abstract: Intermittent social defeat stress induces psychostimulant cross-sensitization, as well as long-lasting social avoidance behavior. Previous data reveal heightened expression of AMPA receptor (AMPAR) GluA1 subunits in rat ventral tegmental area (VTA), which occurs concurrently with social stress-induced amphetamine (AMPH) cross-sensitization. These studies described herein examined whether VTA GluA1 AMPARs are important for the behavioral consequences of social stress and investigated the role of the infralimbic (IL) to VTA pathway in the induction of these responses. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization without affecting social avoidance behavior, while GluA1 overexpression in VTA DA neurons mimicked the effects of stress on AMPH sensitization. Female rats were more sensitive to the effects of stress on AMPH administration than males, specifically during proestrus/estrus, which is characterized by higher circulating estradiol. Fluorescent immunohistochemistry revealed that females expressed higher GluA1 in VTA DA neurons as a result of intermittent social defeat stress, independent of estrus stage; by contrast, females during proestrus/estrus displayed higher tyrosine kinase receptor type 2 (TrkB) expression, which is the receptor for brain derived neurotrophic factor (BDNF), in VTA DA neurons, independent of stress exposure. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization and overexpression mimicked the effects of stress on AMPH sensitization. This suggests that BDNF-TrkB signaling may work concomitantly with GluA1 signaling in the VTA to drive sex-dependent differences in stress-induced locomotor sensitization effects. Optogenetic inhibition of the IL-VTA pathway in male rats prevented stress-induced AMPH sensitization compared to control animals. In addition, fluorescent immunohistochemistry displayed less Fos labeling in the nucleus accumbens (NAc) of rats with IL-VTA light inhibition compared to control animals. This suggests that the IL-VTA pathway plays a critical role in the induction of stress-induced sensitivity to AMPH, and blocking this pathway prevents mesolimbic DA signaling to the NAc. We conclude that IL glutamate projections onto GluA1-homomeric AMPA receptors in VTA DA neurons play a critical role in driving the stress-induced sensitization response in males and females. Therefore, GluA1 VTA DA neurons could potentially be a therapeutic target to prevent stress-induced drug susceptibility in the future. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2020

Maternal smoking during pregnancy is associated with enduring psychopathology, such as increased likelihood of substance use, in offspring. Various animal models demonstrate that continuous nicotine exposure produces teratogenic effects in offspring, as well. In this experiment, a novel intravenous (IV) exposure model was used to determine if gestational nicotine (GN) treatment produced alterations in methamphetamine-induced sensitization and the expression of brain-derived neurotrophic factor (BDNF) in the mesocorticolimbic dopamine (DA) system of adolescent offspring. Dams were injected with IV saline or nicotine (0.05 mg/kg/injection) three times per day on gestational days 8-21. Habituation was measured on postnatal day (PND) 25-27 and baseline activity on PND 28. On PND 29-35, offspring were injected with saline or methamphetamine (0.3 mg/kg) and locomotor activity was measured after the first and seventh injections. On PND 36, brains were removed, flash frozen, and BDNF protein levels in the nucleus accumbens (NAcc), dorsal striatum (Str), frontal cortex (FC), and hippocampus (Hipp) were analyzed. GN did not affect habituation or the induction of methamphetamine-induced sensitization. Interestingly, GN, but not adolescent methamphetamine treatment, elevated levels of BDNF in the NAcc and Str; however, the GN-induced increase in BDNF in the FC was attenuated by adolescent methamphetamine treatment. Both GN and adolescent methamphetamine treatment increased BDNF in the Hipp. These findings indicate that GN exposure will result in increased levels of BDNF protein throughout the mesocorticolimbic DA system during adolescent development and suggests that methamphetamine abuse will modulate the expression of BDNF in motivational circuitries of adolescent offspring exposed to GN.

