Distinct preoptic‐ BST nuclei dissociate paternal and infanticidal behavior in mice
2015; Springer Nature; Volume: 34; Issue: 21 Linguagem: Inglês
10.15252/embj.201591942
ISSN1460-2075
AutoresYousuke Tsuneoka, Kenichi Tokita, Chihiro Yoshihara, Taiju Amano, Gianluca Esposito, Arthur Huang, Lily Yu, Yuri S. Odaka, Kazutaka Shinozuka, Thomas J. McHugh, Kumi O. Kuroda,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoArticle30 September 2015free access Distinct preoptic-BST nuclei dissociate paternal and infanticidal behavior in mice Yousuke Tsuneoka Yousuke Tsuneoka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Anatomy, School of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Kenichi Tokita Kenichi Tokita Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Chihiro Yoshihara Chihiro Yoshihara Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Taiju Amano Taiju Amano Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan Search for more papers by this author Gianluca Esposito Gianluca Esposito Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Psychology and Cognitive Science, University of Trento, Rovereto, TN, Italy Division of Psychology, School of Humanities and Social Sciences, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Arthur J Huang Arthur J Huang Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Lily MY Yu Lily MY Yu Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Yuri Odaka Yuri Odaka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Kazutaka Shinozuka Kazutaka Shinozuka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Thomas J McHugh Thomas J McHugh Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Kumi O Kuroda Corresponding Author Kumi O Kuroda Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Yousuke Tsuneoka Yousuke Tsuneoka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Anatomy, School of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Kenichi Tokita Kenichi Tokita Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Chihiro Yoshihara Chihiro Yoshihara Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Taiju Amano Taiju Amano Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan Search for more papers by this author Gianluca Esposito Gianluca Esposito Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Department of Psychology and Cognitive Science, University of Trento, Rovereto, TN, Italy Division of Psychology, School of Humanities and Social Sciences, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Arthur J Huang Arthur J Huang Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Lily MY Yu Lily MY Yu Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Yuri Odaka Yuri Odaka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Kazutaka Shinozuka Kazutaka Shinozuka Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Thomas J McHugh Thomas J McHugh Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Kumi O Kuroda Corresponding Author Kumi O Kuroda Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Author Information Yousuke Tsuneoka1,2,‡, Kenichi Tokita1,‡, Chihiro Yoshihara1, Taiju Amano1,3, Gianluca Esposito1,4,5, Arthur J Huang6, Lily MY Yu6, Yuri Odaka1, Kazutaka Shinozuka1, Thomas J McHugh6 and Kumi O Kuroda 1 1Laboratory for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan 2Department of Anatomy, School of Medicine, Toho University, Tokyo, Japan 3Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan 4Department of Psychology and Cognitive Science, University of Trento, Rovereto, TN, Italy 5Division of Psychology, School of Humanities and Social Sciences, Nanyang Technological University, Singapore, Singapore 6Laboratory for Circuit and Behavioral Physiology, RIKEN Brain Science Institute, Saitama, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 48 467 7556; Fax: +81 48 467 6853; E-mail: [email protected] The EMBO Journal (2015)34:2652-2670https://doi.org/10.15252/embj.201591942 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Paternal behavior is not innate but arises through social experience. After mating and becoming fathers, male mice change their behavior toward pups from infanticide to paternal care. However, the precise brain areas and circuit mechanisms connecting these social behaviors are largely unknown. Here we demonstrated that the c-Fos expression pattern in the four nuclei of the preoptic-bed nuclei of stria terminalis (BST) region could robustly discriminate five kinds of previous social behavior of male mice (parenting, infanticide, mating, inter-male aggression, solitary control). Specifically, neuronal activation in the central part of the medial preoptic