AAV expression and fiber optic placement

Fluorescently labeled cell bodies were found in the ILC (Fig. 2A), and fluorescently labeled axon terminals were found innervating the NAcSh (Fig. 2B). Furthermore, fiber optic placement was confirmed histologically (Fig. 2B), and only those mice that expressed correct placement of both the AAV and the fiber optic cannulae were included. Based on these criteria, three females and three males were excluded due to incorrect placement of either the AAV or fiber optic implants.

BaselineBaseline trial

During the initial baseline trial, mice were naive to the arena and therefore no zone should have held any significance. In the absence of stimulation, the number of entries into each zone was quantified, as well as the mean amount of time spent in each zone. At baseline, there were no significant differences in the mean number of entries into any zone between females (38.81 ± 3.12) and males (44.19 ± 1.20), as determined by a Welch’s t-test (p = 0.12). Similarly, the mean time in seconds (s) spent in any zone was not different between females (285.60 ± 11.98 s) and males (298.70 ± 16.44 s), determined by a Student’s t-test (p = 0.52).

AcquisitionComparison measurements within stimulus frequency, across stimulus frequency, and across sex

For the data presented below there were three major analyses. First, differences at a single stimulus frequency within sex, are denoted by an asterisk (*). The second compared within sex the effect of stimulus frequency, and is denoted by different letters (a, b, c). The third comparison determined sex differences within a single frequency (and sex differences are displayed by blue filled bars in the female dataset).

Sex differences in active zone entries during acquisition trials

During the acquisition trial, we subtracted the average of the number of entries made into the three inactive zones from the number of entries into the acquisition zone to better assess the number of entries attributable to optogenetic stimulation (Fig. 3A, B). At each stimulation parameter we also compared the number of entries into the acquisition zone with the average number of entries into the inactive zones. Across all frequencies, female mice sought the acquisition zone significantly more than the inactive zones, determined by Student’s t-tests and indicated by asterisks on the graph. Specifically, during the 10-Hz trial, females entered the acquisition zone on average 62.67 ± 6.99 times, versus 39.92 ± 3.80 times (p = 0.009) on average for the inactive zones. At 20 Hz, the difference was 144.90 ± 13.88 times for the active zone versus an average of 53.50 ± 4.81 times (p < 0.0001) for the three inactive zones. At 30 Hz, the difference was 214.00 ± 14.36 times in comparison to 65.20 ± 10.04 (p < 0.0001).

Fig. 3

Sex differences in entries made and time spent in the acquisition zone during acquisition. A At all stimulation frequencies, the total number of entries into the acquisition zone were significantly greater than the average number of entries into the inactive zones (indicated by asterisks). There were also significant differences in the number of entries made into the active zone between 10 and 20 Hz, 10 Hz and 30 Hz, and 20 Hz and 30 Hz (denoted by different letters above each bar). At 30 Hz, there were significant differences between the sexes, shown as the colored bar. B Males entered the acquisition zone significantly more than the inactive zones only at 30 Hz. Males displayed no significant differences across frequencies in the number of entries into the acquisition zone. C In females, the total time spent in the acquisition zone was greater than the time spent in the inactive zones across all frequencies. There were also significant differences in the time spent in the acquisition zone between 10 and 20 Hz, and between 10 and 30 Hz. There were sex differences in the amount of time spent in the acquisition zone at 20 Hz and 30 Hz. D Males spent more total time in the acquisition zone versus the inactive zones at all frequencies. However, there were no significant differences in the time spent in the acquisition zone between any of the stimulation frequencies. Representative track plots illustrate travel throughout the duration of the 30-Hz acquisition trial for E females and F males. The acquisition zone is indicated by the blue square. Data presented as mean ± SEM

For comparison of stimulus frequencies, a Welch’s one-way ANOVA was used, as a Brown–Forsythe test detected significant differences in the variance across stimulation frequencies (F(2, 31) = 4.76; p = 0.02). The ANOVA indicated that there were significant differences in the number of entries made into the active zone between the stimulation parameters (W(2, 17.28) = 30.70; p < 0.0001). A Dunnett’s T3 multiple comparisons test found that there were significant differences in the number of entries between 10 Hz (22.75 ± 6.70) and 20 Hz (91.42 ± 12.40; p = 0.0004), between 10 and 30 Hz (148.80 ± 16.44; p < 0.0001), and between 20 and 30 Hz (p = 0.03). Again, differences are denoted by different letters in Fig. 3A.

