The role of central amygdala neuroinflammation in alcohol use and alcohol withdrawal-induced anxiety
Restricted (Penn State Only)
- Author:
- Melkumyan, Mariam
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 13, 2023
- Committee Members:
- Nicholas Graziane, Major Field Member
Salvatore Stella, Major Field Member
Yuval Silberman, Chair & Dissertation Advisor
Alistair Barber, Program Head/Chair
Jennifer Nyland, Major Field Member
Aron Lukacher, Outside Unit & Field Member - Keywords:
- Central Amygdala
Alcohol
Astrocyte
Microglia
Glutamatergic transmission
Neurotransmission
Binge Drinking
Calcium Transients
Alcohol Withdrawal-Induced Anxiety
Cannabinoids
Cannabidiol (CBD)
Tetrahydrocannabinol (THC)
Neuroimmune Activity
Neuroinflammation - Abstract:
- Alcohol use disorder (AUD) is a medical condition affecting around 15 million individuals in the US annually. There are several treatment options for AUD, including medications and therapy, however, none of these options work for everyone. One of the reasons for the limited treatment efficacy is that alcohol affects various brain regions differently, and there is a need for extensive studies understanding the role of alcohol in these regions. The focus of this dissertation is the central amygdala (CeA), a region in the brain classically involved in fear and anxiety, but also highly relevant in binge intake behaviors and risk for AUD and other substance use disorders. While the CeA is known to play a critical role in binge behaviors and development of AUD, the mechanisms by which acute and chronic alcohol alter CeA neurocircuits is not fully understood. As described in Chapter 1, one limitation of the previous research is the focus on the medial subdivision of the CeA with limited understanding of alcohol effects on other CeA subdivisions or how these effects across subdivisions interact to produce deleterious behavioral outcomes. The previous work exploring the role of acute alcohol on inhibitory neurotransmission in the medial subdivision of the CeA show an increase in inhibitory neurotransmission after alcohol application to the brain slice due to a variety of potential mechanisms that may be sex- and strain-specific. Chronic alcohol exposure has also been shown to increase inhibitory neurotransmission and potentially sensitize the effect of subsequent alcohol application. In terms of excitatory neurotransmission in the medial CeA, the findings are mixed, with some studies showing increased excitatory neurotransmission after alcohol use, while others showing decreased or unaffected excitatory neurotransmission. Findings in the lateral subdivision of the CeA are limited, mainly to previous work from the Silberman Lab. While no studies to date have explored the effects of alcohol on inhibitory neurotransmission in the lateral CeA, the previous work shows an increase in excitatory neurotransmission after acute alcohol application. As discussed in Chapter 1, the changes in both excitatory and inhibitory neurotransmission may be mediated by numerous neuropeptides and neuromodulators. Specifically for this dissertation, we sought to examine the role of cannabinoids and neuroinflammatory activity through astrocytes and microglia in mediating alcohol effects on lateral CeA excitatory neurotransmission, as alcohol use has been shown to increase neuroinflammatory activity in the brain, including in the CeA. Our first study sought to examine the effects of alcohol on excitatory neurotransmission in the lateral subdivision of the CeA and identify whether any changes in neurotransmission are due to neuroinflammatory activity. In chapter 2 of the dissertation, we utilized whole-cell patch-clamp electrophysiology to record changes in excitatory neurotransmission in response to alcohol with and without the presence of pharmacologic and chemogenetic manipulations of glia. We show that bath application of alcohol on a CeA containing mouse brain slice leads to an increase in excitatory neurotransmission in the lateral subdivision. When we used pharmacologic and chemogenetic approaches to inhibit astrocytes and microglia, we found that astrocytes, but not microglia, attenuated the effect of alcohol on excitatory neurotransmission. Therefore, in chapter 2 we show that alcohol increases glutamatergic neurotransmission in the lateral subdivision of the CeA through astrocytic mediated mechanisms. Changes in brain activity ex vivo are not necessarily indicative of changes in behavior. Therefore, in chapter 3, we sought to examine whether manipulating CeA astrocytes will lead to a reduction in alcohol consumption. Our lab has developed a model of binge alcohol consumption through a combination of limited high fat diet intake and limited alcohol intake. We have shown that one day/week of high fat diet significantly increases binge alcohol intake during a 4-hour/day drinking period in the subsequent days. For chapter 3, we performed two experiments examining the impact of astrocyte inhibition on high fat diet and/or alcohol intake. In the first experiment, we inhibited the astrocytes on the high fat diet consumption day. In the second experiment, astrocytes were inhibited on the first alcohol intake day of each week. Overall, we found an increase in alcohol consumption when the astrocytes were inhibited on the high fat diet day but found a cumulative decrease in alcohol consumption on the first alcohol intake session of each week in the second experiment, suggesting that astrocytes in the CeA play a role in modulating alcohol consumption. Our findings of chapter 2 were extended when we tested the effect of calcium activity of astrocytes during alcohol exposure and saw significant increases in calcium activity in response to alcohol compared to baseline firing, with a sensitized effect in mice that underwent the binge eating and binge drinking paradigm. While the CeA is a region known for the regulation of anxiety-like behaviors, it is also known that alcohol withdrawal can induce anxiety, potentially leading to further alcohol use. Due to the involvement of CeA in anxiety, we wanted to test whether we can reduce alcohol withdrawal-induced anxiety via modulation of CeA neuroimmune function. Cannabinoids are currently being used for the treatment of anxiety, however, the mechanism of action of cannabinoids in reducing anxiety is highly debated. Cannabidiol (CBD) has been shown to reduce neuroinflammatory activity in the brain, potentially leading to reduced anxiety. Mixtures of CBD and Δ9-tetrahydrocannabinol (THC) have been shown to be highly effective against anxiety, however, the previous findings are mixed. Therefore, in chapter 4, we sought to examine the effect of CBD and a mixture of CBD and low THC (3:1 CBD:THC) on alcohol withdrawal-induced anxiety at two different withdrawal timepoints and determine if this treatment alters CeA neuroimmune activity. We found limited significant effect of cannabinoids on anxiety-like behavior, with 3:1 CBD:THC having increased psychoactive effect at 24-hour withdrawal period in non-stressed males. We also found that CBD and THC are metabolized differently in 24-hour withdrawal compared to air control. Lastly, we found decreased neuroinflammatory activity as evidenced by decreased astrocytic activity and decreased microglial density at 4-hour, but not 24-hour withdrawal. Overall, this dissertation covers the role of CeA neuroinflammation in alcohol's effect on excitatory neurotransmission, alcohol consumption, and alcohol withdrawal-induced anxiety. Findings from this dissertation will advance the field of alcohol by uncovering the relationship between CeA astrocytes and alcohol-induced effects. To our knowledge, these are the first studies that explored alcohol's effects on calcium transients of CeA astrocytes. To our knowledge, these studies were first to examine the efficacy of CBD:THC mixture on alcohol withdrawal-induced anxiety. Therefore, this dissertation may open new avenues for exploring alcohol-induced neuroinflammatory activity and can lead to the development of new therapeutics targeting the neuroinflammatory activity in the brain to reduce alcohol consumption.