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, 17 (2), 269-79

Cell Type-Specific Genetic and Optogenetic Tools Reveal Hippocampal CA2 Circuits

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Cell Type-Specific Genetic and Optogenetic Tools Reveal Hippocampal CA2 Circuits

Keigo Kohara et al. Nat Neurosci.

Abstract

The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit entorhinal cortex layer II (ECII)→dentate gyrus→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the primary substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, have only recently come to light. Using highly cell type-specific transgenic mouse lines, optogenetics and patch-clamp recordings, we found that dentate gyrus cells, long believed to not project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells through abundant longitudinal projections. CA2 innervated CA1 to complete an alternate trisynaptic circuit, but, unlike CA3, projected preferentially to the deep, rather than to the superficial, sublayer of CA1. Furthermore, contrary to existing knowledge, ECIII did not project to CA2. Our results allow a deeper understanding of the biology of learning and memory.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Molecular, cellular, anatomical, and electrophysiological definition of the CA2 region of the hippocampus
a–d, Representative images of the putative CA2 marker gene expressions, RGS14 (a), PCP4 (b), STEP (c), and a superposed image (d). e–h, Confocal images of CA2 from dotted line-box in (d). i–j, Zeta-projected confocal images of RGS14-positive CA2 neurons (i) and dendritic spine morphologies of CA2 neurons visualized by DiI (j). k–l, Typical dendritic morphologies of CA2 neurons showing a lack complex spines (k) and CA3 neurons with complex spines (l). m–p, Axonal dye tracer BDA was injected into the supramammillary nucleus (SUM). Injection site and diagram (bottom) showing axonal projections from SUM to CA2 and DG (m). Zeta-projected confocal image of BDA-positive terminals (n). Magnified image from the dotted line-box in (n) showing overlay between PCP4 and BDA (o). Single confocal stack from the dotted line-box in (o) showing overlay between PCP4-dendrites and BDA-positive terminals (p). q, Representative z-projected confocal image of a biocytin stained CA2 pyramidal cell. Inset from the dotted-line box: single confocal stacks showing biocytin (top) and RGS14 staining (bottom). r, Patch-clamp recording from a CA2 pyramidal cell (same as q) in current clamp configuration showing the response to a positive step current injection. Note the late firing preceded by a slow depolarizing ramp. Inset: magnification of an action potential showing the fast hyperpolarizing potential. s, I–V curve, traces from the same cell in (q). Double arrowhead: note the slow depolarizing ramp. t, I–V for n=11 cells (mean±SEM), linear fit in red.
Figure 2
Figure 2. Dentate mossy fiber (MF) projections to CA2
a, Confocal image showing PCP4 and calbindin staining in CA2. b, Magnification (top) of dotted line-box in (a) and quantification (bottom) of the number of PCP4-positive CA2 cells receiving calbindin-positive MFs (MF+) and not receiving MFs (MF–). Data are represented as mean ± SEM. Dotted line indicates MFs ending point. c, Confocal image showing ZnT3 and RGS14 staining in CA2. d, Superposed image of ZnT3, VGluT1 and RGS14. Dotted lines in (c) and (d) enclose stratum lucidum. e, Confocal image showing RGS14-positive dendrites in CA2. f, Confocal image showing RGS14-positive dendrites and ZnT3-positive clusters. g, Confocal image showing RGS14-positive dendrites and PSD95-positive clusters. h, Superposed image showing ZnT3-PSD95 double-positive clusters on RGS14-positive dendrites. Bottom: magnification from the inset showing ZnT3-positive, PSD95-positive and ZnT3-PSD95 double-positive clusters (arrowheads). i, ChETA-YFP HSV construct (top). Confocal image of MFs visualized by ChETA-YFP (bottom). i–k, Z-projection from dotted line-boxes in (e) centered on CA3 (j) and CA2 (k). l, Cumulative histogram of MF terminal diameters for CA2 (n=60 boutons) and CA3 (n=43 boutons).
Figure 3
Figure 3. Longitudinal projections of Dentate mossy fibers (MFs) within CA2
