bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discriminative Stimulus Effects of Naltrexone and Haloperidol in Rats with Chronic, Intermittent Access to Sucrose
Authors: Marek Morgan , Moline Adam , Altendorf Luke , Vogelsang Jenna , Cyra Matthew , James Kimberly , Herrmann Jody , Levine Allen , Jewett David 
Published: November 2023

Previous research demonstrated chronic, intermittent sucrose access increased endorphin and dopamine function, and naltrexone elicited some withdrawal‐like behaviors in rats. We determined the naltrexone serves as a discriminative stimulus (3.2 mg/kg, s.c.) in subjects with 12 hr access to sucrose (10–32%) followed by 12 hr access to water, but not in subjects with 24 hr access to water. We wondered if smaller naltrexone doses could be established as discriminative stimuli. Groups of rats were injected with naltrexone (0.1–1.0 mg/kg) or saline 1 hr after sucrose access and 15 min prior to discrimination training. Left lever presses were reinforced under a FR 15 schedule following drug administration, and right lever presses were reinforced following saline administration. Daily training session continued until rats emitted 80% or greater condition‐appropriate responses both prior to the first consequence and for the total session. Most subjects trained with either 0.32 or 1.0 mg/kg acquired the discrimination. Naltrexone (0.1 mg/kg) served as a discriminative stimulus in approximately one‐third of the subjects. We also attempted to establish the dopamine antagonist haloperidol as a discriminative stimulus. Haloperidol (0.056 mg/kg, 30 min PT) was established as a discriminative stimulus in 4 of 4 subjects with chronic, intermittent sucrose access (M=49 sessions) and 5 of 6 subjects with 24 hr water access (M=82 sessions). Two of 5 subjects with chronic, intermittent sucrose access discriminated haloperidol (0.018 mg/kg) from saline (M=84 sessions). Other subjects failed to acquire the discrimination in 120 sessions under these conditions. Haloperidol (0.056 mg/kg) significantly reduced rates of lever pressing. This effect of haloperidol was independent of liquid consumption. In sum, chronic sucrose consumption alters opioid function, naltrexone's ability to serve as a discriminative stimulus depends on the training dose, and sucrose consumption does not consistently alter the ability of haloperidol to serve as a discriminative stimulus.Support or Funding InformationThe University of Wisconsin‐Eau Claire Office of Research and Sponsored Programs and the Minnesota Obesity Center
CRISPR/Cas9‐Mediated Genome Editing of the Murine Pulmonary Circulation
Authors: Marsboom Glenn , Yuan Yang , Rehman Jalees , Malik Asrar B. 
Published: November 2023

CRISPR/Cas9 is increasingly being tested for in vivo genome editing and we describe here the optimization, pharmacokinetic properties, and efficiency of this system to target the pulmonary vasculature in mice. We used cationic liposomes to deliver a PX458 vector expressing Cas9, eGFP, and gRNA to the pulmonary circulation and studied both its bio‐distribution and time needed to knock down proteins.Different amounts of liposomes were injected containing 25, 50, 75, and 100μg of plasmid DNA. After 4 days, mice were sacrificed and several organs were studied for eGFP expression. We found a dose‐dependent increase in pulmonary expression. The only other organ with detectable expression was the liver, but expression was at least 7‐fold higher in the lungs compared to the liver even at the highest dose tested. We found no eGFP expression in kidney, heart, brain, or skeletal muscle (n=4 animals/dose). Within the lung, we used immunohistochemistry to show that endothelial cells and to a lesser degree pulmonary artery smooth muscle cells were selectively targeted. We did not detect any expression in airway epithelial cells or airway macrophages. Lung expression was very temporary, with expression peaking at 4 days and returning to baseline after 28 days (n=4 mice/group), minimizing the chances of side effects, more specifically non‐specific alterations in the DNA that could result from continued expression of the endonuclease Cas9. Using mT mice that ubiquitously express tandem dimer Tomato fluorescent protein, were able to show a downregulation of fluorescence specifically in the majority of the pulmonary blood vessels after just 7 days (n=3 mice/group). Genomic alterations were confirmed by the T7 endonuclease assay and Next‐Generation Sequencing. Importantly, we did not observe any thrombosis or other adverse side‐effects that might be associated with expression of Cas9 in the pulmonary endothelium.In conclusion, we used a non‐viral delivery method to specifically deliver CRISPR/Cas9 to the pulmonary vasculature. The temporary expression of CRISPR/Cas9 led to a pronounced downregulation of the targeted fluorescent protein within 7 days, but could easily be adapted to target almost any gene. Currently available animal models do not allow to knock out a gene specifically in the pulmonary endothelial cells and effects on the pulmonary circulation might be compounded by effects in the systemic circulation. Therefore, our work provides a novel tool to specifically target and study genes in the pulmonary circulation.Support or Funding InformationThis work was supported in part by a Parker B. Francis fellowship and American Heart Scientist Development grant 15SDG23250002 (GM), NIH‐R01‐GM094220 (JR) and NIH‐R01‐HL045638, R01‐HL090152, R01‐HL118068, P01‐HL060678, P01‐HL077806 (ABM).
Histone deacetylase 9 (HDAC9) is associated with increased lysine acetylation levels in diabetic mice kidneys: a potential mechanism underlying renal impairment in type 2 diabetes
Authors: Polce Simran A , Carrillo‐Sepulveda Maria Alicia 
Published: November 2023

