The ventral CA2 region of the hippocampus and its differential contributions to social memory and social aggression

Abstract

The dorsal and ventral regions of the CA1 field of the hippocampus play distinct roles in the encoding of cognitive vs. emotional behaviors, respectively. Whether this distinction applies to other hippocampal fields and other behaviors is unclear. Here, we focus on the hippocampal CA2 field and compare the properties and behavioral roles of its dorsal (dCA2) and ventral (vCA2) regions. Although dCA2 is known to be required for social memory and to promote social aggression, the role of vCA2 is unknown. We report that a defined CA2 region extends to the extreme ventral pole of the hippocampus, with certain distinctions to dCA2. Unlike dCA2, chemogenetic silencing of vCA2 pyramidal neurons did not impair social memory. Similar to dCA2, vCA2 was required to promote social aggression. Thus, consistent with the CA1 region, CA2 may be differentially tuned to support cognitive compared with emotional processes along its dorsal to ventral axis.

Keywords: CA2; CP: Cell biology; CP: Neuroscience; dorsal; hippocampus; social aggression; social memory; social recognition; ventral.

Figure 1.. Horizontal slices from Avpr1b-Cre^+/− x Ai14 mice reveal tdTomato expression along the entire dorsoventral axis

tdTomato (Avpr1b) expression was present along the entire axis from the most dorsal (1) to most ventral (7) slices. Numbers on images correspond to arrows in inset. Scale bar, 200 μm.

Figure 2.. Distinct patterns of co-expression of prototypical CA2 markers and Avpr1b-Cre mediated expression in dorsal compared with ventral hippocampus

(A–E) Dorsal hippocampal transverse slices from Avpr1b-Cre^+/− × Ai14 mice labeled with RGS14 (mouse n = 3) or STEP (mouse n = 4), co-labeled with PCP4 and DAPI. (F–J) Ventral hippocampal transverse slices from Avpr1b-Cre^+/− × Ai14 mice labeled with RGS14 (mouse n = 3) or STEP (mouse n = 2), co-labeled with PCP4 and DAPI. Top white arrow indicates an example cell expressing two markers plus tdTomato (triple-labeled). Scale bar in A, B, F, and G, 200 μm. Bottom white arrow indicates an example cell that expressed PCP4 but not tdTomato (Avpr1b) or RGS14. (K) Percentage of cells that expressed one or more of the three CA2 markers was significantly less in the vCA2 region than in the dCA2 region. Mann-Whitney test, p = 0.0025. Data represented as mean ± SEM. (L) Percentage of cells that expressed the indicated combination CA2 marker(s) relative to the total subpopulation of cells in the vCA2 region that expressed at least one CA2 marker (PCP4, tdTomato/Avpr1b, and/or RGS14/STEP). In the dCA2 region, 98.0% ± 1.3% of cells expressed all three prototypical CA2 markers; none expressed only PCP4 (0.0% ± 0.0%). Significantly fewer cells expressed three markers (43.4% ± 16.5%) in vCA2 than dCA2 (note greater variance); significantly more vCA2 cells expressed only PCP4 (36.1% ± 6.3%) compared with dCA2 (two-way ANOVA Marker × Region, F(4,44) = 79.51, p < 0.0001). Šídák’s multiple comparisons test for dCA2 vs. vCA2; RGS14 only, p = 0.99; tdTomato only, p > 0.99; PCP4 only, p = 0.0011; PCP4 and tdTomato, p = 0.15; triple-labeled, p = 0.0087). Central line is the median. Outer limits of box represent first and third quartiles. Whiskers represent max and min. ns = not significant, **p < 0.01.

Figure 3.. vCA2 PNs receive stronger excitatory inputs from EC than from CA3

(A) A transverse ventral hippocampal slice with a single biocytin-filled CA2 PN (white) from an Avpr1b-Cre^+/− × Ai14 mouse (left). Higher magnification view showing the morphological features of the same cell (right). Scale bars, 200 μm, left; 30 μm, right. (B) Sample traces of compound PSPs (baseline), EPSPs recorded after applying GABA antagonists (GABAblock), and inferred IPSPs obtained by subtracting EPSP from baseline PSP (difference). EPSPs elicited with stimulating electrodes placed in SR to stimulate CA3 inputs (blue traces) or SLM to stimulate EC inputs (red traces). (C and D) The amplitude of the PSP (C) and EPSP (D) evoked from SLM (red) was significantly larger than that evoked from CA3 (blue). PSP: two-way ANOVA F(1,420) = 81.04, p < 0.0001, SR: n_mice = 9, SLM: n_mice = 10. EPSP: two-way ANOVA F(1,196) = 16.73, p < 0.0001, SR: n_mice = 5, SLM: n_mice = 6. (E) IPSPs evoked from SLM and SR were similar in amplitude. Two-way ANOVA F(1,196) = 1.973, p = 0.1617. SR, n = 5 mice; SLM, n = 6 mice. (F) The EPSP/IPSP amplitude ratio evoked from SLM was greater than that from SR. Two-way ANOVA, F(1,196) = 19.09, p < 0.0001. SR, n = 5 mice, SLM, n = 6 mice. ****p < 0.0001. Data are represented as mean ± SEM.

