Browsing by Author "McKinzie, David"

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    The Behavioral Role of Mu Opioid Receptors in Glutamatergic Neurons
    (2021-10) Reeves, Kaitlin C.; Sheets, Patrick; Baucum, Anthony II; Yamamoto, Bryan; McKinzie, David; Yoder, Karmen
    Mu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.
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    Contributions of the Presynaptic Protein Bassoon to Tau Pathogenesis and Neurodegeneration
    (2024-05) Patel, Henika Sanjaybhai; Oblak, Adrian; Lasagna-Reeves, Cristian; McKinzie, David; Kim, Jungsu; Webb, Ian; Murray, Melissa
    Neurodegenerative tauopathies, characterized by the aggregation of misfolded tau protein, pose a significant clinical and scientific challenge. A high-molecular-weight (HMW) tau species is known to be involved in spreading tau pathology. However, the nature and composition of this species remain elusive, hindering targeted interventions. There are four main chapters in this dissertation. The first chapter highlights the existing knowledge about tau and its role in neurodegenerative tauopathies and discusses the possible contribution of protein interactors in the pathogenesis of tau pathology. The second chapter investigates the association between pathological hallmarks and functional deficits in the aged PS19 tauopathy model. The findings indicate that a diverse spectrum of pathological tau species may underly different symptoms and that neuroinflammation might contribute to functional deficits independent of tau pathology. In the third chapter, we isolated and characterized the HMW tau species with seeding capabilities from the PS19 brains. Using unbiased quantitative mass spectrometry analysis, we identified Bassoon (BSN), a presynaptic protein, as a crucial interactor of the HMW tau seed. BSN overexpression exacerbated tau-seeding and toxicity both in vitro and in the Drosophila model of tauopathy. Conversely, the downregulation of BSN reduced tau spreading and overall disease pathology in the PS19 mice, indicating the important role of BSN in taumediated pathogenesis. In chapter four, we studied the disease-associated p.Pro3866Ala missense mutation in BSN and further evaluated the mechanisms through which BSN could induce toxicity and neurodegeneration. Using CRISPR-Cas9 technology, we developed a knock-in mouse model harboring the BSN P3866A missense mutation in the endogenous murine Bsn. We observed somatic BSN accumulation suggesting that the P3866A mutation might be enhancing the aggregation propensity of BSN and provide a conducive environment to promote tau aggregation. Furthermore, we observed dysregulation in protein degradation pathways, neuroinflammation, and enhanced synapse elimination by microglia. These findings underscore the pivotal role of BSN in providing a favorable environment for tau aggregation and influencing the properties of the tau seed, thereby contributing to neurodegenerative processes. Overall, our results indicate that targeting BSN could be a potential therapeutic intervention for neurodegenerative diseases.
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    The Enduring Consequences of Prenatal Opioid Exposure
    (2022-02) Grecco, Gregory Giovanni; Sheets, Patrick; Atwood, Brady; Yamamoto, Bryan; McKinzie, David; Yoder, Karmen
    The opioid crisis has resulted in an unprecedented number of neonates born with prenatal opioid exposure; however, the long-term effects of opioid exposure on offspring behavior and neurodevelopment remain relatively unknown. I developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. PME produced substantial impairments in offspring growth, sensorimotor milestone acquisition, and activity in an open field. Furthermore, these behavioral alterations were associated with significant disruptions in the primary motor cortex (M1). Notably, layer 5 pyramidal neurons of the M1 displayed significantly increased voltage sag which is primarily mediated by HCN1 channels. Interestingly, the α2-adrenergic receptor, a known modulator of HCN1 channels, displayed significantly increased expression in the M1 of PME animals. The locomotor activity in an open field was significantly reduced following in vivo pharmacological activation of the α2-adrenergic receptor with clonidine in PME offspring suggesting this may be therapeutic target for the hyperactivity associated with prenatal exposure to opioids. Previous work has also described an association between prenatal opioid exposure and alterations in opioid reward-related behavior; however, the effect of PME on alcohol reward remains undetermined. Given the widespread accessibility and usage, alcohol represents the most likely addictive substance the growing population of opioid exposed neonates will encounter as they age. I discovered that PME disrupts conditioned preference for alcohol, enhances the locomotor stimulating effects of alcohol, and increases alcohol consumption in a sex-dependent manner. This alcohol-reward phenotype in PME offspring was associated with altered excitatory neurotransmission and disrupted cannabinoid-mediated long-term depression (CB-LTD) in the dorsolateral striatum, an important substrate involved in compulsive drug use. Further work is required to determine the specific inputs at which CB-LTD is disrupted and if restoring this form of plasticity in PME animals prevents the enhanced alcohol addiction phenotype.
