The hippocampus is critical for memory formation. The hypothalamic supramammillary nucleus (SuM) sends long-range projections to hippocampal area CA2. While the SuM-CA2 connection is critical for social memory, how this input acts on the local circuit is unknown. Using transgenic mice, we found that SuM axon stimulation elicited mixed excitatory and inhibitory responses in area CA2 pyramidal neurons (PNs). Parvalbumin-expressing basket cells were largely responsible for the feedforward inhibitory drive of SuM over area CA2. Inhibition recruited by the SuM input onto CA2 PNs increased the precision of action potential firing both in conditions of low and high cholinergic tone. Furthermore, SuM stimulation in area CA2 modulated CA1 activity, indicating that synchronized CA2 output drives a pulsed inhibition in area CA1. Hence, the network revealed here lays basis for understanding how SuM activity directly acts on the local hippocampal circuit to allow social memory encoding. The hippocampus is critical for memory formation and spatial navigation , yet basThe hypothalamic supramammillary (SuM) nucleus sends projections to both area CA2 and the dentate gyrus (DG) . These lEven with the anatomical and in vivo data, the properties and consequences of SuM activation on area CA2 activity remain unexplored. In this study, we use a combination of approaches to specifically examine the effects of SuM input stimulation on neuronal activity in hippocampal area CA2. Here, we show that the SuM-evoked post-synaptic excitation of CA2 PN is controlled by SuM-driven inhibition. We identified PV-expressing basket cells as the neuronal population most strongly excited by SuM input in area CA2, and thus likely responsible for the feedforward inhibition evoked by SuM in CA2 PNs. We found that recruitment of this inhibition enhances the precision of AP firing by area CA2 PNs in conditions of low and high cholinergic tone. Finally, we observed that the resulting synchronized CA2 PN activity drives inhibition in area CA1, thereby providing a circuit mechanism through which SuM can modulate hippocampal excitability by controlling area CA2 output.Its small size and cellular heterogeneity have made the SuM a difficult region to study. It has been shown that the source of vesicular glutamate transporter 2 (VGluT2)-immunopositive boutons in area CA2 originate from the SuM . In ordeTg(Slc17a-icre)10Ki line . The SuM fiber projection area was clearly restricted to area CA2, as defined by expression of the CA2-specific markers PCP4 -YFP (ChR2-EYFP) under the control of Cre into the SuM of a transgenic mouse line with Cre expression controlled by the VGluT2 promoter, the 0Ki line . In para the SuM . We foun the SuM . Further the SuM  where thers PCP4  and RGS1ers PCP4 , and diders PCP4 .We performed whole-cell current and voltage clamp recordings of PNs across the hippocampal CA regions and activated projecting axons with pulses of 488 nm light in acute hippocampal slices. Following all recordings, we performed post-hoc anatomical reconstructions of recorded cells and axonal fibers, as well as immunohistochemical staining for CA2-area markers. Additionally, injection sites were examined post hoc to ensure correct targeting of the SuM.stratum lucidum where the DG mossy fibers (MF) project, some PNs clearly had thorny excrescences (TE) while others had very smooth apical dendrites  or superficial  subpopulations . We found that the SuM-PN connectivity was not different between CA2 and CA3 PNs (We observed that photostimulation of SuM axons elicited excitatory post-synaptic responses in 63% of PNs (n = 166 of 263 cells) located in area CA2. PNs in this region shared similar overall dendritic morphologies and electrophysiological properties  but diffendrites . Based o CA3 PNs  that wer CA3 PNs . We are  CA3 PNs . The purA and GABAB receptor antagonists SR95531 and CGP55845A led to a significant increase of light-evoked PSP amplitude recorded in area CA2 PNs (Using whole-cell voltage clamp recordings in area CA2 and the dentate gyrus (DG), we have previously shown that the CA2-targeting and DG-targeting SuM neurons have contrasting neurotransmitter modalities . Our resstratum radiatum (SR). Stimulation of CA3 inputs evokes a very strong feedforward inhibition, such that it is exceptionally difficult to evoke action potential firing in CA2 pyramidal neurons when inhibitory