CRISPR-Cas9 editing of the arginine–vasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters. Jack H. Taylor et al. Proceedings of the National Academy of Sciences, May 5, 2022. Vol. 119 | No. 19, e2121037119. https://doi.org/10.1073/pnas.2121037119
Significance: Arginine–vasopressin (AVP) acting on V1a receptors (Avpr1as) represents a key signaling mechanism in a brain circuit that increases the expression of social communication and aggression. We produced Syrian hamsters that completely lack Avpr1as (Avpr1a knockout [KO] hamsters) using the CRISPR-Cas9 system to more fully examine the role of Avpr1a in the expression of social behaviors. We confirmed the absence of Avpr1as in these hamsters by demonstrating 1) a complete lack of Avpr1a-specific receptor binding throughout the brain, 2) a behavioral insensitivity to centrally administered AVP, and 3) an absence of the well-known blood-pressure response produced by activating Avpr1as. Unexpectedly, however, Avpr1a KO hamsters displayed more social communication behavior and aggression toward same-sex conspecifics than did their wild-type (WT) littermates.
Abstract: Studies from a variety of species indicate that arginine–vasopressin (AVP) and its V1a receptor (Avpr1a) play a critical role in the regulation of a range of social behaviors by their actions in the social behavior neural network. To further investigate the role of AVPRs in social behavior, we performed CRISPR-Cas9–mediated editing at the Avpr1a gene via pronuclear microinjections in Syrian hamsters (Mesocricetus auratus), a species used extensively in behavioral neuroendocrinology because they produce a rich suite of social behaviors. Using this germ-line gene-editing approach, we generated a stable line of hamsters with a frame-shift mutation in the Avpr1a gene resulting in the null expression of functional Avpr1as. Avpr1a knockout (KO) hamsters exhibited a complete lack of Avpr1a-specific autoradiographic binding throughout the brain, behavioral insensitivity to centrally administered AVP, and no pressor response to a peripherally injected Avpr1a-specific agonist, thus confirming the absence of functional Avpr1as in the brain and periphery. Contradictory to expectations, Avpr1a KO hamsters exhibited substantially higher levels of conspecific social communication (i.e., odor-stimulated flank marking) than their wild-type (WT) littermates. Furthermore, sex differences in aggression were absent, as both male and female KOs exhibited more aggression toward same-sex conspecifics than did their WT littermates. Taken together, these data emphasize the importance of comparative studies employing gene-editing approaches and suggest the startling possibility that Avpr1a-specific modulation of the social behavior neural network may be more inhibitory than permissive.
Discussion
Here, we report the successful use of CRISPR-Cas9 for the generation of Avpr1a KO Syrian hamsters. Through the breeding of a CRISPR-Cas9–edited founder and heterozygote progeny, we were able to successfully produce M and F Syrian hamsters completely lacking a functional Avpr1a gene. Avpr1a KO hamsters exhibited 1) a complete lack of Avpr1a binding throughout the brain, 2) insensitivity to the behavioral effects of centrally administered AVP, and 3) no changes in blood pressure in response to a peripherally injected Avpr1a agonist. Despite these expected changes, it is remarkable that Avpr1a KO hamsters expressed double the levels of odor-stimulated flank marking and aggression toward same-sex conspecifics than WTs.
Importantly, we showed haploinsufficiency in flank marking and aggressive behaviors and in Avpr1a binding in the brain, indicating that heterozygotes will be useful for investigating the effects of reduced, but not absent, Avpr1a expression. The translational relevance of behavioral genetic approaches should improve by increasing the variety of animal models and approaches, and these results demonstrate the utility of CRISPR-Cas9 gene editing in Syrian hamsters to interrogate gene function. Comparison of behavioral genetic data obtained from nontraditional model species with data obtained in KO mice (e.g., effects on aggression) provides important context that may help generalize findings to other rodents or to humans. During the generation and phenotyping of the hamsters described here, a KO was generated via CRISPR-Cas9 in prairie voles (
27). Notably, these voles lack a functional
Oxtr gene, and due to the relationship between Oxtrs and Avpr1a, they provide a useful comparison to our results in hamsters. In both species, CRISPR-Cas9 induced mosaics in edited animals. Mosaicism after Cas9-mediated gene editing is not uncommon (
28–
30), and, when generating a new KO model, it underscores the value of careful selective breeding of edited founders and descendants. Comparisons across species will likely provide new insight into the function and evolution of the various molecular substrates of behavior.
