Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus

Abstract

Acetylcholine (ACh) is an important neurotransmitter in the mammalian brain; it is implicated in arousal, learning, and other cognitive functions. Recent studies indicate that nicotinic receptors contribute to these cholinergic effects, in addition to the established role of muscarinic receptors. In the hippocampus, where cholinergic involvement in learning and memory is particularly well documented, alpha7 nicotinic acetylcholine receptor subunits (alpha7 nAChRs) are highly expressed, but their precise ultrastructural localization has not been determined. Here, we describe the results of immunogold labeling of serial ultrathin sections through stratum radiatum of area CA1 in the rat. Using both anti-alpha7 nAChR immunolabeling and alpha-bungarotoxin binding, we find that alpha7 nAChRs are present at nearly all synapses in CA1 stratum radiatum, with immunolabeling present at both presynaptic and postsynaptic elements. Morphological considerations and double immunolabeling indicate that GABAergic as well as glutamatergic synapses bear alpha7 nAChRs, at densities approaching those observed for glutamate receptors in CA1 stratum radiatum. Postsynaptically, alpha7 nAChRs often are distributed at dendritic spines in a perisynaptic annulus. In the postsynaptic cytoplasm, immunolabeling is associated with spine apparatus and other membranous structures, suggesting that alpha7 nAChRs may undergo dynamic regulation, with insertion into the synapse and subsequent internalization. The widespread and substantial expression of alpha7 nAChRs at synapses in the hippocampus is consistent with an important role in mediating and/or modulating synaptic transmission, plasticity, and neurodegeneration.

Fig. 1.

Distribution of α7 nAChR-LIR in rat hippocampus.A, Low magnification micrograph of the hippocampal formation shows that all areas display α7 nAChR-LIR. The strongest immunoreactivity is present in the cell body layers (1, dentate gyrus; 2, CA3; 3, CA1) and in the molecular layer of the dentate gyrus (arrows). B, C, Higher magnification of the CA3 (B) and CA1 (C) regions show that the apical dendrites of the pyramidal cells (arrowheads) display α7 nAChR-LIR. Scale bar: A, 1 mm; B, C, 170 μm.

Fig. 2.

A, The specificity of the mitochondrial labeling seen with immuno-electron microscopy was addressed by Western blot analysis of hippocampal proteins. The monoclonal anti-α7 nAChR antibody M220 labels two bands in the homogenate and low-speed pellet, with molecular masses of 56 and 46 kDa (nAChRα7, i). The 56 kDa protein, which corresponds to the α7 nAChR subunit, is not present in the mitochondrial fraction (Mito). The 46 kDa immunoreactivity is sensitive to binding conditions and is not present when the binding buffer contains 3% nonfat milk powder (nAChRα7, ii). The mitochondrial protein Hsp-60 is enriched in the mitochondrial fraction.B, Electron micrographs of the mitochondrial fraction tested in A demonstrate that these organelles were highly enriched in the pellet (arrowheads).C–E, Double labeling of different epitopes of α7 nAChR with the monoclonal antibody M220 (5 nm particles) and a polyclonal antiserum SC5544 (10 nm particles) demonstrates that both reagents label synaptic sites (∗) and are often colocalized (arrows). Scale bar: B, 370 nm; C–E, 280 nm.

Fig. 3.

A–C, Light microscopic double labeling for αBgtx (green) and synaptophysin (red) demonstrates the abundance and substantial correspondence of both labeled sites. D, Higher magnification of the area outlined in C reveals the predominant colocalization (arrow) or close apposition of both labeled sites. A small proportion of synaptophysin-labeled sites (double arrowhead) and a fraction of αBgtx-labeled sites (arrowhead) show no colocalization. Scale bar: A–C, 130 μm; D, 30 μm.

Fig. 4.

Electron micrographs of the CA1 stratum radiatum area in αBgtx-labeled intact brain preparations.A–E, Labeling for αBgtx is present at presynaptic (arrowheads) and postsynaptic sites (arrows). b, Presynaptic boutons;s, dendritic spines. Scale bar, 200 nm.

Fig. 5.

Electron micrographs of the CA1 stratum radiatum region in anti-α7 nAChR-labeled intact brain preparations.A–F, Most synaptic contacts contain numerous gold particles at synaptic membranes (arrowheads). Gold particles are also found at presynaptic vesicles (small arrow) and attached to membranous structures in the postsynaptic cytoplasm (double arrowheads). Inset, Gold particles were often found at nonsynaptic membranes at positions corresponding to synapses in adjacent sections (arrowheads), indicating a perisynaptic localization of the α7 nAChR subunits. b, Presynaptic boutons; s, dendritic spines. Scale bar:A–F, 200 nm; inset, 90 nm.

Fig. 6.

Electron micrographs of serial sections through anti-α7 nAChR labeled synaptic contacts in CA1 stratum radiatum.A–I, Some synaptic contacts show persistent labeling at synaptic membranes throughout all serial sections (A–C,D–F, arrowheads), whereas others lack synaptic labeling in at least one of the sections (G–I). Labeling is found at presynaptic vesicles (double arrowheads) and at vesicular structures in the postsynaptic cytoplasm (arrows). b, Presynaptic boutons;s, postsynaptic spines. Scale bar:A–I, 250 nm.

Fig. 7.

Quantitative analysis of CA1 stratum radiatum synapses treated with a monoclonal anti-α7 nAChR antibody.A, Frequency histogram of total number of gold particles lying over the synaptic membrane, measured over three serial sections through each synaptic profile. B, Scatter plot of total number of gold particles lying over the synaptic membrane versus total sampled area of the synapse for each of the 158 synapses investigated. The particle number is weakly but significantly correlated with synaptic area (solid line, linear regression slope; slope significantly different from zero, p < 0.0001; correlation coefficient r = 0.372;dotted lines, 95% confidence limits; runs test for deviation from linearity, p = 0.689, not significant.). C, Frequency histogram of gold particles over background areas equivalent to those measured for synapses, demonstrating that background areas were largely devoid of labeling. Beneath each bar of the histogram, the top number indicates the number of gold particles per area, and thebottom number indicates the number of areas represented by the individual bars.

Fig. 8.

Electron micrographs through anti-α7 nAChR-immunolabeled synapses. A, B, Synapses showing gold particles at the synaptic membranes (arrowhead), presynaptic vesicles (double arrowhead), and postsynaptic stacked membranous structures resembling spine apparatus (arrows). b, Presynaptic boutons; s, postsynaptic spines. Scale bar:A, B, 200 nm.

Fig. 9.

Glutamate/α7 nAChR double labeling at CA1 stratum radiatum synapses. A, A glutamate-like immunoreactive synaptic terminal (1,arrow, 10 nm particles) contacts a postsynaptic spine (s). The synaptic membranes show α7 nAChR-LIR (arrowhead, 5 nm particles).B, A presynaptic terminal in a glutamate-labeled preparation (2) reveals no glutamate labeling at presynaptically located vesicles (arrow). The synaptic membranes, however, show labeling for α7 nAChR (arrowhead, 5 nm particles). Scale bar (shown inB for A and B): 190 nm.

Fig. 10.

GABA/α7 nAChR double labeling at CA1 stratum radiatum synapses. A, B, GABA-LIR profiles (∗) with numerous 10 nm particles (arrows) show also α7 nAChR-LIR at synaptic membranes (arrowheads, 5 nm particles). C, Some GABA-LIR profiles showed no α7 nAChR-LIR. Star, GABA-immunonegative presynaptic profile. Scale bar (shown inC for A–C): 240 nm.