Epigenesis And Mechanisms Of Drug Addiction Biology Essay

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Drug addiction is defined as a chronic, relapsing brain disorder that is characterized by compulsive, uncontrollable drug taking and drug seeking behaviour that persists despite adverse consequences, such as the emergence of negative behavioral states (e.g. anxiety, dysphoria) and cognitive deficits (Koob and Le Moal, 2008).

Drug abuse and addiction pose a huge problem for the modern world; placing an enormous medical, financial and social burden on all levels of society. In fact the 2012 World Drug Report (Austria. UNODC, 2012) concludes that approximately 230 million people worldwide use illicit drug and at least 27 million of these suffer from drug addiction. The economic cost of drug abuse and addiction is estimated to be nearly $600 billion annually in the U.S. alone (NIDA, 2010). Though these figures appear generally stable, in truth they are on the rise due to a yearly increase in prescription drug abuse as well as drug abuse by teenagers (NIDA, 2010). These data indicate that the need to combat drug abuse is becoming increasingly more pressing. However, stopping drug abuse is not enough to stop addictive behaviour.

Addiction is a very complex disease that is determined by an array of different risk factors. Which makes it very difficult to treat and prevent. The addictive phenotype develops gradually and progressively following prolonged drug use, and is defined by long-term structural changes in the brain.

Unfortunately, there are relatively few effective therapeutic interventions available for the treatment of drug addiction. Most modern therapeutic approaches involve detoxification, alleviation of the negative symptoms associated with drug withdrawal and the promotion of abstinence via psychotherapy (A.D.A.M. Medical Encyclopedia, 2011). However, neither of these methods address the addiction-induced structural modifications nor effectively prevent potential relapse.

This has led to a joint effort to uncover the molecular and cellular basis of addiction (Nestler 2004, 2011; Koob and Le Moal 2005; Kalivas and Volkow 2005; Kauer and Malenka 2007) in hope of treating and preventing addiction in the future. More insight into drug-induced structural modifications could help uncover novel targets for the treatment and prevention of addictive disorders (Robison and Nestler, 2011).

It is generally accepted that compulsive drug-seeking behavior is a defining hallmark of drug addiction. Such behaviour can persist for years after the cessation of drug administration (Nestler, 2004). This has led to the hypothesis that drugs of abuse have the ability to induce long lasting structural changes in the brain that lead to uncontrolled drug-seeking behaviour. Further investigations showed that repeated drug administration caused long-lasting restructuring of neuronal arbors and dendritic spines (Robinson et al., 2001). This investigation strongly correlates with findings that chronic exposure to drugs of abuse results in an altered pattern of gene expression in key brain reward areas (Golden and Russo, 2012).

The brain's reward circuit is a key substrate for drug induced structural remodeling. It is centered on dopaminergic projections from the nucleus accumbens (NAc) to the ventral tegmental area (VTA), amygdala, hippocampus as well as parts of the prefrontal cortex (Koob and Le Moal, 2005; Kalivas and Volkow, 2005). The reward pathway is a highly conserved feature of the mammalian brain; it is key in promoting activities that are essential to species survival, such as sex, feeding behaviors and social interactions.

Acute drug administration allows addiction drugs to hijack this natural reward pathway causing increased dopaminergic (DA) signaling between NAc and associated structures; this increased DA signaling leads to the emergence of short term changes in the brain. Chronic drug use induces long-term structural changes that underlie uncontrolled addiction-related behavioral phenotypes.

Fig 1: Brain circuits and structures implicated in drug addiction.

The NAc is important in identifying stimulants by assessing reward and saliency. The orbitofrontal cortex is involved in decision-making and determining the expected rewards of a stimulus. The amygdala and hippocampus are involved in memory formation of the reward. Addictive drugs cause dopaminergic neurons from the VTA to release DA in the NAc. This in turn is believed to regulate activity in the frontal cortical regions thus driving different aspects of addiction. (Adapted from Lee et al. 2012).

