Main Question:

How might alcohol induced glutamatergic neuroadaptations, specifically of α-amino-3‑hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic receptors (AMPAR) and N-methyl-D-aspartate receptors (NMDAR) in the amygdala, affect relapse potential in individuals with alcohol use disorder (AUD)?

Significance: 

Alcohol contains ethanol, which is a toxic substance that can cause dependence and a plethora of both physical and mental health issues. However, it remains one of the most unregulated, normalized, and widely distributed drugs worldwide.

According to World Health Organization (WHO), about 7% of the world's population (400 million people) live with AUD as of 2019. Amongst these, 209 million are alcohol dependent. While small doses of ethanol may not cause immediate dependence, chronic heavy alcohol consumption and dependence create countless health concerns. Some of the more commonly known concerns include liver disease, cardiovascular disease, depression, and cancer. Although the severity and danger of these countless life-threatening noncommunicable diseases is obvious, it is also imperative to be aware of less considered AUD-related deaths. These include:

• Deaths caused by alcohol-related injuries (an estimated 700 000 deaths in 2019)
  • Injuries are commonly sustained through scenarios such as drunk driving, alcohol-related domestic violence, and alcohol-related reckless behaviours
• Deaths caused by communicable diseases (an estimated 300 000 deaths in 2019)
  • Drinking impairs judgement
    • This can lead to risky sexual behavior, which increases chances of transmitting sexually transmitted diseases and infections (STDs and STIs) including chlamydia and syphilis
  • Drinking weakens the immune system
    • Increases the chances of catching a communicable disease and not being able to respond appropriately

In order to help prevent some of these health concerns and to improve quality of life, individuals with AUD will often try to "quit" their addiction. While there have been many successful clean individuals, the relapse rate for AUD is extremely high. There are multiple factors at play for these rates, including the widespread and normalized use of ethanol, withdrawal symptoms, and environmental factors. However, studies₁ have also shown that chronic ethanol consumption alters neurons in the brain and rearranges the brain structure which creates increased alcohol dependence. In this project, we will specifically investigate the role of the amygdala AMPA receptors and NMDA receptors and how their neuroadaptions can affect relapse potentials. This will open possibilities of psychobiology treatments that could potentially reduce relapse rates.

Hypothesis

We propose a multi-step hypothesis that could potentially explain the role of glutamatergic receptors in the amygdala which lead to AUD relapse.

1. If chronic ethanol operant self-administration occurs, then there will be an upregulation of AMPAR and NMDAR in the amygdala because of a lack of excitatory neurotransmission received by the postsynaptic neurons. In the amygdala, this will then lead to an emotional association to ethanol.
2. During withdrawal, there will be a brief upregulation of receptors as the brain continues to register the lack of ethanol. Soon after, there will be an excess of receptors and an increased reception of glutamate which could explain hypersensitivity and negative moods associated with withdrawal.
3. Eventually, downregulation of these receptors will occur as the brain adjusts to maintain synaptic homeostasis
4. If relapse occurs, then AMPAR and NMDAR remain deprived of glutamate and may induce a surge of upregulation. This may result in feelings of guilt, anxiety, and general emotional distress in relation to memory of alcohol.

Our project consists of three constituents: Background Research, Research and Data Analysis, and a Applied Research Proposed Experiment.

Constituent 1: Background Research

Through background research, we gained a general idea of the impacts of alcohol use disorder (AUD) on glutamate's two main ionotropic receptors: NMDA and AMPA, in response to ethanol dependency, withdrawal and relapse. 

Constituent 2: Research and Data Analysis

We analyzed the effects of ethanol exposure and dependency on relapse rates as a result of changes in AMPA receptor and NMDA receptor sensitivity as well as genetic expression rates in proteins. 

Constituent 3: Applied Research and Proposed Experiment

We gathered our research and formed an experiment that could be conducted with a sample population under the pretense of complete ethical permission and unlimited resources. The proposed experiment is made to address the gap identified in the analyzed studies. 

Background Research

What is Alcohol Use Disorder?

