Our understanding of the brain’s beautiful and complex machinery is largely driven by our ability to measure the chemical and electrical signals that govern its convoluted circuitry. As such, our knowledge is only as good as the tools we have at our disposal. The study of neurochemistry and neuropharmacology research has largely been driven by the use of microdialysis and electrochemical methods to measure targets of interest in the brain. While these techniques are powerful and have contributed immensely to our conceptualization of brain activity, they are not without their limitations. Briefly, microdialysis suffers from poor temporal resolution and lacks the potential for real-time data delivery, while previous electrochemical methods suffer from debatable specificity and limited generalizability for the detection of many critical targets. In response to these limitations, I have applied electrochemical aptamer-based (E-AB) sensors to the field of in-brain molecular sensing. The modular E-AB platform does not rely on the reactivity of its intended target, is generalizable to the detection of a wide range of analytes and has been shown to support real-time detection of small molecules directly in the living body. By modifying the existing E-AB platform, which has demonstrated success for peripheral measurements in blood, I have developed a novel E-AB sensor platform for making in-brain measurements of small molecules. In-brain E-AB sensing opens a path towards measuring a wide range of targets in the brain. To showcase this, I have applied this platform to model drug transport (using simultaneous measurements in blood and CSF), as well as study neuropsychopharmacology (using simultaneous measurements of a psychoactive drug and on-going behavior). Together these studies lay the foundation for a powerful new tool with which to study neurochemistry and neuropharmacology.
Personalized medicine, a rapidly evolving field of healthcare, aims to improve therapeuticoutcomes by individualizing patient care. Therapeutic drug monitoring (TDM) presents a vast improvement to personalized medicine, enabling clinicians to optimize dosing regimens to improve therapeutic outcomes while minimizing toxicity. The state of the art of TDM is significantly limited by the current techniques employed to perform it. Existing technologies are limited by reliance on ex vivo quantification that generally results in single time point or low temporal resolution measurements and the inability to measure drug levels in different physiological compartments simultaneously. Electrochemical aptamer-based (EAB) sensors, a novel biosensing platform, present a powerful means of overcoming these limitations, providing seconds-resolved, cross-compartment measurements of drug distribution in real time. Centered around the focus of advancing TDM, this work first utilizes EAB sensors to better elucidate drug transport from blood to solid tissue, with the ultimate goal of improving transport into the brain. Using doxorubicin as a testbed, I first demonstrate that EAB sensors can capture the distribution of chemotherapeutics from the bloodstream to the peripheral subcutaneous tissue. I then utilize these measurements to perform high-precision feedback-controlled drug delivery over plasma drug levels. After careful evaluation of the permeation of drugs into tissue not separated by a physiological barrier, I then demonstrate the in-brain EAB platform can explore how pharmacological manipulations and drug encapsulation methods may improve drug permeation into the brain. Finally, this work utilizes individual, subject-specific measurements to suggest EAB sensors could be used to inform inter-patient pharmacokinetic variability. Collectively, this work argues that EAB sensors could significantly advance both our understanding of drug transport to the brain and peripheral tissues and revolutionize personalized medicine by enabling high-precision therapeutic drug monitoring.
Similar to the pattern observed in people with substance abuse disorders, laboratory animals will exhibit escalation of cocaine intake when the drug is readily available and will exhibit increased drug-seeking behaviors after long periods of abstinence. Additionally, there are long term changes in neuron structure, receptor function, and neurotransmission associated with abstinence from cocaine in humans and animals. DNA methylation is an epigenetic modification to the DNA structure that mediates mRNA expression to confer different cell types, but has recently been implicated in learning and memory mechanisms. The long-term control that DNA methylation has over gene expression in animals makes it a prime candidate for controlling gene expression over the course of abstinence in animals with previous drug experience. Therefore, here, I investigated the contribution of behavioral contingency of cocaine administration on escalation of cocaine intake and re-exposure to cocaine cues as well as DNA methylation and gene expression within the dorsal medial prefrontal cortex (dmPFC) in adult male Sprague-Dawley rats. I exposed rats to daily training for saline (1 h/ day) or cocaine (0.25 mg/kg/inf) in limited- (1 h access per day), prolonged- (6 h access per day), or limited + yoked-access (1 h contingent + 5 h non-contingent access per day) for 15 days. Rats were then put through forced abstinence for 1, 14, or 60 days, and then the dmPFC was dissected out. Saline- and prolonged-access rats were additionally separated into cue- and no cue- conditions after 60 days of abstinence, where cue rats were re-exposed to the operant chamber without cocaine delivery for 2 h. These studies led to 4 main findings. 1) cocaine contingency affects mRNA expression for glutamatergic genes, 2) DNA methylation changes dynamically throughout abstinence, 3) re-exposure to cocaine cues rapidly alters DNA methylation and mRNA expression, and 4) DNA methylation, hydroxymethylation, and transcription factor binding all contribute to altered mRNA expression.
Gene by environment interactions may be important etiological factors that confer risk of numerous psychiatric disorders. Psychiatric disorders are found to be heritable, indicating genetic variants contribute to risk and resilience. In addition to genetics, early life stress confers significant risk. Prenatal stress (PNS) is associated with numerous disorders and alterations to affect and cognition that suggest profound and enduring consequences. Preclinical studies causally indicate the deleterious effects of PNS on models of psychiatric disorders, including effects on prepulse inhibition (PPI) and cocaine reward and locomotion. The intersection of genetics and PNS has been explored and PNS was found to interact with genetic background. PNS differentially alters PPI and cocaine reward and locomotion in the C57/6J (B6) and DBA/2J (D2) inbred mouse strains. These strains may serve as progenitors for populations that can be utilized in forward genetic studies for discovery of quantitative trait loci (QTLs) that will facilitate discovery of PNS interacting variants. The following will present studies that utilized the BXD recombinant inbred mouse panel, derived from the B6 and D2 strains, to discover QTLs that interact with PNS to alter sensorimotor and cocaine-induced behaviors. A QTL by PNS interaction was discovered for PPI and acute cocaine locomotion. The BXD panel is a genetic reference population that allows for extensive accumulation and sharing of data across studies. Following discovery of these QTLs, publicly available BXD mRNA expression data was utilized to prioritize positional candidate genes. These efforts prioritized several positional candidate genes. In addition to offspring phenotypes, the maternal stress corticosterone response and effects of stress on dam-pup contact were assessed, as heritable maternal stress responses may contribute to strain differences in offspring phenotype, with implications for the interpretation of QTLs. Strain differences in the maternal corticosterone response and the maternal behavior response to stress associated with strain differences in PNS effects on male offspring cocaine phenotypes, suggesting a potential role for genetic variants that moderate the maternal stress response. The results obtained are a preliminary step in identifying genes that interact with PNS to confer risk of psychiatric disease.
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