The Scripps Research Institue

Short version: Understanding how drugs of abuse affect the brain at the molecular, cellular, and circuit level. To study how addiction changes the brain, I used patch-clamp electrophysiology, single-cell proteomics, and molecular techniques like RNAscope to look at ion channel activity, GPCR signaling, and gene/protein expression in animal models of addiction/stress. I’ve co-authored papers looking at sex differences in ethanol sensitivity and have studied the role of glucocorticoid receptors and adrenergic signaling in GABAergic CeA synapses.

Long version: Alcohol use disorder (AUD), affects over 18 million people in the United States alone and can be attributed to an economic burden of over $200 billion per year. The development of AUD is characterized as a transition from social drinking, where the primary motivation to consume is hedonic/pleasurable, to dependence-induced drinking, where the primary motivation is to alleviate negative emotional symptoms (e.g. anxiety and depression) that arise during abstinence. Excessive ethanol intake facilitates this shift in reinforcement mechanisms (i.e. from positive to negative) by inducing changes in the brain, such as the dysregulation of executive function and the recruitment of stress systems. These adaptations contribute to relapse in clinical settings, as prolonged abstinence is characterized by lowered mood and a negative affective state. Stress and anxiety play a crucial role in the etiology of alcoholism, as stress can exacerbate withdrawal-related anxiety. Indeed, ethanol-dependent animals display enhanced stress responses and increased ethanol self-administration during acute withdrawal and protracted abstinence. Unfortunately, the neural mechanisms that are responsible for negative affective states are still not completely understood. Worse yet, few effective pharmacological treatments exist for AUD, and new therapeutic targets struggle to show efficacy in humans despite strong preclinical evidence, underscoring the complexity of this disease. More work is obviously needed to understand the neural maladaptations that support alcohol dependence. However, an important yet unresolved problem in the field of neuroscience is the role of sex on neural processes. Psychiatric disorders such as depression and posttraumatic stress disorder are more prevalent in women than men, yet most preclinical studies in the field have excluded females in their models.

We have used electrophysiology to show that acute ethanol (44 mM) has no effect on spontaneous inhibitory postsynaptic currents (sIPSC) in naïve and dependent females, while naïve and dependent males showed significant increases in GABA release. Surprisingly, the concentration-dependent increase in sIPSC frequency in response to acute ethanol (11-88 mM) routinely found in males was absent in naïve female rats. However, 88 mM ethanol does increase the sIPSC frequency in the dependent females.

Furthermore, preliminary studies on several neuropeptide systems (pro-stress and anti-stress) have produced distinct responses in neurotransmission between in females than males. As such, I hypothesize that distinct molecular characteristics exist between males and females, which are responsible for sex-specific mechanisms and adaptations that contribute to/drive addiction and promote relapse/prevent abstinence. To test this hypothesis, I have two aims: 1) Identifying the unique molecular signature of brain regions in each sex and 2) Determining the adaptations that promote and drive anxiety-like behavior. I am using electrophysiology to characterize the properties/activity of neurons, while collecting these neurons for further molecular characterization (i.e. single-cell RNA sequencing and proteomic analysis). This strategy will allow me to determine which molecular adaptations/pathways are associated with ethanol dependence and withdrawal, as well as the synaptic [dys]function of distinct cell populations in each region. The targets identified will ultimately be subject to pharmacological/genetic modulation during a battery of behavioral tests, which will determine their contribution to with anxiety-like behavior, and thus how they promote addiction.

USC

For my dissertation, I studied the interactions between P2X4Rs and NMDA receptors, as it relates to the regulation of ethanol intake. I focused on NMDA receptors because they are a primary regulator of dopamine neuron activity, they are an important component of addiction circuitry, and they are known to be expressed in the ventral tegmental area (VTA), along with dopamine receptors and P2X4Rs.  The VTA region of the brain is part of the mesolimbic pathway, which is strongly associated with reward and addiction. 

Several receptors have already been characterized for their individual roles in the field of alcohol research, but we don’t know as much about the importance of receptor-receptor interactions, or “cross-talk.”  These phenomenon are theorized to be responsible for various behavioral and sensorimotor function associated with addiction.  In fact, P2X4KO mice showed not only cognitive-behavioral changes, but also dysregulation of several receptors (including GluN1) within various regions of the brain as well as aberrant signaling within the mesolimbic pathway. Changes in cell-surface expression of specific glutamate receptor subunits have been shown to dysregulate physiological and behavioral function, which strongly suggested a novel regulatory function for P2X4Rs. For example, P2X4 receptors have been shown to physically interact with GABA receptors, although the physiological relevance of such interactions are currently unknown. Together, these data indicate that P2X4Rs and their interactions are integral in neuronal signaling, as well as disease states, including AUD.

In Vitro experiments: X. laevis oocytes

In vitro characterization of these receptor interactions are performed using two-clamp electrophysiology recordings on x. laevis oocytes, a system used by neuroscientists to model synaptic excitation and inhibition. Manipulation of Xenopus laevis oocytes by mRNA injection leads to expression of receptors on the cell surface, emulating synaptic currents within the brain.  In initial studies, I cloned and characterized various combinations of NMDA receptors, and then co-expressed both P2X4Rs and NMDA receptors in oocytes. I also examined the molecular bases for these [P2X4] interactions by mutating the residues responsible for internalization and ethanol sensitivity (see publication.)

P2X4 Channel Function and Ethanol Sensitivity

The findings of this study provide new insight into the role of residues in the TM1 segment in receptor activity and ethanol action in P2X4Rs. In summary, using electrophyisology, molecular biology, and modeling, we demonstrate that amino acid position 49 contributes to the channel function by providing flexibility/stability of the upper portion of the alpha‐ helix during channel opening. These findings also suggest that R33 in the lower part of the TM1 segment is involved in ethanol sensitivity at lower, behaviorally relevant ethanol concentrations. Moreover, interactions between R33 in the TM1 segment of one subunit (S1) and D354 in the TM2 segment of the neighboring subunit (S2) may be important in affecting the channel transition from closed to open conformation and thus affect ion conduction, as well ethanol sensitivity. These results identify new residues that are important for ethanol action on P2X4Rs and, in combination with modeling studies, provide new information for the development of a pharmacophore for AUD drug discovery.

Si-Lent Slice electrophysiology

To validate these receptor interactions, I performed ex vivo extracellular brain-slice electrophysiology recordings, which give insight into the physiological relevance of P2X4Rs awithin the brain.  Brain-slice electrophysiology recordings operate under similar principles as oocytes, except that receptors are in their native environment. 

In the summer of 2018, I spent 10 weeks in the laboratory of Dr. Mark Brodie at the University of Illinois, Chicago (UIC), and learned to perform a novel brain slice electrophysiology technique that functionally knocks down expression of target genes in neurons. Dr. Brodie pioneered this technique, which we call Si-LENT slice, which stands for SiRNA-Loaded Electrodes kNocksdown Target.

While at UIC, my studies suggested that P2X4Rs regulate dopaminergic (DA) neuron firing within the reward pathway of the brain, yet in the presence of ethanol, P2X4R regulation of DA neuron firing is suppressed. These studies support the hypothesis that P2X4Rs play a role in the mesolimbic system and alcohol addiction (see publication.)