Light Activation of Calcein Inhibits Vesicle Release of Catecholamines.

Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of calcein. We hypothesized that calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.

Basic Hip Arthroscopy: Diagnostic Hip Arthroscopy.

Hip arthroscopy is increasing in popularity for the diagnosis and management of hip preservation. The basics of hip arthroscopy positioning, fluoroscopic assessment, and portal establishment are reviewed in the first 2 parts of this series. This article is the third installment in which we describe a systematic approach to performing a diagnostic hip arthroscopy. A mastery of diagnostic arthroscopy is necessary for surgeons treating hip disorders.

Nicotinic modulation of descending pain control circuitry.

Along with the well-known rewarding effects, activation of nicotinic acetylcholine receptors (nAChRs) can also relieve pain, and some nicotinic agonists have analgesic efficacy similar to opioids. A major target of analgesic drugs is the descending pain modulatory pathway, including the ventrolateral periaqueductal gray (vlPAG) and the rostral ventromedial medulla (RVM). Although activating nAChRs within this circuitry can be analgesic, little is known about the subunit composition and cellular effects of these receptors, particularly within the vlPAG. Using electrophysiology in brain slices from adult male rats, we examined nAChR effects on vlPAG neurons that project to the RVM. We found that 63% of PAG-RVM projection neurons expressed functional nAChRs, which were exclusively of the α7-subtype. Interestingly, the neurons that express α7 nAChRs were largely nonoverlapping with those expressing µ-opioid receptors (MOR). As nAChRs are excitatory and MORs are inhibitory, these data suggest distinct roles for these neuronal classes in pain modulation. Along with direct excitation, we also found that presynaptic nAChRs enhanced GABAergic release preferentially onto neurons that lacked α7 nAChRs. In addition, presynaptic nAChRs enhanced glutamatergic inputs onto all PAG-RVM projection neuron classes to a similar extent. In behavioral testing, both systemic and intra-vlPAG administration of the α7 nAChR-selective agonist, PHA-543,613, was antinociceptive in the formalin assay. Furthermore, intra-vlPAG α7 antagonist pretreatment blocked PHA-543,613-induced antinociception via either administration method. Systemic administration of submaximal doses of the α7 agonist and morphine produced additive antinociceptive effects. Together, our findings indicate that the vlPAG is a key site of action for α7 nAChR-mediated antinociception.