Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Abstract

Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

Figure 1

Photomicrographs of reelin-immunostaining through the motor FPC of WTM (Left) and HRM (Right) of 20-μm coronal sections taken 0.74 mm anterior to Bregma. Note that there is a decrease in the number of reelin-immunopositive neurons as well as an apparent decrease in the expression of reelin in the neuropil. (Bar = 200 μm.)

Figure 2

GAD₆₇-immunoreactive neurons in FPC of HRM and WTM. (Upper Left) A schematic representation of a coronal section of FPC (0.74 mm anterior to Bregma) illustrating the subdivisions of superficial layers (I–IV) and deep cortical layers (V–VI) of the M2, Cg1, and Cg2 regions of the FPC, and Right is the percentage of GAD₆₇-immunoreactive cells in the HRM, as compared with the WTM. Student's t test comparing HRM with WTM. *, P < 0.01;**, P < 0.001. (Lower) Photomicrographs of 40-μm sections through the FPC of the WTM (Left) and HRM (Right) immunolabeled for GAD₆₇. Note that in the HRM there is a decreased number of GAD₆₇-immunopositive cells in the Cg1 and M2 regions, as well as a decrease in the surrounding neuropil expression of GAD₆₇. Each value represents the mean of eight to nine animals. Standard error range from 2–10% of the mean. (Bar = 300 μm.)

Figure 3

Photomicrographs showing a Golgi-impregnated FPC layer III pyramidal cell from a WTM (A) and a HRM (B). C and D are higher magnifications of the area boxed in A and B, respectively. Note the decreased number of spines on the dendrites of the HRM. High magnification of B2 dendritic branches shows that the spines of the HRM (F) appear to be smaller and have a shorter neck than the spines of the WTM (E). (Bars in A and B = 20 μm; in C and D = 10 μm; in E and F = 2.5 μm.)

Figure 4

Number of dendritic spines expressed on basal or apical branches of motor FPC layer III pyramidal neurons (Left) and on basal or apical branches of hippocampal (HIP) CA1 pyramidal neurons (Right). Coronal FPC sections 0.5–1 mm anterior to the Bregma, □, WTM; ■, HRM. Each value is the mean ± SEM of five animals. Student's t test comparing HRM with WTM *, P < 0.02.

Figure 5

Laser confocal microscope fluorescent image of FPC pyramidal neurons stained with Δ⁹ 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate. (A and C) Pyramidal neuron of motor FPC area (1.0 mm anterior to Bregma) of WTM. (B and D) Pyramidal neuron from the homologous FPC area of HRM. (Lower C and D) are the higher magnification of the area boxed in A and B. (Bars: A and B = 10 μm; C and D = 5 μm.)