Article Figures & Data
- Fig. 1.
Representative image of a PN26 Golgi-impregnated neuron. A Golgi-impregnated neuron was imaged at 40× magnification. The cell body is outlined, and a dendrite, node, and spine are indicated to illustrate the parameters of neuronal morphology that were included in our analysis.
- Fig. 2.
Activation of both CB1R and CB2R is necessary to masculinize the total frequency of female play behavior. A, B, Administration of ACEA (a CB1-specific agonist) or GP1a (a CB2-specific agonist) to neonatal (PN0–3) females did not alter the total frequency of juvenile rough-and-tumble play behavior (total of all pouncing, pinning, and boxing events from PN28-37). C–E, Neonatal administration of WIN55,212-2 (WIN, a nonspecific endocannabinoid receptor agonist) or coadministration of ACEA and GP1a increased the frequency of play by juvenile females to the level observed in males but did not cause a further increase in male play. B, D, E, Insets show the average frequency of rough-and-tumble play events for each day of analysis. *p < 0.05, **p < 0.01, n = 6–9.
- Fig. 3.
Activation of both CB1R and CB2R is necessary to masculinize female pouncing behavior. A, B, Administration of ACEA, GP1a, or WIN to neonatal (PN0–3) females did not alter the frequency of pouncing events. C–E, Neonatal coadministration of ACEA and GP1a increased female pouncing and decreased male pouncing. Frequency of pouncing is an average over all days of analysis with the average for each individual day shown in the insets (B, D, E). *p < 0.05, n = 6–9.
- Fig. 4.
Activation of both CB1R and CB2R is necessary to masculinize female pinning behavior. A, B, Administration of ACEA or GP1a to neonatal (PN0–3) females did not alter the frequency of pinning events. C–E, Neonatal coadministration of ACEA and GP1a or treatment with WIN increased the frequency of pinning by females to the level observed in males and had no effect on male pinning behavior. Frequency of pinning is an average over all days of analysis with the average for each individual day shown in the insets (B, D, E). **p < 0.01, ***p < 0.001, n = 6–9.
- Fig. 5.
Combined antagonism of CB1R and CB2R is necessary to feminize the total frequency of male play behavior. A, B, Administration of AM281 (a CB1-specific antagonist) or AM630 (a CB2-specific antagonist) to neonatal males (PN0–3) did not alter the total frequency of rough-and-tumble play events. C–E, Coadministration of AM281 and AM630 decreased the total frequency of rough-and-tumble play events by males to the level observed in females and had no effect on female play. Frequency of play is an average over all days of analysis, with the average for each individual day shown in the insets (B, D, E). **p < 0.01, ***p < 0.001, n = 6–10.
- Fig. 6.
Combined antagonism of CB1R and CB2R is necessary to feminize pouncing behavior by males. A, B, Administration of AM281 or AM630 to neonatal males (PN0–3) did not alter the frequency of pouncing behavior. C–E, Coadministration of AM281 and AM630 decreased pouncing behavior by males to the level observed in females and had no effect on female play. Frequency of pouncing is an average over all days of analysis, with the average for each individual day shown in the insets (B, D, E). **p < 0.01, ***p < 0.001, n = 6–10.
- Fig. 7.
Combined antagonism of CB1R and CB2R is necessary to feminize pinning behavior by males. A, B, Administration of AM281 to neonatal males (PN0–3) did not alter the frequency of pinning behavior, whereas treatment with AM630 caused an increase in pinning. C–E, Coadministration of AM281 and AM630 decreased pinning behavior by males to the level observed in females and had no effect on female play. Frequency of pinning is an average over all days of analysis, with the average for each individual day shown in the insets (B, D, E). **p < 0.01, n = 6–10.
- Fig. 8.
Few sex differences are found in neurons in the neonatal medial amygdala. 3D reconstruction of PN4 Golgi-impregnated neurons in the medial amygdala revealed sex differences in the cell body area in both hemispheres (A), but not in number of dendrites (B), number of nodes (C), spine density (D), total dendritic length (E), or average dendritic length (F). *p < 0.05, n = 10 individuals per group, with six neurons per hemisphere reconstructed for each individual.
