Participation of the nucleus accumbens dopaminergic system in the antidepressant-like actions of a diet rich in omega-3 polyunsaturated fatty acids.

The beneficial effects of omega (ω)-3 polyunsaturated fatty acid (PUFA) supplementation on major depressive disorder have been actively studied, but the underlying mechanism remains unknown. The present study examined the involvement of the nucleus accumbens (NAc) dopaminergic systems in behavioral changes in mice fed a diet high in ω-3 PUFAs. Mice fed a diet containing about double the amount of ω-3 PUFAs (krill oil (KO) diet) exerted shorter immobility times in the forced swim test (FST) than mice fed a control diet, containing only α-linolenic acid (ALA) as ω-3 PUFAs. The shorter immobility times were observed in both male and female mice. A dopamine metabolite, 3,4-dihydroxyphenylacetic acid, increased in the NAc in male mice fed the KO diet when compared with those fed the control diet. In addition, dopamine, 3-methoxytyramine, and homovanillic acid increased in the NAc in female mice fed the KO diet. Notably, the effects of the KO diet on the immobility time in the FST were abolished by microinjection of sulpiride, an antagonist of D2-like receptors, into the NAc. A similar microinjection of an antagonist selective for D1-like receptors, SKF83566, also abolished the reduction in immobility in the FST. Moreover, we found that tyrosine hydroxylase-positive cells increased in the ventral tegmental area (VTA) in mice fed the KO diet. These results suggest that modulation of the VTA-NAc dopaminergic pathway is one of the mechanisms by which a KO diet rich in ω-3 PUFAs reduces the immobility behavior in the mouse FST.

Importance of kynurenine 3-monooxygenase for spontaneous firing and pharmacological responses of midbrain dopamine neurons: Relevance for schizophrenia.

Kynurenine 3-monooxygenase (KMO) is an essential enzyme of the kynurenine pathway, converting kynurenine into 3-hydroxykynurenine. Inhibition of KMO increases kynurenine, resulting in elevated levels of kynurenic acid (KYNA), an endogenous N-methyl-d-aspartate and α*7-nicotinic receptor antagonist. The concentration of KYNA is elevated in the brain of patients with schizophrenia, possibly as a result of a reduced KMO activity. In the present study, using in vivo single cell recording techniques, we investigated the electrophysiological characteristics of ventral tegmental area dopamine (VTA DA) neurons and their response to antipsychotic drugs in a KMO knock-out (K/O) mouse model. KMO K/O mice exhibited a marked increase in spontaneous VTA DA neuron activity as compared to wild-type (WT) mice. Furthermore, VTA DA neurons showed clear-cut, yet qualitatively opposite, responses to the antipsychotic drugs haloperidol and clozapine in the two genotypes. The anti-inflammatory drug parecoxib successfully lowered the firing activity of VTA DA neurons in KMO K/O, but not in WT mice. Minocycline, an antibiotic and anti-inflammatory drug, produced no effect in this regard. Taken together, the present data further support the usefulness of KMO K/O mice for studying distinct aspects of the pathophysiology and pharmacological treatment of psychiatric disorders such as schizophrenia.

Neuronal, astroglial and locomotor injuries in subchronic copper intoxicated rats are repaired by curcumin: A possible link with Parkinson's disease.

We aim herein to assess the neurotoxic effects of subchronic Cu-exposition (0125%) for 6 weeks on dopaminergic and astroglial systems then locomotor activity in rats as well as the probable therapeutic efficiency of curcumin-I (30 mg/kg B.W.). We found that intoxicated rats showed a significant impairment of Tyrosine Hydroxylase (TH) within substantia nigra pars compacta (SNc), ventral tegmental area (VTA) and the striatal outputs together with loss expression of GFAP in these structures. This was linked with an evident decrease in locomotor performance. Co-treatment with curcumin-I inverted these damages and exhibited a significant neuroprotective potential, thus, both TH expression and locomotor performance was reinstated in intoxicated rats. These results prove a profound dopaminergic and astroglial damages following subchronic Cu exposition and new beneficial curative potential of curcumin against subchronic Cu-induced astroglial and dopaminergic neurotoxicity. Consequently, we suggest that Cu neurotoxicity may be strengthened in vivo firstly by attacking and weaking the astroglial system, and curcumin could be prized as a powerful and preventive target for the neurodegenerative diseases related metal element, especially Parkinson's disease.

