Inhibitory projections connecting the dentate gyri in the two hemispheres support spatial and contextual memory

•SOM+ cDG projections control DG activity in a cell-type-specific manner•Activation of SOM+ cDG projections is required for memory formation•Activation of SOM+ cDG projections is sufficient to disrupt memory recall The dentate gyrus (DG) receives substantial input from the homologous brain area of the contralateral hemisphere. This input is by and large excitatory. Viral-tracing experiments provided anatomical evidence for the existence of GABAergic connectivity between the two DGs, but the function of these projections has remained elusive. Combining electrophysiological and optogenetic approaches, we demonstrate that somatostatin-expressing contralateral DG (SOM+ cDG)-projecting neurons preferentially engage dendrite-targeting interneurons over principal neurons. Single-unit recordings from freely moving mice reveal that optogenetic stimulation of SOM+ cDG projections modulates the activity of GABAergic neurons and principal neurons over multiple timescales. Importantly, we demonstrate that optogenetic silencing of SOM+ cDG projections during spatial memory encoding, but not during memory retrieval, results in compromised DG-dependent memory. Moreover, optogenetic stimulation of SOM+ cDG projections is sufficient to disrupt contextual memory recall. Collectively, our findings reveal that SOM+ long-range projections mediate inter-DG inhibition and contribute to learning and memory. The dentate gyrus (DG) receives substantial input from the homologous brain area of the contralateral hemisphere. This input is by and large excitatory. Viral-tracing experiments provided anatomical evidence for the existence of GABAergic connectivity between the two DGs, but the function of these projections has remained elusive. Combining electrophysiological and optogenetic approaches, we demonstrate that somatostatin-expressing contralateral DG (SOM+ cDG)-projecting neurons preferentially engage dendrite-targeting interneurons over principal neurons. Single-unit recordings from freely moving mice reveal that optogenetic stimulation of SOM+ cDG projections modulates the activity of GABAergic neurons and principal neurons over multiple timescales. Importantly, we demonstrate that optogenetic silencing of SOM+ cDG projections during spatial memory encoding, but not during memory retrieval, results in compromised DG-dependent memory. Moreover, optogenetic stimulation of SOM+ cDG projections is sufficient to disrupt contextual memory recall. Collectively, our findings reveal that SOM+ long-range projections mediate inter-DG inhibition and contribute to learning and memory. The hippocampus is a key brain area involved in processing episodic memory (Olton et al., 1979Olton D.S. Becker J.T. Handelmann G.E. Hippocampus, space, and memory.Behav. Brain Sci. 1979; 2: 313-322https://doi.org/10.1017/s0140525x00062713Crossref Google Scholar; Tulving and Markowitsch, 1998Tulving E. Markowitsch H.J. 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Long-range-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex.Science. 2012; 335: 1506-1510https://doi.org/10.1126/science.1217139Crossref PubMed Scopus (206) Google Scholar) connect functionally distinct brain areas within one hemisphere, whereas the DG-DG projections that we focus on here (Eyre and Bartos, 2019Eyre M.D. Bartos M. Somatostatin-expressing interneurons form axonal projections to the contralateral hippocampus.Front. Neural Circuits. 2019; 13: 56https://doi.org/10.3389/fncir.2019.00056Crossref PubMed Scopus (10) Google Scholar) ensure interhemispheric connectivity between homologous brain areas. Connectivity between homologous brain areas has been demonstrated at the anatomical and electrophysiological level for many hippocampal subfields, including the DG. Most projections providing the direct interhemispheric interaction between functionally similar hippocampal areas are excitatory in nature. For instance, hilar mossy cells (MCs) provide bilateral connectivity between the left and the right DG (Deadwyler et al., 1975Deadwyler S.A. West J.R. Cotman C.W. Lynch G.S. A neuro-physiological analysis of commissural projections to dentate gyrus of the rat.J. Neurophysiol. 1975; 38: 167-184https://doi.org/10.1152/jn.1975.38.1.167Crossref PubMed Scopus (81) Google Scholar). Interestingly, as MCs preferentially innervate interneurons, the net effect on GCs is primarily inhibitory (Buzsáki and Czéh, 1981Buzsáki G. Czéh G. Commissural and perforant path interactions in the rat hippocampus. Field potentials and unitary activity.Exp. Brain Res. 1981; 43: 429-438https://doi.org/10.1007/BF00238387Crossref PubMed Scopus (60) Google Scholar; Buzsáki and Eidelberg, 1981Buzsáki G. Eidelberg E. 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Their existence was first inferred based on retrograde tracing experiments (Ribak et al., 1986Ribak C.E. Seress L. Peterson G.M. Seroogy K.B. Fallon J.H. Schmued L.C. A GABAergic inhibitory component within the hippocampal commissural pathway.J. Neurosci. 