However, most individuals are exposed to drugs of abuse at least once during their lifetime, but only a small fraction will go on to develop the addictive phenotype. It remains unclear how structural modifications in the brain propel pathological behavioral aspects that are evident in addiction (e.g. drug craving, compulsive drug taking behavior, withdrawal symptoms and relapse) in some individuals but not in others. Genetics and epigenetics may hold the answer to this question.

As mentioned above, the phenomena of addiction is very complex and cannot be accounted for by any one single factor but rather is the result of a complex interaction between genetics and environmental stimuli.

Two studies on twin pairs carried out by Kendler and colleagues explored the role of genetic pre-disposition and addiction. The first study demonstrated that monozygotic (MZ) twins show a much higher risk of developing cocaine addiction if one twin is an addict, compared to dizygotic (DZ) twins. The results for genetic risk of cocaine dependence were 35% and 0% respectively for MZ and DZ (Kendler and Prescott, 1998). The second study incorporated a number of addictive drugs and found that the total heritability varied widely from 34% for caffeine dependence to 73% for nicotine dependence. Hence it was concluded that genetic factors account for approximately 50% of an individual's vulnerability to addiction (Kendler et al., 2007).

Fraga and colleagues also carried out a study on monozygotic (MZ) twins pairs to explore the role of epigenetics in addiction. The study showed that while MZ twins are genetically identical at birth, they develop differences in a wide range of anthropomorphic features such as susceptibility to disease (Fraga et al., 2005). Hence epigenetics have the capacity to impact behavioural states and may be strongly implicated in the development of addictive phenotypes.

The term epigenetics is used to describe heritable changes in gene expression that lead to cellular and molecular changes (Herring. 1993) but cannot be explained by changes in the underlying DNA sequence (Riggs et al., 1996). Epigenesis refers to functionally relevant modifications to the genome through post-translational chromatin remodeling. As revealed in recent research, certain chromatin modifications can influence chromatin structure and gene expression. Several such mechanisms have been identified; the two major ones are post-translation histone modification and DNA methylation (Allis et al., 2007).

Chromatin is a complex of DNA and proteins that makes up the eukaryotic chromosome. The function of chromatin is to ensure the correct packaging, organization, and readout of DNA material during cellular replication and differentiation (Borrelli et al., 2008). The basic unit of chromatin is the nucleosome; each nucleosome is comprised of 147 DNA base pairs wrapped in 1.67 super helical turns around an octameric histone protein (H) core with two copies of each H2A, H2B, H3 and H4 (Luger et al., 1997).

Chromatin modifications are important in regulating gene expression and, consequently, may contribute to drug-induced behaviors. The structure of chromatin is mainly altered by mechanisms of histone modification and DNA methylation; these promote post-translation remodeling and alter levels of chromatin compaction, resulting in either a more transcriptionally active or repressed chromatin state. For example, histone modifications such as acetylation loosen the DNA thus making it more accessible to transcriptional factors, correlate with transcriptionally active states. Whereas modifications that promote DNA compaction, such as methylation are associated with transcriptional repression (Maze and Nestler, 2011).

Drug induced chromatin modifications have been studied in animal models. These studies demonstrated that administration of stimulants such as acute and chronic cocaine and chronic amphetamine increased total cellular levels of histone modification as well as DNA methylation in the NAc. Sedatives were also found to increase histone modification levels in striatal and cortex areas (Graff et al., 2011). These results provide evidence that chromatin modifications may underlie some of the functional abnormalities found in addiction models (Kumar et al., 2005).

Histone Acetylation: Histone acetylation entails the addition of acetyl groups onto lysine residues of the histone tails.

It is controlled by histone acetyltransferase enzymes (HATs) and histone deacetyltransferases (HDACs), which add and remove acetyl groups respectively. Acetylation usually occurs at the NH2-terminal of the histone tail; the addition of acetyl groups negates the positive charge of the N-terminus and hence decreases histone interaction with negatively charged phosphate groups of the DNA molecule. This results in a "loose" chromatin structure which is much more accessible to transcription factors and thus more transcriptionally active.

