bluuebunny/crossref_metadata_2025 huggingface dataset:
Capsaicin interacts with clathrin coated pit dependent mechanisms to protect motor nerve terminals against botulinum neurotoxin A
Authors: Thyagarajan Baskaran , Potian Joseph G , Patel Vishwendra , Garcia Carmen C , Hognason Kormakur , McArdle Joseph J
Published: June 2021

Botulinum neurotoxin A (BoNT/A) cleaves SNAP‐25 and terminates acetylcholine release at the motor nerve endings (MNEs) resulting in neuroparalysis. Recently, we reported that capsaicin, a TRPV1 channel agonist, protected MNEs from the inhibitory actions of BoNT/A (Thyagarajan et al 2009). This study examines the mechanisms of capsaicin's prophylactic action against BoNT/A. In control mice injection of BoNT/A (3 μl; 6.67 pM) near the innervations of the Extensor digitorum longus muscle of both hind limbs inhibited the toe spread reflex (TSR) within 24 hrs. However, when capsaicin (3 μl; 1 mM) was injected either 5 to10 minutes or 4 or 8 hours before BoNT/A, the TSR remained normal. Capsaicin pretreatment in vitro significantly reduced fluorescent BoNT/A uptake into MNEs and protected stimulus evoked twitch tension and acetylcholine release from MNEs while BoNT/A alone inhibited these motor nerve functions. Capsazepine, a TRPV1 antagonist prevented the protective effects of capsaicin. To understand the mechanism of capsaicin‐induced protection against BoNT/A, we studied cholinergic Neuro 2a cells. Acute exposure to capsaicin altered the subcellular localization of clathrin heavy chain (Chc) and AP2, two proteins essential to clathrin coated pit (CCP) formation. Chlorpromazine, an inhibitor of CCP dependent endocytosis (Maksymowych and Simpson, 2004), mimicked the effects of capsaicin on Chc delocalization. Wortmannin, (WMN, nonselective PI5K inhibitor), reduced fluorescent BoNT/A uptake into MNEs and delocalized Chc and AP2 in Neuro 2a cells. WMN (3 μl; 1 mM) injected 6 hours before BoNT/A protected mice from neuroparalysis. We hypothesize that capsaicin reduced the neuroparalytic effects of BoNT/A by inhibiting CCP dependent uptake of BoNT/A into MNEs.
Suppressive effect of fisetin on MPP+‐ induced alpha‐synuclein expression and PC12 neuronal cell death
Authors: Panchal Hirenkumar , Benzeroual Kenza E
Published: June 2021

Recently, it has been shown that alpha‐synuclein oligomers and neuroinflammation can play an important role in the development of Parkinsons disease, Alzheimer's disease, and dementia. This study, investigated the potential cytoprotective effect of Fisetin and Ibuprofen in rat pheochromocytoma (PC12) cells prior to intoxication with MPP+. Cell viability was measured by MTT assay, and alpha‐synuclein expression was assessed by western blot. PC12 cells were pretreated with varying doses of fisetin for 2 hours prior to treatment with MPP+ for 48 hours and 72 hours. Pretreatment with fisetin produced a dose‐dependent increase in cell viability, and showed a maximal increase in cell viability with 2 μg/mL of fisetin. Furthermore, western blot analysis showed a dose‐dependent decrease in the expression of alpha‐synuclein when PC12 cells were pretreated with fisetin prior to treatment with MPP+ for 48 hours. Ibuprofen at 2 mM also decreased alpha‐synuclein expression in PC 12 cells treated with MPP+. The combination of both drugs showed a synergistic effect in the inhibition of alpha‐synuclein expression. The present study indicates that fisetin may be a promising approach for the treatment of neuroinflammation associated with neurodegeneration. In addition, further investigation of the synergistic effect of ibuprofen may elucidate the underlying mechanisms of the neuroprotection.
Cardiovascular effects of self‐administered remifentanil in opiate‐dependent and nondependent rats
Authors: Jimenez‐Gomez Corina , Cooper Ziva D , Shi Yong‐Gong , Woods James H
Published: June 2021

Opiate‐dependence increases the reinforcing effects of remifentanil, a fast‐acting mu‐opioid receptor agonist, in rats. Chronic exposure to opiates also has been associated with changes in the number of opiate receptors in the brain and altered behavioral responses to opiates. Using cardiovascular activity as an endpoint to measure the physiological effects of remifentanil, this study investigated changes in the cardiovascular response to self‐administered remifentanil as a function of opiate dependence. Cardiovascular changes associated with self‐administered remifentanil (10, 32, and 100 ug/kg/inj) were recorded in morphine‐dependent and morphine‐naïve rats using a telemetry system. Self‐administered remifentanil produced dose‐dependent decreases in heart rate, which were immediately followed by compensatory increases. The initial decrease in heart rate was greater in morphine‐naïve rats, whereas the compensatory increases were greater in the morphine‐dependent rats, suggesting tolerance to the direct bradycardic effects of remifentanil and a sensitized response to the compensatory increase in heart rate. These findings suggest that the cardiovascular effects of remifentanil may serve as a potential index of physiological dependency produced by opiates.
A thermostable mutant of a bacterial cocaine esterase reverses acute cocaine toxicity in the rabbit isolated heart
Authors: Ross Brittany Erin , Narasimhan Diwahar , Sunahara Roger , Woods James , Lucchesi Benedict
Published: June 2021

