Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Long-Lasting Impairment of mGluR5-Activated Intracellular Pathways in the Striatum After Withdrawal of Cocaine Self-Administration.

Background: Cocaine addiction continues to be a major heath concern, and despite public health intervention there is a lack of efficient pharmacological treatment options. A newly identified potential target are the group I metabotropic glutamate receptors, with allosteric modulators showing particular promise. Methods: We evaluated the capacity of group I metabotropic glutamate receptors to induce functional responses in ex vivo striatal slices from rats with (1) acute cocaine self-administration, (2) chronic cocaine self-administration, and (3) 60 days cocaine self-administration withdrawal by Western blot and extracellular recordings of synaptic transmission. Results: We found that striatal group I metabotropic glutamate receptors are the principal mediator of the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine-induced cAMP responsive-element binding protein phosphorylation. Both acute and chronic cocaine self-administration blunted group I metabotropic glutamate receptor effects on cAMP responsive-element binding protein phosphorylation in the striatum, which correlated with the capacity to induce long-term depression, an effect that was maintained 60 days after chronic cocaine self-administration withdrawal. In the nucleus accumbens, the principal brain region mediating the rewarding effects of drugs, chronic cocaine self-administration blunted group I metabotropic glutamate receptor stimulation of extracellular signal-regulated protein kinases 1/2 and cAMP responsive-element binding protein. Interestingly, the group I metabotropic glutamate receptor antagonist/inverse-agonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride, led to a specific increase in cAMP responsive-element binding protein phosphorylation after chronic cocaine self-administration, specifically in the nucleus accumbens, but not in the striatum. Conclusions: Prolonged cocaine self-administration, through withdrawal, leads to a blunting of group I metabotropic glutamate receptor responses in the striatum. In addition, specifically in the accumbens, group I metabotropic glutamate receptor signaling to cAMP responsive-element binding protein shifts from an agonist-induced to an antagonist-induced cAMP responsive-element binding protein phosphorylation.

Enhanced Extracellular Glutamate and Dopamine in the Ventral Pallidum of Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats after Morphine.

The purpose of the present study was to investigate the role of ventral pallidal opioidergic mechanisms in the control of ethanol intake by studying the effects of acute administration of morphine on the levels of GABA, glutamate, and dopamine in the ventral pallidum. The study was conducted using the alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding Alko Non-Alcohol (ANA) rat lines that have well-documented differences in their voluntary ethanol intake and brain opioidergic systems. Therefore, examination of neurobiological differences between the lines is supposed to help to identify the neuronal mechanisms underlying ethanol intake, since selection pressure is assumed gradually to lead to enrichment of alleles promoting high or low ethanol intake, respectively. The effects of an acute dose of morphine (1 or 10 mg/kg s.c.) on the extracellular levels of GABA and glutamate in the ventral pallidum were monitored with in vivo microdialysis. The concentrations of GABA and glutamate in the dialyzates were determined with a high performance liquid chromatography system using fluorescent detection, while electrochemical detection was used for dopamine. The levels of glutamate in the rats injected with morphine 1 mg/kg were significantly above the levels found in the controls and in the rats receiving morphine 10 mg/kg. Morphine 10 mg/kg also increased the levels of dopamine. Morphine could not, however, modify the levels of GABA. The rat lines did not differ in any of the effects of morphine. The data suggest that the glutamatergic and dopaminergic systems in the ventral pallidum may mediate some effects of morphine. Since there were no differences between the AA and ANA lines, the basic hypothesis underlying the use of the genetic animal model suggests that the effects of morphine detected probably do not underlie the different intake of ethanol by the lines and contribute to the control of ethanol intake in these animals.

Agonist and antagonist effects of aripiprazole on D2-like receptors controlling rat brain dopamine synthesis depend on the dopaminergic tone.

BACKGROUND: The atypical antipsychotic drug aripiprazole binds with high affinity to a number of G protein coupled receptors, including dopamine D2 receptors, where its degree of efficacy as a partial agonist remains controversial. METHODS: We examined the properties of aripiprazole at D2-like autoreceptors by monitoring the changes of dopamine synthesis in adult rat brain striatal minces incubated ex vivo. The effects of the dopaminergic tone on the properties of aripiprazole were assayed by comparing a basal condition (2 mM K(+), low dopaminergic tone) and a stimulated condition (15 mM K(+), where dopamine release mimics a relatively higher dopaminergic tone). We also used 2 reference compounds: quinpirole showed a clear agonistic activity and preclamol (S-(-)-PPP) showed partial agonism under both basal and stimulated conditions. RESULTS: Aripiprazole under the basal condition acted as an agonist at D2-like autoreceptors and fully activated them at about 10 nM, inhibiting dopamine synthesis similarly to quinpirole. Higher concentrations of aripiprazole had effects not restricted to D2-like autoreceptor activation. Under the stimulated (15 mM K(+)) condition, nanomolar concentrations of aripiprazole failed to decrease dopamine synthesis but could totally block the effect of quinpirole. CONCLUSIONS: Under high dopaminergic tone, aripiprazole acts as a D2-like autoreceptor antagonist rather than as an agonist. These data show that, ex vivo, alteration of dopaminergic tone by depolarization affects the actions of aripiprazole on D2-like autoreceptors. Such unusual effects were not seen with the typical partial agonist preclamol and are consistent with the hypothesis that aripiprazole is a functionally selective D2R ligand.

