Calcium-induced calcium release in noradrenergic neurons of the locus coeruleus.

The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including cognitive and emotional functions. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread brain regions. One of the key regulators is cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat and demonstrate the underlying pathway. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by increased membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological blockers indicated that it was mediated by Ca2+-activated K+ channels of the non-small conductance type. The application of caffeine increased [Ca2+]c, as visualized by fluorescence microscopy. These findings show CICR suppresses LC neuronal activity, and indicate its dynamic role in modulating the LC-mediated noradrenergic tone in the brain.

Examination of synaptic vesicle recycling using FM dyes during evoked, spontaneous, and miniature synaptic activities.

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.