PirB regulates asymmetries in hippocampal circuitry

Left-right asymmetry is a fundamental feature of higher-order brain structure; however, the molecular basis of brain asymmetry remains unclear. We recently identified structural and functional asymmetries in mouse hippocampal circuitry that result from the asymmetrical distribution of two distinct populations of pyramidal cell synapses that differ in the density of the NMDA receptor subunit GluRε2 (also known as NR2B, GRIN2B or GluN2B). By examining the synaptic distribution of ε2 subunits, we previously found that β2-microglobulin-deficient mice, which lack cell surface expression of the vast majority of major histocompatibility complex class I (MHCI) proteins, do not exhibit circuit asymmetry. In the present study, we conducted electrophysiological and anatomical analyses on the hippocampal circuitry of mice with a knockout of the paired immunoglobulin-like receptor B (PirB), an MHCI receptor. As in β2-microglobulin-deficient mice, the PirB-deficient hippocampus lacked circuit asymmetries. This finding that MHCI loss-of-function mice and PirB knockout mice have identical phenotypes suggests that MHCI signals that produce hippocampal asymmetries are transduced through PirB. Our results provide evidence for a critical role of the MHCI/PirB signaling system in the generation of asymmetries in hippocampal circuitry

Laterality defects in hippocampal synapse morphology in PirB KO and <i>iv</i> mice.

<p>Postsynaptic density (PSD) area (A) and percentage of perforated synapses (B) were compared between left and right CA1 pyramidal cell synapses in the PirB KO and <i>iv</i> mice. No significant difference in these parameters between the left and right hippocampus was observed in either PirB KO or <i>iv</i> mice, whereas significant differences were found between PirB KO and <i>iv</i> hippocampi. Error bars represent s.e.m. An asterisk indicates <i>P</i> < 0.05; absence of an asterisk indicates <i>P</i> > 0.05.</p

Comparison of the concentration dependency of Ro 25–6981 inhibition of NMDA EPSCs in hippocampal synapses of PirB KO and <i>iv</i> mice.

<p>Schematic diagrams showing synaptic inputs onto the apical and basal dendrites of CA1 pyramidal neurons and the arrangement of electrodes. In hippocampal slices prepared from naïve mice, whole-cell recordings [Rec. (WC)] were made from CA1 pyramidal neurons. A stimulating electrode was placed in the stratum radiatum [Stim. (SR)] or the stratum oriens [Stim. (SO)] of the CA1 to activate apical or basal synapses, respectively. Sch, Schaffer collateral fibers; Com, commissural fibers. NMDA EPSCs were recorded at a holding potential of +10 mV. Relative amplitudes of NMDA EPSCs in the presence of several concentrations of Ro 25–6981 are expressed as percentages of control responses (mean ± SEM). Filled and open circles represent basal [iv (basal)] and apical [iv (apical)] synapses (<i>n</i> = 7 each), respectively, of CA1 pyramidal neurons in the <i>iv</i> mouse hippocampus. Open triangles represent CA1 apical synapses in the PirB KO hippocampus (<i>n</i> = 10 each).</p

Relationship between synaptic plasticity and stimulation frequency in hippocampal synapses of PirB KO and <i>iv</i> mice.

<p>(A) Schematic diagrams of the arrangement of electrodes for extracellular recording. Using hippocampal slices prepared from naïve mice, fEPSPs were recorded with an extracellular electrode [Rec. (field)] placed either in the stratum radiatum or stratum oriens of the CA1. To activate apical (Apical) or basal (Basal) dendritic synapses, a stimulating electrode was placed in the stratum radiatum [Stim. (SR)] or stratum oriens [Stim. (SO)], respectively. (B) Tetanic stimulation (100 Hz for 1 s, 3 trains, interval of 10 s) applied at time 0 (arrow) elicited LTPs of the fEPSP slope in hippocampal slices from both PirB KO and <i>iv</i> mice. Filled and open circles represent, respectively, basal [iv (basal), <i>n</i> = 7] and apical [iv (apical), <i>n</i> = 7] synapses of CA1 pyramidal neurons in the <i>iv</i> mouse hippocampus. Open triangles represent CA1 apical synapses in the PirB KO hippocampus (<i>n</i> = 10). Symbols and error bars represent means and SEM, respectively. The upper superimposed traces show representative fEPSPs recorded before (1) or 40 min after (2) tetanic stimulation. Scale bars: 1.0 mV (vertical) and 10 ms (horizontal). (C) Low-frequency stimulation (1 Hz for 15 min, thick bar) induced LTD in apical synapses of the <i>iv</i> mouse hippocampus (open circles, <i>n</i> = 7), but not in basal synapses of the <i>iv</i> mouse hippocampus (filled circles, <i>n</i> = 7) or in PirB KO synapses (open triangles, <i>n</i> = 10). The upper superimposed traces are representative fEPSPs recorded before (1) or 40 min after (2) low-frequency stimulation. Scale bars: 1.0 mV (vertical) and 10 ms (horizontal). (D) Stimulation frequency dependency of synaptic plasticity. Relative amplitudes of fEPSP slopes, estimated 40 min after tetanus, were plotted against stimulation frequency (mean ± SEM). Points at 0.1 Hz (test pulse frequency) indicate baseline values (horizontal dashed line). Symbols are the same as those in (B) and (C). *<i>P</i> < 0.05.</p

