Paolo Enrico

The role of afferents to the ventral tegmental area in the handling stress-induced increase in the release of dopamine in the medial prefrontal cortex: a dual-probe microdialysis study in the rat brain

This study was aimed to identify the neuronal pathways that mediate the handling stress induced increase in the release of dopamine in the medial prefrontal cortex (mPFC) of the rat brain. For that purpose a microdialysis probe was implanted in the ventral tegmental area (VTA) and a second probe was placed in the ipsilateral mPFC. Receptor specific compounds acting on GABA(A) (20 microM muscimol), GABA(B) (50 microM baclofen), acetylcholine (100 microM atropine, 100 microM mecamylamine), NMDA (30, 100 and 300 microM CPP; 300 microM AP-5, 1 mM (+)-HA-966) and non-NMDA receptors (500 microM CNQX) were infused into the VTA by retrograde dialysis, whereas extracellular dopamine was recorded in the ipsilateral mPFC. Intrategmental infusion of muscimol, baclofen, CPP, AP-5, (+)-HA-966 and CNQX decreased extracellular dopamine in the ipsilateral mPFC; atropine and mecamylamine were without effect on the basal values. During infusion of the various compounds rats were gently handled for 15 min. The infusions of muscimol, atropine, mecamylamine and (+)-HA-966 did not modify the handling stress induced increase in extracellular dopamine in the mPFC. However, during intrategmental infusion of baclofen, CPP, AP-5 and CNQX the handling stress induced increase in extracellular dopamine (expressed as % of controls) in the mPFC was suppressed. These results indicate that a glutamatergic projection to the VTA, acting via both NMDA and non-NMDA-glutamate receptors, play a major role in the handling stress-induced increase in dopamine release in the mPFC. In addition the results suggest a certain role for GABAergic neurones, acting via GABA(B) receptors, in the handling response.

Acetaldehyde mediates alcohol activation of the mesolimbic dopamine system

Ethanol (EtOH), the main psychoactive ingredient of alcoholic drinks, is widely considered to be responsible for alcohol abuse and alcoholism through its positive motivational properties, which depend, at least partially, on the activation of the mesolimbic dopaminergic system. However, acetaldehyde (ACD), the first metabolite of EtOH, has been classically considered to be aversive and useful in the pharmacological therapy of alcoholics. Here we show that EtOH‐derived ACD is necessary for EtOH‐induced place preference, a pre‐clinical test with high predictive validity for reward liability. We also found that ACD is essential for EtOH‐increased microdialysate dopamine (DA) levels in the rat nucleus accumbens and that this effect is mimicked by intra‐ventral tegmental area (VTA) ACD administration. Furthermore, in vitro, ACD enhances VTA DA neuronal firing through action on two ionic currents: reduction of the A‐type K+ current and activation of the hyperpolarization‐activated inward current. EtOH‐stimulating properties on DA neurons are prevented by pharmacological blockade of local catalase, the main metabolic step for biotransformation of EtOH into ACD in the central nervous system. These results provide in‐vivo and in‐vitro evidence for a key role of ACD in the motivational properties of EtOH and its activation of the mesolimbic DA system. Additionally, these observations suggest that ACD, by increasing VTA DA neuronal activity, would oppose its well‐known peripherally originating aversive properties. Careful consideration of these findings could help in devising new effective pharmacological therapies aimed at reducing EtOH intake in alcoholics.

Acetaldehyde sequestering prevents ethanol-induced stimulation of mesolimbic dopamine transmission.

Acetaldehyde (ACD) has been postulated to mediate some of the neurobehavioral effects of ethanol (EtOH). In this study we sought to evaluate whether the stimulatory effects of EtOH on mesolimbic dopamine (DA) transmission are affected by the administration of ACD-sequestering agent D-penicillamine (Dp). To this end we studied the effect of EtOH and ACD in the rat mesoaccumbens pathway by in vivo microdialysis in the nucleus accumbens shell (NAccs), and by single cell extracellular recordings from antidromically identified mesoaccumbens DA neurons in the ventral tegmental area (VTA). Both EtOH (1g/kg) and ACD (20mg/kg) administration increased DA levels in the NAccs and increased the activity of mesoaccumbens DA neurons. Pretreatment with Dp (50mg/kg i.p. 1h before drug challenge) prevented both EtOH- and ACD-induced stimulation of the DA mesolimbic system without affecting morphine stimulatory actions. These observations add further support to the notion that EtOH-derived ACD stimulates the mesolimbic DA system and is essential in EtOH-induced stimulation of the DA mesoaccumbens system. We conclude that modulation of ACD bioavailability may influence the addictive profile of EtOH by decreasing its psychotropic effects and possibly leading the way to new pharmacological treatments of alcoholism.