Cocaine use is reported to be the most common cause for drug‐related hospital emergency department visits. Currently, there are no effective therapeutic agents available to directly treat cocaine‐associated toxicity. Cardiovascular events associated with cocaine use include angina pectoris, myocardial infarction, arrhythmias and aortic dissection. The goal of this study was to test the ability of a thermostable mutant of a bacterial cocaine esterase (CocE) to reverse the cardiotoxicty associated with acute cocaine use. The effects of cocaine and CocE treatment on cardiovascular functional parameters and markers of injury were examined in the Langendorff perfused rabbit isolated heart. Cocaine, when added to the perfusion buffer, caused a significant decrease in heart rate, an increase in left ventricular end‐diastolic pressure (LVEDP), an increase in coronary perfusion pressure and a decrease in left ventricular developed pressure. CocE infusion was able to completely reverse the effects on heart rate, LVEDP and developed pressure. CocE was also able to reduce the degree of propidium iodide staining for irreversibly damaged cells in isolated heart sections. In conclusion, this thermostable mutant of CocE is effective in reversing acute cocaine cardiotoxicity. Funding for this project is provided by the University of Michigan Cardiovascular Research Fund.
Exercise is Associated with Increased Drug‐Taking in Rats that Self‐Administer Methamphetamine at Low Levels
Authors: Wright Mayo Jerry , Davis Sophia A , Dickinson Glen D , Taffe Michael A
Published: June 2021

Objective

About 1% of young adults in the U.S. report using methamphetamine (METH) within the past year. Although METH is commonly used at dance parties, little is known about how physical activity influences use. Experiments were conducted to determine the relationship between voluntary exercise and METH self‐administration.

Methods

Rats were trained to self‐administer METH in chambers that contained both drug self‐administration apparatus and a running wheel. Rats were initially trained to self‐administer METH without wheel access. Once performance stabilized, rats were permitted access to the running wheel and the METH‐reinforced response lever concurrently.

Results

Rats self‐administered METH at low levels prior to wheel access and the mean number of drug infusions taken during each session more than tripled after wheel‐access was permitted. Wheel running was stable within individual across sessions. The number of responses on the inactive lever was not significantly related to the dose of METH.

Conclusions

In these experiments, voluntary exercise was associated with increased METH self‐administration in rats that had previously demonstrated low levels of drug self‐administration. These results suggest that reinforcer competition is not the sole factor mediating effects of exercise on drug self‐administration. Research supported by National Institute of Drug Abuse grant R01 DA024705.
Pain‐related depression of intracranial self‐stimulation in rats: effects of the kappa opioid agonist U69,593 and the kappa opioid antagonist norbinaltorphimine
Authors: Negus S. Stevens , Morrissey Ember M. , Rosenberg Marisa B. , Altarifi Ahmad , Cheng K. , Rice Kenner C.
Published: June 2021

Selective kappa agonists produce antinociception in many preclinical assays, but unlike mu agonists, they perform poorly as analgesics in humans. This discrepancy may be related to prodepressant effects of kappa agonists. Kappa antagonists do not produce antinociception, but they can produce antidepressant‐like effects. This study tested the hypothesis that the kappa agonist U69,593 and kappa antagonist norbinaltorphimine (norBNI) would produce pronociceptive and antinociceptive effects, respectively, in an assay of pain‐depressed behavior. Effects of U69,593 (0.056–0.56 mg/kg), norBNI (10–32 mg/kg) and morphine (3.2 mg/kg) were evaluated on stimulation of stretching and depression of intracranial self‐stimulation (ICSS) by intraperitoneal administration of dilute lactic acid in rats. U69,593 blocked acid‐stimulated stretching but only exacerbated acid‐induced depression of ICSS. Norbinaltorphimine did not alter either pain‐related behavior, and morphine blocked both pain‐related behaviors. The assay of acid‐depressed ICSS permitted dissociation of mu and kappa agonist effects and failed to reveal an antinociceptive effect of a kappa antagonist. Supported by NIH grant R01‐NS070715 and the Intramural Research Programs of NIDA and NIAAA.
The differential expression of male sexual behavior in the Lewis, Fischer and Sprague‐Dawley rat strains
Authors: Hurwitz Zachary Edward , Riley Anthony Lee
Published: June 2021

Lewis (LEW) and Fischer (F344) rats differ markedly in reactivity to drugs of abuse and mesolimbic dopamine activity, and interestingly sexual behavior is thought to be mediated in part by some common substrates. The following experiment examined sexual behavior of these strains (and outbred Sprague‐Dawley (SD) rats) to assess possible strain differences. Males were given repeated access to a receptive female of their respective strain during which sexual behavior was coded. Males were then exposed to a receptive LEW, F344 or SD female to assess if male behavior was a function of female receptivity. LEW males exhibited fewer mounts and intromissions (frequency and/or percent of subjects) than the F344 and SD strains (which never differed). Latency to first mount or intromission was similar for the F344 and SD strains but both were shorter than the LEW males (an index of sexual motivation). Ejaculation frequency and percent of subjects displaying ejaculation were different among strains (F344> SD>LEW). No LEW male ever ejaculated. For all males, there was no significant change in mounts, intromissions or ejaculations when given access to females of different strains, indicating the initial differences were not a result of strain differences in receptivity. These findings are consistent with a growing literature describing strain differences in motivated behaviors.

Supported by a Mellon Foundation grant to ALR.
Effects of daily morphine treatment on the relative reinforcing strength of milk and heroin in monkeys trained under concurrent schedules of milk and heroin availability
Authors: Paronis Carol A. , Bergman Jack
Published: June 2021

Tolerance develops to many, but not all, effects of mu‐opioid ligands; the present studies addressed the question of whether tolerance develops to the reinforcing and priming effects of mu‐opioid agonists. Monkeys (n=5) were trained to respond on two levers under concurrent FR15 schedules for sweetened milk or i.v. injections of saline or heroin. Group mean responses on the ‘drug‐lever’ (%DLR) increased dose‐dependently from 8±4% to 97±2% when 0.001–0.032 mg/kg/inj heroin was available. Acute morphine, 0.1–1.0 mg/kg (i.m., 10 min pre‐session) increased %DLR; 3.2 mg/kg morphine decreased response rates to 0.02 resp/sec and %DLR was not determined. Daily injection of 3.2 mg/kg morphine resulted in tolerance to the response‐rate decreasing effects with varying effects on %DLR. When 3.2 mg/kg morphine was given prior to the session, %DLR was >90% regardless of the dose available for injection; when morphine was given post‐ session, the heroin %DLR function was shifted 3‐fold to the right. Moreover, in monkeys receiving 3.2 mg/kg/day morphine, the dose of 1 mg/kg morphine did not increase %DLR. These results suggest that a dose of morphine that produces tolerance to response‐rate decreasing effects may produce tolerance to the reinforcing effects of heroin and the priming effects of lower doses of morphine, but these effects are best seen in the absence of the daily morphine injection. (Supported by NIH Grant DA15723)
Evaluation of N‐Substituted Benztropine (BZT) Analogs for Cocaine Antagonist Effects in Rats Trained to Self‐Administer Cocaine
Authors: Hiranita Takato , Kopajtic Theresa , Cao Jianjing , Newman Amy H.
Published: June 2021

