Critical Review of Alcohol, Alcoholism and the Withdrawal Symptoms I. Mechanisms of Addiction and Withdrawal Syndrome

Alcoholic beverages are socially accepted around the world, consumed mostly to socialize, celebrate, and relax. The pleasant effects of alcohol are attributed to (i) an increase in GABAergic (inhibitory signals), OPergic and 5HTergic (euphoric effects) neuronal activities and (ii) a decrease in DAergic (‘want’ signal or craving), Gluergic (excitatory signals),NEergic (stress signals) neuronal, and the HPA axis (stress hormones) activities. If alcohol drinking continues, the receptors are sensitized, resulting in development of tolerance when alcohol drinking must be increased to achieve desired effects. In genetically/Environmentally predisposed subjects, chronic alcohol drinking results in the development of addiction, characterized by a condition when alcohol caseation results in rapid onset of withdrawal symptoms including, but not limited to, alcohol craving and moderate to severe discomfort. Because pharmacotherapy alone or in combination with behavioral approaches is only modestly effective in treating alcoholism symptoms, there is an urgent need to development effective and safe therapies. At present, a lack of clear understanding of the mechanisms underlying addiction hinders possible development of new treatment strategies. Therefore, the aim of this article is to discuss the mechanisms underlying (i) the euphoric, relaxing and adverse effects of alcohol drinking and (ii) addiction and the withdrawal symptoms.


Introduction
Alcoholic beverages are socially accepted drinks, expected to bring pleasure, satisfaction, and relief from stress [1]. In general, most people drink alcohol responsibly, but, continued drinking may serves as a prelude to alcohol abuse and an escape route for social, personal or career pressures [2]. In the presence of a genetic predisposition and environmental cues, persistent drinking may result in the development of tolerance and addition or alcoholism (defined as a cluster of behavioral, cognitive, and physiological abnormalities developing after repeated alcohol use and resulting in a physical withdrawal state upon abstinence) that may interfere with a person's ability to function normally and impairing his/ her daily life [3]. Abnormal alcohol drinking may also be an important risk factor for a number of diseases such as infection, cancer, atrial fibrillation, hypertension, etc [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. In the United States alone, about 17 million people of different age suffer from alcoholism [18]. Thus, alcoholism may exert tremendous economic consequences not only for the drinking individuals, but also for the society at large [19][20][21]. The journey from responsible alcohol drinking to alcoholism involves four stages listed in Table 1.
The transition from the responsible use of alcohol to an excessive, uncontrolled alcohol consumption (alcohol addiction) results from a complex neuro adaptations involving various excitatory and inhibitory pathways in different brain regions. In alcohol-naïve subjects, the adaptation pathways such as aversive hangover response may keep the social intake of alcohol in check, while, in subject practicing abnormal alcohol drinking, the neuro adaptations in the brain may cause behavioral transitions, resulting in uncontrolled alcohol drinking [22]. Although, there are compelling evidence in support of causal relationship between the Review articles (listed for information only), case reports. English language articles.
Non-English language articles. Human populations diagnosed as addicted-imaging and blood-chemistry studies.
Therapeutic studies in humans or animals.
Animal studies-BAC toxicokinetics, acute and chronic alcohol drinking, alcohol consumption as primary outcome, behavioral abnormalities.
Studies with insufficient number of animals or animals exposed to non-alcohol drugs (opioid addiction may be included in limited cases). Animal brain studies-whole animal imaging, stimulatory and inhibitory signaling, genomics, proteomics, metabolomics, epigenetics and alcohol metabolism at different stages (acute euphoric effects, tolerance, and addiction and withdrawal) of alcohol abnormalities.
Secondary analyses of randomized control trials in which the major outcome of interest was not alcohol consumption.
In vitro studies using animal tissue (neural and non-neural) or cell line samples exposed to ethanol for characterization of cell-signaling, gene expression, epigenetics and alcohol metabolism.
In vitro studies using animal tissue (neural and nonneural) or cell line samples exposed to non-alcohol drugs.
The rising BAC activates the rewarding pathways that further (i) activates the synthesis and release of of β-endorphin, (ii) inhibits GABA ergic neurons and (iii) activates orbitofrontal (OFC) cortex [26,27]. A decrease in GABA ergic signaling disinhibits DA ergic neurons, while an increase in β-endorphine activated DA ergic neurons, resulting in releases DA. β-Endorphin and DA activate the Hedonic hot spotes and ensuing sensory pleasures. DA also induces serotonin (5-HT) levels that induces enkephalin release. DA and enkephalin together may further inhibit GABA ergic neurons and initiate alcohol 'craving'. Activagtion of the Hedonic hot spotes and OFC may develop a conditioned response. The 'stimulatent' effects of alcohol may be mediated via release of norepinephrine (NE) from adrenergic neurons and corticosteroids via the HPA axis [28][29][30][31]. As shown in Figure 3, GABA ergic , ACh ergic, Glu ergic , 5-HT ergic and NA ergic activities, directly or indirectly, are dynamically regulated via the NMDA receptors present on GABA ergic , 5-HT ergic and NA ergic neurons [30,31]. Glu may regulate DA, opioid peptides, GABA, glutamate and 5-HT concentrations that mediates alcohol reinforcement by differentially modulating the GABA ergic neurons. During the rising phase of BAC, the rewarding effects of alcohol drinking may also be associated with an inhibition of NMDA and DA receptors and HPA axis [30,31].