Hippocampal asymmetry and abnormalities in the <i>iv</i> and β2m KO mice.

<p>Left and right CA3 pyramidal neurons and their axons are colored red and blue, respectively. A postsynaptic CA1 pyramidal neuron is in the center, outlined in black, and it represents postsynaptic neurons in both left and right hemispheres. Closed and open circles represent ε2-dominant and ε2-non-dominant synapses, respectively. Apical, apical dendrites; Basal, basal dendrites; WT, wild-type; KO, knockout.</p

Asymmetry defects in the PirB KO mouse hippocampus.

<p>A postsynaptic CA1 pyramidal neuron is in the center, outlined in black, and it represents postsynaptic neurons in both left and right hemispheres. Left and right CA3 pyramidal neurons and their axons are colored red and blue, respectively. Filled circles represent ε2-dominant synapses. Note that ε2-non-dominant synapses are absent and circuit asymmetry is lost in the PirB KO hippocampus. This asymmetry defect is the same as that in the β2m KO hippocampus (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179377#pone.0179377.g001" target="_blank">Fig 1</a>).</p

Inhibitory effect of Ro 25–6981 on NMDA EPSCs at CA1 pyramidal neuron synapses.

<p>(Aa) Schematic diagrams showing synaptic inputs onto the apical dendrites of CA1 pyramidal neurons and the positioning of electrodes. In slices from naïve and VHCT mice, electrical stimuli applied at the stratum radiatum [Stim. (SR)] of area CA1 selectively activated apical synapses. Whole-cell recordings [Rec. (WC)] were made from CA1 pyramidal neurons. Sch, Schaffer collateral fibers; Com, commissural fibers. (Ab) Effects of Ro 25–6981 on NMDA EPSCs at CA1 apical synapses in the PirB KO hippocampus. Representative superimposed traces indicate NMDA EPSCs recorded in the absence (Control) or presence of Ro 25–6981 (Ro, 0.6 μM). The levels of inhibition were maximal after exposure to Ro 25–6981 for 50 to 60 min. “Left” and “Right” indicate recordings from left and right hippocampal slices, respectively. Each trace is the average of five consecutive recordings. Scale bars: 25 pA (vertical) and 100 ms (horizontal). Relative amplitudes of NMDA EPSCs in the presence of Ro 25–6981 are expressed as percentages of control responses. Error bars represent SEM (<i>n</i> = 7 each; <i>P</i> > 0.05 for all combinations, Student’s <i>t</i>-test). (Ac) Effects of Ro 25–6981 on NMDA EPSCs at CA1 apical synapses in the WT hippocampus. Relative amplitudes of NMDA EPSCs in the presence of Ro 25–6981 are expressed as percentages of control responses (mean ± SEM; <i>n</i> = 7 each; *<i>P</i> < 0.05; absence of an asterisk indicates <i>P</i> > 0.05). (Ba) Schematic diagrams showing synaptic inputs onto the basal dendrites of CA1 pyramidal neurons and the positioning of electrodes. Whole-cell recordings [Rec. (WC)] were made from CA1 pyramidal neurons. To activate basal synapses, electrical stimuli were applied at the stratum oriens [Stim. (SO)] of area CA1. (Bb) Effects of Ro 25–6981 on NMDA EPSCs at CA1 basal synapses in the PirB KO hippocampus. Representative superimposed traces indicate NMDA EPSCs recorded in the absence (Control) or presence of Ro 25–6981 (Ro, 0.6 μM). “Left” and “Right” indicate recordings from left and right hippocampal slices, respectively. Scale bars: 25 pA (vertical) and 100 ms (horizontal). Relative amplitudes of NMDA EPSCs in the presence of Ro 25–6981 are expressed as percentages of control responses (mean ± SEM; <i>n</i> = 7 each; <i>P</i> > 0.05 for all combinations). (Bc) Effects of Ro 25–6981 on NMDA EPSCs at CA1 basal synapses in the WT hippocampus. Relative amplitudes of NMDA EPSCs in the presence of Ro 25–6981 are expressed as percentages of control responses (mean ± SEM; <i>n</i> = 7 each; *<i>P</i> < 0.05; absence of an asterisk indicates <i>P</i> > 0.05).</p

