The medial preoptic area (mPOA) differs between males and females in nearly all species examined to date, including humans. Here, using fiber photometry recordings of Ca2+ transients in freely behaving mice, we show ramping activities in the mPOA that precede and correlate with sexually dimorphic display of male-typical mounting and female-typical pup retrieval. Strikingly, optogenetic stimulation of the mPOA elicits similar display of mounting and pup retrieval in both males and females. Furthermore, by means of recording, ablation, optogenetic activation, and inhibition, we show mPOA neurons expressing estrogen receptor alpha (Esr1) are essential for the sexually biased display of these behaviors. Together, these results underscore the shared layout of the brain that can mediate sex-specific behaviors in both male and female mice and provide an important functional frame to decode neural mechanisms governing sexually dimorphic behaviors in the future.
Mating and parental behaviors, though highly dependent on learning and experience in humans and nonhuman primates, are under strong instinctual control in many other species. Males and females of these species often behave differently during mating and toward the young as a result of sexually differentiation of the nervous system1,2,3. In mammals, gonadal secreted steroid hormones, including estrogen and testosterone, act via their receptors to specify irreversible changes in neural structures such as neuronal numbers and connections and to support sex-specific gene expression patterns and neural functions4,5,6,7,8. Yet, despite extensive characterization of sex differences in the brain at molecular and anatomical levels9,10,11,12 and our increased understanding of hormonally regulated genetic and epigenetic programs8, 13,14,15 that drive such sex differences, neural mechanisms governing sexually dimorphic display of behaviors remain poorly understood.
The medial preoptic area (mPOA) of the hypothalamus is a central node in a conserved neural network that regulates social behaviors and social reward16,17,18,19,20, and is anatomically sexually dimorphic in many species, including humans21,22,23. The mPOA expresses at high levels gonadal steroid hormone receptors that regulate sexually differentiation8, 18 and connects to other sexually dimorphic brain areas4, 24,25,26,27. Indeed, neuronal numbers or densities7, 28,29,30,31, synaptic organizations32,33,34, distributions of innervating fibers35, 36, and gene expression patterns8 in the mPOA are all found to be sexually dimorphic. Notably, sexually dimorphic features of the mPOA are sensitive to perinatal hormonal manipulations that also modify sex-specific behaviors4, 32, 36, therefore perhaps reflect sex-specific wirings of the underlying neural circuits that bound an animal to sex-appropriate patterns of behaviors37. Consistent with this notion, the mPOA is found by c-Fos studies to be activated during male-typical copulation and pup care16, 20, 38, both of which are sexually dimorphically displayed1. Furthermore, lesions of the mPOA disrupt these behaviors20, 39,40,41,42,43,44,45.
On the other hand, sex differences in male-typical copulation and parental care are also readily modified by hormonal, sensory, and experiential factors in adult animals5. For example, adult female mice treated with male-typical hormones46 or defect in the processing of vomeronasal information47 copulate females at levels comparable to intact males46. Likewise, male mice, which typically ignore or even attack pups as virgins, do transiently care for them as fathers16. These observations raise the question as how a sexually differentiated brain remains plastic for sex-atypical behaviors. Answers to this question have been largely hindered by the fact that the vast majority of functional studies often included subjects of only one sex.
In this study, we took an unbiased approach to record in vivo activities of the mPOA as well as functionally manipulated genetically defined populations of mPOA neurons during male-typical copulation and pup care in both male and female mice. Intriguingly, we find ramping activities in the mPOA that precede and correlate with sexually dimorphic display of male-typical mounting and female-typical pup retrieval, thereby establishing a direct link between mPOA neural dynamics and sexually dimorphic display of behaviors. Next, we show that optogenetic stimulation of the mPOA elicits similar display of mounting and pup retrieval in both males and females. While two recent studies16, 17 investigating the role of the mPOA in parental care and social reward also used optogenetic approaches to stimulate subsets of mPOA neurons, we here show that the mPOA can drive mounting and pup retrieval in an adult animal. Finally, by means of recording, ablation, optogenetic activation, and inhibition, we demonstrate that mPOA neurons expressing Esr1 are essential for sexually biased display of these behaviors. Together, these results shed new lights on the function of the mPOA and neural organization of sexually dimorphic behaviors in general.
mPOA neural dynamics during sexually dimorphic behaviors
Consistent with previous studies16, we observed stable and quantifiable sex differences in the display of male-typical copulation and pup care in adult virgin C57BL/6 mice that correlated with sex-specific patterns of c-Fos staining in the mPOA (Supplementary Figure 1). To visualize mPOA neural dynamics during behaviors with better temporal resolution, we injected adneno-associated viruses (AAVs) encoding the fluorescent Ca2+ sensor GCaMP6s48 driven by the human synapsin promoter (hSyn) unilaterally into the mPOA of adult C57BL/6 mice and implanted optic fibers (Fig. 1a). This allows us to monitor Ca2+ transients as indicators of neural activities in freely behaving mice, a strategy that has proven to work in the mPOA17 and other brain regions49, 50. Control animals were injected with AAVs encoding EGFP. All recording sites were confirmed post hoc to lie within the mPOA (Supplementary Figure 2a–b).
In experimental animals of both sexes but not the controls, we observed a sharp rise in mPOA activities (average ΔF/F, 32.0 ± 2.7%) upon introduction of females or pups but not inanimate objects into the cage (Supplementary Figure 2c–f), suggesting that the mPOA preferentially processes socially relevant cues. A recent study17 using in vivo two-photon imaging in head fixed female mice has also identified mPOA neurons that encode social cues. After habituation of this initial spike, mPOA activities increased reliably when GCaMP6s injected animals, either male or female, physically or chemo-investigated (sniffed) a female, actions we termed collectively as “social investigations” (Fig. 1b). In complement, we found that social investigations of a female restrained under a barrier also increased mPOA activities (Supplementary Figure 2g). Importantly, investigations of an empty crib or an inanimate object did not change mPOA activities (Supplementary Figure 2g), suggesting that the observed increases in mPOA activities were unlikely motion artifacts. Intriguingly, social investigations that led to subsequent mounting were associated with larger mPOA activities than those that did not (Fig. 1c, d). Since an event of mounting was almost always preceded by an event of social investigation, mPOA activities therefore ramped before the onset of mounting in both sexes and decayed more slowly if mounts transitioned into rhythmic pelvic thrusts in males (Fig. 1e). Moreover, once mounting was initiated, the numbers of mPOA ramping events, as calculated by finding “activity bumps” that were >3 standard deviations above the baseline and lasted for >2 s, strongly correlated with the numbers of mounts, with both being more numerous in males than females (Fig. 1f, g).