Diabetic nephropathy is one of the major complications faced by patients with type 2 diabetes (T2DM). It is characterized by fibrosis, mesangial matrix expansion, loss of proper glomerular filtration, and marked renal vascular dysfunction. The mechanism by which diabetic milieu promotes renal dysfunction is not entirely understood. Recent studies have demonstrated that nephropathy in type 1 diabetes is correlated with high levels of renal lysine acetylation. Importantly, histone deacetylase 9 (HDAC9), a class II subclass IIa mammalian HDAC that catalyze the removal of acetyl groups from ɛ‐amino groups of lysine's, has been linked to T2DM complications. Thus, we hypothesize that T2DM increases levels of renal lysine acetylation which is associated with downregulation of HDAC9. To address our hypothesis, male fourteen‐weeks‐old db/db mice, an experimental model of T2DM, and same age‐matched heterozygous nondiabetic (ND) mice (db/m+) were utilized in this current study. As expected, db/db mice exhibited robust increase in body weight (39.8g vs. 19.5g ND, p<0.05, n=7), kidney weight (238.8mg vs. 163.9mg ND, p<0.05, n=6) and blood glucose levels (265 mg/dl vs. 104.5mg/dl ND, p<0.05, n=7). Together these results confirm the diabetic state in the model utilized. Further, to determine whether HDAC9 and lysine acetylation are altered in kidneys from diabetic mice, western blot analysis was performed. Kidneys from diabetic mice exhibited markedly increases in lysine acetylation levels (80% increase vs. ND, p<0.001, n=5), which was correlated with significant reduction in HDAC9 expression (60% reduction vs. ND, p<0.05, n=5), suggesting that decreased HDAC9 may contribute to the elevated levels of lysine acetylation. Of note, no changes in others HDAC class II subclass IIa, including HDAC4, HDAC5 and HDAC7 were found in kidneys from db/db mice. Our results demonstrate that diabetic milieu increases lysine acetylation in kidney and downregulated HDAC9 epigenetic enzyme expression. Thus, targeting HDAC9 may offer novel therapeutic strategies for treating diabetic nephropathy. Studies using overexpression of HDAC9 has been initiated to confirm the role of HDCA9 in the diabetic renal dysfunction.Support or Funding InformationNIH
Regulator of G‐protein Signaling 10 (RGS10) Expression is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity
Authors: Alqinyah Mohammed , Maganti Nagini , Ali Mourad , Yadav Ruchi , Weng Han‐Rong , Hooks Shelley 
Published: November 2023