transmission is intact (stratum lacunosum molecular (SLM). These inputs are very distal but relatively less attenuated in CA2 PNs in comparison to distal inputs in CA1  and SLM . This observation that concomitant SuM activity is reducing the level of facilitation of several pulses in a train indicates that the short-term dynamics of the SuM-driven excitation and feedforward inhibition are playing a role to prevent cellular excitation from other inputs.We also examine the summation ratio for a train of 4 PSPs at 10 Hz from SR and SLM synaptic inputs stimulation with and without simultaneous SuM input stimulation . We obsestratum oriens (SO) and nine in stratum radiatum (SR). Like non-BCs, these INs did not receive strong excitation from SuM fibers (Because the hippocampus hosts a variety of interneurons (INs) that are involved in controlling specific aspects of PN excitability, we wished to establish which kind of IN was targeted by the SuM input to area CA2. We performed whole-cell recordings from INs in this area and assessed post-synaptic excitatory responses to SuM axons stimulation in these cells . In contM fibers . This dapost hoc test, p=0.022). Consequently, EPSPs recorded at −70 mV were of larger amplitude in BCs than in PNs and non-BCs (M), photostimulation of SuM axons was able to evoke AP firing  or parvalbumin (PV) . We founM level and action potential firing to SuM input stimulation before and after CNO application . While this confirmed the relevance of our silencing strategy, it highlighted the possibility that Gi-DREADD-expressing CA2 PV+ INs may not be fully silenced by CNO. Indeed, we observed residual SuM-evoked AP firing in these cells after CNO application  inhibitory Gi-DREADD in area CA2 of PV-Cre mice together with AAVs expressing ChR2 with a pan-neuronal promoter in the SuM . While wlication . We founlication . These d1, IPSC amplitudes were 245.5 ± 92.6 pA in control and 35.0 ± 15.4 pA in ω-agatoxin TK hence a 81.8 ± 3.9% block, n = 5; paired-T test, p<0.001), suggesting a major contribution from PV+ INs to SuM-driven feedforward inhibition consistent with our previous results. However, we observed that excitatory transmission from SuM axons was also partially blocked by ω-agatoxin TK application, as SuM input-evoked EPSCs were significantly reduced although not abolished . This observation indicates that glutamate release from SuM axons relies on P/Q-type voltage-gated calcium channels to some degree, thereby complicating the interpretation of the reduction of IPSC amplitude in CA2 PNs.We used a pharmacological strategy to selectively manipulate PV+ INs by targeting their GABA release machinery. In the neocortex, P/Q-type voltage-gated calcium channels are necessary for GABA release from PV+ fast spiking INs onto PNs . In cont1; IPSC amplitudes were 343 ± 123 pA in control and 31 ± 12.4 pA in DAMGO hence a 88 ± 5.0% block, n = 6 PNs; Wilcoxon signed-rank test, p=0.031), while direct excitatory transmission remained unaffected . While this result is in agreement with our DREADD and ω-agatoxin TK results showing a major contribution of PV +INs to the SuM-driven feedforward inhibition, it should be noted that the dichotomy between PV+ versus CCK+ INs sensitivity to DAMGO has not been directly verified in area CA2.It has previously been demonstrated that PV+ BC transmission can be strongly attenuated by mu opioid receptor activation (MOR) while CCK+ BC transmission is insensitive to MOR activation . Thus, w1; IPSC amplitudes were 168 ± 28 pA in control and 64 ± 22 pA in DPDPE hence a 61 ± 14% block by DPDPE, n = 7; paired-T test, p=0.015), while leaving direct EPSCs unaffected . Further confirming the PV+ nature of INs responsible for the SuM feedforward inhibition, this result reveals that both the local CA3 and long-range SuM inputs converge onto an overlapping population of INs to inhibit area CA2 PNs, thus enabling cross-talk between these routes through synaptic plasticity of PV+ INs. However, since DORs are only expressed in a fraction of PV+ INs and therefore only reduces but does not fully block PV+ IN-mediated GABA release (It has recently been shown that delta opioid receptors (DORs) are specifically expressed in a fraction of PV+ cells in the hippocampus . Further release , it is dAltogether, these four methods strongly suggest that SuM inputs selectively recruit PV +interneurons to inhibit