The Diagnostic and Statistical Manual of Mental Disorders: Fifth Edition Text Revision (DSM-V-TR) defines alcohol use disorder as follows:

"A problematic pattern of alcohol use leading to clinically significant impairment or distress, as manifested by at least two of the following, occurring within a 12-month period:

1. Craving, or a strong desire or urge to use alcohol.
2. There is a persistent desire or unsuccessful efforts to cut down or control alcohol use.
3. Alcohol is often taken in larger amounts or over a longer period than was intended.
4. Recurrent alcohol use resulting in a failure to fulfill major role obligations at work, school, or home.
5. A great deal of time is spent in activities necessary to obtain alcohol, use alcohol, or recover from its effects.
6. Alcohol use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by alcohol.
7. Continued alcohol use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of alcohol.
8. Important social, occupational, or recreational activities are given up or reduced because of alcohol use.
9. Tolerance, as defined by either of the following: 
   • A need for markedly increased amounts of alcohol to achieve intoxication or desired effect.
   • A markedly diminished effect with continued use of the same amount of alcohol.
10. Withdrawal, as manifested by either of the following:
    • The characteristic withdrawal syndrome for alcohol.
    • Alcohol (or a closely related substance) is taken to relieve or avoid withdrawal symptoms.
11. Recurrent alcohol use in situations in which it is physically hazardous."

NMDA

NMDA (N-methyl-D-aspartate) receptors are one of the ionotropic receptors that glutamate interacts with. It is responsible for synaptic plasticity that occur in response to learning and memory. It is formed from a tetramer, composed of four subunits/four proteins. The genes that code for these proteins are: Glu N1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B. Two GluN1 subunits are always present in the heterotetramer, and it never binds with glutamate, only with glycine in the ligand binding domain, which is required for NMDAR activation. Two non-GluN1 subunits are needed to allow glutamate to bind to their ligand binding domains. 

There are mainly found in the central nervous system, which consists of the brain and the spinal cord. In terms of cellular location, they are found in the dendritic spines of the postsynaptic membrane of neurons. They may also (but uncommonly) be found in the presynaptic membrane.

As an ionotropic receptor, it is a Ca2+ ion channel and activates when glycine and glutamate bind to it. The influx of Ca2+ into the neuron causes the activation of cAMP response element-binding protein (CREB), a transcription factor heavily involved in synaptic plasticity. 

Connection with Ethanol

Ethanol is thought to bind onto non-GluN1 subunits to prevent glutamate from binding. As a result, the NMDAR is inhibited which causes a decrease in excitatory neurotransmission. This contributes to the sedative and impairing effects of alcohol as action potentials are decreased and significantly reduces the strength of electrical signals in the brain. In withdrawal, NMDAR becomes overactivated which causes an excessive influx of Ca2+ which can lead to neuronal death (excitotoxicity). This notably leads to neurodegenerative disorders and seizures.

AMPA

AMPA receptors, or α-amino-3‑hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic receptors, are also ionotropic glutamatergic receptors responsible for synaptic plasticity and learning. In the amygdala, their role in excitatory signalling is particularly significant because it manages emotional memory and habit formation. Similar to NMDA, it is also a tetramer protein composed of subunits GluA1, GluA2, GluA3, and GluA4. 

AMPARs are essential to the central nervous system, which is why they are found in most parts of the brain and surrounding the spinal cord. These include: the limbic system, specifically the amygdala and the hippocampus; the motor system, in the cerebellum and the basal ganglia; the CNS, in the central cortex and the basal ganglia again; as well as in the spinal cord.

When AMPA with GluA2 are activated by glutamate, they allow sodium ions to flow into the postsynaptic neuron and cause depolarization. However, they are impermeable to calcium ions due to a change post-transcription where glutamine is converted to arginine in the subunit RNA. When a small amount of glutamate is released, the AMPARs stay open for a brief amount of time and the small influx of sodium ions create a minor post-synaptic neuron depolarization. When the quantity of glutamate released from the presynaptic neuron is larger, then AMPARs stay activated and open for longer. This results in a larger influx of sodium ions and a more pronounced depolarization in the post-synaptic neuron. The accumulation of positive charge from the sodium ions can also expel the positive magnesium ions that block NMDA. This will allow the larger and more significant calcium ions to flow into the post-synaptic neuron and cause further depolarization.

Connection with Ethanol 

One way ethanol significantly impacts AMPA is how it decreases the amount of GluA2 permeable to calcium. It downregulates the gene expression for the enzyme ADAR2 (Adenosine Deaminase Acting on RNA 2), which edits the RNA of GluA2 to be impermeable to calcium. The consequence of permeability in GluA2 subunit-containing receptors is a faster rate of neuron depolarization, which increases neuron excitation. It can lead to large calcium influx, which often leads to excitotoxicity. 

Glutamate

Glutamate is the most abundant excitatory neurotransmitter in the brain. It contributes to learning and memory, and is responsible for most excitatory connections and synapses in the brain. It strengthens the transportation of chemical messages throughout the brain. 

