Withdrawal is a collection of unpleasant symptoms that derives from the absence of substance use, after its preceding chronic use. This physical discomfort propels users back into a cycle of dependence giving them momentum to misuse once again. Being able to understand the mechanisms by which withdrawal works may provide insight on how to intercept these mechanisms and prevent onset of symptoms. This in turn may be a stepping in stone in recovery for users, helping them regain control of their physical and mental aptitudes. The physiologic structures involved often include members of the mesolimbic pathway, a pathway with a well-established link to pleasure[1] . The VTA, nucleus accumbens, and in some cases the amygdala are the structures mostly associated with withdrawal [1][2] . General changes in protein level within these areas, as well as neuronal changes, are of a primary focus [1][2][3] . At the cellular level changes within dopaminergic, GABAergic, and glutamatergic neurons are the ones thought to be involved in the withdrawal processes [2]. At an even deeper macromolecular level, changes to specific receptors including NMDA, GABA, and nACh receptors have also been correlated with withdrawal [2]. These include changes to composition and level of expression of these receptors.

Physical Effects of Withdrawal

The physical effects of withdrawal, based on animal studies, reports and observation, tend to vary depending on a number of factors including where the substance exerts its affects, the type of substance, duration of use, level of use during that time and a number of subject intrinsic factors [4] . With respect to these findings, the intensity, onset, and duration of withdrawal can generally be correlated with dependence. Some of the more common physical symptoms include "wet dog shakes", teeth chattering, mastication, rearing (seen in animal studies), jumping, piloerection, hyperactivity, ptosis and eye twitch. There have also been reports of symptoms such as diarrhea, salivation, lacrimation and rhinorrhea, however these aren't as readily reproduced in animal studies [4]. Other common physical symptoms that have been reported include vomiting, night sweats, muscle aches, diarrhea, headaches, restlessness, insomnia, feeling cold and anxiety [5] . These are just a collection of symptoms that are commonly observed over a wide spectrum of substance withdrawal, and more specific symptoms are more associated only with certain substances.

Overview of Molecular Effects

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Precipitation of the physical symptoms mentioned above, stems primarily from the macromolecular changes made to the affected cells. These can include receptor subunit modifications, altered receptor representation on cellular membranes, changes to protein levels within the cell, and even whole cell morphological changes. These minute changes can culminate in larger scale signaling changes that ultimately alter functionality of brain systems.

Image from:http://www.squishgames.com/wp-content/uploads/2011/02/RedNeuronal.jpg

Structural Changes to VTA Neurons

A. VTA neurons in control. B. Chronic morphine C. Spontaneous Withdrawal D. Naloxone Withdrawal
The VTA is strongly associated with the rewarding effects of drugs, and subsequently in withdrawal also. Morphological changes to VTA neurons have been observed in vitro. upon morphine withdrawal. Animal studies using confocal laser scanning microscopes have shown size reductions in area and perimeter of presumably dopamine containing neurons (tyrosine-hydroxylase positive) within the VTA
[1]. The neurons of the naloxone-induced withdrawal group showed a size reduction in its area of nearly 50%. The numbers of these cells were also found to increase per field, possibly a compensatory mechanism elicited by the drug-withdrawn animal. The same naloxone- induced withdrawal group had more than doubled in number within its field. These findings were observed the period of withdrawal immediately following chronic morphine use, rather than during chronic use, distinguishing this mechanism as one of withdrawal. Along with size reduction of their somata, VTA neurons that synapse on the nucleus accumbens, have also been observed to exhibit a reduction in firing rate as well as extended refractory periods during periods of withdrawal [6] . These studies that induced morphine withdrawal inhave successfully demonstrated a correlate between the morphologic and functional abnormalities characteristic of morphine withdrawal [1].Altered VTA neurons, whose projections lead to prefrontal limbic structures, also play a role in this withdrawal-mediated system, as the prefrontal cortex is another of the key structures of opiate dependence. Reports have also observed similar changes in withdrawal from cocaine and amphetamine also [1].
The corresponding data to the figure on the right are shown below [1].[Pictures from [1]]

Functional Changes to the Nucleus Accumbens

As mentioned before, downstream of the VTA in the mesolimbic pathway of reward is the nucleus accumbens. In terms of changes here, animal studies eliciting nicotine withdrawal have been able to show a correlate between withdrawal onset and alterations in basal and nicotine-induced DA signals [7] . Microdialysis measurements of dopamine have shown reductions in the neurotransmitter concentration within the NAc, which lasted for a length of time that was proportional to the duration at which nicotine was used [7]. The longer the nicotine treatment, the longer the DA concentrations remained low post-treatment [7]. Furthermore upon re-treatment with nicotine, dopamine concentrations were elevated once more [7]. The low dopamine levels that were observed are thought to initiate drug-seeking behaviour (craving), and with the gratification of using again, relapse becomes increasingly difficult to avoid.

Biochemical Changes

NAch Receptor

The nAChR, the obvious target of nicotine is composed of many subunits, one of which has particular implications in withdrawal. The alpha6 subunit has been shown to affect nicotine-stimulated dopamine release in the striatum and consequently its expression is connected to nicotine dependence [8] . Studies using an alpha6 selective antagonist (a- conotoxin) have shown that despite its affect on dopamine release in the striatum, receptors containing the alpha6 subunit selectively operate on affective nicotine withdrawal but not the physical withdrawal behaviors [8]. These affective behaviours refer to the anxiety related symptoms observed during spontaneous withdrawal, and were measured by time spent in the arms of an elevated plus maze [8]. Expression of somatic or hyperalgesic signs were not altered upon antagonist treatment, suggesting that these effects are largely independent of a6 expression [8]. However it was also seen to be well integrated in the rewarding effects of nicotine and may be a potential target in more effective nicotine cessation treatments [8]. These receptors are also highly localized to catecholamine structures including the VTA, substantia nigra, and the locus coeruleus, all areas strongly affiliated with reward [8].