Mechanisms of alcohol's aversive effects during declining BAC
The descending phase of BAC, as shown in Figures 2, initially. While hangover keeps a check on alcohol drinking, tolerance motivates people to drink more for desired effects.
The following sections discuss the mechanisms underlying hangover and tolerance.

Tolerance
Three basic types of tolerance have been described in literature [55].
• Cellular tolerance occurs at the level of a neuron or a network of many neuronal and supportive cells including astrocytes and glial cells, • Molecular tolerance involves adaptation processes developed by individual molecules (e.g., ion channels) during exposure to ethanol and, • Behavioral tolerance that involves the level of the activity of an entire animal.
is characterized by an increase in acetaldehyde that (1) deactivates the NMDA receptors, resulting in an attenuation of GABA ergic -mediated neuroinhibition and (2) react with DA and synthesized salsolinol that is shown to depolarize and disinhibit DA ergic neurons, decrease GABA ergic activity and enhance nitric oxide production [32,33]. Thus, acetaldehyde and salsolinol together may participate in development of alcohol-related negative reinforcement. In addition, the aversive response of the declining phase of BAC may block the rewarding effects as described below [34].

Hangover and Tolerance
Hangover (a feeling of general misery comprising of drowsiness, concentration problems, dry mouth, dizziness, gastro-intestinal complaints, sweating, nausea, hyper-excitability, and anxiety) is always associated to acute alcohol intoxication and/or heavy episodic drinking [35][36][37]. In addition to unpleasant feeling, hangover also has several social and clinical negative consequences such as work and academic absenteeism and neurocognitive impairments [38][39][40]. Tolerance is defined as a person's diminished euphoric response to alcohol when used repeatedly over time. As tolerance develops, person may need relatively higher quantity of alcohol to achieve the same level of response achieved In this section, participation of genetic and environmental factors in development of alcoholism will be discussed in detail.
Alcohol tolerance can also be classified as acute, rapid, and chronic, based on how long after exposure to alcohol tolerance develops. Possible molecular mechanisms proposed to explain the development of different types of tolerance are shown in Figure 5.  Table 4). The µ-opioid receptor A 118 G (OPRM1) having Asn40Asp SNP has been studied extensively, however, the results have been controversial. Some studies support an association between A 118 G (OPRM1) and addiction [65][66][67][68], while other studies do not [69][70][71][72].