Drugs of abuse induce changes in histone acetylation in the brain. For example acute and chronic exposure of rats to cocaine induced H3 and H4 acetylation in NAc and striatum. These changes in acetylation are thought to underlie some of the functional abnormalities seen in cocaine addiction.

Histone methylation: As the addition of acetyl groups in acetylation; methyl groups can also be added to the lysine and arginine residues of the NH2-terminal tail of the histone. This epigenetic modification is known to have a bi-directional effect on transcriptional activity; methylation is associated with both active and silenced genes.

As with histone acetylation, drugs of abuse can also affect histone methylation.

DNA methylation: DNA methylation involves enzymatic addition of methyl groups to DNA cytosine bases.

DNA methylation occurs at 5′-CpG-3′ (cytosine-phosphate-guanine) sequences. The addition of methyl groups results in the conversion of cytosine to 5methyl-cytosine, which acts very similarly to the original molecule and binds readily to guanine.

DNA methylation is required for proper organism development, tissue-specific gene expression, as well as other vital activities. Therefore human DNA is normally highly methylated. Changes in the levels of methylation for example under-methylation can lead to the development of abnormalities such as cancer and obesity.

DNA methylation is believed to be a transcriptionally repressive modification; it inhibits the binding or transcription factors to the DNA by recruiting co-repressor molecules such as methyl-CpG binding proteins (MBPs). However, highly methylated regions of DNA have also been found at promoter sites of actively transcribed genes, hence the functional impact of DNA methylation on transcriptional activity is not clear (Suzuki and Bird, 2008; Graff et al., 2011).

Drugs of abuse induce changes in DNA methylation. For example repeated cocaine administration was shown to increase DNA methylation in NAc through the upregulation of MeCP2 (Host et al., 2011).

In recent years, these epigenetic mechanisms have become significantly implicated in a wide range of diseases including drug addiction. Addictive drugs drive epigenetic mechanisms that alter gene expression leading to structural change in the brain.

A large number of works have focused on epigenetic mechanisms and their contribution to the development of drug addiction, in hope that insights into such mechanisms may prove beneficial in the search for novel therapeutic targets for the prevention or reversal of drug induced neuronal modifications implicated in addictive phenotypes (Maze and Nestler, 2011; Kauer and Malenka, 2007).

Weaver and colleagues showed that chromatin restructuring can be induced by a socio-environmental factor such maternal behaviour and subsequently reversed using pharmacotherapy. Weaver showed that an increase in pup licking and grooming (LG) by rat mothers increased histone acetylation and DNA methylation in the offspring. Also LG upregulates the expression of glucocorticoid receptors (GR) through a serotonin signaling cascade, as a result of this structural change the offspring of high LG mothers are less anxious and less stressed compared to the offspring of low LG mothers. This indicated that structural changes at the synapse lead to altered behavioural states. Remarkably, Weaver's findings revealed that synaptic changes could potentially be reversed by the administration of histone deacetylase inhibitors (HDACi). This suggests that therapeutic agents may have the capacity to reverse structural changes that drive disease states (Weaver et al., 2004).

However, the search for novel therapies is made harder by the fact that different classes of addictive drugs act on different parts of the reward pathway and promote opposite structural effects on dendritic spines (Fig. 2).

Fig. 2. Action of stimulants and opiates on neurons of the VTA and NAc.

A VTA dopamine neuron is shown innervating a NAc medium spiny neuron. Opiates and stimulants produce generally opposite effects on gene expression and dendritic spine structure of these two neuronal cell types while inducing the same behavioral phenotype. Stimulants such as cocaine primarily act in the NAc whereas sedatives such as heroin mainly stimulate neurons of the VTA. Stimulants increase spine density and dendritic branching in dopamine neurons of the VTA and MSNs of the NAc; whereas sedatives decrease the number and complexity in these same neuronal cells (Taken from Russo et al. 2009).

