The Influence of the Estrous Cycle on Neuropeptide S Receptor-Mediated Behaviors

Abstract

The neuropeptide S receptor (NPSR) has been identified as a potential therapeutic target for anxiety and post-traumatic stress disorder. Central administration of neuropeptide S (NPS) in male mice produces anxiolytic-like effects, hyperlocomotion, and memory enhancement. Currently, the literature is limited in the number of studies investigating the effects of NPS in female test subjects despite females facing a higher prevalence of anxiety-related pathology, as well as greater risk for adverse effects while taking psychoactive drugs. Moreover, no previous studies have considered the influence of estrous cycle on the effects of NPS. The present study investigates whether NPS-mediated behavioral phenotypes seen in males translate to females, and whether they are affected by estrous cycle stage. Female C57BL/6NCr mice were intracerebroventricularly cannulated and underwent behavioral paradigms to test locomotion, anxiety, and memory. Estrous cycle stage was determined through examination of vaginal cytology. Our results provide evidence that NPS-mediated behaviors are influenced by the estrous cycle. Administration of NPS decreased anxiety-like behaviors more robustly when the female mice were in high estrogen stages of the estrous cycle. Therefore, the desired anxiolytic-like effects of targeting the NPSR are intact in female mice. However, these effects may to be influenced by the stage of the estrous cycle. The NPSR remains a strong potential drug target for new anxiolytic compounds and based on our initial observations further studies exploring the interaction of estrous cycle and the NPS system are warranted. SIGNIFICANCE STATEMENT: The neuropeptide S (NPS) receptor has been identified as a potential target for treating anxiety, a condition that is most prevalent in females. Therefore, the potential interaction of estrous cycle with the NPS system described in the present study is an important first step in understanding the function of the NPS system in females.

Fig. 1.

Experimental timelines. Experiment 2—establish dose-responsiveness of NPS in female mice (aCSF, NPS – 1, 0.1, 0.01 nmol). Experiment 3—test the efficacy of the biased NPSR agonist RTI-263 (aCSF, NPS 1 nmol, RTI-263 1 nmol). Experiment 4—a larger cohort of female mice were used to test the effects of free estrous cycling on NPS administration (aCSF, NPS 1 nmol). After the completion of the ASR paradigm, 20 mice were removed from the main study to verify the voltage necessary to produce reliable effects in the inhibitory avoidance paradigm. The voltage–response relationship, where we test groups of animals with different intensities of electric shock, is completed to guard against cohort-to-cohort variation in the responsiveness to the shock. The remainder of the mice proceeded into the inhibitory avoidance using the optimal voltage as determined by the voltage–response test (see below). Those mice that are used as part of the voltage–response testing are not used for any subsequent studies.

Fig. 2.

NPS-mediated hyperlocomotion. Mice were habituated to the apparatus for 90 minutes. They were then administered vehicle or test compound and returned to the apparatus for testing. Their overall movement was recorded for 60 minutes. Mice that received NPS treatment (1 nmol i.c.v.) displayed significantly higher distance traveled than mice aCSF-treated at the 100-, 110-, 120-, 130-, 140-, and 150-minute time points, P = 0.0027, 0.0005, 0.0003, <0.0001, 0.0002, and 0.0152, respectively (see Results section for analysis details). aCSF, n = 18; NPS, n = 18.

Fig. 3.

