Del Rio R et al. (2023), Cell-autonomous and differential endocannabinoi...

XB-ART-59842

Front Synaptic Neurosci 2023 Jan 01;15:1176864. doi: 10.3389/fnsyn.2023.1176864.

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Cell-autonomous and differential endocannabinoid signaling impacts the development of presynaptic retinal ganglion cell axon connectivity in vivo.

Del Rio R , Serrano RG , Gomez E , Martinez JC , Edward MA , Santos RA , Diaz KS , Cohen-Cory S .

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Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ9-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CB1R, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CB1R and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CB1R plays a central role in neural development. During development, most CB1R localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CB1R-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CB1R and the effects of CB1R-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in Xenopus. We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CB1R via morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CB1R knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CB1R levels through CB1R MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CB1R-mediated eCB signaling.

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	FIGURE 1. CB1R expression in the developing Xenopus visual system. (A,B) Coronal section of a retina of a stage 45 tadpole shows localization of strongest CB1R immunoreactivity (green) in the ganglion cell layer (gcl), inner nuclear layer (inl), and the inner plexiform layer (ipl). The cellular layers are clearly denoted by the DAPI staining in (A) (blue). Note the absence of CB1R immunoreactivity in the ciliary margin (cm). (C) Strong CB1R immunoreactivity is also observed in the optic nerve head (ONH) where RGC axons exit the retina, and along the optic nerve (ON). (D–F) CB1R expression in the brain is illustrated by the coronal sections of midbrain (D), caudal midbrain (E,F) and rostral hindbrain (G) where CB1R immunoreactivity (green) localizes to cell bodies that lay medially and in the adjacent neuropil of stage 43 to stage 45 tadpoles. Note the individual neuronal cell bodies and processes projecting to the neuropil with strong CB1R immunoreactivity (G, arrow). In (D,E,G), the DAPI staining highlights the lower CB1R immunoreactivity in the proliferative zones near the ventricle (V) and its absence in the dorsal-most portion of the brain. Scale bars = 50 μm. Similar patterns of CB1R immunoreactivity were obtained with two commercial antibodies to CB1R (A,B,E–G; Cayman Chemicals, and C,D; Calbiochem).
	FIGURE 2. CB1R downregulation alters RGC axon morphology. (A) Projections of three individual RGC axons transfected with Control MO (top) or CB1R MO (bottom) imaged in vivo by two-photon confocal microscopy. While axons from RGCs transfected with the CB1R MO targeted normally within the tectal neuropil, their branching patterns and morphologies differed from those of RGCs in tadpoles transfected with Control MO. Qualitatively, axons from RGCs with CB1R knockdown had more branches that terminated in growth cones (arrows) and/or took abnormal turns (arrowheads). The tracing for the third CB1R MO sample axon better illustrates the abnormal turn (red; curved arrow) taken by the axon. The asterisk in the third Control MO sample points to the growth cone of a targeting RGC axon. Scale bars = 50 μm. (B) Quantitatively, axons from RGCs with CB1R knockdown had significantly fewer branches when compared to axons from RGCs transfected with Control MO 48 h after transfection (n = 23 axons per condition, one axon per tadpole). (C) When imaging over the course of 3 days, axon branch number was significantly lower in RGCs with CB1R MO knockdown at the initial imaging (stage 45), with a trend at the 24-h imaging interval (+24 h) and remaining significantly lower when compared to controls at the end of the imaging period (+48 h). Control MO n = 12; CB1R MO n = 11. Mean ± SEM. p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 3. Rapid and transient branching response by RGC axons to acute tectal URB597 treatment. (A) Tadpoles with RGC axons co-expressing tdTomato and GFP-synaptobrevin received an acute, localized injection of URB597 into the optic tectum at stage 45 and were imaged by confocal microscopy 6, 12 and 24 h after initial imaging (stg 45, time 0 h). Two sample arbors in a control tadpole (top panel) and in a tadpole with tectal injection of URB597 (bottom panel) illustrate the changes in dynamic branching and in the number and localization of GFP-labeled pre-synaptic sites in the axon arbors. In the control sample, one axon expresses tdTomato only (arrow). Scale bar = 40 μm. (B) Quantitative analysis of total branch number shows that RGC axons had significantly more branches in the URB597-treated tadpoles during first 6 h after treatment than RGC axons in control tadpoles, a significant difference that was maintained for 24 h. (C) The rate of axon branching, expressed as the change in branch number per imaging interval, was significantly higher during the first 6 h after URB597 tectal injection and returned to a similar rate to those in control tadpoles at the 6–12- and 12–24-h imaging intervals. Control n = 12 axons, URB597 n = 7 axons. Analysis by Student’s t-tests. Mean ± SEM. p ≤ 0.05, **p ≤ 0.001.
