FURA-2 is a fluorescent dye used by neurophysiologists to measure the concentration of free Ca2+\text{Ca}^{2+}Ca2+ ions inside presynaptic neurones.
FURA-2 contains five negatively charged carboxylate groups (COO−\text{COO}^-COO−) and has a molecular mass of approximately 830 g mol−1830\text{ g mol}^{-1}830 g mol−1. Use this information to explain why FURA-2 is unable to cross cell membranes without chemical modification.
Scientists used FURA-2 to investigate the role of Ca2+\text{Ca}^{2+}Ca2+ ions in synaptic transmission at the Calyx of Held, a giant synapse in the mammalian brainstem. FURA-2 was microinjected into the presynaptic terminal, and its fluorescence was monitored using a laser scanning confocal microscope.
Explain one advantage of using a confocal microscope rather than a standard widefield light microscope to image synapses inside a thick brain tissue slice.
The presynaptic neurone was stimulated electrically at three different frequencies: Low, Medium, and High. At a Low stimulation frequency, the FURA-2 fluorescence ratio inside the presynaptic terminal was Low and the peak membrane potential of the postsynaptic neurone was −65 mV-65\text{ mV}−65 mV. At a Medium stimulation frequency, the fluorescence ratio was Medium and the peak potential was +35 mV+35\text{ mV}+35 mV. At a High stimulation frequency, the fluorescence ratio was High and the peak potential was +35 mV+35\text{ mV}+35 mV.
The fluorescence ratio of FURA-2 is directly proportional to the concentration of free Ca2+\text{Ca}^{2+}Ca2+ ions. The scientists concluded that an increase in the concentration of Ca2+\text{Ca}^{2+}Ca2+ ions in the presynaptic neurone causes an action potential in the postsynaptic neurone. Evaluate and explain the scientists' conclusion.
