An increased propensity for alcoholism has been associated with stress-related anxiety and
dysphoria, a state of general unease or dissatisfaction. The experience of various types of stress, including severe acute stress and chronic stress, can lead to the onset of dysphoria. Ethanol consumption promotes the release of
dopamine into the
nucleus accumbens (NAc) which is translated as a “reward". Thus, to cope with negative emotions, individuals often turn to alcohol as a form of temporary self-medication. Unfortunately, repeated ethanol use results in diminishing returns which prompts increased intake and dependence. A specific subunit of NMDA receptors, NR2B, shows particularly high sensitivity to ethanol as exemplified by increased NR2B expression in response to ethanol. Another family of receptors, metabotropic glutamate receptors (
mGluR), may also contribute by activating MAPK pathways and increasing intracellular Ca2+. Antagonism of mGluR5 showed a decrease in ethanol consumption suggesting mGluR5's role in alcoholism. Furthermore, voltage-gated calcium channels (
VGCCs) were shown to be inhibited by ethanol resulting in reduced influx of Ca2+. Yet, repeated ethanol intake, or chronic ethanol use, increases expression of the slow-inactivating L-type VGCCs known to sustain Ca2+ influx. When these channels are inhibited with an antagonist, ethanol consumption is reduced. ====
Adenylyl Cyclase==== Adenylyl cyclase (
AC) plays a role in ethanol induced signaling pathways. Acute ethanol may increase AC activity resulting in increased levels of
cAMP and altered activity of cAMP targets. Of the cAMP targets,
protein kinase A (PKA) has been associated with ethanol use. While acute ethanol use increases the activity of AC, chronic use tends to desensitize AC such that more simulation, increased ethanol consumption, is required to elicit the same response.
Kinases Ethanol transduction pathways involve several protein kinases known to phosphorylate substrates linked to alcoholism, namely cAMP response element-binding protein (
CREB). CREB plays a central role in ethanol responses making its activation an important step in the pathway. Some of the kinase families currently linked to alcoholism are Ca2+/calmodulin-dependent protein kinases (CaMKs), protein kinase A (PKA), and mitogen-activated protein kinases (MAPKs). ;
CamK :Rapid changes in Ca2+ concentration, influenced by receptors such as those described above, regulate the activity of CaMKs. Withdrawal following chronic ethanol use as well as voluntary ethanol intake, consuming ethanol when both an ethanol solution and water are offered, in rats showed a decrease in CaMKIV and consequently p-CREB. In contrast, ethanol activates CaMKII resulting in phosphorylation of CaMKII targets such as BK potassium channels. ;
PKA :As discussed above, cAMP levels rise following ethanol-induced activation of AC. This rise in cAMP activates PKA. In response to acute ethanol exposure, activated PKA is transported to the nucleus where it phosphorylates CREB. While chronic ethanol use has not been shown to affect the levels of the catalytic domain PKA-Cα, voluntary ethanol intake does increase PKA-Cα in the central nucleus of the amygdala (
CeA) and medial nucleus of the amygdala (MeA) in P rats. The increase in PKA levels following acute ethanol use may induce negative feedback mechanisms to reduce PKA activity. Chronic ethanol exposure has been shown to reduce PKA activity in the
nucleus accumbens and the amygdala due to increased levels of PKA inhibitor α. ;
MAPK :MAPK proteins, especially
Erk1/2, have been linked to ethanol use. While there is not a consensus, acute and chronic ethanol exposure may increase p-Erk1/2 levels in the CeA and MeA of rats. In contrast, a decrease in Erk1/2 is observed during withdrawal. This likely serves as a negative-feedback mechanism to prevent excessive ethanol use. ====
CREB==== CREB may play a significant role in alcohol addiction. CREB is a
transcription factor known to influence CNS functioning. This protein is activated by phosphorylation via the kinase families CaMK, PKA, and MAPK. CREB binds a DNA sequence called CREB Response Element (CRE) in promoter regions and activates transcription via recruitment of CREB binding protein (CBP) and other transcription factors. Some CREB-target genes, relevant for understanding alcoholism, include NPY, BDNF, Arc, and CRF. Levels of CREB and p-CREB (a highly phosphorylated CREB protein) play a dynamic role in the preference for, consumption of, and dependence on ethanol. When comparing alcohol-preferring (P) to –nonpreferring (NP) rats, lower levels of CREB, p-CREB, and CRE-DNA binding activity were observed in the CeA and MeA of P rats. Withdrawal following chronic exposure decreases levels of CRE-DNA binding and p-CREB.
