Right Isomerism of the Brain in Inversus Viscerum Mutant Mice

Left-right (L-R) asymmetry is a fundamental feature of higher-order neural function. However, the molecular basis of brain asymmetry remains unclear. We recently reported L-R asymmetry of hippocampal circuitry caused by differential allocation of N-methyl-D-aspartate receptor (NMDAR) subunit GluRε2 (NR2B) in hippocampal synapses. Using electrophysiology and immunocytochemistry, here we analyzed the hippocampal circuitry of the inversus viscerum (iv) mouse that has a randomized laterality of internal organs. The iv mouse hippocampus lacks L-R asymmetry, it exhibits right isomerism in the synaptic distribution of the ε2 subunit, irrespective of the laterality of visceral organs. This independent right isomerism of the hippocampus is the first evidence that a distinct mechanism downstream of the iv mutation generates brain asymmetry.


Introduction
Previously we demonstrated that the synaptic distribution of NMDAR e2 subunits in the adult mouse hippocampus is asymmetrical between apical and basal dendrites of individual neurons and between the left and right hemispheres [1,2]. These asymmetrical allocations of e2 subunits affect the properties of NMDARs in hippocampal synapses and generate two populations of synapses. The NMDAR-mediated excitatory postsynaptic currents (NMDA EPSCs) of the 'e2-dominant' population are highly sensitive to Ro 25-6981, a e2 subunit-selective antagonist [3][4][5]. The plasticity of these synapses develops rather early. The other population is 'e2-nondominant'. The NMDA EPSCs of these synapses are less sensitive to low concentrations of Ro 25-6981 and the plasticity of these synapses develops slowly. These two populations of synapses are distributed asymmetrically between the left and right hippocampus. These findings represent the first example of L-R asymmetries in synaptic composition and the function of neuronal networks within the brain.
The precise specification of L-R asymmetry is essential for vertebrate development. The molecular mechanisms that generate asymmetrical patterning are well understood for the internal organs [6][7][8] but not for the brain. To investigate the molecular basis of the formation of brain asymmetry, we examined the asymmetry of the distribution of e2 subunits in the iv mouse [9]. Iv is a spontaneous mouse mutant that possesses a mutation in the gene encoding the motor protein, Left-right dynein (Lrd) [10]. In embryos 7.5 days postcoitum, the leftward nodal flow generated by the rotation of cilia initiates the L-R axis determination process [11,12]. However, in iv homozygous (iv/iv) embryos, the nodal cilia are immotile and fail to produce constant leftward flow, resulting in a randomized laterality of visceral organs [13][14][15]. Fifty percent of iv/iv mice exhibit reversed asymmetry (situs inversus), whereas the rest are normal (situs solitus).
Here, we report that the iv mouse hippocampus exhibits right isomerism of the synaptic distribution of the e2 subunit. This laterality defect of the hippocampus is independent of the laterality of visceral organs. Therefore, the mechanisms responsible for the specification of L-R asymmetry differ between visceral organs and the brain.