area (cMPOA) and rhomboid nucleus of the BST (BSTrh) retroactively detected paternal and infanticidal motivation with more than 95% accuracy. Moreover, cMPOA lesions switched behavior in fathers from paternal to infanticidal, while BSTrh lesions inhibited infanticide in virgin males. The projections from cMPOA to BSTrh were largely GABAergic. Optogenetic or pharmacogenetic activation of cMPOA attenuated infanticide in virgin males. Taken together, this study identifies the preoptic-BST nuclei underlying social motivations in male mice and reveals unexpected complexity in the circuit connecting these nuclei. Synopsis Depending on mating status, male mice show paternal nurturing or aggression toward infant mice. The authors could predict and manipulate the balance of paternal versus aggressive behavior based on neural activity in the forebrain nuclei cMPOA and BSTrh. Virgin male males are aggressive to pups while fathers show paternal nurturing. Neuronal activation patterns in two forebrain nuclei, cMPOA and BSTrh, predict paternal and aggressive behavior in male mice with 97% fidelity. Parental care requires the cMPOA, while pup-directed aggression is facilitated by the BSTrh. A cMPOA to BSTrh inhibitory projection may mediate the behavioral switch from aggression to parenting. Introduction Mammalian neonates are born immature, and thus, postpartum mothers are equipped with innate motivation to care for them (Lonstein & Fleming, 2002; Numan, 2015). Previous studies established the critical importance of the medial preoptic area (MPOA) for maternal behavior (Numan, 1974), and more specifically, the central MPOA in female mice (Tsuneoka et al, 2013). In male mice, however, paternal behavior is not spontaneous. Virgin males often attack and kill newborn pups (infanticide) as an adaptive reproductive strategy that increases the males' mating opportunity (Trivers, 1972; Elwood, 1977; vom Saal & Howard, 1982). After mating and cohabitation with a pregnant female, male mice shift their pup-directed behavior from infanticide to paternal care, even toward non-biological offspring. We have recently shown that the accessory olfactory system is highly activated during infanticide, and surgical ablation of the vomeronasal organ in virgin males abolished infanticide and induced paternal behavior (Tachikawa et al, 2013). However, while a similar behavioral transition from infanticide to paternal care can be observed in many species, from Old World monkeys Hanuman langur (Presbytis entellus) (Sugiyama, 1965; Hrdy, 1974) to African lions (Panthera leo) (Schaller, 1972), not all have a well-developed vomeronasal system (Zhang & Webb, 2003). Therefore, a common central mechanism may exist to mediate the behavioral switch from infanticide to parenting after mating, even though the sensory modality for peripheral input differs among species. Here we investigated the forebrain areas responsible for these social behaviors in male laboratory mice (Mus musculus, strain C57BL/6J), to gain insight into the neural mechanism underlying the adaptive transition of pup-directed behaviors. Results Neurochemical delineation of the bed nucleus of stria terminalis in mice Our previous analyses of the neuronal activation pattern during pup-directed behaviors in mice (Kuroda et al, 2007; Tachikawa et al, 2013) suggested that the spatial expression pattern of c-Fos transcription factor in the preoptic area and its dorsolateral neighbor, the bed nuclei of stria terminalis (BST), could be used to classify transpired pup-directed behavior. To achieve high accuracy and reproducibility of histological analyses when testing this hypothesis, we needed a more rigorous anatomical delineation of each BST nucleus than our previous study (Tachikawa et al, 2013). Therefore, we first conducted careful neurochemical mapping of the BST (Fig 1) in male C57BL/6J mice and identified the BST nuclei relevant to pup-directed behaviors. Based on studies in rats (Ju & Swanson, 1989; Ju et al, 1989; Dong et al, 2001), the principal nucleus of the BST (BSTpr) in mice was identified as the cocaine- and amphetamine-regulated transcript (CART) mRNA-positive area with dense Nissl staining, surrounded by dense galanin-immunoreactive (-ir) fibers and cell bodies (Fig 1C, F, I and O). The oval nucleus of the BST (BSTov) was identified as the Neurotensin mRNA-positive area with dense vasoactive intestinal peptide (VIP)-ir fibers (Fig 1D, E, M and N). The rhomboid nucleus of the BST (BSTrh) was defined as the area with dense staining of tyrosine hydroxylase (TH)-ir fibers and with a sparse distribution of VIP-ir and galanin-ir