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    A Monoallelic DNM1L Mutation presenting with Epilepsia Partialis Continua: A Case Report
    (2022) Park, Chanhyun
    Background: Variants in DNM1L are reported as a rare cause of refractory epilepsy and status epilepticus. We report a patient with epilepsia partialis continua (EPC) secondary to a monoallelic DNM1L mutation. Case Information: An 11-year-old boy with prior history of speech delay and well-controlled absence epilepsy on valproate presented in clinic with status epilepticus and posterior frontal diffusion restriction on MRI. Seizures were characterized by hemifacial clonus consistent with EPC. Extensive workup including EEG, MRI, cytokine, and encephalitis panels were unrevealing for etiology. Genetic peroxisomal panel revealed a monoallelic missense mutation (R403C) in the DNM1L locus as cause for his EPC. GDF15 was also elevated, reaffirming the presence of mitochondrial disease. This DNM1L mutation was determined to be the underlying etiology for his presentation. Lacosamide, clobazam and phenobarbital, among other interventions, were ultimately used to control the patient's epilepsy; he was sent home after extensive stays in the PICU and inpatient rehabilitation unit. Conclusions: DNM1L mutations can cause cerebral dysmyelinations, abnormal gyral patterns, microcephaly, and death within the first year of life. Yet several recent cases, including ours, have linked DNM1L variants with other neurological phenotypes, including a late onset of symptoms such as intractable epilepsy, myoclonus, and developmental delay. This case is strikingly like that of a previous report but with additional clinical features such as aphasia and EPC. The presentation of EPC in our patient, as well as the difficulty finding its etiology, exemplifies the unclear clinical pattern that remains with DNM1L mutations. The clinical ambiguity of this mutation complicates diagnosis and demonstrates the importance of prompt genetic testing.
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    (2022) De La Cruz, Daniel; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, Katalin
    Purpose: Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) is a small peptide with numerous neuro-modulatory impacts beyond its role within the neuroendocrine system. TRH's broad central nervous system (CNS) effects, acting as a neurotransmitter and neuromodulator, emphasize a great potential to treat many neurological and psychological disorders. However, its pharmacological applications remain unrealized due to brain delivery shortcomings following its systemic administration. Previously, our laboratory's novel prodrug design, relying on two highly brain-expressed enzymes for prodrug metabolism to TRH, successfully delivered the metabolically highly unstable peptide into the brain. Consequently, an in vivo therapeutic safety assessment was conducted to further validate our prodrug approach through a comparative study that capitalizes on TRH's stimulatory release of thyroid hormones. Likewise, TRH's ability to trigger acetylcholine release is also well documented, and here, this neurochemical marker has been utilized to determine the extent to which TRH is delivered to the CNS via our prodrug approach. Moreover, our laboratory has recently identified pGlu-βGlu-Pro-NH2 ([βGlu2]TRH) as the first functional antagonist of the central cholinergic actions of TRH, and as such, we have explored the receptor-associated mechanism responsible for this antagonism by utilizing a human TRH receptor (hTRH-R) homology model. Ultimately, our extensive computational chemistry-based studies revealed a novel receptor allosteric site that exhibits a selective and high-affinity binding for [βGlu2 ]TRH, while also demonstrating our prodrug's inability to bind and activate this hTRH-R. Methods: Lead TRH prodrugs and various TRH analogues were designed in silico for docking experiments with the hTRH-R using SeeSAR and AutoDock Vina software. A TRH challenge in CD-1 mice, utilizing systemically administered TRH and an equimolar concentration of a TRH prodrug, measured downstream effector levels of thyroid hormones at several subsequent time points analyzed by LC-MS/MS. Microdialysis studies, in the frontal cortex of SD rats, compared each animal's baseline acetylcholine concentration to subsequent levels, following the perfusion of a TRH prodrug and TRH, as a positive control, at equimolar concentrations. This neurochemical survey quantifies acetylcholine turnover using LC-MS/MS, as a surrogate measure of the extent to which TRH is delivered into the brain via our prodrug approach. Results: Compared to TRH, prodrugs were unable to dock to the hTRH-R's active site, and when systemically administered, the TRH prodrug failed to elicit a thyroid response while simultaneously triggering a profound release of acetylcholine in the brain. Conclusions: The inability of TRH prodrugs to elicit a thyroid response was predicted by its in vitro metabolic stability, as well as computational chemistry studies, that demonstrate TRH prodrugs exhibit physiochemical properties that prohibit the direct activation of the hTRH-R. Furthermore, based on [βGlu2]TRH as a template, the design of novel hTRH-R inhibitors will be conducted in a follow-up study to further substantiate our prodrug approach and aid the elucidation of TRH activity and pathways.