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    Follow Your Heart: Heart Rate Variability Reveals Sex Differences in Autonomic Regulation During Anxiety-Like Behavior and During Alcohol Drinking in Rats
    (2024-02) Frasier, Raizel Michele; Yoder, Karmen; Hopf, Woody; McKinzie, David; Lukkes, Jodi; Conroy, Susan
    Mental health conditions remain a substantial and costly challenge to society. Of note, women have a higher prevalence of anxiety disorders than men, and alcohol misuse in women has risen sharply in recent years. However, critical mechanisms underlying these observed sex differences remain incompletely understood. Measures of cardiac function, including heart rate (HR) and HR variability (HRV), reflect dynamic balance between the two opposing branches of the autonomic nervous system: sympathetic (SNS, “fight or flight”) and parasympathetic (PNS, “rest and digest”). Furthermore, recent evidence strongly suggests these measures are potential biomarkers for pathological states, including mental health conditions. To better understand sex differences in autonomic mechanisms related to pathological anxiety and alcohol misuse, we utilized cardiac telemetry to measure HR and HRV. This allowed observation of real-time autonomic tone in awake, freely behaving Wistar rats of both sexes. At baseline, female rats had greater HR and lower SNS influence than males, which concords with human studies. In both anxiety-like behavior and alcohol drinking studies, we observed that females tend to utilize a higher PNS influence to overcome challenge, whereas males tend to utilize higher SNS. Furthermore, female (but not male) baseline HR and HRV are related to within-task behavior, suggesting that baseline state impacts drinking and anxiety-like behavior in a sex-specific way. Again, these data concord with human research suggesting a similar PNS bias in women and SNS bias in men when responding, especially under challenge. Taken together, these data have contributed new knowledge to sex differences in autonomic engagement, especially for anxiety states and alcohol drinking. Importantly, these findings are likely translationally relevant for the development of novel, and more personalized, therapies.
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    The M1/M4 preferring muscarinic agonist xanomeline modulates functional connectivity and NMDAR antagonist-induced changes in the mouse brain
    (Springer Nature, 2021) Montani, Caterina; Canella, Carola; Schwarz, Adam J.; Li, Jennifer; Gilmour, Gary; Galbusera, Alberto; Wafford, Keith; Gutierrez-Barragan, Daniel; McCarthy, Andrew; Shaw, David; Knitowski, Karen; McKinzie, David; Gozzi, Alessandro; Felder, Christian; Pharmacology and Toxicology, School of Medicine
    Cholinergic drugs acting at M1/M4 muscarinic receptors hold promise for the treatment of symptoms associated with brain disorders characterized by cognitive impairment, mood disturbances, or psychosis, such as Alzheimer's disease or schizophrenia. However, the brain-wide functional substrates engaged by muscarinic agonists remain poorly understood. Here we used a combination of pharmacological fMRI (phMRI), resting-state fMRI (rsfMRI), and resting-state quantitative EEG (qEEG) to investigate the effects of a behaviorally active dose of the M1/M4-preferring muscarinic agonist xanomeline on brain functional activity in the rodent brain. We investigated both the effects of xanomeline per se and its modulatory effects on signals elicited by the NMDA-receptor antagonists phencyclidine (PCP) and ketamine. We found that xanomeline induces robust and widespread BOLD signal phMRI amplitude increases and decreased high-frequency qEEG spectral activity. rsfMRI mapping in the mouse revealed that xanomeline robustly decreased neocortical and striatal connectivity but induces focal increases in functional connectivity within the nucleus accumbens and basal forebrain. Notably, xanomeline pre-administration robustly attenuated both the cortico-limbic phMRI response and the fronto-hippocampal hyper-connectivity induced by PCP, enhanced PCP-modulated functional connectivity locally within the nucleus accumbens and basal forebrain, and reversed the gamma and high-frequency qEEG power increases induced by ketamine. Collectively, these results show that xanomeline robustly induces both cholinergic-like neocortical activation and desynchronization of functional networks in the mammalian brain. These effects could serve as a translatable biomarker for future clinical investigations of muscarinic agents, and bear mechanistic relevance for the putative therapeutic effect of these class of compounds in brain disorders.