It is hypothesized that different drug classes promote dendritic restructuring through processes of long-term potentiation (LTP) and long-term depolarization (LTD). A number of recent studies has supported this hypothesis and showed that excitatory synapses in brain reward regions can express LTP and LTD-like changes in response to addictive drugs (Nagerl et al. 2004; Tada and Sheng, 2006). This suggests that the translation to addiction involves the disruption of plasticity mechanisms (Nugent and Kauer, 2008; Nugent et al., 2007). In general, LTD causes shrinkage and retraction of dendritic spines, whereas LTP promotes new spine formation and enlargement of preexisting spines (Nagerl et al. 2004).

Nester has written extensively on drug induced synaptic plasticity and the idea that addictive drugs activate signaling pathways that are involved in cytoskeleton formation and thus promote dendritic restructuring. Numerous factors have been implicated in the drug-induced synaptic plasticity drive: including transcription factors ΔFOSB and CREB, enzymes such as HDACs and MeCP2. All of these regulate transcriptional targets such as NFkB and Cdk5 consequentially leading to various structural alterations (Nestler, 2004; Robison and Nestler, 2011).

PHASE II

However, until now the phenomena of drug-induced dendritic restructuring has only been studied using only in-vitro experiments; a search of the literature also shows that all these studies focused almost exclusively on stimulant drugs and never on sedatives.

In-vitro studies show that stimulant administration induces persistent increases in dendritic spine density and arbourisation in pyramidal neurons of the PFC and of MSNs throughout the NAc. In stark contrast, sedatives decrease the spine density and arbourisation in NAc and PFC (Robison and Kolb, 2004; Russo et al., 2009; Russo et al., 2010; Ferrario et al., 2005).

As mentioned above the majority of data available on drug-induced structural plasticity comes from in-vitro studies, in particular from Golgi-Cox analysis of MSNs in the NAc and pyramidal neurons in the PFC. In one such study Kolb showed that cocaine self-administration in rats caused an increase in dendrite arbourisation and density in pyramidal neurons of the PFC (Robison and Kolb, 2001). Interestingly, rats that were administered stimulants by a lab technician exhibited a lower degree of dendrite arbourisation compared to rats that self-administered, thus self administration is thought to be an important factor in drug induced synaptic plasticity. Kolb also showed the presence of malformed pyramidal dendrites in the PFC of self-administration rats, these malformations might underlie some of the cognitive deficits seen in addiction such as impaired decision making.

Similarly in the NAc, when assessed by Golgi-Cox method, both amphetamine and cocaine show increases in dendritic spine density and dendritic arbourisation (Robison and Kolb, 2001; Robinson and Kolb, 2004). These changes appear to be specific to Drd1-receptor-expressing MSNs in the NAc. These dendritic alterations have been shown to persist long-term after the cessation of drug administration (Lee at al., 2006). Since Drd1-receptors are known to regulate neuronal growth and development as well as mediate some behavioral responses, these changes in dendritic are thought to underlie pathological behaviour that is seen in addiction.

The Golgi-Cox is now considered to be low-resolution methodology of structural analysis (Golden and Russo, 2011). Another limitation of the Golgi-Cox staining method is that doesn't offer any insight into whether the growth and retraction of spines in response to drug administration correspond the creation of new synapses and synapse elimination respectively.

The recent emergence of more advanced methods such as two-photon microscopy (Denk et al. 1990) may allow for a higher degree of understanding of drug induced structural plasticity in brain areas that are proximal to the brain surface such as the PFC (Golden and Russo, 2011). Two-photon microscopy (TPM) is one of the few methods that allow for in vivo imaging of the nervous system. In vivo imaging techniques are becoming new and exciting tools in neurological research as they offer a unique insight into behavioural adaptations or experience-dependent synaptic plasticity of the intact nervous system (Trachtenberg et al., 2002).