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    Low-dose methotrexate exposure induced long-term cognitive deficits in mice
    (2022) Trinh, Oanh; Sumien, Nathalie; Vann, Philip; Davis, Delaney; Luedtke, Robert R.; Basha, Riyaz; Singh, Meharvan
    Purpose: Chemotherapy-related cognitive impairment (CRCI) remains a mysterious morbidity that threatens the quality of life of up to 70% cancer survivors in the United States. Longitudinal studies of CRCI highlighted deficits in memory, learning, attention, motor, and executive functions for up to 20 years after the completion of chemotherapy paradigm. These deficits, especially if happened during childhood, can negatively impact educational achievement, employment, self-independence, and life expectancy of approximately 500,000 adult survivors currently living in the U.S. Given that acute lymphoblastic leukemia (ALL) is the most common diagnosis of childhood cancers worldwide, the folate-inhibitor methotrexate (MTX) has been at the backbone of ALL-treatment with a substantial risk of neurotoxicity. The purpose of this study was to establish a tumor-free mouse model representative of MTX-induced CRCI in childhood ALL survivors and study the long-term effects of chemotherapy treatment on brain function. We hypothesized that MTX administration at a very young age will induce long-term cognitive impairments. Methods: At post-natal day 15, male and female C57BL6/J pups received intraperitoneal injections of either saline (n=12) or MTX (2 mg/kg; n=12) once a day for 3 days. The pups were weaned at post-natal day 21 and allowed to age. At 8-month-old, animals underwent behavioral tests to assess motor, affective and cognitive functions. Results: MTX administration impaired cognitive flexibility in males and impaired spatial learning and memory in females, indicating potential sex- and test-dependent behavioral outcomes. MTX increased performance in coordinated-running test, and increased swimming speed. Anxiety-like behaviors were not affected by the treatment. Conclusions: These preliminary results suggested that low dose MTX-treatment induced sex-dependent cognitive deficits while affective and motor functions were not negatively affected. This study will be repeated, and behaviors will be assessed at other time points to establish complete neurobehavioral profiles of mice affected by MTX chemotherapy.
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    Characterizing Region-Specific Glucose Metabolic Profile of the Rodent Brain Using Seahorse XFe96 Analyzer
    (2022) Wang, Linshu; Chaudhari, Kiran; Winters, Ali; Sun, Yuanhong; Liu, Ran; Yang, Shaohua
    Purpose: The brain is highly complex with diverse structural characteristics in accordance with specific functions. Accordingly, differences in regional function, cellular compositions, and active metabolic pathways may link to differences in glucose metabolism at different brain regions. A recent study using imaging mass spectrometry demonstrated that some of the glucose metabolism enzymes and ATP level vary dramatically cross the brain. Disruption of glucose metabolism forms the pathophysiological basis for many brain disorders. Therefore, the brain spatial metabolic signatures are of high relevance in our understanding of the normal brain physiology and neuropathology of neurological diseases. Method: We optimized an acute biopsy punching method and characterized region-specific glucose metabolism of rat and mouse brain by a Seahorse XFe96 analyzer. Results: In the current study, we demonstrated that 0.5 mm diameter tissue punches from 180-µm thick brain sections allow metabolic measurements of anatomically defined brain structures using Seahorse XFe96 analyzer. We found that the cerebellum displays a more quiescent phenotype of glucose metabolism than cerebral cortex, basal ganglia, and hippocampus. In addition, the cerebellum has higher AMPK activation than other brain regions evidenced by the expression of pAMPK, upstream pLKB1, and downstream pACC. Furthermore, rodent brain has relatively low mitochondrial oxidative phosphorylation efficiency with up to 30% of respiration linked to proton leak. Conclusions: The present study determined the region-specific glucose metabolic profile of rodent brain using acute biopsy punches and Seahorse XFe96 analyzer. The metabolic flux analysis indicated that the cerebellum has a more quiescent phenotype of glucose metabolism as compared with the cerebrum. In addition, glucose metabolism might be less efficient in the brain than we expected, with relatively large component of proton leak-linked respiration.
