Figure 6. Simultaneous measurements of [Ca²⁺]_c and ΔΨ_m in individual neurons using fura-2FF and Rh123 allow dissociation of the change in [Ca²⁺]_c before and after mitochondrial depolarisation.

A, trace showing simultaneous measurements of Rh123 (grey line) and fura-2FF (black line) fluorescence in a typical neuron during exposure to 100 μM glutamate. Measurements were taken with a CCD camera, as described in Methods. Note that there was an initial change in fura-2FF ratio on glutamate challenge, which plateaued briefly, and during which the Rh123 fluorescence began to increase. This was followed by a sharp secondary rise in fura-2FF ratio, occurring concurrently with further loss of ΔΨ_m. B, measurement of Rh123 (grey) and fura-2FF (black) fluorescence changes, in a typical neuron, using a microfluorimetry system for improved temporal resolution. The neuron was initially stimulated with 50 mM KCl, causing a rapid increase in fura-2FF ratio but no increase in Rh123 fluorescence. Application of 100 μM glutamate to the same neuron caused an increase in fura-2FF ratio and an increase in Rh123 fluorescence. During the profound increase in Rh123, a sharp, secondary rise in fura-2FF ratio occurred. C, concentrating on the initial part of the neuronal response to 100 μM glutamate, this example of a typical neuron illustrates that there was an initial [Ca²⁺]_c increase (marked as ‘X’), seen as an increase in fura-2FF ratio, which at a later time point was superseded by a secondary increase in fura-2FF ratio. The initial fura-2FF ratio change occurred prior to loss of ΔΨ_m, whereas the secondary fura-2FF ratio increase was associated with profound loss of ΔΨ_m. D, comparison of the initial fura-2FF ratio increase – which occurred prior to mitochondrial depolarisation (indicated as a grey line on trace Da) – between a population of neurones challenged with 100 μM glutamate (Da) and another challenged with 50 mM KCl (Db). The initial fura-2FF ratio increase in both the glutamate- and KCl-treated populations varied, but there is no significant difference between these two populations.