Requirement of cannabinoid CB(1) receptors in cortical pyramidal neurons for appropriate development of corticothalamic and thalamocortical projections

Abstract

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.

Figure 1. Abnormal fasciculation of corticothalamic and thalamocortical axons in E16.5 CB₁R KO mice

(A) A schematic view illustrates DiI crystal placement in the presumptive S1 area (arrow) of one hemisphere and DiI placement in the dorsal thalamus of the other hemisphere in an E16.5 brain. The two parallel lines indicate the location of two representative planes (rostral and caudal) shown in this figure for each genotype. (B) A cartoon illustrates a coronal section taken from the more caudal section indicated in A. (C–D) The DiI-labeled corticothalamic tracts originating from the S1 cortex cross through the striatum and reach the thalamic nuclei in CB₁R KO (D) and littermate control (C) mice. Scattered, abnormally large fascicles (arrow heads) were found in CB₁R KO mice (D1; panel 2) but not in littermate control mice (C1; panel 1). (E–F) The DiI-labeled thalamocortical axons pass through internal capsule, the striatum, and target the cortical plate. Several abnormally large fascicles (arrow heads) and misrouted fibers (arrows) were found in CB₁R KO thalamocortical axonal trajectories within the PSPB and cortical plate (F and panels 5, 6). (Panels 1–6) Projected images of 3-D confocal image stacks for the areas indicated in C–F. A few axons have been high-lighted with dashed white lines placed slightly below the fibers as illustrative examples of misrouted axons. Abbreviations: cp, cortical plate; DT, dorsal thalamus; ge, ganglionic eminence; hc, hippocampus; HY, hypothalamus; ic, internal capsule; lv, lateral ventricle; PSPB, the pallial-subpallial boundary; RT, reticular nucleus; S1, primary somatosensory cortex; st, striatum; TE, thalamic eminence; th, thalamus; VT, ventral thalamus.

Figure 2. Normal corticothalamic and thalamocortical trajectories in E16.5 GPR55 KO mice

(A,B) The DiI-labeled corticothalamic tracts reach the thalamus and the morphology of the axonal tracts appears normal in GPR55 KO mice (B) and no different from their wild type littermates (A). (C,D) DiI-labeled thalamocortical axons target the cortical plate and the axonal fascicles appear normal in the GPR55 KO (D), and their littermate control (C) mice. (Panels 1–6) Projected images of 3-D confocal image stacks for the areas indicated in A–D. Abbreviations: cp, cortical plate; ge, ganglionic eminence; ic, internal capsule; lv, lateral ventricle; RT, reticular nucleus; st, striatum.

Figure 3. Quantitative difference in fascicle size and number of E16.5 CB₁R KO thalamocortical axons

(A–B) Sample images show L1-NCAM (L1) and CTFL double labeling with coronal brain sections taken from the rostral forebrain areas (as indicated in Figures 1) of control (A) and E16.5 CB₁R KO mice (B). Aberrant axonal fascicles were detected by both L1 and CTFL immunoreactivity at the PSPB (B1–B3; arrows heads indicate large fascicles and arrows indicate misrouted axons; two misrouted axons were high-lighted with dashed green lines placed slightly below the fibers observed). The color of single channel fluorescence images was inverted to provide better illustrations. (C–H) The diameter and number of CTFL-positive TCA fascicles were quantified in a 400×100 µm² area located in the striatum (white dashed rectangles in C, D, panels 1–2) and 100 µm away from the PSPB (yellow dashed lines in C, D). These measurements were conducted in 3–5 different PSPB areas per animal and 3 animals per genotype. (E–H) Both the diameter and number of TCA fascicles are significantly different between CB₁R KO and control mice. There was a shift towards larger fascicles in the distribution of thalamocortical axon fascicle size in CB₁R KO mice (E, F). The number of fascicles per area measured was significantly reduced (G), while the mean fascicle size was significantly increased (H) in CB₁R KO mice compared to control. * Student’s t-test, p <0.05.

