Dendritic integration in olfactory bulb granule cells: Thresholds for lateral inhibition and role of active conductances upon 3D multi-site photostimulation of spines using a holographic projector module

The inhibitory axonless olfactory bulb granule cells (OB GCs) form reciprocal dendrodendritic synapses with mitral and tufted cells (MCs and TCs) via large spines, mediating recurrent and lateral inhibition. Rat GC dendrites are excitable by local Na⁺ spine spikes and global Ca²⁺- and Na⁺-spikes. Since reaching global threshold potentials also represents the onset of lateral inhibition, the goal of my work was to investigate the exact transition from local to global signalling: How many spines, in which position and distribution on the dendritic tree have to be activated to trigger global spikes and what are the molecular key players, i.e. which ion channels are involved. In the first part of this study we have integrated a holographic projector into the existing commercial two-photon (2P) Galvanometer-based 2D laser scanning microscope with an uncaging unit (Uncaging: Activation of photolabile biologically inactive derivatives of neurotransmitters by photolysis), which allows the simultaneous photostimulation of several spines in three dimensions (3D) in acute brain slices. Patterned 2P photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron’s dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope’s built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume of interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 μm² within which a holographic VOI of 70×70×70 μm³ can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in APs. At the same time, it is possible to perform 2P Ca²⁺ imaging in 2D in the dendrite and thus to monitor synaptic Ca²⁺ entry in selected spines and also local regenerative events such as dendritic APs. In the second part of this study we applied the system to study dendritic integration in GCs. Less than 10 coactive reciprocal spines were sufficient to generate diverse regional and global signals that also included local dendritic Ca²⁺- and Na⁺-spikes (D-spikes). Individual spines could sense the respective signal transitions as increments in Ca²⁺ entry. Dendritic integration was mostly linear until a few spines below global Na⁺-spike threshold, where often D-spikes set in. NMDARs strongly contributed to active integration, whereas morphological parameters barely mattered. In summary, thresholds for GC-mediated bulbar lateral inhibition are low.