N‐substituted BZT analogs with high dopamine transporter (DAT) affinity and slow onsets of effect decreased cocaine self administration (SA) in rats (JPET 329:677). The present study examined additional N‐substituted 4′,4″‐diF‐BZT selective DAT inhibitors, with DAT affinities of 5.59 to 29.2 nM, and relatively faster onsets of action [JHW 013 (N‐methyl‐cyclopropyl‐diF‐BZT), GA 1–69 (N‐ethyl‐3‐indole‐diF‐BZT), GA 2–50 (N‐(R)‐2″‐amino‐3″‐methyl‐n‐butyl‐diF‐BZT) and GA 2–99 (N‐2″‐aminoethyl‐diF‐BZT)]. Rats were trained with cocaine SA (0.032–1.0 mg/kg/injection, IV) under fixed‐ratio 5‐response schedules of reinforcement. Maximal response rates were maintained by 0.32 mg/kg/inj of cocaine or by a standard DAT inhibitor, nomifensine (0.1 mg/kg/inj) when substituted for cocaine. Lower response rates were maintained at higher and lower doses. Responding was virtually eliminated when responses did not produce injections, and none of the BZT analogs maintained responding above those levels. Pretreatment (IP) with nomifensine dose‐dependently shifted the cocaine SA dose‐effect curve leftward. In contrast, the BZT analogs (IP) dose‐dependently decreased cocaine SA at lower doses than those that decreased response rates maintained by food reinforcement. The present study further supports the development of N‐substituted BZT analogs as medications to treat cocaine abuse. Supported by NIDA IRP.
Receptor systems involved in self‐administration of the tobacco smoke constituent, norharmane, alone and combined with nicotine
Authors: Arnold Monica M , Perez Jessica M , Belluzzi James D , Leslie Frances M
Published: June 2021

Cigarette smoking is the leading cause of preventable death in the U.S., with low cessation rates. Although drugs that decrease responding in the nicotine self‐administration (SA) model have been marketed as cessation aids for smoking, they have limited efficacy. Their minimal usefulness despite success in animal studies could be because the SA model does not include other tobacco smoke constituents, which may interact with nicotine to create tobacco addiction. Previously, we have shown that the monoamine oxidase inhibitor and tobacco smoke constituent, norharmane is self‐administered alone and increased reinforced responding when combined with nicotine. The aim of the present study was to determine the mechanisms underlying SA of nicotine (nic), norharmane (NH) and the combination (nicNH). Adult male rats were allowed to self‐administer (i.v.) nic (7.5 μg/kg/inj), NH (2.5 μg/kg/inj) or nicNH (7.5+2.5 μg/kg/inj) for 12 days at FR1. Once baseline responding occurred, rats received a 15‐minute pretreatment (s.c.) of mecamylamine (0.75–3.0 mg/kg), ketanserin (0.5–2 mg/kg) or naloxone (0.75– 5.0 mg/kg) prior to SA. Preliminary results indicate that different receptor systems contribute to NH and nic reinforcement. These data suggest that inclusion of other constituents when testing drugs as potential cessation aids may yield more efficacious treatments.
Relative reinforcing efficacy of high and low affinity NMDA channel blockers in rhesus monkeys
Authors: Nicholson Katherine Lynn , Balster Robert L.
Published: June 2021

Preclinical self‐administration (SA) procedures provide face validity and in may cases predictive validity regarding the abuse potential of drugs. A number of NMDA channel blocking antagonists are self‐administered off a fixed‐ratio (FR) phencyclidine (PCP) baseline. To assess relative abuse liability, the high affinity channel blockers PCP, ketamine and dextrorphan and the low affinity channel blockers memantine and dextromethorphan were re‐evaluated using a FR/progressive ratio (PR) procedure. Adult rhesus monkeys participated in daily 1‐h FR 20 SA sessions for 3 to 10 μg/kg/infusion PCP. Test drug solutions serving as positive reinforcers under the FR schedule were evaluated under a PR schedule in which the ratio for each infusion was increased by 10 or 20 (dependent on subject) during each subsequent session. One or more doses of all of the channel blockers, high affinity and low affinity, served as positive reinforcers of behavior under the FR schedule. Under a PR schedule, the high affinity compounds demonstrated much greater break points than the low affinity channel blockers, suggesting a higher reinforcing efficacy and abuse potential for the former compounds. Overall this approach appears to provide a refinement on previous substitution procedures for evaluating the abuse liability of NMDA antagonist drugs that is most predictive of human abuse potential. Supported by NIDA grant DA‐01442.
Individual Differences to Cocaine‐Induced Activation in Rats: Striatal Dopamine D1 and D2 Receptors
Authors: Rorabaugh Jacki Marie , Larson Gaynor A , Zahniser Nancy R
Published: June 2021