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    NMDAR-PSD95-nNOS Axis-Mediated Molecular Mechanisms in the Basolateral Amygdala Underlying Fear Consolidation
    (2021-05) Patel, Jheel; Sheets, Patrick; Shekhar, Anantha; McKinzie, David; Yamamoto, Bryan; Liu, Yunlong
    Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories.
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    Pathological tau and reactive astrogliosis are associated with distinct functional deficits in a mouse model of tauopathy
    (Elsevier, 2022) Patel, Henika; Martinez, Pablo; Perkins, Abigail; Taylor, Xavier; Jury, Nur; McKinzie, David; Lasagna-Reeves, Cristian A.; Anatomy, Cell Biology and Physiology, School of Medicine
    Pathological aggregation of tau and neuroinflammatory changes mark the clinical course of Alzheimer’s disease and related tauopathies. To understand the correlation between these pathological hallmarks and functional deficits, we assessed behavioral and physiological deficits in the PS19 mouse model, a broadly utilized model of tauopathy. At 9 months, PS19 mice have characteristic hyperactive behavior, a decline in motor strength, and deterioration in physiological conditions marked by lower body temperature, reduced body weight, and an increase in measures of frailty. Correlation of these deficits with different pathological hallmarks revealed that pathological tau species, characterized by soluble p-tau species, and tau seeding bioactivity correlated with impairment in grip strength and thermal regulation. On the other hand, astrocyte reactivity showed a positive correlation with the hyperactive behavior of the PS19 mice. These results suggest that a diverse spectrum of soluble pathological tau species could be responsible for different symptoms and that neuroinflammation could contribute to functional deficits independently from tau pathology. These observations enhance the necessity of a multi-targeted approach for the treatment of neurodegenerative tauopathies.
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    Spinophilin Cell Type-Specifically Mediates Metabotrophic Glutamate Receptor 5-dependent Excessive Grooming
    (2022-09) Morris, Cameron W.; Truitt, William; Atwood, Brady; Baucum, Anthony J., II; Ma, Yao-Ying; McKinzie, David
    Compulsive and repetitive behaviors in obsessive-compulsive spectrum disorders (OCSDs) are associated with perturbations in the sensorimotor striatum. Repetitive behaviors are associated with cell type-specific adaptations in striatal direct- and indirect-pathway medium spiny neurons (dMSNs and iMSNs, respectively). Furthermore, preclinical models for understanding OCSDs, such as constitutive knockout of disks large associated protein 3 (SAPAP3), suggest repetitive motor dysfunction, such as excessive grooming, is associated with increased metabotropic glutamate receptor 5 (mGluR5) activity that increases dMSN function relative to iMSNs in the sensorimotor striatum. However, MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Reversible phosphorylation of mGluR5’s C-terminal domain is one mechanism to regulate mGluR5 signaling, however, unlike kinases, promiscuous phosphatases require targeting proteins to shuttle them into contact with their targets. Therefore, phosphatase targeting proteins may be intimately involved in mediating mGluR5-dependent striatal adaptions underlying repetitive behaviors, such as excessive grooming in SAPAP3 deficient mice. Spinophilin, a major striatal postsynaptic phosphatase targeting protein, regulates striatal function, mGluR5 signaling, and forms a protein-protein interaction with SAPAP3 that is increased by mGluR5 co-expression. Therefore, we hypothesized that spinophilin expression in striatal medium spiny neurons mediates mGluR5-dependent excessive grooming. To test this, we used a novel conditional spinophilin mouse line combined with functional, behavioral, and molecular approaches to elucidate spinophilin's MSN subtype-specific contributions to rodent excessive grooming behavior associated with increased mGluR5 function. We found that loss of spinophilin in either MSN subtype abrogated plasticity in the sensorimotor striatum associated with increased mGluR5 function and decreased two models of excessive grooming associated with increased mGluR5 function—SAPAP3 deficient mice and global administration of a mGluR5-specific positive allosteric modulator (VU0360172). Additionally, we found that spinophilin’s protein interaction with mGluR5 correlates with grooming behavior and loss of spinophilin shifts mGluR5 interactions from lipid-raft associated proteins toward postsynaptic density proteins implicated in psychiatric disorders. Collectively, these results identify spinophilin as a novel striatal signaling hub molecule in MSNs that MSN subtype-specifically mediates striatal adaptations associated with repetitive motor dysfunction in psychiatric disorders.