KCC2 and Inhibitory Balance in Opioid Addiction
Supervised by: Dr. Alexey Ostroumov, Principal Investigator within the Department of Pharmacology & Physiology at Georgetown University.
Project Background
Opioid use disorder remains a pervasive public health issue in the US, with current treatments susceptible to high relapse rates and negative emotional states. Identifying the neural mechanisms underlying opioid use disorder is critical for understanding its pathology and developing sustainable, effective therapeutics. Previous research has demonstrated that the chronic use of opioids, amongst several other substances of abuse (2, 3, 4, 5) dysregulates inhibitory signalling in reward circuitry, in part through disrupting the maintenance of intracellular chloride ion homeostasis (1, 2). Further studies have elucidated a specific relevance of potassium chloride cotransporter isoform 2 (KCC2), which plays an essential role in regulating intracellular chloride in VTA GABA neurons (1, 2, 3, 4, 5). KCC2 dysfunction can overactivate brain circuits and contribute to addictive neuroplasticity. Notably, it is downregulated in response to drug exposure, inducing long-lasting impairment (1) of chloride homeostasis regulation and downstream disinhibition of brain regions involved in reward processing (2). Simultaneously, KCC2 restoration has been shown to reduce compulsive consumption of substances including benzodiazepines (3) and alcohol (4, 5, 6.) Considering KCC2’s role in maladaptive inhibitory plasticity across a range of substances including opioids, and its demonstrated potential in reducing addictive behaviors (4), KCC2 is a promising therapeutic target for substance use disorders. By studying its effects on morphine-self administration, I aim to clarify KCC2’s role in opioid addiction specifically. (206)
Research Questions
My proposed project seeks to determine whether improving normal inhibitory signaling in the brain, particularly through enhancing neuronal chloride extrusion, can reduce opioid use and addiction-related behaviors in a rat model. By using CLP290, a KCC2 enhancer, to upregulate KCC2 within a rat morphine self-administration model, I will further test KCC2’s effects on drug intake, motivation for drug seeking, compulsive responding, and affective behaviors associated with opioid use. By examining how modulation of inhibitory signaling influences multiple behavioral domains associated with opioid reinforcement and negative affect, I hope to clarify the role of KCC2-dependent inhibitory control in vulnerability to opioid use disorder. This project will provide insight into how restoring inhibitory balance via KCC2 may impact both drug consumption and addiction-related behavioral outcomes.
Methodology
To upregulate KCC2, I will be administering the small-molecule drug CLP290 via acute intraperitoneal injections. This administration method will enhance KCC2 function in a controlled and time-limited manner, allowing me to test how increases in inhibitory signaling directly affect opioid intake and related behaviors. I will employ an established rodent morphine-self administration model7 to study behaviors associated with opioid use, including drug intake, compulsive responding, and affective behaviors. This paradigm involves rat handling, jugular catheterisation, morphine self-administration training, and a battery of behavior tests to measure affective behavior during withdrawal, including open field, marble burying, elevated plus maze, sucrose preference test, and social interaction. Statistical analysis will be performed via two-way ANOVA for self-administration records, and independent t-test for affective testing.
Works Cited
1. Pearson, A. C., Kimmey, B. A., Taormina, M. B., Holden, W. M., & Ostroumov, A. (2025). Cocaine and morphine converge to disrupt chloride homeostasis in ventral tegmental area GABA neurons. Addiction Biology, 30(12), e70104.
2. Pearson, A. C., & Ostroumov, A. (2024). Midbrain KCC2 downregulation: Implications for stress-related and substance use behaviors. Current Opinion in Neurobiology, 88, 102901.
3. Ostroumov, A., Wittenberg, R. E., Kimmey, B. A., Taormina, M. B., Holden, W. M., McHugh, A. T., & Dani, J. A. (2020). Acute nicotine exposure alters ventral tegmental area inhibitory transmission and promotes diazepam consumption. eneuro, 7(2).
4. Ostroumov, A., Thomas, A. M., Kimmey, B. A., Karsch, J. S., Doyon, W. M., & Dani, J. A. (2016). Stress increases ethanol self-administration via a shift toward excitatory GABA signaling in the ventral tegmental area. Neuron, 92(2), 493-504.
5. Thomas, A. M., Ostroumov, A., Kimmey, B. A., Taormina, M. B., Holden, W. M., Kim, K., ... & Dani, J. A. (2018). Adolescent nicotine exposure alters GABAA receptor signaling in the ventral tegmental area and increases adult ethanol self-administration. Cell reports, 23(1), 68-77.
6. Ostroumov, A., Thomas, A. M., Kimmey, B. A., Karsch, J. S., Doyon, W. M., & Dani, J. A. (2016). Stress increases ethanol self-administration via a shift toward excitatory GABA signaling in the ventral tegmental area. Neuron, 92(2), 493–504.
7. Thomas, A. M., Ostroumov, A., Kimmey, B. A., Taormina, M. B., Holden, W. M., Kim, K., … Dani, J. A. (2018). Adolescent nicotine exposure alters GABAA receptor signaling in the ventral tegmental area and increases adult ethanol self-administration. Cell Reports, 23(1), 68–77.