Testosterone and Corticosterone in the Mesocorticolimbic System of Male Rats: Effects of Gonadectomy and Caloric Restriction.

Steroid hormones can modulate motivated behaviors through the mesocorticolimbic system. Gonadectomy (GDX) is a common method to determine how steroids influence the mesocorticolimbic system, and caloric restriction (CR) is often used to invigorate motivated behaviors. A common assumption is that the effects of these manipulations on brain steroid levels reflects circulating steroid levels. We now know that the brain regulates local steroid levels in a region-specific manner; however, previous studies have low spatial resolution. Using ultrasensitive liquid chromatography tandem mass spectrometry, we examined steroids in microdissected regions of the mesocorticolimbic system (ventral tegmental area, nucleus accumbens, medial prefrontal cortex). We examined whether GDX or CR influences systemic and local steroids, particularly testosterone (T) and steroidogenic enzyme transcripts. Adult male rats underwent a GDX surgery and/or CR for either 2 or 6 weeks. Levels of T, the primary steroid of interest, were higher in all brain regions than in the blood, whereas corticosterone (CORT) was lower in the brain than in the blood. Importantly, GDX completely eliminated T in the blood and lowered T in the brain. Yet, T remained present in the brain, even 6 weeks after GDX. CR decreased both T and CORT in the blood and brain. Steroidogenic enzyme (Cyp17a1, 3ß-hydroxysteroid dehydrogenase, aromatase) transcripts and androgen receptor transcripts were expressed in the mesocorticolimbic system and differentially affected by GDX and CR. Together, these results suggest that T is synthesized within the mesocorticolimbic system. These results provide a foundation for future studies examining how neurosteroids influence behaviors mediated by the mesocorticolimbic system.

Tyrosine hydroxylase in the ventral tegmental area of rams with high or low libido-A role for dopamine.

Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (P<0.05) neurons were positive for tyrosine hydroxylase in the VTA. Sexually inactive rams had fewer (P<0.05) FRA and tyrosine hydroxylase positive neurons in the VTA than sexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine.

Is catalase involved in the effects of systemic and pVTA administration of 4-methylpyrazole on ethanol self-administration?

The oxidative metabolism of ethanol into acetaldehyde involves several enzymes, including alcohol dehydrogenase (ADH) and catalase-hydrogen peroxide (H2O2). In this regard, while it is well known that 4-methylpyrazole (4-MP) acts by inhibiting ADH in the liver, little attention has been placed on its ability to interfere with fatty acid oxidation-mediated generation of H2O2, a mechanism that may indirectly affect catalase whose enzymatic activity requires H2O2. The aim of our investigation was twofold: 1) to evaluate the effect of systemic (i.p. [intraperitoneal]) and local (into the posterior ventral tegmental area, pVTA) administration of 4-MP on oral ethanol self-administration, and 2) to assess ex vivo whether or not systemic 4-MP affects liver and brain H2O2 availability. The results show that systemic 4-MP reduced ethanol but not acetaldehyde or saccharin self-administration, and decreased the ethanol deprivation effect. Moreover, local intra-pVTA administration of 4-MP reduced ethanol but not saccharin self-administration. In addition, although unable to affect basal catalase activity, systemic administration of 4-MP decreased H2O2 availability both in liver and in brain. Overall, these results indicate that 4-MP interferes with ethanol self-administration and suggest that its behavioral effects could be due to a decline in catalase-H2O2 system activity as a result of a reduction of H2O2 availability, thus highlighting the role of central catalase-mediated metabolism of ethanol and further supporting the key role of acetaldehyde in the reinforcing properties of ethanol.

PDE4 Inhibition Restores the Balance Between Excitation and Inhibition in VTA Dopamine Neurons Disrupted by Repeated In Vivo Cocaine Exposure.