Performing the same analysis in males, we used Student’s t-tests to compare the number of entries into the acquisition zone with the average of the number of entries into the inactive zones. During the 10-Hz trial, there were no significant differences between entries into the acquisition zone (86.83 ± 20.81) and into the inactive zones (44.08 ± 2.72), but there was a trend toward significance (p = 0.06). The same held true during the 20-Hz trial, with no significant differences, but a trend toward significance, between entries into the acquisition zone (96.88 ± 21.42) versus the inactive zones (50.25 ± 6.74; p = 0.07). Only at 30 Hz did the males enter the acquisition zone (131.60 ± 22.29) significantly more than the inactive zones (60.22 ± 6.29; p = 0.01) (Fig. 3B) Regarding the effects of stimulus frequency, there were no significant differences in the number of entries made into the active zone across the 10-Hz (42.75 ± 20.22), 20 Hz (49.71 ± 17.98), and 30 Hz (71.33 ± 20.28) stimulation parameters (F(2, 25) = 0.56; p = 0.58) (Fig. 3B).

We then sought to determine if there were sex differences in the increased number of entries into the acquisition zone at each stimulation frequency. Student’s t-tests indicated that there were no sex differences at 10 Hz (p = 0.36), and a trend toward significance at 20 Hz (p = 0.06). At 30 Hz stimulation, the difference in the number of entries made into the active zone was significantly greater in females than in males (p = 0.008), as indicated by the colored bar in Fig. 3A.

Sex differences in time in active zone during acquisition trials

As with the number of entries, we subtracted the average of the time spent in the three inactive zones from the time spent in the acquisition zone, and then compared the time in the active zone with the average time spent in the inactive zones across each stimulation parameter (Fig. 3C, D).

In females, at each stimulation frequency, mice spent significantly more time in the acquisition zone as compared with the inactive zones, determined by Student’s t-tests and indicated by asterisks on the graph. During the 10-Hz trial, females spent 403.30 ± 26.97 s in the acquisition zone, versus 271.50 ± 18.52 s (p = 0.0006) in the inactive zones. At 20 Hz the difference was 905.50 ± 56.69 s, in comparison to 168.90 ± 15.63 s (p < 0.0001), and at 30 Hz, 964.90 ± 75.87 s, versus 119.40 ± 20.97 s (p < 0.0001) (Fig. 3C).

For comparison across stimulus frequencies, a one-way ANOVA indicated that there were significant differences in the increase in time spent in the acquisition zone between the stimulation parameters (F (2, 31) = 30.99; p < 0.0001) (Fig. 3C). Tukey’s multiple comparisons test found that the time spent in the active zone was different between 10 Hz (131.80 ± 40.30 s) and 20 Hz (736.60 ± 71.37 s; p < 0.0001) and between 10 and 30 Hz (845.50 ± 94.76 s; p < 0.0001). There were no statistical differences detected between 20 and 30 Hz. Again, the differences across frequencies are indicated by letters.

The same analyses were performed in males, using Student’s t-tests to compare the amount of time spent the acquisition zone, again with significant differences indicated by asterisks. During the 10-Hz trial, there were significant differences between the amount of time spent in the acquisition zone (499.70 ± 74.35 s) as compared with the inactive zones (234.00 ± 26.96 s; p = 0.005), as determined by a Welch’s t-test. The same was found during the 20-Hz trial, with male mice spending more time in the acquisition zone (505.70 ± 85.48 s) than in the inactive zones (256.30 ± 34.62 s; p = 0.02). Finally, a Student’s t-test indicated significant differences between the time spent in the acquisition zone (642.60 ± 85.85 s) as compared with the inactive zones (217.30 ± 33.58 s; p = 0.0009) during the 30-Hz trial (Fig. 3D).

Comparing across frequencies, a one-way ANOVA indicated that there were no significant differences in the increase in time spent in the acquisition zone between the stimulation frequency parameters (F (2, 26) = 0.89; p = 0.42) (Fig. 3D). There were no significant differences between 10 Hz (265.70 ± 88.36 s) and 20 Hz (249.40 ± 114.50 s), between 10 and 30 Hz (425.30 ± 102.50 s), or between 20 and 30 Hz.

