mGluR5 in cortical excitatory neurons exerts both cell-autonomous and -nonautonomous influences on cortical somatosensory circuit formation

Abstract

Glutamatergic neurotransmission plays important roles in sensory map formation. The absence of the group I metabotropic glutamate receptor 5 (mGluR5) leads to abnormal sensory map formation throughout the mouse somatosensory pathway. To examine the role of cortical mGluR5 expression on barrel map formation, we generated cortex-specific mGluR5 knock-out (KO) mice. Eliminating mGluR5 function solely in cortical excitatory neurons affects, not only the whisker-related organization of cortical neurons (barrels), but also the patterning of their presynaptic partners, the thalamocortical axons (TCAs). In contrast, subcortical whisker maps develop normally in cortical-mGluR5 KO mice. In the S1 cortex of cortical-mGluR5 KO, layer IV neurons are homogenously distributed and have no clear relationship to the location of TCA clusters. The altered dendritic morphology of cortical layer IV spiny stellate neurons in cortical-mGluR5 KO mice argues for a cell-autonomous role of mGluR5 in dendritic patterning. Furthermore, morphometric analysis of single TCAs in both cortical- and global-mGluR5 KO mice demonstrated that in these mice, the complexity of axonal arbors is reduced, while the area covered by TCA arbors is enlarged. Using voltage-clamp whole-cell recordings in acute thalamocortical brain slices, we found that KO of mGluR5 from cortical excitatory neurons reduced inhibitory but not excitatory inputs onto layer IV neurons. This suggests that mGluR5 signaling in cortical excitatory neurons nonautonomously modulates the functional development of GABAergic circuits. Together, our data provide strong evidence that mGluR5 signaling in cortical principal neurons exerts both cell-autonomous and -nonautonomous influences to modulate the formation of cortical sensory circuits.

Figure 1.

mGluR5 expression is reduced in a tissue-specific manner in conditional mGluR5 KO mice. A, B, mGluR5 expression was substantially reduced in the cortex of P4 NEX-mGluR5 KO mice (B₁), while its expression in the striatum was similar to littermate controls (mGluR5^f/f) (A₁). A₂ and B₂ show VGluT2 and DAPI staining in the same sections of A₁ and B₁. C, D, High-magnification images in layer IV of P14 NEX-control (C) and NEX-mGluR5 KO S1 cortex (D). Prominent mGluR5-positive puncta outside NeuN-positive somata were seen in the control neuropil, while only residual mGluR5 immunoreactivity was observed in NEX-mGluR5 KO mice. E–J, Similar levels of mGluR5 expression in both NEX-control (E, G, I) and NEX-mGluR5 KO mice (F, H, J) were found in the ventral basal (VB) thalamus (E, F) and the brainstem nuclei Sp5 (G, H) and PrV (I, J). K, No mGluR5 immunoreactivity was detected in mGluR5^−/− mice (K₁), while NeuN staining revealed distinctive structures (K₂). The age of staining is indicated at the bottom left of each panel. cx, Cortex; PrV, principal sensory nucleus; Sp5, spinal trigeminal nucleus; st, striatum; VB, ventral basal thalamic nuclei.

Figure 2.

Abnormal whisker maps in NEX-mGluR5 KO S1 cortex. A–D, Representative images of CO-stained tangential sections through S1 cortical layer IV prepared from control (A) and NEX-mGluR5 KO (B, C) mice. CO staining revealed whisker-related pattern in control mice (A), while this staining showed whisker map deficits in the NEX-mGluR5 KO mice (B, C). D, E, Nissl staining revealed normal barrel cytoarchitecture in control (D), but not in NEX-mGluR5 KO S1 cortex (E). F–I, VGluT2 and NeuN double labeling of tangential sections from control (F, G) and NEX-mGluR5 KO S1 cortex (H, I) were used to simultaneously visualize the distribution of TCA clusters and cortical neurons. Unlike the discrete whisker-related VGluT2-positive patches in control mice (F₁, G₁), fuzzy VGluT2-staining patterns were found in NEX-mGluR5 KO mice (H₁, I₁), particularly for the TCA clusters related to the rostroventral whiskers (H₁). NeuN staining revealed ring-like organizations of cortical neurons in control mice (F₂, G₂), while no clear segregation of cortical neurons was observed in NEX-mGluR5 KO mice (H₂, I₂). A–E indicate the corresponding whiskers; cd, the representation of the caudodorsal whiskers; rv, the representation of the rostroventral whiskers.

