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Figure 1.
Experimental methods and timeline. (A) 24 h RST using a DecapiCone restraint apparatus. Mice were placed in the cone such that their noses poked out the opening. Metal staples were used to tightly secure mice within the cone. Mice could not move around; however, they were easily able to breathe. (B) Experimental timeline for the SPT including the 24 h RST. From 17:00 on day 1 to 17:00 on day 3, all mice were acclimatized to the water and sucrose solution-containing bottles in their home cages for 48 h. From 17:00 on day 3 to 17:00 on day 4, mice habituated to the SPT chambers containing the water and sucrose solution-filled feeders for 24 h. At 21:00 on day 4, mice underwent the first of two baseline sucrose preference measurements that lasted 12 h. At 21:00 on day 5, the second baseline measurement was carried out for 12 h. Immediately prior to the 24 h RST, all mice were injected with their designated drug treatments. From 21:00 on day 6 to 21:00 on day 7, a subset of mice were restrained in DecapiCones for 24 h. Following the 24 h RST, all mice were treated a second time before entering the final sucrose preference measurement, which lasted for 12 h. Euthanasia and tissue collection were completed by 12:00 on day 8.

Figure 1.
Experimental methods and timeline. (A) 24 h RST using a DecapiCone restraint apparatus. Mice were placed in the cone such that their noses poked out the opening. Metal staples were used to tightly secure mice within the cone. Mice could not move around; however, they were easily able to breathe. (B) Experimental timeline for the SPT including the 24 h RST. From 17:00 on day 1 to 17:00 on day 3, all mice were acclimatized to the water and sucrose solution-containing bottles in their home cages for 48 h. From 17:00 on day 3 to 17:00 on day 4, mice habituated to the SPT chambers containing the water and sucrose solution-filled feeders for 24 h. At 21:00 on day 4, mice underwent the first of two baseline sucrose preference measurements that lasted 12 h. At 21:00 on day 5, the second baseline measurement was carried out for 12 h. Immediately prior to the 24 h RST, all mice were injected with their designated drug treatments. From 21:00 on day 6 to 21:00 on day 7, a subset of mice were restrained in DecapiCones for 24 h. Following the 24 h RST, all mice were treated a second time before entering the final sucrose preference measurement, which lasted for 12 h. Euthanasia and tissue collection were completed by 12:00 on day 8.

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Figure 2.
Cannabinoid and orexin drug effects on 24 h RST-induced anhedonia. (A) Sucrose preference of 200 µL of vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male and female C57BL/6 mice. An 8-day SPT, which included a 24 h RST was used to obtain the sucrose preference data expressed as %. (B) Raw sucrose solution consumption of mice is expressed as (mg). (C) Raw water consumption of mice expressed as (mg). (D) Raw total solution consumption of mice is expressed as (mg). n = 6 mice (n = 3 males; 3 females). Means ± SEM were used for statistical significance analysis via a two-way ANOVA followed by a Tukey’s post-hoc analysis. ** p < 0.01 compared between drug treatments within No 24 h RST. @@/@@@ p < 0.01/0.001 compared between drug treatments within 24 h RST. ##/### p < 0.01/0.001 compared between No 24 h RST and 24 h RST within drug treatments. In the figure above “+” refers to with RST and “−“ refers to without RST.

Figure 2.
Cannabinoid and orexin drug effects on 24 h RST-induced anhedonia. (A) Sucrose preference of 200 µL of vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male and female C57BL/6 mice. An 8-day SPT, which included a 24 h RST was used to obtain the sucrose preference data expressed as %. (B) Raw sucrose solution consumption of mice is expressed as (mg). (C) Raw water consumption of mice expressed as (mg). (D) Raw total solution consumption of mice is expressed as (mg). n = 6 mice (n = 3 males; 3 females). Means ± SEM were used for statistical significance analysis via a two-way ANOVA followed by a Tukey’s post-hoc analysis. ** p < 0.01 compared between drug treatments within No 24 h RST. @@/@@@ p < 0.01/0.001 compared between drug treatments within 24 h RST. ##/### p < 0.01/0.001 compared between No 24 h RST and 24 h RST within drug treatments. In the figure above “+” refers to with RST and “−“ refers to without RST.