Similarly, mPOA neural activities increased reliably when GCaMP6s animals, either male or female, contacted pups, and ramped prior to pup retrieval behavior, which were only displayed by female GCaMP6s animals in these experiments (Fig. 1h–j). Furthermore, the numbers of mPOA ramping events showed some correlation with the numbers of pup retrieval behavior (correlation coefficient = 0.477, p = 0.0752). Meanwhile, no consistent changes in mPOA activities were observed during other components of maternal care such as crouching (Fig. 1k). Together, these results show that mPOA neural activities rise following social investigations of a female and pup contacts and ramp prior to the display of male-typical copulation in both sexes and pup retrieval in females.
mPOA activation elicits male-typical mount in both sexes
To further establish the relationship between mPOA activation and behaviors, we injected AAVs encoding channelrhodopsin 2 (ChR2)51 driven by the hSyn promoter unilaterally into the mPOA of adult virgin C57BL/6 mice and implanted optic fibers (Fig. 2a). Control mice were injected with AAVs encoding mCherry. We first showed by whole-cell patch clamping and by calcium imaging in acute brain slices that light pulses induced firing of action potentials in individual ChR2-expressing neurons and activation of the mPOA at a population level at different stimulation frequencies in manners that were similar between males and females (Fig. 2b–d). Post hoc analysis also confirmed that the viral infection rate was comparable between the two sexes and that the majority of ChR2-expressing neurons expressed c-Fos after light stimulation (Supplementary Figure 3a).
With this approach, we monitored animal behaviors in response to various stimuli in a neutral arena while trains of light pulses were randomly delivered. We first observed that photostimulation produced rapid locomotion that tapered gradually afterward in >90% of ChR2 animals but never in the controls (Supplementary Figure 3b–e), consistent with recent studies16, 17. Strikingly, when we introduced a hormonally primed female as the stimulus, a fraction of ChR2 animals (~50%), either male or female, displayed time-locked mounting behavior within seconds of photostimulation, which occasionally transitioned into rhythmic pelvic thrust (Fig. 2e, Supplementary Figure 3f–j, and Supplementary Movies 1–4). In comparison, mounting behavior displayed by control animals rarely coincided with light stimulation (Fig. 2f). The occurrence of light-induced mounting varied considerably among ChR2 animals but did not differ by the sex (Fig. 2e–g, j). Upon quantification, a subtle sex difference emerged as shown by a longer latency on average for the onset of optogenetically induced mounting in female ChR2 animals (6.5 ± 0.4 s, male vs. 8.5 ± 0.4 s, female; p < 0.001, Fig. 2h, i). Similarly, the fraction of animals that displayed optogenetically induced mounting dropped more steeply in female ChR2 animals with lowered stimulation power (Fig. 2k). Remarkably, light-induced mounting persisted when a male or a young rat, but not a toy mouse, was used as the stimulus or when ChR2 animals were castrated, under which conditions spontaneous mounting almost never occurred (Fig. 2l, Supplementary Figure 4a–b, and Supplementary Movies 5–7). Importantly, spontaneous mounting that happened outside of photostimulation remained sexually dimorphic and was comparable between the ChR2 and the control group in either sex (Fig. 2m). Together, these results show that optogenetic stimulation of the mPOA can bypass hormonal and sensory constraints and drive very similar display of male-typical mating in males and females, demonstrating that both sexes are equipped with functional neural circuits connected to the mPOA to execute such behaviors.
mPOA activation elicits pup retrieval in both sexes
Concurrently, when scattered pups were given as the stimuli, a fraction of ChR2 animals (~70%), either male or female, showed time-locked pup retrieval behavior during light stimulation (Fig. 3a and Supplementary Movies 8–9). This behavior was directed specifically toward pups as beddings were never retrieved. In both sexes, pups were carried in most cases by the trunk and less frequently by head/neck or by extremities during optogenetically induced retrieval, in a manner that resembled spontaneous pup retrieval in virgin females (Fig. 3b). Moreover, if a nest was provided during the test, ~70% bouts of optogenetically induced retrieval resulted in a pup being brought to the nest, a rate that was also comparable to spontaneous pup retrieval by virgin females (Fig. 3c and Supplementary Movie 8). In the absence of a nest, pups tended to be grouped together after a series of optogenetically induced retrieval (Fig. 3d and Supplementary Movie 9). Interestingly, fake pups made of rubber blocks when given as the stimuli were also retrieved or grouped during mPOA optogenetic stimulation (Fig. 3d, Supplementary Figure 4c, and Supplementary Movie 10). The occurrence and onset latency of optogenetically induced pup retrieval were similar between the two sexes (Fig. 3e–h) while the average amount of time spent carrying pups during a photostimulation period was slightly longer in ChR2 males than females (8.5 ± 0.6 s, male vs. 6.8 ± 0.4 s, female; p < 0.05; Fig. 3i). Furthermore, in both sexes optogenetically induced retrieval persisted under lowered power of stimulation or after castration (Fig. 3j–k). By contrast, spontaneous pup retrieval behavior outside of photostimulation remained female biased and was comparable between ChR2 and control animals (Fig. 3l). Together, these results show that optogenetic stimulation of the mPOA elicits similar pup retrieval behavior in both males and females despite female-biased display of such a behavior normally, suggesting the existence of mPOA-connected neural substrates supporting pup retrieval in both sexes, which perhaps underlie facilitation or induction of this behavior by hormonal and experiential factors in virgin or pregnant females and in males52.
mPOA activation correlates with induced behaviors
All animals tested for behaviors were analyzed post hoc to verify viral infection with the majority also stained for light-induced c-Fos expression and measured for serum hormone levels. These analysis confirmed that mPOA was consistently activated across ChR2 animals (Fig. 4a, b) and that optogenetic activation did not alter serum testosterone levels (male, 11 control vs. 41 ChR2: 0.46 ± 0.087 vs. 1.38 ± 0.34 ng ml−1, p = 0.17; female, 15 control vs. 25 ChR2: 0.14 ± 0.0073 vs. 0.13 ± 0.012 ng ml−1, p = 0.53), which remained higher in males in both ChR2 and control groups (two-way ANOVA, p < 0.001). Moreover, by calculating the site that had the densest c-Fos staining along the anterior–posterior, dorsal–ventral, and medial–lateral axes, we simulated an activation center for each ChR2 animal (see “Methods” section) and showed that they predominantly clustered within or close to the mPOA as defined by the atlas (Fig. 4c, http://www.brain-map.org/). In fact, the total number of neurons activated in the mPOA and adjacent areas, as measured by the sum-of-light-induced c-Fos signals in these regions, correlated with light-induced behaviors (Fig. 4d, f). Consistently, optogenetically induced behaviors co-occurred at the animal level (Fig. 4g). In a subset of animals that displayed both optogenetically induced mounting and pup retrieval, we stimulated the mPOA at two different power in the presence of both a female and scattered pups. We found that these animals mounted the female or retrieved pups at random during photostimulation with no apparent bias in either sex (Supplementary Table 1). Occasionally, they even displayed both behaviors in tandem within a single light stimulation (Supplementary Movie 11).
