Anthony Phillips
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Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Cognitive impairment is a debilitating feature of psychiatric disorders including schizophrenia, mood disorders and substance use disorders. Despite cognitive impairment being the strongest predictor of functional outcomes in patients, there is a substantial lack of effective cognitive therapies. This thesis examines the pharmacological effects of d-govadine (d-GOV), a tetrahydroprotoberberine recently demonstrated to significantly enhance working memory and behavioural flexibility in several prefrontal cortex (PFC)-dependent rodent tasks. d-GOV also has high, selective affinity for dopamine (DA) D1 receptors (D1DRs), and selectively potentiates DA efflux in the PFC. This unique pharmacology sets it apart from many dopaminergic drugs and likely contributes to its cognitive effects. However, it is unclear how d-GOV selectively elevates PFC DA or what the functional effects of d-GOV binding to D1DRs are in the PFC. The aim of this thesis was to advance our understanding of these key aspects of d-GOV’s mechanism(s) in the PFC. I first found that d-GOV reverse dialyzed in the PFC, but not the ventral tegmental area (VTA), was sufficient to elevate PFC DA levels. Either PFC delivery or systemic administration of d-GOV preferentially potentiated DA vs NE efflux in the PFC, which differentiates d-GOV’s mechanism from known potentiators of PFC DA. Furthermore, the potentiating effects of d-GOV on PFC DA were ameliorated when glutamatergic transmission was disrupted in either the PFC or the VTA. These results suggest that d-GOV primarily acts in the PFC to potentiate local DA efflux, and that this mechanism likely involves the mesocortical feedback loop involving prefrontal glutamatergic projections to a population of VTA DA neurons that project back to the PFC. Moreover, d-GOV significantly potentiated whole-cell NMDA current in PFC slices in a D1DR-dependent manner. Additional evidence for the D1DR-mediated potentiation of NMDA currents by d-GOV was found in HEK293 cells co-expressing D1DRs and NMDARs. Specifically, d-GOV potentiated GluN2A-mediated currents in D1-GluN1/GluN2A-expressing cells when glutamate and d-GOV concentrations were low. d-GOV did not affect currents recorded from D1-GluN1/GluN2B-expressing cells, or when D1Rs were not expressed. These collective mechanisms through which d-GOV modulates PFC DA function are novel and further endorses it as a treatment for cognitive impairment.
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Accumulating evidence implicates dysfunction within the glutamatergic system anddysregulation of synaptic plasticity in the pathophysiology of depression, particularly in thehippocampus (HPC). Ketamine has rapid and sustained antidepressant activity in treatmentresistant depression and various animal models; however, its effects on synaptic plasticity, as well as their contribution to ketamine’s antidepressant action, are still unclear. To address this, we utilized the Wistar-Kyoto (WKY) model of endogenous stress susceptibility and depression. Consistent with the literature, WKY rats exhibited various depressive-like phenotypes compared to Wistar controls. In addition, we revealed that while in vivo hippocampal long-term depression (LTD) at the Schaffer collateral–CA1 (SC-CA1) synapse was not facilitated in the WKY strain, both early and late long-term potentiation (LTP) were significantly impaired. Importantly, both ketamine (5mg/kg, ip), as well as its metabolite (2R,6R)-HNK (5mg/kg, ip), acutely rescued the LTP deficit in WKYs at 3.5h following injection. Consistent with a sustained LTP-like effect,ketamine also increased SC-CA1 basal synaptic transmission at 24h in these rats. Importantly,ketamine, but not (2R,6R)-HNK, was found to have rapid and sustained antidepressant effects inWKY rats in the FST, leading to a dissociation between FST antidepressant-like activity anddorsal HPC synaptic plasticity. However, consistent with the observed SC-CA1 L-LTP deficitand corresponding effects of drug treatment, WKY rats exhibited impaired hippocampaldependent long-term spatial memory compared to Wistar controls (as measured by the novelobject location recognition test at a delay of 24h), which was effectively restored by bothketamine and (2R,6R)-HNK. We propose that, in the WKY rat model, restoring dorsal HPC LTPdoes not underlie ketamine’s antidepressant effects in FST, but may instead mediate reversal ofhippocampal-dependent cognitive deficits, which are also key features of clinical depression.ivThis work supports the theory that ketamine may reverse the stress-induced loss of connectivityin key neural circuits by engaging synaptic plasticity processes to “reset the system”, andhighlights the importance of deconstructing depression-like phenotypes and identifying theneural circuits that mediate them more precisely. Based on our results, the existing hypothesisthat ketamine’s antidepressant effects are solely due to the actions of its metabolite (2R,6R)-HNK is effectively challenged.