Phosphodiesterase type 4 (PDE4) is a family of enzymes that selectively degrade intracellular cAMP. PDE4 inhibitors have been shown to regulate the rewarding and reinforcing effects of cocaine, but the underlying mechanisms remain poorly understood. Here we show that pretreatments with the PDE4 inhibitor rolipram attenuated cocaine-induced locomotor sensitization in mice. Repeated cocaine exposure in vivo caused a decrease in inhibitory postsynaptic currents (IPSCs) and an increase in the AMPAR/NMDAR ratio in ventral tegmental area (VTA) dopamine neurons in midbrain slices ex vivo. Cocaine exposure disrupted the balance between excitation and inhibition as shown by an increase in the excitation to inhibition (E/I) ratio. Rolipram pretreatments in vivo prevented cocaine-induced reductions in GABAergic inhibition but did not further increase cocaine-induced potentiation of excitation, leading to the restoration of a balance between excitation and inhibition and normalization of the E/I ratio. In support of this idea, we found that repeated cocaine exposure led to an increase in the single-unit action potential firing rate in vivo in VTA dopamine neurons, which was blocked by rolipram pretreatments. These results suggest that repeated cocaine exposure in vivo disrupts the balance between excitation and inhibition in VTA dopamine neurons, while PDE4 inhibition reestablishes the balance between excitation and inhibition through distinct mechanisms.

Suppression of the acute upregulation of phosphorylated-extracellular regulated kinase in ventral tegmental area by a µ-opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer.

We investigated the mechanisms underlying the suppression of the rewarding effects of opioids using the femur bone cancer (FBC) mouse model. The rewarding and antinociceptive effects of subcutaneously administered morphine and oxycodone in the FBC model mice were assessed using the conditioned place preference test and the von-Frey test. In FBC mice, antinociceptive doses of morphine (30 mg/kg) and oxycodone (5 mg/kg) did not produce the rewarding effects but excessive doses of morphine (300 mg/kg) and oxycodone (100 mg/kg) did. Western blot analyses revealed a transient and significant increase in phosphorylated-extracellular regulated kinase (p-ERK) levels in ventral tegmental area (VTA) 5 min after the administration of morphine in sham-group. Interestingly, in FBC group, a regular dose of morphine did not increase p-ERK levels but a high dose of morphine caused an increase in p-ERK level 5 min after administration. The rewarding effects of a regular dose of and a high dose of morphine in the sham-operation and FBC model, respectively, were significantly inhibited by the MEK inhibitor. The suppression of p-ERK might result in resistance to these rewarding effects under the conditions of bone cancer.

Nuclear organization of the substantia nigra, ventral tegmental area and retrorubral field of the common marmoset (Callithrix jacchus): A cytoarchitectonic and TH-immunohistochemistry study.

It is widely known that the catecholamine group is formed by dopamine, noradrenaline and adrenaline. Its synthesis is regulated by the enzyme called tyrosine hydroxylase. 3-hydroxytyramine/dopamine (DA) is a precursor of noradrenaline and adrenaline synthesis and acts as a neurotransmitter in the central nervous system. The three main nuclei, being the retrorubral field (A8 group), the substantia nigra pars compacta (A9 group) and the ventral tegmental area (A10 group), are arranged in the die-mesencephalic portion and are involved in three complex circuitries - the mesostriatal, mesolimbic and mesocortical pathways. These pathways are involved in behavioral manifestations, motricity, learning, reward and also in pathological conditions such as Parkinson's disease and schizophrenia. The aim of this study was to perform a morphological analysis of the A8, A9 and A10 groups in the common marmoset (Callithrix jacchus - a neotropical primate), whose morphological and functional characteristics support its suitability for use in biomedical research. Coronal sections of the marmoset brain were submitted to Nissl staining and TH-immunohistochemistry. The morphology of the neurons made it possible to subdivide the A10 group into seven distinct regions: interfascicular nucleus, raphe rostral linear nucleus and raphe caudal linear nucleus in the middle line; paranigral and parainterfascicular nucleus in the middle zone; the rostral portion of the ventral tegmental area nucleus and parabrachial pigmented nucleus located in the dorsolateral portion of the mesencephalic tegmentum. The A9 group was divided into four regions: substantia nigra compacta dorsal and ventral tiers; substantia nigra compacta lateral and medial clusters. No subdivisions were made for the A8 group. These results reveal that A8, A9 and A10 are phylogenetically stable across species. As such, further studies concerning such divisions are necessary in order to evaluate the occurrence of subdivisions that express DA in other primate species, with the aim of characterizing its functional relevance.

Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area.

Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug-induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up-regulated by both drugs was serum- and glucocorticoid-inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N-myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug-related behaviors, we over-expressed constitutively active (CA) SGK1 in the VTA. SGK1-CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1-CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug-induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction. We find that repeated, but not acute, morphine or cocaine administration induces an increase in serum- and glucocorticoid-inducible kinase (SGK1) gene expression and activity in the ventral tegmental area (VTA). This increase in SGK1 activity may play a role in drug-dependent behaviors and suggests a novel signaling cascade for potential intervention in drug dependence and addiction.

Increased burst-firing of ventral tegmental area dopaminergic neurons in D-amino acid oxidase knockout mice in vivo.

d-Amino acid oxidase (DAO) degrades the N-methyl-d-aspartate (NMDA) receptor co-agonist d-serine, and is implicated in schizophrenia as a risk gene and therapeutic target. In schizophrenia, the critical neurochemical abnormality affects dopamine, but to date there is little evidence that DAO impacts on the dopamine system. To address this issue, we measured the electrophysiological properties of dopaminergic (DA) and non-DA neurons in the ventral tegmental area (VTA) of anaesthetised DAO knockout (DAO(-/-) ) and DAO heterozygote (DAO(+/-) ) mice as compared with their wild-type (DAO(+/+) ) littermates. Genotype was confirmed at the protein level by western blotting and immunohistochemistry. One hundred and thirty-nine VTA neurons were recorded in total, and juxtacellular labelling of a subset revealed that neurons immunopositive for tyrosine hydroxylase had DA-like electrophysiological properties that were distinct from those of neurons that were tyrosine hydroxylase-immunonegative. In DAO(-/-) mice, approximately twice as many DA-like neurons fired in a bursting pattern than in DAO(+/-) or DAO(+/+) mice, but other electrophysiological properties did not differ between genotypes. In contrast, non-DA-like neurons had a lower firing rate in DAO(-/-) mice than in DAO(+/-) or DAO(+/+) mice. These data provide the first direct evidence that DAO modulates VTA DA neuron activity, which is of interest for understanding both the glutamatergic regulation of dopamine function and the therapeutic potential of DAO inhibitors. The increased DA neuron burst-firing probably reflects increased availability of d-serine at VTA NMDA receptors, but the site, mechanism and mediation of the effect requires further investigation, and may include both direct and indirect processes.

Assessment of cytochrome C oxidase dysfunction in the substantia nigra/ventral tegmental area in schizophrenia.

Perturbations in metabolism are a well-documented but complex facet of schizophrenia pathology. Optimal cellular performance requires the proper functioning of the electron transport chain, which is constituted by four enzymes located within the inner membrane of mitochondria. These enzymes create a proton gradient that is used to power the enzyme ATP synthase, producing ATP, which is crucial for the maintenance of cellular functioning. Anomalies in a single enzyme of the electron transport chain are sufficient to cause disruption of cellular metabolism. The last of these complexes is the cytochrome c oxidase (COX) enzyme, which is composed of thirteen different subunits. COX is a major site for oxidative phosphorylation, and anomalies in this enzyme are one of the most frequent causes of mitochondrial pathology. The objective of the present report was to assess if metabolic anomalies linked to COX dysfunction may contribute to substantia nigra/ventral tegmental area (SN/VTA) pathology in schizophrenia. We tested COX activity in postmortem SN/VTA from schizophrenia and non-psychiatric controls. We also tested the protein expression of key subunits for the assembly and activity of the enzyme, and the effect of antipsychotic medication on subunit expression. COX activity was not significantly different between schizophrenia and non-psychiatric controls. However, we found significant decreases in the expression of subunits II and IV-I of COX in schizophrenia. Interestingly, these decreases were observed in samples containing the entire rostro-caudal extent of the SN/VTA, while no significant differences were observed for samples containing only mid-caudal regions of the SN/VTA. Finally, rats chronically treated with antipsychotic drugs did not show significant changes in COX subunit expression. These findings suggest that COX subunit expression may be compromised in specific sub-regions of the SN/VTA (i.e. rostral regions), which may lead to a faulty assembly of the enzyme and a greater vulnerability to metabolic insult.