	FIGURE 4. Global URB597 treatment at stage 45 increases the complexity of actively branching RGCs in vivo. (A–C) Sample RGC axons in stage 45 tadpoles transfected with tdTomato and GFP-synaptobrevin plasmids. Tadpoles imaged in vivo at stage 45 before (0 h), and 24, and 48 h after vehicle (A; Control), URB597 (B), or JZL184 (C) bath treatment. RGC axons gradually increased their number of branches over a period of 48 h. Arrowhead in (A), points to a second axon. Arrow in (C) points to an area of the arbor obscured by a pigment cell. Scale bars = 50 μm. (D) Quantitative analysis of branch number in RGC axons in URB597-treated tadpoles shows a faster and significant increase in branch number during the first 24 h of treatment, resulting in a higher number of branches by 24 and 48 h vs. controls (n = 10 axons per condition). No significant difference in the number of branches was observed for axons in JZL184 treated tadpoles at any observation interval when compared to axons in control tadpoles. (E) The density of GFP-synaptobrevin puncta per arbor was calculated as the number of puncta per 20 μm. No significant difference in puncta density before and 24 h after treatment (change in GFP-synaptobrevin puncta/length 0–24 h) was observed in axons from URB597- or JZL184-treated tadpoles when compared to controls. Analysis by ANOVA with Sidak’s multiple comparisons tests and Student’s t-test. Mean ± SEM, p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 5. URB597 or JZL184 treatment during early axon pathfinding and targeting results in RGC axons with much simpler morphologies as they branch at their target. Tadpoles were treated with URB597 or JZL184 beginning at stage 38 until stage 45, when tadpoles with RGC axons expressing tdTomato and GFP-synaptobrevin were imaged for three consecutive days in the absence of the drug. (A–C) Sample RGC axons in tadpoles treated with vehicle solution (A; Control), URB597 (B) or JZL184 (C) imaged in vivo by confocal microscopy at stage 45, and 24, and 48 h after first imaging. Arrow in (C) points to the first branching point of the axon. Scale bars = 50 μm. (D) Quantitative analysis of branch number in RGC axons of tadpoles exposed to the drugs from stage 38 to stage 45 showed that RGC axons in both JZL184 and URB597-treated tadpoles failed to increase their branch number over the course of 48 h when compared to controls (n = 11 axons per condition). Axons in control-treated tadpoles significantly increased their branch number over the 48-h imaging period. (E) The density of GFP-synaptobrevin puncta per arbor was calculated as the number of puncta per 20 μm. When compared to controls, axons in URB597-treated tadpoles had a significant higher puncta density at all imaging intervals. Analysis by ANOVA with Sidak’s multiple comparisons tests. Mean ± SEM, p ≤ 0.05, *p ≤ 0.005.
	FIGURE 6. Targeting errors in arborizing RGC axons induced by early JZL184 treatment. (A–C) Confocal projections of RGC axons branching in the optic tectum of control (A,B) and JZL184-treated (C) tadpoles imaged at stage 45. (A,B) The examples of control tadpoles with individual axons at stage 45 and +24 h (A), or multiple axons at stage 45 (B) illustrate how RGC axons enter the optic tectum through the optic tract (arrows) with similar directionality before they branch. (C) Confocal images of the optic tectum of a tadpole treated with JZL184 beginning at stage 38 show RGC axons with apparent targeting errors within the contralateral hemisphere at stage 45 (left panel, curved arrow), and reveal abnormal crossing of axons to the ipsilateral hemisphere 24 h after initial imaging (right panel; stitched image). The dashed lines delineate the two midbrain hemispheres. Scale bars = 100 μm. (D) While quantitatively RGC axon arbors in tadpoles treated with URB597 and JZL184 at stage 38 differ in the overall number of branches they possessed at stage 45 (Figure 5), in proportion axon arbors were also observed to make abnormal turns (local turns) within the neuropil and/or to branch more locally (tight branch) when treatment began at stage 38. Moreover, in proportion more RGC axons in tadpoles treated with JZL184 projected aberrantly, showing abnormal ipsilateral crossing (targeting errors). Control n = 14, URB597 at stage 45 n = 16, JZL184 at stage 45 n = 11, URB597 at stage 38 n = 19, JZL184 at stage 38 n = 28. Statistical analysis by chi-square, difference in outcome among groups p ≤ 0.0001.