In the past number of years several transcranial in-vivo imaging studies that employed TPM have been conducted (Trachtenberg et al., 2002; Bonhoeffer and Yuste, 2002; Grutzendler et al., 2002; Zuo et al. 2005; Yang et al., 2009; Sigler and Murphy, 2010). They were able to provide images of individual dendritic spines in multiple regions of the PFC over time intervals (Grutzendler et al., 2002; Zuo et al., 2005).

through a thinned skull is a minimally invasive method that allows for longitudinal studies of cortical structures at high optical resolution over intervals ranging from minutes to years (Grutzendler et al. 2002; Yoder and Kleinfeld 2002; Stosiek et al. 2003; Tsai et al. 2004; Davalos et al. 2005; Nimmerjahn et al. 2005; Zhang et al. 2005; Zuo et al. 2005a,b; Haynes et al. 2006; Majewska et al. 2006; Nishiyama et al. 2007; Xu et al. 2007; Kim et al. 2009; Wake et al. 2009; Wu et al. 2009; Yan et al. 2009; Yang et al. 2009). By creating a thinned skull (thickness _20 μm) cranial window, it has been possible to image fluorescently labeled structures located up to _400-μm deep within the cortex (Grutzendler et al. 2002; Tsai et al. 2004; Davalos et al. 2005; Zuo et al. 2005a; Yang et al. 2009). In recent years, the transcranial two-photon imaging approach has been used to study the development and plasticity of synaptic connections (Grutzendler et al. 2002; Tsai et al. 2004; Zuo et al. 2005a,b; Majewska et al. 2006; Nishiyama et al. 2007; Xu et al. 2007; Wu et al. 2009; Yang et al. 2009), neuronal network activity (Stosiek et al. 2003), cerebral microvascular occlusion (Yoder and Kleinfeld 2002), amyloid plaque deposition (Christie et al. 2001; Tsai et al. 2004; Yan et al. 2009), and microglial dynamics and function (Davalos et al. 2005; Nimmerjahn et al. 2005; Haynes et al. 2006; Wake et al. 2009).

Two-photon imaging through a thinned and intact skull is a relatively straightforward and minimally invasive method for high-resolution study of structural and functional changes in the living cortex. In addition to thinned skull preparations, the cortex can also be visualized through open-skull windows after performing a craniotomy and replacing the skull with a cover glass and/or a layer of agarose on top of the dural layer (see Svoboda et al. 1997; Trachtenberg et al. 2002; Holtmaat et al. 2005, 2006, 2009; De Paola et al. 2006; Lee et al. 2006; Keck et al. 2008; Hofer et al. 2009; Rose 2011). It is important to point out that skull removal can induce a significant inflammatory reaction, and thus could confound efforts to elucidate changes of neurons and glia in the intact brain (Xu et al. 2007; Holtmaat et al. 2009; Yan et al. 2009). The thinned skull window approach not only causes minimal perturbation, but also allows imaging of the cortex immediately after surgery, as opposed to the open-skull method, in which optimal imaging quality is achieved many days after craniotomy (Grutzendler et al. 2002; Trach- tenberg et al. 2002; Holtmaat et al. 2005; Zuo et al. 2005a,b; Yang et al. 2009). Therefore, we suggest that two-photon imaging through a thinned skull window, rather than through an open-skull window, should be the method of choice for studying structure and function of the living cortex. In experiments that cannot easily be performed without removing the skull, it is important to interpret the data carefully in the context of the confounding factors associated with open-skull preparations.

However, even with the use of TPM it remains extremely difficult to perform imaging of synaptic plasticity in the NAc.

Trachtenberg et al carried out one such study; they were able to demonstrate that in experience-dependent synaptic plasticity spine sprouting is associated with synapse formation, and spine retraction with synapse elimination (Trachtenberg et al., 2002).

Therefore, it would be beneficial to a central question in addiction studies is to what extent dendritic spines change in response to addictive drugs in-vivo and how this leads to the development of addictive behaviour. The search for the answer has led to a collaborative effort to uncover the drug-induced neuroadaptations underlying addiction.