Properties of NMDA EPSCs of hippocampal CA1 pyramidal neurons
First we examined properties of NMDARs in mice heterozygous for iv (iv/+). The nodal cilia rotate as rapidly in these heterozygotes as in wild-type mice (WT) [15] and the laterality of visceral organs develops normally [9]. To measure NMDA EPSCs, whole-cell recordings were made from CA1 pyramidal neurons in the presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX, 20 mM) and bicuculline (30 mM) at a holding potential of +10 mV [1]. To discriminate between excitatory synapses on the apical and basal dendrites of CA1 pyramidal neurons, NMDA EPSCs were independently elicited by electrical stimuli applied either at the stratum radiatum or at the stratum oriens of area CA1 ( Figure 1A). In naïve iv/+ mice, Ro 25-6981 (0.6 mM) reduced peak amplitude of NMDA EPSCs to a similar extent in the left and right hippocampus in both the basal and apical dendrites of CA1 pyramidal neurons (Basal; left, 58.763% of control, n = 7 from 7 animals; right, 6162% of control, n = 7 from 7 animals; Apical; right, 6463% of control, n = 7 from 7 animals; left, 5963% of control, n = 7 from 7 animals)( Figure 1B).
Next we examined hippocampal slices prepared from mice in which the ventral hippocampal commissure had been transected (VHCT). These mice lack commissural fibers [1]. Five days after VHCT, Ro 25-6981 sensitivity of CA1 pyramidal neurons was asymmetrical. In basal synapses, Ro 25-6981 diminished NMDA EPSCs more in right hippocampal slices than in left hippocampal slices (left, 7263% of control, n = 7 from 7 animals; right, 4163% of control, n = 7 from 7 animals; P,0.01, t-test)(Basal, Figure 1C). Conversely, in apical synapses, hippocampal slices from the left exhibited a greater sensitivity to Ro 25-6981 compared to the right (right, 7664% of control, n = 7 from 7 animals; left, 3864% of control, n = 7 from 7 animals; P,0.01)(Apical, Figure 1C). These results in iv/+ hippocampus are similar to those previously reported for WT hippocampus [1]. Thus, the laterality of hippocampal circuitry develops normally in iv/+ mice.

Development of NMDAR-dependent synaptic plasticity
To characterize the features of synapses in iv/iv mice that show the high and low sensitivity to Ro 25-6981, we analyzed the development of long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs). We measured the amplitude of LTP in 7-to 9-week-old adult mice and compared it with LTP in 9-to 11day-old pups. The e2 subunit is the major e subunit in the hippocampus at early postnatal ages [16,17]. We examined slices from the left hippocampus in this experiment. fEPSPs were recorded with an extracellular electrode placed either in the stratum oriens or in the stratum radiatum of area CA1 (Figure 2). In basal synapses, the amplitudes of hippocampal LTP were similar between pups and adults (pups, 17463%, n = 7 from 7 animals; adults, 18963%, n = 9 from 9 animals, P.0.05) (Basal, Figure 2). In contrast, in apical synapses, the amplitudes of hippocampal LTP were greater in adults than pups (pups, 13265%, n = 7 from 7 animals; adults, 19264%; n = 9 from 9 animals, P,0.01) (Apical, Figure 2). Thus, LTP of Ro 25-6981 high-sensitivity synapses developed earlier than LTP in Ro 25-6981 low-sensitivity synapses. These differences in synaptic function are likely the consequence of the differential distribution of e2 subunits at these two populations of synapses.

Immunocytochemical analysis of the synaptic distribution of the e2 subunit
To investigate directly the synaptic distribution of e2 subunits, we compared postembedding immunogold labeling for e2 subunit between the apical and basal dendrites of CA1 pyramidal neurons. We examined 9-day-old mice in this experiment because the expression of e1 subunits in the hippocampus at this age is absent or very low, whereas the e2 subunit is already expressed at high levels [16,17]. Therefore, at this stage of development, we consider the number of functional NMDARs to be proportional to the number of e2 subunits at each synapse. Thus, immunogold labeling reflects both the e2 subunit content and the number of functional NMDARs [2] ( Figure 3A). The labeling density for the e2 subunit in iv/iv mice was higher in the stratum oriens than in the stratum radiatum bilaterally (iv/iv, left, oriens/radiatum ratio = 1.5060.1, n = 3 animals, P = 0.04; iv/iv, right, oriens/radiatum ratio = 1.5960.2, n = 3 animals, P = 0.08) ( Figure 3B & D), whereas no significant difference was detected in iv/+ mice (iv/+, right, oriens/radiatum ratio = 0.9760.1, n = 3 animals, P.0.10) (Figure 3C & D).  in iv/iv and iv/+ mice at postnatal day 9. This distribution is significantly different (Kolmogorov-Smirnov test, p,0.05) between the stratum oriens and the stratum radiatum in iv/iv but not in iv/+ mice. (D) Averaged ratios (stratum oriens/stratum radiatum) of e2 particle density at pyramidal synapses (n = 3 each, t-test *P = 0.08 **P = 0.04, absence of an asterisk indicates P.0.10). doi:10.1371/journal.pone.0001945.g003

