The hippocampal CA2 region is essential for social memory

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Abstract

The hippocampus is critical for encoding declarative memory, our repository of knowledge of who, what, where and when1. Mnemonic information is processed in the hippocampus through several parallel routes involving distinct subregions. In the classic trisynaptic pathway, information proceeds from entorhinal cortex (EC) to dentate gyrus to CA3 and then to CA1, the main hippocampal output2. Genetic lesions of EC (ref. 3) and hippocampal dentate gyrus (ref. 4), CA3 (ref. 5) and CA1 (ref. 6) regions have revealed their distinct functions in learning and memory. In contrast, little is known about the role of CA2, a relatively small area interposed between CA3 and CA1 that forms the nexus of a powerful disynaptic circuit linking EC input with CA1 output7. Here we report a novel transgenic mouse line that enabled us to selectively examine the synaptic connections and behavioural role of the CA2 region in adult mice. Genetically targeted inactivation of CA2 pyramidal neurons caused a pronounced loss of social memory—the ability of an animal to remember a conspecific—with no change in sociability or several other hippocampus-dependent behaviours, including spatial and contextual memory. These behavioural and anatomical results thus reveal CA2 as a critical hub of sociocognitive memory processing.

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Figure 1: Genetic targeting of the CA2 subfield using the Amigo2-Cre mouse line.
Figure 2: Genetically targeted tracing of the CA2 circuit.
Figure 3: Electrophysiological verification of CA2 inactivation with TeNT.
Figure 4: Inactivation of CA2 impairs social memory.

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Acknowledgements

We thank T. R. Reardon for providing the rabies virus; J. Kupferman and F. Lema for experimental assistance; and C. Denny, Z. Donaldson, R. Hen, J. Gordon, J. Basu and M. Russo for discussions and comments on the manuscript. This work was supported by a Ruth L. Kirschstein F30 National Research Service Award from the National Institute of Mental Health (F.L.H.) and the Howard Hughes Medical Institute (S.A.S.).

Author information

F.L.H. planned and performed the experiments, analysed the data and wrote the manuscript. S.A.S. oversaw the overall execution of the project, contributed to the experimental design and the interpretation of the data, provided financial support and helped write the manuscript.

Correspondence to Steven A. Siegelbaum.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of Amigo2-Cre mouse line.

λ Red-mediated homologous recombination with galK positive and negative selection was used to make seamless changes to the bacterial artificial chromosome (BAC). PCR cassettes are shown in orange, and Amigo2 locus is shown in blue. The PCR cassette contained two homology arms (H1, 58 nucleotides; H2, 62 nucleotides) that flanked the galactose kinase (galK) cassette. The homology arms flanked the Amigo2 start codon. Recombination followed by positive selection was used to obtain the galK integrate. Recombination of the modified BAC with a PCR cassette containing the Cre open reading frame (ORF) and poly(A) (PA) flanked by the same homology arms yielded the final BAC used to generate the transgenic line.

Extended Data Figure 2 Amigo2-Cre mice express Cre in a genetically defined population of CA2 PNs.

Coronal sections of hippocampus from Amigo2-Cre mice injected in dorsal hippocampus with a Cre-dependent AAV to express YFP (shown in green) in CA2. a, Coronal section of ventral hippocampus (about 2.8 mm caudal to bregma; see Fig. 54 of ref. 9 for a reference image) showing CA2 axons (green) from dorsal CA2. Note absence of YFP from ventral CA2 neurons (RGS14 stain in red). b, 97.22 ± 0.46% of YFP+ cells (n = 4 mice, 2,948 cells) express the CA2 marker PCP4 (red). c, 98.45 ± 0.33% of YFP+ cells (n = 4 mice, 2,870 cells) express the CA2 marker STEP (red). d, Almost no YFP+ cells (0.17 ± 0.13%; n = 4 mice, 2,870 cells) express the CA1 marker WFS1 (red). eg, Magnification of boxed area in b, showing YFP signal (e), PCP4 staining (f) and a merge of the two (g). hj, Magnification of boxed area in c, showing YFP signal (h), STEP staining (i) and a merge of the two (j). km, Magnification of boxed area in d, showing YFP signal (k), WFS1 staining (l) and a merge of the two (m). Nissl stain shown in blue. Scale bars, 400 μm (ad); 100 μm (em).

Extended Data Figure 3 Amigo2-Cre mice express Cre in RGS14+ CA2 PNs but not in GABA+ inhibitory neurons.