CA2 PNs. Although individually each method does not conclusively demonstrate that SuM input exclusively targets PV+ INs, the consistent reduction of SuM-driven feedforward inhibition of CA2 PNs observed with every approach allows us to conclude that PV + cells are predominantly targeted by SuM inputs in area CA2.A and GABAB receptor antagonists abolished these effects of SuM stimulation ; however, the inter-burst time interval was reduced. Indeed, AP bursts with SuM input activation were followed more rapidly by another burst of APs than the ones without SuM input activation . Animal sample sizes were estimated using power tests with standard deviations and ANOVA values from pilot experiments. A 15% failure rate was assumed to account for stereotaxic injection errors and slice preparation complications. Every effort was made to reduce animal suffering.slc17a6 gene coding the vesicular glutamate transporter isoform 2 (VGluT2).We used the Tg(Slc17a-icre)10Ki mouse line that was previously generated  and exprCsf2rb2-Cre mouse line that was recently generated .We used the Pvalbtm1(cre)Arbr/J mouse line that was previously generated  and expr8 vg, the AAV.Synapsin.DIO.hM4D(Gi).mCherry was used at 3.6 × 109 vg and the AAV2/9.hSyn.hChR2(H134R).EYFP.WPRE.hGH was used at 3.7 × 1013 vg. The retrograde tracer CAV2-cre virus was used at 2.5 × 1012 vg. 500 nL of virus was unilaterally injected into the brain of 4-week-old male wild type C57BL/6, Tg(Slc17ab-icre)10Ki (VGluT2-Cre), csf2rb2-cre (SuM-Cre) or Pvalbtm1(cre)Arbr/J (PV-Cre) mice at 100 nL/min and the injection cannula was left at the injection site for 10 min following infusion. In the case of AAV.Synapsin.DIO.hM4D(Gi)-mCherry injection in PV-Cre mice, bilateral injections were performed in dorsal CA2. The loci of the injection sites were as follows: anterior–posterior relative to bregma: −2.8 mm for SuM, −1.6 mm for CA2; medial-lateral relative to midline: 0 mm for SuM, 1.9 mm for CA2; dorsal-ventral relative to surface of the brain: 4.75 mm for SuM, 1.4 mm for CA2.Animals were anaesthetized with ketamine (100 mg/kg) and xylazine (7 mg/kg). The adeno-associated viruses AAV9.EF1a.DIO.hChR2(H134R).EYFP and AAV9.hSynapsin.EGFP.WPRE.bGH were used at 3 × 102PO4 1.25, NaHCO3 30, HEPES 20, glucose 25, thiourea 2, Na-ascorbate 5, Na-pyruvate 3, CaCl2 0.5, MgCl2 10. Brains were then rapidly removed, hippocampi were dissected out and placed upright into an agar mold and cut into 400-µm-thick transverse slices (Leica VT1200S) in the same cutting solution at 4°C. Slices were transferred to an immersed-type chamber and maintained in artificial cerebro-spinal fluid (ACSF) containing the following (in mM): NaCl 125, KCl 2.5, NaH2PO4 1.25, NaHCO3 26, glucose 10, Na-pyruvate 2, CaCl2 2, MgCl2 1. Slices were incubated at 32°C for approximately 20 min then maintained at room temperature for at least 45 min prior to patch-clamp recordings performed with either potassium- or cesium-based intracellular solutions containing the following (in mM): K- or Cs-methyl sulfonate 135, KCl 5, EGTA-KOH 0.1, HEPES 10, NaCl 2, MgATP 5, Na2GTP 0.4, Na2-phosphocreatine 10 and biocytin (4 mg/mL).Transverse hippocampal slices were prepared at least 3 weeks after viral injection and whole-cell patch-clamp recordings were performed from PNs and INs across the hippocampal CA regions. In the case of PV-Cre mice injected with AAV.Synapsin.DIO.hM4D(Gi)-mCherry, slices were prepared 6 weeks after viral injection. Animals were deeply anesthetized with ketamine (100 mg/kg) and xylazine (7 mg/kg), and perfused transcardially with a N-methyl-D-glucamin-based (NMDG) cutting solution containing the following (in mM): NMDG 93, KCl 2.5, NaHstratum radiatum (SR) of CA1 to antidromically excite CA3-CA2 synapses and in stratum lacunosum moleculare (SLM) to stimulate distal dendritic inputs in area CA2. Synaptic currents were evoked with a constant voltage stimulating unit (Digitimer Ltd.) set at 0.1 ms at a voltage range of 5–10 V. For the patch-clamp recordings in area CA1 with stimulation of SuM axons in area CA2, 50 ms long light stimulation pulses were delivered every 10 s. We used a light intensity of 25 mW/mm² which was experimentally determined as the lowest irradiance allowing TTX-sensitive maximal responses in all cell types and conditions. Data were obtained using a Multiclamp 700B amplifier, sampled at 10 kHz and digitized using