How much was previously known about the molecular mechanisms implicated in binge drinking and what did your recent study reveal?
Alcohol binge drinking is mostly driven by positive reinforcement, a process in which a rewarding experience (e.g., the euphoria one feels when intoxicated) strengthens the behaviour leading to this experience (e.g., going to a bar).
A key neural circuit in the processing of rewarding stimuli by the brain is the mesocorticolimbic dopaminergic pathway, which originates in a midbrain area called the ventral tegmental area (VTA) and projects to two forebrain areas: the ventral striatum and the prefrontal cortex.
The release of dopamine in these target regions signals “incentive salience”, i.e. it contributes to the motivational effects of alcohol-associated cues (e.g., when entering a bar triggers an urge to consume alcohol).
Alcohol, and even the expectation that alcohol is soon going to be available, activates this pathway. However, the exact molecular mechanism underlying the stimulatory effect of alcohol on the activity (“firing”) of VTA neurons is still unknown.
Our study sheds some light on this mechanism by showing that removing one subunit of a neuronal potassium channel (GIRK3) completely blocks the ability of alcohol to activate the mesolimbic dopaminergic pathway.
It is important to keep in mind that the mesocorticolimbic dopaminergic pathway is not the only circuit processing reward in the brain. Other neurotransmitters implicated in the positive reinforcing effects of alcohol include the endogenous opioid peptides, the inhibitory neurotransmitter GABA (γ-amminobutyric acid) and the excitatory neurotransmitter glutamate.
These neurotransmitters act in brain regions such as the VTA, the ventral striatum and the prefrontal cortex, but also in distinct areas, such as the amygdala, the bed nucleus of the stria terminalis or the ventral pallidum.
What is the “G protein-gated inwardly rectifying potassium channel” (GIRK) family and why did you decide to focus on the GIRK3 subunit?
GIRK channels are potassium channels found in excitable cells in the brain (neurons) and in the heart (cardiac myocytes). The opening of these channels lets potassium ions go through, which silences the cell (it lowers the membrane potential, which makes the cell less likely to “fire”).
GIRK channels are tetramers, i.e. they are made up of an assembly of four subunits. There are four types of GIRK subunits but only three of them are found in neurons (GIRK1, GIRK2 and GIRK3), while cardiac myocytes only express GIRK1 and GIRK4.
GIRK channels are normally activated by seven-transmembrane domain receptors coupled to inhibitory G-proteins, such as opioid receptors or GABAB receptors. But they can also be directly activated by alcohol, which binds to a cytoplasmic hydrophobic pocket in GIRK channels.
We were interested in determining whether the action of alcohol on GIRK channels matters for its effects in vivo, i.e. how tipsy we feel or how motivated we are to drink alcohol.
To address this question, we studied mice that are lacking the GIRK3 subunit (GIRK3 knockout mice). We focused on GIRK3 because the deletion of the other two neuronal subunits (GIRK1 and GIRK2) leads to behavioural abnormalities, which would have complicated the interpretation of alcohol-related phenotypes.
GIRK3 knockout mice behave normally in the absence of alcohol, and we sought to determine whether they respond differently to alcohol.
How did you investigate the influence of GIRK3 on mouse behavior and neuronal function in the presence of alcohol? What role did “knockout” mice play?
To assess the ataxic and sedative effects of alcohol, we used tests of motor coordination (rotarod) and sleepiness (righting reflex). We also measured body temperature, as alcohol causes hypothermia. To evaluate the propensity to drink, we used several protocols, which model different drinking patterns.
When mice are given access to alcohol for only few hours per day, at a time of the day when they are most motivated to drink alcohol (three hours into the dark phase of the circadian cycle - mice are nocturnal animals), they drink to the point of intoxication, i.e. they “binge drink”. These limited-access sessions can be likened to a happy hour in a bar.
By contrast, if mice are given continuous access to alcohol throughout the day, they drink alcohol sporadically and never become intoxicated.
To evaluate the effect of alcohol on the mesolimbic dopaminergic pathway, we used two complementary techniques. First, the firing of VTA neurons was measured in brain slices using electrophysiological recordings. Second, the release of dopamine was monitored by microdialysis in awake mice implanted with a probe in the ventral striatum.
GIRK3 knockout mice were tested in all these assays to provide a complete picture of the role of GIRK3 in the in vivo effects alcohol.
We also examined the effect of the opposite manipulation (GIRK3 overexpression) by infusing a viral vector directly into the VTA. Using this procedure, we measuring the impact of reintroducing GIRK3 in the VTA of knockout mice, or expressing higher levels of GIRK3 in the VTA of normal mice, on binge drinking.
What were your main findings and how can they be explained?