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The NMDA receptor is a glutamatergic ionotropic receptor key in mediating neuronalplasticity across virtually all synaptic circuits in the brain. An increasing list of neurologicaldisorders have implicated NMDA receptor hypofunction as an integral part of pathogenesis,necessitating the production of NMDA receptor potentiators as therapeutics. To date, most ofthese attempts have used increased co-agonism at the glycine binding site of NMDA receptors,but this strategy has been plagued by low specificity and efficacy. Specific allosteric modulationof NMDA receptors is an ideal solution, but until recently, no known drugs were capable ofdoing so. Building off previous work in our lab that discovered a novel family of compoundscapable of modulating NMDA receptor activity through its apical N-terminal domain, weidentified and characterized a drug candidate, Npam59, predicted to potentiate both GluN2A- and 2B-containing NMDA receptors. Npam59 was shown to potentiate NMDA currentsmediated by both subtypes with EC50 in the low-micromolar range. Npam59 also potentiated d-amphetamine-induced dopamine release in the ventral striatum in an NMDA receptor-dependent manner, but had no observable effect when administered alone. Finally, Npam59 potentiated d-amphetamine-induced hyperlocomotion in Sprague-Dawley rats. These results demonstrate that Npam59 can potentiate the function of NMDA receptors, including both GluN2A- and 2B-containing ones, suggesting its potential as a research tool and drug candidate for further development. Npam59 is the first known NMDA receptor allosteric potentiator with specificity for both GluN2A and GluN2B. Its characterization provides the foundation for therapeutic development and novel insights into the interaction of dopamine-glutamate signaling in the ventral striatum.
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The anterior cingulate cortex (ACC) has been implicated in a myriad of different functions. Converging evidence suggests that the ACC continuously monitors and evaluates actions and their consequences. Such functions are essential in representing action sequences which are the building blocks of all complex behaviors. This dissertation seeks to delineate how ACC neuronal ensembles represent different types of information with special emphasis on action sequences. Chapter 2 shows that the ACC ensembles represents different action sequences via unique activity patterns that change if the order of the actions are altered or if the locations of the actions is changed. Interestingly such shifts are achieved when overall levels of activity remain fixed. Chapter 3 reveals a very different arrangement in which progression through a sequence of actions towards a goal is associated with a change in the overall level of neural activity without a significant change in the patterns of activity. Specifically, ACC ensembles display a smooth progressive change in overall activity over three lever press actions that culminate in a reward. In contrast, the dorsal striatal (DS) ensembles recorded simultaneously from the same animals display fluctuations in activity level that are tightly linked to each action. Together these two chapters show that the ACC may use two different firing rate-related codes to convey categorical versus continuous forms of information.Chapter 4 provides a further examination of the mechanisms which allows the ACC ensembles to encode multiple types of categorical information. While the DS neurons encode both the sequence and the location of the levers in a somewhat synchronized fashion, ACC neurons encoded both of these types of information but kept them functionally segregated. As a result, even though ACC single neurons were no better than the DS in sequence decoding, sequence decoding by ACC ensembles was far superior to DS ensembles. The last chapter attempts to produce a unified theory of ACC function based on its coding properties. I will argue that the ACC monitors many aspects of experience while evaluating the current state with reference to a goal. Its multiple coding schemes efficiently serve both monitoring and evaluating functions.