Flank marking plays a critical role in social communication in hamsters. M and F hamsters flank mark in response to odors of same-sex conspecifics or to hypothalamic injection of AVP without conspecific odors (
9,
22,
31). In the present study, we again showed that intracerebroventricular (ICV) injection of AVP or a selective Avpr1a agonist in WT hamsters produced robust flank marking in odor-free environments; however, ICV injection of AVP or a selective Avpr1a agonist in
Avpr1a KO hamsters had no effect on the expression of flank marking, demonstrating insensitivity of KOs to exogenous AVP. Surprisingly, we found that odor-stimulated flank marking was twofold higher in
Avpr1a KO hamsters than in WT hamsters. This increase was specific to the presence of conspecific odor, as
Avpr1a KO hamsters marked at the same low levels as WT hamsters in a clean cage. These findings indicate that Avpr1a activation is not necessary for the expression of odor-stimulated flank marking. Indeed, the present data are not alone in indicating that a disassociation between the number of Avpr1as and the expression of flank marking can occur. Housing in “summer-like” photoperiods (i.e., >12 h of light per day) dramatically increases the expression of hypothalamic Avpr1as compared with “winter-like” photoperiods (
32). Interestingly, however, odor-stimulated and AVP-induced flank marking are expressed at the same levels, regardless of photoperiod (
11,
33). The mechanisms responsible for the uncoupling of flank marking from the number of hypothalamic Avpr1as in short-photoperiod-exposed hamsters are not known, although photoperiod-induced compensatory changes in the response to neurochemical signals, such as serotonin or galanin, that can influence flank marking do not appear to be involved (
34). Certainly, the investigation of the compensatory mechanisms that mediate the robust, odor-stimulated flank marking in
Avpr1a KO hamsters is a necessary next step. Taken together, these data indicate that, although the presence of Avpr1as are necessary for exogenous AVP to induce flank marking, Avpr1as are not necessary for the expression of odor-stimulated flank marking; thus, it is clear that the neurochemical mechanisms regulating flank marking are more complex than previously thought.
Another intriguing and surprising finding emerged when we examined the result of eliminating functional Avpr1as on aggressive behavior. Previous studies have found that injection of AVP into the AH stimulates aggression in M hamsters and inhibits aggression in F hamsters and that injection of a selective Avpr1a antagonist into the AH inhibits aggression in M and stimulates aggression in F hamsters (
13,
24,
25). Therefore, we predicted that the absence of Avpr1as would reduce aggression in M and increase aggression in F hamsters. As predicted, aggression was higher in
Avpr1a KO F than in WTs, and aggression in heterozygote F was intermediate to that seen in KO and WT F. In
Avpr1a KO M, however, aggression was unexpectedly higher than in heterozygotes or WTs. Indeed, aggression was twofold higher in M
Avpr1a KO hamsters than it was in WT M. Thus, the elimination of functional Avpr1as eliminated the previously established sex differences in AVP-mediated aggression. These data are consistent with the hypothesis that Avpr1as have inhibitory effects on aggression in F and raise the possibility that the global loss of functional Avpr1as can increase aggression in M. Interestingly, aggression significantly increases in M and F hamsters exposed to short photoperiods compared with hamsters housed in long photoperiods (
35,
36), under which there is a reduction of Avpr1as within key sites of the SBNN (
32,
33). Similar to the increase in aggression seen in
Avpr1a KO M hamsters, increased aggressiveness in short-photoperiod-exposed M hamsters does not depend on activation of Avpr1as (
37). The short-photoperiod-induced increase in aggression may be the result of changes in the secretion of pineal melatonin that serve to increase aggression (
38–
42), and it will be interesting to investigate if M
Avpr1a KOs have similar compensatory increases in melatonin secretion.
