The placebo effect represents a fascinating example of how cognition can influence the physiology of the brain and body. The expectation of therapeutic benefit elicited by a placebo given in the guise of active medication has been proposed to be a form of reward expectation, and is associated with activation of brain reward circuitry. Prominent placebo effects occur in Parkinson’s disease (PD), where the expectation of symptom improvement stimulates dopamine release in the striatum. In the work described in this dissertation, positron emission tomography with [¹¹C] raclopride was used to investigate the relationship between the strength of expectation of benefit and the degree of dopamine release in PD, and how this relationship corresponds to current models of dopamine function in reward. Chapter 3 describes a pilot study conducted in patients who had undergone subthalamic nucleus deep-brain stimulation (STN-DBS) in which we examined how awareness of stimulator status (ON or OFF) affected synaptic dopamine levels compared to when subjects were blind. No difference was detected between conditions; however, it proved to be difficult to maintain blinding due to the profound effects of STN-DBS. Chapter 4 describes the development of the methodology for the analysis of high-resolution PET data, in which we utilized the combined efforts of neuroscience and imaging physics to optimize the analysis of [¹¹C] raclopride PET data. In Chapter 5, I describe the use of verbal instructions to manipulate patients’ expectations in order to investigate how the likelihood of receiving levodopa influenced dopamine release when the patients were in fact given placebo. Placebo-induced dopamine release was differentially modulated by expectation in the dorsal and ventral striatum: dopamine release in the putamen was related monotonically to expected reward value, whereas dopamine released in the ventral striatum reflected the uncertainty of benefit or the salience of the expectation. The placebo effect in PD therefore involves at least two related but separate mechanisms: the expectation of benefit itself, which is scaled to reflect the value of the drug to the patient and is mediated by nigrostriatal dopamine, and the uncertainty or salience of benefit that is mediated by mesolimbic dopamine. / Medicine, Faculty of / Graduate

While selective serotonin (5-HT) reuptake inhibitors (SSRIs) are utilized as the first-line strategy in treating depression, new approaches are still desired. Using in vivo electrophysiological techniques, the effects of co-administration of bupropion with the SSRI escitalopram on the firing rate of dorsal raphe 5-HT and locus coeruleus norepinephrine (NE) neurons were investigated. Escitalopram significantly decreased the firing of 5-HT and NE neurons at day 2. The 5-HT firing rate, unlike that of NE, recovered after the 14-day escitalopram regimen. Bupropion did not increase 5-HT firing but decreased that of NE after 2 days. Following 14-day bupropion, 5-HT firing was markedly enhanced, and NE firing was back to baseline. Co-administration of escitalopram and bupropion doubled 5-HT firing after 2 and 14 days, whereas NE neurons were inhibited after 2, but partially recovered after 14 days. Although sustained bupropion administration did not alter the sensitivity of 5-HT1A receptors in hippocampus, the tonic activation of postsynaptic 5-HT1A receptors was enhanced in 14-day bupropion-treated rats to a greater extent than in the 2-day and control rats. The function of terminal 5-HT1B autoreceptors was not changed. The inhibitory action of α2-adrenergic receptors on 5-HT terminals was, however, diminished. The function of terminal α2-adrenergic autoreceptors was also attenuated in rats given bupropion for 14 days. Administration of the antidepressant trazodone suppressed the 5-HT firing at day 2, which recovered to baseline following 14 days. Prolonged trazodone-administration enhanced the tonic activation of postsynaptic 5-HT1A receptors in hippocampus, and decreased the function of terminal 5-HT1B autoreceptors. Finally, a novel psychotropic agent asenapine showed potent antagonistic activity at 5-HT2A, D2, and α2-adrenoceptors. Asenapine, however, acted as a partial agonist at 5-HT1A receptors in dorsal raphe and hippocampus. Overall, the therapeutic effects of various antidepressants may be, at least in part, due to the enhancement of 5-HT and/or NE neurotransmission.