Dose–response of NPS effects in female mice. (A and B) After cannulation, but before treatments, mice were used to assess conditions for the light–dark box paradigm. (C) The highest dose of NPS produced a nonsignificant decrease in marble burying in the Hi-E stages. The aCSF-treated mice in the Lo-E stages had a numerically lower number of marbles buried (Hi-E: aCSF, n = 5; NPS–1 nmol, n = 2; 0.1 nmol, n = 5; 0.01 nmol, n = 4; Lo-E aCSF, n = 2; NPS – 1 nmol, n = 7; 0.1 nmol, n = 6; 0.01 nmol, n = 4). (D and E) Inhibitory avoidance: there were no numerical or statistically significant effects observed during training day. There was a significant increase in latency during the Lo-E stages with the highest dose of NPS. However, this appears to be driven by the fact that the Lo-E aCSF mice did not learn the association (Hi-E: aCSF, n = 3; NPS – 1 nmol, n = 8; 0.1 nmol, n = 6; 0.01 nmol, n = 2; Lo-E aCSF, n = 3; NPS – 1 nmol, n = 2; 0.1 nmol, n = 2; 0.01 nmol, n = 3). (F and G) ASR: when mice were acclimatized to the behavioral apparatus 24 hours prior to testing, there were numerical differences in the amplitude of the startle. This was not observed in animals that did not have the opportunity to acclimatize to the apparatus (acclimatized: aCSF, n = 4; NPS – 1 nmol, n = 3; 0.1 nmol, n = 4; 0.01 nmol, n = 4; nonacclimatized: aCSF, n = 5; NPS – 1 nmol, n = 5; 0.1 nmol, n = 6; 0.01 nmol, n = 6). (H) After calculating the AUC for responses in acclimatized mice, there is a numerical decrease in startle amplitude in the highest NPS dose group (∼50%). (I) All the doses of NPS used produced robust hyperlocomotion when administered after a 90-minute habituation period (aCSF, n = 11; NPS – 1 nmol, n = 10; 0.1 nmol, n = 12; 0.01 nmol, n = 12).

Fig. 4.

Comparison of the effects of NPS and RTI-263 in female mice. Comparison of the effect of NPS to that of RTI-263 (aCSF, NPS, 1 nmol; RTI-263, 1 nmol). (A) Hyperlocomotion: female mice were injected intracerebroventricularly after a 90-minute habituation phase. NPS produced the expect hyperlocomotion, and RTI-263 produced an unexpected increase in locomotor activity during the first 20 minutes after injection as compared with aCSF-injected mice (aCSF, n = 18; NPS, n = 18; RTI-263, n = 17). (B) Marble burying: there were no significant effects. However, the expected NPS-mediated decrease in marble burying appeared to be only present in the Hi-E mice. Hi-E: aCSF, n = 7; NPS, n = 8; RTI-263, n = 12; Lo-E: aCSF, n = 2; NPS, n = 5; RTI-263, n = 5. (C and D) ASR: mice previously treated with NPS had lower startle amplitudes during Hi-E stages of estrous. There were no effects of prior treatment in those animals in Lo-E stages of estrous during the testing. Hi-E: aCSF, n = 5; NPS, n = 11; RTI-263, n = 6; Lo-E: aCSF, n = 5; NPS, n = 4; RTI-263, n = 5.

Fig. 5.

Estrous cycle interacts with short-term and long-term effects of NPS administration. (A) Marble burying: mice administered vehicle or test compound 10 minutes before testing. The number of marbles buried was recorded after 45 minutes. Hi-E mice that received NPS treatment buried significantly less marbles than mice treated with aCSF (**P = 0.0021) (aCSF Hi-E, n = 17; NPS Hi-E, n = 18; aCSF Lo-E, n = 17; NPS Lo-E, n = 12). (B and C) Light–dark box: mice were administered vehicle or test compound ten minutes before testing. The time spent on the light side and the number of entries into the light side was measured. (B) NPS-treated mice spent significantly more time on the light side, regardless of estrous stage. (****<0.00001). (C) NPS-treated mice showed a significant increase in the number of entries into the light side, regardless of estrous stage (*P = 0.0024) (aCSF Hi-E, n = 15; NPS Hi-E, n = 9; aCSF Lo-E, n = 14; NPS Lo-E, n = 14). (D–F) ASR: magnitude of startle elicited from 64 sound trials between 70 dB and 125 dB in (D) Lo-E mice and (E) Hi-E mice that had been microinjected with NPS or aCSF twice prior (1 and 2 weeks prior). (F) Previous NPS treatment reduced startle response in female mice, and the mice in Lo-E stages appear to have higher startle magnitudes (aCSF Hi-E, n = 11; NPS Hi-E, n = 10; aCSF Lo-E, n = 5; NPS Lo-E, n = 7). (G and H) Inhibitory avoidance: on training day, mice were trained to associate an electrical shock with the dark compartment and were then administered vehicle or test compound 20 minutes later. Before that day’s treatment, those mice which were in Lo-E age and previously treated with NPS had a significantly higher latency to enter the dark compartment. After 48 hours, on test day, the latency to enter the dark compartment was measured again (aCSF Hi-E, n = 7; NPS Hi-E, n = 11; aCSF Lo-E, n = 9; NPS Lo-E, n = 8).