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction in neurons and other excitable cells, causing them to be important targets of psychoactive drugs. Structural data for these complex proteins are limited, particularly among eukaryotic family members and for the functionally critical open state. These data limitations cause knowledge gaps regarding the mechanisms of ion channel opening, gating, and modulation. However, a newly discovered bacterial family member, known as sTeLIC, shares numerous structural features with its eukaryotic relatives in our central nervous system. A recently solved electron microscopy structure depicts sTeLIC in an apparent open state with binding pockets in its extracellular domain, compatible with binding a drug with structural similarities to amphetamines, like the 4-bromoamphetamine. This project aims to provide the first structure-function evidence for direct modulation of a pentameric ligand-gated ion channel by an amphetamine. The two most essential tools used in this project to examine the effects of 4-bromoamphetamine on sTeLIC were Xenopus laevis oocytes and two-electrode voltage-clamp. These tools were necessary for the collection of gating and modulation data. Ion channel activities can be analysed by clamping sTeLIC injected Xenopus laevis oocytes into the two-electrode voltage-clamp since it can artificially control the membrane voltage of oocytes. Modulation data show that 4-bromoamphetamine is a bimodal allosteric potentiator, as well as an allosteric agonist. Residues Y104 and W75, located in the binding pocket, were selected by comparing the published open state model with an AlphaFold-generated non-conducting model. Mutating these into valine or alanine reduces the potentiation. One explanation may be that removing tyrosine's aromatic ring complicates retaining essential interactions in the binding pocket while swapping tryptophan for smaller residues could make it easier for the drug to stabilise the closed state.

For many years, the norepinephrine transporter (NET) was considered a 'static' protein that contributed to the termination of the action of norepinephrine in the synapse of noradrenergic neurons. The concept that the NET is dynamically regulated, adjusting noradrenergic transmission by changing its function and/or expression, was considered initially in the mid 1980s. Since that time, a plethora of studies demonstrate that the NET is regulated by several intracellular and extracellular signaling molecules, and that phosphorylation of the NET is a major pathway regulating its cell surface expression and thereby its function. The NET is a target of action of a number of drugs that are used long-term therapeutically or abused chronically. This has driven numerous investigations of how the NET and its function are regulated by long-term exposure to drugs. While repeated exposure to many drugs has been shown to affect NET function and expression, the intracellular mechanisms for these effects remains elusive.

This thesis sets out to address the challenges with the comparison of Amphetamine material in determining whether they originate from the same source or different sources using pairwise ratios of peak areas within each chromatogram of material and then modeling the difference between the ratios for each comparison as a basis for evaluation. The evaluation of an existing method that uses these ratios to determine the sum of significant differences between each comparison of material that is provided is done. The outcome of this evaluation suggests that there the distributions for comparison of samples originating from the same source and the comparison of samples originating from different sources have an overlap leading to uncertainties in conclusions. In this work, the differences between the ratios of peak areas have been modeled using a feature-based approach. Because the feature space is quite large, Discriminant Analysis methods such as Linear Discriminant Analysis (LDA) and Partial least squares Discriminant Analysis (PLS-DA) have been implemented to perform classification by dimensionality reduction. Another popular method that works on the principle of nearest centroid classifier called as Nearest shrunken centroid is also applied that performs classification on shrunken centroids of the features. The results and analysis of all the methods have been performed to obtain the classification results for classes +1 (samples originate from the same source) and  ́1 (samples originate from different sources). Likelihood ratios of each class for each of these methods have also been evaluated using the Empirical Cross-Entropy (ECE) method to determine the robustness of the classifiers. All three models seem to have performed fairly well in terms of classification with LDA being the most robust and reliable with its predictions.

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 μg ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 μg ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 ± 17 s in a 600 s session, vs. 127 ± 13 s in the 6-OHDA group and <75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D1 receptor supersensitivity and further sensitized 5-HT2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA+5,7-DHT-lesioned rats to 76 ± 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.