1986; 6: 3492-3498https://doi.org/10.1523/jneurosci.06-12-03492.1986Crossref PubMed Scopus (108) Google Scholar) and corroborated much later by employing an anterograde viral-tracing approach (Melzer et al., 2012Melzer S. Michael M. Caputi A. Eliava M. Fuchs E.C. Whittington M.A. Monyer H. Long-range-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex.Science. 2012; 335: 1506-1510https://doi.org/10.1126/science.1217139Crossref PubMed Scopus (206) Google Scholar). A more detailed anatomical characterization of these somatostatin-expressing (SOM+) cells ensued (Eyre and Bartos, 2019Eyre M.D. Bartos M. Somatostatin-expressing interneurons form axonal projections to the contralateral hippocampus.Front. Neural Circuits. 2019; 13: 56https://doi.org/10.3389/fncir.2019.00056Crossref PubMed Scopus (10) Google Scholar), but the identity of the target cells in the contralateral hemisphere and their functional role in vivo have remained unknown. In this study, we investigate the synaptic transmission of the contralateral DG (cDG) projections and their role in DG-dependent behavioral tests using a combination of optogenetic and ex vivo and in vivo electrophysiological approaches that allow the selective manipulation of cDG projection terminals. We demonstrate that connections formed by GABAergic projection neurons, although weak and sparse, are sufficient to modulate neuronal activity in the cDG and contribute to memory formation and retrieval. To establish the identity of GABAergic source cells that project to the cDG, we performed viral-tracing experiments. An adeno-associated virus (AAV) allowing the Cre-dependent transduction of ChR2-mCherry was injected unilaterally into the dorsal DG of genetically modified mice, expressing Cre recombinase in distinct GABAergic neurons (Figures 1A, 1B , and S1A–S1D). Three weeks and more following the viral injection, mCherry-labeled axons were visualized in the cDG of GAD2- and SOM-Cre mice, and also, but to a lesser extent, in VIP- and PV-Cre mice (Figures 1C and S1B–S1E). There was no labeling of neurons in the contralateral hemisphere, excluding both transsynaptic spread or retrograde transport of viral particles (Figure S2A). Given that the SOM+ projections were denser than the other 2 GABAergic cell subtypes, we analyzed these projections in more detail. Previous immunohistochemical studies demonstrated that in the dorsal DG, SOM+ cells localized to the hilus (Figure S2B; Amaral et al., 1988Amaral D.G. Insausti R. Campbell M.J. 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Neural Circuits. 2019; 13: 56https://doi.org/10.3389/fncir.2019.00056Crossref PubMed Scopus (10) Google Scholar), at the injection site, mCherry-labeled neurons were located in the hilus (Figures 1B and S2A), and axon terminals were observed mostly in the hilus and the outer molecular layer (OML), with a clear preference for the latter. In the contralateral hemisphere, mCherry-labeled axons were found in the homologous areas (Figures 1C and S2C), namely the hilus (Figure 1C, boxed region 2) and the OML (Figure 1C, boxed regions 1 and 3). To further support our findings, we injected the retrograde tracer CTB-555 into one DG (Figure S1F). Upon inspection of the contralateral hippocampus 5 days post injection, we detected CTB+ cells in the hilus (Figure S1G, top) but not in the CA1 and CA3 (Figure S1G, bottom). The retrogradely labeled CTB+ neurons in the hilus significantly outnumbered the anterogradely labeled mCherry+ neurons (Figures 1B and S1G; CTB+ cells, ∼45 cells/slice, 8 slices from 2 mice; mCherry+ cells, ∼6 cells/slice, 16 slices from 4 mice), which is not surprising, as MCs are known to project to the cDG. In agreement with the notion that MCs selectively express GluR2/3 but not SOM (Fujise and Kosaka, 1999Fujise N. Kosaka T. Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis.Brain Res. 1999; 816: 500-511https://doi.org/10.1016/s0006-8993(98)01202-5Crossref PubMed Scopus (0) Google Scholar; Lein et al., 2007Lein E.S. Hawrylycz M.J. Ao N. Ayres M. Bensinger A. Bernard A. Boe A.F. Boguski M.S. Brockway K.S. 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Using a high Cl− internal solution, brief photostimulation of ChR2-expressing axons evoked inward currents within 5 ms in GCs and morphologically defined interneurons of the cDG. The responses were comparable to the baseline condition after applying kynurenic acid (2 mM), but were completely blocked by the GABAA receptor antagonist gabazine (1 μM) (Figures 1E and S3A–S3C), providing evidence for the inhibitory nature of the optogenetically activated synaptic terminals. Light-evoked responses were detected also in MCs and in various types of dendrite-targeting interneurons (D-INs) but not in basket cells (BC) (Figures 1F, left, and S3D–S3I; Table S1). Further analysis of the light-evoked responses revealed that, despite the large variation in individual cell types, the inhibitory postsynaptic conductance (IPSG) of most D-INs was greater than that of GCs (Figure 1F, right). To control for conductance differences in cells recorded from different slices, we analyzed evoked responses from recorded D-INs and GCs in the same slice. Indeed, in most instances (20 of 23), the IPSG of the current induced in the D-IN was greater than that of the simultaneously recorded GC (Figure 1G). To test the effect of SOM+ cDG projection activation in freely moving mice, we injected a ChR2-mCherry virus into the hilar region of one DG of SOM-Cre mice, and stimulated axon terminals in the cDG. Tetrodes and an optic fiber were implanted above the cDG (Figures 2A , top, and S4A) and the tetrodes were advanced gradually after each recording. Single-unit activity was recorded while the mice were foraging in an open-field environment containing two scented objects (Figures 2A, bottom, and S4B). Such an enrichment of the environment was previously reported to increase the activity of DG neurons known to exhibit normally sparse firing (Heale and Vanderwolf, 1994Robert Heale V. 