Visualising the role of presynaptic calcium in the cerebellar cortex using a novel transgenic mouse

Evidence suggests that CB1Rs and NMDARs may be expressed at PF presynaptic terminals and that these receptors may modulate synaptic transmission at synapses formed with cerebellar PCs. Presynaptic effects of receptors and signalling pathways are hard to establish because synaptic terminals are generally too small to allow direct electrical recording. We have explored the use of a transgenic mouse (SyG37) that expresses a ratiometric calcium indicator selectively in presynaptic terminals. Using a combination of fEPSP recordings, fluorescence measurements of presynaptic calcium and pharmacological manipulation, we have explored the use of SyG37 mice for measuring presynaptic activity and synaptic modulation in the cerebellar cortex. We first established that this sensor was widely expressed in the cerebellum but crucially within PF terminals. In response to different patterns of electrical stimulation, calcium dependent responses were reliably detected within ensembles and even single presynaptic boutons. The effects of changing the intensity, frequency and number of stimuli to PF terminals were studied. SyGCaMP2 responses were calcium dependent and inhibited by application of TTX. Direct activation of the GCL produced responses that propagated directly above the point of stimulation through ascending axons. Signals then bifurcated to spread in either direction along PFs. Direct activation of the ML produced responses that propagated along PFs. Activation of NMDARs reduced synaptic transmission through a direct effect on PF terminals since the effect was maintained when all excitatory and inhibitory synaptic transmission was blocked. This effect was due to a decrease in the number of presynaptic boutons that responded to stimulation. Activation of CB1Rs also produced a reduction in synaptic activity that remained when inhibitory and excitatory synaptic transmission was blocked, also suggesting a direct effect on PF terminals. In this case, both the number of boutons responding to stimulation and the amount of calcium entering single terminals was reduced.