Finally, t-tests were utilized to determine if there were sex differences in the increase in time spent in the acquisition zone. During the 10-Hz trial, a Welch’s t-test indicated no differences between females and males (p = 0.18). However, Student’s t-tests indicated significant differences at both 20 (p = 0.001) and 30 Hz (p = 0.008), indicated by the colored bars in Fig. 3C. Representative 30-Hz track plots for female and male mice are shown in Fig. 3E,F.

Sex differences in bouts of optogenetic stimulation during acquisition trials

Similar to the results seen when analyzing the average number of entries, increasing the stimulation frequency significantly increased the number of stimulation trains females earned in the acquisition zone, as determined by a one-way ANOVA (F(2, 31) = 42.68; p < 0.0001) (Fig. 4A). Tukey’s multiple comparisons test found a significant difference in the number of stimulations received between 10 Hz (65.50 ± 6.53) and 20 Hz (149.60 ± 13.57; p < 0.0001), between 10 and 30 Hz (218.80 ± 13.82; p < 0.001), and between 20 and 30 Hz (p = 0.0007). In males, however, increasing the stimulation did not affect the number of stimulations received in the active zone, as determined by a one-way ANOVA (F(2, 26) = 1.19; p = 0.32) (Fig. 4B). There were no significant differences between 10 Hz (90.42 ± 21.01) and 20 Hz (99.25 ± 21.36), between 10 and 30 Hz (135.30 ± 22.49), or between 20 and 30 Hz. Sex differences in the number of stimulation trains received in the acquisition zone were found at both 20 Hz (p = 0.05) and 30 Hz (p = 0.005). In both cases, females received significantly more stimulation. A Welch’s t-test indicated that there were no differences between the number of stimulations received between the sexes at 10 Hz (p = 0.28).

Fig. 4

Sex differences in optogenetic stimulation during acquisition. A In females, there were significant differences in the number of optogenetic stimulation trains received during each of the three frequencies. Sex differences in the number of stimulation trains occurred at both 20 Hz and 30 Hz. B There were no differences in the number of optogenetic stimulation trains received by males across frequencies. C Across all stimulus conditions, there were significant differences in the amount of time female received optogenetic stimulation. At both 20 Hz and 30 Hz, there were also sex differences in optogenetic stimulation time. D In males, increasing stimulation frequency had no effect on the amount of time optogenetic stimulation was received

Sex differences in total stimulation duration during acquisition trials

In females, increasing stimulation frequency significantly increased the total time stimulation was received, as determined by an ordinary one-way ANOVA (F(2, 31) = 32.96; p < 0.0001) (Fig. 4C). Tukey’s multiple comparisons test found significant differences in the amount of time optogenetic stimulation was received between 10 Hz (260.00 ± 25.11 s) and 20 Hz (551.70 ± 39.32; p < 0.0001), between 10 and 30 Hz (716.60 ± 55.33 s; p < 0.0001), and between 20 and 30 Hz (p = 0.02). An ordinary one-way ANOVA indicated that increasing stimulation had no effect in males regarding the amount of time of optogenetic stimulation was received (F(2, 26) = 1.10; p = 0.35) (Fig. 4D). There were no significant differences between 10 Hz (312.20 ± 55.98 s), 20 Hz (338.30 ± 71.50 s), or 30 Hz (437.70 ± 65.46). Comparing between sexes, a Student’s t-test indicated that at both 20 Hz (p = 0.01) and 30 Hz (p = 0.004), females received optogenetic stimulation for a longer amount of time than did males (Fig. 4C). There were no differences at 10 Hz (p = 0.41), as determined by a Welch’s t-test.