Extensive efforts of varying injection sites and viral amounts failed to further segregate optogenetic-induced behaviors, indicating that neurons responsible for these behaviors likely intermingle closely and may even overlap. Nevertheless, animals whose activation center lied away from the mPOA were less likely to display optogenetically induced mounting or pup retrieval (Fig. 4h), supporting the notion that stimulation of the mPOA drives the display of male-typical mounting and female-typical pup retrieval in both sexes.
mPOA Esr1+ neurons activated during mount and pup retrieval
Given that pan-neuronal activation of the mPOA elicited similar behaviors in both males and females, to explore possible neural mechanisms underlying sexually dimorphic display of mounting and pup retrieval, we targeted a subset of mPOA neurons that express estrogen receptor α (ERα or Esr1), a gene that regulates sexual differentiation and reproductive behaviors4. Esr1+ cells constitute ~50% of mPOA neurons (male, N = 3, 53 ± 5.5%; female, N = 3, 53 ± 3.3%) and overlap with both Vgat and Vglut2, with Vgat+ neurons making up ~80% of all NeuN+/HuCD+ cells in this region (male, N = 3, 78 ± 3.0%; female, N = 3, 76 ± 4.9%) (Supplementary Figure 5a–b). In addition, a fraction of Esr1+ neurons (15–30%), higher in males, express the neuropeptide Galanin and account for about ~50% of the mPOA Galanin+ neurons (Supplementary Figure 5c), which has been shown previously to regulate parental care16.
Utilizing a knock-in Cre line (Esr1-2A-Cre, Esr1Cre) generated previously53, we first validated the fidelity of Cre expression and then injected AAVs encoding Cre-inducible Gcamp6s into the mPOA of Esr1Cre animals to record activities of Esr1+ neurons (Fig. 5a, b, Supplementary Figure 5d, and Supplementary Figure 6a). Similar to mPOA pan-neuronal recordings, activities of Esr1+ neurons increased reliably following social investigations of a female and pup contacts and ramped prior to the onset of mounting in both males and females and pup retrieval in females (Fig. 5c–f). Additionally, events of ramping activities in mPOA Esr1+ neurons strongly correlated with events of mounting, more prevalent in males, and pup retrieval, more prevalent in female (Fig. 5g, h). In comparison, when we injected Cre-silenced Gcamp6s to monitor activities of mPOA non-Esr1+ neurons, much less signals were detected during any phase of social interactions with a female or pups (Supplementary Figure 6b–f, h–i), despite robust activation observed in control wild-type animals injected with the same virus, which now labeled both Esr1+ and non-Esr1+ neurons (Supplementary Figure 6g, j).
In addition, using the method of cellular compartmental analysis of temporal activity by fluorescent in situ hybridization (catFISH)16, 38, we compared populations of mPOA Esr1+ neurons that were activated in response to a female or scattered pups in the same animal. Briefly, animals were allowed two 5-min episodes of social interactions with either a female or scattered pups, separated apart by 30 min, and processed immediately afterward to examine the expression of nuclear and cytoplasmic c-Fos transcripts along with Esr1 (Fig. 6a). Approximately 50% of c-Fos+ cells were Esr1+. By analyzing the percentage of Esr1+ neurons that expressed c-Fos transcripts, either nuclear or cytoplasmic, it was first estimated that ~10–20% Esr1+ neurons were activated by either stimulus. Notably, in animals exposed to the same stimulus twice, ~70–80% of mPOA Esr1+ neurons with nuclear c-Fos also stained positive for cytoplasmic c-Fos, indicating activation of similar neural ensemble (Fig. 6a, b). By contrast, in animals exposed to two different stimuli, only ~30–40% of co-localization was observed (Fig. 6b), which though higher than expected by chance (10–20%) was much lower than the co-localization rate observed in animals presented with the same stimulus, indicating that mPOA Esr1+ neurons activated during social interactions with a female or scattered pups were largely distinct. Interestingly, such a separation was not apparent in non-Esr1+ neurons (Fig. 6c). Taken together, these results show activation of distinct mPOA Esr1+ neurons during social interactions with a female or pups.
mPOA Esr1+ neurons key for sexually dimorphic behaviors
To prove functionally that mPOA Esr1+ neurons regulate mounting and pup retrieval, we injected AAVs encoding Cre-dependent ChR2 or control virus unilaterally into the mPOA of Esr1Cre animals (Fig. 7a, b). Indeed, optogenetic stimulation of mPOA Esr1+ neurons elicited similar display of male-typical mounting and pup retrieval in both males and females with an efficacy that was comparable to pan-neuronal activation (Fig. 7c). Next, we injected AAVs encoding Cre-dependent taCasp354 bilaterally into Esr1Cre animals to ablate mPOA Esr1+ neurons (Fig. 7d). Control animals included Cre mice injected with AAVs encoding mCherry and wild-type littermates injected with the taCasp3 virus. Successful targeting was confirmed by post hoc staining showing reduction of Esr1+ signals in experimental animals compared to the controls (Fig. 7e). Strikingly, ablation of Esr1+ neurons essentially abolished sex-biased display of mounting and pup retrieval by more severely impairing mounting in males and pup retrieval in females (Fig. 7f). Importantly, locomotion, chemoinvestigation, pup contacts, and sexually dimorphic display of territorial aggression were not affected after ablation of mPOA Esr1+ neurons (Supplementary Figure 7). In comparison, ablation of mPOA Vgat + or Vglut2 + neurons, though also greatly reduced the occurrence of male-typical mounting and pup retrieval, failed to affect the sexually dimorphic nature of these behaviors such that pup retrieval remained female biased after ablation of Vgat+ neurons and mounting remained male biased after ablation of Vglut2+ neurons (Supplementary Figure 8). With the caveats of chronic and non-specific effects inherent to lesion studies55 (Supplementary Figure 9), these functional studies strongly implicate mPOA Esr1+ neurons as the key population that underlie sexually dimorphic display of mounting and pup retrieval.
Acute inhibition of mPOA Esr1+ neurons disrupts behaviors
Finally, to test whether activities of mPOA Esr1+ neurons acutely regulate mounting or pup retrieval, we expressed the Guillardia theta anion channelrhodopsin 1 (GtACR1)56 into Esr1+ neurons by injecting AAVs encoding Cre-inducible GtACR1 bilaterally into the mPOA of Esr1Cre animals and implanted optic fibers (Fig. 8a, b). We confirmed by whole-cell patch clamp recording in brain slices that GtACR1-expressing Esr1+ neurons were effectively silenced by repeated delivery of continuous blue light with no detectable rebound activities or cytotoxicity (Fig. 8c, d). Control Esr1Cre animals were injected with AAVs encoding Cre-inducible EYFP. Blue light was delivered either following appetitive approaching behavior or after the initiation of consummatory acts to assess the role of activities in Esr1+ neurons at different phases of social interactions.
