Roberto Rimondini

Upregulation of Voluntary Alcohol Intake, Behavioral Sensitivity to Stress, and Amygdala Crhr1 Expression Following a History of Dependence

BACKGROUND A history of alcohol dependence recruits increased voluntary alcohol intake and sensitivity to stress. Corticotropin-releasing hormone (CRH) has been implicated in this transition, but underlying molecular mechanisms remain unclear. METHODS A postdependent state was induced using intermittent alcohol exposure. Experiments were carried out following > or =3 weeks of recovery to eliminate contributions of acute withdrawal. Voluntary alcohol consumption was assessed in a two-bottle, free choice procedure. Behavioral sensitivity to stress was examined using fear suppression of behavior in a punished drinking (Vogel) conflict test. Effects of forced swim stress on voluntary alcohol intake were examined as a function of exposure history. Expression of Crh, Crhr1, and Crhr2 transcripts was analyzed by in situ hybridization histochemistry. RESULTS Alcohol drinking was upregulated long-term following a history of dependence. Fear suppression of behavior was selectively potentiated in postdependent animals. This persisted 3 months after alcohol exposure and was reversed by the selective CRH-R1 antagonist 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) (10 mg/kg). Forced swim stress increased alcohol intake in postdependent animals but not in control animals. Behavioral changes were paralleled by an upregulation of Crhr1 transcript expression within basolateral (BLA) and medial (MeA) amygdala and Crh messenger RNA (mRNA) in central amygdala (CeA). In contrast, Crhr2 expression was down in the BLA. CONCLUSIONS Neuroadaptations encompassing amygdala CRH signaling contribute to the behavioral phenotype of postdependent animals.

Genetic Impairment of Frontocortical Endocannabinoid Degradation and High Alcohol Preference

Endocannabinoid signaling has recently been implicated in ethanol-seeking behavior. We analyzed the expression of endocannabinoid-related genes in key brain regions of reward and dependence, and compared them between the alcohol-preferring AA (Alko Alcohol) and nonpreferring ANA (Alko Non-Alcohol) rat lines. A decreased expression of fatty acid amidohydrolase (FAAH), the main endocannabinoid-degrading enzyme, was found in prefrontal cortex (PFC) of AA rats, and was accompanied by decreased enzyme activity in this region. Binding of the endocannabinoid-cannabinoid 1 (CB1) receptor ligand 3[H]SR141716A, and [35S]GTPγS incorporation stimulated by the CB1 agonist WIN 55,212-2 were downregulated in the same area. Together, this suggests an overactive endocannabinoid transmission in the PFC of AA animals, and a compensatory downregulation of CB1 signaling. The functional role of impaired FAAH function for alcohol self-administration was validated in two independent ways. The CB1 antagonist SR141716A potently and dose-dependently suppressed self-administration in AA rats when given systemically, or locally into the PFC, but not in the striatum. Conversely, intra-PFC injections of the competitive FAAH inhibitor URB597 increased ethanol self-administration in nonselected Wistar rats. These results show for the first time that impaired FAAH function may confer a phenotype of high voluntary alcohol intake, and point to a FAAH both as a potential susceptibility factor and a therapeutic target.

Buprenorphine Reduces Alcohol Drinking Through Activation of the Nociceptin/Orphanin FQ-NOP Receptor System

BACKGROUND Activation of the NOP receptor by its endogenous ligand nociceptin/orphanin FQ reduces ethanol intake in genetically selected alcohol preferring Marchigian Sardinian alcohol preferring (msP) rats. Here we evaluated whether buprenorphine, a partial agonist at micro-opioid and NOP receptors, would reduce ethanol consumption in msP rats via activation of NOP receptors. METHODS Marchigian Sardinian alcohol preferring rats trained to drink 10% alcohol 2 hours/day were injected with buprenorphine (.03, .3, 3.0, or 6.0 mg/kg intraperitoneally [IP]) 90 min before access to ethanol. RESULTS Similar to prototypical micro-agonists, the two lowest doses of buprenorphine significantly increased ethanol consumption (p < .01); in contrast, the two highest doses reduced it (p < .05). Pretreatment with naltrexone (.25 mg/kg IP) prevented the increase of ethanol intake induced by .03 mg/kg of buprenorphine (p < .001) but did not affect the inhibition of ethanol drinking induced by 3.0 mg/kg of buprenorphine. Conversely, pretreatment with the selective NOP receptor antagonist UFP-101 (10.0 or 20.0 microg/rat) abolished the suppression of ethanol drinking by 3.0 mg/kg of buprenorphine. CONCLUSIONS Buprenorphine has dualistic effects on ethanol drinking; low doses increase alcohol intake via stimulation of classic opioid receptors, whereas higher doses reduce it via activation of NOP receptors. We suggest that NOP agonistic properties of buprenorphine might be useful in the treatment of alcoholism.