As discussed above, the diversity and complexity of social behaviors across species and among individuals is hypothesized to emerge from the functional interactions among the multiple nodes of SBNN circuitry, and not from the activity of its individual components (
3,
5). Investigation of SBNN neurocircuitry has been restricted almost exclusively to studies of how individual nodes influence social behavior because of the inability to manipulate the entire circuit concurrently. Global KOs like those employed in the present study provide one approach, albeit an imperfect one, to manipulate key neurochemical signals across the entire circuit. The dramatic differences in aggression and social communication between the KO and WT hamsters seen in the present study were not predicted by studies employing pharmacological inhibition of Avpr1a activity within specific SBNN nodes. Therefore, elimination of Avpr1a activity throughout the entire SBNN circuit can impact social behavior very differently than does inhibiting Avpr1a activity in individual nodes of the circuit. As such, these data support the hypothesis that social behavior can be an emergent property coming from the interactions across nodes of the entire circuit. It is, however, important to emphasize the possibility that these differences could also be due to elimination of Avpr1as in regions outside the SBNN or as the result of developmental compensation. Nevertheless, the data obtained by using the KO approach suggest some fascinating questions about the putative functions of Avpr1a at a circuit level that are deserving of future study. For example, do Avpr1as operating across the circuit serve to inhibit the expression of at least some social behaviors, even though their activation in specific individual nodes can induce those behaviors? Are sex differences in aggression the result of sex differences in the effects of Avpr1a across the SBNN with activation of Avp1ar reducing aggression in M hamsters and enhancing aggression in F hamsters?
The phenotype of
Avpr1a KO hamsters also differs greatly from the phenotype of
Avpr1a KO mice, indicating that there are important species differences in Avpr1a function and/or compensatory mechanisms. M and F
Avpr1a KO hamsters were more aggressive than were WT hamsters, whereas
Avpr1a KO M mice display no differences in aggression from WTs (
14). The increase in aggression in
Avpr1a KO hamsters relative to WTs is informed by the concomitant increase in odor-stimulated flank marking, suggesting the possible existence of a hypersensitivity to and/or hyperresponsiveness to social olfactory stimuli in the main olfactory system (
43). In contrast,
Avpr1a KO mice exhibit reduced social investigation and impaired olfactory processing compared with WTs (
14,
44). Taken together, these comparisons suggest that as CRISPR-Cas9 and related gene-editing technologies continue to be applied to new species and gene targets, more species-specific differences in gene function and developmental compensation will continue to be discovered. These data certainly emphasize the necessity of using species in addition to mice to interrogate the role of specific neurochemical signaling pathways in generating social behavior.
One critical aspect of genetic models is determining the influence of a gene (or its lack) throughout development (reviewed in ref.
45). It is difficult or impossible to separate the acute effects of eliminating a gene from its developmental effects in mammalian KO models, perhaps most notably in potential developmental compensation. For instance, it is possible that odor-stimulated flank marking in
Avpr1a KO hamsters is “rescued” by other receptors. Two obvious candidates for compensation are Oxtr or Avpr1b. However, given that AVP, which binds and activates Oxtr and Avpr1b (
46,
47), did not stimulate flank marking in KOs, this explanation is unlikely. Though we found no differences in Oxtr binding density in the brain nuclei examined, considering the strong link between the OT and AVP systems, it is possible that some developmental disruptions occurred in the Oxtr system in hamster
Avpr1a KOs. Viral rescue strategies offer the potential to parse developmental and activational influences of
Avpr1a KO (see ref.
48 for an example). The potential developmental consequences from the lack of Avpr1a will be an important target and consideration in future research using
Avpr1a KO hamsters.
In conclusion, the unexpected behavioral phenotypes of Avpr1a KO Syrian hamsters reveal insight into the function of Avpr1as in facilitating social behavior. It was long thought that activation of Avpr1a was both necessary and sufficient for the expression of flank marking, but it now appears that flank marking can occur in the absence of Avpr1a activation in certain situations. This raises new questions regarding non-Avpr1a-mediated mechanisms of social communication. Gene-edited hamsters will provide an important tool in these future studies.