Development of Alcohol Addiction or Alcoholism
Ray, et al. [73] showed that Asp40 carriers in adolescents

Genetic/Epigenetic predisposition to alcoholism
The evidence for genetic predisposition arises from studies involving the 'twins' of alcoholic parents that are more susceptible to develop alcoholism than children of normal parents, even when they were adopted and brought up in different environment (one adaptive parent abused alcohol while the other did not) [61-64]. Several import- CAMKII may be essential for the development and consolidation of addiction.
According to the incentive salience theory of addiction [92], repeated exposure to addictive drugs including alcohol can, in susceptible individuals and under particular circumstances, persistently change brain cells and circuits, a psychological process involved in motivated behavior ( Figure 8). Individuals with the DR Val158Met may mediated the association between OPRM1/Asp40 genotype and alcoholism and that a significantly higher frequency (51.9%) of the Asp40 allele occurred among youth with alcohol-related disorder (ARD), as compared to non-ARD controls (16.3%). In addition to genetics, earlier studies have also provided strong evidence for epigenetic predisposition to addiction in people who have higher probably to develop addiction upon chronic alcohol drinking [74][75][76][77][78][79].
Li, et al. [80] analyzed data using a linkage association in chromosome regions of addiction and identified 1,500 addiction-related genes and five molecular pathways (neuro-active ligand-receptor interaction, long-term potentiation, GnRH signaling pathway, MAPK signaling pathway and gap-junction pathways) that were significantly enriched for alcoholism. They connected the common pathways into a hypothetical common molecular network for addiction. As shown in Figure 7, fast and slow positive feedback loops were interlinked through CAMKII, which may provide clues to explain some of the irreversible features of addiction. Activation of CAMKII may play a central role in the development and maintenance of addiction states. The fast and slow positive feedback loops interlinking through  enzyme (ALDH2 * 1/2 * 2) increased between 1979 and 1992 in Japanese population. ALDH2 genetic polymorphisms have been shown to develop hypertension in a prospective cohort and that alcohol intake significantly modified the conferred risk [97]. These data strengthen GxE interaction in Asian populations.

5-HT transporter gene x environment:
Nilsson, et al. [98] have shown that adolescents (aged 16 to 19) who had the heterozygous 5-HTTLPR l/s genotype and came from families with neutral or poor relationships had over 10-fold increased risk for high intoxication frequency, compared with heterozygous adolescents who had a good relationship with their families. Preadolescents or early adolescents having the heterozygous 5-HTTLPR l/s genotype and were maltreated exhibited a 40% greater risk for alcoholism then those who were not abused. Covault, et al. [99] reported that the 5-HTTLPR s-allele also may be linked with increased use of alcohol and other drugs among college students who have had multiple negative life events. Thus, an interaction between stress and 5-HTTLPR s/l or s/s alleles may predispose a person to abuse alcohol with increased risk of alcoholism.

GABRA x environment:
A review of literature has provided substantial evidence for a key role the GABA receptors play in development of alcohol addiction [100][101][102][103][104]. An interaction between α 1 (A > G SNP) and α 6 (C > T SNP) subunits of GABRA 1 and GABRA 2 and the environment polymorph and CNR1-C allele of rs2023239 may carry a genetic vulnerability that affects respective receptor-mediated signaling in the mesocorticolimbic structures, resulting in incentive salience to alcohol cues.

Gene-Environment (GxE) interaction
Although genetic predisposition (G) is associated with development of alcoholism, the GxE interaction potentiates ( Figure 9) the effects of genetic factors. The joint effects of GxE are significantly greater than would be predicted from the sum of the separate effects [93]. The genetic influences on alcohol drinking behavior is commonly studied among sibling pairs reared in the same family and environmental influence, while the GxE is studied among sibling pair reared in different families and environmental influence [93].
Some of the studies reporting GxE interaction in development of alcoholism are described below.

ADH/ALDH x environment:
Earlier studies [94][95][96] have shown that liver may contain active and inactive alleles of liver mitochondrial aldehyde dehydrogenases (ALDH 2s ). The active forms are encoded by the gene ALDH (2 * 1/2 * 1), while the inactive form is encoded by the genes ALDH (2 * 1/2 * 2) or ALDH (2 * 2/2 * 2). The inactive ALDH form provides a genetic deterrent of heavy drinking and alcoholism among Asians. As shown in Figure 10, the normal active enzyme (ALDH2 * 1/2 * 1) decreased, while the partially inactive ity, and inattention) than did adolescents with other genotypes or those with the same genotypes who grew up in less adverse family conditions. However, Vandenbergh, et al. [112] failed to show any correlation between DAT1 alleles and risk to alcoholism. More research is needed to clarify the role of DAT in alcohol predisposition. Taken together; these observations indicate that the gene-environmental interaction may play a key role in development of alcoholism phenotypes.