Figure 3.

EMX-mGluR5 KO mice have a cortex-specific reduction in mGluR5 expression and a defective cortical whisker map. A, B, mGluR5 expression was substantially reduced in the cortex of P8 EMX-mGluR5 KO mice (B), while its expression in subcortical areas, including the striatum, was similar to their littermate controls (mGluR5^f/f) mice (A). C, D, Representative images of CO-stained tangential sections through cortical layer IV prepared from EMX-control (C) and EMX-mGluR5 KO (D) S1 cortex. E, F, Nissl staining revealed classic barrel cytoarchitecture in control (E) but not in EMX-mGluR5 KO S1 cortex (F). 2 m, 2 months old; A–E indicate the corresponding whiskers; LV, lateral ventricle; st, striatum.

Figure 4.

Quantitative comparison of mGluR5 expression in S1 cortex of the cortex-specific knock-outs using Western blotting. A, Examples of the Western blots used to quantify the abundance of mGluR5 and β-actin in P7 S1 cortex from NEX- and EMX-mGluR5 KO mice and their littermate controls. B, Summary of the normalized ratios of mGluR5/β-actin.

Figure 5.

Layer IV neurons in NEX-mGluR5 KO mice are evenly distributed within the barrel field. A1–A3, B1–B3, Representative immunostaining images show the distribution of TCAs and cortical neurons in tangential slices prepared from P8–P9 NEX-control (A) and NEX-mGluR5 KO mice (B). The VGluT2-positive area (A1, B1) was defined as the barrel hollow. The 20-μm-thick belt encircling the hollow was defined as the barrel wall. The area between barrel walls was defined as the septum. NeuN-positive cells (A2, B2) within the three domains were marked and counted using the Neurolucida program. A4, B4, Schematic images reconstructed from A1–A3 and B1–B3 with the Neurolucida program show the distribution of neurons within barrel hollows (red dots), walls (green dots), or septa (blue dots). C, Bar graph showing neuron density within these three compartments for both genotypes. In NEX-control mice, the density of layer IV cortical neurons was highest in barrel walls [neuron densities in # of neurons/(100 μm)²: barrel hollow, 20.0 ± 2.8; barrel wall, 30.0 ± 2.5; barrel septa, 10.6 ± 1.5; n = 3; p = 0.002 between wall and hollow; p < 0.001 between wall and septa; p < 0.001 between hollow and septa]. This differential distribution of cortical layer IV neurons was absent in NEX-mGluR5 KO mice [neuron densities in # of neurons/(100 μm)²: barrel hollow, 27.7 ± 1.8; barrel wall, 25.1 ± 0.8, barrel septa, 23.9 ± 0.5; n = 3; p = 0.228 between wall and hollow; p = 0.152 between wall and septa; p = 0.106 between hollow and septa]. D, The ratios of wall-to-barrel hollow neuron densities were significantly lower in both NEX-mGluR5 KO mice (NEX-control, 1.45 ± 0.07, n = 3; NEX-mGluR5 KO, 0.91 ± 06, n = 3; p = 0.04 between NEX-control and NEX-mGluR5 KO) and EMX-mGluR5 KO mice (EMX-control, 1.74 ± 0.17, n = 5; EMX-mGluR5 KO, 0.96 ± 0.06, n = 4; p = 0.016, Mann–Whitney rank-sum test) compared to their littermate controls. Asterisks were used to indicate significant differences between the control group (**p < 0.01, ***p < 0.001).

Figure 6.