Discussion
Two distinct populations of synapses in the iv mouse hippocampus The adult WT hippocampus contains two populations of synapses with distinct distributions of e2 subunits [1,2]. The NMDA EPSCs of the 'e2-dominant' population are highly sensitive to Ro 25-6981 and LTP of these synapses develops rather early. In contrast, the NMDA EPSCs of the 'e2nondominant' population are less sensitive to Ro 25-6981 and LTP of these synapses develops slowly. In this study, we discover that the iv mouse hippocampus also contains two separable populations of synapses on apical and basal dendrites of CA1 pyramidal neurons. These two populations of synapses have complementary properties. First, the NMDA EPSCs of basal synapses exhibit a higher sensitivity to Ro 25-6981 than those of apical synapses (Figure 1). Second, LTP of basal synapses develops earlier than that of apical synapses ( Figure 2). Third, the concentration of the e2 subunits, examined in the hippocampus of neonates, is higher in basal synapses than in apical synapses ( Figure 3). The distinct Ro 25-6981 sensitivity of NMDA EPSCs in adult hippocampal synapses indicates that differential distribution of the e2 subunit is maintained until adulthood. On the basis of these findings, we conclude that the iv mouse hippocampus also contains e2-dominant and e2-nondominant synapses and their functional properties are very similar to those in WT mice.
The developmental difference observed in LTP can be explained by the difference in the number of functional NMDA receptors at these synapses. In early postnatal animals such as 9-to 11-day-old mice, the expression of e1 subunits in the hippocampus is absent or very low, whereas the e2 and f1 subunits are already expressed at high levels [16,17]. In this circumstance, the differential allocation of e2 subunits produces distinct numbers of NMDA receptors in these synapses, resulting in differential ability to express synaptic plasticity [1,2]. Therefore, LTP of the e2-dominant basal synapses develops earlier than that of the e2nondominant apical synapses ( Figure 2).

Laterality defects of the iv mouse hippocampus
In WT mice, the localization of e2-doninant and e2-nondominant synapses within hippocampal circuitry is asymmetrical (Figure 4, WT) [1,2]. Inputs from CA3 pyramidal neurons residing in the hippocampus of the left hemisphere form e2-dominant synapses on the apical dendrites and e2-nondominant synapses on the basal dendrites of CA1 pyramidal neurons in both hemispheres. In contrast, inputs from CA3 pyramidal neurons in the right hippocampus form e2-dominant synapses on the basal dendrites and e2-nondominant synapses on the apical dendrites of CA1 pyramidal neurons. However, unlike WT hippocampus, the iv/iv hippocampus lacks L-R asymmetry in localization of these two populations of synapses (Figure 4, iv/iv). In iv/iv mice, synapses formed on the apical and basal dendrites of CA1 pyramidal neurons are e2-nondominant and e2-dominant, respectively. This localization is bilateral and independent of the laterality of visceral organs.
Even more surprisingly, this laterality defect seems to be characterized by right isomerism of the iv/iv hippocampus because CA3 neurons in both hemispheres form e2-dominant and e2nondominant synapses on the basal and apical dendrites, respectively (Figure 4, iv/iv), similar to CA3 neurons in the WT right hemisphere (Figure 4, WT). Thus, it appears as if the 'left' hippocampus is lost and both hemispheres contain a 'right' hippocampus. Thus, we conclude that the iv mouse hippocampus exhibits right isomerism with regard to the synaptic distribution of the e2 subunit.
Distinct mechanisms generating L-R asymmetry of the brain Studies of early murine development reveal that embryos are initially bilaterally symmetric. Subsequently, the symmetry-breaking process in L-R determination is initiated by the leftward nodal flow on the surface of the ventral node, so that the cells in the left side of embryo adopt properties different from the right side [6][7][8]10]. That is, the default properties are those of the right side. Although the precise mechanisms for generating the right isomerism of the iv/iv mouse hippocampus remain elusive, one possible explanation is that the iv/iv mouse brain lacks the expression of factors that distinguish the left hemisphere and thus neurons retain the default characteristics of the 'right' side.
The mutation of Lrd may be the primary cause of the laterality defect observed in the iv/iv mouse brain. We used iv/+ mice in control experiments and there is roughly a 25% genetic background difference between the iv/iv and iv/+ mice. Therefore, experiments using congenic animals will be essential for determining whether the mutation of Lrd is a definite cause of this laterality defect. However, the laterality defect in the iv/iv mouse hippocampus is independent of the laterality of visceral organs, suggesting that a distinct mechanism downstream of the iv mutation generates brain asymmetry.
Compared to recent progress in our understanding of the laterality of visceral organs [6,7], our knowledge about molecular asymmetry of the brain is still limited [18][19][20][21]. This is in part a consequence of the lack of an index for investigating brain asymmetry at the molecular level with experiments in vitro. Our present study demonstrates that the synaptic distribution of e2 subunits and the properties of NMDAR-mediated synaptic functions are sensitive and quantitative indices for detecting abnormalities in the L-R asymmetry of the brain. These indices in combination with the iv mutant mouse that lacks L-R asymmetry are a useful model system for exploring the molecular basis of brain asymmetry.

