CaMKIIa+ neurons in the bed nucleus of the stria terminalis modulate pace of natural reward seeking depending on internal state

Abstract

This study aims to investigate the underlying neurobiological mechanisms that regulate natural reward seeking behaviors, specifically in the context of sexual behavior and sucrose self-administration. The role of CaMKIIa+ neurons in the bed nucleus of the stria terminalis (BNST) was explored using chemogenetic silencing and -stimulation. Additionally, the study examined how these effects interacted with the internal state of the animals. Through detailed behavioral analysis, it was demonstrated that CaMKIIa+ neurons in the BNST play a significant role in the regulation of both sexual behavior and sucrose self-administration. Although the behavioral outcome measures differed between the two behaviors, the regulatory role of the CaMKIIa+ neurons in the BNST was found to converge on the modulation of the pacing of engagement in these behaviors in male rats. Moreover, our study confirmed that the internal physiological state of the animal affects how the BNST modulates these behaviors. These findings suggest that different types of natural rewards may recruit a similar brain circuitry to regulate the display of motivated behaviors. Overall, this research provides valuable insights into the neural mechanisms underlying natural reward seeking and sheds light on the interconnected nature of reward-related behaviors in male rats.

Keywords: Bed nucleus of stria terminalis; CAMKIIa; Chemogenetics; Motivation; Natural reward; Sexual behavior; Sucrose self-administration.

Fig. 1

Effects of DREADDs on sexual incentive motivation. (A) Bilateral viral targeting of the bed nucleus of the stria terminalis (BNST), and experimental timeline. Subject males were tested in the sexual incentive motivation (SIM) and copulation test after vehicle and clozapine-n-oxide (CNO) injection. (B) Example of a heatmap of the time spent in each area of the SIM arena with a receptive female stimulus or male control stimulus rat on each side. (C) Example images of DREADD expression. Dotted lines delineate BNST. LV; lateral ventricle, aca; anterior commissure, 3 V; third ventricle. (D) Number of c-Fos+ cells after homecage control condition (HC) or after copulation to one ejaculation (COP). (n = 6) (E) Time spent in each of the stimulus zones in the SIM test. +p < 0.05 compared to female zone. (F) Preference score (time spent in female zone/total time spent in female + male zones). +p < 0.05 compared to 0.50. (G) Time to performance of first copulatory behavior (i.e., mount or intromission). (E, F, G)n = 12 for Sham and Gi bars, n = 11 for Gq bars. (All panels) *p < 0.05

Fig. 2

Effects of DREADD on copulation parameters. (A) Schematic representation of a sexual behavioral cycle of male rats with mounts (M), intromissions (I), ejaculations (E), and post-ejaculatory interval (PEI). The behaviors are divided into mount bouts interrupted by the time-outs (TO). (B) Total number of ejaculations in 30-minute test. (C) Latency to first ejaculation. On an additional note: 1 Sham-CNO, and 3 Gi-CNO rats did not reach an ejaculation and got assigned 1800 s (maximum test duration). Theoretically this could mean that the effect on ejaculation latency could have been underestimated. (D) Number of mounts preceding the first ejaculation (Series 1). (E) Number of intromissions preceding the first ejaculation. (F) Intromission ratio (number of intromissions/number of mounts + intromissions) preceding the first ejaculation. (G) Mean number of intromissions in a mount bout in the first ejaculation series. (H) Mean duration of time-outs in the first ejaculation series. (I) Duration of the first post-ejaculatory interval (time from ejaculation to first copulatory behavior). (J) Percentage of time until first ejaculation spent on non-copulation-oriented behaviors (i.e., every behavior except for copulations, genital grooming, anogenital sniffing, chasing, and head directed to the female). (All panels) Left to right: n = 11, 12, 12, 12, 10, 10; (exception: (I) n = 10, 11, 11, 8, 10, 10). *p < 0.05

Fig. 3

Effects of DREADD on sucrose self-administration. (A) Bilateral viral targeting of the bed nucleus of the stria terminalis (BNST) and experimental timeline. Subject males were tested in sucrose self-administration test under FR1 and PR schedules after vehicle and clozapine-n-oxide (CNO) injections. (B) Number of c-Fos+ cells after homecage control condition (HC) or after 30 min of FR1 engagement (FR1). (HC, n = 4, FR1 n = 5 Sham rats) (C) Number of obtained rewards during 30 min FR1 test. (D) Cumulative rewards in bins of 1 s, plotted as group mean ± s.e.m. in FR1 test. (E) Cumulative distribution of percentage of total obtained rewards over time in bins of 10% of total rewards in FR1 test. (F) Total number of lever pressing bouts in the FR1 test. (G) Average number of active lever presses per lever pressing bout in FR1 test. (H) Mean interval between rewards, calculated for the time between first and last reward in FR1 test. (I) Total number of obtained rewards under progressive ratio (PR). (J) Cumulative rewards in bins of 1 s, plotted as group mean ± s.e.m. in PR test (K) Mean interval time between active lever presses over the total duration of the PR test (C-K)n = 9 for Sham, 11 for Gi, 12 for Gq. Data points FR1 are averages of 2 vehicle- and 2 CNO-tests. (All panels) *p < 0.05

Fig. 4

Effects of short-term social- and food- deprivation on DREADD effect on sexual behavior and sucrose self-administration, respectively (A) Time spent in each of the stimulus zones in the SIM test. +p < 0.05 compared to female zone. (B) Preference score (time spent in female zone/total time spent in female + male zones). +p < 0.05 compared to 0.50. (C) Time to performance of first copulatory behavior (i.e., mount or intromission). (D) Total number of ejaculations in 30-minute test. (E) Latency to first ejaculation. On an additional note: 1 sham_VEH, 3 Gq-VEH, and 2 Gi-CNO rats did not reach an ejaculation and got assigned 1800 s (maximum test duration). Theoretically this could mean that the effect on ejaculation latency could have been underestimated. (F) Mean duration of time-outs in the first ejaculation series. (G) Duration of the first post-ejaculatory interval (time from ejaculation to first copulatory behavior). §p < 0.05 compared to pair housing condition (Fig. 2I) (H) Intromission ratio (number of intromissions/number of mounts + intromissions) preceding the first ejaculation. (I) Mean number of intromissions in a mount bout in the first ejaculation series. (J) Percentage of time until first ejaculation spent on non-copulation oriented behaviors (i.e., every behavior except for copulations, genital grooming, anogenital sniffing, chasing, and head directed to the female). (K) Total number of obtained rewards under progressive ratio. (L) Mean interval time between active lever presses over the total duration of the PR test. (A-H, J) n = 12 for Sham, 12 for Gi, 10 for Gq. (I) from left to right: n = 11, 12, 11, 8, 8, 11 (K, L) n = 8 for Sham, 11 for Gi, 12 for Gq. §p < 0.05 compared to food ad libitum condition (see Fig. 3). (All panels) *p < 0.05