Anatomical Changes in Alcohol-Addicted Brain
Alcohol addiction is characterized by compulsion to seek and take alcohol, loss of control in limiting intake, and emergence of withdrawal syndrome when access to the drug is prevented [113]. As discussed earlier, the positive has been implicated in the development of alcoholism [105][106][107]. In family-based studies, GABRA 2 has been associated with alcohol dependence exhibiting 13-28 Hz β electroencephalographic frequency in children of alcoholic parents, not in children of control parents [106,[108][109][110]. People with the high-risk allele of GABRA 2 and unhappy marriage had higher likelihood of developing alcoholism than people with the allele or marriage alone [106].

Dopamine transporter 1 gene × environment:
There is some but not compelling evidence for involvement of dopamine transporter genes in development of alcoholism. Laucht, et al. [111] have shown that in adolescents who were homozygous for either of the DAT1 gene variants and grew up in psychosocially adverse familial conditions exhibited higher risk factors for alcoholism (impulsivity, hyperactiv- posing effects of alcohol drinking [115,116]. In general, the addiction related pathology can be classified as 'complicated' and 'uncomplicated' disorders.
In complicated neuropathy, the addicted subjects, in addition to the cognitive deficits, also suffer from liver damage and vitamin B1 deficiency, a condition known as Wernicke-Korsakoff syndrome-WKS, [117][118][119] consisting of two separate syndromes, a short-lived and severe condition called Wernicke's encephalopathy and a long-lasting and debilitating condition known as Korsakoff's psychosis. Wernicke's encephalopathy [120] includes mental confusion, oculomotor disturbances, and difficulty with muscle coordination. Korsakoff's psychosis is a chronic and debilitating syndrome characterized by persistent learning and memory problems [121]. Patients with Korsakoff's psychosis are forgetful and quickly frustrated, have difficulty with walking and coordination and exhibit anterograde amnesia [122][123][124]. Abstinent WKS patients show significant impairment in neuropsychological tests compared to controls [125]. This suggests that the neurological effects of alcohol with thiamine deficiency may be more severe and permanent.
Uncomplicated (non-WKS) alcoholic patients display mostly neuropsychological and behavioral disorders that exhibit significant recovery of functions upon long alcohol abstentions, although some components of these functional reinforcing effects (euphoria and reward) of acute alcohol ingestion is mediate through the cortico-mesolimbic DA ergic pathways, extending from the ventral VTA to the NAc, AMY, HIP, PFC, SN, CP and related structures. The opposing effects (anxiety and stress), mediated through the HIP, muscarinic cholinergic neurons, NE ergic neurons and HPA axis, remain inhibited during this phase [113,114]. The brain of alcoholic patients exhibited abnormalities in many of the brain regions associated with the rewarding and op-The image part with relationship ID rId2 was not found in the file. Earlier studies [158][159][160] have shown age related recovery of alcoholics, younger alcoholics improving to the level of the control groups in the area of visual-spatial functions (which are among those most sensitive to the effects of chronic alcohol abuse), whereas the older alcoholics continued to show deficits. Sclafani, et al. [161] have shown a positive relationship between age and ventricular volume (as % of total brain volume). Studies have shown relatively greater CSF filled spaces in frontal cortices and cerebellum of alcoholics, especially those with thiamine deficiency (Table 5). Shrinkage of Superior Cerebellar Vermis associated with Purkanje cells loss (complicated > uncomplicated) after chronic alcohol intake have been frequently reported [162]. They reported a 21%-40% reduction of Purkinje cell density in the cerebellar vermis with shrinkage of the molecular and granular layers.