a Digidata. The pClamp10 software was used for data acquisition. Series resistance were <20 MOhm and were not compensated in voltage-clamp, bridge balance was applied in current-clamp. An experimentally determined liquid junction potential of approximately 9 mV was not corrected for. Pharmacological agents were added to ACSF at the following concentrations (in µM): 10 NBQX and 50 D-APV to block AMPA and NMDA receptors, 1 SR95531 and 2 CGP55845A to block GABAA and GABAB receptors, 1 DAMGO to activate µ-opioid receptors (MOR), 0.5 DPDPE to activate δ-opioid receptors (DOR), 10 clozapine N-oxide (CNO) to activate hM4D(Gi) DREADDs, 10 CCh to activate cholinergic receptors, 0.2 tetrodotoxin (TTX) to prevent sodic action potential generation, 200 nM ω-agatoxin TK to block P/Q-type voltage-gated calcium channels.ChR2 was excited by 488 nm light delivered by a LED attached to the epifluorescence port of the microscope. Light stimulations trains consisted of 2–10 pulses, 0.5 ms long, delivered at 10 Hz, repeated every 20 s for at least 20 sweeps. Stimulating pipettes filled with ACSF were placed in All surgeries were performed in a stereotaxic frame (Narishige). Csf2rb2-cre male mice from 3 to 6 months of age were anaesthetized using 500 mg/kg Avertin. pAAV.DIO.hChR2(H134R).EYFP was injected into the SuM  using a 10 μL Hamilton microsyringe  with a beveled 33 gauge needle ). A microsyringe pump  with controller  were used to set the speed of the injection (100 nl/min). The needle was slowly lowered to the target site and remained in place for 5 min prior to start of the injection and the needle was removed 10 min after infusion was complete. Following virus injection, a custom-built screw-driven microdrive containing six independently adjustable nichrome tetrodes (14 μm diameter), gold-plated to an impedance of 200–250 kΩ was implanted, with a subset of tetrodes targeting CA1, and an optic fiber  targeting CA2 . Following recovery, the tetrodes were slowly lowered over several days to CA1 pyramidal cell layer, identified by characteristic local field potential patterns (theta and sharp-wave ripples) and high amplitude multiunit activity. During the adjustment period the animal was habituated every day to a small box in which recording and stimulation were performed.2 and pulse width 50 ms) were delivered to the CA2 at 10 Hz. The pulse train was repeated every 10 s for at least 20 times as the animals freely explored the box. Multiunit activity was recorded using a DigitalLynx 4SX recording system running Cheetah v.5.6.0 acquisition software . Broadband signals from each tetrode were filtered between 600 and 6000 Hz and recorded continuously at 32 kHz. Recording sites were later verified histologically with electrolytic lesions as described above and the position of the optic fiber was also verified from the track.Recording was commenced following tetrodes reaching CA1. To examine the impact of SuM terminal stimulation in CA2 the mice were returned to the small familiar box and trains of 10 light pulses  and spikes which occurred 50 ms before and 100 ms after each laser pulse were extracted. Raster plots were generated using a 1 ms bin size. Similar results were obtained using 5 ms and 10 ms bin size (data not shown). Firing rate histograms were calculated by dividing total number of spikes in each time bin by that bin’s duration. Each firing rate histogram was normalized by converting it into z-score values. Mean standard deviation values for the z-score calculation were taken from pre-laser pulse time period. To average the response across all mice, for each tetrode the firing rate in each bin was normalized to the average rate in the pre-laser period.Midbrains containing the injection site were examined post-hoc to ensure that infection was restricted to the SuM.Post-hoc reconstruction of neuronal morphology and SuM axonal projections were performed on slices and midbrain tissue following overnight incubation in 4% paraformaldehyde in phosphate buffered saline (PBS). Midbrain sections were re-sliced sagittally to 100 µm thick sections. Slices were permeabilized with 0.2% triton in PBS and blocked overnight with 3% goat serum in PBS with 0.2% triton. Primary antibody (Life technologies) incubation was carried out in 3% goat serum in PBS overnight at 4°C. Channelrhodopsin-2 was detected by chicken primary antibody to GFP (Life technologies)  