Nitric oxide (NO) is a neurotransmitter which is synthesized on demand from L-arginine by the enzyme nitric-oxide-oxidase, and is implicated in a variety of physiological functions, including release and uptake of dopamine. Amphetamine induces stereotyped behavior via release of dopamine from dopaminergic neurons in the striatum and related structures, while apomorphine induces such behavior via activation of central dopaminergic receptors. Recently we have demonstrated that a NO donors and a NO-synthase inhibitor modify the response of some central dopaminergic receptors to their agonists and antagonists. In the present study we examined the effect of two NO donors and one NO-synthase inhibitor on stereotyped behavior induced in rats by amphetamine and apomorphine, and the sex-selectivity of this effect. A highly significant dose-dependent sexual differentiation was recorded in the stereotyped behavior of amphetamine, as the duration and intensity of this effect was shortened by L-NAME but not by L-arginine and Molsidomine. Differences in the stereotyped behavior between female and male rats administered apomorphine were dose-dependent, but were not affected by any of the three drugs tested. It is concluded that while nitric oxide is involved in the reactivity of central dopamine receptors, the intensity and duration of this effect is drug- and sex-dependent.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was studied in the rat spinal cord, sympathetic ganglia and adrenal glands by immunohistochemical methods, utilizing a polyclonal antiserum raised against the CART peptide fragment 55-102. CART-LI was detected in nerve fibers and in basket-like terminals surrounding many postganglionic neurons of the superior cervical ganglion (SCG), stellate, paravertebral and prevertebral ganglia. Postganglionic neurons exhibited low or non-detectable levels of CART-LI. Surgical sectioning of the cervical sympathetic trunk for 6-7 days resulted in a nearly complete loss of CART-LI fibers and terminals in the SCG. In the adrenal gland, CART-LI nerve fibers formed a plexus underneath the capsule, some of which bifurcated and made a sharp turn toward the adrenal medulla, where clusters of chromaffin cells were intensely labeled. The detection of CART-LI in sympathetic ganglia and adrenal glands extends the previous observation of the presence of CART-LI in sympathetic preganglionic neurons and further supports the notion that CART peptide(s) may function as a signaling molecule in the sympatho-adrenal axis.

The neonatally 6-hydroxydopamine (n6-OHDA)-lesioned rat has been the standard for 40 years, as an animal model of attention-deficit hyperactivity disorder (ADHD). Rats so lesioned during postnatal ontogeny are characterized by ∽99% destruction of dopaminergic nerves in pars compacta substantia nigra, with comparable destruction of the nigrostriatal tract and lifelong ∽99 % dopaminergic denervation of striatum, with lesser destructive effect on the ventral tegmental nucleus and associated lesser dopaminergic denervation of nucleus accumbens and prefrontal cortex. As a consequence of striatal dopaminergic denervation, reactive serotoninergic hyperinnervation of striatum ensues. The striatal extraneuronal milieu of DA and serotonin is markedly altered. Also, a variety of sensitization changes occur for dopaminergic D1 and D2 receptors, and for serotoninergic receptors. Behaviorally, these rats in adulthood display spontaneous hyperlocomotor activity, attentional deficits, and cognitive impairment-all of which are acutely attenuated by the psychostimulants amphetamine (AMPH) and methylphenidate (MPH) (i.e.,opposite to the acute effects of AMPH and MPH in intact control rats). The acute behavioral effects of AMPH and MPH in intact and lesioned rats are analogous to their respective acute effects in non-ADHD and in ADHD humans. The neurochemical template of brain, and behavioral series of changes in n6-OHDA-lesioned rats, is described in the review. Despite the fact that nigrostriatal damage is not an underlying pathophysiological process of human ADHD (i.e.,lacking construct validity), the described animal model has face validity (behavioral profile) and predictive validity (mirror of ADHD/MPH effects, as well as putative and new ADHD treatment effects). Also described in this review is a modification of the n6-OHDA rat, produced by adulthood partial lesioning of the serotoninergic fiber overgrowth. This ADHD model has even more accentuated hyperlocomotor and attentional deficits, counteracted by AMPH-thus providing a more robust means of animal modeling of ADHD. The n6-OHDA rat as a model of ADHD continues to be important in the search for new ADHD treatments.

Progressive-ratio schedules are useful for studying the reinforcing effects of drugs. Earlier human laboratory studies showed that d-amphetamine significantly increased break points relative to placebo. However, the magnitude of the increase was modest, which may be attributable to rather high levels of placebo responding. We used novel response requirements in a modified progressive-ratio procedure and hypothesized that the altered range of response requirements would decrease responding for placebo and increase responding for d-amphetamine. Eight participants completed the study. The participants first sampled oral doses of d-amphetamine (0, 8, 16, and 24mg). In subsequent sessions, the participants were offered the opportunity to work for the sampled dose on a modified progressive-ratio procedure with response requirements ranging from 400 to 1800 mouse clicks. A battery of participant-rated drug-effect questionnaires, a performance measure, and cardiovascular measures were included to more fully characterize the effects of d-amphetamine. Placebo maintained low levels of responding. The intermediate dose of d-amphetamine increased responding significantly above placebo levels. d-Amphetamine produced prototypical subject-rated effects that were an orderly function of dose. These data suggest that the modified response requirements resulted in lower levels of placebo taking and a larger separation between the number of placebo and d-amphetamine capsules earned. Behavioural Pharmacology.