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Browsing by Author "Sheets, Patrick L."
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Item Altered Excitability and Local Connectivity of mPFC-PAG Neurons in a Mouse Model of Neuropathic Pain(SfN, 2018) Cheriyan, John; Sheets, Patrick L.; Anesthesia, School of MedicineThe medial prefrontal cortex (mPFC) plays a major role in both sensory and affective aspects of pain. There is extensive evidence that chronic pain produces functional changes within the mPFC. However, our understanding of local circuit changes to defined subpopulations of mPFC neurons in chronic pain models remains unclear. A major subpopulation of mPFC neurons project to the periaqueductal gray (PAG), which is a key midbrain structure involved in endogenous pain suppression and facilitation. Here, we used laser scanning photostimulation of caged glutamate to map cortical circuits of retrogradely labeled cortico-PAG (CP) neurons in layer 5 (L5) of mPFC in brain slices prepared from male mice having undergone chronic constriction injury (CCI) of the sciatic nerve. Whole-cell recordings revealed a significant reduction in excitability for L5 CP neurons contralateral to CCI in the prelimbic (PL), but not infralimbic (IL), region of mPFC. Circuit mapping showed that excitatory inputs to L5 CP neurons in both PL and IL arose primarily from layer 2/3 (L2/3) and were significantly reduced in CCI mice. Glutamate stimulation of L2/3 and L5 elicited inhibitory inputs to CP neurons in both PL and IL, but only L2/3 input was significantly reduced in CP neurons of CCI mice. We also observed significant reduction in excitability and L2/3 inhibitory input to CP neurons ipsilateral to CCI. These results demonstrating region and laminar specific changes to mPFC-PAG neurons suggest that a unilateral CCI bilaterally alters cortical circuits upstream of the endogenous analgesic network, which may contribute to persistence of chronic pain.Item CaMKII Phosphorylation of the Voltage-Gated Sodium Channel Nav1.6 Regulates Channel Function and Neuronal Excitability(2021-01) Zybura, Agnes Sara; Cummins, Theodore R.; Hudmon, Andy; Baucum II, Anthony J.; Sheets, Patrick L.Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability.Item Cardiac sodium channel palmitoylation regulates channel function and cardiac excitability with implications for arrhythmia generation(2016-12-09) Pei, Zifan; Cummins, Theodore R.; Oxford, Gerry S.; Hudmon, Andy; Rubart-von der Lohe, Michael; Sheets, Patrick L.The cardiac voltage-gated sodium channels (Nav1.5) play a specific and critical role in regulating cardiac electrical activity by initiating and propagating action potentials in the heart. The association between Nav1.5 dysfunctions and generation of various types of cardiac arrhythmia disease, including long-QT3 and Brugada syndrome, is well established. Many types of post-translational modifications have been shown to regulate Nav1.5 biophysical properties, including phosphorylation, glycosylation and ubiquitination. However, our understanding about how post-translational lipid modification affects sodium channel function and cellular excitability, is still lacking. The goal of this dissertation is to characterize Nav1.5 palmitoylation, one of the most common post-translational lipid modification and its role in regulating Nav1.5 function and cardiac excitability. In our studies, three lines of biochemistry evidence were shown to confirm Nav1.5 palmitoylation in both native expression background and heterologous expression system. Moreover, palmitoylation of Nav1.5 can be bidirectionally regulated using 2-Br-palmitate and palmitic acid. Our results also demonstrated that enhanced palmitoylation in both cardiomyocytes and HEK293 cells increases sodium channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation by 2-Br-palmitiate increases closed-state channel inactivation and reduces myocyte excitability. Our computer simulation results confirmed that the observed modification in Nav1.5 gating properties by protein palmitoylation are adequate for the alterations in cardiac excitability. Mutations of potential palmitoylation sites predicted by CSS-Palm bioinformatics tool were introduced into wild-type Nav1.5 constructs using site-directed mutagenesis. Further studies revealed four cysteines (C981, C1176, C1178, C1179) as possible Nav1.5 palmitoylation sites. In particular, a mutation of one of these sites(C981) is associated with cardiac arrhythmia disease. Cysteine to phenylalanine mutation at this site largely enhances of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Therefore, C981 might be the most important site that regulates Nav1.5 palmitoylation. In summary, this dissertation research identified novel post-translational modification on Nav1.5 and revealed important details behind this process. Our data provides new insights on how post-translational lipid modification alters cardiomyocyte excitability and its potential role in arrhythmogenesis.Item The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain(Wiley, 2018) Li, Jun-Nan; Sheets, Patrick L.; Pharmacology and Toxicology, School of MedicineA major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA‐PAG pathway has proved to be a component of descending anti‐nociceptive circuitry, the functional organization of CeA‐PAG neurons remains unclear. We identified CeA‐PAG neurons in C57BL/6 mice of both sexes using intracranial injection of a fluorescent retrograde tracer into the PAG. In acute brain slices, we investigated the topographical and intrinsic characteristics of retrogradely labelled CeA‐PAG neurons using epifluorescence and whole‐cell electrophysiology. We also measured changes to CeA‐PAG neurons in the complete Freund's adjuvant (CFA) model of inflammatory pain. Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL‐PAG neurons consist of a relatively homogeneous population of intrinsically distinct neurons while CeM‐PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM‐PAG subtypes are altered 1 day after induction of the CFA inflammatory pain model. Collectively, our results provide insight into pain‐induced changes to a specific population of CeA neurons that probably play a key role in the integration of noxious input with endogenous analgesia and behavioural coping response.Item Characterizing Effects of Sphingosine-1-Phosphate Receptor 1 Activation in Subtypes of Central Amygdala Neurons and Effects of Prenatal Methadone Exposure on Motor Cortex Neurons in Mice(2021-04) Mork, Briana E.; Atwood, Brady K.; Sheets, Patrick L.; Cummins, Theodore R.; Fehrenbacher, Jill C.; McKinzie, David L.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. S1P receptor (S1PR) ligands have been utilized as an effective immunosuppressant, treatment in multiple sclerosis and studied as a treatment for pain. The primary cellular response to S1P is thought to be elicited through S1PR type 1 (S1PR1). My first goal was to understand how S1PR1 signaling affects neuronal excitability in the central amygdala (CeA), a supraspinal node of the descending pain pathway. The CeA is made up of a heterogenous population of neurons which form complex local and long-range circuits. The central lateral amygdala (CeL) consists of two major populations of inhibitory neurons identified by expression of the peptides somatostatin (Sst) and protein kinase Cδ (PKCδ). Sst neurons have been shown to maintain control over local circuits within the CeL and play a critical role in pain modulation. I utilized transgenic breeding strategies to fluorescently label Sst-expressing CeL neurons for whole-cell electrophysiology in acute brain slice. This strategy allowed me to study the effects of S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR on the cellular physiology of CeL Sst neurons. My findings reveal intrinsically distinct subtypes of CeL Sst neurons that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal pain pathways. My second goal was to assess the physiology of motor cortex neurons in mice exposed to prenatal methadone. Methadone is a synthetic μ-opioid agonist used for opioid maintenance therapy and chronic pain management. Methadone treatment for opioid use disorder in pregnant women can result in structural changes within the brain of their offspring causing and developmental delays to their children, including poorer motor performance. Using a mouse model of prenatal methadone exposure (PME), whole-cell electrophysiology, and analyses of cellular morphology, I elucidated the effects of PME on primary motor cortex (M1) output layer 5 (L5) neurons, which encompass the major cortical output pathway for motor control. My findings provide the first evidence that PME disrupts neuronal firing, subthreshold properties, and strength of local inputs onto M1 L5 neurons in prepubescent mice.Item Dissecting the Effects of Different Pain Modalities and Oxycodone on Prodynorphin Expressing Neurons in the Mouse Prelimbic Cortex(2022-11) Zhou, Shudi; Atwood, Brady K.; Sheets, Patrick L.; McKinzie, David L.; Truitt, William A.; Jin, XiaomingCurrently, changes to endogenous opioid circuits in various pain modalities, including surgical and neuropathic pain, remain unclear. Dynorphin, which is released by prodynorphin-expressing neurons (Pdyn+ neurons), is the endogenous opioid ligand to kappa opioid receptors (KOR). Moreover, a recent study has shown an increase in prodynorphin (Pdyn) mRNA expression in the prelimbic cortex (PL) in a mouse model of chronic pain. However, alterations in the activity of PL Pdyn-expressing neurons (PLPdyn+ neurons) in postoperative and chronic pain have never been explored. Firstly, I found that the population of PLPdyn+ neurons consists of both pyramidal and inhibitory subtypes. Secondly, I found that one day after surgical incision of the mouse hind paw, the excitability of pyramidal PLPdyn+ neurons was increased in both male and female mice, while the excitability of inhibitory PLPdyn+ neurons was unchanged. However, when postoperative pain behavior subsided, inhibitory PLPdyn+ neurons were hyperexcitable in male mice, while pyramidal PLPdyn+ neurons were hypoexcitable in female mice. Lastly, I dissected electrophysiological changes to PLPdyn+ neurons in the spared nerve injury (SNI) model of chronic neuropathic pain. At both early and late stages of SNI pain development, increased excitability of pyramidal PLPdyn+ neurons was detected in both male and female mice. However, in both male and female mice, the excitability of inhibitory PLPdyn+ neurons decreased 3 days after SNI but was conversely increased when measured 14 days after SNI. My findings suggest that different subtypes of PLPdyn+ neurons manifest distinct alterations in the development of different pain modalities in a sex-specific manner.Item Electrophysiological and Pharmacological Properties of the Neuronal Voltage-gated Sodium Channel Subtype Nav1.7(2007-12) Sheets, Patrick L.; Cummins, Theodore R.; Nicol, Grant D.; Oxford, Gerry S.; Vasko, Michael R.; Schild, John H.Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for the initiation of action potentials in excitable tissues by selectively allowing Na+ to flow through the cell membrane. VGSC subtype Nav1.7 is highly expressed in nociceptive (pain-sensing) neurons. It has recently been shown that individuals lacking the Nav1.7 subtype do not experience pain but otherwise function normally. In addition, dysfunction of Nav1.7 caused by point mutations in the channel is involved in two inherited pain disorders, primary erythromelalgia (PE) and paroxysmal extreme pain disorder (PEPD). This indicates Nav1.7 is a very important component in nociception. The aims of this dissertation were to 1) investigate if the antipsychotic drug, trifluoperazine (TFP), could modulate Nav1.7 current; 2) examine changes in Nav1.7 properties produced by the PE mutation N395K including sensitivity to the local anesthetic (LA), lidocaine; and 3) determine how different inactivated conformations of Nav1.7 affect lidocaine inhibition on the channel using PEPD mutations (I1461T and T1464I) that alter transitions between the different inactivated configurations of Nav1.7. Standard whole-cell electrophysiology was used to determine electrophysiological and pharmacological changes in WT and mutant sodium currents. Results from this dissertation demonstrate 1) TFP inhibits Nav1.7 channels through the LA interaction site; 2) the N395K mutation alters electrophysiological properties of Nav1.7 and decreases channel sensitivity to the local anesthetic lidocaine; and 3) lidocaine stabilizes Nav1.7 in a configuration that decreases transition to the slow inactivated state of the channel. Overall, this dissertation answers important questions regarding the pharmacology of Nav1.7 and provides insight into the changes in Nav1.7 channel properties caused by point mutations that may contribute to abnormal pain sensations. The results of this dissertation on the function and pharmacology of the Nav1.7 channel are crucial to the understanding of pain pathophysiology and will provide insight for the advancement of pain management therapies.Item Gpr17 deficiency in POMC neurons ameliorates the metabolic derangements caused by long-term high-fat diet feeding(Springer Nature, 2019-10-14) Reilly, Austin M.; Zhou, Shudi; Panigrahi, Sunil K.; Yan, Shijun; Conley, Jason M.; Sheets, Patrick L.; Wardlaw, Sharon L.; Ren, Hongxia; Medicine, School of MedicineBACKGROUND: Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) control energy homeostasis by sensing hormonal and nutrient cues and activating secondary melanocortin sensing neurons. We identified the expression of a G protein-coupled receptor, Gpr17, in the ARH and hypothesized that it contributes to the regulatory function of POMC neurons on metabolism. METHODS: In order to test this hypothesis, we generated POMC neuron-specific Gpr17 knockout (PGKO) mice and determined their energy and glucose metabolic phenotypes on normal chow diet (NCD) and high-fat diet (HFD). RESULTS: Adult PGKO mice on NCD displayed comparable body composition and metabolic features measured by indirect calorimetry. By contrast, PGKO mice on HFD demonstrated a sexually dimorphic phenotype with female PGKO mice displaying better metabolic homeostasis. Notably, female PGKO mice gained significantly less body weight and adiposity (p < 0.01), which was associated with increased energy expenditure, locomotor activity, and respiratory quotient, while males did not have an overt change in energy homeostasis. Though PGKO mice of both sexes had comparable glucose and insulin tolerance, detailed analyses of liver gene expression and serum metabolites indicate that PGKO mice could have reduced gluconeogenesis and increased lipid utilization on HFD. To elucidate the central-based mechanism(s) underlying the better-preserved energy and glucose homeostasis in PGKO mice on HFD, we examined the electrophysiological properties of POMC neurons and found Gpr17 deficiency led to increased spontaneous action potentials. Moreover, PGKO mice, especially female knockouts, had increased POMC-derived alpha-melanocyte stimulating hormone and beta-endorphin despite a comparable level of prohormone POMC in their hypothalamic extracts. CONCLUSIONS: Gpr17 deficiency in POMC neurons protects metabolic homeostasis in a sex-dependent manner during dietary and aging challenges, suggesting