Regulator of G‐protein Signaling 10 (RGS10) has emerged as a key regulator of pro‐inflammatory cytokine production in microglia, functioning as an important neuroprotective factor. While RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor‐activation. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 expression in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is silenced in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation‐dependent silencing of RGS10 expression in microglia. We demonstrate that RGS10 is endogenously expressed in spinal cord microglia, and expression is suppressed in the spinal cord in a nerve injury induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor Trichostatin A blocks the ability of Lipopolysaccharide (LPS) to suppress RGS10 transcription in BV‐2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of RGS10. Further, we used chromatin immunoprecipitation to demonstrate that H3 histones at the RGS10 proximal promoter are deacetylated in BV‐2 microglia following LPS activation, and HDAC1 association at the RGS10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1‐phosphate, an endogenous microglial signaling mediator which inhibits HDAC activity, enhances basal RGS10 expression in BV‐2 microglia, suggesting that RGS10 expression is dynamically regulated in microglia in response to multiple signals.Support or Funding InformationThis work was supported by grants from the National Multiple Sclerosis Society (SBH) and the National Institutes of Neurological Disorders and Stroke [RO1NS064289] (HRW).
Regulation of the Expression and Function of Exchange Protein Directly Activated by cAMP (EPAC) by Hypoxia
Authors: Olah Mark , Justus Lillian , Kiefer Erika , Shelley Jessica 
Published: November 2023

Exchange protein directly activated by cAMP (EPAC) is a signaling protein implicated in several physiologic and pathophysiologic processes. Following binding of cAMP, EPAC activates small GTPases such as Rap1 to induce downstream effects. In vascular endothelial cells, EPAC may have a role in the regulation of permeability, vascular tone and angiogenesis. As low oxygen tension initiates many vascular events including those modulated by EPAC, we hypothesized that hypoxia regulates EPAC expression and function in human microvascular endothelial cells (HMVEC). HMVEC maintained in 2.0% oxygen for 48 hours demonstrated a significant decrease in expression of EPAC protein relative to normoxic cells or HMVEC exposed to hypoxia for shorter periods. At 48 hours of hypoxia, EPAC expression was 53.9 ± 7.4% of that in normoxic cells. To begin to delineate the mechanism underlying this downregulation of EPAC, we examined the role of the transcription factor hypoxia‐inducible factor‐1α (HIF‐1α). Deferoxamine, an iron chelator, and IOX‐2, an inhibitor of prolyl hydroxylase, both increased expression of HIF‐1α at short time periods as did exposure to 2.0% oxygen. At 48 hours of treatment, deferoxamine and IOX‐2 mimicked the effects of hypoxia by decreasing EPAC expression to levels 40.5 ± 12.1% and 43.0 ± 12.7% of control, respectively. To assess the functional significance of hypoxia‐induced downregulation of EPAC, we examined activation of the downstream effector Akt by determining levels of phospho‐Akt (Ser473) by Western blotting. The A2B adenosine receptor agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) and the adenylyl cyclase activator forskolin, both of which increase intracellular cAMP levels, as well as the EPAC specific activator 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine 3′,5′‐cyclic monophosphate (8‐CPT) induced a decreased Akt stimulation in hypoxic HMVEC relative to normoxic cells. In conclusion, hypoxia downregulates protein expression of EPAC in HMVEC and this may be mediated by HIF‐1α. This decrease may result in reduced EPAC‐mediated Akt activation. Studies are currently further exploring the mechanism of the hypoxia‐induced downregulation of EPAC and examining effects on Akt‐mediated functional responses such as cell survival.Support or Funding InformationSupported by funds from The Raabe College of Pharmacy, Ohio Northern University
UCH‐L1 as a Susceptibility Factor for Nigrostriatal and Mesolimbic Dopamine Neurons: Neurotoxicant Exposure and Aging
Authors: Winner Brittany Michele , Zhang Harue , Lookingland Keith , Goudreau John 
Published: November 2023