No sex differences in locomotor distance and speed during acquisition trials

During all acquisition trials, the mean distance traveled, in meters (m) and mean travel speed, in meters per second (m/s), were analyzed. There were no significant differences in either the distance traveled or rate of travel between females and males during any trial, as determined by Student’s t-tests (Fig. 5). At baseline, there were no differences in distance traveled between females (74.90 ± 4.99 m; Fig. 5A) and males (81.00 ± 2.92 m; p = 0.30; Fig. 5B), nor were there differences in the travel speed between females (0.041 ± 0.003 m/s; Fig. 5C) and males (0.045 ± 0.002 m/s; p = 0.28; Fig. 5D). During the 10-Hz acquisition trial, there were no significant differences in the distance traveled between females (80.92 ± 6.99 m) and males (97.83 ± 9.93 m; p = 0.18). Similarly, the travel speed was not different between females (0.045 ± 0.004 m/s) and males (0.054 ± 0.006 m/s; p = 0.18). During the 20-Hz acquisition trial, again there were no significant differences in the distance traveled between females (137.10 ± 9.67 m) and males (108.50 ± 18.02 m; p = 0.15). Again, the travel speed was not different between females (0.076 ± 0.005 m/s) and males (0.060 ± 0.010 m/s; p = 0.15). Finally, during the 30-Hz acquisition trial, there were no differences in the distance traveled between females (177.10 ± 14.61 m) and males (147.30 ± 12.06 m; p = 0.16). The travel speed was not different between females (0.098 ± 0.008 m/s) and males (0.082 ± 0.007 m/s; p = 0.16).

Fig. 5

No sex differences in distance traveled or travel speed during acquisition. Heightening the stimulation frequency increased the mean distance traveled in A females and B males, though there were no sex differences at any frequency. Similarly, increasing stimulation frequency also increased the travel speed in both C females and D males, again without differences between the sexes

In contrast, increasing the stimulation frequency significantly increased both the mean distance traveled and the mean travel speed within both sexes, as compared with the baseline trial. In females, a Welch’s one-way ANOVA was used, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 46) = 3.99; p = 0.01). The Welch’s ANOVA indicated that there were significant differences in the distance traveled between the stimulation parameters (W(3, 21.90) = 25.50; p < 0.0001). A Dunnett’s T3 multiple comparisons test found significant differences between baseline and 20 Hz (p = 0.0001), between baseline and 30 Hz (p = 0.0002), between 10 and 20 Hz (p = 0.0008), and between 10 and 30 Hz (p = 0.0003) (Fig. 5A). Similarly, a Welch’s one-way ANOVA was used to analyze differences in the travel speed in females, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 46) = 3.95; p = 0.01). The ANOVA indicated that there were statistically significant differences in the travel speed between the stimulation parameters (W(3, 21.89) = 21.75; p < 0.0001). A Dunnett’s T3 multiple comparisons test found that the travel speed was significantly different between baseline and 20 Hz (p = 0.0002), between baseline and 30 Hz (p = 0.0002), between 10 and 20 Hz (p = 0.0009), and between 10 and 30 Hz (p = 0.0003) (Fig. 5C).

Males responded similarly to increases in stimulation frequency. A Welch’s one-way ANOVA was used to analyze the distance traveled, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 41) = 4.08; p = 0.01). The Welch’s ANOVA indicated that there were significant differences in the distance traveled between the stimulation parameters (W(3, 15.09) = 5.08; p = 0.01). A Dunnett’s T3 multiple comparisons test found significant differences between baseline and 30 Hz (p = 0.03) (Fig. 5B). Again, a Welch’s one-way ANOVA was used to analyze differences in the travel speed in males, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 41) = 4.11; p = 0.01). The ANOVA indicated that there were significant differences in the travel speed between the stimulation parameters (W(3, 15.07) = 5.01; p = 0.01). A Dunnett’s T3 multiple comparisons test found significant differences between baseline and 30 Hz (p = 0.03) (Fig. 5D).

ReversalNo sex differences in active zone entries during reversal trials

During the reversal day trials, to better assess the difference in the number of entries made into the previous day’s active zone (“Zone 1”) versus the reversal day’s active zone (“Zone 2”), we subtracted the average of the entries made into Zone 1 from the average of the entries made into Zone 2 (Fig. 6A, B). This aided in analyzing if the mice were able to learn to seek the new active zone, and to begin to parse out behavioral strategies involved.

