Saturnino Spiga

Acetaldehyde increases dopaminergic neuronal activity in the VTA.

Acetaldehyde is the first and principal metabolite of ethanol administered systemically. To its rise in blood, after administration of disulfiram, is ascribed the aversive reaction that should discourage alcoholics from drinking. In the present study, we sought to determine the effect of acetaldehyde on the electrophysiological properties of dopamine (DA)-containing neurons in the ventro tegmental area (VTA) of rats in vivo. Intravenous (i.v.) administration of acetaldehyde (5–40 mg/kg) readily and dose-dependently increased the firing rate, spikes/burst, and burst firing of VTA neurons. Ethanol (250–1000 mg/kg/i.v.) administration produced similar increments in electrophysiological parameters. In addition, a second group of rats was pretreated with the alcohol-dehydrogenase inhibitor 4-methyl-pyrazole (90 mg/kg) intraperitoneally (i.p.), and ethanol and acetaldehyde were administered i.v. at the same doses, 48 h later. In this group, ethanol effects were drastically reduced and the firing rate, spikes/burst, and burst firing were not significantly altered. In contrast, acetaldehyde fully retained its capacity to stimulate electrophysiological indices. The results indicate that acetaldehyde produces electrophysiological actions on VTA neurons in vivo, similar to those produced by ethanol, and significantly participate in ethanol-induced increment in DA neuronal activity. These results also suggest that acetaldehyde, by increasing DA neuronal activity in the VTA, may significantly contribute to the centrally mediated positive motivational properties of ethanol, which would oppose the well-known peripherally originating aversive properties.

Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-γ agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease

Neuroinflammatory changes play a pivotal role in the progression of Parkinsons disease (PD) pathogenesis. Recent findings have suggested that activated microglia may polarize similarly to peripheral macrophages in the central nervous system (CNS), assuming a pro-inflammatory M1 phenotype or the alternative anti-inflammatory M2 phenotype via cytokine production. A skewed M1 activation over M2 has been related to disease progression in Alzheimer disease, and modulation of microglia polarization may be a therapeutic target for neuroprotection. By using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-probenecid (MPTPp) mouse model of progressive PD, we investigated dynamic changes in the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and anti-inflammatory cytokines, such as transforming growth factor (TGF)-β and IL-10, within Iba-1-positive cells in the substantia nigra compacta (SNc). In addition, to further characterize changes in the M2 phenotype, we measured CD206 in microglia. Moreover, in order to target microglia polarization, we evaluated the effect of the peroxisome-proliferator-activated receptor (PPAR)-γ agonist rosiglitazone, which has been shown to exert neuroprotective effects on nigral dopaminergic neurons in PD models, and acts as a modulator of cytokine production and phenotype in peripheral macrophages. Chronic treatment with MPTPp induced a progressive degeneration of SNc neurons. The neurotoxin treatment was associated with a gradual increase in both TNF-α and IL-1β colocalization with Iba-1-positive cells, suggesting an increase in pro-inflammatory microglia. In contrast, TGF-β colocalization was reduced by the neurotoxin treatment, while IL-10 was mostly unchanged. Administration of rosiglitazone during the full duration of MPTPp treatment reverted both TNF-α and IL-1β colocalization with Iba-1 to control levels. Moreover, rosiglitazone induced an increase in TGF-β and IL-10 colocalization compared with the MPTPp treatment. CD206 was gradually reduced by the chronic MPTPp treatment, while rosiglitazone restored control levels, suggesting that M2 anti-inflammatory microglia were stimulated and inflammatory microglia were inhibited by the neuroprotective treatment. The results show that the dopaminergic degeneration was associated with a gradual microglia polarization to the inflammatory over the anti-inflammatory phenotype in a chronic mouse model of PD. Neuroprotective treatment with rosiglitazone modulated microglia polarization, boosting the M2 over the pro-inflammatory phenotype. PPAR-γ agonists may offer a novel approach to neuroprotection, acting as disease-modifying drugs through an immunomodulatory action in the CNS.