Cre+ neurons expressing YFP (shown in green) co-label with RGS14 staining (shown in red), but do not co-label with GABA staining (shown in red in separate images). a, Reproduction of section −1.06 mm shown in Fig. 1b. b, e, Magnification of boxed area in a. c, RGS14 staining of section shown in b. d, Merge of b and c, showing YFP and RGS14 co-labelling. f, GABA staining of section shown in e. g, Merge of e and f, showing no overlap of GABA and YFP. h, Reproduction of section −1.46 mm shown in Fig. 1b. i, l, Magnification of boxed area in h. j, RGS14 staining of section shown in i. k, Merge of i and j, demonstrating YFP and RGS14 co-labelling. m, GABA staining of section shown in l. n, Merge of l and m, showing no overlap of GABA and YFP. o, Reproduction of section −2.18 mm shown in Fig. 1b. p, s, Magnification of boxed area in o. q, RGS14 staining of section shown in p. r, Merge of p and q, demonstrating YFP and RGS14 co-labelling. t, GABA staining of section shown in s. u, Merge of s and t, showing no overlap of GABA and YFP. Scale bars, 200 μm. Nissl stain shown in blue.

Extended Data Figure 4 Specificity of the pseudotyped rabies virus.

a, b, No labelled cells were observed (n = 3 mice) after injection of the (EnvA)SAD-ΔG-mCherry virus when TVA was not expressed in CA2. b, Magnification of boxed area in a. Rabies labelling would have appeared in magenta; Nissl stain shown in green. Scale bars, 200 μm.

Extended Data Figure 5 Inactivation of CA2 does not alter locomotor activity or anxiety-like behaviour.

a, There was no significant difference (P = 0.31, two-tailed unpaired Student’s t-test) between CA2-YFP and CA2-TeNT groups in the distance travelled in the open field (OF) test (YFP, 53.14 ± 4.62 m, n = 8; TeNT, 47.04 ± 3.70 m, n = 10). b, There was also no significant difference (P = 0.55, two-tailed unpaired Student’s t-test) between the groups in the number of rearing events recorded during the OF session (YFP, 378.0 ± 17.36, n = 8; TeNT, 354.7 ± 30.99, n = 10). c, d, Inactivation of CA2 did not alter anxiety-like behaviour measured in the elevated plus maze (EPM). The number of open arm entries was not significantly different (P > 0.99, two-tailed unpaired Student’s t-test) between the groups (YFP, 14.00 ± 1.46, n = 8; TeNT, 14.00 ± 1.54, n = 10). Additionally, the time spent in the open arms (YFP, 163.7 ± 10.43 s, n = 8; TeNT, 155.1 ± 16.38 s, n = 10) did not differ significantly (P = 0.68, two-tailed unpaired Student’s t-test) between the groups. Results are means ± s.e.m.

Extended Data Figure 6 Spatial learning and memory assayed with the Morris water maze task is unaltered by CA2 inactivation.

a, Diagram of the experimental design. On days 1 and 2 mice were trained to find a platform with a visible flag. On days 3–7 mice were trained to find a hidden platform located in the southwest quadrant of the water maze. Spatial memory was assayed on day 8 with the platform removed. Reversal training was conducted on days 9–13 with the platform now hidden in the northwest quadrant. Spatial memory of the novel location was tested on day 14. b, Path length to the platform was not altered significantly by CA2 inactivation (two-way repeated-measures ANOVA: treatment × time F(11,770) = 0.67, P = 0.77; time F(11,770) = 21.87, P < 0.0001; treatment F(1,70) = 2.85, P = 0.10). c, Latency to find the platform did not differ significantly between the two groups (two-way repeated-measures ANOVA: treatment × time F(11,770) = 0.78, P = 0.66; time F(11,770) = 25.23, P < 0.0001; treatment F(1,70) = 2.84, P = 0.10). YFP, n = 8; TeNT, n = 10. d, Spatial memory during the probe trial was unaffected by CA2 inactivation. The percentage of time spent in the target quadrant (YFP, 33.00 ± 2.66%; TeNT, 38.6 ± 4.79%) was not significantly different between the two groups (P = 0.36, two-tailed unpaired Student’s t-test). e, Spatial memory after reversal training was unaffected by CA2 inactivation. There was no significant difference between the groups in the percentage of time spent in the target quadrant during the probe trial after reversal training (YFP, 36.38 ± 5.75%; TeNT, 36.40 ± 2.92%; P > 0.99, two-tailed unpaired Student’s t-test). Results are means ± s.e.m.

Extended Data Figure 7 Contextual fear-conditioning memory and auditory fear-conditioning memory are unaffected by inactivation of CA2.