Alcohol Withdrawal Syndrome
Earlier studies have shown that many of the alcohol-induced neurological abnormalities listed above (section 8.2.) were partially reversed with maintained abstinence [142,163]. However, in alcohol addicted subjects, abstinence (alcohol withdrawal) results in severe physical and emotional distress (paleness, excessive perspiration, nausea, stomach discomfort, heart palpitations, headaches, appetite loss, shakiness, seizures, nervousness, and, in extreme cases, death) that may increase alcohol craving [145]. Alcohol drinking rapidly reverses the withdrawal symptoms [164]. The aim of this section is to discuss the mechanisms underlying the development of the withdrawal symptoms in response to alcohol withdrawal.
In general, a state of neural hyper-excitability, lasting for at least 24 h to 72 h following withdrawal may be central to the development of the withdrawal symptoms [165]. Geisler, et al. [148] have shown that neural hyper-excitability to primary electrical stimulation may exist for at least 72 h and then subsiding at 1 week after alcohol withdrawal in alcoholics. A secondary stimulation given a week after the first stimulation resulted in a state of hypo-excitability (Figure 11). This shows longterm effects of alcohol withdrawal in rats.
Earlier studies have suggested that chronic ethanol exposure induces a net excitation state in Dorsal Raphe (DR) neurons that contributes to enhanced anxiety and excitation during ethanol withdrawal [166][167][168]. The neurotransmit-domains recover faster or more fully than others [126][127][128][129][130][131][132][133][134][135][136]. Kopera, et al. [137] have shown only partial recovery in brain function of alcoholic patients abstinent for more than a year. Thus there may be some processes in the brain that do not easily recover over time.
The proceeding sections describe some of the permanent, reversible and dementia-like abnormalities associated with alcoholic brains.

Permanent brain damage
Chronic alcohol use may initiate neuronal loss in different brain regions (larger neurons (greater than 90 µm) are more sensitive than smaller neurons) and reduce the white matter volume in cortex and cerebellum. This may compromise the cerebellum-cerebral cortex loop (complicated >> uncomplicated) [138][139][140][141]. Neuronal loss in cortex, hypothalamus and cerebellum may result in lasting impairment in behavior such as decision-making activities [142,143]. Loss of GFAP in neurons (complicated >> uncomplicated) occurred without gross changes in brain pathology or brain weight and was not restricted to pathologically susceptible brain regions [144]. Alcoholic patients having vitamin B1 deficiency exhibits symptoms of alcoholism-related poly neuropathy (ALPN), a potentially debilitating disease associated with sensory, motor, and autonomic nerve dysfunctions [145,146]. Although B1 deficiency is believed to be the prime cause of ALPN, many studies have shown that ALPN was not significantly abated or reversed by thiamine repletion [147][148][149]. ALPN progresses slowly with burning pain and superficial loss of sensation due to irregular segmental [150], whereas thiamine deficiency result in acutely progressive deficits in superficial and deep sensation, due to degeneration of large fiber axons and sub-perineurial edema [151].