CC-chemokine ligand 2 facilitates conditioned place preference to methamphetamine through the activation of dopamine systems.

Methamphetamine addiction is characterized by drug craving caused by stimulation of the reward system. Because neuroinflammation underlies several neurological disorders, we investigated whether CC-chemokine ligand 2 (CCL2) participates in the methamphetamine dependence using mice. Upregulation of CCL2 but not CC-chemokine receptor 2 (CCR2), a dominant receptor for CCL2, mRNA in both the prefrontal cortex (PFC) and nucleus accumbens (NAC) was observed after methamphetamine (3 mg/kg, s.c.) administration. Using immunohistochemistry, high CCL2 protein levels localized to neurons in the PFC and NAC. In the conditioned place preference (CPP) test, methamphetamine (0.3 - 3 mg/kg, s.c.) induced a CPP, reflecting psychic dependence on methamphetamine, in a dose-dependent manner. The CPP to methamphetamine was attenuated by RS504393 (1 mg/kg, s.c.), a CCR2 antagonist. Moreover, methamphetamine increased phosphorylated tyrosine hydroxylase (pTH) levels in the ventral tegmental area (VTA). Increased levels of pTH in the VTA by methamphetamine was also suppressed by RS504393. Furthermore, intracerebroventricular injection of recombinant CCL2 increased pTH levels in the VTA. Taken together, we demonstrate that activation of dopamine neurons, which enhances reward-system activity, via the CCL2-CCR2 axis plays a crucial role in psychic dependence on methamphetamine. Novel treatments targeting this machinery may be effective for drug addiction.

Role of ERK1, 2, and 5 in dopamine neuron survival during aging.

Extracellular signal-regulated kinases (ERKs) 1, 2, and 5 have been shown to play distinct roles in proliferation, differentiation, and neuronal viability. In this study, we examined ERK1, 2, and 5 expression and activation in the substantia nigra (SN), striatum (STR), and ventral tegmental area (VTA) during aging. An age-related decrease in phosphorylated ERK5 was observed in the SN and STR, whereas an increase in total ERK1 was observed in all 3 regions. In primary cultures of the SN and VTA, inhibition of ERK5 but not ERK1 and 2 decreased dopamine neuronal viability significantly. These data suggest that ERK5 is essential for the basal survival of SN and VTA dopaminergic neurons. This is the first study to examine ERK1, 2, and 5 expression and activation in the SN, STR, and VTA during aging, and the relative roles of ERK1, 2, and 5 in basal survival of SN and VTA dopaminergic neurons. These data raise the possibility that a decline in ERK5 signaling may play a role in age-related impairments in dopaminergic function.

Morphine-induced apoptosis in the ventral tegmental area and hippocampus after the development but not extinction of reward-related behaviors in rats.

Some data suggest that morphine induces apoptosis in neurons, while other evidences show that morphine could have protective effects against cell death. In this study, we suggested that there is a parallel role of morphine in reward circuitry and apoptosis processing. Therefore, we investigated the effect of morphine on modifications of apoptotic factors in the ventral tegmental area (VTA) and hippocampus (HPC) which are involved in the reward circuitry after the acquisition and extinction periods of conditioned place preference (CPP). In behavioral experiments, different doses of morphine (0.5, 5, and 10 mg/kg) and saline were examined in the CPP paradigm. Conditioning score and locomotor activity were recorded by Ethovision software after acquisition on the post-conditioning day, and days 4 and 8 of extinction periods. In order to investigate the molecular mechanisms in each group, we then dissected the brains and measured the expression of apoptotic factors in the VTA and HPC by western blotting analysis. All of the morphine-treated groups showed an increase of apoptotic factors in these regions during acquisition but not in extinction period. In the HPC, morphine significantly increased the ratio of Bax/Bcl-2, caspases-3, and PARP by the lowest dose (0.5 mg/kg), but, in the VTA, a considerable increase was seen in the dose of 5 mg/kg; promotion of apoptotic factors in the HPC and VTA insinuates that morphine can affect the molecular mechanisms that interfere with apoptosis through different receptors. Our findings suggest that a specific opioid receptor involves in modification of apoptotic factors expression in these areas. It seems that the reduction of cell death in response to high dose of morphine in the VTA and HPC may be due to activation of low affinity opioid receptors which are involved in neuroprotective features of morphine.