NMDA Receptor

Another receptor, known as the N-methyl-D-aspartate receptor (NMDAR), is also thought to be modified in withdrawal mechanisms, particularly to that of ethanol[9] . Alterations either to the expression levels or localization are thought to contribute to the neurotoxicity and seizures characteristic of severe alcohol withdrawal [9]. A number of changes were seen, one being within the receptor itself with greater coupling of the NR1 and NR2B subunits [9]. There was also an increase in co-expression of the receptors with the synaptic proteins synaptophysin and PSD protein 95 [9]. This receptor clustering suggests that normally extrasynaptic NMDA receptors are localized on synapses in response to the depression of activity caused by ethanol [9]. It acts as an adaptation to the decrease in activity and only upon cessation of ethanol activity are the NMDA receptors seen to migrate back to their extrasynaptic loci [9]. This relocalization is mediated by phosphorylation events involving kinases, and tends to decrease in activity upon chronic ethanol treatment [9]. It's possible that either inhibition of a kinase or activation of a phosphatase decreases the rate at which the NMDA receptors are phosphorylated, immobilizing the receptors in the synapse, subjecting the neurons to cytotoxicity [9]. Neuroadaptations, like this, where substances abuse cellular machinery that later elicits discomfort in withdrawal, are the basis of drug dependence. The image below shows the promotion or receptor clustering at synapses upon ethanol exposure [9]. [Picture from [9]]



The GABA neurotransmitters' role in withdrawal is mediated primarily through its release and trafficking of its receptor within the VTA[10] . Using wild type and mu- opiod receptor knockout mice, morphine withdrawal was induced and the results were as follows [10]. In wild type mice, there was an increased probability of GABA release within the VTA, in a cAMP-dependent manner, induced by morphine [10]. Somatic signs of withdrawal, including tremors, jumps, wet-dog shakes and rears were observed, and correlated with the increase in GABA release during naloxone treatment [10]. Modified GABA release wasn't observed in the knockout mice however, suggesting that trafficking of the mu-opiod receptor is involved in this withdrawal mechanism [10]. During morphine use, GABA release is shown to decrease, heightening effects of dopamine in the VTA [10]. This is counteracted in withdrawal when GABA release acts in elevated concentrations inhibiting dopamine neurons the reward circuit [10]. The second messenger cAMP drives the increase in GABA release and it was shown that treatment with cAMP inhibitors were able to attenuate withdrawal symptoms [10]. This further solidifies the idea that GABA release is linked to dependence in the mesolimbic reward pathway. This hyperactivation of the cAMP-GABA pathway is likely a compensatory mechanism of the cell, a common trend in withdrawal, in which the cell increases adenylyl cyclase activity, a process seen in vivo, within regions such as the locus coeruleus and striatum[11] . Failed endocytosis of mu-opiod receptors can be held accountable for this hyperactivation, since its desensitization and receptor recycling, prevent cAMP hyperactivation, and the subsequent increase in GABA release[12] . This understanding of receptor trafficking helps define yet another mechanism of withdrawal in which synaptic adaptations set the basis for dependence.

Protein Level Changes

BDNF and DeltaFosB

Two proteins that seem to help mediate withdrawal effects are BDNF and deltaFosB. BDNF levels seem to be modifiable in the VTA, nucleus accumbens (NAc), amygdala, hippocampus and the medial prefrontal cortex[13] . All key structures of substance misuse, the medial prefrontal cortex is particularly active in relapse tendencies [13]. Elevated BDNF levels in the mPFC, which have been observed in cocaine withdrawal, induce LTP in the pyramidal neurons of layer V [13]. This long-term synaptic plasticity may mediate the craving and relapse as well as enhance responsiveness to drug associated cues [13]. Depression of GABAergic neuronal activity and reduced expression of GABA receptors on cell membranes, are two other mechanisms by which elevated BDNF exerts its effects [13. This disinhibition through suppression of GABA results in more compulsive drug seeking behaviour and may be one of the underlying causes.

The second protein involved is deltaFosB with similar outcomes. This transcription factor offers promise in understanding the long-term effects of addictive drugs, since it is likely that some form of gene transcription is involved[14] . Following chronic use of addictive drugs, deltaFosB is found to build up in striatal brain regions and the orbitofrontal cortex [14]. It also tends to withstand degradation and remains in the brain for extended periods of time following drug exposure [14]. This overexpression and persistence of deltaFosB is thought to function in enhancing the rewarding effects of addictive drugs, as well as impulsivity during withdrawal [14].Along with the more generic structures of addiction recent research also shows that that actions of deltaFosB may extend to other circuits, like the stress system, which is thought to have a direct relation to addiction[15] . Increased expression of FosB/DeltaFosB in hypothalamic and extrahypothalamic structures such as the PVN, NTS and amygdala during morphine withdrawal, may be the culprit causing long term synaptic plasticity that induces relapse [15]. The mechanisms are still not well understood, but the implications are vast.

Big Picture

Withdrawal is a highly idiosyncratic state that varies greatly in duration, intensity, time of onset, and depends heavily on the character of the compound that elicits its development. Attempting to understand it any level is a difficult task to undertake, but progress is being made, and with such profound implications, the goal to gain knowledge on the mechanisms is likely to persist.


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