that Gpr17 could be an effective anti-obesity target in specific populations with poor metabolic control.Item Highly differentiated cellular and circuit properties of infralimbic pyramidal neurons projecting to the periaqueductal gray and amygdala(Frontiers Media S.A., 2015-04-28) Ferreira, Ashley N.; Yousuf, Hanna; Dalton, Sarah; Sheets, Patrick L.; Department of Pharmacology and Toxicology, IU School of MedicineThe infralimbic (IL) cortex is a key node in an inter-connected network involved in fear and emotion processing. The cellular and circuit-level mechanisms whereby IL neurons receive, filter, and modulate incoming signals they project onward to diverse downstream nodes in this complex network remain poorly understood. Using the mouse as our model, we applied anatomical labeling strategies, brain slice electrophysiology, and focal activation of caged glutamate via laser scanning photostimulation (glu-LSPS) for quantitative neurophysiological analysis of projectionally defined neurons in IL. Injection of retrograde tracers into the periaqueductal gray (PAG) and basolateral amygdala (BLA) was used to identify cortico-PAG (CP) and cortico-BLA (CA) neurons in IL. CP neurons were found exclusively in layer 5 (L5) of IL whereas CA neurons were detected throughout layer 2, 3, and 5 of IL. We also identified a small percentage of IL neurons that project to both the PAG and the BLA. We found that L5 CP neurons have a more extensive dendritic structure compared to L5 CA neurons. Neurophysiological recordings performed on retrogradely labeled neurons in acute brain slice showed that CP and CA neurons in IL could be broadly classified in two groups: neuronal resonators and non-resonators. Layer 2 CA neurons were the only class that was exclusively non-resonating. CP, CA, and CP/CA neurons in layers 3 and 5 of IL consisted of heterogeneous populations of resonators and non-resonators showing that projection target is not an exclusive predictor of intrinsic physiology. Circuit mapping using glu-LSPS revealed that the strength and organization of local excitatory and inhibitory inputs were stronger to CP compared to CA neurons in IL. Together, our results establish an organizational scheme linking cellular neurophysiology with microcircuit parameters of defined neuronal subclasses in IL that send descending commands to subcortical structures involved in fear behavior.Item Increased Excitability of Pyramidal Neurons in the Secondary Motor Cortex Enhances Cocaine-Seeking(2023-09) Huang, Donald; Atwood, Brady K.; Baucum, AJ; Hopf, Woody; Ma, Yao-Ying; Sheets, Patrick L.Cocaine addiction is a brain disorder characterized by chronic relapse. Although drug-seeking behaviors have been recognized to be associated with relapse, the role of the motor cortex, including the primary (M1) and secondary (M2) motor cortex, which are functionally important mediators of complex behaviors remains unclear in addiction. Here we use a rat cocaine intravenous self-administration (IVSA) model to investigate the intrinsic excitability of pyramidal neurons in the medial prefrontal cortices and motor cortices during withdrawal. Cocaine IVSA-trained rats performed a cocaine-seeking test on withdrawal day (WD) 1 or WD 45. Relative to WD 1 an increase in cocaineseeking was detected on WD 45. Whole-cell patch clamp recordings revealed an increase in intrinsic excitability in pyramidal neurons in Layer 2 of the secondary motor cortex (M2-L2) in cocaine trained rats on WD 45. Using a pharmacological approach, bath application of GABAA receptor agonist, muscimol, dosedependently (0.1 mM, 0.3 mM, and 1.0 mM) decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats on WD 45. Pharmacological inactivation of M2-L2 by bilateral intra-M2 injection of muscimol (324 ng/1.0 μl) attenuated cocaine-seeking on WD 45. A chemogenetic approach was used to validate that M2-L2 pyramidal neurons play a contributing role in the increase in cocaine-seeking, a microinjection of rAAV5-CaMKIIa-hM4di-mCherry was performed to express Gi-DREADD receptors on M2-L2 pyramidal neurons. Activating Gi-DREADD with an intraperitoneal injection of compound 21 on WD 45 attenuated cocaine-seeking. To elucidate the mechanism that contributes to the increased excitability of M2-L2 pyramidal neurons, an analysis of the action potential kinetics revealed that calcium-activated small conductance potassium (SK) channel-mediated medium afterhyperpolarization amplitude decreased in cocaine vs. saline IVSA-trained rats on WD 45. SK channel activation by 1-EBIO (300 μm) increased the medium afterhyperpolarization amplitude and decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats. Furthermore, intra-M2 injection of 1-EBIO on WD 45 attenuated cocaine-seeking. These experiments suggest that cocaine IVSA-training-induced persistent changes in M2-L2 pyramidal neurons' intrinsic excitability contributes to enhanced cocaine-seeking. Our results provide evidence targeting the SK channels in the superficial layer for M2 could be an important therapeutic approach for preventing cocaine relapse.
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