Neuroimaging reveals functionally distinct neuronal networks associated with high-level alcohol consumption in two genetic rat models.

Human imaging data suggest that the motivational processes associated with alcohol reward are reflected in the patterns of neural activation after alcohol or alcohol-related cues. In animal models of alcohol drinking, however, the changes in brain activation during voluntary alcohol ingestion are poorly known. In order to improve the translational utility of animal models, we examined alcohol-induced functional brain activation in Alko Alcohol (AA) and Marchigian-Sardinian alcohol-preferring (msP) rats that drink voluntarily high levels of alcohol, but exhibit widely different neurochemical and behavioral traits cosegregated with alcohol preference. Brain imaging was performed using manganese-enhanced MRI (MEMRI), which is based on accumulation of Mn2+ ions in activated neurons, allowing the identification of functional neuronal networks recruited during specific behaviors in awake animals during a subsequent imaging session under anesthesia. MEMRI was performed following 4 weeks of voluntary alcohol drinking, using water drinking as the control. Despite similar levels of alcohol drinking, strikingly different alcohol-induced neuronal activity patterns were observed in AA and msP rats. Overall, functional activation in the AA rats was more widespread, involving large cortical areas and subcortical structures, such as the bed nucleus of the stria terminalis, preoptic area, hypothalamus, periaqueductal grey, and substantia nigra. In the msP rats, however, alcohol-related activation was largely confined to prefrontal cortical regions and insular cortex, and olfactory areas. Overlapping areas of activation found in both rat lines included the nucleus accumbens, prelimbic, orbital, and insular cortex. In conclusion, our data reveal strikingly different brain circuits associated with alcohol drinking in two genetically different rat lines and suggest innately different motivational and behavioral processes driving alcohol drinking. These findings have important implications for the use of these lines in translational alcohol research.

Continuous delivery of naltrexone and nalmefene leads to tolerance in reducing alcohol drinking and to supersensitivity of brain opioid receptors.

Opioid antagonist treatments reduce alcohol drinking in rodent models and in alcohol-dependent patients, with variable efficacy across different studies. These treatments may suffer from the development of tolerance and opioid receptor supersensitivity, as suggested by preclinical models showing activation of these processes during and after subchronic high-dose administration of the short-acting opioid antagonist naloxone. In the present study, we compared equipotent low and moderate daily doses of naltrexone and nalmefene, two opioid antagonists in the clinical practice for treatment of alcoholism. The antagonists were given here subcutaneously for 7 days either as daily injections or continuous osmotic minipump-driven infusions to alcohol-preferring AA rats having trained to drink 10% alcohol in a limited access protocol. One day after stopping the antagonist treatment, [35 S]GTPγS autoradiography on brain cryostat sections was carried out to examine the coupling of receptors to G protein activation. The results prove the efficacy of repeated injections over infused opioid antagonists in reducing alcohol drinking. Tolerance to the reducing effect on alcohol drinking and to the enhancement of G protein coupling to µ-opioid receptors in various brain regions were consistently detected only after infused antagonists. Supersensitivity of κ-opioid receptors was seen in the ventral and dorsal striatal regions especially by infused nalmefene. Nalmefene showed no clear agonistic activity in rat brain sections or at human recombinant κ-opioid receptors. The findings support the as-needed dosing practice, rather than the standard continual dosing, in the treatment of alcoholism with opioid receptor antagonists.

Alcohol preference and consumption are controlled by the caudal linear nucleus in alcohol-preferring rats.

