Gap junctions in hippocampal principal neurons.

Abstract

Gap junctions in principal neurons of the mammalian brain have been postulated to exist based on experimental and computer simulation studies. The subcellular location of these gap junctions have been suggested to be intersomatic, interdendritic and most recently between the axons of cortical principal neurons. However, definitive ultrastructural evidence for gap junctions in cortical principal neurons has been lacking. The development of methods such as confocal laser scanning microscopy (CLSM) and digital reconstruction of fluorescent dye-filled neurons in lightly perfusion-fixed brain tissue allow for the delineation of neuronal fine structure at near the limits of light microscopic resolution. We applied such methods to the study of CA3 pyramidal neurons in the adult rats. Using CLSM, in 4 out of 500 dye-loaded CA3 neurons (~1%), we found nonspiny processes contacting the proximal segment of CA3 principal neuron apical dendrites. These processes were not part of the injected neuron, but nevertheless filled with dye. Based on these dye coupling studies and the similarity of nonspiny processes to mossy fiber axons, we hypothesized that the mossy fiber synapses onto proximal dendrites of CA3 pyramidal neurons may support structures such as gap junctions, allowing the passage of low-molecular weight dyes from the CA3 pyramidal neurons to the mossy fiber axon. Using thin-section transmission electron microscopy (TEM) we found two classes of putative axodendritic gap junctions onto CA3 pyramidal dendrites. The first class was found between mossy fiber boutons and the thorny excrescences of CA3 pyramidal neurons, as was originally hypothesized based on our dye-coupling experiments. The second class of putative gap junctions was found between an inhibitory terminal and a positively identified CA3 pyramidal dendrite. We also found one example of a putative gap junction between a dendritic spine and another spiny dendrite in the CA3 region. While scanning hippocampal stratum lucidum under high magnification (20,000-30,000 ×) TEM, we found six close membrane appositions resembling gap junctions (~30-70 nm in diameter) between pairs of hippocampal mossy fiber axons (~100-200 nm in diameter) in the CA3b field of the adult rat ventral hippocampus. Using freeze-fracture replica immunogold labeling (FRIL) TEM, one axonal gap junction (~100 connexons) was found on a mossy fiber axon in the CA3c field of the adult rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data suggest that axodendritic synapses from both excitatory and inhibitory terminals onto CA3 pyramidal neurons are mixed (chemical and electrical) at least in some instances. They also support computer modeling and in vitro electrophysiological studies suggesting that axoaxonic gap junctions may play an important role in the generation of very fast (≻70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy.