Molecular mechanisms underlying alcohol-drinking behaviours

Abstract

The main characteristic of alcohol use disorder is the consumption of large quantities of alcohol despite the negative consequences. The transition from the moderate use of alcohol to excessive, uncontrolled alcohol consumption results from neuroadaptations that cause aberrant motivational learning and memory processes. Here, we examine studies that have combined molecular and behavioural approaches in rodents to elucidate the molecular mechanisms that keep the social intake of alcohol in check, which we term 'stop pathways', and the neuroadaptations that underlie the transition from moderate to uncontrolled, excessive alcohol intake, which we term 'go pathways'. We also discuss post-transcriptional, genetic and epigenetic alterations that underlie both types of pathways.

Figure 1. Signalling pathways underlying the go pathways

Repeated cycles of excessive alcohol exposure and withdrawal lead to aberrant activation of specific intracellular signalling cascades (the go pathways), the nature of which varies depending on the brain region. Collectively, these alcohol-related changes in intracellular signalling cascades drive long-lasting, detrimental behavioural phenotypes associated with alcohol abuse, such as excessive consumption, alcohol seeking, craving and relapse. Protein kinase A (PKA) has a central role in the go pathways. PKA is activated by adenylyl cyclase (AC), which hydrolyses ATP to cyclic AMP and is activated by alcohol through several mechanisms, including the inhibition of equilibrative nucleoside transporter 1 (ENT1) (inactivated proteins are shown in dark green) and subsequent activation of the Gsα-coupled adenosine A2A receptors (A2ARs) and/or the activation of the Gsα-coupled dopamine D1 receptor (D1R). cAMP binds to the regulatory subunit of PKA, thus freeing the catalytic subunit of the kinase to phosphorylate its substrates, which include striatum-enriched protein-tyrosine phosphatase (STEP). Activation of FYN requires the recruitment of protein-tyrosine phosphatase-α (PTPα), which dephosphorylates an inhibitory phosphorylation site. STEP is inactivated by PKA phosphorylation, which enables the sustained activation of FYN. Alcohol might also activate FYN by additional mechanisms (as indicated by the dashed line). When FYN is activated, it phosphorylates the GluN2B subunit of NMDA-type glutamate receptors (NMDARs), resulting in enhancement of NMDAR activity. Calcium entry via the NMDARs activates calcium/calmodulin-dependent protein kinase type II (CaMKII), resulting in autophosphorylation of the kinase. CaMKII phosphorylates the AMPA-type glutamate receptor (AMPAR) subunit GluA2, resulting in forward trafficking of these receptors to the synaptic membrane. Another target of PKA is RAS-specific guanine nucleotide-releasing factor 1 (RAS-GRF1), which, when activated, promotes the transition of the small GTP-binding proteins HRAS and KRAS from inactive GDP-bound forms to active GTP-bound forms. Activation of HRAS leads to the activation of phosphoinositide 3-kinase (PI3K). PI3K, in turn, activates AKT, which phosphorylates glycogen synthase kinase 3β (GSK3β), thus inhibiting its activity. When GSK3β activity is reduced, collapsin response mediator protein 2 (CRMP2) binds to tubulin, enabling microtubule assembly. AKT, through intermediate proteins, also activates mechanistic target of rapamycin complex 1 (mTORC1) (as indicated by the dashed line). mTORC1 phosphorylates its substrates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase (S6K), which then phosphorylates its substrate S6. 4E-BP1, S6K and S6 are part of the ribosomal translational machinery, and activation of mTORC1 initiates the translation of postsynaptic density protein 95 (PSD95), Homer, CRMP2 and GluA1. HRAS also activates mitogen-activated protein kinase kinase 1 (MKK1), which in turn activates extracellular signal-regulated kinases 1 and 2 (ERK1/2), inducing gene transcription. Moreover, HRAS indirectly activates (as indicated by the dashed line) phospholipase Cγ (PLCγ), which in turn activates protein kinase Cε (PKCε).