When it interacts with its receptors on the post-synaptic neuron, it causes an influx of ions which leads to neuron depolarization. This increases the likelihood that the post-synaptic neuron will fire an action potential. Its 3 main ionotropic glutamate receptors are: NMDAR, AMPAR, and Kainate Receptors. When activated, positively charged sodium ions flow into the postsynaptic neuron which depolarizes it and fires an action potential. It also interacts with 3 metabotropic glutamate receptors that have more varied effects than its ionotropic receptors. 

Glutamate is stored in synaptic vesicles that are located in the nerve endings, when a glutamatergic neuron is activated to pass a signal to other neurons, the synaptic vesicles fuse with the membrane of the terminal region to release the neuron. Glutamate crosses the synaptic cleft to interact with the receptors on the postsynaptic neuron.

The postsynaptic neutron sends glutamate back into the presynaptic neuron and glial cells to prevent the excessive excitation of the postsynaptic neutron. The glial cells converts glutamate into the glutamine.

Glutamate and Ethanol

Ethanol is found to inhibit glutamate’s excitatory effects as glutamate uptake is significantly reduced. This further inhibits synaptic plasticity, particularly LTP which is important for memory and habit formation. In withdrawal, as glutamate was previously suppressed by ethanol, the glutamate levels become hyperactive which causes excitoxicity and contributes to symptoms of withdrawal, such as, seizures and anxiety.

Detailed Literature Review

A control system analysis of the dynamic response of N-methyl-D-aspartate glutamate receptors to alcoholism and alcohol withdrawal | Theoretical Biology and Medical Modelling | Full Text

Abstract

Ethanol inhibits NMDAR during chronic ethanol use. These changes result in significant dependence to ethanol to feel “normal”. To add, if ethanol was removed from the system this results into excitotoxic withdrawal (severe effects of withdrawal). To control extracellular glutamate concentration, the brain attempts to adjust the number of NMDARs at the synapses. The brain is able to adjust them in three ways: making and inserting new receptors, removing receptors and placing then into another area, or changing these receptors. 

Study created a mathematical model to express how nmdar changes, looked into the effect of different drinking patterns on receptors during withdrawal, and measured how bad the withdrawal was based on changes in NMDAR. 

Chronic alcoholism leads to heavy dependance on ethanol. It leads to homeostatic adaptations in the brain. To add, the brain will not function unless there is a high concentration of ethanol in the bloodstream. If individuals decide to withdraw from ethanol, the body’s homeostasis is now disrupted, which causes really bad symptoms that make you feel sick. 

NMDAR plays a significant role in dependency and withdrawal symptoms. They become dysfunctional as the brain tries to balance glutamate levels. NMDAR are not fixed in number or composition, they adapt to neuronal activity and sensory input. When they are blocked, they rapidly respond by doing phosphorylation (specifically in NR2 subunits) which allows them to adapt to that short-term inhibition effect.

When long-term glutamate levels are reduced, NMDAR increases to compensate for this reduction in glutamate. Increasing NMDAR does not increase glutamate, it enables brain cells to feel more sensitive to glutamate. There was an increase in specific parts of nmdar in response to ethanol → NR2B subunit mRNA and NR1 subunit polypeptide.

When ethanol is removed, NMDAR becomes hyper activated and can lead to brain damage, seizures and increased sensitivity of the receptors.

Results and Discussion

Study developed a mathematical model to understand the behavior of NMDAR when exposed to ethanol. This is regulated as a negative feedback system. The model has two parts, activity controller and density controller. 

Activity controller: part that allow brain’s synapses to stay active by increasing number of nmdar at synapseDensity controller: part that adjusts number of nmdar by removing activer nmdar

Using mathematical models it showed that:

1. Increased number of NMDARs at synapses when exposed to ethanol
2. Severity of withdrawal symptoms are influenced by different patterns of alcohol consumption
3. When withdrawal happens, NMDARs are overactivated
4. Over time, NMDAR levels go back to normal after withdrawal

Limitations 

Model is based on hypothesis. No experimental data. Doesn’t provide information on how mechanisms work.

Chronic Ethanol and Withdrawal Differentially Modulate Pre- and Postsynaptic Function at Glutamatergic Synapses in Rat Basolateral Amygdala | Journal of Neurophysiology

Abstract

Withdrawal anxiety is associated with relapse in alcohol. The basolateral amygdala (BLA) deals with emotion. It regulates expression of fear and anxiety. Researchers looked into the effect of chronic intermittent ethanol and withdrawal on glutamatergic synaptic transmission in the BLA. NMDAR increase in glutamatergic synaptic transmission in BLA relates to fear-learning behaviors. NMDA-dependant changes in synaptic plasticity in BLA and hippocampus have similar molecular processes. Used chronic intermittent ethanol inhalation model (CIE). Repeated withdrawal is known to trigger seizures in response to convulsants. 

