AfaSci (United States)

Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg2+) concentration is critical to activation of OTR, and low serum Mg2+ concentration is predictive of migraine headache. We, therefore, examined the functional impact of Mg2+ concentration on OT-OTR binding efficacy using two complimentary bioassays. Current clamp recordings of rat trigeminal ganglia neurons (TG) measured the impact of Mg2+ on OT-induced reduction in excitability. Secondly, we assessed the impact of Mg2+ on intranasal OT-induced craniofacial analgesia in rats. Key Results: OT alone dose-dependently hyperpolarized TG neurons, decreasing their excitability; the addition of 1.75mM Mg2+ significantly enhanced this effect. Similarly, while intranasal application of OT produced dose-dependent craniofacial analgesia; Mg2+ significantly enhanced these effects. Conclusion and Implications: Mg2+ concentration is critical to OT-OTR signaling, and OT efficacy may be limited by low ambient Mg2+ levels. The addition of Mg2+ to OT formulations may improve its efficacy in reducing headache pain as well as for other oxytocin-dependent processes.

Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg2+) concentration is critical to activation of OTR, and low serum Mg2+ concentration is predictive of migraine headache. We, therefore, examined the functional impact of Mg2+ concentration on OT-OTR binding efficacy using three complimentary bioassays. Experimental Approach: Bioluminescence resonance energy transfer assay was used to investigate the impact of Mg2+ on G-protein activation secondary to OT-OTR binding. Current clamp recordings of rat trigeminal ganglia neurons (TG) measured the impact of Mg2+ on OT-induced reduction in excitability. Finally, we assessed the impact of Mg2+ on intranasal OT-induced craniofacial analgesia in rats. Key Results: Mg2+ is an essential cofactor for full OT-OTR agonism and concentration-dependently increased Gq and G11 activation in OTR-transfected HEK cells, although OT-OTR binding did not reach maximal efficacy at physiologic concentrations. OT alone dose-dependently hyperpolarized TG neurons, decreasing their excitability; the addition of 1.75mM Mg2+ significantly enhanced this effect. Finally, while intranasal application of OT produced dose-dependent craniofacial analgesia; Mg2+ significantly enhanced these effects. Conclusion and Implications: Mg2+ concentration is critical to OT-OTR signaling, and OT efficacy may be limited by low ambient Mg2+ levels. The addition of Mg2+ to OT formulations may improve its efficacy in reducing headache pain as well as for other oxytocin-dependent processes.

A dysregulation in function of neuronal low-voltage-activated T-channel causes neuropathic pain. A group of ten 1,4-disubstituted and 1,4,4-trisubstituted piperidines were synthesized through short sequences of reactions from 4-cyanopiperidine, which may potentially produce a library of analogs. Inhibitions of T-channel in rat dorsal root ganglion neurons and/or Ca v3.2 channel in human embryonic kidney-293 cells were screened, and subsequent analgesic effects and inhibition of seizure in pentylenetetrazole-induced seizure mouse model were examined. The two most promising piperidine molecules showed analgesic effects in rat model using a spared nerve injury protocol. One displayed a long withdrawal latency in response to thermal stimulation, and an 80% increase in mechanical threshold assessment studies.

Neuropathic pain caused by nerve injury is debilitating and difficult to treat. Current systemic pharmacological therapeutics for neuropathic pain produce limited pain relief and have undesirable side effects, while current local anesthetics tend to nonspecifically block both sensory and motor functions. Calcitonin gene related peptide (CGRP), a neuropeptide released from sensory nerve endings, appears to play a significant role in chronic neuropathic pain. In this study, an analgesic microneedle (AMN) patch was developed using dissolvable microneedles to transdermally deliver selective CGRP antagonist peptide in a painless manner for the treatment of localized neuropathic pain. Local analgesic effects were evaluated in rats by testing behavioral pain sensitivity in response to thermal and mechanical stimuli using neuropathic pain models such as spared-nerve injury and diabetic neuropathy pain, as well as neurogenic inflammatory pain model induced by ultraviolet B (UVB) radiation. Unlike several conventional therapies, the AMN patches produced effective analgesia on neuropathic pain without disturbing the normal nociception and motor function of the rat, resulting from the high specificity of the delivered peptide against CGRP receptors. The AMN patches did not cause skin irritation or systemic side effects. These results demonstrate that dissolvable microneedle patches delivering CGRP antagonist peptide provide an effective, safe, and simple approach to mitigate neuropathic pain with significant advantages over current treatments.