Figure 4.. Dual fluorescent protein expression in dCA2 and vCA2 reveals a topographic projection pattern along the hippocampal dorsoventral axis and in lateral septum

(A) Experimental scheme. Cre-dependent GFP- or YFP-expressing AAV injected in dCA2 and Cre-dependent RFP-expressing AAV injected in vCA2 in Avpr1b-Cre^+/− mouse. (B–J) Transverse slices from dorsal hippocampus (B–D) and ventral hippocampus (E–J). Scale bar in B, E, and H, 200 μm. (B and E) Composite tiled images of injection site in dCA2 (B) and vCA2 (E). (C and F) Red channel, vCA2 projections to dorsal (C) and ventral (F) hippocampus. Green channel, dCA2 projections to dorsal (D) and ventral (G) hippocampus. (H) Zoomed image of vCA1 from composite image in (E) showing distinct projection pattern of dCA2 and vCA2 to vCA1. (I) vCA2 projection to vCA1 (red channel) was predominantly located in stratum orients. (J) dCA2 projection to vCA1 (green channel) was predominantly located in stratum radiatum. Scale bar, 200 μm. White arrow indicates magnified vCA1 region in (H)–(J). (K) 5x composite image of a coronal slice from an Avpr1b-Cre^+/− mouse injected with Cre-dependent AAV to express EYFP in dCA2 and mCherry in vCA2. Images show projections from dCA2 (green) to anterior dorsal lateral septum and from vCA2 (red) to posterior ventral lateral septum. Scale bar in K, 200 μm. (L) Left: 20x merged image of projections from dCA2 (green) and vCA2 (red) to lateral septum. Center: dCA2 projections only (green channel). Right: vCA2 projections only (red channel). Scale bar in L (left), 200 μm. (M) Left: 10x horizontal tile from Avpr1b-Cre^+/− mouse showing projections from dCA2 (green) to anterior dorsal lateral septum and projections from vCA2 (red) to posterior ventral lateral septum. White A indicates anterior side; white P indicates posterior side. Center: dCA2 projections only (green channel). Right: vCA2 projections only (red channel). Scale bar in M (left ), 200 μm.

Figure 5.. Optogenetic activation of vCA2 evokes larger PSP in PNs in vCA1b/c compared with vCA1a

(A) Avpr1b-Cre^+/− mice were injected with Cre-dependent AAV to express ChR2 in vCA2. Light-evoked PSPs or postsynaptic currents were measured, respectively, in current-clamped or voltage-clamped PNs in vCA1a or vCA1b/c. Membrane held at −70 mV. White neurons in vCA1b show biocytin staining following patch clamp recordings with biocytin in electrode. Scale bar, 100 μm. (B) Proportion of neurons in vCA1a and vCA1b/c with measurable light-evoked synaptic responses (chi-square, z = 2.338, p = 0.019). Cells recorded from voltage clamp and current clamp were pooled. (C) Example traces of light-evoked PSP in PNs in vCA1a and vCA1b/c. (D) Peak current-clamped PSP amplitude as a function of light intensity recorded in vCA1a and vCA1b/c PNs. PSP was significantly larger in vCA1b/c compared with vCA1a. Two-way ANOVA, vCA1a vs. vCA1b/c: F(1, 174) = 21.99, p < 0.0001. (E) Integral of light-evoked PSP was significantly larger in vCA1b/c compared with vCA1a. Two-way ANOVA, vCA1a vs. vCA1b/c: F(1,174) = 9.833, p = 0.002). **p < 0.01, ****p < 0.0001. Data are represented as mean ± SEM.