Cocaine stimulates locomotor activity by inhibiting the dopamine transporter (DAT) in nucleus accumbens (NAc) and dorsal striatum (dSTR) and thereby increasing extracellular dopamine (DA), which then binds to DA D1‐ and D2‐type receptors (D1Rs and D2Rs). Following an acute low dose of cocaine (10 mg/kg; ip), male Sprague‐Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on their drug‐induced locomotor activity. DATs and extracellular DA levels in LCRs and HCRs are differentially affected by acute cocaine, but whether D1Rs and D2Rs also differ has not been investigated. Here we used saturation binding of the D1R antagonist [3H]SCH 23390 to quantify D1Rs and the D2R antagonist [3H]raclopride to quantify D2Rs in NAc and dSTR at 40 min after acute cocaine. While the HCRs (N = 10) exhibited ~4‐fold greater cocaine‐induced locomotor activity than LCRs (N = 10) or saline controls (N = 7), there were no significant differences among the three groups in total numbers or affinities of D1Rs or D2Rs in either striatal region. Our results suggest that basal levels of striatal D1Rs and D2Rs are similar in LCRs and HCRs. However, our findings do not rule out the possibility that differences in D1R and/or D2R coupling/signaling, may help to explain the differential initial cocaine‐induced behavioral activation between LCRs and HCRs. Supported by DA004216 & DA015050.
Methamphetamine Treatment During Development Attenuates the Dopaminergic Deficits Caused by Subsequent High‐Dose Methamphetamine Administration
Authors: Hoonakker Amanda J , Vieira‐Brock Paula L , Stout Kristen A , Sawada Nicole M , Suchy Amanda , Ellis Jonathan D , Allen Scott C , McFadden Lisa M , Walters Elliot T , Nielsen Shannon M , Gibb James W , Alburges Mario E , Wilkins Diana G , Hanson Glen R , Fleckenstein Annette E
Published: June 2021

Methamphetamine (METH) abuse is a serious public health problem as evidenced by findings that high‐dose administration of this stimulant causes persistent dopaminergic deficits in both non‐human and human models. Noteworthy, adolescent (i.e., postnatal day (PND) 40) rats are less susceptible to this damage than young adult (PND90) rats. Further, bi‐weekly treatment with METH, beginning at PND40 and continuing throughout development, prevents the persistent dopaminergic deficits caused by a “challenge” high‐dose METH regimen when administered at PND90. Mechanisms underlying this “resistance” in rats treated with METH during development were thus investigated. Results revealed that METH treatment throughout development attenuated both acute and persistent effects on monoaminergic transporter function caused by a challenge METH treatment. METH treatment during development also attenuated the hyperthermic response caused by a “challenge” METH treatment. Mechanisms underlying these phenomena, including the role of pharmacokinetics, will be discussed. (Supported by: DA09407, 019447, 013367, 00869, 11389, 04222, 00378)
EFFECTS OF CHOLINERGIC COMPOUNDS IN METHAMPHETAMINE‐DISCRIMINATION STUDIES
Authors: Desai Rajeev I , Martin Jared , Traina Wyatt , Bergman Jack
Published: June 2021

Accumulating evidence suggests that acetylcholine nicotinic mechanisms may contribute to the abuse‐related effects of methamphetamine (MA). In the present study, effects of indirect dopamine (DA) agonists, (d‐amphetamine, L‐MA, cocaine, GBR 12909, methylphenidate), full [nicotine (NIC), anabasine (ANA), (+)‐epibatadine, (−)‐epibatadine, isoarecolone] and partial [varenicline (VAR)] nicotinic agonists, and other cholinergic compounds (atropine, scopolamine, rivastigmine, donepezil) were examined in rats that discriminated 0.3 mg/kg i.p. MA from saline. All indirect DA agonists fully substituted for MA in a dose‐related manner. Among cholinergic agonists, NIC fully substituted for MA in a dose‐dependent manner, whereas other nicotinic agonists produced partial MA‐like responding. Other cholinergic compounds, failed to substitute for MA. In additional experiments, nicotinic agonists were examined in squirrel monkeys that discriminated 0.1 mg/kg i.m. MA from saline. NIC fully substituted for MA, whereas ANA and VAR produced partial MA‐lever responding. These results are consistent with the idea that nicotinic activity plays a role in the abuse‐related effects of MA, and may provide a mechanism for anti‐stimulant medication development (supported by ARRA‐NIH‐NIDA DA026548‐01A1).
Effects of GABAA Modulators in Rats Responding Under A Differential Reinforcement of Low Rates (DRL) Schedule
Authors: Amato Russell Joseph , Lewis Peter Blake , He Hongbo , Winsauer Peter John
Published: June 2021

Little is known about the behavioral effects of neurosteroids in comparison to other drugs that modulate the GABAA receptor complex. The present study examined the effects of pregnanolone (1 – 10 mg/kg) and DHEA (32 – 240 mg/kg) in male rats responding under a DRL‐18 sec schedule of reinforcement. Lorazepam (0.01 – 3.2 mg/kg), ethanol (0.32 – 2.4 g/kg), pentobarbital (1.8 – 18 mg/kg), β‐CCM (0.32 – 5.6 mg/kg) and flumazenil (3.2 – 18 mg/kg) were tested for comparison, and pregnanolone was administered in combination with DHEA to determine if there were any antagonistic effects between these substances. Pregnanolone increased responding at intermediate doses and decreased responding at high doses similar to lorazepam, ethanol, and pentobarbital. All four drugs dose‐dependently decreased percent reinforced responding and shifted the response distribution curve downward and to the left. Dose‐effect curves were similar for DHEA, β‐CCM, and flumazenil, but different from those for the other four drugs (i.e., these drugs did not change response rate or shift the response distribution curve). DHEA did not antagonize the disruptive effects of pregnanolone on responding under this schedule. In summary, pregnanolone and DHEA produce effects on DRL responding similar to other drugs that modulate the GABAA receptor complex, and do not produce these effects through a single binding site. Supported by DA 019625
Dizocilpine enhances the discriminative stimulus effects of midazolam in rats discriminating pregnanolone
Authors: Eppolito Amy Katherine , Bai Xiang , Gerak Lisa
Published: June 2021

