carbocyanines and fura-2-am

Morphine, a mu-opioid agonist, suppressed the Ca(2+)-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by morphine was concentration-dependent and blocked by the nonselective opioid receptor antagonist naloxone. As determined by examining the mechanism of mu-opioid receptor-mediated inhibition of glutamate release, morphine caused a significant reduction in 4-aminopyridine-evoked increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](c)), but failed to alter both 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential and Ca(2+) ionophore (ionomycin)-induced glutamate release. In addition, morphine was not capable of producing further inhibition on 4AP-evoked glutamate release in synaptosomes pretreated with the cannabinoid CB(1) receptor agonist WIN 55212-2, which has been shown to depress glutamate release through a suppression of presynaptic voltage-dependent Ca(2+) channel activity. These data suggest that morphine exerts its inhibitory effect presynaptically, likely through the reduction of Ca(2+) influx into nerve terminals, and thereby inhibits the release of glutamate in the cerebral cortex. This may therefore indicate that mu-opioid receptor agonists have neuroprotective properties, especially in the excessive glutamate release that occurs under certain pathological conditions.

Topics: 4-Aminopyridine; Analgesics, Opioid; Animals; Benzothiazoles; Benzoxazines; Calcium; Calcium Channel Blockers; Carbocyanines; Cerebral Cortex; Dose-Response Relationship, Drug; Exocytosis; Fluorometry; Fura-2; Glutamic Acid; Ionomycin; Ionophores; Male; Membrane Potentials; Morphine; Morpholines; Naloxone; Naphthalenes; Narcotic Antagonists; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Synaptosomes

There is strong evidence for the presence of nucleotide (P2) receptors in sensory neurons, which might play a role in the transmission of pain signals. In contrast, virtually nothing is known about P2 receptors in satellite glial cells (SGCs), which are the main glial cells in sensory ganglia. We investigated the possibility that P2 receptors exist in SGCs in murine trigeminal ganglia, using Ca(2+) imaging, patch-clamp recordings, and immunohistochemistry. We found that ATP caused an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in SGCs. As adenosine had no effect on [Ca(2+)](i), and the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid largely blocked the response to ATP we conclude that P1 receptors did not contribute to the responses. We obtained the following evidence that the responses to ATP were mediated by metabotropic P2Y receptors: (i) persistence of the responses in Ca(2+)-free solution, (ii) inhibition of the response by cyclopiazonic acid, (iii) [Ca(2+)](i) increases in response to the P2Y agonists uridine triphosphate, adenosine thiodiphosphate, and 2-methylthio ADP, and (iv) failure of the P2X agonist alpha,beta-methylene ATP to elicit a response. Agonists of P2Y(1) receptors and uridine triphosphate, an agonist at P2Y(2) and P2Y(4) receptors, induced [Ca(2+)](i) increases suggesting that at least these P2Y receptor subtypes are present on SGCs. Using an antibody against the P2Y(4) receptor, we found immunopositive SGCs. Patch-clamp recordings of SGCs did not reveal any inward current due to ATP. Therefore, there was no evidence for the activation of ionotropic P2X receptors under the present conditions. The results indicate the presence of functional nucleotide (P2Y) receptors in SGCs.

Topics: Adenosine Triphosphate; Animals; Calcium; Carbocyanines; Cytophotometry; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Female; Fura-2; Glutamate-Ammonia Ligase; Immunohistochemistry; In Vitro Techniques; Indoles; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; Neuroglia; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Receptors, Purinergic P2; Trigeminal Ganglion

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.

Topics: Action Potentials; Animals; Calcium; Carbocyanines; Cell Survival; Cells, Cultured; Colon; Fluorescent Dyes; Fluorometry; Fura-2; Guinea Pigs; Male; Microscopy, Fluorescence; Myenteric Plexus; Neurons, Afferent; Patch-Clamp Techniques