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Figure 3.
Change in body weight of cannabinoid and orexin drug-treated mice throughout the SPT and 24 h RST. (A) Daily change in body weight since day 1 of No 24 h RST groups. (B) Daily change in body weight since day 1 of RST groups. (C) Body weight change between days 1–8 of all drug and stress treatment groups. (AC) The body weights of adult male and female C57BL/6 mice treated with either 200 µL of vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185 were measured in the morning of days 1, 3, 5, 6, and 8. Each day’s body weight was compared to that of day 1. Change in body weight is expressed as (g). n = 6 mice (n = 3 males; n = 3 females) as each experimental group is reported as a mean ± SEM. Statistical significance for graphs A and B was assessed by a two-way ANOVA followed by a Tukey’s post-hoc analysis with repeated measurements within groups between days and across groups within days. Significant differences within panel C were evaluated via Tukey’s post-hoc analysis without repeated measures. In the figure above “+” refers to with RST and “−“ refers to without RST.

Figure 3.
Change in body weight of cannabinoid and orexin drug-treated mice throughout the SPT and 24 h RST. (A) Daily change in body weight since day 1 of No 24 h RST groups. (B) Daily change in body weight since day 1 of RST groups. (C) Body weight change between days 1–8 of all drug and stress treatment groups. (AC) The body weights of adult male and female C57BL/6 mice treated with either 200 µL of vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185 were measured in the morning of days 1, 3, 5, 6, and 8. Each day’s body weight was compared to that of day 1. Change in body weight is expressed as (g). n = 6 mice (n = 3 males; n = 3 females) as each experimental group is reported as a mean ± SEM. Statistical significance for graphs A and B was assessed by a two-way ANOVA followed by a Tukey’s post-hoc analysis with repeated measurements within groups between days and across groups within days. Significant differences within panel C were evaluated via Tukey’s post-hoc analysis without repeated measures. In the figure above “+” refers to with RST and “−“ refers to without RST.

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Figure 4.
The effect of cannabinoid and orexin drugs on plasma corticosterone levels of mice that underwent the SPT and 24 h RST. After the final sucrose preference measurement on day 8, adult male and female C57BL/6 mice were euthanized, and their blood was collected for quantification of the circulating corticosterone using an ELISA kit. Data from 200 µL vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55, 940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated mice are expressed as pg/mL. n = 6 mice (n = 3 males, n = 3 females) for Vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185 treatment groups. n = 4 mice (n = 2 males, n = 2 females) for 0.3 mg/kg CP55,940, 1 mg/kg TCS-1102, 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102 treatment groups. Means ± SEMs were used to evaluate statistical significance by a two-way ANOVA followed by a Tukey’s post-hoc analysis. In the figure above “+” refers to with RST and “−“ refers to without RST.

Figure 4.
The effect of cannabinoid and orexin drugs on plasma corticosterone levels of mice that underwent the SPT and 24 h RST. After the final sucrose preference measurement on day 8, adult male and female C57BL/6 mice were euthanized, and their blood was collected for quantification of the circulating corticosterone using an ELISA kit. Data from 200 µL vehicle, 0.3 mg/kg CP55,940 (cannabinoid receptor agonist), 1 mg/kg TCS-1102 (DORA), 0.3 mg/kg CP55, 940 + 1 mg/kg TCS-1102, 3 mg/kg SR141716A (CB1R antagonist), 40 mg/kg YNT-185 (OX2R agonist), and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated mice are expressed as pg/mL. n = 6 mice (n = 3 males, n = 3 females) for Vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185 treatment groups. n = 4 mice (n = 2 males, n = 2 females) for 0.3 mg/kg CP55,940, 1 mg/kg TCS-1102, 0.3 mg/kg CP55,940 + 1 mg/kg TCS-1102 treatment groups. Means ± SEMs were used to evaluate statistical significance by a two-way ANOVA followed by a Tukey’s post-hoc analysis. In the figure above “+” refers to with RST and “−“ refers to without RST.