The discriminative stimulus effects of the neuroactive steroid pregnanolone are thought to be mediated by multiple neurotransmitter systems including positive modulation at GABAA receptors and antagonism of NMDA receptors. This study investigated the contribution of different receptor mechanisms by establishing pregnanolone as a discriminative stimulus and studying the NMDA receptor antagonist dizocilpine alone and in combination with the positive GABAA modulator midazolam. Male Sprague‐Dawley rats (n=10) discriminated pregnanolone (7.5 mg/kg, i.p.) and responded under a fixed ratio 10 schedule for food. Pregnanolone and midazolam produced >80% pregnanolone‐lever responding. In contrast, dizocilpine produced >80% pregnanolone‐lever responding in two rats and <20% pregnanolone‐lever responding in the remaining eight rats, while the mu opioid receptor agonist morphine produced predominantly vehicle‐lever responding. When administered 30 minutes before sessions, dizocilpine shifted the midazolam dose‐effect curve leftward 2‐ to 6‐fold; however, morphine did not alter the midazolam dose‐effect curve. Although dizocilpine did not substitute for pregnanolone, enhancement of the effects of midazolam by dizocilpine suggests that the pregnanolone discriminative stimulus has both GABAA and NMDA components.
Effect of ovariectomy on the receptor mechanisms contributing to the discriminative stimulus effects of ethanol
Authors: Helms Christa Marie , Buckert Aubrey D , Grant Kathleen A
Published: June 2021

The progesterone metabolites allopregnanolone and pregnanolone produce ethanol‐like discriminative stimulus effects in male Long‐Evans rats and in male and female cynomolgus monkeys. Fluctuations in endogenous ovarian‐derived neuroactive steroids alter ethanol and allopregnanolone substitution for ethanol in drug cynomolgus monkeys. This study determined whether absence of ovarian hormones alters the receptor mechanisms mediating the discriminative stimulus effects of ethanol. Sham (n = 13) and ovariectomized (OVX, n = 12) female Long‐Evans rats were trained to discriminate 1.0 g/kg ethanol (i.g., 15% w/v) from water with a 30‐min pre‐treatment interval. Ovariectomy increased pregnanolone and decreased 5‐HT receptor agonist substitution efficacy (Table 1). Compared to studies in males, neuroactive steroids showed lower substitution efficacy in female rats. Ovarian steroids in females affect specific receptor mechanisms mediating the discriminative stimulus effects of ethanol.

Percentage of rats showing complete substitution of test drugs for 1.0 g/kg ethanol and mean (± SEM) ED50 (mg/kg).

Sham
OVX

Ethanol
100
683 ± 37
100
620 ± 48

Allopregnanolone
54
6.8 ± 0.9
64
5.9 ± 1.1

Pregnanolone
38
6.3 ± 1.9
75
6.1 ± 1.7

Pentobarbital
85
5.1 ± 0.9
67
3.3 ± 0.7

Midazolam
75
1.2 ± 0.6
63
0.6 ± 0.3

Dizocilpine
62
0.036±0.006
88
0.036±0.008

TFMPP
62
0.6 ± 0.1
29
0.11±0.001

RU 24969
85
0.9 ± 0.1
57
0.4 ± 0.2

NIH AA017040
Modulation of the cocaine cue by predictable and unpredictable stress in rats: relation to monoamine transporter levels
Authors: Kohut Stephen J , DeCicco‐Skinner Kathleen L , Riley Anthony L
Published: June 2021

Discriminative stimuli (SD) play an important role in the acquisition, maintenance and reinstatement of drug‐taking behavior and are altered by a variety of manipulations, including stress. The present study tested whether two different schedules of stressor presentation (predictable; PS and unpredictable; UPS) for 10 days can modify the discriminative effects of cocaine in male rats trained to discriminate cocaine (10 mg/kg) from saline. Dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporter levels in mesolimbic areas were also measured after stress to see if the relative ratio of these proteins may explain differences in behavior. In handled (H) rats, ED50s for cocaine‐like responding were stable after 10 days of handling compared to baseline. Animals exposed to PS and UPS displayed shifts in the cocaine dose‐response curve after stress but with different patterns of responding. PS produced a time‐dependent left‐ward shift in cocaine‐like responding, indicating increased sensitivity to the cocaine cue after stress. ED50s after UPS did not differ from baseline, although maximal cocaine‐like responding was lower at the two highest doses of cocaine tested where UPS rats switched from cocaine to saline‐like responding. DAT and SERT levels were different in select brain regions in UPS compared to P/H and in P compared to H which may explain the differences in behavior.
The Human Cytomegalovirus Encoded GPCR US28 Exhibits Constitutive Signaling in Productively Infected Glioblastoma Cells
Authors: Avery Diana L , Miller William E
Published: June 2021

The Human cytomegalovirus (HCMV) is found in a high percentage of glioblastoma multiforme brain tumors. However, a causal relationship between the virus and glioblastoma has not been proven. Interestingly, HCMV encodes a gene termed US28 that is homologous to members of the GPCR superfamily. US28 functions as a constitutively active GPCR and is coupled to the Gαq/PLC‐β pathway. Recent studies indicate that US28 may activate pathways associated with cellular proliferation, suggesting that there may be a link between US28 and glioblastoma progression. In the present study, we aimed to utilize HCMV/US28 recombinant viruses to examine US28 expression and signaling activity in HCMV infected U373 glioblastoma cells. Our results indicate that US28 is expressed in HCMV‐infected glioblastoma cells with early kinetics and that it constitutively signals through the Gαq/PLC‐β pathway. These data support the hypothesis that constitutive signaling activity via the US28 GPCR could play a role in HCMV‐associated glioblastoma development. Future studies will need to define the downstream targets of US28 and determine if/how these targets regulate glioblastoma growth. These studies may facilitate the design of novel therapeutics targeted against this viral protein that could be ultimately used to slow or prevent HCMV‐associated disease.

This project was supported by NIH Grant R01 AI058159 and by the ASPET SURF Program.
Modulation of Cannabinoid Receptor Interacting Protein 1a (CRIP1a) Expression Using Adeno‐Associated Virus and Effects on Striatal Neuropharmacology and Neuronal Cells in Culture
Authors: Blume Lawrence C. , Bass Caroline E. , Dalton George D. , Roberts David C.S. , Selley Dana E. , Howlett Allyn C.
Published: June 2021

CB1 cannabinoid receptors and the D2 dopamine receptors are both GPCRs that associate with interacting proteins (CRIP1a and DRIP) that bind to the C‐terminus of the receptor. The DRIP neuronal calcium sensor‐1 is involved in Ca2+ mediated regulation of the D2 receptor, whereas the recently discovered cannabinoid receptor interacting protein (CRIP1a), has been shown to suppress the tonic inhibition of voltage‐gated Ca2+ channels induced by CB1 (Niehaus et al., 2007). Therefore, CRIP1a may provide a new avenue for modulation of the endocannabinoid system.