On the mechanism of d-amphetamine-induced changes in glutamate, ascorbic acid and uric acid release in the striatum of freely moving rats

The effects of systemic, intrastriatal or intranigral administration of d‐amphetamine on glutamate, aspartate, ascorbic acid (AA), uric acid, dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations in dialysates from the striatum of freely‐moving rats were evaluated using microdialysis. d‐Amphetamine (2 mg kg−1) given subcutaneously (s.c.) increased DA, AA and uric acid and decreased DOPAC+HVA, glutamate and aspartate dialysate concentrations over a 3 h period after d‐amphetamine. 5‐HIAA concentrations were unaffected. Individual changes in glutamate and AA dialysate concentrations were negatively correlated. d‐Amphetamine (0.2 mM), given intrastriatally, increased DA and decreased DOPAC+HVA and aspartate dialysate concentrations, but failed to change those of glutamate, AA uric acid or 5‐HIAA, over a 2 h period after d‐amphetamine. Haloperidol (0.1 mM), given intrastriatally, increased aspartate concentrations without affecting those of glutamate or AA. d‐Amphetamine (0.2 mM), given intranigrally, increased AA and uric acid dialysate concentrations and decreased those of glutamate, aspartate and DA; DOPAC+HVA and 5‐HIAA concentrations were unaffected. These results suggest that d‐amphetamine‐induced increases in AA and uric acid and decreases in glutamate concentrations are triggered at nigral sites. The changes in aspartate levels may be evoked by at least two mechanisms: striatal (mediated by inhibitory dopaminergic receptors) and nigral (activation of amino acid carrier‐mediated uptake).

Effect of naloxone on morphine-induced changes in striatal dopamine metabolism and glutamate, ascorbic acid and uric acid release in freely moving rats

Recent findings have shown that systemic morphine increases extracellular dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), ascorbic acid (AA) and uric acid concentrations in the striatum of freely moving rats. The morphine-induced increase in DA oxidative metabolism is highly correlated with that of xanthine. In the present study, we evaluated the effects of subcutaneous (s.c.) naloxone (1 mg/kg) on morphine-induced changes in DA, DOPAC, HVA, 5-hydroxyindoleacetic acid (5-HIAA), AA, uric acid and glutamate in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection or (glutamate) ultraviolet detection. Morphine (5-20 mg/kg) given s.c. increased DA, DOPAC+HVA, 5-HIAA, AA and uric acid and decreased glutamate dialysate concentrations over a 3 h period after morphine. Morphine (1 mM), given intrastriatally, did not affect all the above parameters, with the exception of an early short-lasting decrease in AA concentration. Naloxone antagonised all morphine-induced changes with the exception of AA increase and glutamate decrease in dialysate concentrations. Systemic or intrastrial (0.2-2 mM) naloxone increased AA and decreased glutamate dialysate concentrations. When given intranigrally, morphine (1 mM) increased DOPAC+HVA, AA and uric acid and decreased glutamate dialysate concentrations over a 2 h period after morphine; DA and 5-HIAA concentrations were unaffected. These results suggest that: (i) morphine increases striatal DA release and 5-hydroxytryptamine oxidative metabolism by a micro-opioid receptor-mediated mechanism mainly at extranigrostriatal sites; (ii) morphine increases DA and xanthine oxidative metabolism and affects glutamate and AA release by a micro-opioid receptor mediated mechanism acting also at nigral sites; and (iii) a micro-opioid receptor-mediated mechanism tonically controls at striatal sites extracellular AA and glutamate concentrations.

Manganese increases L‐DOPA auto‐oxidation in the striatum of the freely moving rat: potential implications to L‐DOPA long‐term therapy of Parkinson's disease

We have previously shown that manganese enhances L‐dihydroxyphenylanine (L‐DOPA) toxicity to PC12 cells in vitro. The supposed mechanism of manganese enhancing effect [an increase in L‐DOPA and dopamine (DA) auto‐oxidation] was studied using microdialysis in the striatum of freely moving rats. Systemic L‐DOPA [25 mg kg−1 intraperitoneally (i.p.) twice in a 12 h interval] significantly increased baseline dialysate concentrations of L‐DOPA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid, compared to controls. Conversely, DA and ascorbic acid concentrations were significantly decreased. A L‐DOPA oxidation product, presumptively identified as L‐DOPA semiquinone, was detected in the dialysate. The L‐DOPA semiquinone was detected also following intrastriatal infusion of L‐DOPA. In rats given L‐DOPA i.p., intrastriatal infusion of N‐acetylcysteine (NAC) significantly increased DA and L‐DOPA dialysate concentrations and lowered those of L‐DOPA semiquinone; in addition, NAC decreased DOPAC+HVA and uric acid dialysate concentrations. In rats given L‐DOPA either systemically or intrastriatally, intrastriatal infusion of manganese decreased L‐DOPA dialysate concentrations and greatly increased those of L‐DOPA semiquinone. These changes were inhibited by NAC infusion. These findings demonstrate that auto‐oxidation of exogenous L‐DOPA occurs in vivo in the rat striatum. The consequent reactive oxygen species generation may account for the decrease in dialysate DA and ascorbic acid concentrations and increase in enzymatic oxidation of xanthine and DA. L‐DOPA auto‐oxidation is inhibited by NAC and enhanced by manganese. These results may be of relevance to the L‐DOPA long‐term therapy of Parkinsons disease.