Rosiglitazone decreases peroxisome proliferator receptor-gamma levels in microglia and inhibits TNF-alpha production: new evidences on neuroprotection in a progressive Parkinson's disease model

Thiazolidinedione (TZD) class of peroxisome proliferator receptor gamma (PPAR-γ) agonists display neuroprotective effects in experimental Parkinsons disease (PD) models. Neurons and microglia express PPAR-γ, therefore both of them are potential targets for neuroprotection, although the role of each cell type is not clear. Moreover, receptor-dependent as well as receptor-independent mechanisms have been involved. This study further investigated mechanisms of TZD-mediated neuroprotection in PD. We investigated the rosiglitazone effect in the progressive MPTP/probenecid (MPTPp) model of PD. C57BL/6J mice received MPTP (25 mg/kg) plus probenecid (100 mg/kg) twice per week for 5 weeks. Rosiglitazone (10 mg/kg) was given daily until sacrifice, starting on the fourth week of MPTPp treatment, in presence of an ongoing neurodegeneration with microgliosis. Changes in PPAR-γ levels were measured by immunofluorescence and confocal microscopy in tyrosine hydroxylase (TH)-positive neurons and CD11b-positive microglia of the substantia nigra pars compacta (SNc). Chronic MPTPp treatment induced a PPAR-γ overexpression in both TH-positive neurons and microglia (139.9% and 121.7% over vehicle, respectively). Rosiglitazone administration to MPTPp-treated mice, reverted PPAR-γ overexpression in microglia without affecting TH-positive neurons. Thereafter, changes in CD11b and tumor necrosis factor α (TNF-α) immunoreactivity in microglia were evaluated in the SNc. MPTPp progressively increased CD11b immunoreactivity, conferring to microglia a highly activated morphology. Moreover, TNF-α levels were increased (457.38% over vehicle) after MPTPp. Rosiglitazone administration counteracted the increase in CD11b immunoreactivity caused by MPTPp. Moreover, rosiglitazone reverted TNF-α expression to control levels. Nigrostriatal degeneration was assessed by high pressure liquid chromatography (HPLC) measurement of striatal dopamine, and counting of TH-positive neurons in the SNc. MPTPp treatment caused a severe decline of striatal dopamine and a partial degeneration of the SNc. Rosiglitazone arrested the degenerative process in both areas. Results suggest that PPAR-γ expression in microglia and TNF-α production by these cells are crucial changes by which rosiglitazone exerts neuroprotection in PD.

Astroglial in vivo response to cocaine in mouse dentate gyrus: a quantitative and qualitative analysis by confocal microscopy.

Astrocytes have been proved to play a critical role in neuromodulation, neuroprotection, pH maintenance, axon guidance control during development, homeostasis preservation and blood brain barrier maintenance in the CNS (Kimmelberg and Norenberg, 1989). Quantitative changes in the expression of glial fibrillary acidic protein (GFAP), a cytoskeletal intermediate filament protein exclusively expressed in astrocytes (Bignami et al, 1972), have been observed after administration of alcohol (Framke, 1995), morphine (Beitner-Johnson et al., 1993), amphetamine and its derivates (Aguirre et al., 1999), cannabinoids (Suarez et al., 2000), nicotine (Janson and Moller, 1993), caffeine (Marret et al., 1993) and prenatal exposure to cocaine (Clarke et al., 1996; Nassogne et al., 1998). However, the general astrocytic response to drugs of abuse is still far from being defined. In the present study we examined the in vivo astroglial response to cocaine in mouse dentate gyrus, the hippocampus being a common target of neurotoxic agents (Walsh and Emerich, 1988) which has a prominent effect on learning and memory processes (Eichenbaum et al., 1992). Quantitative changes in immunoreactivity of GFAP were investigated 24 h after acute and repeated daily administration of intraperitoneal cocaine (20 mg/kg). Drug-induced morphological alterations and spatial distribution of astrocytes were evaluated by means of confocal microscope. The results show that, compared to control animals, GFAP expression is two-fold enhanced after a single cocaine injection, still significantly higher after seven consecutive daily administrations, but not statistically different after prolonged (14 days) drug treatment. Moreover, morphological and morphometric analyses reveal significant modifications in astrocytic numbers, cell size and shape complexity. These data demonstrate that in mouse dentate gyrus, cocaine exposure differently affects the expression of GFAP and induces strong changes in astrocytes proliferation rate and cell morphology. Taken together, our findings provide the first in vivo quantitative and qualitative evaluation of astrocytic response to several regimens of cocaine in adult animals brain.