Fig. 6

Entries made and sex differences in time spent in the reversal zone. A Females entered the reversal zone versus the previously active zone a greater number of times at 20 Hz and 30 Hz. The number of entries into the reversal zone was also greater at 30 Hz than 10 Hz. B Males entered the reversal zone significantly more only at 10 Hz and showed no differences in entries across stimulation frequencies. C Females spent more time in the reversal zone versus the acquisition zone at all three stimulus frequencies. They also spent more time in the reversal zone at 20 Hz and 30 Hz in comparison to 10 Hz. At 30 Hz, there was a sex difference in the time spent in the reversal zone. D In males, there were significant differences in the time spent in the reversal zone as compared with time spent in the acquisition at all stimulus frequencies. However, there were no differences across frequency. Representative track plots illustrate travel throughout the duration of the 30-Hz reversal trial for both E females and F males. The reversal zone is indicated by the solid blue square, and the previous acquisition zone is indicated by the dashed blue square

In females, a Welch’s t-test indicated no differences in the number of entries between Zone 2 (73.50 ± 13.06) and Zone 1 (50.33 ± 6.09; p = 0.13) in the 10-Hz trial. However, Welch’s t-tests indicated that at 20 Hz females entered Zone 2 136.80 ± 17.23 times, versus 70.42 ± 7.17 (p = 0.003) into Zone 1, and at 30 Hz, females entered Zone 2 194.30 ± 24.61 times, as compared with 59.30 ± 7.34 (p = 0.0003) entries into Zone 1 (Fig. 6A).

Comparing across frequencies, a Welch’s one-way ANOVA was used, as a Brown–Forsythe test detected significant differences in the variance across stimulation frequencies (F(2, 31) = 6.17; p = 0.006). The ANOVA indicated that there were statistically significant differences in the number of entries into Zone 2 between the stimulation parameters (W(2, 17.78) = 8.82; p = 0.002) (Fig. 6A). A Dunnett’s T3 multiple comparisons test found significant differences in the number of entries into Zone 2 between 10 Hz (23.17 ± 10.38) and 30 Hz (135.00 ± 26.10; p = 0.005), but not between 10 and 20 Hz (66.42 ± 15.06) or between 20 and 30 Hz (Fig. 6A).

Repeating these analyses in males, Student’s t-tests indicated significant differences between the number of entries made into Zone 2 versus Zone 1 during both the 10-Hz and 30-Hz trials. At 10 Hz, males entered Zone 2 an average of 77.55 ± 7.42 times, versus 47.91 ± 4.96 (p = 0.003) entries into Zone 1. During the 30-Hz trial there was a trend toward significance; males entered Zone 2 an average of 139.40 ± 31.55 times, as compared with 71.78 ± 8.56 entries into Zone 1 (p = 0.06). There were no differences in the number of entries between Zone 2 (99.33 ± 23.05) and Zone 1 (57.38 ± 8.31) during the 20-Hz trial (p = 0.14).

To compare across frequencies, a Welch’s one-way ANOVA was used, as a Brown–Forsythe test detected significant differences in the variance across stimulation frequencies (F(2, 23) = 5.86; p = 0.009). The ANOVA indicated that there were not statistically significant differences in the number of entries into the new active zone between the stimulation frequency parameters (W(2, 10.56) = 1.77; p = 0.22) (Fig. 6B). There were no significant differences between 10 Hz (29.64 ± 8.01) and 20 Hz (41.50 ± 16.66), between 10 and 30 Hz (89.11 ± 30.44), or between 20 and 30 Hz. Student’s t-tests indicated that, unlike during acquisition trials, there were no sex differences in the number of entries at 10 Hz (p = 0.63), 20 Hz (p = 0.32), or 30 Hz (p = 0.27).

Sex differences in time in active zone during reversal trials

We subtracted the average amount of time spent in Zone 1 from the average amount of time spent in Zone 2 during the reversal day to assess the difference in the amount of time spent in the new active zone as compared with the previous active zone (Fig. 6C, D). Comparing within frequencies in females, Welch’s t-tests found significant differences between the amount of time spent in Zone 2 as compared with Zone 1 at all stimulation frequencies. During the 10-Hz trial, females spent 450.00 ± 46.24 s in Zone 2, versus 221.10 ± 19.20 s in Zone 1 (p = 0.0004). During the 20-Hz trial, mice spent 865.50 ± 91.46 s in Zone 2, versus 203.90 ± 34.76 s in Zone 1 (p < 0.0001). Finally, during the 30-Hz trial, females spent 923.40 ± 83.78 s in Zone 2, as compared with 136.20 ± 33.73 s in Zone 1 (p < 0.0001) (Fig. 6C).

Comparing across frequencies, a one-way ANOVA indicated significant differences in the amount of time spent in Zone 2 across stimulation frequencies (F(2, 31) = 8.298; p = 0.0008) (Fig. 6C). Tukey’s multiple comparisons test found significant differences between 10 Hz (228.90 ± 55.68 s) and 20 Hz (661.60 ± 117.80 s; p = 0.008) and between 10 and 30 Hz (787.20 ± 111.80; p = 0.001). There were no differences between 20 and 30 Hz (Fig. 6C).

