negative correlation with antidepressant dose (imipramine-equivalent dose) (ρ = -0.55, p < 0.01) and significantly decreased in antidepressant-treated group (AD-treated group) compared to controls (p < 0.05). CSF CART levels showed significant negative correlations with psychomotor retardation, somatic anxiety, and general somatic symptoms (all p < 0.05) and a positive correlation with obsessive and compulsive symptoms (p < 0.05). LIMITATIONS: In our analysis, all classes of antidepressants were combined together and the effects of medication use in a longitudinal manner did not confirm. CONCLUSIONS: We report for the first time that CSF CART peptide levels are reduced in patients with MDD compared with healthy controls. The CART levels showed negative correlations with antidepressant dose and some symptoms, supporting the possibility that CART peptide is involved in the development of depressive symptoms.

Relationships of Cerebrospinal Fluid Monoamine Metabolite Levels With Clinical Variables in Major Depressive Disorder.

OBJECTIVE: Many studies have investigated cerebrospinal fluid (CSF) monoamine metabolite levels in depressive disorders. However, their clinical significance is still unclear. We tried to determine whether CSF monoamine metabolite levels could be a state-dependent marker for major depressive disorder (MDD) based on analyses stratified by clinical variables in a relatively large sample. METHODS: Subjects were 75 patients with MDD according to DSM-IV criteria and 87 healthy controls, matched for age, sex, and ethnicity (Japanese). They were recruited between May 2010 and November 2013. We measured homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in CSF samples by high-performance liquid chromatography. We analyzed the relationships of the metabolite levels with age, sex, diagnosis, psychotropic medication use, and depression severity. RESULTS: There was a weak positive correlation between age and 5-HIAA levels in controls (ρ = 0.26, P < .016) and a similar trend in patients, while sex was unrelated to any metabolite. All monoamine metabolites in moderately to severely depressed patients (17-item Hamilton Depression Rating Scale score > 12) were significantly lower than those in controls (P < .0005 for all 3 metabolites). We found that antidepressants decreased the levels of 5-HIAA (ρ = -0.39, P < .001) and MHPG (ρ = -0.49, P < .0001) and that antipsychotics increased levels of HVA (ρ = 0.24, P < .05). There was a strong correlation between HVA and 5-HIAA levels (controls: ρ = 0.79, P = .000001; MDD: ρ = 0.66, P = .000001). HVA levels (ρ = -0.43, P < .001) and 5-HIAA levels (ρ = -0.23, P < .05), but not MHPG levels (ρ = -0.18, P > .1), were related to depression severity. CONCLUSIONS: CSF 5-HIAA and HVA levels could be state-dependent markers in MDD patients. Since 5-HIAA levels greatly decrease with the use of antidepressants, HVA levels might be more useful in the clinical setting.

Cocaine inhibits leptin-induced increase of the cocaine- and amphetamine-regulated transcript peptide in the nucleus accumbens in rats.

Two well-known appetite-regulatory peptides, leptin and cocaine- and amphetamine-regulated transcript (CART), are known to be involved in the brain rewarding pathway. However, it is not yet known how they interact in the nucleus accumbens, an important region mediating the rewarding effects of drugs of abuse. Using the immunoassay method, we found that a microinjection of leptin into the nucleus accumbens core induces an immediate and transient increase of the CART peptide in this site, whereas these effects were inhibited by cocaine. These results expand the role of accumbal leptin to the regulation of the CART peptide and further suggest that possible interaction of these appetite-regulating peptides may be involved in cocaine-mediated rewarding effects.

The expression of methiopropamine-induced locomotor sensitization requires dopamine D2, but not D1, receptor activation in the rat.

Methiopropamine (MPA) is a structural analog to methamphetamine and is categorized as a novel psychoactive substance that needs to be controlled. However, no study has been performed to determine whether MPA actually develops an addiction-like behavior similar to those arising from other psychomotor stimulants. Thus, we attempted to determine whether MPA produces locomotor sensitization in a manner similar to amphetamine. In the first experiment, rats were pre-exposed to either saline or one of three different doses of MPA (0.2, 1.0, or 5.0mg/kg, IP) with a total of four injections, respectively. After a 2-week withdrawal period, when they were challenged with the same dose of MPA, only the group that was pre-exposed to high dose of MPA (5.0mg/kg) showed sensitized locomotor activity. In the second experiment, all rats were pre-exposed to MPA (5.0mg/kg) only. Interestingly, the expression of MPA-induced locomotor sensitization was inhibited by a pre-injection of a dopamine D2 receptor antagonist, eticlopride (0.05mg/kg, IP), though not by a dopamine D1 receptor antagonist, SCH23390 (0.01mg/kg, IP). These results suggest that repeated injection of MPA in the rat provokes certain neuronal changes involving specific, likely D2, dopamine receptor-mediated pathways that contribute to the expression of MPA-induced locomotor sensitization.