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    Assessment of Sex Differences Following Repeated Mild Head Injuries
    (2022) Duggal, Aakaash; Vann, Philip; Metzger, Daniel; Ahmed, Affan; Sumien, Nathalie; Schreihofer, Derek
    Background: Traumatic brain injury (TBI) is a major cause of disability, morbidity, and mortality in the U.S. Although there is a growing understanding of the effects of moderate and severe TBI, less is understood about the effects of repetitive mild TBI (rmTBI). Nevertheless, some studies show that long term participants in contact sports have an increased risk for neurodegenerative disease. In addition, there is limited information about sex differences in TBI, despite some studies suggesting females participating in contact sports experience more head injuries than males. With an increasing number of females participating in contact sports, it's important to explore the effects of rmTBI in females. Purpose: This study will test the hypothesis that rmTBI will lead to more severe neurological deficits in female mice than in male mice. Methods: C57BL/6 female mice were assigned to sham and rmTBI groups (n=30/group). Lightly anesthetized mice received 25 mild head injuries, once a day (M-F) over 5 weeks using a weight drop model that included a free fall with rotational injury. Acute effects of injury were assessed by righting reflex and balance beam tests weekly. Chronic effects were tested with rotarod, Morris water maze (MWM), elevated plus maze (EPM), and T-maze beginning 5 or 25 weeks after the last injury. Effects in female mice will be compared to previously collected data in male mice. Inflammation and white matter injury will be assessed with western blotting and immunohistochemistry, respectively. Results: Acutely, rmTBI female mice performed worse than sham injured mice on the balance beam (F (1,28) =4.309, P=0.0472) whereas there was no difference in males. Five weeks after injury, both male and female mice in the rmTBI group performed significantly (T-test P< 0.01) worse on the Rotarod. Neither males nor females displayed deficits in cognition on the T-Maze or learning phase of the MWM, although males had a significant impairment on MWM memory (Probe T-test P< 0.05). Neither sex showed deficits in the EPM. Fifteen weeks after injury, male mice displayed significant deficits in learning in the MWM (T-test P< 0.05) and EPM (T-test P< 0.05). Male mice in the rmTBI group also showed increased astrogliosis and Tau phosphorylation in the cerebral cortex compared to sham injured mice. Additional assessments of white matter injury are planned, but assessment of female mice 15 weeks after injury is incomplete at this time. Conclusion: Acutely, female mice showed balance deficits that were not apparent in males. Five weeks after injury, both sexes continued to show motor deficits on the rotarod, but only males had mild deficits in cognition. Ongoing studies will assess whether these differences persist or new differences between males and females appear chronically. AUP: 2021-0035
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    Changes in PVN Neurons after Low-Frequency Acute Optogenetic Stimulation
    (2022) Paundralingga, Obed; Jia, Shuping; Cunningham, Joseph
    The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN-projecting MnPO neurons discharge frequency is < 5Hz. To gain more insight into the acute change in PVN neuron properties after low-frequency stimulation, whole-cell patch-clamp recordings were performed on PVN neurons. Adult 250-350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno-associated virus encoding Channelrhodopsin (AAVrg-CaMKIIa-hChR2(H134R)-mCherry) or with AAVrg-CAG-tdTomato as control. Three weeks after the injections, rats were anesthetized with isoflurane (2-3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. With the bath perfused with normal aCSF (2-3 ml/min), postsynaptic currents were recorded from PVN neurons in whole-cell voltage clamp configuration (Vhold=-60 mV). Axon terminals in the PVN were stimulated with 5Hz LED-generated blue light (470nM) pulses of 50-ms duration, 5 seconds off/on, for a total of 1 min. Evoked currents were measured every 5 min before and after the photo-stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic-evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post-stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre-stimulation amplitude 100±0.62 % vs. post-stimulation amplitude 86.50±0.54 %, P=0.0371; pre-stimulation frequency 3.82±1.7 Hz, post-stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low-frequency MnPO photostimulation differentially might reduce spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.