and an alexa488-conjugated goat-anti chick secondary. Other primary antibodies used were mouse anti-RGS14 (Neuromab) (1:300 dilution), rabbit anti- PCP4 (Sigma) (1:600 dilution), guinea pig anti-VGluT2 antibody (Milipore) , rabbit anti-parvalbumin antibody (Swant) (1:2000 dilution). Alexa-546-conjugated streptavidin (Life technologies), secondary antibodies and far-red neurotrace (Life technologies) incubations were carried out in block solution for 4 hr at room temperature. Images were collected with a Zeiss 710 laser-scanning confocal microscope.stratum oriens) or superficial (closest to stratum radiatum) based on the radial position of their soma in the pyramidal layer. CA1, CA2, and CA3 PNs were identified based on their somatic localization, dendritic arborization and presence of thorny excrescences (TE). Among INs with somas located in the pyramidal layer , discrimination between BCs and non-BCs was achieved based on the restriction of their axons to SP or not, respectively. When available, firing patterns upon injection of depolarizing current step injection, action potential (AP) half-width, amount of repolarizing sag current upon hyperpolarization from −70 mV to −100 mV by current step injection, membrane resistance (RM) and capacitance (CM) were additionally used for cell identification. CA2 and CA3a PNs as well as superficial and deep PNs displayed similar firing patterns, AP width, sag current, RM and CM; the only statistically difference being a larger RM of CA3a compared to CA2 PNs which is consistent with previous studies  and Clampfit (Molecular devices) software. For accurate measurements of the kinetics and latencies of post-synaptic responses, the following detection process was used. For each cell, average traces were used to create a template waveform that was then fitted to individual traces and measurements were performed on the fitted traces. When only amplitudes of responses were needed, traces were baselined and amplitudes were simply measured at the peak of the responses. Firing adaptation indexes were computed as the ratio of the initial firing rate (instantaneous firing frequency of the first spikes) over the steady-state firing rate (average instantaneous firing frequency for spikes occurring after 500 ms in the 1 second-long current step). Results are reported ± SEM. Statistical significance was assessed using χ² test, Student’s T test, Mann-Whitney U test, Wilcoxon signed-rank test, Kruskal-Wallis test, one-way or two-way ANOVA where appropriate. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The study describes the properties of inputs from the supramammillary nucleus (SuM) to the CA2 area of the hippocampus. This is an important area of study given the increasing importance of SuM inputs and area CA2 in social memory encoding.Novel information is presented in this paper about the influence of the SuM input on the local hippocampal network in CA2 and the effects of this input on network activity in CA1. The authors use complimentary methods to address this question including patch-clamp recordings and optogenetics. The study will be of interest to readers who study hippocampal function, supramammillary nucleus function, social memory, or neuropsychiatric disorders in which CA2 circuits are affected.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Laura Colgin as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Sreedharan Sajikumar (Reviewer #2).Thank you for submitting your article "Local circuit allowing hypothalamic control of hippocampal area CA2 1 activity and consequences for CA1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at The study describes the properties of inputs from the supramammillary nucleus (SuM) to the CA2 area of the hippocampus. Novel information is presented by the influence of the SuM input on the local hippocampal network in the CA2 and what the effect of this input is on network activity in the CA1. The authors use complimentary methods to address this question including patch-clamp recordings and optogenetics. Overall the reviewer found this study important, the experiments well-designed and the data of high quality. However, there are several key points raised by the reviewers to strengthen the data in order to fully support the authors' conclusions, addressing these will require additional experimental work. The list below summarizes the list of