In males, Welch’s t-tests also indicated significant differences in the amount of time spent in Zone 2 as compared with Zone 1 at all frequencies. During the 10-Hz trial, males spent 560.80 ± 62.35 s in Zone 2 compared with 273.40 ± 37.02 in Zone 1 (p = 0.0008). During the 20-Hz trial, males spent 622.80 ± 135.60 s in Zone 2, versus 226.00 ± 40.46 s in Zone 1 (p = 0.03), and during the 30-Hz trial, mice spent 501.80 ± 88.62 s in Zone 2 as compared with 220.50 ± 26.97 s in Zone 1 (p = 0.01) (Fig. 6D).

Comparing across frequencies, a one-way ANOVA indicated no significant differences in the increase in the amount of time spent in Zone 2 over that spent in Zone 1 across stimulation frequencies (F(2, 23) = 0.30; p = 0.74). There were no significant differences between 10 Hz (287.50 ± 80.52 s) and 20 Hz (396.80 ± 158.20 s), between 10 and 30 Hz (281.30 ± 101.80 s), or between 20 and 30 Hz (Fig. 6D).

Student’s t-tests indicated no differences in the increase in the amount of time spent in Zone 2 between females and males during the 10-Hz (p = 0.55) or the 20-Hz (p = 0.21) trials. Females spent significantly more time in the new active zone than males did during the 30-Hz trial (p = 0.004) (Fig. 6C).

No sex differences in bouts of optogenetic stimulation during reversal trials

A one-way ANOVA indicated that increasing stimulation frequency during the reversal trials significantly increased the number of stimulations females received in the reversal zone (F(2, 30) = 12.51; p = 0.0001) (Fig. 7A). Tukey’s multiple comparisons test found significant differences in the number of stimulations received between 10 Hz (77.75 ± 12.24) and 20 Hz (144.30 ± 16.27; p = 0.02) and between 10 and 30 Hz (205.80 ± 25.71; p < 0.0001). There was a trend toward significance between 20 and 30 Hz (p = 0.06). Performing the same analysis in males indicated that increasing stimulation frequency during the second day of testing significantly had no effect on the number of stimulations received in the reversal zone (F(2, 23) = 1.95; p = 0.16) (Fig. 7B). Tukey’s multiple comparisons test found no significant differences in the number of stimulations received between 10 Hz (84.64 ± 6.84) and 30 Hz (141.90 ± 32.64; p = 0.14), between 10 and 20 Hz (101.70 ± 37.42; p = 0.86) or between 20 and 30 Hz (p = 0.48). There were no differences in the number of stimulations received in the reversal zone between females and males at 10 Hz (p = 0.64), 20 Hz (p = 0.15), or 30 Hz (p = 0.14) stimulation parameters.

Fig. 7

Optogenetic stimulation during reversal trials. A In females, there were significant differences in the number of optogenetic stimulation trains received during reversal trials between 10 Hz versus 20 Hz and 30 Hz. B In males, increasing the optogenetic stimulation frequency did not increase the number of stimulations. C Increasing stimulation frequency in females resulted in differences in the mean amount of time optogenetic stimulation was received, again between 10 Hz versus 20 Hz and 30 Hz. At 30 Hz stimulation, females received significantly more optogenetic stimulation than did males. D In males, there were no significant differences in mean time optogenetic stimulation

Sex differences in total stimulation duration during reversal trials

In females, a one-way ANOVA indicated that increasing stimulation frequency resulted in significant differences in average amount of time optogenetic stimulation was received in the reversal zone (F(2, 30) = 12.02; p = 0.0001) (Fig. 7C). Tukey’s multiple comparisons test found that there were significant differences in the amount of time that optogenetic stimulation was received in the active zone between 10 Hz (284.90 ± 36.68 s) and 20 Hz (522.50 ± 56.30 s; p = 0.007) and between 10 and 30 Hz (656.00 ± 69.42 s; p = 0.0001), but not between 20 and 30 Hz.