Animals and Chronic Ethanol Exposure

Male Sprague-Dawley rats were housed with a 12-h light/dark cycle and given food and water unlimitedly hehe. They were exposed to an ethanol vapor chamber! 4-6 rats per cage, ethanol vapor was pumped into chambers at a rate of 16 L/min. Animals were exposed to vapor or room air (there was a control group) for 12 h/day for 10 days. Rats were killed right after the 10th exposure (CIE). Another group was made, where they removed some of CIE rats from ethanol exposure for 24 hours then killed them (WD) group. Rats were anesthetized and decapitated and studied. 

Behavioral Assays

Anxiety-like behaviors were measured by placing rats into a two-compartment light/dark box. All rats start on the light section. They used two infrared sensors to analyze general locomotor activity, time spent in the light and dark compartments, number of light-dark transitions and more. 

There was increased anxiety in the WD group as rats spent less time on the light side, made few movements between light and dark areas and took longer to reenter light area when moving from the dark side.

Understanding Anxiety-like Behaviours

First microinjection experiment: A group of adult male rats were injected with muscimol, a drug that activates GABA_A receptors, and behaviour was examined in the light/dark box. The goal was to see if activating GABA_A receptors can calm down neuronal activity. Second microinjection experiment: A group young male rates were injected with DNQX, a drug that blocks AMPA receptors, and behaviour was also examined in the light/dark box. Goal was to see if AMPA could reduce neuronal activity as well. These two drugs show that they reduce anxiety-like behaviours in rats.

Results 

Both CIE and WD had an increased NMDA/AMPA current ratio in BLA neurons compared to control. NMDAR were more active than AMPAR. They are more responsive than the control group. To measure NMDA synaptic responses, they used a chemical called DNQX to block AMPAR and measure NMDAR activity. To measure spontaneous glutamatergic events, they used a toxin called TTX to stop neurons from firing action potentials.

CIE and WD group had an increased activity of NMDAR in BLA and showed stronger responses to stimulation. CIE and WD had more frequent sEPSCs. CIE and WD had increased presynaptic function. 

Overall results show that increase anxiety may be caused from the increase in glutamatergic function. NMDAR increases in function and expression in BLA neurons. It also increases AMPAR expression too.

Chronic Ethanol Exposure and Protracted Abstinence Alter NMDA Receptors in Central Amygdala | Neuropsychopharmacology

Abstract

Chronic ethanol treatment (CET) and early withdrawal changed glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of amygdala. NMDA-mediated EPSCs in CET rats were inhibited in CET rats in comparison to rats that have not been exposed to ethanol (naive rats) and had decreased paired-pulse facilitation (a measure of synaptic strength)

Rats were subjected to chronicle ethanol use and their brain slices were studied after stopping ethanol for 1 or 2 weeks. After 1 week without ethanol, CET brain slices were subjected again to ethanol and they had a similar reaction to rats that never had ethanol. The brain’s postsynaptic effects were reversed.

CET rats increased levels of certain NMDAR subunits → NR1 and NR2B compared to control rats

Materials and Methods

Slices: Amygdala slices from male Sprague-Dawley rats were anesthetized with halothane and decapitated.   

CET: Ethanol inhalation method. 2-4 male Sprague-Dawley rats per cage with a 0600 - 1800 light cycle and had unlimited access to food and water. Divided into two groups, one was subjected to ethanol vapor chambers, another into air-only chamber. CET rats were exposed to ethanol vapors for at least 2 weeks.

Blood alcohol levels of the CET and control rats were taken three times per week. CET rats showed no signs of abnormalities, they were pretty healthy.  

Results

Prolonged Abstinence

Depressant effect of acute ethanol on NMDA-EPSP/C amplitudes was stronger (increased sensitivity) in CeA neurons from CET rats than the control group. A 44mM ethanol concentration resulted in maximal inhibition of NMDA-EPSCs in both groups. For control rats, ethanol reduced NMDA-EPSCs by 15-20% and in CET rats, NMDA-EPSCs was reduced by 35-42%.

PPF indicates the synaptic activity → changes in glutamate release. Decreased PPF = increase in glutamate release. 

In control rats, PPF showed that there was little change in glutamate release after exposure to ethanol. In CET rats, PPF showed that ethanol increased glutamate release during the 1 week withdrawal period, but then went back to its normal level of glutamate release after the 2 week withdrawal period.