RASA3 (Ras p21 GTPase Activating Protein 3) is required for D2SR (Dopamine D2 Short Receptor) induced ERK1/2 inhibition in pituitary lactotroph GH4ZR7 cells. We hypothesized that RASA3 may be important for D2SR signaling to inhibit ERK1/2 in dopamine neurons, and thus negatively regulate TH (Tyrosine Hydroxylase) expression and activity. We designed and made shRASA3 lentivirus and showed that it inhibits RASA3 expression. Lentivirus mediated RASA3 knockdown can partially reverse the D2SR mediated ERK1/2 inactivation in GH4ZR7 cells. We then showed that knockdown of RASA3 in dopamine-secreting PC12 cells increased NGF-stimulated ERK1/2 in cells expressing D2SR, but not in cells lacking D2SR, thus implicating RASA3 plays a role in D2SR-mediated inhibition of ERK1/2 signaling. We also found that knockdown of RASA3 increased TH protein levels in cells expressing D2R receptors but not those without D2SR, suggesting that D2SR tonically inhibits the synthesis of TH. We also found preliminary indication that mutant RASA3 mice show increased level of TH in SN compared to WT mice. RASA3 mutant mice showed no striking changes in basal locomotion, anxiety or depression phenotypes, but further studies are needed to specifically address dopamine-driven behaviors. In summary, our data support the role of RASA3 in mediating D2SR-induced inhibition of ERK1/2 in dopamine neurons to negatively regulate TH expression and activity.

Aromatase is the only enzyme that converts androgens into estrogens, which is found in the brain, testes and ovaries. In teleosts, brain aromatase is exclusively expressed in radial glial cells, which are the abundant stem-like non-neuronal progenitors involved in neuroendocrine functions and neurogenesis in the central nervous system. With little information about radial glial cell regulation by neurotransmitters and neurohormones available, the overall goal of this thesis is to investigate the interactions between dopamine neurons and radial glial cells in the adult goldfish (Carassius auratus) forebrain. Immunocytochemistry and confocal imaging revealed a close anatomical relationship between dopamine neurons and radial glial cells along the ventricular surface in the telencephalon. Transcriptional regulation of brain aromatase by dopamine indicated a brain region-specific pattern and suggested the involvement of other regulators in the goldfish forebrain. A novel goldfish primary radial glial cell culture model was established and characterized for brain aromatase regulation studies. Pharmacological studies demonstrated that specific activation of dopamine D1 receptors up-regulates brain aromatase through a cAMP-dependent molecular mechanism, which can be enhanced or attenuated by the product of aromatase action, 17β-estradiol. Proteome profiling and the response following treatment with the specific dopamine D1 receptor agonist SKF 38393 revealed that proteins involved in cell proliferation and growth are regulated through small molecules- and transcription factors-mediated signaling pathways. Analysis of genes related to radial glial cell and dopamine neuron functions demonstrated that glial activation and dopamine neuron recovery are estrogen-dependent in a neurotoxin MPTP-induced goldfish model of Parkinson’s disease. This thesis illustrates novel molecular mechanisms underlying brain aromatase regulation as well as radial glial cell function regulation and provides a framework for future investigation of existing endocrine disruptors modulating neurosteroid levels in the teleost brain.

The activity of neurons in the prefrontal cortex (PFC) may underlie working memory processes in the brain. Both the performance of working memory tasks and the activity of PFC neurons are modulated by dopamine. The goal of the present thesis was to gain insight into the neural basis of working memory by studying the PFC, and the DA system in the PFC, from both a behavioral and cellular perspective. The functional contribution of the PFC to working memory processes in the rat was assessed in Chapter 2 of the present thesis using memory-based foraging tasks on an 8-arm radial maze. The results of these studies indicated that lidocaine-induced inactivations of the PFC selectively disrupted the ability to use mnemonic information to guide foraging, but not the ability to acquire or retain such information. The ability to use mnemonic information to guide foraging was also disrupted by microinjection of a D1 but not D2 receptor antagonist into the PFC. Chapters 3-5 investigated how PFC neurons process synaptic inputs to their dendrites to produce spike output. The intrinsic membrane properties and synaptic responses at the soma and dendrites of deep layer PFC pyramidal neurons were recorded using sharp intracellular or whole-cell patch-clamp techniques in a brain-slice preparation. Different passive and active membrane properties of the soma and dendrites of PFC neurons were observed. The distal dendrites of PFC neurons responded most effectively to strong, highly coincident synaptic inputs. Ca²⁺currents near the soma both amplified the effects of these inputs and modulated the spike output pattern. Spike output at the soma was also controlled by the interplay of slowly-inactivating Na⁺ and K⁺ currents. Chapter 6 investigated the modulation of PFC neurons by DA. DA or a D1 but not D2 receptor agonist increased the evoked firing of PFC neurons via a D1- mediated modulation of slowly-inactivating Na⁺ and K⁺ currents. Concurrently, D1 receptor activation reduced burst firing in PFC neurons, due to a attenuation of Ca²⁺ currents. D1 receptor activation also increased both GABA[sub A] IPSPs and NMDA EPSPs. The final chapter of this thesis integrated these data into a cellular model of PFC function and its modulation by DA. It is proposed that DA may tune PFC neurons such that they respond selectively to strong synchronized inputs from other cortical areas. In the presence of DA, working memory processes mediated by the PFC may be influenced selectively by stimuli of behavioral significance. / Arts, Faculty of / Psychology, Department of / Graduate