In males, a one-way ANOVA found no significant differences in the time optogenetic stimulation was received in the reversal zone across stimulation parameters (F(2, 23) = 0.67; p = 0.52) (Fig. 7D). There were no significant differences between 10 Hz (308.90 ± 24.52 s) and 20 Hz (376.40 ± 86.74 s), between 10 and 30 Hz (389.30 ± 68.82 s), or between 20 and 30 Hz. Finally, there were sex differences in the amount of time optogenetic stimulation was received in the reversal zone only at 30 Hz (p = 0.01), but not at 10 Hz (p = 0.60) or 20 Hz (p = 0.16).

No sex differences in locomotor distance and speed during reversal trials

As during the acquisition trials, the mean distance traveled and the mean travel speed was analyzed during the reversal trials, and again, there were no significant differences between the sexes in either parameter at any stimulation frequency (Fig. 8). During the 10-Hz reversal trial, there were no significant differences in the distance traveled between females (94.26 ± 12.07 m; Fig. 8A) and males (94.30 ± 6.91 m; p = 0.99; Fig. 8B), nor were there differences in travel speed between females (0.052 ± 0.007 m/s; Fig. 8C) and males (0.052 ± 0.004 m/s; p = 0.99; Fig. 8D). There were no differences in the distance traveled between females (137.00 ± 11.65 m) and males (118.50 ± 18.41 m) during the 20-Hz reversal trial (p = 0.39). Similarly, the mean speed was not different between females (0.076 ± 0.006 m/s) and males (0.066 ± 0.010 m/s; p = 0.39). Finally, during the 30-Hz reversal trial, there were no differences in the mean distance traveled between females (161.30 ± 12.72 m) and males (143.10 ± 11.62 m; p = 0.31). Similarly, there were no differences in the travel speed between females (0.090 ± 0.007 m/s) and males (0.079 ± 0.006 m/s; p = 0.31).

Fig. 8

Distance traveled and travel speed during reversal trials. As with the acquisition trials, heightening the stimulation frequency increased the mean distance traveled in both A females and B males, without any differences between the sexes. Similarly, increasing frequency stimulation also increased the travel speed in C females and D males, without sex differences

Similar to the results during the acquisition trials, increasing the stimulation frequency during the reversal trials increased both the mean distance traveled and the mean travel speed within both sexes as compared with the baseline measurements. In females, an ordinary ANOVA indicated that there were significant differences in the distance traveled between the stimulation parameters (F(3, 46) = 15.32; p < 0.0001). A Tukey’s multiple comparisons test found significant differences in the distance traveled between baseline and 20 Hz trials (p = 0.0002), between baseline and 30 Hz (p < 0.0001), between 10 and 20 Hz (p = 0.02), and between 10 and 30 Hz (p = 0.0003) (Fig. 8A). Similarly, an ordinary one-way ANOVA indicated that there were significant differences in the travel speed between the stimulation parameters (F(3, 46) = 15.35; p < 0.0001). A Tukey’s multiple comparisons test found that the travel speed was significantly different between baseline and 20 Hz (p = 0.0002), between baseline and 30 Hz (p < 0.0001), between 10 and 20 Hz (p = 0.02), and between 10 and 30 Hz (p = 0.0003) (Fig. 8C).

In males, a Welch’s one-way ANOVA was used, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 38) = 5.86; p = 0.002). The Welch’s ANOVA indicated that there were statistically significant differences in the mean distance traveled between the stimulation parameters (W(3, 13.09) = 9.56; p = 0.001). A Dunnett’s T3 multiple comparisons test found significant differences between baseline and 30 Hz (p = 0.003) and between 10 and 30 Hz (p = 0.01) (Fig. 8B). Finally, a Welch’s one-way ANOVA was used to analyze differences in the travel speed in males, as a Brown–Forsythe test detected significant differences in the variance of one or more groups (F(3, 38) = 5.72; p = 0.003). The ANOVA indicated that there were significant differences in the travel speed between the stimulation parameters (W(3, 13.07) = 9.37; p = 0.001). A Dunnett’s T3 multiple comparisons test found differences between baseline and 30 Hz (p = 0.003) and between 10 and 30 Hz (p = 0.02) (Fig. 8D) (Additional files 1, 2, 3).