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A hallmark of drug addiction is the compulsive drug taking behaviour that persists despite detrimental and adverse consequences. The gradual sensitization to the effects of drugs has been proposed to be an underlying mechanism for increased drug use and it serves as an animal model of craving and enduring neural plasticity. Evidence from studies conducted in rodents, non-human primates and humans have supported a role for sensitization in the development of states that promote drug taking. Rodent studies have also demonstrated that repeated exposure to drugs producing behavioural sensitization increases dopamine (DA) levels in the nucleus accumbens (NAc) and contributes to the acquisition of drug self-administration behaviour as well as the effort level to actively acquire a drug. The changes in neural functioning following repeated drug exposure is attributed to alterations in synaptic connections that underlie and are crucial for normal brain function as well as mechanisms of learning and memory. There is recent evidence supporting the development of a new class of interference peptides aimed at repairing functional and structural alterations in brain regions implicated in a number of psychiatric disorders. One such interference peptide, Tat-GluA2₃Y, blocks long-term depression (LTD) at glutamatergic synapses and has also been demonstrated to block the expression of behavioural sensitization and significantly reduce cue-induced reinstatement of heroin self-administration. This peptide interferes with the interaction between α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor and Brag2, a clathrin-adaptor protein which initiates the process of AMPA receptor endocytosis, leading to LTD. Results from the studies contained in this dissertation suggest that modulating protein-protein interactions with Tat-GluA2₃Y can influence the synaptic modifications following repeated amphetamine exposure, to effectively block the development and long-term maintenance of behavioral sensitization in a context-dependent manner. Effects of this peptide can be applied to understanding the circuitry and mechanisms involved in the development of psychostimulant sensitization, possibly serving as a platform from which to develop a novel pharmacotherapeutic approach for treating drug addiction.
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Enhanced dopamine efflux in the medial prefrontal cortex is a well-documented response to acute stress and is associated with deficits in cognitive performance. However, the underlying mechanism(s) for this response is unknown. The mesocortical dopamine system is comprised of dopamine neurons in the ventral tegmental area that receive excitatory input from and send reciprocal projections to the medial prefrontal cortex. We hypothesize that glucocorticoid receptors in the medial prefrontal cortex modulate the activity of this descending glutamatergic input to the ventral tegmental area during stress. Using in vivo microdialysis, we demonstrate that blocking glucocorticoid receptors locally within the rat medial prefrontal cortex, but not in the ventral tegmental area, attenuates mesocortical dopamine efflux to acute tail-pinch stress. Acute stress leads to a significant increase in glutamate release in the ventral tegmental area that is prevented by blockade of glucocorticoid receptors in the medial prefrontal cortex. The functional impact of enhanced mesocortical dopamine efflux evoked by acute stress was demonstrated using cognitive tasks measuring executive function. Exposure to acute tail-pinch stress selectively impaired performance on a working memory and set-shifting task. Conversely, performance on a non-delayed random foraging or reversal learning task that do not require the medial prefrontal cortex were unaffected by stress. Notably, stress-induced impairments in working memory were attenuated by blockade of glucocorticoid receptors in the medial prefrontal cortex. Taken together, these data suggest that glucocorticoids act locally within the medial prefrontal cortex to modulate mesocortical dopamine efflux by potentiation of glutamatergic drive onto dopamine neurons in the ventral tegmental area leading to the executive function impairments observed during acute stress.