- Fig. 9.
Factor analysis separates neurons into groups based on observable differences in neuronal morphology. 3D reconstruction of male and female PN26 Golgi-impregnated neurons in the medial amygdala, separation of the neurons into classes based on cell body shape, spine frequency, or numbers of dendrites, and graphing by factor scores for Factor 1 (Dendritic Complexity) vs. Factor 2 (Dendritic Field) shows how classes of neurons cluster together within specific quadrants. Type I neurons (blue triangles) had spindle-shaped cell bodies, 1–3 primary dendrites and clustered within quadrants 3 and 4. Type III neurons (green squares) contained ovoid cell body, 2–5 dendrites, moderate spine density, and clustered within quadrants 2 and 4. Type IV neurons (black circles) had ovoid cell bodies, 3–5 primary dendrites, numerous spines, and clustered within quadrant 1. In each quadrant, tracing for a representative neuron is shown with the corresponding data point indicated with an asterisk.
- Fig. 10.
Juvenile females have greater dendritic complexity in the right hemisphere relative to males. 3D reconstruction of male and female PN26 Golgi-impregnated neurons in the medial amygdala and factor analysis of cell body area, number of dendrites, number of nodes, total dendritic length, average dendritic length, and spine density (A) identified three factors (Dendritic Complexity, Dendritic Field, and Spine Density) depicted with longer aqua (lighter) bars indicating positive loading of a parameter and teal (darker) indicating negative loading. Factor scores for neurons from vehicle males and females were plotted and compared for Dendritic Complexity (B), Dendritic Field (C), and Spine Density (D). **p < 0.01, n = 3–5 individuals per group, with six neurons per hemisphere reconstructed for each individual.
- Fig. 11.
Combined neonatal agonism of CB1R and CB2R uniquely shifted juvenile female dendritic field relative to selective agonism of either receptor. 3D reconstruction of female PN26 Golgi-impregnated neurons in the medial amygdala from animals treated neonatally with ACEA, GP1a, ACEA+GP1a, or vehicle and factor analysis of cell body area, number of dendrites, number of nodes, total dendritic length, average dendritic length, and spine density (A) identified three factors (Dendritic Complexity, Dendritic Field, and Spine Density) depicted with longer light green bars indicating positive loading of a parameter and dark green indicating negative loading. Factor scores for neurons from ACEA, GP1a, ACEA+GP1a, and vehicle females were plotted and compared for Dendritic Complexity (B) and Dendritic Field (C). Exact p values are indicated, n = 3–5 individuals per group, with six neurons per hemisphere reconstructed for each individual.
- Fig. 12.
Neonatal agonism of CB1R and CB2R masculinize juvenile female dendritic field in the right hemisphere. 3D reconstruction of male and female PN26 Golgi impregnated neurons in the medial amygdala from animals treated neonatally with ACEA+GP1a or vehicle and factor analysis of cell body area, number of dendrites, number of nodes, total dendritic length, average dendritic length, and spine density (A) identified three factors (Dendritic Field, Dendritic Complexity, and Nodes) depicted with longer light blue bars indicating positive loading of a parameter and dark navy blue indicating negative loading. Factor scores for neurons from vehicle male and females and ACEA+GP1a-treated females were plotted and compared for Dendritic Field (B) and Dendritic Complexity (C). *p < 0.05, **p < 0.01, n = 3–5 individuals per group, with 6 neurons per hemisphere reconstructed for each individual.
