Browsing by Subject "Electrophysiology"
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Item Aromatase inhibitors augment nociceptive behaviors in rats and enhance the excitability of sensory neurons(Elsevier, 2016-07) Robarge, Jason D.; Duarte, Djane B.; Shariat, Behzad; Wang, Ruizhong; Flockhart, David A.; Vasko, Michael R.; Pharmacology and Toxicology, School of MedicineAlthough aromatase inhibitors (AIs) are commonly used therapies for breast cancer, their use is limited because they produce arthralgia in a large number of patients. To determine whether AIs produce hypersensitivity in animal models of pain, we examined the effects of the AI, letrozole, on mechanical, thermal, and chemical sensitivity in rats. In ovariectomized (OVX) rats, administering a single dose of 1 or 5mg/kg letrozole significantly reduced mechanical paw withdrawal thresholds, without altering thermal sensitivity. Repeated injection of 5mg/kg letrozole in male rats produced mechanical, but not thermal, hypersensitivity that extinguished when drug dosing was stopped. A single dose of 5mg/kg letrozole or daily dosing of letrozole or exemestane in male rats also augmented flinching behavior induced by intraplantar injection of 1000nmol of adenosine 5'-triphosphate (ATP). To determine whether sensitization of sensory neurons contributed to AI-induced hypersensitivity, we evaluated the excitability of neurons isolated from dorsal root ganglia of male rats chronically treated with letrozole. Both small and medium-diameter sensory neurons isolated from letrozole-treated rats were more excitable, as reflected by increased action potential firing in response to a ramp of depolarizing current, a lower resting membrane potential, and a lower rheobase. However, systemic letrozole treatment did not augment the stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP) from spinal cord slices, suggesting that the enhanced nociceptive responses were not secondary to an increase in peptide release from sensory endings in the spinal cord. These results provide the first evidence that AIs modulate the excitability of sensory neurons, which may be a primary mechanism for the effect of these drugs to augment pain behaviors in rats.Item Automatic Detection and Characterization of Autonomic Dysreflexia Using Multi-Modal Non-Invasive Sensing and Neural Networks(Mary Ann Liebert, 2022-11-10) Suresh, Shruthi; Everett, Thomas H., IV; Shi, Riyi; Duerstock, Bradley S.; Medicine, School of MedicineAutonomic dysreflexia (AD) frequently occurs in persons with spinal cord injuries (SCIs) above the T6 level triggered by different stimuli below the level of injury. If improperly managed, AD can have severe clinical consequences, even possibly leading to death. Existing techniques for AD detection are time-consuming, obtrusive, lack automated detection capabilities, and have low temporal resolution. Therefore, a non-invasive, multi-modal wearable diagnostic tool was developed to quantitatively characterize and distinguish unique signatures of AD. Electrocardiography and novel skin nerve activity (skNA) sensors with neural networks were used to detect temporal changes in the sympathetic and vagal systems in rats with SCI. Clinically established metrics of AD were used to verify the onset of AD. Five physiological features reflecting different metrics of sympathetic and vagal activity were used to characterize signatures of AD. An increase in sympathetic activity, followed by a lagged increase in vagal activity during the onset of AD, was observed after inducing AD. This unique signature response was used to train a neural network to detect the onset of AD with an accuracy of 93.4%. The model also had a 79% accuracy in distinguishing between sympathetic hyperactivity reactions attributable to different sympathetic stressors above and below the level of injury. These neural networks have not been used in previous work to detect the onset of AD. The system could serve as a complementary non-invasive tool to the clinically accepted gold standard, allowing an improved management of AD in persons with SCI.Item CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability(American Society for Biochemistry and Molecular Biology, 2020-08-14) Zybura, Agnes S.; Baucum, Anthony J., II.; Rush, Anthony M.; Cummins, Theodore R.; Hudmon, Andy; Biology, School of ScienceNav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.Item CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability(MDPI, 2022-07-04) Zybura, Agnes S.; Sahoo, Firoj K.; Hudmon, Andy; Cummins, Theodore R.; Biology, School of ScienceAberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.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 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 De-Mixing Decision Representations in Rodent dmPFC to Investigate Strategy Change During Delay Discounting(2023-05) White, Shelby M.; Lapish, Christopher; Czachowski, Cristine; Oberlin, Brandon; Seamans, JeremySeveral pathological disorders are characterized by maladaptive decision-making (Dalley & Robbins, 2017). Decision-making tasks, such as Delay Discounting (DD), are used to assess the behavioral manifestations of maladaptive decision-making in both clinical and preclinical settings (de Wit, Flory, Acheson, Mccloskey, & Manuck, 2007). DD measures cognitive impulsivity and broadly refers to the inability to delay gratification (Hamilton et al., 2015). How decisions are made in tasks that measure DD can be understood by assessing patterns of behavior that are observable in the sequences of choices or the statistics that accompany each choice (e.g. response latency). These measures have led to insights that suggest strategies that are used by the agent to facilitate the decision (Linsenbardt, Smoker, Janetsian-Fritz, & Lapish, 2016). The current set of analyses aims to use individual trial data to identify the neural underpinnings associated with strategy transition during DD. A greater understanding of how strategy change occurs at a neural level will be useful for developing cognitive and behavioral strategies aimed at reducing impulsive choice. The rat dorso-medial prefrontal cortex (dmPFC) has been implicated as an important brain region for recognizing the need to change strategy during DD (Powell & Redish, 2016). Using advanced statistical techniques, such as demixed principal component analysis (dPCA), we can then begin to understand how decision representations evolve over the decision- making process to impact behaviors such as strategy change. This study was the first known attempt at using dPCA applied to individual sessions to accurately model how decision representations evolve across individual trials. Evidence exists that representations follow