During tests of mating behaviors, light stimulation when the tested animals were within one body length distance to a female greatly reduced the percentage of trials that mounting occurred and the numbers of mounts, without decreasing social investigation, in GtACR1 males but not the controls (Fig. 8e). By comparison, delivery of a 10 s pulse of light right after the initiation of mounting only slightly shifted mount duration to be shorter in GtACR1 males but dramatically decreased the fraction of mounts that transitioned into rhythmic pelvic thrusts (Fig. 8f). Furthermore, a 10 s pulse light after the initiation of a rhythmic pelvic thrust drastically decreased the duration of the behavior, such that it more likely terminated during the stimulation period in GtACR1 males compared to the controls (Fig. 8g). In parallel, blocking activities of mPOA Esr1+ neurons also appeared to reduce male-typical mounting in GtACR1 females but the baseline levels were too low to permit meaningful statistical analysis. To circumvent this, we treated females with subcutaneous injection of testosterone to augment male-typical mounting46 and found that light delivery following approaching essentially eliminated male-typical mounting without affecting social investigation in testosterone-treated GtACR1 females but not the controls (Fig. 8h). Taken together, these results demonstrate that activities of mPOA Esr1+ neurons are required for male-typical mounting behavior in both males and females.
Similarly, during test of maternal behaviors, light stimulation in virgin females when the tested animals were within a pre-specified distance to pups significantly decreased the percentage of trials with retrieval behavior and the numbers of pups that were retrieved to the nest, without affecting pup contacts, in GtACR1 animals but not the controls (Fig. 9a). The same stimulation protocol in GtACR1 fathers also appeared to inhibit pup retrieval (N = 2). In complement, a 5 s pulse light stimulation after the initiation of pup retrieval did not terminate or decrease the duration of retrieval in the GtACR1 females but resulted in a dramatic reduction in the rate that a pup was retrieved to the nest (Fig. 9b). By contrast, photoinhibition during crouching had no obvious effects on the behavior (Fig. 9c). Furthermore, we mated females to deliver pups and tested the effects of photoinhibiting mPOA Esr1+ neurons on maternal behaviors in lactating females. With some minor differences on the effect size, essentially all behavioral deficits observed in virgin GtACR1 females with photoinhibition were also found in lactating females (Fig. 9d–f). Thus, activities in mPOA Esr1+ neurons regulate pup retrieval in both virgin and lactating females and in fathers. Taken together, our data support a model that sexually dimorphic activation of mPOA Esr1+ neurons underlie sex-biased display of mounting and pup retrieval.
Despite extensive characterization of sex differences in the brain at the molecular and anatomical levels, neural organization of sexually dimorphic behaviors remains unclear. Here, by recording in vivo activities of mPOA neurons during male-typical mating and pup care as well as functional manipulating genetically defined neuronal populations, we find that the mPOA in a fully sexually differentiated adult animal maintains the capacity to drive either male-typical mounting and female-typical pup retrieval and that sexually dimorphic engagements of mPOA Esr1+ neurons likely underlie the observed sex differences in these behaviors. Our results serve as a foundation for future investigation on neural mechanisms governing sexually dimorphic display of behaviors.
By applying fiber photometry along with viral expression of a genetically encoded calcium indicator, we found that activities of the mPOA, in particular Esr1+ neurons, increased following social investigations of a female or pup contacts, behaviors that are generally considered as appetitive, and ramped preceding consummatory acts of male-typical mounting or pup retrieval. While previous c-Fos studies16, 20 have demonstrated collective activation of the mPOA during male-typical mating and parental care, such studies lacked the necessary temporal resolution to separate out mPOA neural dynamics during different behavioral components. Indeed, results from our in vivo recording experiments, immediately suggest that the mPOA regulates consummatory actions. Supporting this, ablating mPOA Esr1+ neurons or inhibiting their activities dramatically disrupted male-typical mating and pup retrieval without decreasing appetitive social interactions, while activation of the mPOA or mPOA Esr1+ neurons drove male-typical mating and pup retrieval in both sexes. These results are especially interesting when considered in juxtaposition with a previous study53, which has assigned a more central role for Esr1+ neurons in the ventrolateral division of the ventromedial hypothalamus (VMHvl) in controlling social investigations toward females. VMHvl reciprocally connects with the mPOA. Activation of VMHvl Esr1+ neurons was both necessary and sufficient for close investigations toward a female, and was sufficient but not necessary for male-typical mounting. In fact, VMHvl neural activities declined during consummatory phase of male mating38. Furthermore, unlike the mPOA, acute inhibition of the VMHvl or VMHvl Esr1+ neurons could not disrupt male-typical mating38, 53. Thus, it seems that the mPOA is more important for consummatory acts of mating and pup retrieval while VMHvl, perhaps along with other inputs to the mPOA, controls appetitive social interactions and activation of the mPOA during such phases.
It is rather striking that optogenetic stimulation of the mPOA elicited the display of mounting and pup retrieval that were to the large extents similar between males and females. Importantly, spontaneous display of these behaviors outside of photostimulation periods as well as serum testosterone levels remained sexually dimorphic, suggesting that ChR2 animals still behaved normally according to their sex without photostimulation. Optogenetic stimulation experiments are “gain of function,” therefore should be interpreted with caution. Nevertheless, one revealing implication extended from these results is that an adult animal still maintains the ability to behave like the opposite sex along with sex-specific patterns and connections in the mPOA and other brain regions. Latent but functional neural circuits that are capable of driving behaviors typically observed in the opposite sex has also been found in female fruit flies57, 58, suggesting the possibility of an evolutionarily conserved coding scheme for sexually dimorphic behaviors. Such a neural circuitry scheme can permit stable yet plastic control of sex differences in behaviors amidst motor functions that are largely shared by the two sexes. According to this view, functionality of molecular and anatomical sex differences identified should be viewed more broadly.
While the mPOA targeted in our study is enriched for the expression of Esr1 and is anatomically separable from other preoptic nucleus such as the ventrolateral preoptic nucleus (VLPO) important for sleep and temperature regulation59, 60, one outstanding question remains whether there are subsets of mPOA Esr1+ neurons that are specialized and dedicated to the control of male-typical mounting vs. pup retrieval. Noteworthy, the issue of functional heterogeneity prevails across hypothalamic studies16, 38, 53, 61. Although results from the catFISH experiment suggest that mPOA Esr1+ neurons activated in response to a female or pups are largely distinct, the usage of c-Fos transcripts as indicators of neural activities again imposed a limit on the temporal resolution to separate out neurons engaged during appetitive and consummatory social interactions. Future studies using optogenetics assisted single-unit recordings (Opto-tagging)60 or GRIN lenses recordings62 are required to resolve these issues more precisely. Moreover, as manipulation of mPOA Esr1+ as a whole always affected both male-typical mounting and pup retrieval, further partition of this population along the axes of inputs and outputs, genetic markers or neuronal activities using newly developed tools60, 63,64,65,66,67 and functional studies of each subset will complete the picture on how mPOA Esr1+ neurons regulate the display of behaviors in either sex. Along this line, we will achieve a firmer understanding on how sex-specific wiring in subsets of mPOA Esr1+ neurons or changes in their gene expression patterns or excitabilities could render male-typical mating or pup retrieval pathway more or less accessible in one sex vs. the other, thereby lead to sexually dimorphic display of these behaviors. In summary, our study represents an important initial step in efforts to systematically dissect neural control of sexually dimorphic behaviors in vertebrates.