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Figure 5.
Colocalization between CB1R and OX1R in male mesocorticolimbic brain regions following prolonged restraint stress and drug treatments. (AC), CB1R-OX1R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male C57BL/6 mice. Immediately following the final sucrose preference measurement on day 8, mice were euthanized, and their brains were perfused for immunohistochemical analysis. A Zeiss LSM880 confocal microscope was used to collect the colocalization images. FIJI with ImageJ (version 2.1.0) was used to analyze the images. Receptor colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three male mice. Means ± SEM were used to calculate statistical significance via a two-way ANOVA and Tukey’s post-hoc analysis. **/*** p < 0.01/0.001 compared between drug treatments within No 24 h RST. @/@@/@@@ p < 0.05/0.01/0.001 compared between drug treatments within 24 h RST. # p < 0.05 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative confocal microscopy images of CB1R-OX1R colocalization in the VTA (D), NAC (E), and MPFC (F) of males subjected to RST, which correspond to graphs A, B, and C, respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

Figure 5.
Colocalization between CB1R and OX1R in male mesocorticolimbic brain regions following prolonged restraint stress and drug treatments. (AC), CB1R-OX1R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male C57BL/6 mice. Immediately following the final sucrose preference measurement on day 8, mice were euthanized, and their brains were perfused for immunohistochemical analysis. A Zeiss LSM880 confocal microscope was used to collect the colocalization images. FIJI with ImageJ (version 2.1.0) was used to analyze the images. Receptor colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three male mice. Means ± SEM were used to calculate statistical significance via a two-way ANOVA and Tukey’s post-hoc analysis. **/*** p < 0.01/0.001 compared between drug treatments within No 24 h RST. @/@@/@@@ p < 0.05/0.01/0.001 compared between drug treatments within 24 h RST. # p < 0.05 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative confocal microscopy images of CB1R-OX1R colocalization in the VTA (D), NAC (E), and MPFC (F) of males subjected to RST, which correspond to graphs A, B, and C, respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

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Figure 6.
Cannabinoid receptor type 1 and OX2R colocalization in mesocorticolimbic brain regions of male mice that had undergone 24 h RST and drug treatments. (AC), CB1R-OX2R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male C57BL/6 mice. Succeeding the final sucrose preference measurement on day 8, mice were euthanized for their brains to be perfused for immunohistochemistry. A Zeiss LSM880 confocal microscope obtained the receptor colocalization images. Analysis of these images was done via FIJI and ImageJ (version 2.1.0). Colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three male mice. Means ± SEM were used to determine statistical significance using a two-way ANOVA and Tukey’s post-hoc analysis. * p < 0.05 compared between drug treatments within No 24 h RST. @ p < 0.05 compared between drug treatments within 24 h RST. # p < 0.05 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative images of CB1R-OX2R colocalization in the VTA (D), NAC (E), and MPFC (F) of males subjected to RST, which associate with graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

Figure 6.
Cannabinoid receptor type 1 and OX2R colocalization in mesocorticolimbic brain regions of male mice that had undergone 24 h RST and drug treatments. (AC), CB1R-OX2R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult male C57BL/6 mice. Succeeding the final sucrose preference measurement on day 8, mice were euthanized for their brains to be perfused for immunohistochemistry. A Zeiss LSM880 confocal microscope obtained the receptor colocalization images. Analysis of these images was done via FIJI and ImageJ (version 2.1.0). Colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three male mice. Means ± SEM were used to determine statistical significance using a two-way ANOVA and Tukey’s post-hoc analysis. * p < 0.05 compared between drug treatments within No 24 h RST. @ p < 0.05 compared between drug treatments within 24 h RST. # p < 0.05 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative images of CB1R-OX2R colocalization in the VTA (D), NAC (E), and MPFC (F) of males subjected to RST, which associate with graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

