There is a rapidly growing demand for female animals in preclinical animal, and thus it is necessary to determine animals' estrous cycle stages from vaginal smear cytology. However, the determination of estrous stages requires extensive training, takes a long time, and is costly; moreover, the results obtained by human examiners may not be consistent. Here, we report a machine learning model trained with 2,096 microscopic images that we named the "Stage Estimator of estrous Cycle of RodEnt using an Image-recognition Technique (SECREIT)." With the test dataset (736 images), SECREIT achieved area under the receiver-operating-characteristic curve of 0.962 or more for each estrous stage. A test using 100 images showed that SECREIT provided correct classification that was similar to that provided by two human examiners (SECREIT: 91%, Human 1: 91%, Human 2: 79%) in 11 s. The SECREIT can be a first step toward accelerating the research using female rodents. Knowledge of the precise stages of the estrous cycle is very important for interpretations of female animals' data. Compared to men, women have a high lifetime incidence of several mental illnesses, including depression, posttraumatic stress disorders, generalized anxiety, and eating disorders 1 , but preclinical animal investigations for these illnesses have use mainly males in part because of the volatility of female animals' experimental data that cannot be separated from their estrous cycle. Several research groups have stated that it is difficult to apply the findings from males' preclinical results to women's medicine 2. Indeed, the estrous cycle affects the expression of genes 3 , proteins 4,5 , electrophysiological properties 6,7 , behaviors 8,9 , and drug effects 10. In 2015, the U.S. National Institutes of Health (NIH) announced that when conducting NIH-funded research, researchers should study both sexes 11. The number of preclinical studies using female animals is thus gradually increasing. The estrous cycle in rodents is generally divided into three or four stages 12 , and the cycle is 4-5 days: Diestrus (D) → proestrus (P) → estrus (E) → (metestrus) →. In many studies of rodents, the estrous cycle stage of each animal has been determined by vaginal cytology. Each stage is decided based on the type, number, shape, size, and proportion of cells in a vaginal smear 12-17 (Fig. 1a-c). Briefly, stage D was identified by the presence of leukocytes and nucleated cells with or without a few cornified cells. Stage P was identified by the presence of nucleated epithelial cells and cornified cells without leukocytes. Stage E was identified by the presence of nucleated epithelial cells without leukocytes or cornified cells. Vaginal cytology is also used to decide whether an ovariectomy in a rodent is successful 18,19 (Fig. 1d). However, determining the estrous stage of a rodent by using vaginal cytology evaluated by a human examiner has some problems: (1) a long training period is required in order to become skillful; (2) it takes a long time to determine the estrou...

Feeding behavior is adaptively regulated by external and internal environment, such that feeding is suppressed when animals experience pain, sickness, or fear. While the lateral parabrachial nucleus (lPB) plays key roles in nociception and stress, neuronal pathways involved in feeding suppression induced by fear are not fully explored. Here, we investigate the parasubthalamic nucleus (PSTN), located in the lateral hypothalamus and critically involved in feeding behaviors, as a target of lPB projection neurons. Optogenetic activation of lPB-PSTN terminals in male mice promote avoidance behaviors, aversive learning, and suppressed feeding. Inactivation of the PSTN and lPB-PSTN pathway reduces fear-induced feeding suppression. Activation of PSTN neurons expressing pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide enriched in the PSTN, is sufficient for inducing avoidance behaviors and feeding suppression. Blockade of PACAP receptors impaires aversive learning induced by lPB-PSTN photomanipulation. These findings indicate that lPB-PSTN pathway plays a pivotal role in fear-induced feeding suppression.

The neuronal circuitry for pain signals has been intensively studied for decades. The external lateral parabrachial nucleus (PB) was shown to play a crucial role in nociceptive information processing. Previous work, including ours, has demonstrated that stimulating the neuronal pathway from the PB to the central region of the amygdala (CeA) can substitute for an actual pain signal to drive an associative form of threat/fear memory formation. However, it is still unknown whether activation of the PB–CeA pathway can directly drive avoidance behavior, escape behavior, or only acts as strategic freezing behavior for later memory retrieval. To directly address this issue, we have developed a real-time Y-maze conditioning behavioral paradigm to examine avoidance behavior induced by optogenetic stimulation of the PB–CeA pathway. In this current study, we have demonstrated that the PB–CeA pathway carries aversive information that can directly trigger avoidance behavior and thereby serve as an alarm signal to induce adaptive behaviors for later decision-making.

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