	FIGURE 7. Single-cell CB1R knockdown decreases the branching and growth of tectal neurons in vivo. (A) Tracings of sample neurons in stage 45 tadpoles transfected with Alexa 488 dextran and lissamine-tagged Control MO or CB1R MO and imaged in vivo by two-photon confocal microscopy over the course of 3 days. Dendritic arbors were digitally reconstructed in three-dimensions using the Neuromantic tracing software and rendered for illustration purposes using Adobe Photoshop. (B) Dendritic arbors of neurons with CB1R MO had a similar number of branches as Control MO neurons at stage 45 after but possessed a significantly a lower number of branches than controls by the 48-h imaging time point. (C) Quantifying the rate of branch addition as the change in branch number in every 24 h-imaging intervals showed that neurons transfected with Control MO increased their complexity by adding new branches, neurons transfected CB1R MO failed to increase the complexity of their dendritic arbor. (D,E) Quantification of total dendritic arbor length reveals that tectal neurons with CB1R MO fail to increase their dendritic arbor length (D) and grow at a lower rate (E) than Control MO transfected neurons (n = 29 neurons per condition). Statistical analysis by one-way ANOVA and Student’s t-test. Mean ± SEM. p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 8. URB597 treatment enhances visually guided responses. (A) Schematic of the visual avoidance task. Tadpoles at stage 45, treated with vehicle (0.1% DMSO in rearing solution), URB597 or JZL184, were tested for their ability to alter their swimming behavior upon encountering moving dots (arrow depicts downward movement of dots). (B) The tadpole’s swimming path and responses to advancing stimuli (black and red circles) were tracked and analyzed. A tadpole freezing response upon coinciding with the stimulus (red circles) or changing its swimming direction and speed was considered active avoidance. (C) Tadpoles treated with URB597 had increased avoidance responses to the presentation of the stimulus 24 h post-treatment when compared to vehicle treated controls. Control n = 24 tadpoles, URB597 n = 20 tadpoles, JZL184 n = 20 tadpoles. Mean ± SEM. ****p ≤ 0.0001.

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	FIGURE 1. CB1R expression in the developing Xenopus visual system. (A,B) Coronal section of a retina of a stage 45 tadpole shows localization of strongest CB1R immunoreactivity (green) in the ganglion cell layer (gcl), inner nuclear layer (inl), and the inner plexiform layer (ipl). The cellular layers are clearly denoted by the DAPI staining in (A) (blue). Note the absence of CB1R immunoreactivity in the ciliary margin (cm). (C) Strong CB1R immunoreactivity is also observed in the optic nerve head (ONH) where RGC axons exit the retina, and along the optic nerve (ON). (D–F) CB1R expression in the brain is illustrated by the coronal sections of midbrain (D), caudal midbrain (E,F) and rostral hindbrain (G) where CB1R immunoreactivity (green) localizes to cell bodies that lay medially and in the adjacent neuropil of stage 43 to stage 45 tadpoles. Note the individual neuronal cell bodies and processes projecting to the neuropil with strong CB1R immunoreactivity (G, arrow). In (D,E,G), the DAPI staining highlights the lower CB1R immunoreactivity in the proliferative zones near the ventricle (V) and its absence in the dorsal-most portion of the brain. Scale bars = 50 μm. Similar patterns of CB1R immunoreactivity were obtained with two commercial antibodies to CB1R (A,B,E–G; Cayman Chemicals, and C,D; Calbiochem).