VHC transection
To examine synapses made by ipsilateral Sch fibers, VHC was transected 5 days before electrophysiological recording [1,2]. The iv/iv (SI/Col6C57BL/6J hybrid) and iv/+ (produced by crossing the iv/iv and C57BL/6J) mice (7 to 9 W) were anesthetized with an injection of pentobarbital (60 mg/Kg, i.p.) and positioned with a stereotaxic apparatus. A small piece of a razor blade (2.5 mm wide) was glued onto a rod that was clamped onto a micromanipulator. After removing a portion of the skull (3 mm wide and 4 mm long, including the bregma), the blade was inserted to a depth of 4.0 mm at the midline to transect the VHC. To avoid damaging the sagittal sinus, the blade was initially shifted 0.5 mm to the right and inserted 0.5 mm into the cerebral cortex and was then returned to the midline position as the blade was lowered. After slowly removing the blade, a piece of skull was replaced and the scalp was closed with sutures. Animals receiving this procedure were viable for more than 3 months. All experiments were performed under the guidance of Animal Experiments in Faculty of Sciences, Kyushu University and the law (No.105) and notification (No. 6) of the government.

Electrophysiology
Transverse hippocampal slices (450 mm thick) were cut with a vibrating microtome (VT 1000S) in ice-cold artificial cerebrospinal fluid (ACSF) (in mM: NaCl, 119; KCl, 2.5; CaCl 2 , 2.5; MgSO 4 , 1.3; NaH 2 PO 4 , 1.0; NaHCO 3 , 26; glucose, 10, saturated with 95% O 2 / 5% CO 2 ). Brains were fixed on an agar block, which was made by two pieces of agar (with a slope of 20u) stuck together at a right angle and mounted on the cutting stage. We lowered the left rear or right rear of the brain using the agar slopes when cutting the left or right hemisphere, respectively. Slices from a similar septotemporal level were used for experiments. Recordings were made in a submerged slice chamber perfused with ACSF at room temperature. Electrodes filled with 0.9% NaCl were used for extracellular recording. Synaptic responses were evoked at 0.1 Hz using a bipolar tungsten electrode. An LTP-inducing tetanic stimulus was given at 100 Hz for 1 s at baseline stimulus strength. The fEPSP slope was expressed as a percentage of mean slope value before the tetanic stimulation. Synaptic currents were recorded from CA1 pyramidal neurons using the blind-patch technique [22] in the whole-cell voltage-clamp mode (Axopatch 1D). A high-Mg 2+ and Ca 2+ (4 mM of MgSO 4 and CaCl 2 ) ACSF was used to increase membrane stability in the presence of bicuculline. Patch electrodes (3-5 MV) were filled with an intracellular solution (in mM: cesium gluconate, 122.5; CsCl, 17.5; HEPES buffer, 10; EGTA, 0.2; NaCl, 8; Mg-ATP, 2; Na 3 -GTP, 0.3; pH 7.2). We recorded NMDA EPSCs at +10 mV in the presence of DNQX (20 mM) and bicuculline (30 mM). We adopted a relatively low holding potential to obtain stable recordings of NMDA EPSCs throughout the experiment [1,2]. Series resistance (10-30 MV) was regularly monitored during recordings. Cells were rejected if more than a 20% change occurred during the experiment. All records were filtered at 2 kHz, digitized at 4 kHz, and stored on a computer equipped with an A/D converter (Mac Lab 2e). No failures were detected in our experiments. All data were expressed as means6s.e.m. and analyzed with Student's t-test.

