Two-Photon Na+ Imaging Reports Somatically Evoked Action Potentials in Rat Olfactory Bulb Mitral and Granule Cell Neurites

Dendrodendritic synaptic interactions are a hallmark of neuronal processing in the vertebrate olfactory bulb. Many classes of olfactory bulb neurons including the principal mitral cells (MCs) and the axonless granule cells (GCs) dispose of highly efficient propagation of action potentials (AP) within their dendrites, from where they can release transmitter onto each other. So far, backpropagation in GC dendrites has been investigated indirectly via Ca2+ imaging. Here, we used two-photon Na+ imaging to directly report opening of voltage-gated sodium channels due to AP propagation in both cell types. To this end, neurons in acute slices from juvenile rat bulbs were filled with 1 mM SBFI via whole-cell patch-clamp. Calibration of SBFI signals revealed that a change in fluorescence Delta F/F by 10% corresponded to a Delta[Na+](i) of similar to 22 mM. We then imaged proximal axon segments of MCs during somatically evoked APs (sAP). While single sAPs were detectable in similar to 50% of axons, trains of 20 sAPs at 50 Hz always resulted in substantial Delta F/F of similar to 15% (similar to 33 mM Delta[Na+](i)). Delta F/F was significantly larger for 80 Hz vs. 50 Hz trains, and decayed with half-durations tau(1/2) similar to 0.6 s for both frequencies. In MC lateral dendrites, AP trains yielded small Delta F/F of similar to 3% (similar to 7 mM Delta[Na+](i)). In GC apical dendrites and adjacent spines, single sAPs were not detectable. Trains resulted in an average dendritic Delta F/F of 7% (16 mM Delta[Na+](i)) with tau(1/2) similar to 1 s, similar for 50 and 80 Hz. Na+ transients were indistinguishable between large GC spines and their adjacent dendrites. Cell-wise analysis revealed two classes of GCs with the first showing a decrease in Delta F/F along the dendrite with distance from the soma and the second an increase. These classes clustered with morphological parameters. Simulations of Delta[Na+](i) replicated these behaviors via negative and positive gradients in Na+ current density, assuming faithful AP backpropagation. Such specializations of dendritic excitability might confer specific temporal processing capabilities to bulbar principal cell-GC subnetworks. In conclusion, we show that Na+ imaging provides a valuable tool for characterizing AP invasion of MC axons and GC dendrites and spines

Local Postsynaptic Signaling on Slow Time Scales in Reciprocal Olfactory Bulb Granule Cell Spines Matches Asynchronous Release

In the vertebrate olfactory bulb (OB), axonless granule cells (GC) mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Locally triggered release of GABA from the large reciprocal GC spines occurs on both fast and slow time scales, possibly enabling parallel processing during olfactory perception. Here we investigate local mechanisms for asynchronous spine output. To reveal the temporal and spatial characteristics of postsynaptic ion transients, we imaged spine and adjacent dendrite Ca2+- and Na+-signals with minimal exogenous buffering by the respective fluorescent indicator dyes upon two-photon uncaging of DNI-glutamate in OB slices from juvenile rats. Both postsynaptic fluorescence signals decayed slowly, with average half durations in the spine head of t(1/2-)Delta[Ca2+](i) similar to 500 ms and t(1/2)_Delta[Na+](i) similar to 1,000 ms. We also analyzed the kinetics of already existing data of postsynaptic spine Ca2+-signals in response to glomerular stimulation in OB slices from adult mice, either WT or animals with partial GC glutamate receptor deletions (NMDAR: GluN1 subunit; AMPAR: GluA2 subunit). In a large subset of spines the fluorescence signal had a protracted rise time (average time to peak similar to 400 ms, range 20 to > 1,000 ms). This slow rise was independent of Ca2+ entry via NMDARs, since similarly slow signals occurred in Delta GluN1 GCs. Additional Ca2+ entry in Delta GluA2 GCs (with AMPARs rendered Ca2+-permeable), however, resulted in larger Delta F/Fs that rose yet more slowly. Thus GC spines appear to dispose of several local mechanisms to promote asynchronous GABA release, which are reflected in the time course of mitral/tufted cell recurrent inhibition