	FIGURE 2. CB1R downregulation alters RGC axon morphology. (A) Projections of three individual RGC axons transfected with Control MO (top) or CB1R MO (bottom) imaged in vivo by two-photon confocal microscopy. While axons from RGCs transfected with the CB1R MO targeted normally within the tectal neuropil, their branching patterns and morphologies differed from those of RGCs in tadpoles transfected with Control MO. Qualitatively, axons from RGCs with CB1R knockdown had more branches that terminated in growth cones (arrows) and/or took abnormal turns (arrowheads). The tracing for the third CB1R MO sample axon better illustrates the abnormal turn (red; curved arrow) taken by the axon. The asterisk in the third Control MO sample points to the growth cone of a targeting RGC axon. Scale bars = 50 μm. (B) Quantitatively, axons from RGCs with CB1R knockdown had significantly fewer branches when compared to axons from RGCs transfected with Control MO 48 h after transfection (n = 23 axons per condition, one axon per tadpole). (C) When imaging over the course of 3 days, axon branch number was significantly lower in RGCs with CB1R MO knockdown at the initial imaging (stage 45), with a trend at the 24-h imaging interval (+24 h) and remaining significantly lower when compared to controls at the end of the imaging period (+48 h). Control MO n = 12; CB1R MO n = 11. Mean ± SEM. p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 3. Rapid and transient branching response by RGC axons to acute tectal URB597 treatment. (A) Tadpoles with RGC axons co-expressing tdTomato and GFP-synaptobrevin received an acute, localized injection of URB597 into the optic tectum at stage 45 and were imaged by confocal microscopy 6, 12 and 24 h after initial imaging (stg 45, time 0 h). Two sample arbors in a control tadpole (top panel) and in a tadpole with tectal injection of URB597 (bottom panel) illustrate the changes in dynamic branching and in the number and localization of GFP-labeled pre-synaptic sites in the axon arbors. In the control sample, one axon expresses tdTomato only (arrow). Scale bar = 40 μm. (B) Quantitative analysis of total branch number shows that RGC axons had significantly more branches in the URB597-treated tadpoles during first 6 h after treatment than RGC axons in control tadpoles, a significant difference that was maintained for 24 h. (C) The rate of axon branching, expressed as the change in branch number per imaging interval, was significantly higher during the first 6 h after URB597 tectal injection and returned to a similar rate to those in control tadpoles at the 6–12- and 12–24-h imaging intervals. Control n = 12 axons, URB597 n = 7 axons. Analysis by Student’s t-tests. Mean ± SEM. p ≤ 0.05, **p ≤ 0.001.
	FIGURE 4. Global URB597 treatment at stage 45 increases the complexity of actively branching RGCs in vivo. (A–C) Sample RGC axons in stage 45 tadpoles transfected with tdTomato and GFP-synaptobrevin plasmids. Tadpoles imaged in vivo at stage 45 before (0 h), and 24, and 48 h after vehicle (A; Control), URB597 (B), or JZL184 (C) bath treatment. RGC axons gradually increased their number of branches over a period of 48 h. Arrowhead in (A), points to a second axon. Arrow in (C) points to an area of the arbor obscured by a pigment cell. Scale bars = 50 μm. (D) Quantitative analysis of branch number in RGC axons in URB597-treated tadpoles shows a faster and significant increase in branch number during the first 24 h of treatment, resulting in a higher number of branches by 24 and 48 h vs. controls (n = 10 axons per condition). No significant difference in the number of branches was observed for axons in JZL184 treated tadpoles at any observation interval when compared to axons in control tadpoles. (E) The density of GFP-synaptobrevin puncta per arbor was calculated as the number of puncta per 20 μm. No significant difference in puncta density before and 24 h after treatment (change in GFP-synaptobrevin puncta/length 0–24 h) was observed in axons from URB597- or JZL184-treated tadpoles when compared to controls. Analysis by ANOVA with Sidak’s multiple comparisons tests and Student’s t-test. Mean ± SEM, p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 5. URB597 or JZL184 treatment during early axon pathfinding and targeting results in RGC axons with much simpler morphologies as they branch at their target. Tadpoles were treated with URB597 or JZL184 beginning at stage 38 until stage 45, when tadpoles with RGC axons expressing tdTomato and GFP-synaptobrevin were imaged for three consecutive days in the absence of the drug. (A–C) Sample RGC axons in tadpoles treated with vehicle solution (A; Control), URB597 (B) or JZL184 (C) imaged in vivo by confocal microscopy at stage 45, and 24, and 48 h after first imaging. Arrow in (C) points to the first branching point of the axon. Scale bars = 50 μm. (D) Quantitative analysis of branch number in RGC axons of tadpoles exposed to the drugs from stage 38 to stage 45 showed that RGC axons in both JZL184 and URB597-treated tadpoles failed to increase their branch number over the course of 48 h when compared to controls (n = 11 axons per condition). Axons in control-treated tadpoles significantly increased their branch number over the 48-h imaging period. (E) The density of GFP-synaptobrevin puncta per arbor was calculated as the number of puncta per 20 μm. When compared to controls, axons in URB597-treated tadpoles had a significant higher puncta density at all imaging intervals. Analysis by ANOVA with Sidak’s multiple comparisons tests. Mean ± SEM, p ≤ 0.05, *p ≤ 0.005.
