Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice

Abstract

Development of whisker-specific neural patterns in the rodent somatosensory system requires NMDA receptor (NMDAR)-mediated activity. In cortex-specific NR1 knockout (CxNR1KO) mice, while thalamocortical afferents (TCAs) develop rudimentary whisker-specific patterns in the primary somatosensory (barrel) cortex, layer IV cells do not develop barrels or orient their dendrites towards TCAs. To determine the role of postsynaptic NMDARs in presynaptic afferent development and patterning in the barrel cortex, we examined the single TCA arbors in CxNR1KO mice between postnatal days (P) 1-7. Sparsely branched TCAs invade the cortical plate on P1 in CxNR1KO mice as in control mice. In control animals, TCAs progressively elaborate patchy terminals, mostly restricted to layer IV. In CxNR1KO mice, TCAs develop far more extensive arbors between P3-7. Their lateral extent is twice that of controls from P3 onwards. By P7, CxNR1KO TCAs have significantly fewer branch points and terminal endings in layers IV and VI but more in layers II/III and V than control mouse TCAs. Within expansive terminal arbors, CxNR1KO TCAs develop focal terminal densities in layer IV, corresponding to the rudimentary whisker-specific patches. Given that thalamic NMDARs are spared in CxNR1KO mice, the present results show that postsynaptic NMDARs play an important role in refinement of presynaptic afferent arbors and whisker-specific patterning in the developing barrel cortex.

Fig. 1

Optimal angles for preparation of intact thalamocortical pathway slices at different developmental ages. Oblique planes of sectioning parallel to the thalamocortical pathway were used for different postnatal ages. The forebrains were split in half along the sagittal plane, and each hemisphere was embedded in 2% agar with the medial side down (A). The specimen was placed on an X-Y coordinate, with the occipital pole contacting the Y-axis, and the plane along the olfactory bulb and the base of the forebrain aligned with the X-axis. Different cutting angles (15, 20, 25, or 30° deviating from the X-axis) were tested; the cuts were made through the agar block (B). The specimen was lifted with a spatula from its ventral side. Different oblique angles of cuts (45, 50, 55, or 60° deviating from the Y-axis) were tested (C). The rostral pole of the block was mounted down to the vibratome stage and the tissue block was cut from the caudal pole (D). The gray scale codes are used to illustrate the results. When a single TCA could be traced from the internal capsule to a CO-dense single patch in layer IV, the plane was designated as optimal (the darkest color) (E). A typical example of a thalamocortical slice with DiI implant in the barreloid region of the VB is illustrated for a P5 case (F). Arrows point to barrels in the cortex and barreloids in the VB. Inset shows higher magnification view of the DiI implant (arrowhead). Scale bars = 200 μm in the inset; 1 mm in A–D.

Fig. 2

Development of TCA patches in normal and CxNR1KO barrel cortex as revealed with 5-HTT immunohistochemistry. Tangential sections of flattened cortices at ages P3 (A and D), P5 (B and E), and P7 (C and F) are collected from control (A–C) and CxNR1KO (D–F) mice. Whisker-specific patterns of 5-HTT-positive TCAs appear in the normal somatosensory cortex as early as P3 (A), and become more distinctive as the brain develops (B, C). However, the patterns are less discernible in the CxNR1KO cortex at P3 (D). At later ages, smaller TCA patches become visible in the major whisker representation area (E, F). Scale bar = 100 μm in F (applies to A–F).

Fig. 3

Delineation of cortical laminae at various developmental ages with adjacent sections stained for Nissl (left) and CO (right). Sections were cut according to the optimal angles at P3 (A and B), P5 (C and D), and P7 (E and F) and stained with Nissl and CO. Cortical layers are revealed by both staining methods in control (A, C, E) and CxNR1KO (B, D, F) mouse brains. Note the cell-dense barrel walls in Nissl-stained control cortices (C, E) and their absence in CxNR1KO brains, and normal CO patches in control cortex and smaller CO patches in CxNR1KO mice. Scale bar = 200 μm in F (applies to A–F).

Fig. 4

DiI-labeled single axons in CO-stained thalamocortical slices. A small crystal of DiI was inserted into the barreloid region of a slightly CO stained thalamocortical section; single TCAs were then visualized and traced. Examples of control (A–C) and CxNR1KO (D–F) P7 TCA are presented. Cortical layers and barrel patterns are revealed by CO staining (A, D, asterisks). Single TCAs in the somatosensory cortex are labeled with DiI. Images from different focal planes were collected and superimposed, then converted into black-and-white for better resolution (B, E). Tracings of single TCAs were made manually based on the reconstructed images (C, F). Single patches (gray in C, F) from CO-stained sections (A, D) were pasted on the afferent arbor indicating the relative position of the CO-dense patches with respect to TCA terminal arbor fields. Scale bar = 100 μm in F (applies to A–F).

Fig. 5

Examples of single TCA arbors in control and CxNR1KO cortex between P1–7. At P1, TCAs invade the cortical plate (CP) as simple axons with few small branches in both control and CxNR1KO cases (A). On P3, TCAs display focalized branches in layer IV and a few in layer VI in control mice. Relatively few branches are seen in other layers. In CxNR1KO cortex, TCAs display a wider terminal territory and more branches are present in other layers (B). Normal TCAs in P5 and P7 show their focal terminal arbors in layer IV and some terminal arbors in layer VI but have very few branches in other layers. TCAs of CxNR1KO mice continue their lateral expansion and branching in other layers (C and D). Scale bar = 100 μm.

Fig. 6

Bifurcation points and terminal tips of TCAs in different cortical layers at different postnatal ages. Numbers of bifurcation points (A) and terminal tips (B) of each reconstructed single TCA were counted in different cortical layers in CxNR1KO and control mice. For bifurcation points (A), from P3 and on, TCAs of CxNR1KO mice have a greater number in layers II/III and layer V than control TCAs. On the other hand, in layer IV, CxNR1KO mice have fewer bifurcation points than control mice on P5 and P7. For terminal tips (B) from P3 and on, CxNR1KO TCAs have a greater number in layers II/III and layer V but a lower number in layer IV than control TCAs.

Fig. 7

Mediolateral extent of single TCA arbors between P1–7. Lateral extent of TCAs from control (black) and CxNR1KO (gray) animals were measured and plotted in groups. At P1, there is no difference between control and CxNR1KO cases. However from P3 and on the mediolateral extent of the TCAs in the mutant cortex is significantly greater than those in controls. Mean value of each group is demonstrated by a horizontal bar. Significant differences are indicated by asterisks (***P < 0.001, Student’s t-test).

Fig. 8

Arbor density of control and CxNR1KO TCAs. The 2D density of arbors was determined by counting intersections across the two diagonals of every 25 × 25 μm² (A) and plotted in gray scale (B). The control case has more high-density spots (intersections greater than 5) than the CxNR1KO case (16 and 2, respectively) and the profile matches the CO-stained pattern of the adjacent section (C, asterisk, left). Note there is some relatively higher density spots present in the CxNR1KO case forming a small region matching the smaller CO-stained patches (C, asterisk, right). Scale bars = 100 μm in A; 200 μm in C.