CET increased mRNA levels of NR1 (139%) and N2B (184%) subunits, it also increased protein levels of both subunits and N2A, these are the most-ethanol sensitive NMDAR subunits. These changes correlate to changes in glutamatergic transmission. During withdrawal period, 1 week of withdrawal → mRNA levels of both subunits decreased. 2 weeks of withdrawal → mRNA levels of both subunits returned to normal levels.

Protein levels, NMDA-EPSC amplitudes, NMDA currents and PPF returned to normal after the two week ethanol withdrawal period.

mGlu5 Receptors Creating Neuroadaptations in the Hippocampus

Materials and Method

Male wistar rats were raised and decapitated. Hippocampii were isolated, sliced, scanned for neurodegeneration, and then exposed to an ethanol medium that was refreshed at the same concentration every day to imitate chronic alcohol consumption. After 7 days, slices were placed in a fresh medium without ethanol to imitate withdrawal. All slices were analyzed for miRNA and electrophysiological investigations.

Results:

GluA1 and GluA2 were both significantly downregulated and had decreased numbers on the post-synaptic compartment after ethanol exposure and withdrawal. Scaffolding proteins forming NMDARs were significantly reduced. After ethanol exposure, microtubuli were significantly disorganized.

Discussion:

Ethanol exposure may cause microtubuli disorganization and decrease gene expression of both presynaptic and postsynaptic neurons. It also decreases the amount of post-synaptic excitatory transmission.

GluA1 Subunit and Relation to AUD Relapse Rates:

Purpose: 

To observe the effects of alcohol seeking behaviours after alcohol dependency and withdrawal, with special regard to alcohol deprivation effect (ADE). 

Materials and Methods:

Acamprosate (GYKI52466) is an AMPA antagonist thought to prevent relapse in alcoholic patients by reducing the hyperglutamaterica state of a dependent brain. Relapse behaviour is studied in two behavioural models: cute induced reinstatement and alcohol deprivation effect. A separate experiment used mice deficient in GluA3.

Two-month-old male Wistar rats were used. A group was genetically modified to have GluA3 gene deletions, referred to as group "kick-out" or KO. AMPA receptor subunit expression was evaluated with Western blotting in the hippocampi and cerrebelli because they would express changes most dramatically.

Cue-Induced Reinstatement Model in KO and Control Rats

Chambers had a response lever on each side panel, which would activate a syringe pump into a receptacle next to the lever. A stimulus light and a loudspeaker provided presentation of conditioned stimuli next to reinforcer delivery. A saccharin fading procedure trained the rats to self-administrate alcohol, and the conditioning phase began once the self-administration was stable. Sessions last thirty minutes with five daily sessions per week.

Conditioning Phase

During a conditioning phase, which would last 20 sessions, there would be a positive stimuli (s+) and a conditioned stimuli (CS+) accompanying the delivery of ethanol upon a lever press. The S+ was an orange smell preceding the lever push, and the CS+ was a 5 second light exposure accompanying the ethanol. Rats were also trained with negative stimuli (S-), which was the smell of anise, and conditioned negative stimuli (CS-), which was the a 5 second beep upon pressing the lever. These negative stimuli were associated with the delivery of water.

Extinction Phase

During the extinction phase, which would last twelve sessions, no odors or consequences would commence during lever presses. Rats were assigned to three matched groups according to their responses at the twelfth session.

Reinstatement Tests

Rats were injected with a vehicle (control) or with the antagonist acamprosate. Odors (S+/-) were not present, but responses at the lever were followed by CS+/- conditions. Half the animals in each group were tested under positive conditions, while the other half were tested under negative. The next day, conditions were reversed for both groups.

ADE in Rats

Rats were assigned to groups matched to their baseline consumption before ethanol removal of 20 days (after 40 weeks access). Each received the acamprosate antagonist the day before alcohol reintroduction. Ethanol was reintroduced and water consumption was measured.

ADE in GluA3 KO Mice

An additional group of mice were trained to drink ethanol in a free choice model where they could pick between drinking water and increasing concentrations of alcohol. Water and ethanol intake was calculated per day. ADE was assessed after 14 weeks of 16% alcohol consumption.

Results

Cue Induced Reinstatement and ADE Reduction from GYKI524 Acamprosate Antagonist

Operant responses (lever presses) decreased across extinction sessions. During the positive conditioning sessions, both groups of rats treated with high and low GYKI 52466 showed a higher number of presses that during the negative conditioning and extinction presses. In negative conditioning and extinction presses, there were no significant changes in lever presses and there were no significant differences between groups with different alcohol baselines. 