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Opioid use disorder (OUD) is a major contributor to drug-related deaths worldwide. Opioid use cessation causes severe withdrawal symptoms, including prominent hyperalgesia – a contributor to the negative reinforcement of drug taking. Effective pain control is an underappreciated aspect of managing opioid withdrawal, and its absence presents a significant barrier to successful opioid detoxification. Exploring analgesic interventions for withdrawal-induced hyperalgesia may reveal novel OUD therapies. This thesis describes a model of hyperalgesia in both acute and extended withdrawal in morphine-dependent animals and the effect of two clinical candidate analgesic drugs on withdrawal-induced hyperalgesia. l-Tetrahydropalmatine (l-THP) is a tetrahydroprotoberberine compound and active ingredient of a botanical formulation used in Vietnam for OUD treatment with preclinical efficacy in neuropathic pain models. Z944 is a selective T-type calcium channel antagonist undergoing clinical trials as an anticonvulsive and analgesic. To establish drug dependence modelling intermittent access during abuse scenarios, morphine (15 mg/kg, i.p.) was given once a day, 5 days/week and Von Frey tests were conducted 2-3 times a week ~23 h after morphine injection. Animals subjected to three weeks of morphine treatment experienced a ~30% reduction in pain tolerance. To model hyperalgesia during detoxification, animals entered abstinence after 3 weeks of morphine treatment and with Von Frey testing showing that hyperalgesia was persistent for 14 days before spontaneous recovery. Both l-THP (5 or 7.5 mg/kg, p.o.) and Z944 (10 mg/kg, p.o.) were effective at attenuating hyperalgesia during acute withdrawal. Seven-day treatment with l-THP (5 mg/kg) or Z944 (10 mg/kg) in morphine-dependent animals undergoing extended withdrawal resulted in a significant increase in paw retraction thresholds compared to controls and this effect persisted after the completion of treatment. Importantly, the improvement in pain tolerance remained after treatment completion, hastening pain tolerance recovery to baseline by 61% and 80% (l-THP and Z944, respectively). Neither candidate drug influenced mechanical sensitivity in morphine-naïve animals. Overall, these findings support the hypothesis that pain management during detoxification is necessary for improved treatment outcomes. l-THP and Z944, therefore, may be a valuable addition to the currently limited arsenal of opioid detoxification treatments.
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Mixed L-/T-type calcium channel antagonists attenuate morphine- and amphetamine induced conditioned place preference (CPP). Subtype specific antagonists for T-type calcium channels attenuate nicotine-reinforced behaviours in rats. This thesis investigated the effects of a novel T-type calcium channel antagonist, Z944, on the acquisition, expression, and reinstatement of amphetamine and morphine CPP. Furthermore, we examined Z944 for aversive or rewarding properties, and determined changes in locomotion with Z944 alone and in conjunction with amphetamine or morphine. CPP was induced with either morphine (5.0 mg/kg, IP) or amphetamine (1.5 mg/kg, IP) and Z944 (vehicle, 5.0, 7.5 mg/kg, IP) was administered 15 min prior to conditioning sessions for CPP acquisition experiments. For CPP expression experiments, Z944 was administered prior to the test session. In a related experiment Z944 was administered 15 min prior to the reinstatement injection of morphine after a period of extinction. Aversive and rewarding properties of Z944 were evaluated using a CPP/CPA procedure. Z944 dose-dependently attenuated the acquisition of morphine CPP, the expression of amphetamine CPP, and drug-induced reinstatement of both amphetamine and morphine CPP. Further, Z944 alone had no inherent rewarding or aversive effects, despite causing a decrease in spontaneous locomotor activity. It was also revealed that Z944 attenuated amphetamine-induced hyperlocomotion and potentiated morphine-induced hypolocomotion. These results suggest that T-type calcium channel antagonists differentially attenuate behaviours induced by several classes of drugs of abuse.
