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. 2021 Aug;1(2):101-111.
doi: 10.1016/j.bpsgos.2021.04.005. Epub 2021 May 4.

Modeling intrahippocampal effects of anterior hippocampal hyperactivity relevant to schizophrenia using chemogenetic excitation of long axis-projecting mossy cells in the mouse dentate gyrus

James P Bauer  1 , Sarah L Rader  1 , Max E Joffe  2   3 , Wooseok Kwon  1 , Juliana Quay  4 , Leann Seanez  1 , Chengwen Zhou  5   6 , P Jeffrey Conn  2   3   6   7 , Alan S Lewis  1   5   6   7
Affiliations

Modeling intrahippocampal effects of anterior hippocampal hyperactivity relevant to schizophrenia using chemogenetic excitation of long axis-projecting mossy cells in the mouse dentate gyrus

James P Bauer et al. Biol Psychiatry Glob Open Sci. 2021 Aug.

Abstract

Background: The anterior hippocampus of individuals with early psychosis or schizophrenia is hyperactive, as is the ventral hippocampus in many rodent models for schizophrenia risk. Mossy cells (MCs) of the ventral dentate gyrus (DG) densely project in the hippocampal long axis, targeting both dorsal DG granule cells and inhibitory interneurons. Mossy cells are responsive to stimulation throughout hippocampal subfields, and thus may be suited to detect hyperactivity in areas where it originates such as CA1. Here we tested the hypothesis that hyperactivation of ventral MCs activates dorsal DG granule cells to influence dorsal hippocampal function.

Methods: In CD-1 mice, we targeted dorsal DG-projecting ventral MCs using an adeno-associated virus intersectional strategy. In vivo fiber photometry recording of ventral MCs was performed during exploratory behaviors. We used excitatory chemogenetic constructs to test the effects of ventral MC hyperactivation on long-term spatial memory during an object location memory task.

Results: Photometry revealed ventral MCs were activated during exploratory rearing. Ventral MCs made functional monosynaptic inputs to dorsal DG granule cells, and chemogenetic activation of ventral MCs modestly increased activity of dorsal DG granule cells measured by c-Fos. Finally, chemogenetic activation of ventral MCs during the training phase of an object location memory task impaired test performance 24 hours later, without effects on locomotion or object exploration.

Conclusions: These data suggest that ventral MC activation can directly excite dorsal granule cells and interfere with dorsal DG function, supporting future study of their in vivo activity in animal models for schizophrenia featuring ventral hyperactivity.

Keywords: hippocampus; hyperactivity; learning and memory; mossy cells; psychosis; schizophrenia.