AAV that contain transgenes to express shRNA designed to knock down CB1, D2R, or CRIP1a were created and injected via stereotaxically. Striata from AAV‐D2R and AAV‐CB1 shRNA‐treated rats displayed a significant reduction in D2 and CB1 mRNA levels, respectively. The AAV overexpressing CRIP1a virus increased CRIP1a mRNA levels without altering CB1 levels. However, significant reductions occurred in levels of preprodynorphin(Pdyn) and preproenkephalin‐1(Penk‐1).

In vitro, N18TG2 stably transfected with CRIP1a‐pcDNA3.1 displayed increased levels of CRIP1a, Pdyn, and delta opioid receptor mRNA levels without effecting CB1 levels. Additionally, CRIP1a overexpressers had a significant reduction in CB1 mediated phosphorylation of ERK during Rimonabant‐sensitive basal conditions. However, CB1 agonist mediated effects on maximum ERK phosphorylation were unaltered in CRIP1a overexpressors.

These studies provide both in vivo and in vitro insight into the mechanisms involved in CRIP1a modulation of CB1 receptors.
Involvement of a Novel a1A‐Adrenergic Receptor‐Coupled Signaling Pathway in Cardiac Contractility
Authors: Mohl Marion , Xiao Xiao‐Hui , Prosser Hamish , Iismaa Siiri , Feneley Michael , Graham Robert
Published: June 2021

In the heart under normal conditions the β1‐adrenergic receptors (ARs) predominantly regulate cardiac contractility, with the α1‐ARs playing a minor role. However, when the heart is compromised, e.g. during cardiac hypertrophy, the α1‐ARs play a more prominent role.

Using a model of enhanced cardiac α1A‐AR signaling (α1A‐H) we identified a novel signaling pathway involved in cardiac contractility but not hypertrophy. This pathway involves α1A‐AR‐activated translocation of Snapin and the transient receptor potential channel 6 (TRPC6) to the plasma membrane to form a ternary complex with α1A‐AR. In CMs from adult α1A‐H and WT mice, the α1A‐AR‐Snapin‐TRPC6 complex results in increased [Ca2+]i with stimulation by the α1A‐AR specific agonist A61603, which is dose related and proportional to the level of receptor expression. Blockade of TRPC6 inhibited α1A‐AR‐mediated increases in cardiac contractility in WT and α1A‐H hearts. Given that this model of enhanced α1A‐AR activity displays robust hypercontractility but no hypertrophy, our data hold promise that targeting of the α1A‐AR‐Snapin‐TRPC6‐mechanism may allow dissection of pivotal cardiac phenotypes with imminent therapeutic perspectives.

Sponsor: NHMRC
Hypnotic and Hypotensive Responses to the Alpha2‐adrenergic Agonists Are Enhanced and Accompanied by Increased G Protein Coupling to the Alpha2AAR in Spinophilin Null Mice
Authors: Chen Yunjia , Lu Roujian , Peng Ning , Jiao Kai , Limbird Lee , Wyss J Michael , Wang Qin
Published: June 2021

We have previously identified spinophilin (SP) as a critical regulator of alpha2 adrenergic receptor trafficking and signaling both in vitro and in vivo. Compared to wild type mice, mice lacking SP expression are more sensitive to alpha2‐agonist‐elicited sedation. In the present study, we assessed cardiovascular responses and hypnotic effects elicited by systemic administration of an alpha2‐agonist, UK14,304, in SP null mice. Our data demonstrate that mice lacking SP expression display dramatically enhanced and prolonged hypotensive, bradycardic, and hypnotic responses to alpha2AAR stimulation. These changes in sensitivity to alpha2AR agonist occur independent of any changes in alpha2AAR density or intrinsic affinity for UK14,304 in the brain of SP null mice when compared to wild type controls. In contrast, the data indicate that coupling of the alpha2AAR to cognate G proteins is enhanced in SP null mice, based on changes in guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of alpha2AAR in the high affinity state in preparations from SP null mice when compared to those from wild type mice. This suggests that an enhanced receptor/G protein coupling efficiency in the absence of SP expression contribute to an increased responsiveness of alpha2AARs in SP null mice, indicating that SP may exert ongoing regulation of receptor‐G protein coupling in vivo.
Regulation of α2C Adrenergic Receptor Temperature‐Sensitive Traffic by HSP90
Authors: Filipeanu Catalin
Published: June 2021

Within GPCR class, α2C‐adrenergic receptor (α2C‐AR) has unique characteristics, being poorly transported to the plasma membrane at physiological temperature (37°C). Lowering the temperature enhanced its cell surface expression and this particular trafficking pattern may explain the aberrant vasoconstriction to cold observed in Raynaud Phenomenon. Indeed, we found that at 37°C in HEK293T transfected with GFP‐tagged α2C‐AR, the receptor is extensively colocalized with endoplasmic reticulum (ER) markers, but at 30°C α2C‐AR is mostly present at the plasma membrane. These data were confirmed in radioligand binding which demonstrated an increase by 69 ± 9% in the plasma membrane receptor levels at low‐temperature. Treatment with three distinct HSP90 inhibitors, radicicol (10 μM), macbecin (1 μM) and 17‐DMAG (0.5 μM) significantly enhanced α2C‐AR cell surface levels at 37°C (57 ± 6%, 91 ± 7% and 78 ± 12% respectively). In contrast, these HSP90 inhibitors have no effects on the α2C‐AR cell surface levels at 30°C. Further, co‐immunoprecipitation experiments demonstrated that α2C‐AR interactions with HSP90 are decreased at low‐temperature. In addition, overexpression of GRP94, the ER resident HSP90 isoforms did not change the α2C‐AR cell surface levels at 37°C or at 30°C. In conclusion, the temperature‐sensitive α2C‐AR is under the control of cytosolic HSP90 isoforms. Supported by NIH Grant2P20RR018766‐06.
Generation and Characterization of PRL‐3 Knockout Mice
Authors: Zimmerman Mark W. , Homanics Gregg E. , Lazo John S.
Published: June 2021