The inactivation of extracellular signal-regulated kinase by glucagon-like peptide-1 contributes to neuroprotection against oxidative stress.

Glucagon-like peptide-1 (GLP-1), an insulinotropic peptide secreted from enteroendocrine cells, has been known to have a neuroprotective effect. However, it is not fully understood the intracellular mediator of GLP-1 signaling in neuronal cells. In the present study, we examined the change in intracellular signaling of cortical neurons after GLP-1 application and luminal glucose stimulation in vitro and in vivo. GLP-1 receptor was highly expressed in cultured cortical neurons and brain tissues including the prefrontal cortex and hippocampus. The activation of GLP-1 receptor (5min) significantly decreased levels of phosphorylated extracellular signal-regulated kinase (pERK), which is involved in neuronal cell survival and death, in cultured cortical neurons. Oral glucose administration also rapidly reduced pERK levels in the prefrontal cortex, while intraperitoneal glucose injection did not show such an effect. Further, GLP-1 attenuated hydrogen peroxide-induced cell death and hyperactivity of ERK in cultured cortical neurons. It is possible that increased GLP-1 by luminal glucose stimulation affects cortical system including the maintenance of neuronal cell survival.

Microinjection of cocaine- and amphetamine-regulated transcript 55-102 peptide into the nucleus accumbens could modulate anxiety-related behavior in rats.

Cocaine- and amphetamine-regulated transcript (CART) peptide is abundantly expressed in the nucleus accumbens (NAcc) and is involved in stress, anxiety and reward responses. To examine the role of CART peptide in anxiety-related behavior, naïve rats were bilaterally injected with CART 55-102 peptide (0.5, 1.0 or 2.5 µg/0.5 µl/side) or vehicle into the NAcc. Following this, their anxiety-related behavior was assessed using the elevated plus maze and the open field tests with a one-week interval between the tests. There was no difference in the time spent in open arms, or number of entries into open arms on the elevated plus maze in the CART-treated animals at any dose, when compared with the vehicle-treated group. However, there was a significant increase in the time spent in the center of the open field with administration of the low dose of CART peptide (0.5 µg/0.5 µl/side), although this effect disappeared at the high dose (2.5 µg/0.5 µl/side). None of the doses of CART peptide altered total locomotion in these tests. To further determine the possible anxiety-modulating effect of CART peptide at low dosages, the light and dark test was performed. Additional groups of rats given doses of 0.01 µg/0.5 µl/side or 0.5 µg/0.5 µl/side of CART peptide showed increased exploration time in the light side. These results suggest that accumbal-CART peptide reduces anxiety-like behavior in a dose-dependent manner.

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.

Blockade of ERK Phosphorylation in the Nucleus Accumbens Inhibits the Expression of Cocaine-induced Behavioral Sensitization in Rats.

Repeated administration of psychostimulants such as cocaine leads to the development of behavioral sensitization. Extracellular signal-Regulated Kinase (ERK), an enzyme important for long-term neuronal plasticity, has been implicated in such effects of these drugs. Although the nucleus accumbens (NAcc) is the site mediating the expression of behavioral sensitization by drugs of abuse, the precise role of ERK activation in this site has not been determined. In this study we demonstrate that blockade of ERK phosphorylation in the NAcc by a single bilateral microinjections of PD98059 (0.5 or 2.0µ g/side), or U0126 (0.1 or 1.0µg/side), into this site dose-dependently inhibited the expression of cocaine-induced behavioral sensitization when measured at day 7 following 6 consecutive daily cocaine injections (15 mg/kg, i.p.). Acute microinjection of either vehicle or PD98059 alone produced no different locomotor activity compared to saline control. Further, microinjection of PD98059 (2.0µ g/side) in the NAcc specifically lowered cocaine-induced increase of ERK phosphorylation levels in this site, while unaffecting p-38 protein levels. These results indicate that ERK activation in the NAcc is necessary for the expression of cocaine-induced behavioral sensitization, and further suggest that repeated cocaine evokes neuronal plasticity involving ERK pathway in this site leading to long-lasting behavioral changes.

Cabergoline, a dopamine receptor agonist, has an antidepressant-like property and enhances brain-derived neurotrophic factor signaling.

