Postnatal development of cannabinoid receptor type 1 expression in rodent somatosensory cortex

Abstract

Endocannabinoids are powerful modulators of synaptic transmission that act on presynaptic cannabinoid receptors. Cannabinoid receptor type 1 (CB1) is the dominant receptor in the CNS, and is present in many brain regions, including sensory cortex. To investigate the potential role of CB1 receptors in cortical development, we examined the developmental expression of CB1 in rodent primary somatosensory (barrel) cortex, using immunohistochemistry with a CB1-specific antibody. We found that before postnatal day (P) 6, CB1 receptor staining was present exclusively in the cortical white matter, and that CB1 staining appeared in the gray matter between P6 and P20 in a specific laminar pattern. CB1 staining was confined to axons, and was most prominent in cortical layers 2/3, 5a, and 6. CB1 null (-/-) mice showed altered anatomical barrel maps in layer 4, with enlarged inter-barrel septa, but normal barrel size. These results indicate that CB1 receptors are present in early postnatal development and influence development of sensory maps.

Figure 1

CB1 immunoreactivity in mature (P40) rat barrel cortex. A, Low resolution brightfield image of CB1 staining in 50 micron-thick across-row S1 section. Layer boundaries were determined from neighboring CO-stained section in (B). WM, white matter. B, Neighboring cytochrome oxidase (CO)-stained section, showing barrels from whisker rows C, D and E. C, Same image as A, with barrel boundaries (dashed) projected from panel B. Solid contours are pia and blood vessels used for alignment. Scale in A–C: 500 μm. D, High power brightfield image of area in A marked with white rectangle. Scale: 50 μm. E, Schematic of rodent brain depicting across-row section angle. Relative size of S1 map was expanded to allow visualization. F. Section reacted in parallel without primary antibody. Scale: 500 μm. G. Quantification of CB1 staining intensity for section shown in A. Thin black traces, staining intensity along individual barrel-related transects. Thin grey traces, staining along septa-related transects. Thick traces show averages of individual transects. Locations of transects are shown in the inset. Grey vertical lines indicate layer boundaries, as determined from neighboring CO-stained section.

Figure 2

Representative CB1 immunoreactivity in S1 at different postnatal ages. Layer boundaries were determined from neighboring CO-stained sections (not shown). All sections in this image were stained and photographed in parallel to allow comparison of staining intensity across age. WM, white matter. Scale: 500 μm.

Figure 3

Quantification of CB1 staining luminance as a function of depth below the pia. Lower numbers represent darker staining. Black, barrel-centered transects. Grey, septa-centered transects from the same sections. Bars show mean ± SEM, with each section counted as a single sample (see Experimental Procedures). N indicates number of sections. Light grey rectangles indicate mean depths corresponding to L4 and L5b, as determined by CO staining.

Figure 4

CB1 immunoreactivity in S1 in one wild type and one CB1 null mouse. Layer boundaries were determined from neighboring CO-stained sections (not shown). Scale: 500 μm.

Figure 5

Tangential distribution of large whisker barrels in S1 of mature wild type (CB1^+/+) and CB1 knockout (CB1^−/−) mice. A: Montage of cytochrome oxidase stain of L4 barrels in one CB1^−/−mouse. R, rostral. C, caudal. L, lateral. M, medial. B, Reconstruction of barrel boundaries for the case in (A). Point show centroids of C2, C3, C4, D3, and E3 barrels. Barrel and septal dimensions were measured along lines connecting centroids. C. Barrel maps from CB1^+/+ and CB1^−/− mice. Scale in A–C: 500 μm. D, Barrel width along the arc dimension, for barrels D1–D4. Bars are mean ± SEM. E, Width of septa between D and E barrels, for arc 1–4. Bars are mean ± SEM. Asterisks, p < 0.05