C57BL/6J animals were purchased from Jackson Laboratory, Slac Laboratory Animal (Shanghai) and bred in house. Esr1-2a-Cre (Esr1Cre, #017913) and Vglut2-Ires-Cre (Vglut2Cre, #012569) were purchased from the Jackson Laboratory. The Vgat-Cre (VgatCre) line was previously generated68. All Cre lines were bred onto C57BL/6J background for at least one generation. Animals were housed in the Institute of Neuroscience animal facility on 12 h:12 h light/dark cycle with food and water ad libitum. Only heterozygous animals of Cre alleles were used in the study. All experimental protocols were approved by the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China (IACUC No. NA-016-2016).
Adult mice 2–4 months old were anesthetized with isoflurane (0.8–5%) and placed in a stereotactic frame (David Kopf Instrument, Model 1900). Ophthalmic ointment was applied to prevent dehydration. The skull was exposed with a small incision and holes were drilled to inject virus with glass pipettes (15–25 μm in diameter at the tip) and to implant optic fibers. The coordinates for viral delivery into the mPOA were AP, −0.16 mm; ML, ±0.4 mm; DV, −5.150 mm (Paxios and Franklin mouse brain atlas, 2nd edition). 100–400 nl of virus was injected per side at a flow rate of 70 nl per min using a homemade nanoliter injector or at 40 nl per min using a hydraulic pump (Harvard Apparatus). Glass pipettes were left in place for about ~10 min after the injection before retraction. Mono optic fibers (diameter, 200 μm; N.A., 0.37; length, 6 mm; AniLab Software and Instruments Co., Ltd) or dual optic fibers (DFC_200/245–0.37_6.0mm_DF0.9_FLT; Doric Lense) were placed 400 μm above the virus injection site for optogenetic experiments or 50 μm above for fiber photometry recordings. Optic fibers were secured with dental cement and skull screws. Littermates were randomly chosen to be injected with either experimental or control virus. Castration surgeries were performed with animals anesthetized with intraperitoneal (i.p.) injection of ketamine (80 mg kg−1) and xylazine (8 mg kg−1). Animals were allowed 3–4 weeks to recover after surgeries before subsequent behavioral tests.
The following viruses were used in this study: AAVs-hSyn-ChR2-mCherry (Serotype 2/8, titer 2.26 × 1013 genomic copies per ml), AAVs-hSyn-mCherry (Serotype 2/8, titer 3.38 × 1012 genomic copies per ml), AAVs-Ef1a-DIO-ChR2-mCherry (Serotype 2/8, titer 7.39 × 1012 genomic copies per ml), AAVs-Ef1a-DIO-mCherry (Serotype 2/8, titer 8.93 × 1012 genomic copies per ml), all purchased from Obio Technology Co. (Shanghai); AAVs-Ef1a-DIO-EYFP (Serotype 2/9, titer 8.6 × 1012 genomic copies per ml), AAVs-hSyn-GCaMP6s (Serotype 2/9, titer 8.8 × 1012 genomic copies per ml), AAVs-hSyn-EGFP (Serotype 2/9, titer 4.52 × 1012 genomic copies per ml); AAVs-hSyn-DIO-GCaMP6s (Serotype 2/9, 3.85 × 1012 genomic copies per ml), AAVs-hSyn-DO-GCaMP6s (Serotype 2/9, titer 1.18 × 1013 genomic copies per ml), AAVs-CAG-DIO-GtACR1-P2A-EGFP (Serotype 2/5, titer 7.5 × 1012 genomic copies per ml), all produced by Shanghai Taitool Bioscience, Co. Ltd; and AAVs-Ef1a-DIO-taCasp3.Tevp (Serotype 2/5, titer 4.2 × 1012 genomic copies per ml) purchased from University of North Carolina Gene Therapy Center VectorCore.
All behavioral tests were initiated at least half an hour after the onset of the dark cycle, and were recorded using an infrared camera at a frame rate of 25 Hz and scored by experimenters blinded to the genotype, group information, or photostimulation status of the animals involved. All animals tested for behaviors, except for those used in optogenetic inhibition experiments, were naive virgin mice prior to the test. In optogenetic inhibition experiments, all male mice were co-housed with female mice and some of them became fathers before testing while some females were co-housed with pups, or mated to become mothers, or treated with weeks of testosterone injection before testing. Except for animals used in optogenetic activation experiments, all animals were singly housed 3–7 days before behavioral testing.
For mating behavior tests, a hormonal primed ovariectomized C57BL/6 female was introduced to the home cage and videotaping for 30 min. Mating behavior tests were repeated three times with different stimulus animals at least three days apart. Parental behavior was tested by scattering three pups age between P1 and P4 at the edge away from the nest and videotaping the animal for ~15 min and were repeated 2–3 times. Territorial aggression was tested by introducing an intruder mouse of Balb/c stain purchased from Slac Laboratory Animal (Shanghai) to the home cage of the tested animal and videotaping for ~15 min.
Videos were manually annotated on a frame-by-frame basis using a custom-written MATLAB program as previously described8. Behaviors were scored according to the following criteria: nose-to-face, nose-to-body, or nose-to-urogenital contacts initiated by the tested animals are scored collectively as “social investigation,” of which nose-to-urogenital contact is specifically defined as “chemoinvestigation,” also known as “sniff.” “Mount” is scored when the experimental animal places its forelimbs on the back of the stimulus, climbs on top and moves the pelvis. Rhythmic pelvic movements after mount are scored as “Pelvic thrust.” Actions initiated by the resident toward a male intruder including lunges, bites, tumbling are scored as “Attack.” “Pup contact” is scored when an animal contacted a pup with its nose or mouth. “Pup retrieval” is scored when an animal holds the pup with its mouth and traveled, usually toward the nest. “Crouching” is scored when the animal arches its back and hovers over pups in the nest. Pups were always inspected after behavioral trial for possible wounds. About 2% of behavioral trials resulted in wounded pups. Exclusion of these trials did not influence any conclusion.
Animals used for optogenetic experiments were group housed. On the day of the behavioral test, they were introduced to a fresh cage. An external optic fiber was used to connect a 473 nm laser power source (Shanghai Laser and Optics Century Co. or Changchun New Industries Optoelectronics Tech Co., Ltd.) to the implanted optic fiber in the animal. The external optical fiber was attached to a rotary joint (FRJ_1 × 1_FC_FC, Doric Lense) to allow the animal to freely behave. The tested animal was allowed to explore the cage for ~15 min with the external fiber attached. Afterward, a custom MATLAB program was started to control a Master 9 (A.M.P.I.), which sent a trigger to initiate recording from the camera and signals to the laser to deliver 15 s of photostimulation at 40 Hz 12 mW or 20 Hz 5 mW, spaced 90–120 s apart at random. The laser power was adjusted for each animal according to the luminance transitivity efficiency of the implanted optical fiber measured prior to implantation to assure the power of the light emitted at the tip of the fiber. During laser stimulation, animals were either tested alone for locomotion, or with a hormonal primed ovariectomized female, a C57BL/6 male, a young rat age of P13–P15, pups age of P1–P4, or rubber blocks of similar size of pups as the stimulus. Five–twelve stimulations were given during each assay. One–four assays with each stimulus (3–5 min apart) were performed on any given day for each animal. After completion of all behavioral testes, animals were given trains of photostimulation (15 s, 12 mW, 40 Hz, 10 times, a fixed 105 s interval) and perfused transcardially with 4% paraformaldehyde one hour after light stimulation for histological analysis. Blood was also collected at the time of killing for hormonal measurements.
Animals used for fiber photometry experiments were singly housed 3–7 days before behavioral testing. During the day of testing, the implanted optic fiber was connected to F-scope (Biolink Optics Technology Inc., Beijing), an integrated fiber photometry recording setup, through an external optical fiber. In the F-scope, a 488 nm excitation laser (OBIS 488LS; Coherent) is reflected off a dichroic mirror (MD498, Thorlabs). The emission signals collected through the implanted optic fiber are filtered with a bandpass filter (MF525-39, Thorlabs) and directed onto a photomultiplier tube (PMT, R3896, Hamamatsu). During recordings, the laser power was adjusted for each animal according to the luminance transitivity efficiency of the implanted optical fiber to assure that light emitted at the tip of fiber was ~30 μw and the PMT voltage gain was set to 500 v for GCamp6s animals and 300–350 v for control animals. Once started, the F-scope software sent a trigger signal to initiate video recording from the infrared camera. Emission signals were low-pass filtered at 30 Hz and were sampled at 500 Hz with a data acquisition card (USB6009, National Instrument) using a software provided by the Biolink Optics. For a given trial, animals were first allowed to explore for ~5 min, during which time baseline signals were recorded. Afterward, a stimulus such as a hormonal primed ovariectomized female, a fake plastic mouse toy, pups age of P1–P4 or rubber blocks of similar size of pups were introduced. Animals were allowed to interact and behavior with the stimuli for 15–90 min before removal of the stimuli, after which signals were recorded for another ~5 min.
Animals used for optogenetic inhibition experiments were single housed. Male mice were co-housed with females to be sexually experienced or to become fathers before testing. Virgin female mice were tested as naive animals for male-typical mating and for maternal behaviors. In cases of poor baseline maternal behaviors during the initial test, they were co-housed with pups overnight and tested again for maternal behaviors. After testing of maternal behaviors, some females were treated every other day with subcutaneous injection of testosterone (100 μg in 50 μl sunflower oil, Shanghai Pharm.) for about 3 weeks before being tested again for male-typical mating. Females tested as mothers were mated after viral injection to produce their own pups.
Before the behavioral test, a dual fiber-optical patch cord (DFP_200/230/900–0.37_1m_DF0.9_2FCM, Doric Lense) was used to connect a 473 nm laser power source (Shanghai Laser and Optics Century Co. or Changchun New Industries Optoelectronics Tech Co., Ltd.) to the implanted dual optic fiber (DFC_200/245–0.37_6.0mm_DF0.9_FLT, Doric Lense) in the animal via a rotary joint (FRJ 1 × 2i FC-2FC 0.22, Doric Lense) to allow the animal to behave freely. After ~10 min of habitation, the camera was triggered to record the behavioral process by a signal sent from Master 9, which was controlled by a custom written MATLAB program. To deliver light after approaching, light (~12 mW) was automatically triggered to be continuously delivered as long as the positions of the tested animal were detected by the program to be within one body length to the hormonal primed ovariectomized female in mating behavior tests or within the pup region, which was defined by marking a slight larger area surrounding each scattered pup in maternal behavior tests. At least two light trials and two no light trials were performed alternatively for each test. To deliver lights after initiation of defined behaviors, blue light (~12 mW) of fixed length (5 or 10 s) was manually triggered when the experimenter observed the behavior of interests in real-time. One or two light trials were performed alternatively for each test.
Animals were anesthetized with 10% chloral hydrate and transcardially perfused with PBS followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Afterward, brains were sectioned at 40 μm thickness using a vibratome (VT1000S, Leica). Sections were divided equally into several sets and processed for staining or mounted directly. Brain sections were blocked in 5% goat serum in AT (0.1% Triton and 2 mM MgCl2 in PBS) for 1 h at room temperature and incubated overnight at 4 °C in AGT (0.5% normal goat serum, 0.1% Triton and 2 mM MgCl2 in PBS) containing appropriate primary antibody (rabbit anti-c-Fos, 1:2000, Santa Cruz, Cat# sc‐52; rabbit anti-Esr154, 1:10,000, Millipore, Cat# 06-935). The next day, brain sections were washed with AGT for three times (30 min each) and incubated with appropriate secondary antibodies (Alexa Fluor 488 conjugated, 1:1000; Cy3 conjugated, 1:1000, Jackson ImmunoResearch) at room temperature for 2 h. Brain sections were counterstained with Neuro Trace (Life Technologies, Cat# N21479, 1:300) or DAPI (5 mg ml−1, 1:1000, Sigma) in AT. After several washes in AGT, AT and PBS, brains sections were mounted onto glass slides.
Brain sections were prepared similar to fluorescent immunostaining experiments and were pretreated with 3% H2O2 for 30 min at room temperature to block the endogenous peroxidase, washed twice for 10 min each in PBST (0.3% Triton X-100 in PBS), blocked with 5% goat serum in PBST for 2 h at room temperature, and incubated with primary anti-c-Fos antibody (Rabbit anti-c-fos, 1:2000, Santa Cruz, Cat# sc‐52) in PBST overnight at 4 °C. The next day, sections were washed six times in PBST, 10 min each, and then incubated with the biotin-conjugated goat anti-rabbit secondary antibody (Cat#111-065-003, Jackson ImmunoResearch) in PBST for 2 h at room temperature. After three washes in PBS, 5 min each, brain sections were stained with VECTASTAIN® ABC Reagent for 30 min following the manufacturer’s manual. After two 5-min washes in PBS, brain sections were incubated in 3,3-diaminobenzidine (Cat# D5637-5G, Sigma) solution with nickel intensification until achieving desired staining intensity. Reaction was stopped by rinsing sections in tap water. Brain sections were mounted onto glass slides and captured under ×4 or ×10 objectives with conventional light microscopes.