We found that the absence of GIRK3 did not impact how fast the mice clear alcohol from their body nor how sensitive they are to alcohol intoxication. Alcohol reduced their motor coordination, made them sleepy and lowered their body temperature to the same extent as in normal mice. GIRK3 knockout mice also drank as much alcohol as normal mice when they were given continuous access to alcohol.
However, under limited-access conditions, which emulate “binge drinking”, GIRK3 knockout mice drank more than normal mice. When we reintroduced GIRK3 in the VTA of GIRK3 knockout mice, their alcohol intake dropped down to normal levels. Increasing the levels of GIRK3 in the VTA of normal mice reduced their alcohol consumption even further.
We concluded that GIRK3 in the VTA keeps binge drinking in check: the more GIRK3, the less binge drinking. We then wanted to understand how GIRK3 controls binge drinking: do the GIRK3 knockout mice drink more because alcohol is more rewarding to them, or because more alcohol is needed for them to experience the same level of reward?
How did you evaluate the two possible explanations?
To answer this question, we measured the activity of VTA neurons in brain slices. Alcohol usually make VTA neurons fire more – but in the absence of GIRK3, these neurons were completely insensitive to alcohol, even at a very high concentration.
We also measured the levels of dopamine in the ventral striatum. Injecting mice with a moderate dose of alcohol usually causes a rise in dopamine levels – but again, GIRK3 knockout mice were completely unresponsive.
What did this evaluation reveal about the reward-seeking pathway?
These results may seem paradoxical. If the canonical “reward pathway” of the brain cannot be activated by alcohol, these mice should not have any motivation to drink alcohol. But, as said above, the mesocorticolimbic dopaminergic pathway is not the only brain circuit responsible for the rewarding properties of alcohol, and we think that GIRK3 knockout mice end up drinking more alcohol to activate alternative circuits more strongly than normal mice would.
Do you think this research is likely to lead to a potential way to reduce alcohol consumption in heavy drinkers?
Our hope is that the effect of GIRK3 could be harnessed to help heavy binge drinkers curb their alcohol consumption, as was done by increasing the levels of GIRK3 in the VTA of mice. However, in order to design a viable therapeutic strategy targeting GIRK3, we still need to understand the consequences of removing or increasing GIRK3 at the subcellular level.
GIRK3 is one of three types of neuronal GIRK subunits, and functional GIRK channels can still form in the absence of GIRK3. Indeed, a study performed in cultured cells had shown that GIRK3 promotes the degradation of GIRK subunits that co-assemble with it, which means that GIRK3 knockout mice could actually have more of the other GIRK subunits.
Another possibility that still needs to be explored is that the incorporation of GIRK3 could enhance or reduce the ability of alcohol to activate GIRK channels.
Finally, an important hurdle is that we have yet to find a compound that can selectively activate GIRK3-containing channels. However, with the recent identification of a potent agonist selective for GIRK1-containing channels, this hurdle may soon be overcome.
What further research is needed to better understand the molecular mechanisms implicated in binge drinking?
The mesocorticolimbic dopaminergic pathway is undoubtedly the focus of numerous, if not most, studies of reward processing. But another take-home message from our work is that there are other brain regions deserving just as much attention if we want to fully understand how the urge to drink alcohol is generated.
It would be foolish to design a therapeutic strategy for heavy binge drinking that would concentrate on the effects of alcohol in the VTA while overlooking the role of alternative neurotransmitters and circuits involved in the rewarding effects of alcohol, such as the opioid peptides or the amygdala.
Where can readers find more information?
The website of the National Institute on Alcohol Abuse and Alcoholism is a great source of information on a wide range of alcohol-related topics (http://www.niaaa.nih.gov/). It contains easy-to-read brochures on the deleterious consequences of drinking, epidemiologic data on alcohol use and abuse, and other fact sheets whose content is intended for a lay audience.
For readers seeking additional educative opportunities, the website also contains peer-reviewed scientific publications addressing the neurobiological mechanisms underlying the effects of alcohol, and information on research programs funded by the agency.
About Dr Candice Contet
Candice Contet, Ph.D., is Assistant Professor at The Scripps Research Institute, La Jolla, CA. She is an NIH-funded investigator with 15 years of experience in the study of drug addiction. She is a member of the Integrative Neuroscience Initiative on Alcoholism (INIA-WEST) consortium funded by NIAAA, and directs the Viral Vector Core of TSRI Alcohol Research Center.
She has a documented track record of investigating the molecular changes elicited by drugs of abuse and their relevance to behavioral adaptations. She has authored twenty-two peer-reviewed publications. She is on the editorial board of the journal Frontiers in Molecular Neuroscience.
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