Rationale: Nicotine (NIC) potently increases operant responding for non-NIC reinforcers, and this effect may depend on drug-mediated increases in incentive motivation. According to this hypothesis, NIC should also potently increase approach to Pavlovian-conditioned stimuli associated with rewards. Objective: The present studies explored the effects of NIC on Pavlovian-conditioned approach responses. Method: To do so, liquid dippers were used to deliver an unconditioned stimulus (US; 0.1 ml sucrose) after presentation of a conditioned stimulus (CS; 30 s illumination of a stimulus light) - both the CS and US were presented in receptacles equipped to monitor head entries. Results: In experiment 1, the CS and US were presented in the same receptacle, but NIC pretreatment (0.4 mg/kg base) did not increase conditioned approach responses. Delivery of the sucrose US was then shifted to receptacle in a different location. All rats learned to approach the new US location (goal-tracking) at similar rates. Approach to the CS receptacle (sign-tracking) declined for saline-pretreated rats, but NIC pretreatment increased sign-tracking. In experiment 2, NIC pretreatment increased sign-tracking when the CS and US were spatially separated during acquisition. In experiment 3, NIC pretreatments were replaced with saline, but the effect of NIC persisted for an additional 24 test sessions. Conclusion: The findings suggest that NIC increases incentive motivation and that this effect is long-lasting, persisting beyond the pharmacological effects of NIC.

Rationale: Nicotine (NIC) potently increases operant responding for non-NIC reinforcers, and this effect may depend on drug-mediated increases in incentive motivation. According to this hypothesis, NIC should also potently increase approach to Pavlovian-conditioned stimuli associated with rewards. Objective: The present studies explored the effects of NIC on Pavlovian-conditioned approach responses. Method: To do so, liquid dippers were used to deliver an unconditioned stimulus (US; 0.1 ml sucrose) after presentation of a conditioned stimulus (CS; 30 s illumination of a stimulus light) - both the CS and US were presented in receptacles equipped to monitor head entries. Results: In experiment 1, the CS and US were presented in the same receptacle, but NIC pretreatment (0.4 mg/kg base) did not increase conditioned approach responses. Delivery of the sucrose US was then shifted to receptacle in a different location. All rats learned to approach the new US location (goal-tracking) at similar rates. Approach to the CS receptacle (sign-tracking) declined for saline-pretreated rats, but NIC pretreatment increased sign-tracking. In experiment 2, NIC pretreatment increased sign-tracking when the CS and US were spatially separated during acquisition. In experiment 3, NIC pretreatments were replaced with saline, but the effect of NIC persisted for an additional 24 test sessions. Conclusion: The findings suggest that NIC increases incentive motivation and that this effect is long-lasting, persisting beyond the pharmacological effects of NIC.

The interaction of emotion and memory is necessary for establishing a cognitive map including current context and past experiences, which is used by prefrontal cortex to regulate the internal state and guide goal directed actions and decision making. The amygdala, hippocampus and anterior cingulate cortex (ACC) play critical roles in these processes, but the organization of pathways between them is largely unknown in primates. This issue was addressed using neural tracers in rhesus monkeys to label the bidirectional pathways between amygdala and hippocampus and the unidirectional pathway from hippocampus to ACC. The amygdala sent a robust projection to hippocampus that formed large and closely spaced dual synapses on spines from the same dendritic segment, suggesting a strong influence. Further, amygdalar axon boutons innervated some disinhibitory calretinin neurons in CA1, suggesting enhanced excitatory influence. In contrast, in CA3 the amygdala pathway innervated calretinin and some of the powerful parvalbumin inhibitory neurons, which may help enhance memory of affective events. The reverse pathway from hippocampus densely and mainly targeted the ventro-medial part of the amygdala, including the basolateral (BL) and paralaminar basolateral (PLBL) nuclei. Hippocampal terminations formed synapses mostly on spines vii of presumed excitatory neurons. Some hippocampal terminations innervated inhibitory neurons in BL and PLBL and showed a rank of preference, by targeting mostly calretinin, and then calbindin and least parvalbumin inhibitory neurons. This pattern of innervation may allow contextual information represented by hippocampus to influence affective processes in the amygdala. The hippocampus sent strong projections to ACC (A32, A24a and A25) and targeted particularly A25, suggesting a role in affective and autonomic regulation. About 90% of hippocampal terminations in A25 innervated excitatory neurons, suggesting strong excitatory effects. The hippocampal pathway had a close relationship with postsynaptic D1 receptors in A25, especially in the deep layers. Dopamine has a strong influence in goal-directed actions, rewards, and attention in prefrontal cortex in primates, and may facilitate contextual information from the hippocampus to A25 to influence emotional regulation. The pathways studied were distinct, and suggest specific roles in emotional memory by the amygdala in hippocampus, in flexible learning and forgetting fear based on context transmitted from hippocampus to the amygdala, and in the synthesis of current context and past experience carried out by the hippocampal pathway to ACC to influence adaptive goal directed behavior. / 2021-09-27T00:00:00Z