Figure 4. Aberrant thalamocortical axonal fascicles in postnatal CB₁R KO mice

(A–H) Representative images of CTFL staining of the TCA distributions in P0, P1, P4 and P7 CB₁R KO and control mice using coronal planes of the rostral forebrain. (Panels 1–10) Confocal single plane images indicated in A–H by the white rectangles. In these panels, colors have been inverted to better highlight axonal trajectories. Green arrows indicate aberrant axonal trajectories while red arrow heads indicate abnormal fasciculation. A few axons have been high-lighted with dashed green lines placed slightly below the fibers as illustrative examples. Note that the projections of the high-lighted axons from control (panel 1) and CB₁R KO mice were quite different (panel 3). (I–J) DiI-labeled TCAs in P1 brains reveal the aberrant phenotypes in CB₁R KO mice observed with CTFL staining. (Panels 11–12) Confocal single plane images indicated in I–J. Abbreviations: cx, cortex; hc, hippocampus; ic, internal capsule; st, striatum.

Figure 5. CB₁R is detected in corticothalamic but not in thalamocortical axons during brain development

(A–C) CB₁R expression in developing corticothalamic axons at E12.5 (A), E14.5 (B) and E16.5 (C). A is the inverted image of fluorescence image. B, C are the bright-field images of DAB staining. (D–I) Example images of CB₁R and GFP double staining in E14.5 (D–E), E16.5 (F–H), and P4 (I) TCA^mGFP mice. In TCA^mGFP mice, GFP labels TCAs originating from the primary thalamic relay nuclei. (D) At E14.5, CB₁R positive axons and GFP-TCAs meet in the PSPB area and are intermingled in axon bundles (E). (F–H) By E16.5, CB₁R positive axons have passed through the striatum and internal capsule. (F–G) At the striatum and the PSPB in the rostral forebrain region, CB₁R-corticothalamic and GFP-TCA fibers are mingled within the same axon bundles. (H) Single confocal plane high magnification images show that CB₁R- and GFP-positive fibers remain segregated within individual axonal bundles. D4 is the inverted fluorescent images in D3. (I) By P4, CB₁R expression is down-regulated in axonal tracts, while hippocampal expression remains high. Abbreviations: cp, cortical plate; ge, ganglionic eminence; hc, hippocampus; lv, lateral ventricle; ic, internal capsule; st, striatum; th, thalamus.

Figure 6. DGLβ and MGL are expressed in developing thalamocortical axonal tracts

(A) A schematic diagram shows the distributions of CTAs and TCAs at E14.5. (B, D) At E14.5, DGLβ is expressed in GFP-TCAs (arrow heads, D1, 2, 4, E1–4), and to a lesser extent in CB₁R-CTAs (arrows, D2–4, F1–4). (C,G) At E14.5, MGL is located along both GFP-TCAs (arrow heads, G1, 2, 4, H1–4) and CB₁R-CTAs (arrows, G2–4, I1–4). (J–N) At E16.5, DGLβ is located along the GFP-TCAs in the internal capsule (enlarged in K) and as the fibers traverse through the striatum (arrow head in J). DGLβ is mainly present in GFP-TCAs (yellow in J–N) in the striatum, and as the fibers extend within the white matter (N). (O–Q) At E16.5, a punctate MGL staining pattern was found in both CB₁R-CTAs (arrows) and GFP-TCAs (arrow heads) as the fibers traversed the striatal region (Q1–4) and the internal capsule (P). Abbreviations: cp, cortical plate; ge, ganglionic eminence; hc, hippocampus; ic, internal capsule; st, striatum; th, thalamus.

Figure 7. The absence of CB₁R in developing cortical principal neurons leads to abnormal thalamocortical axon tract development

(A–B) Sample images of L1-NCAM and CTFL double staining reveal aberrant fasciculation and axon trajectories in E16.5 NEX-CB₁R cKO (B) but not their littermate control mice (A). CTFL staining in E16.5 NEX-CB₁R cKO mice (B3) revealed abnormal thalamocortical fasciculation and axon trajectories. Arrows point to aberrant axonal trajectories while arrow heads indicate aberrant fasciculation in the mutants. (Panels 1–2) Enlarged views of the corresponding areas indicated by the white rectangles in A3 and B3. Abbreviations: cp, cortical plate; ge, ganglionic eminence; lv, lateral ventricle; st, striatum.