Parkinson disease (PD) is a debilitating neurodegenerative disorder in which nigrostriatal (NSDA) dopamine neurons die, affecting signaling within the basal ganglia pathway. Loss of dopamine input to the striatum (ST) results in the motor symptoms required to diagnose PD. Although the majority of cases are idiopathic, genes mutated in familial PD can provide clues to highlight important pathways important in pathogenesis and progression of PD. One such pathway is the ubiquitin proteasome system (UPS) which degrades proteins tagged with ubiquitin (Ub) chains. Since accumulation and aggregation of misfolded proteins such as alpha synuclein is a hallmark of PD, enhancing degradation of key substrates is a lucrative target for therapy. One UPS‐related protein mutated in familial PD is UCH‐L1, a deubiquitinating enzyme responsible for maintaining available pools of free monomeric Ub. UCH‐L1 is an abundant neuron‐specific protein that is critical for normal synaptic function and to maintain the structure of the neuromuscular junction. In a mouse model of oxidative damage specific to dopamine neurons, systemic administration of neurotoxicant MPTP resulted in loss of UCH‐L1 expression and function in the substantia nigra after 24 h. Since loss of UCH‐L1 and loss of phenotypic markers both occur concurrently in NSDA neurons, we hypothesized that loss of UCH‐L1 contributes to susceptibility to neurotoxicant administration. Not all dopamine neurons are equally susceptible in PD: mesolimbic dopamine neurons (MLDA) are spared while NSDA neurons degenerate. We compared NSDA to MLDA neurons and found that MLDA neurons are less susceptible to MPTP‐induced loss of ST dopamine and tyrosine hydroxylase (TH), which coincides with maintenance of UCH‐L1 expression in the ventral tegmental area. In addition, we performed an aging study to determine if UCH‐L1 is lost in aged mice versus young mice and if aged mice are more sensitive to loss of UCH‐L1 after MPTP. Finally, we hypothesized that overexpression of UCH‐L1 in NSDA neurons via rAAV 2/5 will be protective against proteasome impairment and loss of dopamine phenotypic markers after MPTP. Preliminary results indicate that aged mice are more sensitive to MPTP‐induced loss of dopamine in the ST compared to young mice, but not more sensitive to loss of TH and dopamine transporter expression. However, aged mice show decreased metabolism of dopamine as evidenced by lower levels of one of its metabolites, DOPAC. Aged mice also show similar levels of UPS impairment compared to young mice. Further study will determine if UCH‐L1 expression is influenced by aging in NSDA neurons, and if UCH‐L1 overexpression rescues UPS impairment in young mice.Support or Funding InformationInstitute for Integrative Toxicology T32 Predoctoral Fellowship National Institutes of Health NINDS
Behavioral and Biochemical Consequences of Simulated Vehicle Exhaust Exposure (SVEE) in Rats
Authors: Salvi Ankita , Patki Gaurav , Liu Hesong , Salim Samina 
Published: November 2023

Air pollution from vehicle exhaust is a serious environmental threat, known to contribute to cardiovascular, respiratory and neurological complications. While the effect of vehicle exhaust exposure on heart and lungs is well known, adverse effects of these emissions on the brain and its psychological impact has been ignored. Here, we examined exhaust‐induced psychological and neurobiological alterations in rats using a simulated vehicle exhaust exposure (SVEE) model. Prolonged exposures to the gaseous constituents of vehicle exhaust, carbon dioxide (CO2), carbon monoxide (CO) and nitrogen dioxide (NO2) are known to increase oxidative stress. And, our previous studies have established a causal link between oxidative stress and behavioral as well as cognitive deficits. Therefore, we hypothesized that prolonged exposures to pro‐oxidants from vehicle exhaust elevate oxidative stress leading to behavioral and cognitive deficits. And, interventions that limit/prevent oxidative stress such as a mitochondria‐permeable antioxidant, MitoQ, can protect SVEE‐induced impairments. Four groups of Sprague Dawley rats were included: Control+Vehicle (C+V), Control+MitoQ (C+M), Exposure+Vehicle (E+V) and Exposure+MitoQ (E+M). The MitoQ groups received 250μM of MitoQ for 4 weeks via drinking water. Vehicle groups received normal drinking water during this period. Following the treatment, exposure rats were exposed to a simulated mixture of vehicle exhaust containing 0.04% CO2, 0.9 ppm NO2 and 3 ppm CO. Exposures were performed in whole body exposure chambers 5h daily for 2 weeks. Duration of exposures was comparable to daily exposure of exhaust levels in areas of high traffic. Control rats were exposed to normal air for the same duration. Following SVEE, anxiety‐ and depression‐like behavior, as well as cognitive function and intelligence quotient (IQ) levels were assessed. An increase in anxiety‐like and depression‐like behavior as well as impaired memory and low IQ was observed in E+V rats as compared to C+V and C+M rats. However these deficits were not seen in E+M rats. At the biochemical level, effect of SVEE on mitochondrial function (one of the targets of increased oxidative stress) was assessed. Mitochondrial oxygen consumption in three brain regions, namely, prefrontal cortex, hippocampus and amygdala was determined using high resolution respirometry. SVEE led to decreased mitochondrial oxygen consumption in rats suggesting SVEE‐induced mitochondrial dysfunction. Oxygen consumption was unaltered in rats that received MitoQ.Thus our study has identified behavioral and biochemical impairments associated with exposure to vehicle exhaust in rats and identified a potential role of the antioxidant MitoQ in preventing these impairments.Support or Funding InformationGrant Support: 2R15MH093918‐02
Live tissue imaging of organic cation and anion transport at the blood‐CSF barrier reveals specific transporter function and distinct transcellular pathways
Authors: Hu Tao , Zha Weibin , Duan Haichuan , Wang Joanne 
Published: November 2023