fibers located posterior to the BSTov (Fig 1F, I, L and O). The transverse nucleus of BST (BSTtr) was located ventral to the BSTrh and identified by Nissl staining as a cluster of neurons that were oriented transversely (Fig 1C and O). The anterolateral part of BST (BSTal) was identified by the distribution of galanin-ir cell bodies (Fig 1G–I and M–O). Figure 1. Neurochemical mapping of the BST subnuclei in virgin male mice A–L. Neurochemical analysis of the BST. All panels are coronal sections arranged from rostral (top) to caudal (bottom). The distances from bregma to the sections were +0.22 mm (top), +0.1 mm (middle) and −0.08 mm (bottom), respectively. ac, the anterior commissure; st, the stria terminalis; fx, fornix; LV, lateral ventricle. Scale bar is 0.2 mm. (A–C) Nissl staining of the BST. (D–L) Triple fluorescent staining of the BST; (D–F) Cart and Neurotensin double ISH with anti-VIP immunohistochemistry (IHC); (G–I) Cart ISH with anti-TH and galanin IHC; (J–L) Gad67 ISH with anti-TH and c-Fos IHC. For (J–L), the male mice were exposed to unfamiliar pups for 2 h to confirm infanticidal behavior. M–O. Delineation of the BST nuclei based on the results of (A–L). Download figure Download PowerPoint For rigorous anatomical analyses using multiple animal samples, the use of conservative contours (smaller areas than the actual nucleus) is required to overcome the variation of individual samples (e.g., see Palkovits & Brownstein, 1992). Therefore, we set a conservative contour for each BST nucleus as a numerically defined closed line on the coronal plane as shown in Fig 2Z–d; for example, the conservative contour of BSTal was set posterolaterally to the anterior commissure (Fig 2c) to unequivocally count c-Fos-ir neurons of BSTal but not of adjacent areas for individual animals, although the BSTal itself is larger and extends rostrocaudally. Figure 2. Distribution of c-Fos-ir neurons in the preoptic-BST nuclei after various social behaviors A–Y. Representative photomicrographs of coronal sections triple-stained for c-Fos (black), VIP (brown) and galanin (pink-red) of control, infanticidal, parenting, mating and intermale aggression groups. Z–d. Conservative contours of the nine subregions where the c-Fos-ir neurons were counted (indicated by dashed lines). The distances from bregma to the sections were +0.22 mm (first row), +0.1 mm (second row), +0.22 mm (third row), +0.1 mm (fourth row) and −0.08 mm (fifth row), respectively. Scale bars = 0.2 mm. e–m. Mean (± SE) density of c-Fos-ir neurons in each area induced by direct social interactions. C: control (stayed alone) (n = 8); I: infanticidal virgin males (n = 7); Pv: paternally behaving virgin males (n = 6); Pf: paternally behaving fathers (n = 11); M: virgin males experienced mating (n = 8); and A: virgin males experienced intermale aggression (n = 13). Letters above each bar indicate significant differences between each behavioral group (i.e., bars with the same letter are not significantly different by Welch's ANOVA). P-values are described separately in Table EV1. n. Mean (± SE) density of c-Fos-ir neurons in the BSTrh induced by direct (white in I, n = 7) or indirect (blue in I, pups within mesh balls, n = 14) pup exposure to infanticidal males, and by exposure to a mesh ball (blue in C, n = 9) or by no exposure but the sham action of opening and closing the cage top (white in C, n = 8). Download figure Download PowerPoint The preoptic subregions, cMPOA anterior (caMPOA) and posterior (cpMPOA), the anterior commissural nucleus (ACN) and the medial part of the medial preoptic nucleus (MPNm) were identified in our previous study in C57BL/6J female mice (Tsuneoka et al, 2013). In this study, the conservative contours of these preoptic regions were set for male C57BL/6J mice (see 4). A total of nine preoptic-BST nuclei (Fig 2Z–d, see also Fig EV1) was used for the c-Fos expression analyses described below. Click here to expand this figure. Figure EV1. Distribution of c-Fos-ir neurons in the preoptic-BST region after various social behaviors (low magnification of Figure 2) A–O. Representative photomicrographs for c-Fos (black), VIP (brown) and galanin (pink-red) triple staining of control, infanticidal, parenting, mating and intermale aggression groups. All panels are coronal sections arranged from rostral (top) to caudal (bottom) order. The distances from bregma to the sections were +0.22 mm (top), +0.1 mm (middle) and −0.08 mm (bottom), respectively. Scale bar = 0.5 mm. P–R. Area names and locations where the c-Fos-ir neurons were counted (indicated by blue lines). Download figure Download PowerPoint