Detecting synaptic connections in the medial nucleus of the trapezoid body using calcium imaging

The study of synaptic transmission in brain slices generally entails the patch-clamping of postsynaptic neurones and stimulation of identified presynaptic axons using a remote electrical stimulating electrode. Although patch recording from postsynaptic neurones is routine, many presynaptic axons take tortuous turns and are severed in the slicing procedure, blocking propagation of the action potential to the synaptic terminal and preventing synaptic stimulation. Here we demonstrate a method of using calcium imaging to select postsynaptic cells with functional synaptic inputs prior to patch-clamp recording. We have used this method for exploring transmission in the auditory brainstem at the medial nucleus of the trapezoid body neurones, which are innervated by axons from the contralateral cochlear nucleus. Brainstem slices were briefly loaded with the calcium indicator fura-2 AM and stimulated with an electrode placed on the midline. Electrical stimulation caused a rise in intracellular calcium concentration in those postsynaptic neurones with active synaptic connections. Since <10% of the medial nucleus of the trapezoid body neurones retain viable synaptic inputs following the slicing procedure, preselecting those cells with active synapses dramatically increased our recording success. This detection method will greatly ease the study of synaptic responses in brain areas where suprathreshold synaptic inputs occur but connectivity is sparse.