Rats treated with GYKI 52466 did not show an increase in ethanol consumption after deprivation. In fact, the decrease of ethanol drinking after reintroduction was very small in lower dosed GYKI 52466 compared to saline injected rats. Physiologically, the KO rats showed no difference from the wild-type rats except a minimally lower locomotive coordination. Psychologically, the number of AMPA subunits was drastically decreased in the cerebellum but seemingly unnaffected in the hippocampus. In KO rats, their increase in alcohol consumption on the first day of the post-extinction phase was significantly blunted. 

GluA3 KO rats and wild-type exhibited a similar amount of reinforced lever presses during operant ethanol self-administration training, and during extinction phase. Ability of noncontingent reintroduction of light reinstated behavior (CS+) after extinction period found that the number of lever presses on the previously designed active lever was significantly higher in wild type rats than GluA3 KO rats.

Conclusion and Discussion

GluA3 has a direct effect on ethanol seeking and relapse behavior. The GYKI 52466 antagonist is likely to be significant to the inhibitory effects on AMPA receptors with GluA3, as it reduces ethanol seeking behavior and decreases the occurence of ADE.

AMPA Receptor Activity and CaMKII Dependent Alcohol Self-Administration

Context and Introduction

Calcium 2+ ion calmodulin dependent kinase (CaMKII) is a protein found in high concentration in synapses that helps form memory in the nervous system by phosphorylating gene regulatory proteins, which changes the transcription of specific genes. Notably, GluA1 is dependent on CaMKII.

Positive feedback drives the initial addiction stage of alcohol intake (binge/intoxication). Ethanol usurps glutamatergic synaptic plasticity in reward circuits, and repeated exposure causes stronger synapses in brain regions that promote positive reinfocement and reward. In partcular, extracellular glutamate is in remarkably high levels in reward-related brain regions. Most relevantly, AMPAR GluA1 is significantly upregulated after both chronic exposure and low-dose operant self-administration. Inhibition of CaMKII in the prefrontal cortex increases reinforcing effects of alcohol, because it dampens senses of critical thinking.

Materials and Methods:

(P-) Adult male alcohol preferring rats were pair-housed and had ad libitum access to food and water.

Experiment 1: Evaluation of effects of operant self-administration on GluA1 post-translation

P- rats were trained to self-administer alcohol or sucrose. Brain tissue was collected after the 28th self-administration, and were processed for GluA1. Immunoreactivity was evaluated as well.

Experiment 2 Method: Evaluation of site-specific positive glutamate modulation activity at AMPA on alcohol self-administration

P- rats received site-specific infusion of aniracetam aimed at the CeA before self-administation sessions. Drug testing began on the 71st day of self-administration when aniracetam was administered an a randomized order, and then rats were assessed for locomotors to see if the positive modulator affected their movement. Sucrose trianed P- rats received CeA dose of aniracetame immediately before operant sucrose self-administration sessions on the 39th day.

Experiment 3: Evaluation for relationships between inhibition of CaMKII phosphorylation and alcohol self-administration

On the 35th day, P- rats received CeA m-AIP inhibitory peptide immediately before the operant alcohol self-administration. A second group of P- rats received intra-amygdala microinjections of vehicla and aniracetam to determine if the aniracetam-induced changes in alcohol self-administration were related to CaMKII.

Results: 

Experiment 1:

Operant performance is similar between sucrose and alcohol self-administration rats, which allowed experimenters to directly compare immunoreactivity between the two groups. Notably, pGluA1 was significantly increased in the BLA and the CeA, but not the LA. Data indicates that operant alcohol self-administration increases GluA1 phosphorylation in the amygdala and nucleus accumbens to a greater extent than a non drug reinforcer such as sucrose would.

Experiment 2

Aniracetam (positive AMPA receptor modulator) injected into the CeA increased the amount of self-administration. This increase was not affected by locomotor activity as intra CeA injections of the modulator had no effect on its activity. Water intake was not affected, and neither was sucrose intake, so the modulator-induced response was, as anticipated, unique to ethanol. Essentially, it was proved that artificial potententiation of the receptor in the amygdala causes increased ethanol self-administration.

This information could be potentially useful when assessing the speed and efficiency of receptor antagonists such as GYKI524 in treatment.

Experiment 3

Inhibiting CaMKII mAIP reduced alcohol reinforced lever responses. This is anticipated, because non-phosphorylated receptors are less efficient and, in the case of AMPA, be less effective at accepting both excitatory neurotransmitters and ethanol. 