Distinct c-Fos expression patterns induced by four types of male social behaviors To examine c-Fos expression induced by pup exposure in virgin males and fathers, these mice were first pre-tested to determine their pup-directed behavior. All fathers exhibited paternal behavior on postpartum day 3. The majority of virgin males committed infanticide, while a small fraction (6.5%) of virgins spontaneously performed paternal behavior in the pre-test, as expected from our previous study (Tachikawa et al, 2013). Clearly, infanticidal virgin males (n = 7), paternal virgin males (n = 6) and fathers (n = 11) were selected and were singly housed for 2 days to allow the c-Fos expression induced by the pre-test to return to baseline levels (Morgan et al, 1987; Numan & Numan, 1994), and then they were exposed to three donor pups for 2 h. The pup-directed behavior during this main experiment was essentially consistent with the pre-test as shown previously (Tachikawa et al, 2013). In addition to these pup-exposed groups, two groups of male mice were subjected to other types of social interactions: mating (with ejaculation) (n = 8), which has been shown to be necessary for the behavioral change in fathers (Kennedy & Elwood, 1988), and inter-male aggression (n = 13), which involved biting and attack toward an unfamiliar adult male intruder in their home cage. The control group males were single-housed and were not exposed to any social encounter, but otherwise treated in the same manner (n = 8). Two hours later, these mice (total n = 53) were perfused with PFA and the brains were subjected to triple immunohistochemical labeling (Figs 2A–Y and EV1) for c-Fos, VIP and galanin, to quantify the c-Fos-ir neurons in each nucleus (Fig 2e–m, see also Table EV1 for P-values). Paternal behavior induced significantly more c-Fos expression in the ACN, the caMPOA and the cpMPOA (Fig 2e–g) in both the fathers (ACN, P < 0.001; caMPOA, P = 0.0285; and cpMPOA, P < 0.001) and paternal virgin males (ACN, P = 0.0013; caMPOA, P = 0.0013; and cpMPOA, P = 0.0015), compared to that of control mice, suggesting a similar pattern of parenting-induced activation regardless of gender or previous reproductive experiences (Tsuneoka et al, 2013). This parenting-induced activation was even more pronounced in paternal virgin males than in fathers (caMPOA, P = 0.0405; and cpMPOA, P = 0.0340; Fig 2f and g), suggesting the novelty of the paternal behavior led to greater activation in the inexperienced virgin males. After mating and ejaculation, a robust increase in c-Fos expression was seen in the MPNm and the BSTpr (MPNm, P < 0.001; and BSTpr, P < 0.001, compared to the control; Fig 2h and i), both of which are sexually dimorphic (larger in males) and have been implicated in male sexual behavior (Simerly et al, 1986; Yahr et al, 1994; Balthazart & Ball, 2007). Interestingly, the areas activated during paternal behaviors were also activated during mating, most prominently cpMPOA (P < 0.001, compared to the control, Fig 2g). In infanticidal virgin males, only the BSTrh was significantly activated compared with all the other types of social encounter (P = 0.0074 compared to the control) (Fig 2J and j). Based on its projection pattern, it has been suggested that the BSTrh is involved in ingestive behavior (Dong & Swanson, 2003). Thus, to address whether activation of the BSTrh after infanticide was caused by ingestion of pups' blood or tissues, we performed an additional experiment where the stimulus pups were protected by wire mesh balls (n = 14). This indirect pup presentation to the previously infanticidal virgin males caused a comparable amount of BSTrh activation to that observed in mice who had physical access to pups and could bite them (t = −6.64, df = 6.17, P < 0.001 for direct pup presentation and t = −6.96, df = 14.72, P < 0.001 for indirect pup presentation, compared to the control groups, respectively; Fig 2n, see also Fig EV2). Therefore, we concluded that the BSTrh activation was correlated with motivation for rather than the commitment of infanticide. Click here to expand this figure. Figure EV2. Comparison between direct and indirect pup presentation and the effect on c-Fos expressionMean (± SE) density of c-Fos-ir neurons in each area induced by direct exposure of pups (white) and indirect exposure of pups within mesh balls (blue). Letters above each bar indicate significant differences between each behavioral group (i.e., bars with a same letter are not significantly different by Welch's ANOVA). Asterisks denote significant differences among exposure types (direct, exposed pups; indirect, pups