Morphine withdrawal‐induced abnormalities in the VTA: confocal laser scanning microscopy

Morphine withdrawal is characterized by functional alterations at the level of the ventrotegmental area. We investigated the effects of chronic morphine administration and withdrawal on the morphological properties of immuno-labelled tyrosine hydroxylase-positive neurons of the rat ventrotegmental area with a confocal laser scanning microscope. Morphological evaluation revealed a reduction in the area and perimeter of tyrosine hydroxylase-positive somata in morphine-withdrawn rats. Conversely, the number of cells per field was found to have increased in the naloxone group. Collectively, the present results indicate that withdrawal from a chronic morphine treatment, and not chronic morphine per se, modifies cellular morphology of tyrosine hydroxylase-positive, presumably dopamine-containing, neurons of the rat VTA. This is consistent with the idea that withdrawal from morphine alters functioning of the mesolimbic dopamine system and provides a direct morphological correlate for the functional abnormalities typical of morphine withdrawal.

Enduring Effects of Chronic Ethanol in the CNS: Basis for Alcoholism

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.

Morphine withdrawal-induced morphological changes in the nucleus accumbens

Morphine withdrawal produces a hypofunction of mesencephalic dopamine neurons that impinge upon medium spiny neurons (MSN) of the forebrain. After chronic treatment (from 20 to 140 mg/kg of morphine twice a day over 14 days at escalating doses) rats were withdrawn from chronic morphine spontaneously and pharmacologically. In these two distinct conditions we studied the effects of withdrawal on the morphology of MSN of the core and shell of the nucleus accumbens (Nacc). MSN were stained with the Golgi–Cox procedure and analysed by a confocal laser‐scanning microscope (CLSM). Our analysis shows that, shell and core MSN differed significantly for perikarya size and spine density, and the various morphine treatments did not affect the perikarya morphometry. Both spontaneous and naloxone‐induced withdrawal produced a similar reduction in spine density in MS shell neurons, as compared with MS core neurons. This effect is selectively localized at the level of second order dendritic trunks where afferents converge. By contrast, spine density counts of accumbens MSN from rats chronically treated with morphine, did not reveal any change. Collectively, the results of the present study are twofold: (i) spontaneous and pharmacologically precipitated withdrawal, but not chronic morphine per se, affects spine density of target structures of a reduced mesolimbic dopamine transmission, and (ii) the reduction of spine density in second order dendritic trunks is selectively segregated in the MSN of the shell of the Nacc. In conclusion, morphine withdrawal dramatically alters spine density, selectively in second order dendritic trunks of Nacc shell MSN, thereby further impoverishing the already abated dopamine (DA) transmission. This is in line with recent views suggesting the hypodopaminergic state as a cardinal feature of opioid dependence.

Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats

Significance This paper examines the intimate neuroarchitecture of the nucleus accumbens shell region and how it affects synaptic plasticity in alcohol-dependent rats. To do so, a simultaneous morphometrical/immunofluorescence method was applied to visualize various types of dendritic spines and patch-clamp techniques to detect changes in synaptic currents. Using these tools, we show a selective loss of “long thin” spines accompanied by an impaired long-term depression (LTD) in alcohol-dependent rats. Dopaminergic and glutamatergic signaling are similarly altered. The results highlight the role of long thin dendritic spines in the genesis of LTD in alcohol dependence. Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that “long thin” but not “mushroom” spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.