a breakdown and remapping at the individual trial level (Karlsson, Tervo, & Karpova, 2012; Powell & Redish, 2016). Furthermore, these representational changes across individual trials have previously been proposed to act as a signal to change strategies (Powell & Redish, 2016). This study aimed to test the hypothesis that a ‘breakdown’ followed by a ‘remapping’ of the decision representation would act as a signal to change strategy that is observable in the behavior of the animal. To investigate the relationship between trials surrounding the breakdown and/or subsequent remapping of the decision representation and trials surrounding strategy changes, sequences of trials surrounding the breakdown and/or remapping were compared to sequences of 9 trials surrounding the strategy-change trial. Strategy types consisted of either exploiting the immediate lever (IM-Exploit), delay lever (DEL-Exploit), or exploring between the two lever options (Explore). Contrary to the hypothesis, an overall relationship between breakdown and remapping trial sequences were not associated with change-trial sequences. In partial support of the hypothesis however, at the 4-sec delay when the subjective value of the immediate reward was high, a relationship between breakdown sequence and strategy change sequence was detected for when the animal was exploiting the delay lever (e.g. DEL-Exploit strategy). This result suggests that a breakdown in decision representation may act as a signal to prompt strategy change under certain contexts. One notable finding of this study was that the decision representation was much more robust at the 4-sec delay compared to the 8-sec delay, suggesting that decisions at the 4-sec delay contain more context that differentiate the two choice options (immediate or delay). In other words, the encoding of the two choice options was more dissociable at the 4-sec delay compared to the 8-sec delay, which was quantified by measuring the average distance between the two representations (immediate and delay) on a given trial. Given that Wistar rats are equally likely to choose between the immediate and delay choice alternatives at the 8-sec delay (Linsenbardt et al., 2016), this finding provides further support for current prevalent theories of how animals use a cognitive search process to mentally imagine choice alternatives during deliberation. If context which differentiates choice options at the 8-sec delay is less dissociable, it is likely that the cognitive search process would be equally likely to find either choice option. If the choice options are equally likely to be found, it would be assumed that the choice alternatives would also be equally likely to be chosen, which is what has been observed in Wistar rats at the 8-sec delay.Item Early-life trauma alters hippocampal function during an episodic memory task in adulthood(2017-05-02) Janetsian-Fritz, Sarine S.; Lapish, Christopher; Sangha, Susan; Goodlett, Charles; Neal-Beliveau, BethanyEarly life trauma is a risk factor for a number of neuropsychiatric disorders, including schizophrenia (SZ) and depression. Animal models have played a critical role in understanding how early-life trauma may evoke changes in behavior and biomarkers of altered brain function that resemble these neuropsychiatric disorders. However, since SZ is a complex condition with multifactorial etiology, it is difficult to model the breadth of this condition in a single animal model. Considering this, it is necessary to develop rodent models with clearly defined subsets of pathologies observed in the human condition and their developmental trajectory. Episodic memory is among the cognitive deficits observed in SZ. Theta (6-10 Hz), low gamma (30-50 Hz), and high gamma (50-100 Hz) frequencies in the hippocampus (HC) are critical for encoding and retrieval of memory. Also, theta-gamma comodulation, defined as correlated fluctuations in power between these frequencies, may provide a mechanism for coding episodic sequences by coordinating neuronal activity at timescales required for memory encoding and retrieval. Given that patients with SZ have impaired recognition memory, the overall objectives of these experiments were to assess local field potential (LFP) recordings in the theta and gamma range from the dorsal HC during a recognition memory task in an animal model that exhibits a subclass of symptoms that resemble SZ. In Aim 1, LFPs were recorded from the HC to assess theta and gamma power to determine whether rats that were maternally deprived (MD) for 24-hrs on postnatal day (PND 9), had altered theta and high/low gamma power compared to sham rats during novel object recognition (NOR). Brain activity was recorded while animals underwent NOR on PND 70, 74, and 78. In Aim 2, the effects of theta-low gamma comodulation and theta-high gamma comodulation in the HC were assessed during NOR between sham and MD animals. Furthermore, measures of maternal care were taken to assess if high or low licking/grooming behaviors influenced recognition memory. It was hypothesized that MD animals would have impaired recognition memory and lower theta and low/high gamma power during interaction with both objects compared to sham animals. Furthermore, it was hypothesized that sham animals would have higher theta-gamma comodulation during novel object exploration compared to the familiar object, which would be higher than the MD group. Measures of weight, locomotor activity, and thigmotaxis were also assessed. MD animals were impaired on the NOR task and had no change in theta or low/high gamma power or theta-gamma comodulation when interacting with the novel or familiar object during trials where they performed unsuccessfully or successfully. However, higher theta and gamma power and theta-gamma comodulation was observed in sham animals depending on the object they were exploring or whether it was a successful or unsuccessful trial. These data indicate altered functioning of the HC following MD and a dissociation between brain activity and behavior in this group, providing support that early life trauma can induce cognitive and physiological impairments that are long-lasting. In conclusion, these data identify a model of early life stress with a translational potential, given that there are points of contact between human studies and the MD model. Furthermore, these data provide a set of tools that could be used to further explore how these altered neural mechanisms may influence cognition and behavior.Item Effects of carbon nanotubes on barrier epithelial cells via effects on lipid bilayers(2013) Lewis, Shanta; Blazer-Yost, Bonnie; Petrache, Horia; Witzmann, F. A. (Frank A.); Atkinson, SimonCarbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.