Figure 6.. Pharmacogenetic inhibition of Cre-expressing cells in dCA2 but not vCA2 of Avpr1b-Cre^+/− mice blocks social memory recall

Avpr1b-Cre^+/− (data shown in magenta) and Cre^−/− (data shown in orange) littermates injected in either dCA2 (B–D) or vCA2 (E–G) with Cre-dependent mCherry-iDREADD AAV. (A) One-trial test for social memory recall. A subject mouse explored a three-chamber arena with a familiar littermate and novel conspecific confined under wire pencil cup cages in opposite chambers for 10 min. (B) Dorsal hippocampal transverse slice co-stained for mCherry and PCP4. Scale bar, 200 μm. (C) Exploration times of littermate and novel mouse in social memory test for Avpr1b-Cre^+/− (n = 16) and Cre^−/− (n = 16) littermates. Both groups administered CNO 30 min prior to test. Cre^−/− but not Cre^+/− littermates injected in dCA2 preferentially explored the novel mouse. Two-way ANOVA genotype × interaction partner F(1,30) = 6.937, p = 0.013; Šídák’s multiple comparisons test: Cre^−/−, littermate vs. novel, p = 0.0003; Cre^+/−, littermate vs. novel, p = 0.76. (D) Discrimination Index measure of preference for novel vs. familiar mice was significantly different from zero for Cre^−/− but not Cre^+/− mice. One-sample t test against zero: Cre^−/−, t = 5.306, df = 15, p < 0.0001; Cre^+/−, t = 0.5603, df = 15, p = 0.56. Unpaired t test between Cre^−/− and Cre^+/− mice, t = 2.687, df = 30, p = 0.012. (E) ventral hippocampal transverse slice co-stained for mCherry and PCP4. (F) Both Cre^−/− (n = 24) and Cre^+/− mice (n = 18) injected in vCA2 interact more with novel compared with familiar littermate. Two-way ANOVA interaction partner F (1,40) = 16.71, p = 0.0002; genotype × interaction partner, F(1,40) = 0.005, p = 0.94; followed by Šídák’s multiple comparisons test for littermate vs. novel: Cre^−/−, p = 0.0078; Cre^+/−, p = 0.018. A greater interaction time with the novel over familiar littermate is only observed in Cre^+/− mice expressing iDREADD in vCA2 compared with dCA2 (two-way ANOVA interaction partner × brain region F(1,32) = 4.938, p = 0.0335; followed by Šídák’s multiple comparisons test for littermate vs. novel: dCA2, p = 0.80; vCA2, p = 0.010). (G) Discrimination index for Cre^−/− and Cre^+/− mice was significantly greater than zero. One-sample t test against zero: Cre^−/−, t = 2.648, df = 23, p = 0.014; Cre^+/−, t = 3.988, df = 17, p = 0.001. Unpaired t test comparing Cre^−/− and Cre^+/−, t = 0.1545, df = 40, p = 0.88. There was a significant difference between discrimination index of Cre^+/− mice injected with Cre-dependent mCherry-iDREADD in dCA2 (data from D) vs. vCA2 (G). Unpaired t test t = 2.265, df = 32, p = 0.030. Central line is the median. Outer limits of box represent first and third quartile. Whiskers represent max and min. *p < 0.05,**p < 0.01, ***p < 0.001, ns = non-significant.

Figure 7.. Inhibition of Cre-expressing neurons in vCA2 of PCP4-Cre^+/− mice decreases social aggression

PCP4-Cre^+/− (data shown in magenta) and Cre^−/− (data shown in orange) littermates injected in vCA2 with Cre-dependent mCherry-iDREADD AAV. (A) Ventral hippocampal transverse slice co-stained for mCherry and PCP4. Scale bar, 200 μm. (B) Three weeks following viral injection, both groups of subjects (residents) were administered CNO 30 min prior to exposure to a novel mouse intruder in a resident-intruder test. Total time spent in attack was significantly lower in Cre^+/− (n = 8) compared with Cre^−/− (n = 8) residents. Median attack duration: Cre^−/− = 12.3 s; Cre^+/− = 0.15 s; Mann-Whitney test, p = 0.0042. (C) Cre^−/− residents showed significantly more attack bouts compared with Cre^+/− residents (median Cre^−/− = 13 attacks, median Cre^+/− = 0.5 attacks, Mann-Whitney test, p = 0.018). (D) Cre^+/− and Cre^−/− subjects displayed similar total duration of all social interactions (total = time spent in aggression + social dominance + social investigation). Median Cre^−/− = 134.3 s, median Cre^+/− = 140.6 s, Mann-Whitney test, p = 0.8517. Central line is the median. Outer limits of box represent first and third quartile. Whiskers represent max and min. **p < 0.01, *p < 0.05, ns = non-significant.