CREB Targets The effects of ethanol on CREB are further manifested in CREB-target genes, namely BDNF, TrkB, Arc, NPY, and CRF. ;
BDNF :BDNF signaling plays a role in dendritic spine formation and synaptic plasticity. The BDNF signaling pathway progresses in the following manner. After its activation, BDNF binds TrkB receptors whose subsequent activation results in dimerization and autophosphorylation of the receptor. In its phosphorylated form,
TrkB receptors recruit and bind adaptor proteins which result in the activation of MAPK. As described previously, MAPK pathways activate CREB. Thus, a feed-forward mechanism leads to the ramping up of p-CREB, BDNF, and TrkB levels which leads to the creation of new set-point, higher stimulus requirements to elicit a response, and may contribute to the downward spiral of alcoholism. In support of this idea, P rats show lower baseline BDNF levels than NP rats. Furthermore, BDNF-haplodeficient mice show higher ethanol preference and decreasing BDNF levels increases ethanol consumption and anxiety. Conversely, increasing BDNF decreases ethanol intake in rats. Acute and voluntary ethanol exposure increases BDNF expression in the dorsal striatum of mice, while chronic exposure tends to decrease BDNF in the hippocampus and cortex. Withdrawal, like chronic ethanol use, is associated with decreased BDNF levels. It is regulated by both BDNF and CREB signaling. For example, administration of BDNF increases Arc levels and promotes dendritic spine expansion. Acute ethanol exposure in rats increases Arc levels and DSD in the CeA and MeA. In juxtaposition, chronic ethanol exposure tends to decrease DSD. Withdrawal also decreases DSD as well as decreasing Arc expression, BDNF signaling, and CREB activation. This molecule binds to GPCRs that lead to the inhibition of AC and thus decrease cAMP levels. While NPY decreases cAMP, it also has been shown to evoke other pathways that increase p-CREB levels. This is yet another example of a feed-forward mechanism associated with alcoholism. Ethanol consumption and anxiety are increased in NPY knockouts and decreased when NPY is overexpressed. In addition, P and HAD rats show lower NPY levels in the CeA when compared to NP and LAD rats. Modulation of the upstream regulators in the pathways discussed influence NPY and ethanol consumption. For example, inhibition of PKA with Rp-cAMP in the
nucleus accumbens decreases NPY expression and increased ethanol preference. Conversely, addition of a PKA activator or NPY into the CeA decreases ethanol consumption and anxiety in P rats. In contrast to NPY, CRF is a peptide that binds GPCRs (CRF-R1 and –R2) that lead to the activation of AC and consequently increase cAMP levels. CRF-R1 and –R2 appear to have opposing functions in the amygdala: while CRF-R1 antagonsim reduces ethanol consumption and anxiety-like behaviors caused by withdrawal, a CRF-R2 activation using an agonist decreases ethanol consumption. Furthermore, an increase in CRF-R1 is correlated with increased sensitivity to stress and propensity to ethanol relapse. Taken together, ethanol consumption influences a wide array of molecules. Many of these are involved in feed-forward mechanisms which further promote alcohol relapse and dependence.