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    Methionine Synthase: A target for novel small molecules to inhibit cocaine and methamphetamine induced neuronal death
    (2022) Young, Olivia; Funk, Arlene; Deb, Biddut; Amankwa, Charles E.; Chintagunta, Anila; Gondi, Sudershan; Forster, Michael; Shetty, Ritu; Acharya, Suchismita
    Purpose: Oxidative stress-induced cell death is involved in the pathology of psychostimulant addiction neuropathies and ischemic stroke. These conditions potentially cause neuronal and functional changes via different mechanisms - epigenetic alterations (DNA hypomethylation) and reactive oxygen species (ROS) accumulation. Current medications for the treatment of psychostimulants (e.g., cocaine and methamphetamine) induced addiction neuropathies are largely ineffective due to the high rate of relapse and marginal alterations of dependency to these diseases. To circumvent this, our laboratory has synthesized novel hybrid antioxidant small molecules: SA-30 and SA-31, with predictive neuroprotective and broad-spectrum reactive oxygen species (ROS) scavenging abilities in mouse hippocampal HT22 neural cells. Our objective here was to test if the compounds increase cell proliferation, superoxide dismutase (SOD) enzyme, as well as methionine synthase (MS) enzyme, a key enzyme largely responsible for DNA methylation, neuronal growth, and survival using human neuroblastoma cells (SH-SY5Y) expressing dopaminergic neurons. Methods: The synthesis and structure characterization of compounds SA-30 and SA-31 were previously synthesized in Acharya lab using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Human neuroblastoma cells SH-SY5Y expressing dopaminergic neurons were purchased (ATCC), cultured, and treated with different concentrations of cocaine hydrochloride or methamphetamine for 8, 24, and 48h for determining EC50 using MTT assay. In the next experiment, either cocaine hydrochloride (1.5mM) or Methamphetamine (METH, 3.5 mM) was added followed by co-treatment with compounds SA-30 and SA-31 and 4-hydroxy tempol (all at 100µM) for 24 hours. Cell viability was assessed using MTT assay. The level of intracellular MS and SOD enzymes was assessed using ELISA. Results: In SHSY5Y cells, the EC50 of cocaine was 1.5 mM, and for METH was 3.5 mM after 24h of treatment. Both compounds SA-30 and SA-31 were not cytotoxic at varying concentrations (0.01, 0.1, 1, 10, 100µM) and rescued cells from both cocaine and METH-induced oxidative stress/cell death at 100µM concentrations. SA-31 at 100µM significantly increased (~1.5 fold) intracellular MS as compared to control. There was a decrease in MS level after METH treatment and treatment with SA-30 and SA-31 increased the level. SOD levels were significantly higher (~3 fold) in METH+SA-31 treated groups than only METH groups. Conclusion: Both novel hybrid small molecules SA-30 and SA-31 are neuroprotective in SH-SY5Y cells from psychostimulants cocaine and METH-induced oxidative stress/neural cell death. Increasing MS and SOD enzyme activities is one of the mechanisms by which neuroprotection was attained. Future studies will address the potential of both SA-30 and SA-31 to progress further in pre-clinical drug development, with a future application for the treatment of substance abuse disorder.
