NMDA receptors control development of somatosensory callosal axonal projections
- Department of Neurology, University of California, San Francisco, United States
- Weill Institute for Neurosciences, University of California, San Francisco, United States
- Department of Neurological Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, China
- Department of Cell and Tissue Biology, Program in Craniofacial Biology and Institute for Human Genetics, University of California, San Francisco, United States
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, United States
- Programs in Neuroscience and Developmental Stem Cell Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Kavli Institute for Fundamental Neuroscience, United States
Figures
Figure 1 with 1 supplement
The callosal somatosensory innervation pattern was disrupted in Emx1cre/+; Grin1fl/fl mice.
(A–E) Postnatal development of callosal projection in S1. (A) EGFP plasmid injected into lateral ventricle of embryo at embryonic day 15.5 (E15.5) and electrical pulse given to enable the plasmid to …
Figure 1—figure supplement 1
The expression of NMDAR in cortex was reduced in Emx1cre/+; Grin1fl/fl mice.
Examples of 12 µm coronal brain sections from P8 Emx1cre/+; Grin1wt/wt (A) and Emx1cre/+; Grin1fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed …
Figure 2 with 3 supplements
The callosal innervation defect was first detected at P6 in Emx1cre/+; Grin1fl/fl mice.
(A, A’) At P6, most axons in control grew into deeper layer VI of S1 (see ‘*’); a few axons grew to layer V from medial to lateral S1 (see arrows). However, axons projecting to lateral S2 had grown …
Figure 2—figure supplement 1
There was no difference between Emx1cre/+; Grin1fl/wt and Emx1cre/+; Grin1fl/fl mice during axonal extension into the ipsilateral CC (P0) and to the contralateral CC (P5).
(A, B) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and GluN1 KO littermates (Emx1cre/+; Grin1fl/wt and Emx1cre/+; Grin1fl/fl mice). The arrows show the …
Figure 2—figure supplement 2
No increased cell death in S1 of Emx1cre/+; Grin1fl/fl mice at P6.
(A) In control mice (Emx1cre/+; Grin1fl/wt), cleaved caspase-3+ cells were mostly detected in layer II/III of M1 (A’), only rare cell death was observed in other cortical regions, such as S1 (A’’). …
Figure 2—figure supplement 3
Callosal axon density analysis by Image J.
(A) This picture was an 8-bit image. (B) Fluorescence signals within threshold in image A turned to red after setting up threshold range. Box I was drawn to encompass only the S1. Box II was used to …
Figure 3 with 1 supplement
NMDAR is required in target neurons for normal callosal innervation.
(A–D) Deleting NMDAR specifically in projecting neurons. Vectors expressing Cre-recombinase (Cre) and EGFP were delivered into S1 of floxed GluN1 mice (Grin1fl/wt x Grin1fl/wt) by in utero …
Figure 3—figure supplement 1
Deleting NMDAR specifically in target neurons.
GluN1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in Grin1fl/fl; Rosa26fs-tdTomato mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. This …
Figure 4 with 1 supplement
Increased callosal innervation in S1 after contralateral but not ipsilateral injection of anti-NMDAR antibodies from P2 to P12.
(A–D) Anti-GluN1 antibodies were injected into the lateral ventricle from P2 to P12 in ipsilateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice did not show …
Figure 4—figure supplement 1
The efficiency of intraventricular antibody injection and the distribution territory in the cortex after 3 hr of last injection.
(A) Anti-GluN1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hr later after last injection. Rabbit IgG served as control. Mouse brains then were stained …
Figure 5
Contralateral injection of anti-NMDAR antibodies from P4 to P8 but not P8 to P14 had increased callosal innervation in S1.
(A–D) Anti-GluN1 antibodies were injected into the lateral ventricle from P4 to P8 in contralateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice show …
Figure 6 with 2 supplements
Emx1cre/+; Grin2bfl/fl but not Emx1cre/+; Grin2afl/fl mice had the same disrupted callosal innervation patterns as Emx1cre/+; Grin1fl/fl at P14.
Callosal innervation patterns in control Emx1cre/+; Grin2afl/wt (A) and Emx1cre/+; Grin2afl/fl mice (B) at P14. ‘*’ points out M1/S1 border. (C) Quantification of fluorescence density. p=0.392. …
Figure 6—figure supplement 1
NR2B (Emx1cre/+; Grin2bfl/fl) but not NR2A (Emx1cre/+; Grin2afl/fl) mice had same disrupted callosal innervation patterns as Emx1cre/+; Grin1fl/fl at P30.
(A) The callosal innervation pattern in S1 at P30 in control mice (Emx1cre/+; Grin2afl/wt) is similar as the pattern in P14 WT control mice, with few axons in S1 but a dense innervation at S1/S2 …
Figure 6—figure supplement 2
The protein expression level of GluN2A and GluN2B in S1 at P8.
(A) Western blot analysis of cortical protein extracts from P8 S1 showed that GluN2A protein has been expressed in S1 at P8. Relative to the loading control GAPDH, the protein levels of GluN2A were …
Figure 7
NMDAR regulates callosal circuit development independent of NMDAR channel activity.
(A–D) Blocking Ca2+ influx through NMDAR by MK-801. MK-801 enters the open NMDAR channel and binds to the ‘blocking site’ located deep in the pore (A). Callosal innervation patterns in Saline (B) …
Figure 8 with 2 supplements
NMDARs cooperate with EPHRIN-B/EPHB in controlling axon targeting in S1.
(A) EPHRIN-B1, expressed by the projecting neuronal axons, signals through EPHB2 and NMDAR, located on the target neurons, regulates axon extension in contralateral cortex. (B, C) Deleting EPHRIN-B1 …
Figure 8—figure supplement 1
The protein but not RNA level of EPHB2 was reduced in Emx1cre/+; Grin1fl/fl mice at P8.
(A, B) EPHB2 protein expression is decreased in Emx1cre/+; Grin1fl/fl mice at P5. In control Emx1cre/+; Grin1wt/wt mice, EPHB2 was expressed both in CC and cortex (A). EPHB2 in Emx1cre/+; Grin1fl/fl …
Figure 8—figure supplement 2
The cell membrane expression of EPHB2 was absent in Emx1cre/+; Grin1fl/fl; Rosa26fs-tdTomato positive cells.
(A) We crossed Emx1cre/+; Grin1fl/fl mice with Cre-reporter Rosa26fs-tdTomato mice to produce GluN1 knockout cells labeled with red fluorescence. (B) 12 µm coronal brain sections from P8 Emx1cre/+; …