Hyperactivation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) by dopamine D₁ receptor (D₁R) in the striatum is a characteristic feature of several neuropsychiatric conditions, including drug addiction and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) within individuals suffering from Parkinson’s disease. However, the current mechanistic gap in understanding of D₁R-mediated regulation of ERK1/2, both in physiology and disease, hampers effective treatment of these conditions. One important factor that is underexplored in this regard is the role played by proteins that physically interact with the intracellular regions of D₁R. Using yeast two-hybrid screens and co-transfected human embryonic kidney 293 (HEK293) cells, our laboratory has recently characterized an interaction between the third intracellular loop of D₁R and synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase family. Moreover, our lab identified endogenous D₁R-SAP102 complex within rat striatum and hippocampus. Interestingly, SAP102 regulates ERK signaling pathway within the hippocampus, and modulates adenosine A2A receptor-mediated ERK1/2 activation within transfected HEK293 cells. Capitalizing on the above findings, I hypothesized a role for SAP102 in controlling D₁R-mediated ERK1/2 activation. Herein, I demonstrate using co-transfected HEK293 cells that SAP102 alters the temporal activation of ERK1/2 by D₁R. Intriguingly, experiments using the protein kinase A (PKA) inhibitors H89 and protein kinase inhibitor 14-22 amide myristoylated show that SAP102 also facilitates a switch in D₁R-mediated ERK1/2 activation from a PKA-independent to PKA-dependent pathway. Furthermore, SAP102 reduces basal ERK1/2 activation in HEK293 cells, reminiscent of a previously documented role of hippocampal SAP102. To the best of my knowledge, my findings are the first to demonstrate scaffolding protein-mediated switching of a G protein-coupled receptor (GPCR) signaling pathway. Future studies aimed at uncovering the details of this process should provide valuable insight on the mechanisms contributing to D₁R-mediated ERK1/2 activation, and may also offer clues to the existence of similar phenomena for other GPCRs. These studies would also aid development of improved pharmacological treatment options for conditions with dysfunctional D₁R-dependent ERK1/2 activation.

Indiana University-Purdue University Indianapolis (IUPUI) / Drinking despite aversive consequences, or compulsive drinking, is a criterion of alcohol use disorder and can be modeled in rodents by adding bitter quinine into alcohol. Previous studies have shown the development of quinine-resistant ethanol (EtOH) drinking following a drinking history, but used animals that achieved relatively low blood alcohol levels. Selectively bred crossed High Alcohol Preferring (cHAP) mice average over 250 mg/dl during a two-bottle choice procedure. Compulsive drinking is hypothesized to be D1-receptor mediated via the dorsolateral striatum (DLS). We hypothesized that 2 weeks of free-choice EtOH would lead to quinine resistance and intra-DLS infusion of a D1-antagonist, SCH23390, would attenuate quinine-resistant alcohol drinking with no effect on non-conflicted EtOH drinking. Infusion of SCH23390 into the DMS would not affect quinine-resistant drinking. cHAP mice had guide cannulae placed in the DLS or DMS and had either two weeks (2W) of EtOH and water two-bottle choice or were EtOH naïve (0W). Mice were infused with either SCH23390 or saline immediately prior to one 10% EtOH and water test day and SCH23390 did not disturb alcohol drinking. The following day, we adulterated the EtOH with 0.32-g/L quinine (0.89 mM), and mice received the same microinjection. For animals cannulated in the DLS, 2W history group infused with saline drank more quinine-adulterated EtOH than the 0W saline mice. While SCH23390 infused 0W animals looked no different from saline treated mice, it attenuated quinine + EtOH intake in the 2W animals to the level of 0W animals. Interestingly, DMS-cannulated mice demonstrated similar behavior, with SCH23390 reducing EtOH + quinine consumption, while leaving EtOH consumption undisturbed. Quinine resistance following 2 weeks of free-choice EtOH consumption is attenuated by acute administration of a D1-antagonist in the DLS, suggesting that an alcohol history induces compulsivity and that dopamine contributes to this behavior. This is unique to compulsive drinking, as non-conflicted EtOH drinking was unaffected.