The neuroanatomical and neurochemical basis of alcohol drinking has been extensively studied, but the neural circuitry mediating alcohol reinforcement has not been fully delineated. In the present experiments, we used both neuroimaging and pharmacological tools to identify neural systems associated with alcohol preference and high voluntary alcohol drinking in alcohol-preferring AA (Alko Alcohol) rats. First, we compared the basal brain activity of AA rats with that of heterogeneous Wistar rats with manganese-enhanced magnetic resonance imaging (MEMRI). Briefly, alcohol-naïve rats were implanted with subcutaneous osmotic minipumps delivering 120 mg/kg MnCl2 over a 7-day period, and were then imaged using a three-dimensional rapid acquisition-relaxation enhanced pulse sequence. MEMRI analysis revealed that the most conspicuous subcortical activation difference was located in the caudal linear nucleus of raphe (CLi), with AA rats displaying significantly lower T1 signal in this region compared to Wistar rats. However, following long-term alcohol drinking, CLi activity was increased in AA rats. In the second experiment, the CLi was targeted with pharmacological tools. AA rats trained to drink 10% alcohol during 2-h sessions were implanted with guide cannulas aimed at the CLi and were given injections of the GABAA receptor agonist muscimol into the CLi before drinking sessions. Muscimol dose-dependently increased alcohol drinking, and co-administration of the gamma aminobutyric acid (GABA)A antagonist bicuculline blocked muscimol's effect. These findings suggest that the mediocaudal region of the ventral tegmental area, particularly the CLi, is important for the propensity for high alcohol drinking and controls alcohol reward via GABAergic transmission.

Brain activation induced by voluntary alcohol and saccharin drinking in rats assessed with manganese-enhanced magnetic resonance imaging.

The neuroanatomical and neurochemical basis of alcohol reward has been studied extensively, but global alterations of neural activity in reward circuits during chronic alcohol use remain poorly described. Here, we measured brain activity changes produced by long-term voluntary alcohol drinking in the alcohol-preferring AA (Alko alcohol) rats using manganese-enhanced magnetic resonance imaging (MEMRI). MEMRI is based on the ability of paramagnetic manganese ions to accumulate in excitable neurons and thereby enhance the T1-weighted signal in activated brain areas. Following 6 weeks of voluntary alcohol drinking, AA rats were allowed to drink alcohol for an additional week, during which they were administered manganese chloride (MnCl2 ) with subcutaneous osmotic minipumps before MEMRI. A second group with an identical alcohol drinking history received MnCl2 during the abstinence week following alcohol drinking. For comparing alcohol with a natural reinforcer, MEMRI was also performed in saccharin-drinking rats. A water-drinking group receiving MnCl2 served as a control. We found that alcohol drinking increased brain activity extensively in cortical and subcortical areas, including the mesocorticolimbic and nigrostriatal dopamine pathways and their afferents. Remarkably similar activation maps were seen after saccharin ingestion. Particularly in the prelimbic cortex, ventral hippocampus and subthalamic nucleus, activation persisted into early abstinence. These data show that voluntary alcohol recruits an extensive network that includes the ascending dopamine systems and their afferent connections, and that this network is largely shared with saccharin reward. The regions displaying persistent alterations after alcohol drinking could participate in brain networks underlying alcohol seeking and relapse.

Manganese-enhanced magnetic resonance imaging reveals differential long-term neuroadaptation after methamphetamine and the substituted cathinone 4-methylmethcathinone (mephedrone).

BACKGROUND: In recent years there has been a large increase in the use of substituted cathinones such as mephedrone (4-methylmethcathinone, 4-MMC), a psychostimulant drug that shows a strong resemblance to methamphetamine (METH). Unlike METH, which can produce clear long-term effects, the effects of 4-MMC have so far remained elusive. We employ manganese-enhanced magnetic resonance imaging (MEMRI), a highly sensitive method for detecting changes in neuronal activation, to investigate the effects of METH and 4-MMC on the brain. METHODS: In Wistar rats we performed a MEMRI scan two weeks after binge treatments (twice daily for 4 consecutive days) of METH (5 mg/kg) or 4-MMC (30 mg/kg). Furthermore, locomotor activity measurements and novel object recognition tests were performed. RESULTS: METH produced a widespread pattern of decreased bilateral activity in several regions, including the nucleus accumbens, caudate putamen, globus pallidus, thalamus, and hippocampus, as well as several other cortical and subcortical areas. Conversely, 4-MMC produced increased bilateral activity, anatomically limited to the hypothalamus and hippocampus. Drug treatments did not affect the development of locomotor sensitization or novel object recognition performance. CONCLUSIONS: The pattern of decreased brain activity seen after METH corresponds closely to regions known to be affected by this drug and confirms the validity of MEMRI for detecting neuroadaptation two weeks after amphetamine binge treatment. 4-MMC, unlike METH, produced increased activity in a limited number of different brain regions. This highlights an important difference in the long-term effects of these drugs on neural function and shows precisely the anatomical localization of 4-MMC-induced neuroadaptation.