within mesh balls). C: control no exposure to pups but a sham action of opening and closing the cage top (white in C, n = 8), exposure to a mesh ball (blue in C, n = 9); I: infanticidal virgin males exposed to pups directly (white in I, n = 7) or indirectly (blue in I, n = 14); Pv: parentally behaving virgin males exposed to pups directly (white in Pv, n = 6) or indirectly (blue in Pv, n = 2); Pf: parentally behaving fathers exposed to pups directly (white in Pf, n = 11) or indirectly (blue in Pf, n = 8). Download figure Download PowerPoint Bilateral lesions of the BSTrh inhibited infanticidal behavior in virgin males Next, we investigated the role of BSTrh in infanticide. Infusion of the amino acid N-methyl-D-aspartic acid (NMDA) is known to overexcite and deplete the local neurons expressing the NMDA receptor, sparing the passing fibers, while its optical isomer N-methyl-L-aspartic acid (NMLA) is non-toxic (Numan et al, 1988). Bilateral excitotoxic lesions in the BSTrh resulted in significant inhibition or delay in performing infanticide in virgin males for all four test days (Fig 3A and B, P = 0.002, 0.033, 0.023 and 0.033, respectively, Fisher's exact probability test; see also Fig EV3 for the individual lesioned areas), compared to the NMLA-injected control males. The positive correlation between the extent of bilateral BSTrh damage and the latency of infanticide was significant from the second to fourth test days (Fig 3F). The latency of the first pup sniffing was largely unaffected (Fig 3E), suggesting that the BSTrh lesions did not produce gross deficits in the sensorimotor system, which may inhibit infanticide non-specifically. Bilateral BSTrh lesions increased c-Fos expression in cMPOA ((t = −2.71, df = 19.40, P = 0.0139; Fig 3C and D). However, the effect of bilateral BSTrh lesions on the latency of first pup retrieval or pup grouping did not reach statistical significance (Fig 3G and H). Unilateral BSTrh lesions did not cause significant behavioral changes for all four test days (Fig 3B, P = 0.148, 0.159, 0.263 and 0.366, Fisher's exact probability test; Fig 3E and F), or changes to the c-Fos expression in the ipsilateral cpMPOA (t = −1.318, df = 11, P = 0.214; Fig 3C and D). Figure 3. Excitotoxic lesions in BSTrh attenuate infanticidal behavior of virgin male mice A, B. The photos and data shown in top, middle and bottom rows, respectively, belong to the control, unilateral and bilateral lesion groups. (A) Representative photomicrographs showing the effects of NMDA injections. The sections were stained by immunohistochemistry (IHC) using anti-NeuN (black) and anti-VIP (brown) antibodies. Dashed line indicates the BSTrh. Scale bar = 0.5 mm. (B) Relative frequencies of responses to pups. Committing infanticide within 3 min (red), committing infanticide in 3–30 min (pink), indifference (neither infanticidal nor parenting) (white) and parenting (retrieving at least one pup within 30 min) (blue). *: significant difference from the control group of the same day. C. Representative photomicrographs showing c-Fos expression in cpMPOA (dashed contour) 2 h after the final behavioral test. The sections were stained by IHC using anti-c-Fos (black), anti-VIP (brown) and anti-galanin (pink-red) antibodies. Scale bar = 0.25 mm. D. Mean (± SE) density of c-Fos-ir neurons in cpMPOA induced by direct pup exposure. Differential letters indicate significant difference among bars. For the unilateral group, the data were analyzed and shown for each side. E–H. Scatter plot showing the relation between the proportion of the BSTrh damage with the latency of the first pup sniffing (E), the latency of infanticide (F), the latency of the first pup retrieval (G) and the latency of the completion of pup grouping (H). For unilateral and bilateral groups, the data contain both mis-targeted (less than half of BSTrh area was destroyed) and successful lesion (half and more than half of BSTrh area was destroyed). Black, red and blue points, respectively, indicate control, mis-targeted and successfully lesioned mice. Horizontal lines show mean and vertical lines show ± SE. r: Spearman's correlation index between the damaged area and the behavioral parameters. *, ** and *** indicate the statistical significance for the correlation (P < 0.05, P < 0.01 and P < 0.001, respectively). Please note that the distribution of each variable, in particular the latency of infanticide, shows a bimodal nature and may skew the correlation index in the scattered plots. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Individual lesion mapping for the BSTrh in virgin malesSuperimposition of the left and right BSTrh lesioned areas of individual male mice with ID numbers, classified into “mis-targeted,” “unilateral” and “bilateral” groups. BSTrh conservative contours are shown as the light blue areas. Numbers are the IDs of individual mouse. Download figure Download PowerPoint Lesions of the cMPOA changed paternal behavior to infanticidal We also examined the effect of excitotoxic lesions in the areas activated during paternal behavior. Our preliminary studies revealed a critical role for the cMPOA, but not the ACN, similar to what was previously seen in females (Tsuneoka et al, 2013). Bilateral cMPOA (= caMPOA + cpMPOA) lesions not only abolished paternal behavior, but drastically shifted a majority of fathers to perform infanticidal behavior on all 4 test days (Fig 4A and B, P < 0.001, = 0.004, < 0.001 and < 0.001, respectively, Fisher's exact probability test; Fig 4F for latency of infanticide; see also Fig EV4 for the individual lesioned areas). Unilateral cMPOA lesions caused a milder effect (Fig 4B, day 1, P = 0.059, Fisher's exact probability test), shown as significantly increased latencies for the first retrieval and pup grouping (Fig 4G and H). On the other hand, the sniffing latency was not grossly affected (Fig 4E), suggesting that the inhibitory effects of cMPOA lesions were likely not due to a non-specific disruption of general sensorimotor responses. Additionally, c-Fos expression in the ipsilateral, but not the contralateral, BSTrh was significantly increased by the unilateral cMPOA lesion (t = −5.014, df = 8, P = 0.001; Fig 4C and D), suggesting ipsilateral inhibitory regulation of the BSTrh activity by cMPOA. Figure 4. Excitotoxic lesions in cMPOA switch behavior in fathers from paternal to infanticidal A, B. The photos and data shown in top, middle and bottom rows, respectively, belong to the control, unilateral and bilateral lesion groups. (A) Representative photomicrographs showing the effects NMDA injections. The sections were stained by immunohistochemistry (IHC) using anti-NeuN (black) and anti-VIP (brown) antibodies. Dashed line indicates cpMPOA. Scale bar = 0.5 mm. (B) Relative frequencies of responses to pups. Committing infanticide within 3 min (red), committing infanticide in 3–30 min (pink), indifference (neither infanticidal nor parenting) (white) and parenting (retrieving at least one pup within 30 min) (blue). *: significant difference from the control group of the same day. #: significant difference from the unilateral group of the same day. C. Representative photomicrographs showing c-Fos expression in BSTrh (dashed contour) 2 h after the final behavioral test. The sections were stained by IHC using anti-c-Fos (black), anti-VIP (brown) and anti-galanin (pink-red) antibodies. Scale bar = 0.25 mm. D. Mean (± SE) density of c-Fos-ir neurons in BSTrh. Different letters indicate significant difference among bars. For the unilateral group, the data were analyzed and shown for each side. E–H. Scatter plot showing the relation between the proportion of the BSTrh damage with the latency to the first pup sniffing (E), the latency to the infanticidal behavior (F), the latency of the first pup retrieval (G) and the latency of the completion of pup grouping (H). For unilateral and bilateral groups, the data contain both mis-targeted (less than half of cpMPOA area was destroyed) and success lesion (half and more than half of cpMPOA area was destroyed). Black, red and blue points, respectively, indicate control, mis-targeted and successfully lesioned mice. Horizontal lines show mean and vertical lines show ± SE. r: Spearman's correlation index between the damaged area and the behavioral parameters. *, ** and *** indicate the statistical significance for the correlation (P < 0.05, P < 0.01 and P < 0.001, respectively). Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Individual lesion mapping for the cMPOA in father malesSuperimposition of the left and right cpMPOA lesioned areas of individual male mice with ID numbers, classified into “mis-targeted,” “unilateral” and “bilateral” groups. cpMPOA conservative contours are shown as the light blue areas. Numbers are the IDs of individual mouse. Download figure Download PowerPoint Social behaviors can be retroactively discriminated by c-Fos expression in the four preoptic-BST nuclei We next tested whether the males' recent social behavior could be deduced simply by measuring the neuronal activation pattern of the preoptic-BST nuclei; this kind of analysis lays the groundwork for possible applications to detect or prevent infanticide b
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