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Pharmacological treatments for drug addiction often relate to the dopamine (DA) system, which is known to play a key role in the development, persistence and relapse to compulsive drug-taking. However, many agents that alter single-receptor DA signaling have been clinically ineffective and there is growing interest in compounds targeting multiple receptors as a more promising therapeutic approach. Tetrahydroprotoberberines (THPB) derived from traditional Chinese herbal medicines have a high affinity for DA D1 and D2 receptors and have potential as novel treatments for drug addiction. This study assessed the effects of the THPB d-govadine on the acquisition, maintenance, expression and reinstatement of amphetamine-induced conditioned place preference (CPP). Furthermore, the effect of d-govadine on the acquisition and maintenance of food CPP was evaluated to gain insight into its action on multiple forms of reward-learning. Amphetamine CPP was established in rats by pairing d-amphetamine (1.5 mg/kg, i.p.) or saline with compartments with distinct contextual cues, and food CPP was induced by pairing Froot loops or no Froot loops with distinct contexts. In separate experiments, rats received d-govadine (0.5, or 1.0 mg/kg, i.p.) or vehicle, a) before each d-amphetamine injection during the conditioning phase, b) before tests for expression of amphetamine-induced CPP, c) before amphetamine-induced reinstatement of CPP, or d) before placement into food-associated compartments during the conditioning phase. CPP was assessed as greater time spent in the amphetamine- as compared to saline-paired contexts. d-govadine did not affect the acquisition of amphetamine CPP. However, groups that were pre-treated with d-govadine dose-dependently extinguished their preference for the amphetamine-associated context at a faster rate compared to vehicle-treated animals. While the expression of amphetamine CPP was not affected by d-govadine administered on the test day, the amphetamine-induced reinstatement of CPP following an extinction period was blocked by d-govadine (1.0 mg/kg). Finally, the intermediate dose of d-govadine (0.5 mg/kg) blocked the acquisition of food CPP and rats pre-treated with the high dose (1.0 mg/kg) extinguished their preference at a faster rate than vehicle-treated animals. These data suggest that d-govadine affects the acquisition of reward-context associations and the ability of amphetamine to reinstate preference for the amphetamine-associated compartment.
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Using a 6-OHDA model of Parkinson’s disease, we have preliminary evidence that L-DOPA-derived dopamine (DA) ceases to be released through conventional mechanisms of exocytosis under severe denervation. This may be problematic, as large, and possibly unregulated release of L-DOPA-derived DA would be expected to cause abnormal patterns of DA stimulation at the postsynaptic receptors, likely contributing to the development of dyskinesia. This issue may be overcome with the Dopamine Transporter (DAT) blocker Methylphenidate (MPD). Ahn and Phillips observed that MPD augmented the L-DOPA-derived DA efflux in a manner consistent with the under-appreciated fact that MPD also acts as a vesicular monoamine transporter (VMAT2) agonist (Volz, 2008), thereby facilitating the sequestration of cytosolic DA into vesicles, where it could enter into a cycle of impulse-dependent release processes. The hypothesis that MPD may influence the sequestration and release of L-DOPA-derived DA into presynaptically-regulated mechanisms of exocytosis may have beneficial therapeutic implications. Thus, the two major objectives of this thesis were first to assess whether L-DOPA-derived DA remained under presynaptic D2 autoreceptor regulation dependent on the severity of striatal denervation, and second, to investigate the mechanism(s) by which MPD may facilitate vesicular DA release, possibly by involvement of the D2 autoreceptor. L-DOPA was reverse-dialyzed into the intact and 6-OHDA lesioned dorsal striatum of the rat, followed by the reverse-dialysis of the D2 autoreceptor agonist and antagonist, Quinpirole and Eticlopride, or the VMAT2 inhibitor, Tetrabenazine. Although L-DOPA-evoked DA efflux remained under D2 autoreceptor control in the intact and moderately lesioned striatum, in the case of severe, 95% denervation, L-DOPA-evoked DA efflux was unaffected by D2 autoreceptor regulation or VMAT2 inhibition. However, despite the apparent loss of autoreceptor regulation, a subsequent study found that inhibitory binding of the D2 autoreceptor by reverse-dialysis of Eticlopride into the severely denervated striatum prior to the administration of MPD, completely blocked MPD-induced augmentation of L-DOPA-derived DA. These results implicate the D2 autoreceptor in a novel mechanism by which MPD can facilitate DA neurotransmission, and suggest that even under conditions of severe denervation, the presynaptic D2 autoreceptor may be manipulated pharmacologically to facilitate the exocytotic release of L-DOPA-derived DA.
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