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    Myalgic Beckers Muscular Dystrophy Due to an Exon 15 Point Mutation: Case Series and Literature Review
    (2022) Tavallaee, Zachary; Hamby, Tyler; Marks, Warren
    Background: Dystrophinopathies result from mutations to the DMD gene. Presentation varies from asymptomatic elevations in creatine kinase levels to early loss of ambulation and significant impairment as seen in Duchenne's muscular dystrophy. We report 5 boys in 3 families with heterogenous phenotypes due to a point mutation in the DMD gene: a hemizygous tyrosine-to-cysteine change in exon 15 (c.1724T>C) resulting in an amino acid substitution of leucine to proline at codon 575. The specific mutation on DMD c.1724T>C (p.Leu575Pro) is listed in the Clinvar database as a variant of unknown significance. Our report provides contributing evidence that this genetic alteration should be classified as pathogenic. Case Information: Our 3 patients above the age of 2 years presented with elevated creatine kinase levels, myalgia after exercise, and occasional muscle cramping. Our 2 patients below the age of 2 years presented with elevated creatine kinase levels and no other findings. This mutation has been reported before, with 3 prior patients presenting with similar clinical findings of myalgia, myoglobinuria, and occasional muscle cramping. Some similarities among all 8 patients include elevated creatine kinase levels, no muscle weakness, no calf hypertrophy, and no Gower sign. Discussion: Of note, these patients can present initially with elevated liver function enzymes, as seen with 1 of our patients, and our report raises awareness that dystrophinopathies should be considered before undergoing costly gastrointestinal testing. Two of the 8 patients presented with neuropsychiatric disorders: 1 with attention-deficit/hyperactivity disorder (ADHD) and 1 with autism. This suggests that ADHD and autism may be a presenting feature of dystophinopathies, and creatine kinase levels should be considered in their evaluation. This report elucidates the clinical presentation of the mutation on DMD c. 1724T>C.
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    Therapeutic Efficacy of ????7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators in Acute Ischemic Stroke
    (2022) Hernandez, Katherine
    Ischemic strokes are a leading cause of disability in humans, which can result in decreased motor function, impaired cognition, and emotional disturbances. Currently, there are only two approved treatment options available: administering a tissue plasminogen activator or performing a surgical thrombectomy. Both treatment options are limited by time constraints, making it difficult to treat stroke patients effectively. PNU 120596, a positive allosteric modulator of the alpha 7 nicotinic acetylcholine receptor (a7 nAChR), has previously exhibited increased motor function in male stroke model rats. However, research on the effects of this novel drug on female rats has not been conducted. We hypothesize PNU 120596 will improve both motor and cognitive function in female rats. We will study this using behavioral tests, such as Rotarod and Barnes maze, on a middle cerebral artery occlusion (MCAO) stroke in both male and female Sprague Dawley rats. Depending on these results, we will further confirm our findings using either Magnetic Resonance Imaging (MRI) or brain tissue staining to support or disprove our hypothesis.