Mock Experiment Method:

Female Representation in Neuroscience and Experimental Research

Before we dive into the methodology of our ideal experiment, we wanted to note the gender representation gap in neuroscience research. We noticed that female rats were almost never used in any of the experiments we explored, and later discovered that this general underrepresentation was because researchers wanted to avoidance of potential discrepancies caused by the hormone fluctuation involved in their menstrual cycles. We discovered that, as a result, an estimated 80% of neurscience research was done exclusively with male rats₂.

We believe that while it's important to control as many variables in order to minimize error, the lack of female sex representation in experiments may lead to results that are inaccurate to female sex humans. Humans have menstrual cycles that could interfere with the variables presented in data collected from only males, making it inaccurate₃. For this reason, we decided to incorporate them into our mock experiment, and avoided potential error by separating their analysis from the male rats.

Methodology

Rat groupings (larger and resizeable version can be printed from the canva link here: flowchart link)

Both female and male rats would be monitored for 6 months, with a separate group's brain slices being examined each week. They would be treated with alcohol vapour sessions to induce a dependency on ethanol, and then a saccharin fading procedure would be used to train an association between fulfilling alcohol dependency and bottles. Blood will be drawn after each session to measure their blood ethanol concentration (BEC) to ensure that the intended level of intoxication is reached. Conditioning phase commences, with use of a sweet smell (CS+) and a bitter smell (CS-). Baseline consumption would be measured and rats would be sorted accordingly once self-administration became stable.

The mice would then be subjected to an extinction phase, variable between a week, two weeks, three weeks, and four weeks. During this phase, no alcohol will be delivered and no consequences will occur when they press the lever. This will form withdrawal symptoms.

After the extinction phase, reinstatement phase will begin. Each group will be split in half between positive and negative stimuli reinstatement for a month.

Alcohol use would be measured through increments of volume, not lever presses. Lever presses cause the delivery into the bottle and is associated with stimuli.

Blood levels

Blood samples would be collected using the tail vein sampling method immediately after ethanol exposure, during extinction phase - to confirm ethanol clearance - and reinstatement phase. Blood would also be routinely drawn from the control rats. Blood samples during CET would measure blood ethanol concentration (BEC) to ensure that a BEC of 0.15-0.25 g/dL intoxication is maintained. The ethanol will be quantified using enzymatic assay, an alcohol dehydrogenase.

Slice preparation 

Rats would be anesthetized with isoflurane and transcardially perfuse with PBS. Then, rats would be decapitated to remove the brains. These brains would be placed into an ice-cold cerebrospinal fluid. The tissue would then be incubated in oxygenated aCSF for 1-2 minutes before slicing. A vibratome would be used to cut 300-400 µm coronal slices then cut 1.5-2.5 mm posterior to Bregma to obtain amygdala slices. Both slices would be placed into an oxygenated aCSF at 32 degrees celsius. For electrophysiology, slices would be kept in oxygenated aCSF, and used within 6-8 hours. While for Western Blot, slices would be homogenized in a buffer.      

Electrophysiology: Study neuronal function and activity (synaptic transmission)

Brain slices are transferred into a recording chamber which has a perfusion system to ensure slices are properly oxygenated and at a constant temperature. A seal will be formed between a glass pipette filled with an intracellular solution (KCl) and the cell membrane, eventually breaking the membrane to achieve whole-cell patch configuration. Using a voltage-clamp, synaptic currents and receptor activity (action potentials) would be measured. Data would be collected using an amplifier and a data acquisition system. Receptors would not be isolated, and studied simultaneously.  

Western Blot: Receptor expression of specific proteins: NR1 and GluA2

Supernatants would be obtained by centrifugation of the homogenized sample. Protein extract would be mixed with a Laemmli sample buffer to boiled to fully denature proteins. Proteins would be separated by size using gel electrophoresis and moved onto a nitrocellulose membrane. Proteins would be stained with antibodies: primary antibodies and secondary antibodies. Then, protein bands would be visualized with chemiluminescence.

Significant Data From Literature Review

Chronic Ethanol Exposure and Protracted Abstinence Alter NMDA Receptors in Central Amygdala | Neuropsychopharmacology

Figure 1a: Shows NMDA-EPSC recordings in a control rat

Figure 1b: Displays recordings in a CET rat

Figure 1c and 1d: Compares baseline NMDA-EPSP responses in control and withdrawn rats

Figure 1e: Shows NMDA-EPSC recordings from 1 week-withdrawn rats

Figure 1f: Shows NMDA-EPSC recordings from 2-week-withdrawn rats

Involvement of the AMPA Receptor GluR-C Subunit in Alcohol-Seeking Behavior and Relapse

Free Bottle Choice Model

Fig. A: Average voluntary ethanol consumption in 26 KO rats and in 32 wild-type, when offered the option between ethanol and water.