RATIONALE: Dopamine agonists have been implicated in the treatment of depression. Cabergoline is an ergot derivative with a high affinity to dopamine D(2)-like receptors; however, there have been few preclinical studies on its antidepressant-like effects. MATERIALS AND METHODS: Behavioral effects of cabergoline were examined in rats using forced swimming (FST), novelty-suppressed feeding (NST), open field (OFT), and elevated-plus maze (EPT) tests. In a single treatment paradigm, behaviors of rats were analyzed 4 h after single injection of cabergoline (s.c., 0-4 micromol/kg). In a repeated-treatment paradigm, OFT, EPT, and FST were conducted on days 11, 12, and 13-14, respectively, during daily cabergoline injections (s.c., 0.5 micromol/kg), and then hippocampus was removed 24 h after the last injection. NST was conducted in a separate experiment at day 14. Western blotting was used for the analysis of the protein levels of brain-derived neurotrophic factor (BDNF) and the activation of intracellular signaling molecules. RESULTS: Single injection of cabergoline demonstrated decreased immobility in FST and distance traveled during 0-10 min in OFT, while time spent and entry into open arms were increased at 4 micromol/kg. When cabergoline was repeatedly administered, immobility in FST and the latency of feeding in NSF were significantly reduced, while vertical movement was increased in OFT. The time in closed arms was tended to be decreased in EPT. Expression of BDNF and activation of extracellular signal-regulated kinase 1 were up-regulated after the chronic administration of cabergoline. CONCLUSIONS: Cabergoline exerts antidepressant- and anxiolytic-like effects, which may be mediated by potentiation of intracellular signaling of BDNF.

Microinjection of CART peptide 55-102 into the nucleus accumbens core inhibits the expression of conditioned hyperactivity in a cocaine-associated environment.

A distinct environment associated with psychomotor stimulants like cocaine and amphetamine can elicit conditioned locomotion in rats. This study examined the contribution of CART 55-102 peptide in the NAcc core to the expression of conditioned locomotion in a cocaine-associated environment. Rats in different groups were administered injections in five 2-day blocks: Paired, cocaine (15 mg/kg, IP) in locomotor activity boxes on day 1 and saline in their home cages on day 2; Unpaired, saline in the activity boxes on day 1 and cocaine in their home cages on day 2; or Control, saline in both environments. One week after the last conditioning block, all rats were tested for their conditioned locomotor response in the activity boxes for 1h following an IP saline injection, which was preceded by a bilateral microinjection into the NAcc core of saline or CART 55-102 (1.0 and 2.5 microg/side). As expected, Paired rats showed both increased locomotor activity and rearing compared to rats in either the Unpaired or Control groups. However, the expression of this conditioned hyper-locomotion was inhibited by microinjection into the NAcc core of CART 55-102. These results suggest that CART 55-102 peptide in the NAcc plays a role in the expression of conditioned locomotion in an environment associated with cocaine, and further extends the notion that CART 55-102 plays an important regulatory role in psychomotor stimulants actions in the NAcc.

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.

CART peptide 55-102 microinjected into the nucleus accumbens inhibits the expression of behavioral sensitization by amphetamine.

CART peptide has been shown to regulate the actions of psychomotor stimulants. Here we have further investigated the role of the biologically active CART 55-102 peptide in the nucleus accumbens (NAcc) in the expression of behavioral sensitization by amphetamine (AMPH). Rats were pre-exposed 5 times to either saline or AMPH (1 mg/kg, i.p.). After 2 weeks of withdrawal, rats were microinjected into the NAcc with saline or CART 55-102 (1.0, or 2.5 microg/0.5 microl/side) followed by AMPH challenge (1 mg/kg, i.p.). The enhanced increase of locomotion and rearing produced by repeated AMPH pre-exposures was dose-dependently inhibited by microinjection into the NAcc of CART 55-102 peptide. These results indicate that CART 55-102 peptide in the NAcc can play a compensatory inhibitory role in the expression of behavioral sensitization by AMPH and further suggest that CART peptide may be a useful target to control the drug addiction by psychomotor stimulants.

Microinjection of CART peptide 55-102 into the nucleus accumbens blocks both the expression of behavioral sensitization and ERK phosphorylation by cocaine.

The role of the biologically active CART 55-102 peptide in the nucleus accumbens (NAcc) in the expression of cocaine-induced behavioral sensitization was investigated. Rats were divided into four groups: one for saline and the other three for cocaine pre-exposures (15 mg/kg, i.p., once daily for 7 days). After 3 weeks of withdrawal, rats were microinjected into the NAcc either saline or CART 55-102 (1.0, or 2.5 microg/0.5 microl/side) followed by cocaine challenge (10 mg/kg, i.p.). Microinjection into the NAcc of CART 55-102 peptide dose-dependently blocked the expression of locomotor sensitization produced by repeated cocaine pre-exposures. Next, we further examined the effect of CART 55-102 microinjection on extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in the NAcc. Additional four groups of rats were all cocaine pre-exposed and, after 3 weeks of withdrawal, they were either saline or cocaine challenged systemically following microinjection into the NAcc of either saline, CART 55-102 (2.5 microg/0.5 microl/side), or the equivalent mole amount of inactive CART 1-27 peptide. The increase of ERK1/2 phosphorylation levels in the NAcc by cocaine was completely blocked by CART 55-102 microinjection in this site, while it remains unaffected by inactive CART 1-27 peptide. These results suggest that CART 55-102 peptide in the NAcc may play a compensatory inhibitory role in the expression of behavioral sensitization by cocaine and these effects may be mediated by its inhibition of ERK1/2 phosphorylation in this site.