CaMKII activity in the ventral tegmental area gates cocaine-induced synaptic plasticity in the nucleus accumbens.

Addictive drugs such as cocaine induce synaptic plasticity in discrete regions of the reward circuit. The aim of the present study is to investigate whether cocaine-evoked synaptic plasticity in the ventral tegmental area (VTA) and nucleus accumbens (NAc) is causally linked. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of long-term synaptic plasticity, learning, and drug addiction. We examined whether blocking CaMKII activity in the VTA affected cocaine conditioned place preference (CPP) and cocaine-evoked synaptic plasticity in its target brain region, the NAc. TatCN21 is a CaMKII inhibitory peptide that blocks both stimulated and autonomous CaMKII activity with high selectivity. We report that intra-VTA microinjections of tatCN21 before cocaine conditioning blocked the acquisition of cocaine CPP, whereas intra-VTA microinjections of tatCN21 before saline conditioning did not significantly affect cocaine CPP, suggesting that the CaMKII inhibitor blocks cocaine CPP through selective disruption of cocaine-cue-associated learning. Intra-VTA tatCN21 before cocaine conditioning blocked cocaine-evoked depression of excitatory synaptic transmission in the shell of the NAc slices ex vivo. In contrast, intra-VTA microinjection of tatCN21 just before the CPP test did not affect the expression of cocaine CPP and cocaine-induced synaptic plasticity in the NAc shell. These results suggest that CaMKII activity in the VTA governs cocaine-evoked synaptic plasticity in the NAc during the time window of cocaine conditioning.

Glycogen synthase kinase-3ß in the ventral tegmental area mediates diurnal variations in cocaine-induced conditioned place preference in rats.

Cocaine sensitization and reward are reported to be under the influence of diurnal rhythm. However, no previous studies have reported brain areas that play a role as modulators and underlie the mechanism of diurnal variations in cocaine reward. We examined (1) the diurnal rhythm of glycogen synthase kinase-3ß (GSK-3ß) activity in the suprachiasmatic nucleus (SCN) and reward-related brain areas in naive rats; (2) the effect of day and night on the acquisition of cocaine-induced conditioned place preference (CPP); (3) the influence of cocaine-induced CPP on GSK-3ß activity in the SCN and reward-related brain areas; and (4) the effect of the GSK-3ß inhibitor SB216763 microinjected bilaterally into the ventral tegmental area (VTA) on cocaine-induced CPP. A significant diurnal rhythm of GSK-3ß activity was found in the SCN and reward-related brain areas, with diurnal variations in cocaine-induced CPP. GSK-3ß activity in the SCN and reward-related brain areas exhibited marked diurnal variations in rats treated with saline. GSK-3ß activity in rats treated with cocaine exhibited distinct diurnal variations only in the prefrontal cortex and VTA. Cocaine decreased the expression of phosphorylated GSK-3ß (i.e. increased GSK-3ß activity) only in the VTA in rats trained and tested at ZT4 and ZT16. SB216763 microinjected into the VTA bilaterally eliminated the diurnal variations in cocaine-induced CPP, but did not affect the acquisition of cocaine-induced CPP. These findings suggest that the VTA may be a critical area involved in the diurnal variations in cocaine-induced CPP, and GSK-3ß may be a regulator of diurnal variations in cocaine-induced CPP.

The alcohol deprivation effect: marked inhibition by anticatalase gene administration into the ventral tegmental area in rats.

