Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Abstract

Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.

Fig. 1.

Morphology of CA1 pyramidal cell apical dendrite spines and synapses is dependent on the laterality of presynaptic CA3 pyramidal cell. (A) Schematic illustration of left (blue) and right (red) CA1 pyramidal cells having NR2B dominant (dark green) and NR2B non-dominant (light green) synapses. The diagram was derived from Kawakami et al (3). (B) GFP-expressing lentivirus was injected unilaterally into the CA3 pyramidal cell layer (arrow in i). Axons and their terminals were heavily labeled for GFP in ipsilateral (ii) and contralateral (iii) CA1 SR. SP, stratum pyramidale. (C) GFP-labeled axon terminals were clearly observed by electron microscopy (Upper), and the spines making synapses (arrows) with labeled terminals were reconstructed using serial ultrathin sections (Lower). Reconstructed spines were classified as thin (i) or mushroom-type (ii) spines. Mushroom spines are defined as those with perforated PSDs (in red). (Scale bars, 300 nm.) (D) Cumulative percentile distributions of PSD area size (i) and spine head volume (ii). Those parameters were measured for the spines making synapses with ipsilateral (L → L, blue filled square, R → R, red filled circle) and contralateral (L → R, blue open triangle, R → L, red open triangle) projections in CA1. L, left; R, right. (E) Average PSD area (i), spine head volume (ii), and percentage of mushroom-type spines (iii) were calculated from three animals. Blue and red bars indicate left and right presynaptic origins, respectively. Statistically significant differences were detected between all combinations of red and blue bar data (mean ± SD; *, P < 0.05; **, P < 0.01). Error bars represent SD. (F) Asymmetrical morphology of CA1 pyramidal cell synapses. Left CA3-originated axons (blue) make synapses more frequently with small thin CA1 spines, whereas right CA3-originated axons (red) make synapses more frequently with large mushroom-type CA1 spines.

Fig. 2.

Asymmetrical expression of NR2B and GluR1 subunits in hippocampal synapses. (A) Conceptual diagram of VHC transected (VHCT) mice model, in which CA3 axons projecting to contralateral CA1 degenerate. The coloring scheme is the same as that in Fig. 1A. (B) Western blot analysis of NR2B and GluR1 subunits in PSD fractions prepared from the left and the right CA1 SR of VHCT or naïve mice. In VHCT mice, NR2B immunoreactivity was higher in the left than in the right, whereas that of GluR1 was higher in the right. The data were obtained from the same acrylamide gels. No asymmetry of GluR1 immunoreactivity was detected in naïve mice. (C) Left/right ratios of NR2B and GluR1 immuno-reactivities were measured in PSD fractions prepared from CA1 SR (n = 3). Ratios of optical densities (left/right) in VHCT mice significantly deviated from 1.0 and from those for GluR1 observed in naïve mice (n = 3 for each;**, P < 0.01, t test). Error bars represent SD. (D) No asymmetry of subunit expression was detected for NR2A, GluR2, and GluR3 in VHCT mice (n = 3).

Fig. 3.

Inverse correlation between NR2B and GluR1 densities in individual synapses. (A) Paired SDS-FRL from GluR1-dense (i) and NR2B-dense (ii) synapses in VHCT mice. Immuno-gold particles for GluR1 (in yellow) and NR1 (in black and arrows) were observed in E-face, and for NR2B (in green) were in P-face. (Scale bars, 100 nm.) (B) Inverse correlation between NR2B and GluR1 labeling densities in individual synapses in a naïve mouse. Synapses in the left and right sides are plotted in blue and red, respectively. (C) Labeling densities for NR2B and GluR1 subunits in VHCT mice. Data from two VHCT mice were pooled. Each mouse showed a significant inverse correlation between GluR1 and NR2B labeling (Rs = −0.597, P < 0.01; Rs = −0.438, P < 0.01).

Fig. 4.

Relationship between expression of synaptic GluR1/NR2B subunits and postsynaptic area as revealed by the post-embedding method. (A) Electron micrographs displaying post-embedding immuno-gold labeling for GluR1 (i) and NR2B (ii) in spine synapses (arrows) in CA1 SR. Note that gold particles for GluR1 are abundant in the large spine (a), whereas NR2B gold particles are more concentrated in the small spine (b) compared with the large spine (c). (B) Relationship between the number of gold particles for synaptic GluR1 (i, red) or NR2B (ii, blue) and postsynaptic area. The total area of each synapse was measured from serial sections. Data from two VHCT mice were pooled. (C) Relationship between the density of synaptic GluR1 (i, red) or NR2B (ii, blue) labeling and postsynaptic area. A significant positive correlation was seen between GluR1 density and postsynaptic area, whereas a hyperbolic (i.e., 1/x) fit described the relation well for NR2B.

Fig. 5.

Left-right asymmetry of hippocampal synapses with differential distributions of glutamate receptors. (A) Asymmetry of CA1 pyramidal cell synapses. Both left (blue) and right (red) CA1 pyramidal cells frequently make NR2B dense, small synapses on thin spines (green) with axon terminals from left CA3 pyramidal cells (blue) and GluR1 dense, large synapses on mushroom-type spines (yellow) with axon terminals from the right (red). (B) Relationship between glutamate receptor density and synaptic area. NR2B (blue) density is negatively correlated to the synapse area, resulting in relatively constant receptor expression regardless of synaptic size. GluR1 (red) density increases as a synapse becomes larger (shown in green to yellow). Expression of other subunits (yellow) is proportional to the synaptic area.