In situ hybridization
DNA templates for generating in situ probes were cloned using the following primer sets for each corresponding gene: Vgat, 5′-gccattcagggcatgttc-3′ and 5′-agcagcgtgaagaccacc–3′; Vglut2, 5′-atcgactagtccaaatcttacggtgctacctc-3′ and 5′-atcgctcgagtagccatctttcctgttccact-3′; Cre, 5′- ccaatttactgaccgtacacca-3′ and 5′-tatttacattggtccagccacc-3′; GAD1, 5′-cattgaggagatagagaggttg-3′ and 5′-agagaagagcgaaggctact-3′; Galanin, 5′-atcctgcactgaccagcc-3′ and 5′-ttggcttgaggagttggc-3′. Anti-sense RNA probes were transcribed with T7 RNA polymerase (Promega, Cat# P207E) and digoxigenin (DIG)-labeled nucleotides. Animals were anesthetized with 10% chloral hydrate and transcardially perfused with DEPC-treated PBS (D-PBS) followed by ice-cold 4% paraformaldehyde (PFA) in D-PBS. Afterward, brains were sectioned at 40 μm thickness using a vibratome (VT1000S, Leica). Brain sections were washed in 2XSSC buffer containing 0.1% triton for 30 min, acetylated in 0.1 M triethanolamine (pH 8.0) with 0.25% acetic anhydride (vol/vol) for 10 min, equilibrated in pre-hybridization solution for 2 h at 65 ℃ and subsequently incubated with 0.5 μg ml−1 of specific RNA probes in hybridization buffer overnight at 65 ℃. The next day, sections were rinsed in pre-hybridization solution and pre-hyb/TBST (TBS with 0.1% tween-20) for 30 min each. Next, sections were washed with TBST for twice and TAE for three times, each for 5 min. Sections were then transferred into wells in 2% agarose gel, which were run in 1XTAE at 60 V for 2 h to remove unhybridized probes. Sections were then washed twice in TBST, and subsequently incubated with sheep anti-digoxygenin-AP (1:2000, Roche, Cat# 11093274910), sometimes together with rabbit anti-Esr1 antibody (1:3000, Millipore, Cat# 06–935) for co-staining purposes, in 0.5% blocking reagent (Roche, 11096176001) at 4 °C overnight. On the second day, for brightfield staining sections were washed and stained with NBT (Roche, Cat# 11383213001) and BCIP (Roche, Cat# 11383221001) for 4–10 h at 37 ℃. For fluorescent in situ hybridization combined with immunohistochemistry, sections were stained with fluorescent secondary antibodies first, followed by staining with fast red (HNPP Fluorescent Detection Set, Roche, Cat# 11758888001) and counterstaining with DAPI (5 mg ml−1, 1:1000, Sigma) in PBS. All sections were washed after staining and mounted on glass slides. Images were captured with ×20 objective using a conventional or confocal microscope.
Most animals used in the catFish experiment were experienced. For the procedure, animals were allowed two 5-min episodes of social interactions with a hormonal primed ovariectomized female or scattered pups, 30 min apart. Immediately after the second episode, animals were anesthetized with 10% chloral hydrate and transcardially perfused with DEPC-PBS followed by ice-cold 4% PFA in PBS. Brains were dissected and post-fixed over night at 4 °C and dehydrated with 30% sucrose in DPEC-PBS. Afterward, brains were sectioned at 20 μm thickness and mounted to SuperFrost Plus® Slides (Fisher Scientific, Cat. No. 12-550-15). After drying in the air, slides were stored in −80 °C before being processed according to RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual (ACD Bio.). Probes against c-Fos intron, c-Fos mRNA, and Esr1 were ordered from ACD Bio. and used in the experiment. Images were captured under a ×60 objective using a confocal microscope.
C57BL/6 animals injected with AAVs encoding ChR2-mCherry in the mPOA or Esr1Cre mice injected with AAVs encoding GtACR1 were anesthetized with isoflurane and transcardially perfused with ice-cold oxygenated (95% O2/5% CO2) high-sucrose solution (composition in mM: 2.5 KCl, 1.25 NaH2PO4, 2 Na2HPO4, 2 MgSO4, 213 sucrose, 26 NaHCO3). After dissection of the brain, coronal sections including the mPOA were cut at 250 μm using a vibratome (VT-1200S, Leica) in ice-cold oxygenated cutting solution. Brain slices were then incubated in artificial cerebrospinal fluid (ACSF; composition in mM: 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.25 Na2HPO4, 2 MgSO4, 10 glucose, 26 NaHCO3, 2 CaCl2) at 34 °C for at least 1 h and recorded at room temperature. Cells were identified under fluorescent microscope and visualized by infrared differential interference contrast (BX51, Olympus). Whole-cell current clamp recordings were accomplished with a MultiClamp700B amplifier and Digidata 1440A interface (Molecular Devices). The patch-clamp electrode (5–8 MΩ) was backfilled with an intracellular solution (composition in mM: 120 K‐gluconate, 4 KCl, 10 HEPES, 10 sodium phosphocritine, 4 Mg‐ATP, and 0.3 Na3‐GTP, pH:7.3, 265 mOsm). For optogenetic activation, blue light (473 nm, 10 ms width, 14 mw mm−2, 40 pulses) was delivered onto slices through ×40 objective using an X-Cite LED light source (Lumen Dynamics). The spike fidelity in ChR2-expressing neurons was measured by counting the number of light pulses that successfully evoked action potentials at different frequencies. Spike fidelity was averaged across five stimulations and plotted. For optogenetic inhibition, the action potential of GtACR1-expressing neurons was induced by withholding the membrane potential −48 mW with a current injection and repeated continuous blue light was delivered onto slices.
Calcium imaging in brain slices
AAVs encoding ChR2 and AAVs encoding GCamp6s, both driven by hSyn, were co-injected into the mPOA of C57BL/6 animals. Acute brain slices including the mPOA were prepared similar to the electrophysiological procedures described above. Calcium imaging was carried out using two photon laser-scanning microscope (Ultima, Prairie Instruments Inc.) with a 20X/0.95-NA XLUMPLFL water-immersion objective (Olympus). The Ti:sapphire laser was tuned to 940 nm, and images of size of 512 × 512 pixel were acquired through 525/70 nm emission filter at 1.2 Hz frame rate. Randomized trains of 470 nm light (10-ms pulse, 15 s, 8.4 mW mm−2) of different frequencies (10 Hz, 20 Hz, 40 Hz) were delivered to the slices with intervals of ~2 min. Images were analyzed using ImageJ.
Trunk blood was collected at the time of killing before perfusion. Serum was prepared. Hormone titers were assayed using a testosterone ELISA kit (DRG Instruments GmbH, Germany, Division of ARG international, Inc, Cat# EIA-1559) according to the manufacturer’s protocols.
All codes were written in MATLAB and used for data analysis, and are available upon request. No sample size calculation was performed. The number of cells recorded in slice or counted in staining and the number of animals used for behavioral tests and staining were chosen according to the published literature of similar experiments. Data points from animals which upon post hoc histological analysis were deemed as mis-hits according to pre-established criteria were excluded from the analysis. In all, three female Esr1Cre mice and one male Esr1Cre mouse injected with GtACR1, one male and female VgatCre and one female Esr1Cre mouse injected with Casp3 were excluded from analysis.