BACKGROUND: Animals that have chronically consumed alcohol and are subsequently deprived of it markedly increase their intake above basal levels when access to alcohol is reinstated. Such an effect, termed the alcohol deprivation effect (ADE), has been proposed to reflect (i) an obsessive-compulsive behavior, (ii) craving, or (iii) an increased reinforcing value of ethanol (EtOH). It has been reported that acetaldehyde, a highly reinforcing metabolite of EtOH, is generated in the brain by the action of catalase. Recent studies show that the administration of an anticatalase (shRNA)-encoding lentiviral vector into the brain ventral tegmental area (VTA) of naïve rats virtually abolishes (85 to 95%) their EtOH intake. It is hypothesized that the antireinforcing effect of the anticatalase vector will also inhibit the ADE. METHODS: Two-month-old Wistar-derived UChB alcohol drinker rats were offered free access to water and 10 and 20% EtOH for 67 days. Thereafter, the animals were deprived of EtOH for 15 days and were subsequently offered access to the EtOH solutions. At the start of the deprivation period, animals were microinjected a single dose of an anticatalase (or control) vector into the VTA. EtOH intake was measured on the first hour of EtOH re-exposure as well as on a 24-hour basis for 7 days. RESULTS: A marked ADE was observed when EtOH intake was measured on the first hour or 24 hours following EtOH re-exposure, compared to the corresponding controls. The administration of the anticatalase vector reduced ADE by 60 to 80% (p < 0.001) on the first hour and by 63 to 80% (p < 0.001) on the initial 24 hours of EtOH re-exposure (first and second ADE, respectively) without changing the total fluid intake, indicating a specific effect on EtOH drinking. CONCLUSIONS: Ethanol intake associated with ADE--a binge-like drinking behavior--is markedly inhibited by the administration of an anticatalase vector into the VTA, which blocks the conversion of EtOH into acetaldehyde, strongly suggesting that the marked increased EtOH intake that follows an alcohol deprivation period is mediated by acetaldehyde and its reinforcing metabolite.

Loss of tyrosine hydroxylase expression within the nigro-striato-cortical pathways in the cirrhotic rat: the possible restorative effect of the neurosteroid dehydroepiandrosterone sulfate.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder occurring as a consequence of both acute and chronic liver failure. Advanced HE is generally accompanied with extrapyramidal symptoms including rigidity and tremor, which may reflect alterations of the dopaminergic system. Recently we reported a beneficial effect of the neuroactive steroid dehydroepiandrosterone sulfate (DHEAS) in cirrhotic rats, however the mechanisms of such an effect by DHEAS were not addressed. In the present study, we describe the changes of the dopaminergic system occurring in the cirrhotic rats and concomitantly we investigated the effect of DHEAS on this system in Sprague-Dawley rats using the expression of tyrosine hydroxylase (TH) as a neuronal marker. Rats were submitted to bile duct ligation (BDL) surgery and TH immunohistochemistry was assessed in the Substantia nigra pars compacta (SNc), striatum, ventral tegmental area (VTA) and the cortex. TH immunoreactivity showed a significant diminution in both SNc and VTA concomitantly with the cortical and the striatal outputs in the BDL rats vs. controls. Three daily injections of 5mg/kg of DHEAS to BDL rats significantly normalized TH expression decrease in both SNc and VTA as well as dopaminergic projections to the striatum and the cortex of BDL rats. The present data support an involvement of the dopaminergic system in mild HE and a possible beneficial effect of the neurosteroid DHEAS as a potential pharmacological treatment of mild HE.

An accurate method for the quantification of cytochrome C oxidase in tissue sections.

Cytochrome oxidase (COX) is the enzyme that constitutes the last step of the mitochondrial electron transport chain for the production of ATP. Measurement of COX activity can be achieved by histochemistry, thus providing information about the metabolic status of the brain. Brain regions with high metabolism will present high COX activity in histochemistry assays and vice versa. Using histochemistry versus biochemistry to assess COX activity presents the advantage of providing a map of the differences in metabolism in discrete brain regions. Moreover, COX histochemistry allows quantifying the activity of a particular brain region, by converting units of optical density into units of activity. In the present work we have devised a methodology that allows not only quantifying differences in COX activity between groups, but also quantifying the amount of COX present in brain tissue sections, by directly relating optical density (OD) measurements to cytochrome C oxidase concentration, something that traditionally is achieved by the use of western blot. For this purpose we created a set of standards of known concentration of COX that were affixed to a nitrocellulose membrane, and this membrane was incubated together with the tissue sections in which COX activity was assessed. A standard curve was created using a gradient of different concentrations of purified bovine heart cytochrome oxidase (from 2µg to 0.1µg in intervals of 0.25µg). This standard curve allowed us to detect changes in optical density as low as 5%, and relate these OD differences with known concentrations of cytochrome C oxidase.