Fig. B: Percentage of KO and wild-type rats that preferred ethanol during the free bottle choice model.

Fig. C: Voluntary ethanol consumption in KO and wild-type rats before and after extinction (withdrawal) period.

Fig. A: Mean number of reinforced ethanol lever presses in KO and wild-type rats during their last 10 self-administration sessions.

Fig. B: The number of presses when stimulated by conditioned stimuli despite lack of desired ethanol delivery.

Rodent models for compulsive alcohol intake - PMC

Fig. (A) and (B): Alcohol and sucrose lever presses were similar enough to allow direct comparison of data.

Fig. (C): Phosphorylated GluA1 is in significantly higher levels in the basolateral amygdala (BLA) after ethanol exposure.

Fig. (D): Phosphorylated GluA1 is in significantly higher levels in the central amygdala (CeA) after ethanol exposure.

Fig. (E): No change in phosphorylated GluA1 levels after ethanol exposure in the lateral amygdala (LA).

 

 

Conclusion

Alcohol abuse continues to be an ongoing problem which ultimately affects both the individual and those around them. There are many interventions that have been used to treat individuals with AUD but there is yet to be a definitively effective solution to relapse. The literature has pointed part of this problem to the dysfunction of glutamate and its receptors: NMDAR and AMPAR.

As a result of chronic ethanol use, withdrawal symptoms emerge partly due to the hyperactive negative feedback loop in the amygdala. From the patterns we observed in our literature review, we gathered that AMPA and NMDA are enhanced during chronic ethanol use and  withdrawal. This happens because the brain detects a lack of excitatory neurotransmitters and attempts to increase the number of glutamatergic receptors in an attempt to return to baseline levels. This causes not only tolerance to inhibitors like ethanol, but also induces withdrawal symptoms which are very likely to influence relapse rates. Overtime, the changes in these receptors may be reversed after a long period of withdrawal.

Specifically in the amygdala, which is the centre of primal emotion and fear responses, we found that glutamate and its receptors have the potential to play a significant role in relapse rates and withdrawal symptoms. Increased excitatory activity explains the anxiety around operant self-administration that many patients have, especially during the beginning of abstinence. It also explains the emotional hypersensitivity associated with withdrawal. Emotional associations and the feelings of anxiety formed in the amygdala may be heightened, encouraging individuals to act upon the brain's physiological need for ethanol. Overall, these findings support our hypothesis.

Application

We wanted to take a step back to acknowledge the enormous behavioural aspect of relapse and withdrawal in regard to not just the amygdala, but to the rest of the brain and to society as a whole. Since ethanol consumption is normalised in most cultures, it is often associated with polite socialization, celebration, and stress relief. While we did not extensively explore environmental influences on relapse behaviour in this project, we recognize that it is an incredibly complex concept and continues to be relevant to relapse.

As for stress relief, we wanted to point out the vicious cycle of ethanol's immediate impact on the amygdala during consumption; ethanol effectively and temporarily numbing feelings of stress and anxiety, but causes them to return intensely after self-administration stops. Oftentimes patients may return to ethanol as a coping mechanism because of a deeply ingrained emotional association between stress relief and ethanol, rather than the rational association between ethanol and elevated feelings of distress. This complex and often conflicting emotional association involves the interwoven system between the psychobiology in multiple parts of the brain, and the structural norms of society.

We also wanted to recognize the lack of female laboratory animal representation in neuroscience. We acknowledge the importance of avoiding experimental error and maintaining efficient experiments; however, we feel that applying results universally across both sexes is innaccurate when data was collected from an exclusively male sample size. While biologically male and female sex humans contain countless similar characteristics, biological differences exist and cannot be ignored in research for cures that will apply to everyone.

Ultimately, much remains unknown about the link between relapse and the psychobiological impacts leading to and resulting from it.  Extensive research still needs to be done to further understand these mechanisms, with inclusion of female rats. No lab experiment can stimulate an individual’s withdrawal experience, and any proposed treatment based on further research done on these receptors will never be a one-size-fits-all to the AUD population.

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We would like to extend our thanks to Mr. Buhler, our science fair coordinator and teacher sponsor for supporting us in our project. We would also like to thank Research Coordinator Ms. Katherine Buhler at the University of Calgary, who helped us with our presentation and trifold. We are also very grateful for the guidance and feedback that we received from the Host-Parasite Interactions group at the University of Calgary and from Dr. E.P. Scarlett's STEM Club. Lastly, we would like to thank our families for their support!