Transient brain damage
Substantial evidence associate alcoholism with global cerebral atrophy and ensuing cognitive dysfunction with age being a critical modulating factor [152][153][154]. Beck, et al. [155] have shown that alcohol addiction is associated with altered density of gray matter and white matter of specific brain regions, thus supporting the assumption that alcohol dependence is associated with both local gray matter dysfunction and altered brain connectivity ( Table 5). The volumetric changes in the brain and certain cognitive impairments can be reversed with abstinence [156,157].  [171][172][173]. An increase in DA in the brain may augment craving for alcohol [174,175], while an increase in NA and activation of the HPA axis increases the stress response and anxiety, while a decrease in opioid and 5-HT receptors enhance the feeling of despair [175,176].
Earlier studies, using linkage-based genome scans of chromosomes, have identified markers of alleles that predispose to alcohol addiction and withdrawal symptoms [177][178][179][180][181][182]. Long, et al. [183] and Buck, et al. [177] have shown that the risk for alcohol withdrawal highly correlat-ter changes occurring in the brain of alcoholics that may be associated with the withdrawal symptoms are shown in Figure 12. GABA ergic neurons in NAc, inter neurons in VTA regions, and postsynaptic GABA receptors, notably GABA A receptors, may play a central role in development of addiction and withdrawal symptoms [113,169].
GABA A receptors are unique because they cause neuro-inhibition in alcohol-naïve subjects, but cause neuro-excitation in alcohol deprived alcoholic subjects by recruiting different pathways [170]. Chronic alcohol drinking may switch GABA A receptor function from inhibitory to excit- increase in GABA ergic (inhibitory signals), OP ergic and 5HT ergic (euphoric effects) neuronal activities and (ii) a decrease in DA ergic ('want' signal or craving), Glu ergic (excitatory signals),NE ergic (stress signals) neuronal, and the HPA axis (stress hormones) activities. If alcohol drinking continues, the receptors are sensitized, resulting in development of tolerance when alcohol drinking must be increased to achieve desired effects (Figure 14). In genetically predisposed subjects, chronic alcohol drinking results in the development of addiction, characterized by a condition when alcohol caseation results in rapid onset of withdrawal symptoms including, but not limited to, alcohol craving and moderate to severe discomfort such as tremors, seizures, hallucinations, DT and, in extreme cases, death. Although mechanisms are not fully understood, in alcohol withdrawal, the neurochemical changes are opposite to those during the acute exposure phase: Glu ergic , DA ergic , NA ergic and HPA activities are elevated, while GABA ergic activity is reduced. Alcohol drinking rapidly reverses the withdrawal symptoms. Taken together, these observations suggest that alcoholism is a multifaceted disease, affecting almost every system in the body, including neurological, physiological/hormonal, and cardiovascular systems. Because of this, there is a great need to improve the success rates of all forms of treatment of alcoholism including prevention of relapse, curbing active alcohol consumption and craving and treatment of the disease. ed with markers located at chromosomes 1, 2, 4 and 11. In chromosome 5, the marker was found at 38-42 cM from the centromere of chromosome 4 ( Figure 13) in mouse that is syntonic with human 9p21-p23 and 1p32-p22.1. Buck, et al. [177] also detected a QTL near Gad 1, α-subunit of brain sodium channels (Scn1a, Scn2a, Scn3a) and a glial-specific sodium channel (Scn7a) of chromosome 2. Gad1 may be related to a candidate gene responsible for GABA synthesis. The differences in alcohol withdrawal severity among mice of different strains could be associated with differences in GAD enzyme activity and/or gene expression. Chromosome 11 contained a QTL in proximity to genes encoding the α 1 , α 6 , and γ 2 subunits of GABA A receptors [184] that correlated with severity of the withdrawal syndrome.
Alcohol withdrawal, in addition to the rapid-onset withdrawal symptoms, also cause long-lasting post-abstinence changes including, but not limited to, sleep disturbances, cognitive deficits, anxiety, mood swings, depression mostly in females, panic disorder and suicide behavior [130,[185][186][187][188][189][190][191][192]. Zorrilla,et al. and Koob [193,194] have shown that protracted withdrawal from ethanol or cocaine is associated with altered limbic CRF-LI and circulating CORT levels beyond the detoxification stage. These may play some role in the development of the delayed symptoms.

Conclusions
Alcohol exposure in alcohol-naive subjects elicits euphoric and relaxing responses that are attributed to (i) an  Figure 13: Application of QTL in search for candidate genes responsible for alcohol withdrawal on chromosomes (Chrs) 1, 2, 4, and 11. Results for the B6D2 F2 mouse population (a cross of alcohol preferring C57BL/6J (B6) and alcohol avoiding DBA/2J (D2) mouse with high and low alcohol withdrawal, HAW and LAW, respectively) are shown. The markers, map positions indicated in centiMorgans from the centromere (at 0 cM). Estimated 1.0 LOD confidence intervals for the positions of QTL on mouse chromosomes 1, 2 (proximal), and 11 are also shown (boxed regions). For chromosome 4, QTL actually extends beyond the range of markers examined. For chromosome 2, the 1.0 LOD confidence intervals is shown only for the proximal QTL. The position of the best correlated marker for each separate experiment (i.e., RI, F2, or S2) is also indicated within each boxed region. Candidate genes located within or near the 1.0 LOD confidence intervals are also shown [177].