	FIGURE 6. Targeting errors in arborizing RGC axons induced by early JZL184 treatment. (A–C) Confocal projections of RGC axons branching in the optic tectum of control (A,B) and JZL184-treated (C) tadpoles imaged at stage 45. (A,B) The examples of control tadpoles with individual axons at stage 45 and +24 h (A), or multiple axons at stage 45 (B) illustrate how RGC axons enter the optic tectum through the optic tract (arrows) with similar directionality before they branch. (C) Confocal images of the optic tectum of a tadpole treated with JZL184 beginning at stage 38 show RGC axons with apparent targeting errors within the contralateral hemisphere at stage 45 (left panel, curved arrow), and reveal abnormal crossing of axons to the ipsilateral hemisphere 24 h after initial imaging (right panel; stitched image). The dashed lines delineate the two midbrain hemispheres. Scale bars = 100 μm. (D) While quantitatively RGC axon arbors in tadpoles treated with URB597 and JZL184 at stage 38 differ in the overall number of branches they possessed at stage 45 (Figure 5), in proportion axon arbors were also observed to make abnormal turns (local turns) within the neuropil and/or to branch more locally (tight branch) when treatment began at stage 38. Moreover, in proportion more RGC axons in tadpoles treated with JZL184 projected aberrantly, showing abnormal ipsilateral crossing (targeting errors). Control n = 14, URB597 at stage 45 n = 16, JZL184 at stage 45 n = 11, URB597 at stage 38 n = 19, JZL184 at stage 38 n = 28. Statistical analysis by chi-square, difference in outcome among groups p ≤ 0.0001.
	FIGURE 7. Single-cell CB1R knockdown decreases the branching and growth of tectal neurons in vivo. (A) Tracings of sample neurons in stage 45 tadpoles transfected with Alexa 488 dextran and lissamine-tagged Control MO or CB1R MO and imaged in vivo by two-photon confocal microscopy over the course of 3 days. Dendritic arbors were digitally reconstructed in three-dimensions using the Neuromantic tracing software and rendered for illustration purposes using Adobe Photoshop. (B) Dendritic arbors of neurons with CB1R MO had a similar number of branches as Control MO neurons at stage 45 after but possessed a significantly a lower number of branches than controls by the 48-h imaging time point. (C) Quantifying the rate of branch addition as the change in branch number in every 24 h-imaging intervals showed that neurons transfected with Control MO increased their complexity by adding new branches, neurons transfected CB1R MO failed to increase the complexity of their dendritic arbor. (D,E) Quantification of total dendritic arbor length reveals that tectal neurons with CB1R MO fail to increase their dendritic arbor length (D) and grow at a lower rate (E) than Control MO transfected neurons (n = 29 neurons per condition). Statistical analysis by one-way ANOVA and Student’s t-test. Mean ± SEM. p ≤ 0.05, p ≤ 0.005, **p ≤ 0.001.
	FIGURE 8. URB597 treatment enhances visually guided responses. (A) Schematic of the visual avoidance task. Tadpoles at stage 45, treated with vehicle (0.1% DMSO in rearing solution), URB597 or JZL184, were tested for their ability to alter their swimming behavior upon encountering moving dots (arrow depicts downward movement of dots). (B) The tadpole’s swimming path and responses to advancing stimuli (black and red circles) were tracked and analyzed. A tadpole freezing response upon coinciding with the stimulus (red circles) or changing its swimming direction and speed was considered active avoidance. (C) Tadpoles treated with URB597 had increased avoidance responses to the presentation of the stimulus 24 h post-treatment when compared to vehicle treated controls. Control n = 24 tadpoles, URB597 n = 20 tadpoles, JZL184 n = 20 tadpoles. Mean ± SEM. ****p ≤ 0.0001.