a, Diagram of the experimental design. Delay fear conditioning was employed to test hippocampus-dependent contextual fear memory and amygdala-dependent auditory fear memory. b, There was no significant difference in percentage freezing between the groups (two-way repeated-measures ANOVA: treatment × day F(4,68) = 0.31, P = 0.87; treatment F(1,17) = 0.13, P = 0.73; day F(4,68) = 100.8, P < 0.0001; YFP, n = 11; TeNT, n = 8). Before training on day 1, neither group showed a fear response to context A (YFP, 2.45 ± 1.06%; TeNT, 0.75 ± 0.49%) or to the tone (YFP, 3.09 ± 1.31%; TeNT, 1.63 ± 0.84%). On day 2 after training, robust fear responses to context A were measured in both groups (YFP, 24.09 ± 2.88%; TeNT, 26.00 ± 4.10%). Both groups showed low levels of freezing on day 3 in novel context B (YFP, 6.55 ± 1.52%; TeNT, 4.00 ± 0.87%), demonstrating context specificity of the fear memory and a lack of fear generalization. Both groups showed robust freezing to the tone on day 3 (YFP, 35.82 ± 4.93%; TeNT, 34.63 ± 3.96%), demonstrating intact auditory fear memory. c, Freezing data plotted in 30-s bins. Shaded areas represent tone presentation. Red line represents shock delivery. Left: two-way repeated-measures ANOVA revealed no significant difference between groups in freezing on day 1 (treatment × time F(6,102) = 1.135, P = 0.3474; treatment F(1,17) = 1.116, P = 0.3056; time F(6,102) = 6.348, P < 0.0001). Middle: two-way repeated-measures ANOVA revealed no significant difference between groups in freezing on day 2 (treatment × time F(9,153) = 0.9741, P = 0.4637; treatment F(1,17) = 0.1326, P = 0.7203; time F(9,153) = 6.335, P < 0.0001). Right: two-way repeated-measures ANOVA revealed no significant difference between groups in freezing on day 3 (treatment × time F(7,119) = 0.2490, P = 0.9716; treatment F(1,17) = 0.6517, P = 0.4307; time F(7,119) = 50.87, P < 0.0001). Results are means ± s.e.m.

Extended Data Figure 8 Object recognition memory and preference for novelty is preserved in CA2-TeNT animals.

a, Diagram of the experimental design for the novel-object-recognition task. b, The groups did not differ significantly in exploration of object 1 (YFP, 16.75 ± 1.57 s; TeNT, 19.60 ± 2.24 s) or object 2 (YFP, 16.50 ± 1.97 s; TeNT, 15.90 ± 1.66 s) averaged over the course of the first four trials (two-way ANOVA: treatment × object F(1,32) = 0.80, P = 0.38; object F(1,32) = 1.05, P = 0.31; treatment F(1,32) = 0.34, P = 0.56; YFP, n = 8; TeNT, n = 10). c, Both groups explored the novel object (YFP, 21.23 ± 2.37 s; TeNT, 24.37 ± 2.81 s) more than the familiar object (YFP, 7.41 ± 0.92 s; TeNT, 8.57 ± 1.48 s). Statistical analysis revealed a significant effect of object, but not CA2 inactivation or interaction of the two (two-way ANOVA: treatment × object F(1,28) = 0.22, P = 0.64; object F(1,28) = 48.46, P < 0.0001; treatment F(1,28) = 1.02, P = 0.32). Multiple comparison testing revealed a significant difference between exploration of the novel object compared with exploration of the old object for both the YFP group (P = 0.0002) and the TeNT group (P < 0.0001). d, Diagram of the experimental design for another variation of the novel-object-recognition task. e, The groups did not differ significantly in time spent exploring object 1 (YFP, 21.50 ± 2.31 s; TeNT, 22.18 ± 3.57 s) or object 2 (YFP, 22.02 ± 2.23 s; TeNT, 22.36 ± 2.81 s) during trial 1 of day 4 (two-way ANOVA: treatment × object F(1,44) = 0.004, P = 0.95; object F(1,44) = 0.02, P = 0.90; treatment F(1,44) = 0.03, P = 0.85; YFP, n = 12; TeNT, n = 12). f, Both groups explored the novel object (YFP, 21.49 ± 1.91 s; TeNT, 22.73 ± 1.82 s) more than the familiar object (YFP, 13.74 ± 1.83 s; TeNT, 16.53 ± 1.64 s). Statistical analysis revealed a significant effect of object, but not CA2 inactivation or interaction of the two (two-way ANOVA: treatment × object F(1,44) = 0.18, P = 0.67; object F(1,44) = 15.02, P = 0.0004; treatment F(1,44) = 1.25, P = 0.27). Multiple comparison testing revealed a significant difference between exploration of the novel object compared with exploration of the old object for both the YFP group (P = 0.008) and the TeNT group (P = 0.02). Results are means ± s.e.m.

Extended Data Figure 9 Olfaction is unaffected by CA2 inactivation.

a, There was no significant difference between the groups in latency to find a buried food pellet (YFP, 63.93 ± 8.22 s, n = 15; TeNT, 67.06 ± 9.42 s, n = 16; P = 0.81, two-tailed unpaired Student’s t-test). b, There was no significant difference between the groups (YFP, n = 15; TeNT, n = 14) in performance on the olfactory habituation/dishabituation task (two-way repeated-measures ANOVA: treatment × trial F(11,297) = 0.933, P = 0.51; treatment F(1,27) = 0.08, P = 0.78; trial F(11,297) = 60.21, P < 0.0001). Results are means ± s.e.m.

Extended Data Table 1 Electrophysiological properties of Cre+ neurons

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Hitti, F., Siegelbaum, S. The hippocampal CA2 region is essential for social memory. Nature 508, 88–92 (2014). https://doi.org/10.1038/nature13028

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