The Phosphatase of Regenerating Liver‐3 (PRL‐3), along with its family members PRL‐1 and PRL‐2, are small, prenylated, protein tyrosine phosphatases with more than 75% amino acid identity. PRL‐3 was discovered to be overexpressed in metastases derived from colorectal cancer, and further studies have revealed that PRLs may enhance cell migration and invasion. High levels of PRL‐3 expression also correlate with poor prognostic outcome in patients with cancers of the liver, ovary, breast, and pancreas. Despite significant information implicating PRL‐3 as a novel target for the treatment of malignant disease, the substrates and downstream targets of PRL‐3 remain unclear. In order to further elucidate the biology of PRL‐3, as well as its potential roles in cancer and metastasis, we have created a conditional knockout mouse model for PRL‐3 ablation. In our gene‐targeted model, Cre recombinase activity is predicted to result in a functionally inactive gene product with no homology to the wildtype protein. To model the effect of PRL‐3 ablation on a cellular level mouse embryonic fibroblasts possessing the PRL‐3+/flox and PRL‐3flox/flox alleles will be transfected with Cre recombinase. Current studies are also aimed at characterizing the phenotypic effect of heterozygous and homozygous knockout in the whole animal as well as dissecting the cellular signaling pathways affected by PRL‐3 knockout.
Subcellular localization and activation of CaMKII delta splice variants
Authors: Mishra Shikha , Zhang Tong , Brown Joan Heller
Published: June 2021

Ca2+/Calmodulin dependent protein kinase II delta (CaMKII D) is the predominant isoform in the heart. There are two splice variants, deltaB (DB) and deltaC (DC) which differ only by a nuclear localization sequence in DB. We previously reported that cardiac DB and DC transgenic (TG) mice both develop hypertrophy but only DC TG mice show altered Ca handling and transition to heart failure. The inclusion of an NLS and our findings from immunostaining suggest distinct localization of DB and DC which could affect the targets and consequences of transgene expression. Surprisingly, subcellular fractionation of hearts from DB and DC mice revealed that CaMKII D localization is not distinct. In DB mice, more than 50% of total CaMKII D was located outside the nucleus, and in DC mice, almost 25% of total CaMKII is in the nucleus. Since CaMKII is multimeric, we hypothesized that this apparent “mislocalization” could reflect formation of heteromultimers. Accordingly, we crossed DB and DC TG mice with recently generated CaMKII D null mice. In the single isoform TG mice, subcellular fractionation revealed that the distribution of DB and DC was nearly identical to that described above. Further studies using these mice are underway to determine whether there is a differential activation of CaMKII DB and DC at distinct subcellular sites and how this dictates target phosphorylation, cellular responses and heart failure progression.
Pharmacological characteristics of ATI‐9242, a Novel Atypical Antipsychotic
Authors: Choppin Agnes , Lach Leang , Lee Alexander , Nunez Oscar , Becker Cyrus , Palme Monica P , Paliard Xavier , Druzgala Pascal , Jasper Jeffrey R
Published: June 2021

ATI 9242 is a dibenzo(b,f)(1,4)oxazepine derivative designed with the goal of developing an oral drug that is effective for neuropsychiatric disorders with an improved side effect profile.

The in vitro receptor profiling demonstrated that ATI 9242 has high affinity for 5‐HT2A and D2 receptor and submicromolar affinity for other dopamine (D1, D3, D4 and D5), serotonin (5‐HT1A, 5‐HT1B, 5‐HT1D, 5‐HT2B, 5‐HT2C, 5‐HT6 and 5‐HT7), histamine (H1) and alpha‐adrenergic (α1B and α2C) receptors.

In cell‐based functional assays, ATI 9242 (10 μM) had no measurable agonist activity at dopamine (D1, D2S), histamine (H1), alpha‐adrenergic and serotonin (5‐HT2A, 5‐HT2B, 5‐HT2C, 5‐HT6 and 5‐HT7) receptors. At the 5‐HT1A receptor, ATI 9242 appeared to be a partial agonist (Ki=147 nM).

ATI‐9242 prevented 5‐HT2A receptor‐mediated rat aortic ring contractions in a concentration‐dependent manner (ED50 = 3 nM), data consistent with the receptor binding results.

In vivo, ATI 9242 exhibited an atypical antipsychotic‐like activity against apomorphine‐induced disruption of swimming in mice, but also in models of prepulse inhibition (PPI), without cataleptic effect (up to 30mg/kg IP), sedation (up to 10mg/kg IP), nor motor coordination impairment.

Overall, the data presented herein indicate that ATI‐9242 is a novel atypical antipsychotic designed for multi‐target interactions with reduced side effects.
Relationship between norepinephrine transporter (NET) potency and hemodynamic responses to monoamine reuptake inhibitors in an anesthetized rat model
Authors: Pulido‐Rios M. Teresa Ramos , Zamora Frederick , Tsuruda Pamela , Obedencio Glenmar , Schweigert Kevin , Kwan Keith , Cui Liping , Kanta Anne , Smith Jacqueline AM , Hegde Sharath S , Martin William J
Published: June 2021