Ethanol and acetaldehyde action on central dopamine systems: Mechanisms, modulation and relationship to stress

There has been a great deal of activity in recent years in the study of the direct effects of ethanol on the dopamine reward system originating in the ventral tegmental area (VTA). In addition, recent evidence suggests that acetaldehyde formed from ethanol in the brain or periphery may be a crucial factor in the central effects of ethanol. This critical review examines the actions of ethanol and acetaldehyde on neurons of the VTA and the possible interactions with stress, with a focus on electrophysiological studies in vivo and in vitro. Ethanol has specific effects on dopamine neurons and there is recent evidence that some of the in vivo and in vitro effects of ethanol are mediated by acetaldehyde. Stress has some analogous actions on neuronal activity in the VTA, and the interactions between the effects of stress and alcohol on VTA neurons may be a factor in ethanol-seeking behavior. Taken together, the evidence suggests that stress may contribute to the activating effects of ethanol on dopamine VTA neurons, that at least some actions of ethanol on dopamine VTA neurons are mediated by acetaldehyde, and that the interaction between stress and alcohol could play a role in susceptibility to alcoholism. The link between acetaldehyde and ethanol actions on brain reward pathways may provide a new avenue for the development of agents to reduce alcohol craving.

Correlation between 1-methyl-4-phenylpyridinium ion (MPP+) levels, ascorbic acid oxidation and glutathione levels in the striatal synaptosomes of the 1-methyl-4-phenyl-1,2,3-6-tetrahydropyridine (MPTP)-treated rat

In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the crude striatal synaptosomal fraction after single injections of MPTP 35 mg/kg i.p. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced a 32.5% death rate within 15 min to 10 h. Groups of surviving rats were sacrificed 1, 3, 8 and 24 h after MPTP. MPTP significantly increased levels of DHAA and uric acid and decreased levels of DOPAC and GSH. Individual synaptosomal levels of MPP+ were correlated inversely with DOPAC (r = -0.601, P < 0.002) and GSH levels (r = -0.496, P < 0.02) and directly with levels of uric acid (r = +0.627, P < 0.001); these latter, in turn, were correlated with DHAA (r = +0.418, P < 0.05) and GSH levels (r = -0.357, P = 0.07). In conclusion, the response of the endogenous antioxidant system (increase in AA oxidation, decrease in GSH levels) correlates well with the MPTP-induced increase in uric acid levels and provides further evidence for a mechanism of MPTP neurotoxicity involving oxidative stress produced by xanthine oxidase.

Crucial role of acetaldehyde in alcohol activation of the mesolimbic dopamine system.

Ethyl alcohol (EtOH), the main psychoactive ingredient of alcoholic drinks, is widely considered responsible for alcohol abuse and alcoholism through its positive motivational properties, which depend, at least partially, on the activation of the mesolimbic dopaminergic system. On the other hand, acetaldehyde (ACD), EtOHs first metabolite, has been classically considered aversive and useful in the pharmacologic therapy of alcoholics. Here we show that EtOH‐derived ACD is necessary for EtOH‐induced place preference, a preclinical test with high predictive validity for reward liability. We also found that ACD is essential for EtOH‐increased microdialysate dopamine (DA) levels in the nucleus accumbens (NAcc), and that this effect is mimicked by ACD administration to the intraventral tegmental area (VTA). Furthermore, in vitro, ACD enhances VTA DA neuronal firing. Coherently, EtOH‐stimulating properties on DA neurons are prevented by pharmacologic blockade of local catalase: the main metabolic step for biotransformation of EtOH into ACD in the central nervous system. These results provide in vivo and in vitro evidence for a key role of ACD in EtOH motivational properties and its activation of the mesolimbic DA system. Additionally, these observations suggest that ACD, by increasing VTA DA neuronal activity, would oppose its well‐known peripherally originating aversive properties. These findings could help in devising new effective pharmacologic therapies in alcoholism.

Effect of morphine on striatal dopamine metabolism and ascorbic and uric acid release in freely moving rats

Recent ex vivo findings have shown that morphine increases dopamine (DA) and xanthine oxidative metabolism and ascorbic acid (AA) oxidation in the rat striatum. In the present study, we evaluated the effects of subcutaneous daily morphine (20 mg/kg) administration on DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), AA and uric acid in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection. On the first day, morphine administration caused a significant increase in extracellular DA, DOPAC, HVA, AA and uric acid concentrations over a 3 h period after morphine. In all treated rats (n = 7), individual concentrations of DOPAC + HVA were directly correlated with individual AA and uric acid concentrations. Last morphine administration on the 4th day increased DOPAC, HVA, AA and uric acid concentrations but failed to increase those of DA. Individual DOPAC + HVA concentrations were still directly correlated with individual AA and uric acid concentrations. These results suggest that systemic morphine increases both striatal DA release and DA and xanthine oxidative metabolism. Only the former effect undergoes tolerance. The increase in DA oxidative metabolism is highly correlated with that of xanthine. The subsequent enhancement in reactive oxygen species production may account for the increase in extracellular AA.