Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca²⁺ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.

New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca²⁺ responses in GFP-tagged neurons, a non-green fluorescent Ca²⁺ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca²⁺ indicator fura-2 has been used in combination with GFP-tagged neurons¹. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals². Therefore, we decided to use a red fluorescent Ca²⁺ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons³. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons⁴. Fura-red based Ca²⁺ imaging was also successfully combined with GFP-tagged pancreatic β-cells and GFP-tagged receptors expressed in HEK cells^5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium⁷. Therefore, the fluorescence of resting neurons with low Ca²⁺ concentration has relatively high intensity. It should be noted, that other red Ca²⁺-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.