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Figure 7.
Colocalization of CB1R and OX1R in female mesocorticolimbic brain regions as a result of prolonged restraint stress and drug treatments. (AC), CB1R-OX1R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult female C57BL/6 mice. On day 8, following the final sucrose preference measurement, mice were anesthetized and perfused in order to collect brain tissue for immunohistochemical analysis. Colocalization images were taken using a Zeiss LSM880 confocal microscope. Images were then analyzed on the FIJI extension of ImageJ (version 2.1.0). Colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three female mice. Means ± SEM were calculated, and a two-way ANOVA followed by a Tukey’s post-hoc analysis was used to determine statistical significance. @@@ p < 0.001 compared between drug treatments within 24 h RST. #/### p < 0.05/0.001 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative confocal microscopy images of CB1R-OX1R colocalization in the VTA (D), NAC (E), and MPFC (F) of females subjected to RST, which correspond to graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

Figure 7.
Colocalization of CB1R and OX1R in female mesocorticolimbic brain regions as a result of prolonged restraint stress and drug treatments. (AC), CB1R-OX1R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult female C57BL/6 mice. On day 8, following the final sucrose preference measurement, mice were anesthetized and perfused in order to collect brain tissue for immunohistochemical analysis. Colocalization images were taken using a Zeiss LSM880 confocal microscope. Images were then analyzed on the FIJI extension of ImageJ (version 2.1.0). Colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three female mice. Means ± SEM were calculated, and a two-way ANOVA followed by a Tukey’s post-hoc analysis was used to determine statistical significance. @@@ p < 0.001 compared between drug treatments within 24 h RST. #/### p < 0.05/0.001 compared between No 24 h RST and 24 h RST within drug treatments. (DF), Representative confocal microscopy images of CB1R-OX1R colocalization in the VTA (D), NAC (E), and MPFC (F) of females subjected to RST, which correspond to graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

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Figure 8.
Cannabinoid receptor type 1 and OX2R colocalization throughout mesocorticolimbic brain regions of female mice subjected to 24 h RST and cannabinoid and orexin drug treatments. (AC), CB1R-OX2R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult female C57BL/6 mice. Mice were euthanized, and their perfused brains were collected following the final sucrose preference measurement on day 8. Immunohistochemical analysis was performed on perfused brain slices, which were then imaged using a Zeiss LSM880 confocal microscope. Receptor colocalization analysis was done using FIJI and ImageJ (version 2.1.0). Data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three female mice. Means ± SEM allowed for statistical significance analysis using a two-way ANOVA and Tukey’s post-hoc analysis. (DF), Representative images of CB1R-OX2R colocalization in the VTA (D), NAC (E), and MPFC (F) of females subjected to RST, which associate with graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

Figure 8.
Cannabinoid receptor type 1 and OX2R colocalization throughout mesocorticolimbic brain regions of female mice subjected to 24 h RST and cannabinoid and orexin drug treatments. (AC), CB1R-OX2R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult female C57BL/6 mice. Mice were euthanized, and their perfused brains were collected following the final sucrose preference measurement on day 8. Immunohistochemical analysis was performed on perfused brain slices, which were then imaged using a Zeiss LSM880 confocal microscope. Receptor colocalization analysis was done using FIJI and ImageJ (version 2.1.0). Data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three female mice. Means ± SEM allowed for statistical significance analysis using a two-way ANOVA and Tukey’s post-hoc analysis. (DF), Representative images of CB1R-OX2R colocalization in the VTA (D), NAC (E), and MPFC (F) of females subjected to RST, which associate with graphs (AC), respectively. In the figure above “+” refers to with RST and “‒“ refers to without RST.

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