Alcohol Co-Administration Changes Mephedrone-Induced Alterations of Neuronal Activity.

Mephedrone (4-MMC), despite its illegal status, is still a widely used psychoactive substance. Its effects closely mimic those of the classical stimulant drug methamphetamine (METH). Recent research suggests that unlike METH, 4-MMC is not neurotoxic on its own. However, the neurotoxic effects of 4-MMC may be precipitated under certain circumstances, such as administration at high ambient temperatures. Common use of 4-MMC in conjunction with alcohol raises the question whether this co-consumption could also precipitate neurotoxicity. A total of six groups of adolescent rats were treated twice daily for four consecutive days with vehicle, METH (5 mg/kg) or 4-MMC (30 mg/kg), with or without ethanol (1.5 g/kg). To investigate persistent delayed effects of the administrations at two weeks after the final treatments, manganese-enhanced magnetic resonance imaging brain scans were performed. Following the scans, brains were collected for Golgi staining and spine analysis. 4-MMC alone had only subtle effects on neuronal activity. When administered with ethanol, it produced a widespread pattern of deactivation, similar to what was seen with METH-treated rats. These effects were most profound in brain regions which are known to have high dopamine and serotonin activities including hippocampus, nucleus accumbens and caudate-putamen. In the regions showing the strongest activation changes, no morphological changes were observed in spine analysis. By itself 4-MMC showed few long-term effects. However, when co-administered with ethanol, the apparent functional adaptations were profound and comparable to those of neurotoxic METH.

Brain activation induced by chronic psychosocial stress in mice.

Chronic psychosocial stress is a well-established risk factor for neuropsychiatric diseases. Abnormalities in brain activity have been demonstrated in patients with stress-related disorders. Global brain activation patterns during chronic stress exposure are less well understood but may have strong modifying effects on specific brain circuits and thereby influence development of stress-related pathologies. We determined neural activation induced by chronic social defeat stress, a mouse model of psychosocial stress. To assess chronic activation with an unbiased brain-wide focus we used manganese-enhanced magnetic resonance imaging (MEMRI) and immunohistochemical staining of ∆FOSB, a transcription factor induced by repeated neural activity. One week after 10-day social defeat we observed significantly more activation in several brain regions known to regulate depressive and anxiety-like behaviour, including the prefrontal cortex, bed nucleus of stria terminalis, ventral hippocampus and periaqueductal grey in stressed compared to control mice. We further established that the correlation of ∆FOSB positive cells between specific brain regions was altered following chronic social defeat. Chronic activation of these neural circuits may relate to persistent brain activity changes occurring during chronic psychosocial stress exposure, with potential relevance for the development of anxiety and depression in humans.

Modulation of nucleus accumbens connectivity by alcohol drinking and naltrexone in alcohol-preferring rats: A manganese-enhanced magnetic resonance imaging study.

The nonselective opioid receptor antagonist naltrexone is now used for the treatment of alcoholism, yet naltrexone's central mechanism of action remains poorly understood. One line of evidence suggests that opioid antagonists regulate alcohol drinking through interaction with the mesolimbic dopamine system. Hence, our goal here was to examine the role of the nucleus accumbens connectivity in alcohol reinforcement and naltrexone's actions using manganese-enhanced magnetic resonance imaging (MEMRI). Following long-term free-choice drinking of alcohol and water, AA (Alko Alcohol) rats received injections of MnCl2 into the nucleus accumbens for activity-dependent tracing of accumbal connections. Immediately after the accumbal injections, rats were imaged using MEMRI, and then allowed to drink either alcohol or water for the next 24h. Naltrexone was administered prior to the active dark period, and the second MEMRI was performed 24h after the first scan. Comparison of signal intensity at 1 and 24h after accumbal MnCl2 injections revealed an ipsilateral continuum through the ventral pallidum, bed nucleus of the stria terminalis, globus pallidus, and lateral hypothalamus to the substantia nigra and ventral tegmental area. Activation was also seen in the rostral part of the insular cortex and regions of the prefrontal cortex. Alcohol drinking resulted in enhanced activation of these connections, whereas naltrexone suppressed alcohol-induced activity. These data support the involvement of the accumbal connections in alcohol reinforcement and mediation of naltrexone's suppressive effects on alcohol drinking through their deactivation.