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The mice do not have a preference for the scented objects during the recording sessions (Figures S4C–S4E). Of the recorded DG neurons (n = 1,052 neurons) only a subset (n = 61 neurons) responded to the 10 Hz photostimulation. We utilized peristimulus time histograms (Figure 2B) to further categorize responsive neurons considering the change in firing rate and the response latency. Three groups were identified. Specifically, we distinguished neurons that were inhibited early (IE; n = 7 neurons, latency 3.5–6.5 ms) or late (IL; n = 44 neurons, latency 10.5–28.5 ms) and neurons that were excited (E; n = 10 neurons, latency 4.5–17.5 ms) following photostimulation (Figure S4F). Based on the distribution of response latencies, the cutoff value between IE and IL cells was set to 7 ms. We wondered how the three groups of neurons correlate with previously established criteria for functionally distinct DG neuronal subtypes (Senzai and Buzsáki, 2017Senzai Y. Buzsáki G. 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Most IE and IL neurons mapped onto quadrants IV and II/III, thus corresponding to wide- and narrow-waveform interneurons, respectively, while E neurons could be any cell type (Figure 2C). Narrow-waveform inhibitory neurons exhibit higher firing rates than wide-waveform inhibitory neurons (Morales et al., 2021Morales C. Morici J.F. Espinosa N. Sacson A. Lara-Vasquez A. García-Pérez M.A. Bekinschtein P. Weisstaub N.V. Fuentealba P. Dentate gyrus somatostatin cells are required for contextual discrimination during episodic memory encoding.Cereb. Cortex. 2021; 31: 1046-1059https://doi.org/10.1093/cercor/bhaa273Crossref PubMed Scopus (6) Google Scholar; Senzai and Buzsáki, 2017Senzai Y. Buzsáki G. Physiological properties and behavioral correlates of hippocampal granule cells and mossy cells.Neuron. 2017; 93: 691-704.e5https://doi.org/10.1016/j.neuron.2016.12.011Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Indeed, IL neurons exhibited significantly higher mean firing rates during light-off sessions than IE or E neurons (Figure 2D). The firing-rate maps of both IE and IL neurons showed little spatial selectivity, whereas some E neurons exhibited spatial firing specificity (Figure 2B). Some GCs and MCs were shown to exhibit spatial selectivity (GoodSmith et al., 2017GoodSmith D. Chen X. Wang C. Kim S.H. Song H. Burgalossi A. Christian K.M. Knierim J.J. Spatial representations of granule cells and mossy cells of the dentate gyrus.Neuron. 2017; 93: 677-690.e5https://doi.org/10.1016/j.neuron.2016.12.026Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, GoodSmith et al., 2019GoodSmith D. Lee H. Neunuebel J.P. Song H. Knierim J.J. Dentate gyrus mossy cells share a role in pattern separation with dentate granule cells and proximal CA3 pyramidal cells.J. Neurosci. 2019; 39: 9570-9584https://doi.org/10.1523/jneurosci.0940-19.2019Crossref PubMed Scopus (0) Google Scholar). Accordingly, we found four E cells with higher information scores than what was expected by chance (Figure 2E). The information score of the spatially tuned E cells, however, was lower than that typically reported for GCs and MCs (Gil et al., 2018Gil M. Ancau M. Schlesiger M.I. Neitz A. Allen K. De Marco R.J. Monyer H. Impaired path integration in mice with disrupted grid cell firing.Nat. Neurosci. 2018; 21: 81-91https://doi.org/10.1038/s41593-017-0039-3Crossref PubMed Scopus (62) Google Scholar; GoodSmith et al., 2017GoodSmith D. Chen X. Wang C. Kim S.H. Song H. Burgalossi A. Christian K.M. Knierim J.J. Spatial representations of granule cells and mossy cells of the dentate gyrus.Neuron. 2017; 93: 677-690.e5https://doi.org/10.1016/j.neuron.2016.12.026Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, GoodSmith et al., 2019GoodSmith D. Lee H. Neunuebel J.P. Song H. Knierim J.J. Dentate gyrus mossy cells share a role in pattern separation with dentate granule cells and proximal CA3 pyramidal cells.J. Neurosci. 2019; 39: 9570-9584https://doi.org/10.1523/jneurosci.0940-19.2019Crossref PubMed Scopus (0) Google Scholar), and may depend on the exact recording conditions. Of all neurons responding to laser stimulation, the majority was categorized as IL (44 of 61 responsive cells, 72%). Since IL neurons were characterized by high mean firing rates and narrow spike waveforms, they are very likely fast-spiking inhibitory neurons. Considering the chosen cutoff value of 7 ms (> IE and < IL), it is safe to assume that the