Neuron Class and Target Variability in the Three-Dimensional Localization of SK2 Channels in Hippocampal Neurons as Detected by Immunogold FIB-SEM

Abstract

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Keywords: 3D-reconstruction; FIB/SEM; SK channels; dendritic shafts; electron microscopy; spines; synapses.