Monoamine reuptake inhibitors (MRI) that inhibit transporters for serotonin (SERT) and/or norepinephrine (NET) produce hemodynamic effects of varying magnitudes in the clinic. Here, we used an anesthetized rat model to compare the potency to increase heart rate (HR) and mean arterial pressure (MAP) and to inhibit tyramine pressor response (TPR) of intraperitoneally‐dosed MRIs with a range of SERT and NET potencies and selectivities. In vitro SERT and NET potencies (IC50) were determined using a [3H]neurotransmitter uptake assay with rat synaptosomal preparations. With parallel pharmacokinetic studies, we estimated the plasma free drug concentrations required to produce 50% inhibition of TPR (Tyr EC50) or to induce a 10 mm Hg increase in MAP (MAP PC10) and a 25 bpm increase in HR (HR PC25). Esreboxetine, desipramine, atomoxetine, milnacipran, duloxetine and venlafaxine produced dose‐dependent decreases in TPR and increases in MAP and HR. TPR EC50 positively correlated (r2 = 0.9) with in vitro NET IC50 but not SERT IC50 (r2 = 0). Moreover, NET IC50 of the molecules also positively correlated with their HR PC25 (r2= 0.9) but not MAP PC10 (r2= 0.6). Milnacipran was an exception in that NET potency correlated with TPR EC50, but not with HR PC25. This model offers a simple and sensitive in vivo method for assessing the potency of MRIs to evoke mechanism‐based hemodynamic effects and to inhibit NE transporters in the periphery.
SU11248, a selective tyrosine kinases inhibitor suppresses breast tumor angiogenesis and growth via targeting both tumor vasculature and breast cancer cells
Authors: Gu Jian‐Wei , Tucker Kevan B , Huang Min , Young Emily , Wells Jeremy , Pan Zhi‐Jun
Published: June 2021

SU11248 is a selective inhibitor of certain protein kinases including VEGF receptors. Present study determines whether the anti‐tumor activity of SU11248 results from anti‐angiogenesis, direct anti‐proliferation and anti‐migration effects on breast tumor. Eight wk old female mice (C57BL/6J) were given SU11248 at 20–40 mg/kg/d in drinking water for 4 wks. Control mice received drinking water only. In the 2nd wk, 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored. At the end, tumors were isolated for measuring tumor size and intratumoral microvessel density (IMD) using CD31 immunohistochemistry. SU11248 significantly reduced tumor weight over the control (1.22±0.28 vs. 3.28±0.31 g; n=6; P<0.01) and IMD (111±10 vs. 155±6 IM#/mm^2; P<0.01). Western blot indicated that ERα, VEGF‐R1 and −R2 were expressed in cultured E0771 cells. 3H‐thymidine incorporation showed that SU11248 (10 and 20 μmol/L) significantly suppressed the proliferation of cultured E0771 cells. Migration assay showed that SU11248 (10 μmol/L) significantly inhibited the migration of cultured E0771 cells. These findings support the hypothesis that the anti‐tumor activity of SU11248 on breast cancer is possibly mediated by targeting the paracrine and autocrine effects of VEGF on breast cancer to suppress tumor angiogenesis, proliferation, and migration. (HL51971 & AA013821)
Role of Caveolin‐1 in Mouse Lung Endothelial Cell Proliferation and Differentiation
Authors: Temple Anton Lee , Shinin Vasily , Minshall Richard D.
Published: June 2021

Caveolin‐1 null (Cav‐1−/−) mice exhibit pulmonary hypertension, dysfunctional microvasculature networks, and paradoxically, increased lung microvascular density. To elucidate the cellular basis of these alterations, wild‐type (WT) and Cav‐1−/− mouse lung ECs were isolated in parallel from juvenile male mice for characterization of growth and angiogenic properties in vitro. Pure populations of ECs were investigated in 2D (on plastic) and 3D (cells grown on beads and plated in collagen gels) cultures. Cav‐1−/− ECs proliferated 3‐times faster and initiated colony formation at a 50‐fold lower density than WT ECs. In gel‐bead tubulogenesis assays, sprout number was 15‐times greater and sprout length was 5‐times longer for Cav‐1−/− ECs compared to WT ECs after 11 days in culture. However, only 15% of the Cav‐1−/− EC sprouts formed lumens whereas EC tube and lumen formation was observed in 88% of WT EC 3D cultures. Therefore, EC growth, sprouting, and sprout length were significantly greater for Cav‐1−/− mouse lung ECs compared to WT ECs, but these cells differentiated less into endothelial tubes. Thus, 2D and 3D EC cultures support the histological findings of hypercellularity and high density‐dysfunctional microvasculature networks in the Cav‐1−/− mouse lung. Cav‐1 is therefore an important negative regulator of EC proliferation and positive regulator of EC differentiation and thus angiogenesis.
Vitamin D regulates cathelicidin (CAMP) but not interleukins (IL12A or 27) mRNA, levels in HTR‐8/SVneo human placental trophoblasts in vitro
Authors: Caty Jane T , Jones Sara , Brannon Patsy M.
Published: June 2021

The placenta synthesizes active 1,25‐dihydroxyvitamin D [1,25(OH)2D], which may suppress the maternal immune system by regulating placental immunomodulatory peptides and placental hormonal function. Whether it also regulates placental growth is unknown. To determine its regulation of placental immunomodulatory peptides and cell growth, HTR‐8/SVneo human trophoblast cells were cultured with 100–1000 nmol/L of 1,25(OH)D3 or 25(OH)D3 or 0.1% ethanol (C) for 24 h. VEGF, Ki‐67 (marker of cell proliferation), interleukins (IL‐12A and IL‐27) and cathelicidin antimicrobial peptide (CAMP) mRNA levels were measured by RT‐PCR. Cellular protein and DNA were measured using Lowry and spectrofluorometric assays. 1,25(OH)2D3 and 25(OH)D3 increased CAMP mRNA levels 1 fold (p<0.001 by ANOVA), but did not affect IL‐27 and IL‐12 A mRNA levels. 1,25(OH)2D3 (100‐1000 nmol/L) and 25(OH)D3 (500–1000 nmol/L) were equally effective in regulating CAMP mRNA, suggesting that HTR‐8/SVneo human trophoblasts activate 25(OH)D3. Neither 1,25(OH)2D3 nor 25(OH)D3 altered cellular DNA, protein‐to‐DNA ratio or Ki67mRNA levels, suggesting that it does not alter growth in 24 h. Neither altered hCG mRNA levels in 24 h. Vitamin D regulates placental CAMP expression and may, thus, modulate maternal innate immunity, which might be important in protecting maternal and placental tissue from microbial infection.