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. 2023 Jan 19;16(1):10.
doi: 10.1186/s13041-023-00993-4.

Role of Calcr expressing neurons in the medial amygdala in social contact among females

Kansai Fukumitsu  1 , Arthur J Huang  2 , Thomas J McHugh  2 , Kumi O Kuroda  3
Affiliations

Role of Calcr expressing neurons in the medial amygdala in social contact among females

Kansai Fukumitsu et al. Mol Brain. .

Abstract

Social animals become stressed upon social isolation, proactively engaging in affiliative contacts among conspecifics after resocialization. We have previously reported that calcitonin receptor (Calcr) expressing neurons in the central part of the medial preoptic area (cMPOA) mediate contact-seeking behaviors in female mice. Calcr neurons in the posterodorsal part of the medial amygdala (MeApd) are also activated by resocialization, however their role in social affiliation is still unclear. Here we first investigated the functional characteristics of MeApd Calcr + cells; these neurons are GABAergic and show female-biased Calcr expression. Next, using an adeno-associated virus vector expressing a short hairpin RNA targeting Calcr we aimed to identify its molecular role in the MeApd. Inhibiting Calcr expression in the MeApd increased social contacts during resocialization without affecting locomotor activity, suggesting that the endogenous Calcr signaling in the MeApd suppresses social contacts. These results demonstrate the distinct roles of Calcr in the cMPOA and MeApd for regulating social affiliation.

Keywords: Calcitonin receptor; Medial amygdala; RNA interference; Social contacts.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sexual dimorphic Calcr expression and gene profiles of Calcr + cells in the medial amygdala. A Representative coronal brain sections of both sexes, showing the distribution of Calcr immunoreactivity (Calcr-ir, black) and NeuN counterstaining (red) in subregions of the amygdala in female or male mice. MeAad anteroventral part of the medial amygdala, MeApd posterodorsal part of the medial amygdala, MeApv posteroventral part of the medial amygdala, CeM medial part of the central amygdala, CeL lateral part of the central amygdala, CeC capsular part of the central amygdala, AA anterior amygdaloid area, LA lateral amygdala, BLA basolateral amygdala, BMA basomedial amygdala. Scale bars, 500 μm. B Number of Calcr-ir neurons in each subregion of female or male mice (n = 4 mice per group). C Sections stained with Gad67 mRNA (blue) by ISH together with anti-Calcr (brown) antibody. Arrowheads indicate double-labeled cells. Scale bars, 500 μm (left) and 50 μm (right). D Sections stained with anti-ERα (black) by IHC together with anti-Calcr (brown) antibody. Scale bars, 500 μm (left) and 50 μm (right). E Sections stained with anti-amylin (black) by IHC together with anti-NeuN (red) antibody. Scale bars, 500 μm (left) and 50 μm (right). Asterisks in (B) denote significant differences between two groups (Welch’s unpaired t-test, *P < 0.05). Graphs represent mean ± SEM. See Additional file 1 for detailed statics
Fig. 2
Fig. 2
Targeted Calcr knockdown in the MeApd does not affect behavioral responses to social isolation. A Timeline of behavioral experiments. At 2 weeks before testing, female mice were administered an AAV expressing shRNA-EGFP for specific Calcr knockdown or with a shRNA virus carrying scrambled shRNA (Cont shRNA) into the MeApd region. An AAV-injected adult female mouse was subjected to a somatic isolation test and reunion tests. B Representative coronal sections of shRNA-EGFP viral expression in the MeApd showing the efficacy of Calcr shRNAs. Scale bars, 2.5 mm (Left), 500 μm (Middle and Right). A shRNA virus carrying scrambled shRNA was used as control. C Quantification of fold changes in the number of Calcr + cells distributed in the MeApd (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). D Time course of biting responses (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). E Quantification of the total number of biting responses over 30 min (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). F Number of various behaviors per min observed under somatic isolation for 30 min (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). Time bins 15 s (D–F). Asterisks denote significant differences between the two groups (C, E, F: Welch’s unpaired t-test, D: 2-way repeated measure ANOVA tests followed by Sidak's post hoc tests; ***P < 0.001). Graphs represent mean ± SEM. See Addi for detailed statics
Fig. 3
Fig. 3
Targeted Calcr knockdown in the MeApd facilitates behavioral responses to the reunion. A Number of various behaviors per min observed under social reunion for 30 min (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). B Time course of the percentage of social contacts (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). (C) Total percentage of social contacts over 30 min (Cont shRNA: n = 15 mice and Calcr shRNA: n = 12 mice). D Raster plots showing the effects of Calcr knockdown in the MeApd on contact occurrence. Time bins 15 s (A), 5 min (B–D). Asterisks denote significant differences between the two groups (A, C: Welch’s unpaired t-test, B: 2-way repeated measure ANOVA tests followed by Sidak's post hoc tests; *P < 0.05, **P < 0.01). Graphs represent mean ± SEM. See Additional file 1 for detailed statics
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