Line Data analyzed Results Data structure Type of test Observed power or confidence interval a Total play: female vehicle, female ACEA, female GP1a F(2, 224) = 0.28, p = 0.756 Normal ANOVA 0.094 b Pouncing: female vehicle, female ACEA, female GP1a F(2, 222) = 0.2.713, p = 0.069 Normal ANOVA 0.533 c Pinning: female vehicle, female ACEA, female GP1a F(2, 210) = 0.699, p = 0.498 Normal ANOVA 0.167 d Number of gridlines crossed: female vehicle, female ACEA, female GP1a F(2, 23) = 1.008, p = 0.383 Normal ANOVA 0.201 e Time spent in the center zone: female vehicle, female ACEA, female GP1a F(2, 23) = 0.262, p = 0.772 Normal ANOVA 0.086 f Total play: male vehicle, male AM281, male AM630 F(2, 208) = 1.314, p = 0.271 Normal ANOVA 0.282 g Pouncing: male vehicle, male AM281, male AM630 F(2, 208) = 2.103, p = 0.125 Normal ANOVA 0.429 h Pinning: male vehicle, male AM281, male AM630 F(2, 187) = 5.483, p = 0.005 Normal ANOVA 0.845 i Pinning: male vehicle, male AM281 p = 0.675 Tukey post hoc –0.4573 to 0.2162 j Pinning: male vehicle, male AM630 p = 0.004 Tukey post hoc –0.7868 to –0.1227 k Number of gridlines crossed: male vehicle, male AM281, male AM630 F(2, 21) = 1.150, p = 0.339 Normal ANOVA 0.221 l Time spent in the center zone: male vehicle, male AM281, male AM630 F(2, 21) = 1.568, p = 0.236 Normal ANOVA 0.289 m Total play: male vehicle, female vehicle, male WIN, female WIN, male ACEA+GP1a, female ACEA+GP1a F(2, 360) = 4.449, p = 0.012 Normal 2-Way ANOVA: sex × treatment interaction 0.762 n F(1, 360) = 4.108, p = 0.043 Main effect of sex 0.524 0 F(1, 360) = 2.275, p = 0.104 Main effect of treatment 0.461 p Total play: male vehicle, female vehicle t(109.2) = 2.666, p = 0.0089 t-test 0.5331 to 3.624 q Total play: female vehicle, female WIN t(127.3) = 2.01, p = 0.0465 t-test 0.023 to 2.919 r Total play: female vehicle, female ACEA+GP1a t(114.7) = 2.071, p = 0.0406 t-test 0.07583 to 3.415 s Total play: male vehicle, female WIN t(112.5) = 0.7378, p = 0.4622 t-test –1.024 to 2.24 t Total play: male vehicle, female ACEA+GP1a t(116.8) = 0.3607, p = 0.719 t-test –1.497 to 2.163 u Total play: male vehicle, male WIN t(83.32) = 0.882, p = 0.3803 t-test –1.182 to 3.066 v Total play: male vehicle, male ACEA+GP1a t(109) = 1.711, p = 0.0899 t-test –3.2 to 0.2348 w Pouncing: male vehicle, female vehicle, male WIN, female WIN, male ACEA+GP1a, female ACEA+GP1a F(2, 359) = 6.914, p = 0.001 Normal 2-Way ANOVA: sex × treatment interaction 0.923 × F(1, 359) = 3.474, p = 0.63 Main effect of sex 0.46 y F(2, 359) = 3.7, p = 0.026 Main effect of treatment 0.677 z Pouncing: male vehicle, female vehicle t(118.7) = 2.347, p = 0.0206 t-test 0.2161 to 2.551 aa Pouncing: female vehicle, female WIN t(131) = 1.358, p = 0.1767 t-test –0.3653 to 1.966 bb Pouncing: female vehicle, female ACEA+GP1a t(119.8) = 2.123, p = 0.0358 t-test 0.09501 to 2.724 cc Pouncing: male vehicle, female WIN t(116.3) = 0.947, p = 0.3456 t-test –0.6366 to 1.803 dd Pouncing: male vehicle, female ACEA+GP1a t(115.3) = 0.038, p = 0.9698 t-test –1.388 to 1.336 ee