To analyze data from Ca2+ imaging experiments in brain slices, images were quantified in ImageJ. Briefly, GCaMP6s+ cells were outlined manually and the relative fluorescence changes (ΔF/F) were calculated for each cell with the equation ΔF/F = (F−F0)/F0, in which F0 is the average pixel intensity in the last 10 frames before the photostimulation and F is the average pixel intensity in the first 6 frames after the photostimulation. Images acquired during the photostimulation periods were excluded from the analysis. An activated cell was operationally defined as cells showing increase in ΔF/F that was 5 standard deviations away from average ΔF/F values measured in 30 frames before the first photostimulation.
To analyze light-evoked locomotion, motion traces were first extracted using custom MATLAB codes, which recognize the centroid of an animal. Motion speed was then calculated in 1 s time bins by measuring the distance traveled. The 15 motion speed data points during a photostimulation period were compared with previous 15 points to test whether that light stimulation induced speed increase in that trial. At the animal level, averages of motion speeds during each photostimulation period were compared with averages of motion speeds immediately prior to each photostimulation period to determine whether light-induced locomotion increases in that animal. For optogenetic-induced mounting and pup retrieval, videos were first scored blindly. Video logs were then aligned to photostimulation logs to extract the trial percentage, the onset latency and the total duration of evoked behaviors. Only behaviors that occurred during light stimulation were counted as optogenetically induced behaviors. If a behavior occurred prior to but nevertheless ran into a photostimulation period, such behavior was not counted as optogenetically induced behaviors. Parameters were always averaged across photostimulation periods in the same behavioral trial and then across trials if there were multiple. To plot time distribution of optogenetically evoked behaviors, only photostimulation periods in which behaviors occurred were used to average. In behavioral trials with a male, a young rat or fake pups were used as the stimulus or when ChR2 animals were castrated, optogenetically induced behaviors were compared with spontaneous behaviors that occurred in the 15 s period immediately preceding photostimulation periods.
To analyze the effect of optogenetically inhibition on specific behavior, videos were first scored blindly. Video logs were then aligned to photostimulation logs to extract behavior events that encountered light stimulation. To plot various parameters of specific behaviors, behavioral events encountered light stimulus were averaged on the trial level if multiple trials existed and then on the animal level. To plot the accumulation distribution of specific behaviors, only behavior events that encountered light stimulation were analyzed.
To simulate an activation center for a ChR2 animal, a 1100×1500 μm area with one side close to the midline and the perpendicular side close to the bottom of the brain was cropped from each coronal brain section and was resized to 1 μm per pixel. c-Fos signals in each 100×100 μm were extracted semi-automatic using MATLAB program along with ImageJ and summed into a 11 × 15 matrix according to their locations within each section. The average number of NeuN/HuCD+ cells within a 100 μm2 square was estimated to be ~25. To calculate the coordinates of the activation center along the lateral–medial and dorsal–ventral axis, numbers within the 11 × 15 matrix were averaged across a series of brain sections that spanned anteriorly and posteriorly for each animal and fitted with two-dimension Gaussian function in MATLAB. To calculate the anterior–posterior coordinate of the activation center, the total c-Fos number was summed for each brain section and fitted with a single Gaussian function.
For fiber photometry experiments, raw fluorescence signals were further adjusted according to the overall trend to account for photo bleaching. Trials with jitter, square waves of signals or of extremely low signal to noise ratio were excluded from further analysis. For initial response, the values of fluorescence change (ΔF/F) was derived by calculating (F−F0)/F0, where F0 is the median of the baseline fluorescence signal. For the fluorescence signals aligned to behaviors, data were segmented based on behavioral events within individual trials and average signals 10–15 s before behavior initiation were used as F0. To analyze the statistical significance of the two event-related fluorescence signals, t test with false discovery rate (FDR) of 0.05 was used. To correlate Ca2+ transients and behaviors, a ramping Ca2+ event was counted when the ΔF/F values were 3 standard deviation above the baseline and lasted for longer than 2 s. The numbers of Ca2+ events were counted between the first and last behavior and correlated with the numbers of behavioral events during that period. Only behavioral trials that had more than two mounts and more than one retrieval were used for correlation analysis.
Histological images were captured with ×20 objective on a confocal microscope unless otherwise specified. Images were processed and counted in ImageJ with custom-written MATLAB codes. All counting was done by experimenters blind to the sex or the test condition of the animal. To analyze the viral expression of ChR2, the co-labeling of ChR2, c-Fos and Nissl in center injection area was counted manually. To analyze co-labeling of Esr1 and Vglut2, Vgat or Galanin, five relatively fixed areas in five brain sections of similar positions were selected to count manually. To test the specificity of Cre expression in Esr1-cre animals, the co-labeling of Esr1 and Cre was counted manually in various areas of multiple slices obtained from one animal. To quantify the images of catFISH experiments, which were taken under a ×60 objective, cells positive for cytoplasmic or nuclear c-Fos and for Esr1 were recognized with DAPI as the counterstaining and counted manually. To quantify the effects of viral ablation of Esr1+ neurons, Esr1+ signals in the mPOA as defined by the Allen Brain Atlas were counted for both sides using unbiased stereology with Stereo Investigator software (MBF bBioscience). Specifically, using an optical fractionator probe, Esr1+ signals were enumerated in a 30×30 μm counting box within a 100×100 μm sampling grid. To quantify the effects of viral ablation of Vgat+ or Vglut2+ neurons, brain sections were imaged with ×10 objective via Olympus VS120. Areas that stained with Vgat+ or Vglut2+ signals within the mPOA and adjacent regions were extracted semi-automatic using MATLAB codes.
For bar graphs, data are presented as mean ± s.e.m. In box and whiskers plots, data are presented as min to max whiskers and the mean indicated by “+” and the median indicated by a horizontal line. Unless specified otherwise, the following statistical tests were performed and all tests were specified as two-sided. Categorical data were analyzed by Fisher’s exact test. Paired data were analyzed by paired test. To analyze the statistic difference of accumulation distribution, two-sample Kolmogorov–Smirnov test was used. Data with one independent variables were analyzed by one-way ANOVAs followed by post hoc test with Bonferroni correction. Data with two independent variables were analyzed by two-way ANOVAs followed by post hoc test with Bonferroni correction. For other comparisons, data were tested for the distribution with Lilliefors’ goodness-of-fit normality test. If data passed the normality test, parametric test (Student’s t test) was used. Otherwise, nonparametric Wilcoxon rank sum test was used. The p value and the correlation coefficient for Pearson’s correlation were computed by using a Student’s t distribution for a transformation of correlation. *p < 0.05, **p ≤ 0.01, ***p < 0.001.
All data in this study are available on request.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank Chen X.K., Chen X.W., Shen W., and members of the Xu Lab for moments on the manuscript. We thank Poo M.M. and Xu N.L. for insightful discussions. We thank Lu B. for help with the RNAscope assay. This work was supported by grants from the National Nature Science Foundation of China (31471065), Ministry of Science and Technology office China 973 program (2015CB559201), the Thousand Young Talents Program of China, the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB02030005), and as part of the CAS interdisciplinary innovation team.