Mixed Electrical–Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36

Farid Hamzei-Sichani,Roger D. Traub,Kimberly G. V. Davidson,John E. Rash,Patrick R. Hof,Ole P. Ottersen,Rafael Gutiérrez,William G. M. Janssen,Thomas Yasumura,Susan L. Wearne

doi:10.3389/fnana.2012.00013

Abstract

Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for “mixed” (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328–1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.

Highlights

Electrical coupling and connexin-36 (Cx36)-containing gap junctions have been demonstrated between interneuronal dendrites in other regions of neocortex and olfactory bulb (Rash et al, 2005; Fukuda et al, 2006), neither electrical coupling nor the connexin composition of neuronal gap junctions has been adequately examined in hippocampus between mossy fiber (MF) terminals and CA3 pyramidal neurons (CA3pyr), between glutamatergic interneurons, between glutamatergic interneurons and principal cells, or between granule cells and their innervating axons of the perforant path
During image analysis, Lucifer Yellow (LY) was occasionally noted to reveal apparent CA3pyr-to-MF dye-coupling, apparently at MF axon contact areas within thorny excrescences (Figure 1), as we later confirmed by thin-section transmission electron microscopic (tsTEM) of conventionally fixed samples (Figure 2)
The dye-coupled MF axons established direct contacts with dye-injected CA3pyr dendrites (Figure 1B) or with their smaller spine-covered dendrites (Figure 1E, lower left arrowhead), potentially revealing the sites for gap junctional dye transfer. [Note: As in CA1pyr (Megías et al, 2001), the proximal 10–20 μm of CA3pyr dendrites are essentially devoid of spines (Figure 1A inset, yellow brackets), providing a potential confounding factor in the fracture replica immunogold labeling (FRIL) identification of mixed synapses on proximal dendritic shafts of CA3pyr vs. aspiny interneurons.] Overall, the relatively low frequency of dye transfer detected may reflect the use of the classical dye, LY (457 mw), which is slightly above or very near the cutoff molecular weight for gap junction-permanent dyes in vertebrate species

Summary

Introduction

Electrical synaptic transmission and the ultrastructural correlate of electrical synapses – gap junctions – are accepted featuresof neuronal circuitry in many areas of the mammalian central nervous system (CNS; Bennett and Zukin, 2004; Hormuzdi et al, 2004). Electrical coupling and connexin-36 (Cx36)-containing gap junctions have been demonstrated between interneuronal dendrites in other regions of neocortex and olfactory bulb (Rash et al, 2005; Fukuda et al, 2006), neither electrical coupling nor the connexin composition of neuronal gap junctions has been adequately examined in hippocampus between MF terminals and CA3pyr, between glutamatergic interneurons, between glutamatergic interneurons and principal cells, or between granule cells and their innervating axons of the perforant path This pathway, which arises from the entorhinal cortex, forms the major input to the DG, which receives no extrinsic inputs from other cortical structures (Do et al, 2002). Because evidence indicates that CA3pyr synthesize Cx36 (Belluardo et al, 2000), and presumably form gap junctions, local recurrent collaterals of CA3pyr may provide additional sources of glutamatergic mixed synapses

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