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    Optimization of benzo[d]oxazol-2(3H)-one activators of Slack potassium channels
    (2022) Mishra, Nigam; Du, Yu; Spitznagel, Brittany; Weaver, C.; Emmitte, Kyle
    Introduction: Fragile X syndrome (FXS) is an inherited genetic disorder that causes intellectual disability (ID) and other comorbid conditions, including cognitive disabilities, behavioral conditions, and autism spectrum disorder (ASD). FXS is caused by silencing of the FMR1 gene, which leads to an absence of the fragile X mental retardation protein 1 (FMRP). FMRP has multiple important functions and among these is activation of Slack potassium (K+) channels. Slack is a member of the Slo family of channels, which are critical regulators of electrical activity in the nervous system. Thus, dysregulation of Slack activity leads to abnormal neuronal activity. Preclinical studies have shown that Slack activity is critical for higher brain functions including cognitive flexibility, learning, and memory. Hence, selective activation of Slack by a small molecule may represent a viable approach to treating certain cognitive deficits in FXS and potentially other forms of ID. Objective: Currently there are no available selective small-molecule activators of Slack, so the objective of our work is to develop such tools to investigate them as a potential new therapeutic approach to FXS. Methods: Hit compound VU0609159 was identified via high-throughput screening (HTS) using thallium (Tl+) flux assay in HEK-293 cells stably expressing Slack and confirmed in an automated electrophysiology assay. Library synthesis based, iterative hit optimization strategy around hit VU0609159 was employed. Classical and state-of-the-art synthetic chemistry techniques including microwave assisted organic synthesis and flow chemistry were employed for synthesis of target compounds. Purification was done by automated liquid chromatography. Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS were utilized to obtain NMR and HRMS, respectively. Results: VU0609159 is a member of a series of benzo[d]oxazol-2(3H)-ones and has multiple regions amenable to rapid Structure-activity relationship (SAR) development through the preparation of small libraries of compounds. During SAR study, we found cyclic and acyclic branched linkers optimal for the activity. Substitution of benzo[d]oxazol-2(3H)-one ring with benzo[d]thiazol-2(3H)-one gave encouraging results. Conclusion: SAR for multiple regions around Slack activity in the VU0609159 series has been identified utilizing this approach and our Tl+ flux assay. Studies combining optimal functional groups within each region of the scaffold are ongoing.
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    Sex Specific Effects of Salt Loading on GABAA Responses in Oxytocin Neurons from the Supraoptic Nucleus
    (2022) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, Joseph
    Arginine Vasopressin (AVP) and oxytocin (OXY) contribute to body fluid balance homeostasis. Salt loading (2% NaCl for 7 days) increases both AVP and OXY release in rats. The chronic increase in AVP release is associated with a change in the sensitivity of AVP neurons in the supraoptic nucleus (SON) to GABA so that GABAA receptor activation becomes excitatory. It is not clear if a similar mechanism is associated with chronic OXY release in this model. Our hypothesis is that changes in chloride homeostasis associated with salt loading occur in OXY neurons. To test this hypothesis, we used a chloride imaging approach with a ratio metric chloride sensitive dye, ClopHensorN (Addgene #50758) combine with an AAV with an oxytocin specific promoter (pFBOT563, Addgene # 40864). Adult, intact, Sprague Dawley rats of both sexes were anesthetized with isoflurane (2-3%) and were bilaterally injected with the AAV2-pFBOT--ClophensorN virus directly into the SON. Rats of both sexes were salt loaded by providing them with only 2% NaCl to drink for 7 days. Later, the animals were sacrificed and the brains were rapidly removed. The SON was dissected away from the brain and the cells were dissociated, plated on cover slips, and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging on an inverted microscope. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells and then allowed to wash off. Background fluorescence was subtracted. In cells from males, muscimol resulted in chloride influx in 70% OXY of the cells tested while chloride influx was observed in all OXY cells from females. The results suggest that salt loading may influence GABA responses of OXY neurons in males but not females.