Tissue preparation for electron microscopy
The iv/iv and iv/+ mice at postnatal day 9 were anesthetized with an injection of pentobarbital (60 mg/Kg, i.p.) and transcar-dially perfused with 25 mM PBS pH7.4 followed by a fixative containing 4% paraformaldehyde, 0.05% glutaraldehyde and 0.5% picric acid in 0.1 M phosphate buffer (PB) pH 7.4 for 15 min. After perfusion, the brains were removed and 100 and 500 mm-thick coronal slices were alternately cut from the left and right dorsal hippocampus.

Postembedding immunogold labeling
For postembedding labeling, the middle CA1 areas (0.561.0 mm) were trimmed from 500-mm-thick slices of the left and right hippocampus and cryoprotected in 10, 20, and 30% glycerol in 0.1 mM PB pH 7.4 overnight. Samples were then frozen with liquid propane (2185uC) in a cryofixation unit (EM CPC). Freeze substitution and low-temperature embedding in Lowicryl HM20 were performed as described previously [23]. Briefly, the samples were immersed in 1% uranyl acetate dissolved in anhydrous methanol (290uC, 24 h) in a cryosubstitution unit (EM AFS). The temperature was then raised (4uC/h) from -90uC to -45uC. The samples were washed three times with anhydrous methanol and infiltrated with Lowicryl HM20 resin (Polysciences) at -45uC with a progressive increase in the ratio of resin to methanol. Polymerization was performed with ultraviolet light (360 nm) at -45uC for 24 h and 0uC for 36 h. Postembedding immunogold reaction was performed as described previously [24]. Lowicryl-embedded ultrathin sections (90-nm thickness) were picked up onto grids (nickel 400 mesh) coated with coat-quick ''G'' medium (Daido Sangyo). The sections were incubated in blocking solution (2% human albumin serum in TBS with 0.1% Triton X-100) for 30 min, and then in the same solution containing an anti-NR2B antibody [2] overnight at room temperature. After several washes with TBS for 30 min, the sections were incubated with 5-nm gold anti-rabbit IgG secondary antibody (British Biocell International) diluted (1:100) in blocking solution containing polyethyleneglycol (molecular weight, 7500 kDa, 5 mg/ml) for 3 h. Sections were washed in ultrapure water, contrasted with uranyl acetate and lead citrate, and examined with a Jeol 1010 electron microscope. Mean oriens/ radiatum ratios of e2 labeling densities (n = 3 animals) were calculated by determining the oriens/radiatum ratio for each animal by dividing mean densities of immunogold particles (n = 101 to 179) in postsynaptic membrane specializations in the stratum oriens by those in the stratum radiatum.