Comparison of fluorescence biosensors and whole-cell patch clamp recording in detecting ACh, NE, and 5-HT.

The communication between neurons and, in some cases, between neurons and non-neuronal cells, through neurotransmission plays a crucial role in various physiological and pathological processes. Despite its importance, the neuromodulatory transmission in most tissues and organs remains poorly understood due to the limitations of current tools for direct measurement of neuromodulatory transmitters. In order to study the functional roles of neuromodulatory transmitters in animal behaviors and brain disorders, new fluorescent sensors based on bacterial periplasmic binding proteins (PBPs) and G-protein coupled receptors have been developed, but their results have not been compared to or multiplexed with traditional methods such as electrophysiological recordings. In this study, a multiplexed method was developed to measure acetylcholine (ACh), norepinephrine (NE), and serotonin (5-HT) in cultured rat hippocampal slices using simultaneous whole-cell patch clamp recordings and genetically encoded fluorescence sensor imaging. The strengths and weaknesses of each technique were compared, and the results showed that both techniques did not interfere with each other. In general, genetically encoded sensors GRABNE and GRAB5HT1.0 showed better stability compared to electrophysiological recordings in detecting NE and 5-HT, while electrophysiological recordings had faster temporal kinetics in reporting ACh. Moreover, genetically encoded sensors mainly report the presynaptic neurotransmitter release while electrophysiological recordings provide more information of the activation of downstream receptors. In sum, this study demonstrates the use of combined techniques to measure neurotransmitter dynamics and highlights the potential for future multianalyte monitoring.

Desflurane Post-treatment Reduces Hypoxic-ischemic Brain Injury via Reducing Transient Receptor Potential Ankyrin 1 in Neonatal Rats.

Perinatal hypoxic-ischemic (HI) brain injury leads to mortality and morbidity in neonates and children. There are no effective and practical methods to attenuate this brain injury. This study determined whether desflurane, a volatile anesthetic with limited effect on the cardiovascular system, protected against HI-induced brain damage and the role of transient receptor potential ankyrin 1 (TRPA1), a mediator for simulated ischemia-induced myelin damage, in this protection. Seven-day-old male and female Sprague-Dawley rats had brain HI. They were exposed to 4.8%, 7.6% or 11.4% desflurane immediately or 4.8% desflurane at 0.5, 1 or 2 h after the HI. Brain tissue loss was evaluated 7 days later. Neurological functions and brain structures of rats with HI and 4.8% desflurane post-treatment were evaluated 4 weeks after the HI. TRPA1 expression was determined by Western blotting. HC-030031, a TRPA1 inhibitor, was used to determine the role of TRPA1 in the HI-induced brain injury. HI induced brain tissue and neuronal loss, which was attenuated by all tested concentrations of desflurane. Desflurane post-treatment also improved motor function, learning and memory in rats with brain HI. Brain HI increased the expression of TRPA1 and this increase was inhibited by desflurane. TRPA1 inhibition reduced HI-induced brain tissue loss and impairment of learning and memory. However, the combination of TRPA1 inhibition and desflurane post-treatment did not preserve brain tissues, learning and memory better than TRPA1 inhibition or desflurane post-treatment alone. Our results suggest that desflurane post-treatment induces neuroprotection against neonatal HI. This effect may be mediated by inhibiting TRPA1.

Attenuating oxygen-glucose deprivation-caused autophagosome accumulation may be involved in sevoflurane postconditioning-induced protection in human neuron-like cells.

Application of the commonly used volatile anesthetic sevoflurane after brain ischemia (sevoflurane postconditioning) attenuates ischemic brain injury. It is not known whether autophagy plays a role in this sevoflurane postconditioning-induced neuroprotection. Human SH-SY5Y cells were induced to become neuron-like cells. These cells were subjected to 1 h oxygen-glucose deprivation (OGD) and then exposed to sevoflurane for 1 h. Chloroquine, an inhibitor of autolysosomes, rapamycin, an autophagy inducer, or 3-methyladenine (3-MA), an autophagy inhibitor, were incubated with cells during OGD and sevoflurane exposure. OGD and the subsequent simulated reperfusion increased lactate dehydrogenase (LDH) release from the cells. This increase was dose-dependent inhibited by sevoflurane postconditioning. OGD increased the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3I and the expression of beclin-1 and p62. These increases were attenuated by sevoflurane. Sevoflurane alone did not have any effects on the expression of p62, beclin-1 and the ratio of LC3II to LC3I. Sevoflurane also enhanced the co-location of autophagosomes and lysosomes. Chloroquine increased the ratio of LC3II to LC3I, p62 and LDH release in cells subjected to OGD. Sevoflurane postconditioning attenuated OGD-induced inactivation of Akt and mechanistic target of rapamycin (mTOR). Inducing autophagosome generation by rapamycin attenuated sevoflurane postconditioning-reduced LDH release. Inhibition of autophagosome generation by 3-MA decreased OGD-induced LDH release. These results suggest that OGD increase autophagosome accumulation via increased formation of autophagosomes and reduced autophagosome clearance and that attenuation of OGD-induced autophagosome accumulation may contribute to sevoflurane postconditioning-induced cell protection.