Barrel walls are absent in NEX-mGluR5 KO mice. NeuN (green) and VGluT2 (red) double labeling of coronal sections prepared from P7 NEX-control (A, B) and NEX-mGluR5 KO mice (C, D). B, D, Enlarged views from A and C. Similar cortical layering patterns between NEX-control (B1) and NEX-mGluR5 KO (D1) mice were revealed by NeuN labeling. VGluT2 immunoreactivity was enriched in cortical layers IV and VI of both genotypes (B2, D2). The distinctive barrel walls within cortical layer IV of control mice (arrows in B3) were absent in NEX-mGluR5 KO mice (D3). Panel 3 images are the enlarged views from B₁ and D₁ (dashed boxes). II–VI, Cortical layers; cx, cortex; hi, hippocampus; st, striatum; th, thalamus.

Figure 7.

The absence of mGluR5 function in layer IV spiny stellate neurons leads to abnormal dendritic patterning. A, A low-magnification image shows Golgi-stained S1 cortex of a coronal slice prepared from a P35 NEX-control mouse. The high-magnification view of the barrel field (red dashed box) is shown in B1. B, C, Example images of Golgi-positive spiny stellate neurons from P35 NEX-control (B1) and P35 NEX-mGluR5 KO (C1) S1 cortex. B2, C2, Their computer-aided reconstructions. D, Pie charts showing the percentage of cells with polarized or nonpolarized distributions of dendrites (neurons with a dendritic asymmetry value >0.75 were considered to be polarized). E, The degree of dendritic asymmetry is significantly lower in NEX-mGluR5 KO neurons (NEX-control, 0.80 ± 0.02, n = 34; NEX-mGluR5 KO, 0.70 ± 0.02, n = 31; p < 0.001 between NEX-control and NEX-mGluR5 KO). Points indicate the data from individual tracings. F, The total number of dendritic segments is significantly higher in NEX-mGluR5 KO neurons (NEX-control, 17.26 ± 4.25, n = 19; NEX-mGluR5 KO, 22.62 ± 6.91, n = 26; p = 0.002 between NEX-control and NEX-mGluR5 KO, Mann–Whitney rank-sum test). G, The total length of dendritic segments is also significantly greater in NEX-mGluR5 KO neurons (NEX-control, 525.63 ± 36.74 μm, n = 19; NEX-mGluR5 KO, 736.79 ± 30.29 μm, n = 26; p < 0.001 between NEX-control and NEX-mGluR5 KO). H, Summary of mean segment number per branch order. I, Summary of mean segment length per branch order. Asterisks are used to indicate significant differences from the control group (*p < 0.05, **p < 0.01, ***p < 0.001, t test).

Figure 8.

Abnormal TCA arborization patterns in mGluR5^−/− mice and NEX-mGluR5 KO mice. Single TCAs of control (including mGluR5^f/f, mGluR5^+/−, and NEX-mGluR5^{f /+} mice), mGluR5^−/−, and NEX-mGluR5 KO mice were labeled with DiI and reconstructed. A–C, Two-dimensional projected images of single TCAs reconstructed in three dimensions. The majority of control TCAs (A) had extensive arborizations in cortical layer IV (layer IV boundaries are indicated by dashed lines). In mGluR5^−/− (B) or NEX-mGluR5 KO (C) mice, many TCAs had simple arborization patterns. D, Summary of mean segment number per branch order. E, Summary of mean segment length per branch order. F, Summary of total branch nodes. In mGluR5^−/− and NEX-mGluR5 KO mice, the total branch number (from the first bifurcation point) of the TCAs was significantly reduced compared to that in control mice (control, 34.32 ± 1.18, n = 25; mGluR5^−/−, 17.32 ± 2.47, n = 25; NEX-mGluR5 KO, 17.03 ± 1.73, n = 32; p < 0.001 between control and mGluR5^−/− with Mann–Whitney rank-sum test; p < 0.001 between control NEX-mGluR5 KO with t test). G, Summaries of total length. The total axonal length of mGluR5^−/− and NEX-mGluR5 KO TCAs were also significantly reduced compared to those from control mice (control, 2.94 ± 0.15 mm, n = 25; mGluR5^−/−, 1.96 ± 0.20 mm, n = 25; NEX-mGluR5 KO, 2.00 ± 0.15 mm, n = 32; p < 0.001 between control and mGluR5^−/−, and p < 0.001 for between control and NEX-mGluR5 KO; Mann–Whitney rank-sum test). H, Summary of axonal spans. The lateral extent of the axonal coverage of mGluR5^−/− and NEX-mGluR5 KO TCAs was significantly wider than control TCAs (control, 196.13 ± 8.94 μm, n = 25; mGluR5^−/−, 232.79 ± 14.34 μm, n = 25; NEX-mGluR5 KO, 222.65 ± 9.36 μm, n = 32; p = 0.034 between control and mGluR5^−/−; p = 0.022 between control NEX-mGluR5 KO; Mann–Whitney rank-sum test for both comparisons). I, Summary of the highest branch order. The highest branch order in mGluR5^−/− and NEX-mGluR5 KO mice was also significantly less than in control mice (control, 12.6 ± 0.4, n = 25; mGluR5^−/−, 9.0 ± 0.5, n = 25; NEX-mGluR5 KO, 8.6 ± 0.4, n = 32; p < 0.001 for comparisons between control and mGluR5^−/−, and for between control and NEX-mGluR5 KO; Mann–Whitney rank-sum test). Results are mean ± SEM. Asterisks are used to indicate significant differences from the control group (*p < 0.05, **p < 0.01, ***p < 0.001, t test).