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    The effects of previous exposure to chronic methamphetamine on drug-seeking behavior and neurodegeneration in male and female mice
    (2022) Davis, Delaney; Metzger, Daniel; Vann, Philip; Wong, Jessica; Shetty, Ritu; Forster, Michael; Sumien, Nathalie
    Purpose: Recreational and medical use of stimulants among young adults have gained popularity in the United States over the last decade, with amphetamine compounds becoming the second most common illicit drug used in college students. Although amphetamine stimulants have proven to be safe and efficacious in children and adults with Attention Deficit and Hyperactivity Disorder (ADHD) when used as prescribed, these drugs can have significant adverse side effects such as an increased potential of recreational abuse liability, dependence, and neurotoxicity. There are known sex differences in drug abuse, in which women have lower rates of illicit drug use, but use more of the drug, reach dependence faster and have more adverse effects. We hypothesize that females may be more vulnerable to the reinforcing effects of METH as well as METH-induced neurotoxicity and dopaminergic dysregulation. Our study investigated the effects of early chronic exposure to the prototypical stimulant, methamphetamine (METH), at a dose designed to emulate human therapeutic dosing, on abuse potential and biochemical markers of dopaminergic function and neurodegeneration in male and female mice. Methods: Groups of 4-month-old male and female C57BL/6J mice were administered non-contingent intraperitoneal injections of either saline or METH (1.4 mg/kg) twice a day for 4 weeks. METH (0.5 mg/kg)-induced conditioned place preference (CPP) was tested in mice to determine the reinforcing effects of previous METH exposure. Mice were randomly assigned to either: Experiment I (short-term) in which male and female mice underwent CPP 13 days after injection cessation or Experiment II (long-term) in which female mice underwent CPP 5 months after injection cessation. Following behavioral testing, the animals were euthanized and striatum and midbrain were collected for biochemical testing of dopaminergic function and neurodegeneration. Results: In Experiment I, chronic METH exposure induced drug preference for subsequent doses of METH especially in males, and downregulated dopaminergic markers in males and induced apoptosis in females. In Experiment II, when CPP was performed 5 months after injection cessation, females with prior exposure to METH did not exhibit drug preference to subsequent doses of METH and there were no effects on markers of neurodegeneration or dopaminergic function. Conclusion: Previous exposure to METH induced a heightened sensitivity to subsequent doses of METH especially in males. While the effect in females was smaller, it disappeared in the long-term suggesting that this heightened sensitivity does not last over time. The increase in sensitivity was supported by alterations in the dopaminergic system in males. These outcomes suggest sex differences in response to prior METH exposure, and that these effects may not be long-lasting.
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    Impact of PINK1 knockout on expression and phosphorylation of mitochondrial proteins and dopamine regulation: insight into early-stage Parkinson's Disease in the rat model.
    (2022) John, Joshia; Salvatore, Michael
    Purpose: Mutations in the gene for PINK1, a mitochondrial protein, causes recessively inherited young-onset Parkinson's Disease (PD). This first report indicated the mutation when mapped to the PARK6 locus in a Spanish family. To model the impact of the PINK1 mutation, a rat model has been developed, but ambiguous results on dopamine regulation and motor function have been reported early in the lifespan. Here we investigated the impact of this mutation on Parkin, a mitochondrial protein that functions with PINK1 in mitophagy and dopamine regulation in early development. We hypothesized that compensatory changes in Parkin expression or its phosphorylation would occur, coinciding with changes in dopamine regulation due to the loss in expression or function of PINK1. Methods: In five-month-old PINK1 KO and age-matched wild-type (WT) rats, we confirmed the genotype by PCR, indicating the 26-base pair deletion as shown in the human mutation. Protein expression of PINK1, Parkin, phosphorylated Parkin, and TOM-20 were evaluated in comparison with dopamine tissue content, dopamine turnover, and tyrosine hydroxylase expression using quantitative Western Blotting. Results: In the PINK1 KO, dopamine tissue content decreased 43% in the striatum and 26% in the substantia nigra as compared to wild-type rats, commensurate with increased dopamine turnover in both regions. Parkin, phosphorylated Parkin, and TOM-20 protein expression were not significantly different in the PINK1 KO rat. Conclusion: This study suggests that compensatory changes in Parkin expression or phosphorylation are not modified in the PINK1 KO rat, though there is increased dopamine turnover and loss in both regions. Our findings indicate that dopamine regulation is affected early in the lifespan of the PINK1 rat, which may portend the overt loss of dopamine that leads to the parkinsonian signs with increasing age. The five-month-old rats used in this study correlate to a human age of approximately 20 years. Given that individuals with the PINK1 knockout mutation begin seeing motor impairments around 34 years of age, targeting mitochondrial dysfunction prior to when symptoms arise could diminish nigrostriatal dopamine loss and be a promising approach to the development of future treatment.