Figure 2. Signalling pathways underlying the stop pathways

Moderate, but not excessive, alcohol consumption causes activation of intracellular signalling cascades that lead to the expression of genes encoding growth factors or neuropeptides. These signalling molecules have a protective role by promoting downstream cascades that prevent the dominance of the go pathway and the establishment of detrimental behavioural phenotypes. Moderate intake of alcohol increases the levels of brain-derived neurotrophic factor (Bdnf) mRNA. Binding of BDNF to tropomyosin-related kinase B (TRKB) activates extracellular signal-regulated kinase 1 and 2 (ERK1/2) signalling, thus promoting the expression of Drd3 (which encodes the dopamine D3 receptor) and Pdyn (which encodes preprodynorphin). Similarly, binding of glial cell line-derived neurotrophic factor (GDNF) to its receptors RET and GDNF family receptor α1 (GFRα1) results in the activation of ERK1/2 and the induction of the transcription of Gdnf. Activated protein kinase A (PKA) phosphorylates the transcription factor cyclic AMP response element (CRE)-binding protein (CREB). PKA-mediated phosphorylation of CREB results in the activation of the transcription machinery and in the induction of expression of genes, including neuropeptide Y (Npy). Activity of PKA is terminated by 3′,5′-cyclic nucleotide phosphodiesterase (PDE), which hydrolyses cAMP to 5′ AMP. Alcohol-induced inhibition of PDE activity enables the sustained activation of PKA. Activity of HRAS and KRAS is terminated by GTPase-activating proteins, including neurofibromin (NF1). Circadian locomoter output cycles protein kaput (CLOCK) drives the transcription of Per1 and Per2, which encode the period proteins. PER1 and PER2 suppress the transcription of Clock. The casein-kinases Iε and Iδ (CKIε/δ) phosphorylate PER1 and PER2, leading to the proteasomal degradation of these proteins. AC, adenylyl cyclase; MKK1, mitogen-activated protein kinase kinase 1.

Figure 3. Signalling, neural circuits and alcohol

The go (part a) and stop (part b) pathways affect the function of several neural circuits. In the schematics, up arrows depict activation or increased expression of a molecule, whereas down arrows depict decreases in expression and/or activity of a molecule. The mesocorticolimbic circuitry (blue) mediates reward processing. Glial cell line-derived neurotrophic factor (GDNF), signalling via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and circadian locomoter output cycles protein kaput (CLOCK), acts as a stop-pathway molecule in the ventral tegmental area (VTA) and dampens alcohol drinking, whereas HRAS–phosphoinositide 3-kinase (PI3K)–mechanistic target of rapamycin complex 1 (mTORC1), calcium/calmodulin-dependent protein kinase type II (CaMKII), protein kinase Cε (PKCε) and microRNA-382 (miR-382) act as go-pathway molecules in the nucleus accumbens (NAc) and promote alcohol drinking. In the prefrontal cortex (PFC), mTORC1 (go pathway) is activated in a reconsolidation of an alcohol-seeking session, and brain-derived neurotrophic factor (BDNF) levels are reduced after excessive drinking in response to increases in miR-206 and miR-30a-5p levels (go-pathway molecules). The nigrostriatal circuitry (purple) has a role in goal-directed behaviours (dorsomedial striatum (DMS)), habitual learning and compulsive behaviour (dorsolateral striatum (DLS)). The tyrosine-protein kinase FYN–protein-tyrosine phosphatase-α (PTPα)–GluN2B signalling pathway is centred in the DMS (go pathway), and BDNF–ERK1/2 signalling mainly occurs in the DLS (stop pathway). The reduction in activity of striatum-enriched protein-tyrosine phosphatase (STEP) in the DMS also contributes to the go pathway. The extended amygdala (grey) is implicated in the negative emotional state that characterizes alcohol withdrawal and relapse. The go-pathway actions of PKCε–corticotropin-releasing factor (CRF) and 3′,5′-cyclic nucleotide phosphodiesterase 10A (PDE10A) are centred in the central amygdala (CeA) and basolateral amygdala, respectively; CaMKII and ERK1/2 also have a role in go-pathway signalling in the CeA. These signalling molecules promote anxiety-like and alcohol-drinking behaviours. By contrast, the BDNF–ERK1/2 (stop) pathway in the CeA and medial amygdala (MeA) dampens these phenotypes. In addition, malfunctioning of cyclic AMP-dependent protein kinase A (PKA)–cAMP response element (CRE)-binding protein (CREB) signalling in the CeA and MeA also contributes to the interplay between stress and heightened drinking. Finally, mTORC1 (go pathway) in the CeA mediates alcohol-associated memory reconsolidation. ALK, anaplastic lymphoma kinase; DS, dorsal striatum; LMO3, LIM domain only protein 3; NPY, neuropeptide Y; PKMζ, protein kinase Mζ; SNc, substantia nigra pars compacta.