Figure 9.

Whisker maps are normal in the subcortical relay stations of NEX-mGluR5 KO mice. CO staining of coronal sections through the ventrobasal thalamus (A–C), the principal nucleus (D–F), and the spinal trigeminal nucleus (G–I) of the brainstem trigeminal complex reveals barreloid (A–C) and barrelette patterns (D–I). In mGluR5^−/− mice, CO patches corresponding to rostroventral whiskers were indistinct, while CO patches corresponding to caudodorsal whiskers were evident (B, E, H). Normal whisker presentations for both rostroventral and caudodorsal whiskers in the subcortical relay stations were observed in mGluR5^+/− (A, D, G) and NEX-mGluR5 KO (C, F, I). Dashed circles enclose the area representing the rostroventral whiskers. cd, The representation of the caudodorsal whiskers; fp, forepaw; hp, hindpaw; lj, lower jaw; PrV, principal sensory nucleus; rv, the representation of the rostroventral whiskers; Sp5, the spinal trigeminal nucleus; VB, the ventrobasal thalamus.

Figure 10.

mGluR5 deletion alters functional inputs onto layer IV neurons. A, B, Example recordings of mEPSCs (A) and mIPSCs (B) from layer IV neurons of mGluR5^+/− and mGluR5^−/− mice. Representative single mEPSCs and mIPSCs are shown on the right. C, Summaries of mEPSC frequencies from mGluR5^+/− and mGluR5^−/− mice (mGluR5^+/−, 0.75 ± 0.15 events/s, n = 11; mGluR5^−/−, 1.48 ± 0.13 events/s, n = 9; p = 0.002, t test). D, Summary of mIPSC frequencies from mGluR5^+/− and mGluR5^−/− mice (mGluR5^+/−, 0.94 ± 0.12 events/s, n = 14; mGluR5^−/−, 0.51 ± 0.13 events/s, n = 9; p = 0.023, t test). E, Summaries of mEPSC frequencies from control and NEX-mGluR5 KO mice (control, 1.00 ± 0.26 events/s, n = 11; NEX-mGluR5^−/−, 0.82 ± 0.17 events/s, n = 15; p = 0.88, Mann–Whitney U test). F, Summaries of mIPSC frequencies from NEX-control and NEX-mGluR5 KO mice (control, 1.57 ± 0.42 events/s, n = 8; NEX-mGluR5 KO, 0.89 ± 0.11 events/s, n = 12; p = 0.042 by t test). Points indicate the data from individual recordings.

Figure 11.

Normal intrinsic membrane properties in mGluR5 KO layer IV regular-spiking neurons. A, Example recordings show synaptic responses of regular-spiking neurons recorded from layer IV of NEX-control and NEX-mGluR5 KO S1 cortex triggered by different amount of currents. APs were triggered upon injections of suprathreshold depolarizing current at resting membrane potential. B, The summary of the input–output relationships between the number of evoked APs and the amount of injected current. C, Summary of the amplitudes of the first to the fifth AP triggered by current pulses. D, Summary for the interspike intervals (ISI) between APs.