PHASE III

The goal of the proposed experiment is to characterize the effects of cocaine and heroin self-administration in rodents on dendritic spine density in the prefrontal cortex (PFC) in-vivo using two-photon microscopy and to subsequently carry out a quantitative analysis of synapses to determine whether spine sprouting is associated with synapse formation, and spine retraction with synapse elimination.

Prior to two photon imaging rodents have to be trained to self-administer drugs in an operant chamber apparatus. Once rodents are trained they will be subjected to minor surgical operation, which involves the thinning of the skull to make it optically transparent to the two-photon microscope. After in-vivo two-photon imaging of the PFC, rodents will be euthanized and PFC pyramidal neurons will be studied using electron microscopy.

Rats are the recommended animal models in this this experiment.

Self-administration training:

Prior to self-administration training rats should be outfitted with an intra-jugular catheter using procedures described previously (Thomsen and Caine, 2005), in particular Basic protocol 3 is recommended. Once the catheter is in place it is flushed with sterile saline solution containing antibiotic (Thomsen and Caine, 2005; Ferrario et al., 2005) to prevent infection and to make sure that the catheter is functioning properly. Control rats should also be outfitted with a jugular vein catheter. After all the animals are fitted they are allowed a few days of rest and acclimation.

Self-administration training methodology recommended for use be one described by C.R. Ferrario (Ferrario et al., 2005). However, it is proposed that the operant chamber is fitted with a bar press as opposed to a nose-poke port. This is due to the fact that a bar press is a more typical manipulandum for rats.

Rats fitted with catheter should be divided into three groups: (A) will receive cocaine upon pressing the active lever (B) will receive heroin (C) control group will receive saline solution. Self-administration training should be conducted in standard operant chambers that are located inside soundproof cabinets. This prevents the effects of environmental stimuli on the experiment. Each operant chamber should be fitted with two lever presses with a light above each lever as well as all the standard features (Koob et al., 2007). Both lever presses are then connected to a computer and each lever press during the self-administration training session is recorded. Pressing one of the levers (inactive) had no consequences but is recorded. Alternatively pressing the active lever results in the delivery of either cocaine or heroin to the rat accompanied by the illumination of a light above the active lever.

Control rats are placed into an operant chamber identical to the self-administration chamber. They were administered a dose of saline solution following each lever press. Thus, the control group experiences were identical to those of animals with self-administration experience, one exception being that they weren't administered a drug and hence didn't experience drug-related reward. In the control group lever presses per day are expected to decrease as days go on, this can be explained by the fact that rats get accustomed to the fact that nothing happens in the operant chamber or to the rat following a lever press, so quickly enough they stop pressing the lever. However a small number of lever presses a day is still expected, these are taken to be accidental.

Upon completion of the last self-administration session, all rats were returned to normal housing conditions and were left undisturbed for 1 month. Following this period of abstinence, a subset of rats was chosen from each of the three groups to undergo drug-seeking screening. Rats that exhibited drug-seeking behaviour as well as control rats were taken for an in-vivo two-photon microscopy study.

Two Photon Microscopy:

Rats that were selected after self-administration training had to undergo a surgical procedure, which involves thinning of the rodent skull to facilitate in-vivo two-photon imaging. Two-photon imaging through a thinned intact skull is preferential to partial skull removal as it is a minimally invasive method for studying the rodent brain in-vivo (Grutzendler et al., 2011). In this case two-photon imaging is used to provide insight into dendritic spine dynamics in stimulant and sedative addiction in vivo. Hopefully, the results from this study will reveal new information about in-vivo spine dynamics and show whether these findings correlate with in-vitro studies (Robison and Kolb, 2001; Robison and Kolb, 2004; Russo et al., 2009; Russo et al., 2010).

This thinned skull preparation as well as the subsequent imaging of the PFC has been described previously (Grutzendler et al., 2011). This is the recommended protocol.

Synapse Quantification:

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