Date of Award

5-31-2022

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Developmental and Brain Sciences

First Advisor

S. Tiffany Donaldson

Second Advisor

Zsuzsa Kaldy

Third Advisor

Richard Hunter

Abstract

Mammalian adolescence can be a difficult transition from childhood to adulthood, where increases in impulsivity and novelty- and risk-seeking are combined with heightened affect and elevated sensitivity to stress. Indeed, during adolescence, first drug use patterns emerge and in the continental United States, increasing misuse of amphetamines has been observed in adolescent youth. Myriad neural mechanisms underlie this shift in adolescence, including the dynamic remodeling of the mesocorticolimbic (MCL) pathway. Repeated drug administration affects neuroimmune substrates within the MCL circuit including toll-like receptor 4 (TLR4)Advances in addiction neuroscience indicate that drugs of abuse activate neural TLR4 and implicate glial TLR4 activation in neuroplasticity associated with the progressive stages of drug addiction. The present dissertation explores the roles of MCL TLR4 activity and extreme trait anxiety-like behavior in the expression and maintenance of amphetamine sensitization in male adolescent Long-Evans rats through a systematic review and two experimental studies.

We prospectively registered our systematic review in PROSPERO to minimize bias, to increase transparency, and to avoid duplication of our review aims. PROSPERO allows for the authors to identify the intended processes, and to serve as guideposts for readers to assess whether these processes were followed. We followed a PECO (Population, Exposure, Comparator, Outcomes) framework for developing our research question: For in vivo rodent models of psychostimulant abuse, do variations in sex, anxiety-like profiles, and neuroinflammatory signaling affect addiction-like behavior during adolescence? Results from the systematic review revealed that (1) there is poor standardization of studies using females, (2) there are relatively few studies that include relative parameters when they do include females (e.g., stage of estrous, monitoring cycling, removing and replacing hormones), (3) adolescent age, stress and anxiety are also not standardized or operationalized in studies (4) dopamine, GABA, glutamate, and 5-HT and their associated receptors, enzymes, and metabolites are implicated in addiction models as well as (5) pro-inflammatory marks linking (6) to key regions of the brain reward pathway and those involved in anxiety. To further examine the neuroinflammatory role in adolescent addiction and anxiety, we devised two experimental studies. In the first experiment, we developed a low dose repeated amphetamine paradigm to distinguish locomotor sensitization to amphetamine between adolescent Long Evans male rats showing high (HAn) and low (LAn) anxiety-like behavior. Our results show that from a purchased line of rats, individual differences emerged for HAn and LAn profiles; we were able to separate adolescent males along these extreme lines. Using a 4-day repeated amphetamine regimen, we found that HAn adolescent males developed sensitization over the treatment days, and after a 7-day withdrawal. Within these same animals, the medial prefrontal cortex (mPFC), caudate putamen (cPU), nucleus accumbens (NAcc), and basolateral amygdala (BLA) were stained for anti- TLR4, NFkB, TNFa, and GDNF. AMPH animals expressed more mPFC TLR4 protein than SAL controls. For NFkB, within the mPFC HAn animals expressed more NFkB protein than LAn animals; within the cPU we observed a trait effect, where HAn SAL-treated animals expressed more NFkB than LAn SAL-treated controls. We also observed interaction effects, where HAn Sal-treated expressed more NFkB relative to LAn drug-treated animals. Finally, we observed within-subjects effects, where for HAn animals, SAL-treated animals expressed more NFkB than AMPH-treated animals. For LAn animals, this was reversed, where AMPH-treated animal expressed more NfkB than SAL-treated controls. For TNFa, in the mPFC we saw a trait effect where HAn SAL-treated animals expressed more TNFa than LAn drug-treated animals. Finally, for GDNF we observed main trait effects across all regions of interest, where HAn animals expressed more GDNF than LAn animals. GDNF is a neurotrophic factor canonically associated with the survival of midbrain dopamine neurons and motor neurons and is upregulated in response to an immune challenge via NF-kB signaling. In the final experiment, we evaluated whether the downstream mitogen-activated protein kinase (MAPK) pathway might contribute to the elevated response to amphetamine in the HAn lines. MAPK is important for proinflammatory and plasticity cellular response and the potent inhibitor, SB239063, was administered prior to amphetamine on the challenge day of the regimen. Our results indicate that this MAPK inhibition effectively attenuated the expression of sensitization in the HAn lines. Furthermore, there was increased BLA TLR4, a region implicated in the neuroplasticity and exaggerated learning that is a hallmark of addiction. Together, our work on the systematic review and subsequent experimental studies implicates pro-inflammatory cascades as important targets for further study as they may hold promise as novel therapeutic targets for disrupting the learned associations of addiction.

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