Targeted disruption of the <i>Pirb</i> gene does not alter basal synaptic transmission.

<p>(A) Schematic diagrams showing the arrangement of electrodes for extracellular recording. Using hippocampal slices prepared from naïve mice, fEPSPs were recorded with an extracellular electrode [Rec. (field)] placed in the stratum radiatum of area CA1. To activate apical synapses, a stimulating electrode was placed in the stratum radiatum of area CA1 [Stim. (SR)]. Sch, Schaffer collateral fibers; Com, commissural fibers. (B) Input–output relationship of fEPSP amplitude as a function of PFV amplitude in PirB KO mice (<i>n</i> = 8) and WT mice (<i>n</i> = 8). Inset traces show typical fEPSPs obtained with various stimulus intensities. Scale bars: 0.5 mV (vertical) and 5 ms (horizontal). (C) PPF induced by pairs of stimulus pulses delivered at interpulse intervals of 50 ms. Each trace is the average of three consecutive recordings. Scale bar: 0.5 mV (vertical) and 25 ms (horizontal). PPFs are expressed as the ratio of the second fEPSP divided by the first fEPSP (mean ± SEM; <i>n</i> = 11 each; <i>P</i> > 0.05, Student’s <i>t</i>-test).</p

Effect of PirB deficiency on the amplitude of NMDA EPSCs evoked in CA1 pyramidal neuron synapses.

<p>Schematic diagrams showing the arrangement of electrodes for recording. A stimulating electrode was placed in the stratum radiatum [Stim. (SR)] or the stratum oriens [Stim. (SO)] of the CA1 to activate apical or basal synapses, respectively. Whole-cell patch recordings [Rec. (WC)] were made from CA1 pyramidal neurons. Sample superimposed traces of representative EPSCs recorded in hippocampal slices prepared from PirB KO and WT mice. The top traces show NMDA EPSCs at +30 mV in the presence of DNQX and bicuculline. The bottom traces show non-NMDA EPSCs at −90 mV in the presence of bicuculline. Each trace was averaged from five consecutive recordings. Scale bars: 50 pA (vertical) and 100 ms (horizontal). Relative amplitudes of NMDA EPSCs are expressed as percentages of non-NMDA EPSCs. Columns and error bars represent means and SEM, respectively (<i>n</i> = 7 each; <i>P</i> > 0.05, <i>t</i>-test). Api, apical synapses; Bas, basal synapses.</p

Inhibitory effects of Ro 25–6981 on NMDA EPSCs in the basal dendrites of CA3 pyramidal neurons.

<p>Schematic diagrams showing synaptic inputs onto the basal dendrites of CA3 pyramidal neurons in the PirB KO and WT mice. In slices from naïve mice, whole-cell recordings [Rec. (WC)] were made from CA3 pyramidal neurons. A stimulating electrode was placed in the ventral fimbria [Stim. (Fim)] to activate commissural fibers (Com). Representative superimposed traces indicate NMDA EPSCs recorded in the absence (Control) or presence of Ro 25–6981 (Ro, 0.6 μM). “Left” and “Right” indicate recordings from left and right hippocampal slices, respectively. Each trace is the average of five consecutive recordings. Scale bars: 25 pA (vertical) and 200 ms (horizontal). Relative amplitudes of NMDA EPSCs in the presence of Ro 25–6981 are expressed as percentages of control responses (mean ± SEM; <i>n</i> = 7 each; *<i>P</i> < 0.05; absence of an asterisk indicates <i>P</i> > 0.05).</p