Pouncing: male vehicle, male WIN t(77.85) = 1.917, p = 0.0589 t-test –0.06252 to 3.323 ff Pouncing: male vehicle, male ACEA+GP1a t(108.9) = 2.069, p = 0.0409 t-test –2.554 to –0.05488 gg Pinning: male vehicle, female vehicle, male WIN, female WIN, male ACEA+GP1a, female ACEA+GP1a F(2, 329) = 3.171, p = 0.043 Normal 2-Way ANOVA: sex × treatment interaction 0.605 hh F(1, 329) = 4.158, p = 0.042 Main effect of sex 0.529 ii F(2, 329) = 3.53, p = 0.03 Main effect of treatment 0.655 jj Pinning: male vehicle, female vehicle t(70.79) = 3.206, p = 0.002 t-test 0.2814 to 1.207 kk Pinning: female vehicle, female WIN t(99.25) = 5.11, p < 0.0001 t-test 0.5536 to 1.256 ll Pinning: female vehicle, female ACEA+GP1a t(89.6) = 2.911, p = 0.0045 t-test 0.1803 to 0.9554 mm Pinning: male vehicle, female WIN t(96.71) = 0.6144, p = 0.5404 t-test –0.6797 to 0.3584 nn Pinning: male vehicle, female ACEA+GP1a t(103.2) = 0.6438, p = 0.5211 t-test –0.367 to 0.7198 oo Pinning: male vehicle, male WIN t(94.97) = 0.002952, p = 0.9977 t-test –0.576 to 0.5777 pp Pinning: male vehicle, male ACEA+GP1a t(105) = 0.4127, p = 0.6807 t-test –0.4773 to 0.7281 qq Number of gridlines crossed: male vehicle, female vehicle, male WIN, female WIN, male ACEA+GP1a, female ACEA+GP1a F(2, 45) = 2.497, p = 0.095 Normal 2-Way ANOVA: sex × treatment interaction 0.471 rr F(2, 45) = 0.39, p = 0.536 Main effect of sex 0.094 ss F(1, 45) = 1.145, p = 0.329 Main effect of treatment 0.237 tt Time spent in the center zone: male vehicle, female vehicle, male WIN, female WIN, male ACEA+GP1a, female ACEA+GP1a F(2, 45) = 0.701, p = 0.502 Normal 2-Way ANOVA: sex × treatment interaction 0.16 uu F(1, 45) = 1.592, p = 0.215 Main effect of sex 0.234 vv F(2, 45) = 0.936, p = 0.401 Main effect of treatment 0.2 ww Total play: male vehicle, female vehicle, male AM281+AM630, female AM281+AM630 F(1,240) = 7.081, p = 0.008 Normal 2-Way ANOVA: sex × treatment interaction 0.755 xx F(1, 240) = 11.592, p = 0.001 Main effect of sex 0.924 yy F(1, 240) = 5.207, p = 0.023 Main effect of treatment 0.623 zz Total play: male vehicle, female vehicle t(126) = 4.011, p = 0.0001 t-test –9.707 to –3.293 aaa Total play: male vehicle, male AM281+AM630 t(105.5) = 3.195, p = 0.0018 t-test –8.584 to –2.01 bbb Total play: female vehicle, male AM281+AM630 t(106.1) = 0.7202, p = 0.473 t-test –4.515 to 2.109 ccc Total play: female vehicle, female AM281+AM630 t(106.2) = 0.2976, p = 7.666 t-test –2.3 to 3.113 ddd Pouncing: male vehicle, female vehicle, male AM281+AM630, female AM281+AM630 F(1, 240) = 0.7072, p = 0.008 Normal 2-Way ANOVA: sex × treatment interaction 0.754 eee F(1, 240) = 13.742, p < 0.0001 Main effect of sex 0.958 fff F(1, 240) = 6.151, p = 0.014 Main effect of treatment 0.695 ggg Pouncing: male vehicle, female vehicle t(124,9) = 4.191, p < 0.0001 t-test –7.361 to –2.639 hhh Pouncing: male vehicle, male AM281+AM630 t(107.5) = 3.225, p = 0.0017 t-test –4.036 to 1.252 iii Pouncing: female vehicle, male AM281+AM630 t(103,6) = 0.8051, p = 0.4226 t-test –3.337 to 1.41 jjj Pouncing: female vehicle, female AM281+AM630 t(107.8) = 0.1472, p = 0.8833 t-test –1.754 to 2.035 kkk Pinning: male vehicle, female vehicle, male AM281+AM630, female AM281+AM630 F(1, 235) = 10.073, p = 0.002 Normal 2-Way ANOVA: sex × treatment interaction 0.885 lll F(1, 235) = 1.538, p = 0.216 Main effect of sex 0.235 mmm F(1, 235) = 2.716, p = 0.101 Main effect of treatment 0.375 nnn Pinning: male vehicle, female vehicle t(122.1) = 3.015, p = 0.0031 t-test –2.532 to –0.5248 ooo Pinning: male vehicle, male AM281+AM630 t(106.5) = 3.251, p = 0.0015 t-test –2.687 to –0.6514 ppp Pinning: female vehicle, male AM281+AM630 t(101.2) = 0.2961, p = 0.7678 t-test –0.8049 to 1.087 qqq Pinning: female vehicle, female AM281+AM630 t(121.8) = 1.189, p = 0.2368 t-test –0.3514 to 1.408 rrr Number of gridlines crossed: male vehicle, female vehicle, male AM281+AM630, female AM281+AM630 F(1, 32) = 0.119, p = 0.733 Normal 2-Way ANOVA: sex × treatment interaction 0.063 sss F(1, 32) = 0.001, p = 0.872 Main effect of sex 0.05 ttt F(1, 32) = 0.071, p = 0.792 Main effect of treatment 0.058 uuu Time spent in the center zone: male vehicle, female vehicle, male AM281+AM630, female AM281+AM630 F(1, 32) = 0.377, p - 0.544 Normal 2-Way ANOVA: sex × treatment interaction 0.091 vvv F(1, 32) = 0.008, p = 0.93 Main effect of sex 0.051 www F(1, 32) = 0.406, p = 0.529 Main effect of treatment 0.094 xxx PN4 cell body area, left hemisphere: male, female t(56.06) = 0.0325, p = 0.0325 Normal t-test –311.4 to –14.03 yyy PN4 cell body area, right hemisphere: male, female t(48.57) = 2.088, p = 0.042 Normal t-test –353 to –6.739 zzz PN4 number of dendrites, left hemisphere: male, female t(73.5) = 0.9388, p = 0.3509 Normal t-test –1.024 to 0.368 aaaa PN4 number of dendrites, right hemisphere: male, female t(68.86) = 1.385, p = 0.1705 Normal t-test –0.2165 to 1.2 bbbb PN4 number of nodes, left hemisphere: male, female t(73.09) = 0.9446, p = 0.348 Normal t-test –1.935 to 0.6905 cccc PN4 number of nodes, right hemisphere: male, female t(71.89) = 0.643, p = 0.5223 Normal t-test –0.5776 to 1.128 dddd PN4 spine density, left hemisphere: male, female t(72.17) = 0.2635, p = 0.7929 Normal t-test –0.01184 to 0.01545 eeee PN4 spine density, right hemisphere: male, female t(72.52) = 0.06693, p = 0.9468 Normal t-test –0.01593 to 0.01489 ffff PN4 total dendrite length, left hemisphere: male, female t(74) = 1.527, p = 0.1309 Normal t-test –64.47 to 8.518 gggg PN4 total dendrite length, right hemisphere: male, female t(72.09) = 0.2125, p = 0.8323 Normal t-test –28.12 to 34.83 hhhh PN4 average dendrite length, left hemisphere: male, female t(71.66) = 1.452, p = 0.1509 Normal t-test –14.13 to 2.221 iiii PN4 average dendrite length, right hemisphere: male, female t(71.1) = 0.82898, p = 0.4094 Normal t-test –11.26 to 4.641 PN26 factor analysis: sex differences in neuronal morphology jjjj Factor 1, left hemisphere: male, female t(40.31) = 0.4664, p = 0.6435 Normal t-test –0.4694 to 0.7511 kkkk Factor 1, right hemisphere: male, female t(28.31) = 3.454, p = 0.0018 