PCP4 immunoreactivity suggests the presence of hippocampal 1 region CA2 in solitary, social and eusocial mole-rat species 2

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Introduction
African mole-rats (family Bathyergidae) are subterranean rodents that are endemic to sub-Saharan Africa.This family is remarkable in several ways.Spending their lives almost entirely in extensive underground tunnel systems, they have attracted attention due to their long life expectancy (Edrey et al., 2012(Edrey et al., , 2013;;Dammann et al., 2011), resistance to tumors (Liang et al., 2010), adaptations to low oxygen concentrations (Peterson et al., 2012;Park et al., 2017), and diverse social behaviors, ranging from strictly solitary to social and eusocial species.This work considers four mole-rat species covering the full spectrum of sociality: The strictly solitary Cape mole-rat (Georychus capensis), the social Highveld mole-rat (Cryptomys hottentotus), as well as the eusocial Damaraland mole-rat (Fukomys damarensis) and naked mole-rat (Heterocephalus glaber).While Cape mole-rats meet each other with deadly aggression outside brief periods for mating and maternal care (Bennett and Jarvis, 1988), Highveld mole-rats form colonies with one breeding pair and up to 12 subordinates (Bennett and Faulkes, 1 2000).Damaraland mole-rat and naked mole-rats live in larger and more complex colonies with social hierarchies, division of labor, and cooperative breeding (Jarvis, 1981;Jarvis and Bennett, 1993).Thus, due to their evolutionary proximity, similar ecological niche, and life history, these species are prime candidates for comparative studies to understand the effect of sociality on brain circuit evolution (Kalamatianos et al., 2010;Amrein et al., 2014;Coen et al., 2015;Kverková et al., 2018).
Both recognition and aggression depend on the vasopressin receptor 1b (Wersinger et al., 2002;Leroy et al., 2018), which is expressed almost exclusively in the dorsal CA2 (Young et al., 2006).
The importance of CA2 for social behavior motivated us to investigate the prominence of the hippocampal region CA2 in solitary, social and eusocial mole-rat species.As a first step, we characterized the CA2 region by staining for Purkinje Cell Protein 4 (PCP4).PCP4 is one of several established molecular markers for CA2 pyramidal cells in mice (San Antonio et al., 2014;Kohara et al., 2014).We observed prominent PCP4 immunoreactivity in the central part of the cornu Ammonis for all mole-rat species.These data suggest that the extend of CA2 does not drastically deviate despite extreme differences in social behavior.However, given the superficial nature of this analysis, further verification is warranted.

Animals
We used remnant mole-rat slices from two previous studies (Amrein et al., 2014, Highveld, Cape and naked molerats) and (Oosthuizen and Amrein, 2016, Damaraland mole-rats).Each mole-rat species had a different origin: Cape mole-rats were trapped near Darling, Western Cape, South Africa, and Highveld mole rats in Centurion, Gauteng, South Africa.Damaraland mole-rats originated from Blackrock, Northern Cape, South Africa.Naked mole-rats were derived from a colony at the University of Nairobi, Kenya.Trapping and treatment were in accordance with the ethical guidelines of the respective universities.Neither queens nor pregnant or lactating animals were included in the study.After weighing and determining sex, the animals were anesthetized with pentobarbital, 50 mg / kg body weight, and perfused transcardiacally with phosphate buffered saline (PBS), followed by sodium sulfide solution and fixed with 4% paraformaldehyde (PFA).The brains were extracted, post-fixed overnight and divided into the right and left hemispheres, which were cryoprotected in 30% sucrose and processed for immuno-2 histochemistry, or stored in PFA until methacrylate embedding, respectively.Teeth and femurs were extracted for subsequent age determination.

Results
To characterize CA2 in mole-rat species throughout the full spectrum of sociality, we investigated PCP4 expression in hippocampal slices of strictly solitary Cape mole-rat, the social Highveld mole-rat, as well as the eusocial Damaraland mole-rat and naked mole-rat.We detected spatially restricted PCP4 expression in a central part of the cornu Ammonis pyramidal cell layer in all four mole-rat species, similar to the common laboratory mouse strain C57BL/6 (see Fig. 1).These findings suggest the presence of CA2 in all investigated mole-rat species.

Discussion
In this study, we used PCP4 immunostaining to label hippocampal region CA2 in hippocampal slices of four different mole-rat species with diverse levels of sociability and compared them to the common house mouse.In all species, we found prominent staining in a central part of the cornu Ammonis region -which indicates the presence of CA2.However, to corroborate these findings future work should combine staining with multiple CA2 specific markers, such as RGS14, STEP, and α -actinin 2 (Lee et al., 2010;Kohara et al., 2014;Chevaleyre and Siegelbaum, 2010), and classical anatomical markers, such as the presence of pyramidal cells with mossy fiber input but without complex spines (Kohara et al., 2014).
Given the broad range of sociality in mole-rats, differences in CA2 would not come as a surprise.Previous investigations found that neurogenesis is generally low in mole-rats, but the amount of neurogenesis in the dentate gyrus differs between social and solitary species.Proportions of young neurons are lower in solitary Cape molerats, compared to social Highveld and naked mole-rats (Amrein et al., 2014).Furthermore, in both eusocial naked and Damaraland mole-rats an inverse relationship between social status and neurogenesis has been observed, with queens showing the least and workers the most neurogenesis (Peragine et al., 2014 2016).It seems plausible that the observed differences in neurogenesis reflect distinct cognitive requirements, both in habitat and social interactions (Oosthuizen, 2017), as has been observed in rats (Kozorovitskiy and Gould, 2004) and mice (Monteiro et al., 2014).Along similar lines, it can be argued that requirements for social recognition memory likely differ in solitary and social mole-rat species and, thus, should properties of CA2.
A thorough anatomical description of CA2 in mole-rats would open new research avenues.An area of interest 4 may be the potential relationship between species-specific aggression levels and differences in CA2.Mice exhibit social aggression mediated by the activity of pyramidal cells in dorsal CA2 (Wersinger et al., 2002;Leroy et al., 2018).As mice transition from exploratory behavior to aggression, the firing rates of these pyramidal cells gradually increase, activating the lateral septum and subsequently disinhibiting the ventromedial hypothalamus, which is known to initiate an attack (Leroy et al., 2018).The function of this circuit depends on the CA2-specific vasopressin receptor 1b.Infusing an antagonist of the vasopressin receptor 1b has been shown to prevent aggression in mice (Leroy et al., 2018).Thus, it can be hypothesized that in Cape mole-rats the suppression of aggression during mating and maternal care may be regulated by vasopressin receptors in CA2.Using a similar antagonist infusion as in mice, this hypothesis can be directly tested.

Figure 1 :
Figure 1: PCP4 immunoreactivity in a central region of the cornu Ammonis suggests the presence of hippocampal region CA2 in solitary, social and eusocial mole-rats species.Note, the immunostaining in the Highveld mole-rat has been performed in a different subspecies (Cryptomys hottentotus pretoriae) compared to the picture depicting a group of animals (Cryptomys hottentotus hottentotus).Pictures of living animals are adapted from the following sourses: Cape and Damaraland mole-rat Oosthuizen (2017), Highveld mole-rat Elizabeth Archer (personal communication), naked mole-rat Smithsonian's National Zoological Park shared under CC0, house mouse by Boris Heifets shared under CC BY 4.0.