Neuroplasticity in brain reward circuitry following a history of ethanol dependence

Mitogen‐activated and extracellular regulated kinase (MEK) and extracellular signal‐regulated protein kinase (ERK) pathways may underlie ethanol‐induced neuroplasticity. Here, we used the MEK inhibitor 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene (UO126) to probe the role of MEK/ERK signaling for the cellular response to an acute ethanol challenge in rats with or without a history of ethanol dependence. Ethanol (1.5 g/kg, i.p.) induced expression of the marker genes c‐fos and egr‐1 in brain regions associated with both rewarding and stressful ethanol actions. Under non‐dependent conditions, ethanol‐induced c‐fos expression was generally not affected by MEK inhibition, with the exception of the medial amygdala (MeA). In contrast, following a history of dependence, a markedly suppressed c‐fos response to acute ethanol was found in the medial pre‐frontal/orbitofrontal cortex (OFC), nucleus accumbens shell (AcbSh) and paraventricular nucleus (PVN). The suppressed ethanol response in the OFC and AcbSh, key regions involved in ethanol preference and seeking, was restored by pre‐treatment with UO126, demonstrating a recruitment of an ERK/MEK‐mediated inhibitory regulation in the post‐dependent state. Conversely, in brain areas involved in stress responses (MeA and PVN), an MEK/ERK‐mediated cellular activation by acute ethanol was lost following a history of dependence. These data reveal region‐specific neuroadaptations encompassing the MEK/ERK pathway in ethanol dependence. Recruitment of MEK/ERK‐mediated suppression of the ethanol response in the OFC and AcbSh may reflect devaluation of ethanol as a reinforcer, whereas loss of an MEK/ERK‐mediated response in the MeA and PVN may reflect tolerance to its aversive actions. These two neuroadaptations could act in concert to facilitate progression into ethanol dependence.

Long-lasting tolerance to alcohol following a history of dependence.

Tolerance to alcohol effects is one of the defining features of clinical alcohol dependence. Here, we hypothesized that the post‐dependent state may include tolerance to sedative‐hypnotic alcohol actions. To address this question, we used a recently developed animal model in which repeated cycles of alcohol intoxication and withdrawal trigger long‐lasting behavioral plasticity. This animal model shares important features with the clinical condition. Animals were exposed to 7 weeks of intermittent alcohol vapor, allowed to recover for 3 weeks, and tested in protracted abstinence to exclude contributions from acute withdrawal. Post‐dependent and control rats were injected with a hypnotic dose of alcohol (3 g/kg), and the loss of righting reflex (LORR) was recorded, blood alcohol levels were monitored, and the elimination rate was calculated. Post‐dependent animals showed a decrease in LORR. Alcohol metabolism and elimination kinetics did not differ between groups. In conclusion, a history of alcohol dependence induces long‐lasting hypnotic tolerance. This process may play an important role in maintaining the dependent state.

Modulation of voluntary ethanol consumption by beta-arrestin 2

Beta‐arrestin 2 is a multifunctional key component of the G protein‐coupled receptor complex and is involved in μ‐opiate and dopamine D2 receptor signaling, both of which are thought to mediate the rewarding effects of ethanol consumption. We identified elevated expression of the beta‐arrestin 2 gene (Arrb2) in the striatum and the hippocampus of ethanol‐preferring AA rats compared to their nonpreferring counterpart ANA line. Differential mRNA expression was accompanied by different levels of Arrb2 protein. The elevated expression was associated with a 7‐marker haplotype in complete linkage disequilibrium, which segregated fully between the lines, and was unique to the preferring line. Furthermore, a single, distinct, and highly significant quantitative trait locus for Arrb2 expression in hippocampus and striatum was identified at the locus of this gene, providing evidence that genetic variation may affect a cis‐regulatory mechanism for expression and regional control of Arrb2. These findings were functionally validated using mice lacking Arrb2, which displayed both reduced voluntary ethanol consumption and ethanol‐induced psychomotor stimulation. Our results demonstrate that beta‐arrestin 2 modulates acute responses to ethanol and is an important mediator of ethanol reward.—Björk, K., Rimondini, R., Hansson, A. C., Terasmaa, A., Hyytiä, P., Heilig, M., Sommer, W. H. Modulation of voluntary ethanol consumption by beta‐arrestin 2. FASEB J. 22, 2552–2560 (2008)