Innate BDNF expression is associated with ethanol intake in alcohol-preferring AA and alcohol-avoiding ANA rats.

We have shown recently that acute administration of ethanol modulates the expression of brain-derived neurotrophic factor (BDNF) in several rat brain areas known to be involved in the development of addiction to ethanol and other drugs of abuse, suggesting that BDNF may be a factor contributing to the neuroadaptive changes set in motion by ethanol exposure. The purpose of the present study was to further clarify the role of BDNF in reinforcement from ethanol and in the development of addiction to ethanol by specifying the effect of acute administration of ethanol (1.5 or 3.0 g/kg i.p.) on the expression profile of BDNF mRNA in the ventral tegmental area and in the terminal areas of the mesolimbic dopamine pathway in the brain of alcohol-preferring AA and alcohol-avoiding ANA rats, selected for high and low voluntary ethanol intake, respectively. The level of BDNF mRNA expression was higher in the amygdala and ventral tegmental area of AA than in those of ANA rats, and there was a trend for a higher level in the nucleus accumbens. In the amygdala and hippocampus, a biphasic change in the BDNF mRNA levels was detected: the levels were decreased at 3 and 6h but increased above the basal levels at 24h. Furthermore, there was a difference between the AA and ANA lines in the effect of ethanol, the ANA rats showing an increase in BDNF mRNA levels while such a change was not seen in AA rats. These findings suggest that the innate levels of BDNF expression may play a role in the mediation of the reinforcing effects of ethanol and in the control of ethanol intake.

Effects of Lifelong Ethanol Consumption on Brain Monoamine Transmitters in Alcohol-Preferring Alko Alcohol (AA) Rats.

The purpose of the present study was to examine the combined effects of aging and lifelong ethanol exposure on the levels of monoamine neurotransmitters in different regions of the brain. This work is part of a project addressing interactions of aging and lifelong ethanol consumption in alcohol-preferring AA (Alko Alcohol) line of rats, selected for high voluntary consumption of ethanol. Intake of ethanol on the level of 4.5-5 g/kg/day for about 20 months induced only limited changes in the neurotransmitter levels; the concentration of noradrenaline was significantly reduced in the frontal cortex. There was also a trend towards lower levels of dopamine and 5-hydroxytryptamine (5-HT) in the frontal cortex, and towards a lower noradrenaline level in the dorsal cortex. Aging was associated with a decreased concentration of dopamine in the dorsal cortex and with a declining trend in the striatum. The levels of 5-HT in the limbic forebrain were higher in the aged than in the young animals, and in the striatum, there was a trend towards higher levels in older animals. The data suggest that a continuous intake of moderate amounts of ethanol does not enhance the age-related alterations in brain monoamine neurotransmission, while the decline in the brain level of dopamine associated with aging may be a factor contributing to age-related neurological disorders.

Brain-derived neurotrophic factor expression after acute administration of ethanol.

Earlier findings suggest that, in addition to its well-known neurotrophic role, brain-derived neurotrophic factor (BDNF) is also involved in the rewarding and reinforcing effects of drugs of abuse. The purpose of the present study was to examine the effects of acute administration of ethanol (1.25 or 2.5 g/kg i.p.) on the expression profile of BDNF in the rat brain by determining the BDNF mRNA expression in the frontal cortex, nucleus accumbens, amygdala, hippocampus, and ventral tegmental area. Ethanol decreased BDNF mRNA levels dose-dependently in the hippocampus, and after the higher ethanol dose in the frontal cortex, nucleus accumbens and amygdala, while increasing them in the ventral tegmental area. Furthermore, BDNF mRNA expression was found to be regulated in a temporally different manner in all investigated brain areas. These data suggest that BDNF is involved in the acute effects of ethanol, but separate brain areas may be differentially engaged in the mediation of these effects.

Role for ventral pallidal GABAergic mechanisms in the regulation of ethanol self-administration.

