Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Identification and conservation of the axolotl endocannabinoid receptors. (A) Protein sequence alignment of the putative axolotl CB1 sequence with the rat and zebrafish CB1 sequence. (B) Protein sequence alignment of the putative axolotl CB2 sequence with the rat and zebrafish CB2 sequence. Red asterisks and red boxes indicate amino acids that are conserved between all three species. (C) Western blot using the rat CB1 antibody on axolotl tail tissue demonstrates a single prominent band at ~120 kDa ( n = 3). (D) Western blot using the rat CB2 antibody on axolotl tail tissue demonstrates two bands at a similar molecular weight of ~46 kDa ( n = 3). MW = molecular weight (for each band in ladder). (E) Preadsorption control for the CB1 antibody using either CB1 or CB2 antigenic peptides ( n = 3). (F) Preadsorption control for the CB2 antibody using CB1 or CB2 antigenic peptides ( n = 3).

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Sequencing, Western Blot, Molecular Weight, Control

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are upregulated in response to tail amputation. (A) Western blot analysis demonstrates a significant upregulation of CB1 in the first 3 days after tail amputation, compared to uninjured controls ( n = 3; F (4,40) = 5.994, p = .0007, one‐way ANOVA). (B) No change in CB2 expression is shown in the first 3 days post tail amputation ( n = 3; F (4,40) = 2.779, p = .0397, one‐way ANOVA). (C) Western blot analysis demonstrates a significant upregulation of CB1 expression at both 7 and 14 days after tail amputation ( n = 3; F (2,24) = 15.97, p < .0001, one‐way ANOVA). (D) Western blot analysis demonstrates a significant upregulation of CB2 at 7 and 14 days post tail amputation ( n = 3; F (2,24) = 10.84, p = .0004, one‐way ANOVA). Uninj = uninjured tail tissue. hpa = hours post tail amputation; dpa = days post tail amputation. ns = not significant. * p < .05, ** p < .01, *** p < .001, *** *p < .0001 compared to uninjured controls. # p < .05.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Western Blot, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are expressed in ependymoglia and neurons in the regenerating spinal cord. (A) Schematic displays the cell‐type architecture of the axolotl spinal cord. The spinal cord is comprised of ependymoglial cells (blue) that line the central canal (cc) of the spinal cord. These ependymoglia extend GFAP + processes toward the periphery of the spinal cord. The spinal cord also contains NeuN + neurons (green) that surround the ependymoglia and extend axons that express β‐III‐tubulin. (B) Immunohistochemistry ( n = 3) shows the absence of CB1 from neuronal cell bodies (iv), and shows the co‐localization of CB1 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cell processes (xii, blue arrow). (C) Immunohistochemistry ( n = 3) shows the absence of CB2 from neuronal cell bodies (iv), and displays the co‐localization of CB2 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cells (xii, blue arrow). (D) Fluorescent in situ hybridization ( n = 2) demonstrates cb1 mRNA expression in both neurons (yellow arrow) and ependymoglia (blue arrow). Scale bars: 100 μm.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Immunohistochemistry, In Situ Hybridization, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting CB1 and CB2 receptor signaling impairs tail regeneration. (A) Representative images of tail regenerates after a 7‐day treatment with the vehicle (control, i), 1 μM AM251 (ii), or 1 μM AM630 (iii). Black dotted line indicates the original plane of amputation. Scale bar: 1 mm. (B, C) Graphs show that the proportional increase in axolotl body length was significantly reduced following either a 7‐day treatment with either 1 μM AM251 ( n = 8; B) or after a 7‐day treatment with 1 μM AM630 ( n = 8; C) compared to the vehicle control (unpaired t tests). (D) Graph shows a significant reduction in the proportional increase in axolotl body length (7 days after tail amputation) following only a 1‐day pulse treatment with either 1 μM AM251 ( n = 10) or 1 μM AM630 ( n = 10), compared to vehicle controls ( n = 10; F (2,27) = 18.86; p < .0001, one‐way ANOVA). (E) Western blot analyses show that treatment with AM251 prevented the upregulation of CB1 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; Constant 7‐day bath treatment: F (3,32) = 14.69; p < .0001; 1‐day pulse treatment: F (3,32) = 18.60; p < .0001; one‐way ANOVAs). Representative blot for 1‐day pulse treatment shown. (F) Treatment with AM630 prevented the upregulation of CB2 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; constant treatment: F (3,32) = 24.80; p < .0001; 1‐day pulse treatment: F (3,32) = 11.60; p < .0001; one‐way ANOVAs). Representative blot for 7‐day constant treatment shown. * *p < .01, ** *p < .001, *** *p < .0001 compared to vehicle controls. ### p < .001. #### p < .0001.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Control, Western Blot

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting cannabinoid receptor activity reduces ependymoglial cell proliferation and upregulates GFAP + in glial cell processes. (A) Representative images of EdU + cells in the regenerating axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (ii), 1 μM AM630 (iii), or the vehicle (control, i). White dotted circles outline the spinal cord. (B) Graph shows a significant reduction in the proportion of EdU + cells in the axolotl spinal cord at 7‐days post tail amputation after treatment with either 1 μM AM251 ( n = 4) or 1 μM AM630 ( n = 4) in comparison to vehicle controls ( n = 4; F (2,9) = 25.25; p = .0002, one‐way ANOVA). ** *p < .001 compared to vehicle controls. (C) Representative images of GFAP expression in uninjured axolotl tail tissue (i) and in regenerating tail tissue (ii) at 7‐days post tail amputation (dpa). (D) Quantified western blot data demonstrates a significant reduction in GFAP expression in the first 7‐days post tail amputation in comparison to uninjured tail tissue ( n = 3; F (3,32) = 25.97, p < .0001, one‐way ANOVA). ** *p < .001 compared to uninjured controls. (E, F) Immunohistochemistry shows GFAP expression paired with either CB1 (E) or CB2 (F) staining in the axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (Eii), or 1 μM AM630 (Fii) or the vehicle (controls, Ei and Fi). Scale bars = 100 μm.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Activity Assay, Control, Comparison, Expressing, Western Blot, Immunohistochemistry, Staining