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Figures

Figure 1
Figure 1
Intersectional targeting of longitudinal vMC projections. (A) vMC to dDG longitudinal circuit illustrated by the Allen Mouse Brain Connectivity Atlas (81). AAV expressing EGFP was infused into the ventral hilus of wild-type C57BL/6J mouse (top). Serial two-photon tomography shows axonal projections throughout longitudinal extent of the hippocampus, with strong projection to the dDG IML (https://connectivity.brain-map.org/projection/experiment/112672268). While this tracer is not specific for MCs, most of the depicted circuit comprises MC projections. (B) To target vMCs specifically, retrograde-AAV-pgk-Cre was infused into dDG IML, and AAV-hSyn-Flex-GCaMP6f was infused into contralateral vDG hilus, enabling recombination only in vMCs that project to dDG. (C) Fluorescence microscopy from CD-1 mouse following targeting strategy shown in panel (B) reveals GCaMP6f is expressed in vMC somata with organized projections to contralateral vDG and bilateral dDG. (D) GCaMP6f from targeting strategy in panel (B) is highly colocalized with calretinin, a marker for ventral but not dorsal MCs. (E) Quantification of viral targeting strategy in panel (B) from 4 mice reveals that most GCaMP6f+ hilar neurons are calretinin+, with limited off-target expression in nearby ventral granule cells and CA3 pyramidal neurons. AAV, adeno-associated virus; CA3, cornu ammonis 3; dDG, dorsal dentate gyrus; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; IML, inner molecular layer; MC, mossy cell; vDG, ventral dentate gyrus; vMC, ventral MC.
Figure 2
Figure 2
In vivo fiber photometry reveals that vMCs are selectively activated by exploratory rearing. (A) Fluorescence microscopy of GCaMP6f expression in vMCs by intersectional targeting and fiber optic cannula placement. (B) Example individual photometry trace surrounding an exploratory rearing event. Vertical red dashed line corresponds to initial rearing apogee. (C) ΔF/F0z scores of rearing events aligned to time of initial rearing apogee in an individual mouse. Events are ordered chronologically during the recording. $ corresponds to trace in panel (B). (D) ΔF/F0z scores were aligned to time of rearing apogee (perievent time = 0), averaged within mouse, then averaged across mice (68 rearing events from 5 mice). Black line depicts mean ΔF/F0z score, pink depicts SEM. (E, F) To test whether vMCs were activated by abrupt changes in motor behavior or nonrearing exploration, ΔF/F0z scores for the same 5 mice from (D) during the same behavioral session were aligned to time when mice transitioned between XY-plane horizontal movement and no horizontal movement (E, start to stop, 98 transitions) or between no horizontal movement and XY-plane horizontal movement (F, stop to start, 98 transitions). Black line depicts mean ΔF/F0z score; shading depicts SEM. (G) From the averaged ΔF/F0z-score curves, area under the curve was calculated for the 2 seconds before (−2 to 0 s) and after (0 to +2 s) the indicated behavioral event, revealing that vMC calcium activity during rearing was greater than both horizontal motor transitions in the time period immediately after the behavior but not leading up to the behavior (time × behavior interaction: F2,8 = 8.498, p = .011; time: F1,4 = 19.24, p = .012; behavior: F2,8 = 4.197, p = .057). Pairwise comparison p values are shown in the figure. vMC, ventral mossy cell.
Figure 3
Figure 3
AAV1-mediated anterograde transsynaptic tracing and optogenetic excitation of vMC terminals reveals functional synapses between vMCs and dorsal granule cells. (A) AAV1-Cre was infused unilaterally into vDG hilus, and Cre-dependent mCherry (AAV-DIO-mCherry) was infused either contralaterally (box 1) or ipsilaterally (box 2) to dDG to detect anterograde transsynaptic trafficking. (B) AAV-DIO-mCherry does not express mCherry when infused alone (left); however, additional infusion of AAV1-Cre in either contralateral (middle) or ipsilateral (right) DG results in dDG granule cell expression of mCherry, supporting functional synaptic connectivity between the ventral hilus and dDG most consistent with longitudinal MC projections. (C) Channelrhodopsin-mCherry (AAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA) was expressed in vMCs projecting to the contralateral dDG by intersectional targeting. (D) In slices, whole-cell patch-clamp recordings from dDG granule cells were performed while stimulating channelrhodopsin-mCherry-expressing vMC terminals in the dDG IML using a blue laser (473-nm wavelength, 5-ms pulse duration, 20 Hz for 1 s). (E) Action potentials were elicited from dDG granule cells held at approximately −60 mV (traces are shown for each of the three granule cells demonstrating action potentials out of five total recorded granule cells from 2 mice). A horizontal black bar for each trace denotes a 1000-ms duration, 20-Hz blue light stimulation period. AAV, adeno-associated virus; DG, dentate gyrus; dDG, dorsal DG; GC, genome copies; IML, inner molecular layer; MC, mossy cell; vDG, ventral DG; vMC, ventral MC.