a, Schematic of DGGC-specific transgenic Cre mouse, Cre-dependent tTA lentivirus and tTA-dependent ArchT-GFP-AAV9. Cre-recombinase switches on the expression of tTA in DGGCs inducing the expression of ArchT-GFP in DGGCs. b, Dorsorostral injection-site (green) and location of serial sections in (c). c, Serial images of RGS14, ArchT-GFP and synaptoporin (MF marker). DG cells in sections #1–3 express ArchT-GFP. d, Confocal image from the dotted line-box in (c). e, Longitudinal projections of MFs in CA2. f–h, Same approach applied to dorsal (f), intermediate (g) and ventral (h) DG injection sites (green dots) and location of serial sections in (i, j, k) respectively. i–k, Images showing extent and direction of MFs (green) within the RGS14-positive (red) CA2 region for dorsal (i), intermediate (j) and ventral (k) DG injection sites. l–n, Magnified confocal images from the corresponding dotted line-boxes in (i, j, k). o–q, Normalized fluorescence intesity of GFP-positive MFs terminals within dorsal (D), intermediate (I) and ventral (V) CA2, for dorsal (o), intermediate (p) and ventral (q) DG injection sites (n=6 samples per group from n=3 mice per injection site). Data are represented as mean ± SEM.
Figure 4
Figure 4. Functional monosynaptic connection of MF terminals onto CA2 pyramidal cells
a, DGGC-specific Cre transgenic mice were injected with Cre-dependent AAV9-EF1α-ChR2-YFP. Cre-recombinase switches on the expression of ChR2-YFP in DGGCs. b, DGGC-specific expression of ChR2-YFP (top) and enlarged image from the dotted line-box (bottom). c, Optogenetic stimulation of ChR2-YFP-positive DGGCs. d, Zeta-projected confocal image of a recorded CA2 pyramidal cell (yellow) overlapped by ChR2-YFP-positive MFs (green). Bottom: single confocal stacks showing colocalization of biocytin (red) and RGS14 (cyan). e, Patch-clamp recording of CA2 pyramidal cell and optogenetic stimulation of MFs in acute hippocampal slice. f–g, Optogenetic stimulation of MFs elicited an EPSC in a CA2 pyramidal cell (f) which is sensitive to NBQX/AP5 (n=5, two tailed paired t-test: *P<0.05) and DCG-IV (n=5, two tailed paired t-test: *P<0.05) (g). Data are represented as mean ± SEM. h, Representative zeta-projected confocal image of CA1, CA2 and CA3 pyramidal cells recorded in the same slice. i–j, EPSCs recorded in CA1, CA2 and CA3 pyramidal cells elicited by optogenetic stimulation of MFs (i). CA2 and CA3 cells displayed a fast EPSC onset whereas CA1 cells displayed a delayed onset (j, n=9 triplets, for CA1 and CA2, two tailed paired: t-test ***P<0.001, for CA3 and CA2 *P<0.05).
Figure 5
Figure 5. Response of CA2 cells to optogenetic stimulation of mossy fibers (MFs)
a, Z-projected confocal image of biocytin-filled CA2 pyramidal cell. Note ChR2-YFP-positive MFs. Inset from the dotted-line box: single confocal stacks showing biocytin (top) and RGS14 staining (bottom). b, Patch-clamp recording from a CA2 pyramidal cell (same as a) in current mode showing the spiking activity in response to optogenetic stimulation (30 Hz train) of MFs. c, Spiking activity of the same CA2 cell. Bottom: firing probability. d, Average firing probability in response to a 30 Hz train of light pulses for n=14 CA2 pyramidal cells. Note the increased probability in the recovery pulse R. e, Current clamp recording from a CA2 pyramidal cell in response to optogenetic stimulation of MFs showing a large EPSP (average of 30 traces in red) followed by inhibition sensitive to antagonists of GABAergic transmission (blue CPG, black GBZ). f–g, EPSP amplitude and decay, as shown in (e), are affected by GABAa and GABAb antagonists (two-tailed paired t-test: ***P<0.001, *P<0.05, n=4). Average: black lines. h, Z-projected confocal image of a biocytin-filled (violet) CA2 interneuron. Inset from the dotted line-box: CA2 interneuron GAD67-positive and parvalbumin-negative. Note the ChR2-YFP-positive MFs in green. i, Current (black) and voltage clamp recordings (green) of the same CA2 interneuron in (h), in response to optogenetic stimulation of MFs. Note the spiking activity (action potential peak time: green asterisks). Inset: intrinsic electrophysiological properties in response to current steps. j, First action potential peak times from n=5 CA2 interneurons in response to optogenetic stimulation of MFs (average: green dotted-line). k, EPSC amplitudes and onsets from n=11 CA2 interneurons in response to optogenetic stimulation of MFs (average: green dot).