Epigenetic Mechanisms In coordination with the molecules and pathways discussed, epigenetic mechanisms play a role in the development of alcoholism. These mechanisms include
DNA methylation,
histone acetylation and
methylation, and (
microRNA miRNA) action. Methylation of the DNA typically occurs at
CpG sites, or a cysteine nucleotide followed by a guanine nucleotide in the 5’ to 3’ direction. These sites are common promoter and regulatory elements in mammals and methylation of cysteine residues typically inhibits these functions resulting in the repression of gene expression. DNA methylation is carried out by DNA methyltransferases (
DNMTs) which are recruited to CpG sites by methyl-DNA binding proteins, such as
MeCP2. Next, histones can be modified in several ways to increase or decrease gene expression.
Histones are protein complexes used to package DNA into structures known as
nucleosomes. The level of coiling of the DNA around histones is variable and influences transcription levels. Tight coiling, or
heterochromatin, is associated with low gene expression or even silencing. Loose coiling, or
euchromatin, is associated with higher levels of gene expression. Typically, acetylation of histones is associated with euchromatin formation. Acetyl groups are added by histone acetyltransferases (
HATs), such as CBP. In opposition, histone deacetylases (
HDACs) remove acetyl groups, typically leading to the formation of heterochromatin. HDACs are recruited by
scaffolding proteins, such as RACK 1. HDAC inhibitors prevent HDAC functioning which promotes gene expression. Histone methylation, adding a methyl group to specific histone protein amino acids, can both increase or decrease gene expression depending on the histone protein, amino acid, and number of methyl groups used. Gene expression can also be inhibited post-transcriptionally by miRNA, double-stranded RNA, typically formed from hairpin structures, that is used to inhibit translation of proteins. After processing by the RNA interference (
RNAi) molecules
Drosha and
Dicer, a single, guide-strand is loaded into the RNA induced silencing complex (
RISC) which is used to bind mRNA. This binding suppresses protein synthesis and sometimes initiates mRNA degradation. The epigenetic link to several ethanol related molecules has been established. As discussed before, acute ethanol exposure tends to increase CREB and p-CREB levels while withdrawal after chronic ethanol use is associated with decreased CREB and p-CREB. Also, CREB recruits the CBP, a HAT. Increased CREB and CBP activity at the BDNF promoter have been associated with decreased H3 methylation and increased H3 acetylation at lysine 9. In concordance, histone acetylation, particularly at the BDNF promoter II, increases BDNF expression. Similarly, BDNF exon IV expression following depolarization is increased and is associated with increased histone acetylation, reduced DNA methylation and reduced MeCP2 binding at the BDNF promoter. These changes would tend to increase BDNF expression during acute ethanol exposure. Conversely, since CREB levels and subsequent CBP recruitment fall during withdrawal, these types of epigenetic changes would likely reverse upon withdrawal after chronic ethanol use. In particular, lack of CBP likely results in decreased acetylation of the BDNF promoter. Another layer of regulation modulates the activity of MeCP2 via the protein RACK1. RACK1 at H3 and H4 inhibits MeCP2 binding and promotes histone acetylation; thus, resulting in increased BDNF expression. Chronic stress, often linked with a propensity to alcoholism, increases H3 methylation near BDNF promoters which inhibits transcription. To oppose this process, antidepressants have been shown to reduce histone methylation, increase H3 acetylation at the BDNF promoter, and reduce levels of HDAC5. Thus, the increase in NR2B expression in chronic ethanol exposed rats may be mediated by a more open chromatin structure. BK potassium channels are another target: miRNA-9 has been shown to target BK channel transcripts and may influence ethanol tolerance. This will be discussed in more detail in the tolerance section. Finally, a link has been found between ethanol use and histone acetylation during development. After ethanol exposure, adolescent rats showed increased H3 and H4 acetylation in reward centers of the brain, such as the frontal cortex and nucleus accumbens. This effect was not seen in adult rats. Thus, brain chromatin remodeling that increases gene expression in reward centers of developing brains may contribute to an increased propensity toward alcoholism upon and after ethanol exposure. == Tolerance ==