Olanzapine counteracts stress-induced anxiety-like behavior in rats

Atypical antipsychotics, such as olanzapine, have been reported to display anxiolytic properties as shown in several preclinical and clinical studies. Furthermore, several experimental evidences have shown that olanzapine reduces fear and anxiety in activated anxiety-like behavior test such as Geller-Seifter test, ultrasonic vocalization test and stress-induced EtOH consumption. Here, we hypothesized that the anxiolytic action of olanzapine might be due to via an indirect activation of the gamma-amino butyric acid (GABA)-ergic system through 3alpha-hydroxy-5alpha-pregnan-20-one [allopregnanolone (ALLO)], a potent neuroactive steroid that positively modulates the benzodiazepine-gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptors complex. To address this question, we used a preclinical animal test to screen for novel anxiolytic compounds - the elevated plus-maze (EPM) - in basal condition and after 45 min restrain stress after acute or repeated (21 days) administration of olanzapine (0.5mg/kg, i.p.). In this condition, we therefore study the effect of the 5-alpha-reductase inhibitor finasteride (FIN) (50mg/kg) after co-administration with olanzapine. FIN is an inhibitor of steroidogenic enzymes which acts by inhibiting type II 5-alpha reductase, the enzyme that converts into 5-alpha-reduced metabolites like the GABA(A) positive neuroactive steroid ALLO. Results showed an anxiolytic effect of the acute, but not of the chronic, treatment with olanzapine only in stressed rats. This anxiolytic effect was counteracted by the co-administration with FIN. These evidences suggest that the anxiolytic effects of olanzapine might be due to possible action of olanzapine on steroid function via activation of GABA system.

Intermittent exposure to ethanol vapor affects osteoblast behaviour more severely than estrogen deficiency does: In vitro study on rat osteoblasts

With rising rates of alcohol consumption acute and chronic damage from alcohol is expected to increase all over the world. Habitual excessive alcohol consumption is associated with pathological effects on bone. The aim of the present in vitro study was to investigate comparatively the proliferation and synthetic activity of osteoblasts (OB) isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapor, sham-aged rats and long-term estrogen deficient rats. Cell proliferation (WST1) and synthesis of alkaline phosphatase (ALP), osteocalcin (OC), collagen I (CICP), transforming growth factor beta1 (TGF-beta1), interleukin-6 (IL-6), tumor necrosis factor alfa (TNFalpha) were measured at 3, 7 and 14 days of culture. Osteoblast proliferation rate and TGF-beta1, IL-6 and TNFalpha syntheses were significantly affected by alcohol exposure. Estrogen deficiency and alcohol consumption share many common pathophysiological mechanisms of damage to bone, but alcohol affects OB proliferation and TNFalpha synthesis significantly more than menopause does. Therefore, these in vitro data suggest that alcohol has even more deleterious effects on bone than estrogen deficiency does.

Plasticity and impact of the central renin–angiotensin system during development of ethanol dependence

Pharmacological and genetic interference with the renin–angiotensin system (RAS) seems to alter voluntary ethanol consumption. However, understanding the influence of the RAS on ethanol dependence and its treatment requires modeling the neuroadaptations that occur with prolonged exposure to ethanol. Increased ethanol consumption was induced in rats through repeated cycles of intoxication and withdrawal. Expression of angiotensinogen, angiotensin-converting enzyme, and the angiotensin II receptor, AT1a, was examined by quantitative reverse transcription polymerase chain reaction. Increased ethanol consumption after a history of dependence was associated with increased angiotensinogen expression in medial prefrontal cortex but not in nucleus accumbens or amygdala. Increased angiotensinogen expression also demonstrates that the astroglia is an integral part of the plasticity underlying the development of dependence. The effects of low central RAS activity on increased ethanol consumption were investigated using either spirapril, a blood–brain barrier-penetrating inhibitor of angiotensin-converting enzyme, or transgenic rats (TGR(ASrAOGEN)680) with reduced central angiotensinogen expression. Spirapril reduced ethanol intake in dependent rats compared to controls. After induction of dependence, TGR(ASrAOGEN)680 rats had increased ethanol consumption but to a lesser degree than Wistar rats with the same history of dependence. These data suggest that the central RAS is sensitized in its modulatory control of ethanol consumption in the dependent state, but pharmacological or genetic blockade of the system appears to be insufficient to halt the progression of dependence.

The Tyrosine Kinase Receptor RET Interacts in Vivo with Aryl Hydrocarbon Receptor-Interacting Protein to Alter Survivin Availability

Unita di Genetica Medica (M.V., D.F., I.K., G.R., E.B.), Policlinico Universitario S. Orsola-Malpighi, 40138 Bologna, Italy; Bio3/Bioinformatics and Molecular Genetics (Faculty of Biology) (D.D., R.B.), University of Freiburg, 79085 Freiburg, Germany; Center for Biochemistry and Molecular Cell Research (Faculty of Medicine) (R.B.), and Center for Systems Biology (ZBSA) and FRIAS, School of Life Sciences (LIFENET) (R.B.), Albert-Ludwigs-University Freiburg, D79104 Freiburg, Germany; Department of Histopathology (I.M.), Ospedale Infantile Regina Margherita, 10126 Torino, Italy; Department of Neurosurgery (A.M.), A.S.O. CTO-CRF-M.Adelaide, 10131 Torino, Italy; and Department of Pharmacology (R.R.), University of Bologna, 40126 Bologna, Italy