Figure 6
Figure 6. Cortico-hippocampal projections to CA2
a, The EC of a MECIII-specific Cre mouse was injected with a Cre-dependent AAV9-EF1α-ChR2-YFP. Parasagittal hippocampal slice stained for RGS14, netrin-G2 a MECII-specific marker and DAPI (white). On the side: enlarged confocal images from the dotted line-box. ChR2-YFP-positive MECIII fibers (green) are restricted to CA1-SLM and do not overlap with CA2. b–d Optogenetic stimulation of MECIII fibers. (b) Confocal image of biocytin-filled (violet) CA1, CA2, and CA3 pyramidal cells. Note ChR2-YFP-positive MECIII fibers (green) in CA1-SLM. Insets from the dotted-line box: CA2 pyramidal cell identified by colocalization of biocytin (violet) with RGS14 (red). (c) The same cells in voltage clamp, in response to optogenetic stimulation of ChR2-YFP-positive MECIII fibers. (d) Only CA1 pyramidal cells displayed an excitatory response. e, The EC of a wild type mouse was injected with a non-Cre-dependent AAVrh8-CaMKIIα-ChR2-YFP. Parasagittal hippocampal slice expressing ChR2-YFP, stained for netrin-G2. On the side: enlarged images from the dotted line-box. YFP-positive MECII fibers costained with netrin-G2 (yellow) running into the DG, CA3 and overlapping with RGS14-positive dendrites of CA2. f, Confocal image of biocytin-filled (violet) CA1, CA2 and CA3 pyramidal cells. Note the ChR2-YFP-positive (green) MECII/III fibers. Inset from the dotted-line box: CA2 pyramidal cell identified by colocalization of biocytin (violet) with RGS14 (red). g, Same cells in voltage clamp, in response to optogenetic stimulation of ChR2-YFP-positive MECII/III fibers. h, Average EPSC amplitudes for CA1, CA2 and CA3 pyramidal cells. Data are represented as mean ± SEM.
Figure 7
Figure 7. Mapping inputs to hippocampal CA2 neurons using the rabies virus-based monosynaptic tracing reveals MECII and LECII as the primary source of entorhinal inputs
a–b, Schematic (a) and sagittal whole brain section (b) from CA2-specific Cre mouse, injected with helper AAV. Insets from the dotted line-box: note the EGFP expression restricted to RGS14-positive CA2 pyramidal cells. c, Sagittal whole brain section from CA2-specific Cre mouse, injected with helper AAV and rabies virus (RV), expressing AAV-specific EGFP signal (green), RV-specific mCherry signal (red) and stained with Nissl (blue). d, Percent of mCherry-only positive cells in layer II and III of MEC (top) and LEC (bottom), respectively (n=6 samples from 3 mice, two tailed paired t-test ***P<0.001 and **P<0.005, average in red). e–f, Horizontal sections showing the mCherry-only positive cells in both MEC (d) and LEC (e). Dotted line indicates ECII/ECIII border.
Figure 8
Figure 8. A preferential connection from CA2 to deep CA1 pyramidal cells establishes a novel trisynaptic circuit: DG-CA2-CA1deep.
a, CA2-specific Cre knock-in mice were injected with Cre-dependent AAV9-EF1α-ChR2-YFP. Cre-recombinase switches on the expression of ChR2-YFP in CA2. b, ChR2-YFP expression in RGS14-positive CA2 cells. c, Optogenetic stimulation of a ChR2-YFP-positive CA2 pyramidal cell. d, Representative image of a recorded CA1 pyramidal cell pair located in different sublayers (dotted line: pyramidal cell layer). Upper side-panels: deep cell CaMKIIα-positive and calbindin-negative. Lower side-panels: superficial cell CaMKIIα-positive and calbindin-positive. e, Patch-clamp recordings of CA1 superficial and deep pyramidal cells combined with optogenetic stimulation of CA2 fibers in acute hippocampal slices. f–g, Optogenetic stimulation of CA2 fibers elicited EPSCs in a CA1 pyramidal cell pair. Deep pyramidal cells displayed a stronger response (n=14 pairs, two tailed paired t-test: ***P<0.001). EPSCs were sensitive to NBQX/AP5 (n=7, two tailed paired t-test: *P<0.05). Data are represented as mean ± SEM.

Comment in

  • Deciphering CA2 connectivity.
    Bayer H. Bayer H. Nat Neurosci. 2014 Feb;17(2):152. doi: 10.1038/nn0214-152. Nat Neurosci. 2014. PMID: 24473262 No abstract available.

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