Normal t-test –1.479 to –0.378 llll Factor 2, left hemisphere: male, female t(43.65) = 0.2945, p = 0.7698 Normal t-test –0.6725 to 0.5011 mmmm Factor 2, right hemisphere: male, female t(30.22) = 0.7176, p = 0.4785 Normal t-test –0.4041 to 0.842 nnnn Factor 3, left hemisphere: male, female t(42.96) = 0.9127, p = 0.3665 Normal t-test –0.3008 to 0.798 oooo Factor 3, right hemisphere: male, female t(46) = 1.431, p = 0.1592 Normal t-test –0.9151 to 0.1546 PN26 factor analysis: effects of specific and dual agonism on neuronal morphology pppp Factor 1, left hemisphere: female vehicle, female ACEA, female GP1a F(2, 60) = 0.533, p = 0.0589 Normal ANOVA 0.134 qqqq Factor 1, left hemisphere, female vehicle: female ACEA+GP1a t(38.27) = 0.4245, p = 0.6735 Normal t-test –0.4879 to 0.747 rrrr Factor 1, right hemisphere: female vehicle, female ACEA, female GP1a F(2, 60) = 2.099, p = 0.132 Normal ANOVA 0.414 ssss Factor 1, right hemisphere: female vehicle, female ACEA+GP1a t(37.95) = 1.639, p = 0.1094 Normal t-test –1.193 to 0.1255 tttt Factor 2, left hemisphere: female vehicle, female ACEA, female GP1a F(2, 60) = 2.576, p = 0.085 Normal ANOVA 0.494 left hemisphere: female vehicle, female ACEA, female GP1a uuuu Factor 2, left hemisphere: female vehicle, female ACEA+GP1a t(36.6) = 0.2349, p = 0.8156 Normal t-test –0.5315 to 0.6708 vvvv Factor 2, right hemisphere: female vehicle, female ACEA, female GP1a F(2, 60) = 0.235, p = 0.791 Normal ANOVA 0.085 wwww Factor 2, right hemisphere: female vehicle, female ACEA+GP1a t(39.38) = 1.888, p = 0.0664 Normal t-test –0.03766 to 1.099 PN26 dual agonism masculinizes neuronal morphology xxxx Factor 1, left hemisphere: male vehicle, female vehicle, female ACEA+GP1a F(2, 72) = 0.257, p = 0.774 Normal ANOVA 0.089 yyyy Factor 1, right hemisphere: male vehicle, female vehicle, female ACEA+GP1a F(2, 72) = 5.689, p = 0.005 Normal ANOVA 0.849 zzzz Factor 1, right hemisphere: male vehicle, female vehicle p = 0.008 Tukey's Post-hoc –1.3958 to –0.1738 aaaaa Factor 1, right hemisphere: female vehicle, female ACEA+GP1a p = 0.012 Tukey’s post hoc 0.1473 to 1.4254 bbbbb Factor 1, right hemisphere: male vehicle, female ACEA+GP1a p = 1.000 Tukey’s post hoc –0.5597 to 0.5628 ccccc Factor 2, left hemisphere: male vehicle, female vehicle, female ACEA+GP1a F(2, 72) = 0.956, p = 0.775 Normal ANOVA 0.089 ddddd Factor 2, right hemisphere: male vehicle, female vehicle, female ACEA+GP1a F(2, 72) = 1.991, p = 0.144 Normal ANOVA 0.398
- Movie 1.
Representative example of male juvenile social play behavior. Two juvenile males (PN28) were allowed to acclimate to the chamber for 2 minutes, after which video recording was initiated. Instances of pouncing, pinning, and boxing were recorded from time 0 to 10:00.
- Movie 2.
Representative example of female juvenile social play behavior. Two juvenile females (PN28) were allowed to acclimate to the chamber for 2 minutes, after which video recording was initiated. Instances of pouncing, pinning, and boxing were recorded from time 0 to 10:00.
In this issue
