Newborn mice form lasting CA2-dependent memories of their mothers

Abstract

Some of the most enduring social connections begin when infants first recognize their caregivers, memories that form the basis of many family relationships. It remains unknown whether these early social memories persist into adulthood in mice and, if so, which brain regions support them. Here we show that mice form memories of their mother within days after birth and that these memories persist into adulthood. Pups display greater interest in the mother than in an unfamiliar dam before weaning, after which this preference reverses. Inhibition of CA2 neurons in the pup temporarily blocks the ability to discriminate between the mother and an unfamiliar dam, whereas doing so in adulthood prevents the formation of short-term memories about conspecifics, as well as social discrimination related to long-term memories of the mother. These results suggest that the CA2 supports memories of the mother during infancy and adulthood with a developmental switch in social preference.

Keywords: CA2; DREADDs; chemogenetic; development; hippocampus; immediate early gene; kin memory; mother memory; social investigation; social memory; social preference.

Figure 1.. Mice discriminate between the caregiving mother and a novel mother in infancy and adulthood, and exhibit reversed social preference and CA2 IEG expression

(A) Pups placed in a neutral corner of a testing box with the caregiving mother and a novel mother in wire mesh containers for a 2-choice social test, and postweaning offspring undergoing direct social interaction testing. (B) Offspring demonstrate a preference for the caregiving mother from P3 to P14 (P3 n = 19, P6 n = 24, P14 n = 7) and a preference for a novel mother from P30 to P150 (P30 n = 24, P60 n = 20, P100 n = 20, P150 n = 20) (F(6,127) = 10.03, p < 0.0001; Table S1). (C) Pups were exposed to the caregiving mother or a novel mother in a wire mesh container, and postweaning offspring were exposed to the caregiving mother or a novel mother directly and perfused 1 h later. (D) Pups exhibit more Zif268+ CA2 neurons after exposure to the caregiving mother (n = 6) than after exposure to a novel mother (n = 6), whereas adult offspring exhibit the opposite effect (caregiving n = 5, novel n = 5) (F(1,18) = 15.04, p < 0.0001; Table S1). (E) Confocal images of Zif268+ cells (green, arrowheads) in the RGS14 labeled (red) CA2 from pup (top) and adult (bottom) exposed to a caregiving mother (pup) or novel mother, respectively. Scale bars, 200 μm. *p < 0.05 compared with groups within brackets or the same-age caregiving mother group; bars show mean + SEM; sec, seconds.

Figure 2.. Molecular markers of the adult mouse CA2 emerge over the first 2 postnatal weeks

(A–C) NECAB2 labeling appears in the CA2 as early as P3 but with less regional specificity than observed at P60. At P60, NECAB2 was distinctly localized to all layers of the CA2, including the stratum oriens (so), the stratum pyramidale (sp), the stratum radiatum (sr), and the stratum lacunosum moleculare (slm). (D–F) PCP4 labeling of the CA2 is only present at P14 and P60. (G–I) RGS14 is diffuse at P3, with specific labeling of the CA2 at P14 and P60. (J–L) vGLUT2 immunolabeling is absent at P3 but present at P14 and P60. (M–O) Wisteria floribunda agglutinin (WFA)+ PNNs are absent at P3, with intense staining in the CA2 around pyramidal neurons and parvalbumin (PV)+ interneurons at P14 and P60. Scale bars, 200 μm.

Figure 3.. Activation of inhibitory DREADDs in the CA2 impairs memory of the caregiving mother in pups

(A) P3 pups received bilateral injections of control virus (AAV5-hSyn-mCherry) (n = 13) or inhibitory DREADD virus (AAV5-hSyn-hM4D(Gi)-mCherry) (n = 14) targeting the dorsal CA2 (dCA2). (B) PCP4 staining and endogenous mCherry expression reveal specific infection of CA2 neurons with inhibitory DREADD virus at P10 (Figure S4). Scale bars, 200 μm. (C) Schematic demonstrating control- or DREADD-infected P10–P12 pup behavior after CNO or saline administration. (D) Control-virus-infected pups with saline or CNO and DREADD-virus-infected pups with saline had positive difference scores (time spent investigating the caregiving mother minus time spent investigating the novel mother). DREADD-virus-infected pups with CNO did not prefer the caregiving mother over a novel mother (F(1,50) = 5.831, p = 0.0194; Table S1). (E) No differences were observed in overall investigation times between control-virus-infected mice (n = 13) and DREADD-virus-infected mice (n = 13) with saline or CNO (F(1,48) = 0.1757, p = 0.6770; Table S1). *p < 0.05 compared with either the groups within brackets or the DREADD-infected, saline-injected mice; bars represent mean +SEM; sec, seconds.

Figure 4.. Inhibition of CA2 neurons prevents social discrimination of the caregiving mother and a novel mother in adulthood

(A) Adult mice received bilateral injections of control virus (AAV5-hSyn-mCherry) (n = 15) or inhibitory DREADD virus (AAV5-hSyn-hM4D(Gi)-mCherry) (n = 16) targeting the dCA2. (B) RGS14 staining and endogenous mCherry expression reveal specific infection of CA2 neurons with inhibitory DREADD virus (Figure S4). Scale bars, 250 μm. (C) Timeline for viral infection and behavior testing and schematic demonstrating direct social interaction behavior testing in adult virus-infected mice. (D) Adult mice injected with control virus displayed a preference for the novel mother over the caregiving mother after saline or CNO administration, as did adult mice injected with inhibitory DREADD virus and administered saline. Adult mice injected with DREADD virus and CNO did not discriminate between the caregiving mother and a novel mother (F(1,27) = 10.74, p = 0.0004; Table S1). (E) No differences were observed in overall locomotion times between control-virus-infected mice (n = 7) and DREADD-virus-infected mice (n = 7) with saline compared with CNO (F(1,14) = 0.0003419, p = 0.9855; Table S1). *p < 0.05 compared with either the groups within brackets or the DREADD-infected, saline-injected mice; bars represent mean + SEM; sec, seconds.

Figure 5.. Inhibition of CA2 neurons blocks memory of familiar conspecifics in adulthood, but not sociability

(A) Timeline for control (AAV5-hSyn-mCherry) (n = 15) or inhibitory DREADD (AAV5-hSyn-hM4D(Gi)-mCherry) (n = 14) viral infection and direct social interaction testing in adult mice and schematic demonstrating direct social interaction behavior testing. (B) Adult mice infected with control virus and injected with saline, or CNO, preferred a novel same-age mouse, as did adult mice infected with the DREADD virus and then injected with saline. After CNO injection, mice infected with the DREADD virus showed reduced discrimination between the novel and the familiar mouse (F(1,27) = 16.41, p = 0.0004; Table S1). (C) Schematic demonstrating the sociability 3-chamber test. (D) Investigation times of a stimulus mouse were higher than those of an empty container in both DREADD-virus-infected mice (n = 8) and control-virus-infected mice (n = 7) injected with CNO (F(1,13) = 49.45, p < 0.0001; Table S1). *p < 0.05 compared with either the groups within brackets in (B) or the social chamber for each virus group in (D); bars show mean + SEM; sec, seconds.