Ethanol-induced extracellular signal regulated kinase: role of dopamine D1 receptors.

BACKGROUND Addictive drugs activate extracellular signal regulated kinase (ERK) in brain regions critically involved in their affective and motivational properties. The aim of this study was to demonstrate the ethanol-induced activation of ERK in the nucleus accumbens (Acb) and in the extended amygdala [bed nucleus of the stria terminalis lateralis (BSTL) and central nucleus of the amygdala (CeA)] and to highlight the role of dopamine (DA) D(1) receptors in these effects. METHODS Ethanol (0.5, 1, and 2 g/kg) was administered by gavage and ERK phosphorylation was determined in the nucleus Acb (shell and core), BSTL, and CeA by immunohistochemistry. The DA D(1) receptor antagonist, SCH 39166 (SCH) (50 microg/kg), was administered 10 minutes before ethanol (1 g/kg). RESULTS Quantitative microscopic examination showed that ethanol, dose-dependently increased phospho-ERK immunoreactivity (optical and neuronal densities) in the shell and core of nucleus Acb, BSTL, and CeA. Pretreatment with SCH fully prevented the increases elicited by ethanol (1 g/kg) in all brain regions studied. CONCLUSIONS The results of this study indicate that ethanol, similar to other addictive drugs, activates ERK in nucleus Acb and extended amygdala via a DA D(1) receptor-mediated mechanism. Overall, these results suggest that the D(1) receptors/ERK pathway may play a critical role in the motivational properties of ethanol.

Differential effects of intravenous R,S-(+/-)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its S(+)- and R(-)-enantiomers on dopamine transmission and extracellular signal regulated kinase phosphorylation (pERK) in the rat nucleus accumbens shell and core

R,S(±)‐3,4‐methylenedioxymethamphetamine (R,S(±)‐MDMA, ‘Ecstasy’) is known to stimulate dopamine (DA) transmission in the nucleus accumbens (NAc). In order to investigate the post‐synaptic correlates of pre‐synaptic changes in DA transmission and their relationship with MDMA enantiomers, we studied the effects of R,S(±)‐MDMA, S(+)‐MDMA, and R(−)‐MDMA on extracellular DA and phosphorylated extracellular signal regulated kinase (pERK) in the NAc shell and core. Male Sprague–Dawley rats, implanted with a catheter in the femoral vein and vertical concentric dialysis probes in the NAc shell and core, were administered i.v. saline, R,S(±)‐MDMA, S(+)‐MDMA, or R(−)‐MDMA. Extracellular DA was monitored by in vivo microdialysis with HPLC. Intravenous R,S(±)‐MDMA (0.64, 1, and 2 mg/kg) increased dialysate DA, preferentially in the shell, in a dose‐related manner. S(+)‐MDMA exerted similar effects but at lower doses than R,S(±)‐MDMA, while R(−)‐MDMA (1 and 2 mg/kg) failed to affect dialysate DA. R,S(±)‐ and S(+)‐MDMA but not R(−)‐MDMA increased ERK phosphorylation (expressed as density/neuron and number of pERK‐positive neurons/area) in both subdivisions of the NAc. The administration of the D1 receptor antagonist, SCH 39166, prevented the increase in pERK elicited by R,S(±)‐MDMA and S(+)‐MDMA, while the D2/3 receptor antagonist, raclopride, increased pERK in the NAc core per se but failed to affect the R,S(±)‐MDMA‐elicited stimulation of pERK. The present results provide evidence that the DA stimulant effects of racemic MDMA are accounted for by the S(+)‐enantiomer and that pERK may represent a post‐synaptic correlate of the stimulant effect of R,S(±)‐MDMA on D1‐dependent DA transmission.