Formed by the choroid plexus (CP) epithelial cells, the blood‐cerebrospinal fluid (CSF) barrier plays an active role in removing neurotoxins, drugs, and metabolites from the brain. Several organic cation and anion transporters, including the plasma membrane monoamine transporter (PMAT), organic anion transporter 1 and 3 (OAT1/3), and multidrug resistance‐associated protein 1 and 4 (MRP1/4) have been reported to be expressed in CP, but their relative expression and specific function at the blood‐CSF barriers have not been well characterized at the tissue level. The goal of this study is to quantify the expression of organic cation and anion transporters in CP tissues and determine their specific roles in organic cation or anion transport by live tissue imaging using fluorescent substrates. In both human and mouse CPs, real‐time PCR analysis showed highest mRNA expression of PMAT followed by MRP1, OAT3 and MRP4. CP also expresses several drug metabolizing enzymes including the monoamine oxidases (MAOs), catechol‐O‐methyltransferase (COMT), UDP‐glucuronosyltransferases (UGTs) and sulfotransferases (SULT). In CP isolated from wildtype (wt) mouse, the fluorescent organic cation probe IDT307 is transported into CP cells at the apical (CSF‐facing) membrane and is highly accumulated in CP cells with a large storage in mitochondria. There was little efflux at the basolateral membrane. In contrast, IDT307 uptake and intracellular accumulation is attenuated by ~3–4 folds in CPs isolated from Pmat knockout mice. Interestingly, in wt CPs, the organic anion probe fluorescein‐methotrexate (FL‐MTX) is rapidly taken up and efficiently excreted into the capillary side with little intracellular accumulation. MK571, a potent inhibitor of MRP1/4, abolished basolateral efflux of FL‐MTX, resulting in high accumulation within CP cells. Together, our study in live CP tissue demonstrates an important role of PMAT in apical uptake of organic cations and a crucial function of MRP1/4 in basolateral efflux of organic anions. Based on these studies, we propose distinct molecular and cellular pathways for organic cation and anion transport at the blood‐CSF barrier.Support or Funding InformationThis study is supported by National Institutes of Health Grant GM066233.
Identification of transport systems for fluorescent bile salt derivatives
Authors: Stieger Bruno , Leuenberger Michele , Stephanie Häusler , Lochner Martin 
Published: November 2023

Organic anion transporters of hepatocytes are mediating uptake of bile salts as well as drugs into hepatocytes. Consequently, transport of endogenous substrates and drugs by these transporters is routinely investigated during drug development as well as in basic research studying physiology and pathophysiology of bile formation. In order to facility transport measurements, fluorescent transporter substrates are desirable. We therefore aimed to develop novel fluorescent substrates for the bile salt transporters sodium‐taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP) as well as for the hepatocellular organic anion transporting polypeptides (OATPs). Three fluorescent analogs of cholylethylenediamine amides (Cen) were synthesized with the fluorescence moiety coupled to the side chain: CenNBD (Cen with NBD), CenDansyl (Cen with dansyl) and CenPB (Cen with pacific blue). Uptake experiments were performed using CHO cells stably transfected with NTCP, OATP1B1, OATP1B3 or OATP2B1 and insect cell vesicles expressing BSEP with standard substrates as positive control and with fluorescent bile salts. All fluorescent bile salts inhibited the investigated OATPs in a concentration dependent manner. All three fluorescent bile salts were substrates of OATP2B1 and to some extent of OATP1B1 and OATP1B3. In contrast, only CenDansyl was a substrate of NTCP. This bile salt derivative is an inhibitor of BSEP, but not a substrate. Our data confirm that a chemical modification of the side chain of bile salts has a considerable impact on the transporter selectivity and on the transport efficiency of bile salt derivatives.Support or Funding InformationSNF National Center of Competence in Research TransCure, University of Bern, Switzerland