The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.

Nicotine is a heavily used addictive drug acquired through smoking tobacco. Nicotine in cigarette smoke is deposited and absorbed in the lungs, which results in a rapidly peaked slowly declining arterial concentration. This pattern plays an important role in initiation of nicotine addiction. A method and device were developed for delivering nicotine to rodents with lung alveolar region-targeted aerosol technology. The dose of delivery can be controlled by the nicotine aerosol concentration and duration of exposure. Our data showed that, in the breathing zone of the nose-only exposure chamber, the aerosol droplet size distribution was within the respirable diameter range. Rats were exposed to nicotine aerosol for 2 min. The arterial blood nicotine concentration reached 43.2 ± 15.7 ng/ml (mean ± SD) within 1-4 min and declined over the next 20 min, closely resembling the magnitude and early pharmacokinetics of a human smoking a cigarette. The acute inhalation toxicity of nicotine: LC50 = 2.3mg/L was determined; it was affected by pH, suggesting that acidification decreases nicotine absorption and/or bioavailability. A noninvasive method and toolkit were developed for delivering nicotine to rodents that enable rapid delivery of a controllable amount of nicotine into the systemic circulation and brain-inducing dose-dependent pharmacological effects, even a lethal dose. Aerosol inhalation can produce nicotine kinetics in both arterial and venous blood resembling human smoking. This method can be applied to studies of the effects of chronic intermittent nicotine exposure, nicotine addiction, toxicology, tobacco-related diseases, teratogenicity, and for discovery of pharmacological therapeutics.

The combination of molecular manipulation of gene expression and behavior endpoint phenotypic analysis has uncovered numerous novel gene functions and provided unprecedented opportunities for new drug development. To facilitate investigation of diverse behavioral processes, we have developed a home cage monitoring system for automated rodent neurobehavioral phenotypic analysis and higher-throughput in vivo drug screening. This system termed SmartCage™ measures rodents’ behavior in their home cages as a significant endpoint. The SmartCage™ system consists of multiple instrument platforms that interface with ordinary rodent home cages. Each SmartCage™ is comprised of multiple sensors including a floor-vibration sensor, an infrared (IR) matrix and flexible modular devices. This system is noninvasive and allows the animal to be tested in its home cage that has bedding, food, and water, making it appropriate to monitor animals for days or weeks. The automated measurements include wake and sleep/inactive states. The active parameters include locomotion, rearing, and animal movement patterns, for example, rotations (cycling). In validating the system, we first compared it with conventional technologies, e.g., video camera recording. The results indicate a strong correlation between imaging tracking and IR detection. Next we used pharmacological tools (both stimulant and activity inhibitors) to further validate the system, which demonstrated that SmartCage™ is sensitive enough to detect drug-induced increases and decreases in activity levels. Secondly, wake and sleep state automated classification by the SmartCage™ was validated by comparing it with simultaneously recorded electroencephalograph (EEG)/electromyograph (EMG) data. This comparison confirms that the floor-sensor can provide up to 85–90% accuracy in estimating sleep states. Using modular devices incorporated into the SmartCage™ platform, other behavioral measures can be collected. For example, a new rotarod device can be flexibly installed in the SmartCage™ system. The manual rotarod test can be conducted in a similar manner as the conventional rotarod test. That is, the researcher placing the mouse onto the rod and then start the test. Using this new device, spontaneous performance of rotarod test is automatically initiated by the animal at its home cage. The duration of staying on the rotating rod and numbers of spontaneous performance are significantly different between middle cerebral arterial occlusion (MCAO) and sham control mice. Another modular insert includes a dark box that can be placed directly into the animal’s home cage and our initial tests indicate that the SmartCage™ can detect diazepam-induced anxiolytic effects. The result is comparable to those obtained using conventional anxiety tests such as the elevated plus T-maze and Zero-maze. In conclusion, this noninvasive integrated home cage monitoring system, SmartCage™, can provide an automated assessment of rodent behavior to identify gene-altered phenotypes, to facilitate initial in vivo drug screening, and to rapidly filter out neurobehavioral toxic compounds prior to testing in individual disease models.