RATIONALE: The striatopallidal medium spiny neurons have been viewed as a final common path for drug reward, and the ventral pallidum (VP) as a convergent point for hedonic and motivational signaling. The medium spiny neurons are GABAergic, but they colocalize enkephalin. OBJECTIVE: The present study investigated the role of the GABAergic mechanisms of the VP in ethanol consumption. METHODS: The effects of bilateral microinjections of GABA(A) and GABA(B) receptor agonists and antagonists into the VP on voluntary ethanol consumption were monitored in alcohol-preferring Alko alcohol rats given 90 min limited access to ethanol in their home cages every other day. The influences of coadministration of GABA and opioid receptor modulators were also studied. RESULTS: The GABA(A) receptor agonist muscimol (1-10 ng/site) decreased ethanol intake dose-dependently, while administration of the GABA(A) receptor antagonist bicuculline (10-100 ng) had an opposite effect. The GABA(B) receptor agonist baclofen (3-30 ng) also suppressed ethanol intake, but the GABA(B) receptor antagonist saclofen (0.3-3 µg) failed to modify it. Animals coadministered with bicuculline (30 ng) and baclofen (30 ng) consumed ethanol significantly less than those treated with bicuculline alone. Coadministration of the µ-receptor agonist D-Ala2,N-Me-Phe4,Glyol5-enkephalin (DAMGO, 0.1 µg) with bicuculline counteracted, whereas the µ-receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 1 µg) enhanced the bicuculline-induced increase of ethanol intake. When given alone, DAMGO decreased while CTOP increased ethanol intake. CONCLUSIONS: The study provides evidence for the ventral pallidal GABAergic mechanisms participating in the regulation of ethanol consumption and supports earlier work suggesting a role for pallidal opioidergic transmission in ethanol reward.

Opioidergic modulation of ethanol self-administration in the ventral pallidum.

BACKGROUND: Striatopallidal medium spiny neurons have been viewed as a final common path for drug reward and the ventral pallidum as an essential convergent point for hedonic and motivational signaling in the brain. The medium spiny neurons are GABAergic, but they colocalize enkephalin. Purpose of this study was to investigate the role of the opioidergic mechanisms of the ventral pallidum in ethanol self-administration behavior. METHODS: Effects of bilateral microinjections of µ-, δ-, and κ-opioid receptor agonists and antagonists into the ventral pallidum on voluntary ethanol consumption were monitored in alcohol-preferring Alko Alcohol (AA) rats using the 90-minute limited access paradigm. RESULTS: Stimulation of µ-opioid receptors with DAMGO (0.01 to 0.1 µg) or morphine (1 to 10 µg) in the ventral pallidum decreased ethanol intake dose-dependently. Conversely, blocking µ-receptors with CTOP (0.3 to 3 µg) increased ethanol intake significantly. Unlike CTOP, DAMGO also increased locomotor activity. Consumption of ethanol was not modified significantly by a broad-spectrum opioid receptor antagonist naltrexone, by δ-opioid receptor agonist DPDPE or antagonist naltrindole, or by a κ-opioid receptor agonist U50,488H or antagonist nor-BNI. CONCLUSIONS: The study provides evidence for µ- but not δ- or κ-opioid receptors in the ventral pallidum playing a role in the regulation of voluntary ethanol consumption. Furthermore, present findings give support to earlier work, suggesting an essential role of pallidal opioidergic transmission in drug reward.

GABA and glutamate overflow in the VTA and ventral pallidum of alcohol-preferring AA and alcohol-avoiding ANA rats after ethanol.

AIMS: Earlier findings suggest that dopaminergic neurons are probably not critically involved in ethanol self-administration behavior and in the differential intake of ethanol by the alcohol-preferring AA (Alko Alcohol) and non-preferring ANA (Alko Non-Alcohol) rat lines selected for differential ethanol intake. The purpose of the present study was, therefore, to clarify the role of GABAergic and glutamatergic afferents and efferents with the mesolimbic dopamine system in the control of ethanol intake as well as in differential intake of ethanol by AA and ANA rats. METHODS: The effects of an acute dose of ethanol (1 or 2 g/kg i.p.) on the levels of GABA and glutamate in the ventral pallidum and the ventral tegmental area of AA and ANA rats were monitored with in vivo microdialysis. The concentrations of GABA and glutamate in the dialysates were determined with a high performance liquid chromatography system using fluorescent detection. RESULTS: Ethanol significantly decreased the extracellular levels of GABA in the ventral pallidum but not in the ventral tegmental area. The ANA rats were more sensitive than the AA rats to the suppressive effect of ethanol on pallidal GABA levels. Ethanol did not have any effect on the concentrations of glutamate in either rat line. CONCLUSIONS: The suppressive effect of ethanol on the extracellular levels of GABA in the ventral pallidum suggests a role for pallidal GABAergic transmission in the control of ethanol consumption.