Figure 4
Figure 4
Chemogenetic activation of vMCs activates dorsal granule cells. (A) To express hM3Dq-mCherry or mCherry in vMCs, retrograde-AAV-pgk-Cre was unilaterally infused into dDG, and AAV-hSyn-DIO-hM3D(Gq)-mCherry or AAV-hSyn-DIO-mCherry was bilaterally infused into vDG hilus. (B) Fluorescence microscopy showing representative image of bilateral vMC targeting. (C) Whole-cell recordings from vMCs expressing either mCherry (3 cells from 2 mice) or hM3Dq-mCherry (4 cells from 3 mice) revealed that hM3Dq+ neurons showed significantly greater mean inward current after bath application of 10 micromolar CNO than control mCherry+ neurons. (t5 = 4.24, ∗∗p = .0068). (D) Mice with vMCs expressing mCherry (n = 3–4 mice) or hM3Dq-mCherry (n = 4–5 mice) were administered vehicle or 10 mg/kg CNO i.p., then perfused 90 minutes later and immunostained for c-Fos. Only vMCs expressing hM3Dq-mCherry strongly expressed c-Fos, but also strongly expressed c-Fos in neighboring vDG granule cells (treatment × virus: F1,12 = 33.38, p < 10−4, ∗∗∗∗p < 10−4 vs. all other groups). (E) Same method as in panel (D) but with reduced dose of CNO (1 mg/kg) showed increase in c-Fos in vMCs expressing hM3Dq compared with mCherry (n = 3 mice per group; t2.98 = 5.58, ∗p = .012), but no hyperactivation of neighboring granule cells. (F) dDG granule cell c-Fos was also significantly upregulated in mice with vMC expression of hM3Dq-mCherry treated with 1 mg/kg CNO as compared with mice with vMC expression of mCherry (n = 3 mice per group; t3.77 = 3.21, ∗p = .036). CNO, clozapine N-oxide; dDG, dorsal dentate gyrus; vMC, ventral mossy cell; vDG, ventral DG.
Figure 5
Figure 5
vMC hyperactivation during object location training impairs retrieval 24 hours later. (A) Schematic of the object location memory test. After 3 days of handling, mice undergo 6 days of habituation to an arena with spatial cues on the wall. The following day, they undergo a 10-minute training session, during which, they are exposed to two identical objects. Twenty-four hours later, they are returned to the arena for a 5-minute testing session where one object has been moved to a novel location and the other remains in the familiar location. CNO (1 mg/kg) was administered 30 minutes before the training session only. (B) CNO administration during training impaired object location memory in mice expressing hM3Dq in vMCs (n = 10 mCherry, n = 9 hM3Dq; virus × session: F1,17 = 3.147, p = .094; virus: F1,17 = 4.338, p = .053; session: F1,17 = 1.192, p = .29). Pairwise comparison p values are shown in the figure. One-sample t test vs. DI = 0: t9 = 2.20, #p = .028. (C) Total time spent investigating both objects did not significantly differ between the mice expressing mCherry (n = 10) or hM3Dq (n = 9) during the training (left; t15.65 = 0.37, p = .72) or testing session (right; t12.65 = 1.40, p = .18). (D–G) Mice were later tested in a 10-minute open field test in an arena with a novel floor surface 30 minutes after administration of 1 mg/kg CNO (n = 12 mCherry, n = 11 hM3Dq) (D). There was no significant difference between mCherry- and hM3Dq-expressing mice in the total distance traveled (t19.74 = 0.27, p = .79) (E), time spent in the arena center (t15.13 = 0.68, p = .51) (F), or number of rearing events (t20.31 = 0.66, p = .52) (G). For panels (B) and (C) and (E)–(G), individual data points containing “X” represent female mice. No mice were excluded because of lack of vMC targeting. (H) Schematic suggesting that a specific setpoint of vMC activity exists for optimal encoding performance, which is supported by previous work showing that inhibition of vMCs impairs spatial encoding (31) and this study suggesting that hyperactivation of vMCs also impairs spatial encoding. CNO, clozapine N-oxide; DI, discrimination index; vMC, ventral mossy cell.
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