ElectrophysiologySex differences in synaptic strength and intrinsic excitability

Heightened optogenetic self-stimulation behavior in females could be due to sex differences in synaptic strength and/or in intrinsic excitability. First, we examined the strength of the ILC-NAcSh connection via optogenetic local field potentials (oLFP) using ex vivo slice recordings (Fig. 9A). Paradoxical to our behavioral measures, at all tested stimulus durations, male animals exhibited significantly increased oLFP strength in comparison to females (two-way ANOVA, F(1,11) = 15.53, p = 0.002) (Fig. 9B–D). To assess whether the sex differences in glutamatergic synaptic strength were related to differences in MSN excitability, current clamp recordings of NAcSh MSNs were performed in a separate group of animals (Fig. 9E). Contrary to the sex differences in oLFP glutamatergic synaptic strength (males > females), female NAcSh MSN neuronal excitability was greater compared to males across a current injection stimulus response curve (two-way ANOVA, F(1,18) = 4.46, p = 0.0489) (Fig. 9F–H). The maximum firing frequency elicited at + 220 pA current injection was also significantly higher in females compared to males (16.0 ± 1.2 Hz vs 12.5 ± 1.1 Hz, unpaired t-test, p = 0.0435) (Fig. 9I).

Fig. 9

Males exhibit stronger ILC-NAcSh glutamatergic neurotransmission, while females display increased MSN intrinsic excitability. A Neurons in the infralimbic cortex (ILC) in both female and male mice were transfected with an eYFP-labeled AAV expressing channelrhodopsin, and the terminals in the shell of the nucleus accumbens (NAcSh) were optogenetically stimulated ex vivo. The shaded area in the enlarged box indicates recording area. Representative traces of the measured optogenetic local field potentials (oLFPs) in both female (B) and male (C) mice are presented at light stimulation durations of 1 ms (ms) (B: female, light red; C: male, light blue) and 4 ms (B female, dark red; C male, dark blue). D At all stimulus frequencies, males (blue) exhibited a significantly greater glutamatergic response (p = 0.002) than females (red). E Whole-cell current clamp recordings were measured in medium-spiny neurons (MSNs) in NAcSh. The shaded area in the enlarged box indicates recording area. Representative current clamp recordings of female (F) and male G NAcSh MSNs at 160 pA (top) and 200 pA (bottom) depolarizations are presented. Input current elicited significantly more action potentials in females than in males at 160 pA (F top) and 200 pA (F bottom), indicated by the asterisk. H Females (red) exhibited significantly greater intrinsic firing frequencies than males (blue) in response to current injection (p = 0.0489), and post hoc analysis indicated significant differences at 160 pA through 200 pA (p < 0.05). I Females (red) had significantly increased NAcSh MSN action potential firing frequency at maximum current injection (+ 220 pA) compared to males (blue) (p = 0.0435). NAcSh, nucleus accumbens shell; ac, anterior commissure; AAV, AAV adeno-associated virus; ILC infralimbic cortex

Further analysis from the ramp protocol (Fig. 10A–D) revealed sex differences in rheobase, where females required less current injection to fire an action potential (100.6 ± 8.1 pA vs 142.3 ± 10.3 pA, unpaired t-test, p = 0.005) (Fig. 10B). Female NAcSh MSNs also had a shorter duration in time from onset of current injection to firing an action potential (350.3 ± 18.6 ms vs 446.5 ± 23.6 ms, unpaired t-test, p = 0.005) (Fig. 10C). Voltage threshold to firing an action potential (Vt) was not significantly different between females and males (− 37.1 ± 1.0 mV vs − 35.0 ± 0.7 mV, unpaired t-test, p = 0.1064) (Fig. 10D). We also found no sex differences in passive NAcSh MSN membrane properties (capacitance, resting membrane potential, membrane resistance and hyperpolarizing input resistance) (Table 2). Taken together the data indicate that while glutamatergic synaptic inputs from the ILC to the NAcSh may be stronger in males than females, the heightened intrinsic excitability of female NAcSh neurons promotes the observed sex differences in motivated behavior.

Fig. 10

Females have a lower rheobase and shorter time to initiate an action potential compared to males. A Representative ramp current injection traces for females (red, top) and males (bottom, blue). B Females (red) have a significantly lower rheobase compared to males (blue) (p = 0.005). C Females (red) have a significantly shorter time to initiate an action potential compared to males (blue) (p = 0.005). D There is no apparent sex difference in voltage threshold to firing an AP between females (red) and males (blue) (p = 0.1064)