required experiments reviewers agreed would be necessary for having full confidence in the authors' conclusions. Reviewers' individual comments contain additional issues that would require the authors to make changes to the discussion, figure legends etc.1. The authors would need to show the effect of SuM stimulation on on synaptically triggered APs and not only on Aps evoked with a current step.2. The change in the balance of EPCs and IPSCs in a train should be demonstrated in a single cell.3. The properties of monosynaptic/disynaptic events should be compared and the lack of direct GABAergic input from the SuM demonstrated. The authors should quantify the delay time to light-evoked IPSCs to address whether the SuM-CA2 inputs are forming monosynaptic or disynaptic GABAergic connections to pyramidal neurons, as it is possible SuM neurons co-release glutamate and GABA to CA2. Given the importance of the mono vs. disynaptic innervation of different types of cells, the authors should go beyond the TTX experiments (as TTX would block a disynaptic EPSC) and also use 4-AP to recover the TTX blocked current to unequivocally prove that they inputs are monosynaptic.4. The preferential role of PV+ cells should be shown with a more selective pharmacological approach.5. The authors should elaborate on how SuM stimulation influences theta/γ rhythms in the CA1 area.Reviewer #1:In this study Robert et al. describes the properties of long-range projections from the SuM to the CA2 area of the hippocampus. The authors identified direct excitatory and indirect inhibitory drive from SuM inputs on CA2 pyramidal neurons and showed that direct excitatory drive impinges on PV-positive basket cells. The overall effect of the input on CA2 activity was an increased precision of APs. The study also suggests that the input from the CA2 drives inhibition in the CA1 area. The study provides very interesting and new information about the cellular properties of SuM input in the CA2 area. This is an important question given the increasing importance of SuM inputs in social memory encoding. The study is timely, currently we have very limited data about the features and exact cellular profile of this input. The study is using elegant technical approaches o answer the central question of the study. While the study is addressing an important question and provides novel data, the authors central claim about the role of feedforward inhibition would need to be strengthened by the addition of experiments addressing how E-I balance changes in trains in individual neurons and how this can be linked to changes in the temporal precision of synaptically evoked APs.Action potentials are evoked with a current step. Since the study is focused on the network effects of feedforward inhibition, it would be useful to see how the properties of synaptically evoked action potentials change. In the cortex and in the CA1 feed forward inhibition was shown to limit the temporal summation of excitatory inputs which lead to decrease in AP jitter . In order to map these dynamics APs should be evoked via synaptic stimulation and not through current injection.The authors show recordings of monosynaptic EPSCs in pyramidal cells and interneurons. It would be important to know how inhibitory and excitatory PSCs change in a train. Recordings from single cells held at E-GLUT ad E-GABA would allow the authors to monitor excitatory and inhibitory events in a train ad map how their balance changes. Can the change in E-I balance explain the change in AP jitter?What are the characteristics of the SuM-driven inhibitory currents? Does the latency and jitter of monosynaptic EPSCs and disynaptic IPSCs differ? If one is monosynaptic and the other is disynaptic one would expect significant differences in both of these parameters.How do the authors exclude the contribution of feed-back inhibition? feedforward and feed-back inhibition both could have an impact on the temporal precision of APs.Reviewer #2:The article brings to light the functional consequences of the activity of SuM afferents terminating at CA2 neurons in the hippocampus using a combination of a variety of methods like whole-cell voltage clamp and optogenetics. In addition, the authors provide evidence that modulation of the CA2 neurons by SuM afferents affects the activity pattern of CA1 neurons. Specifically, the study reveals that the 'functional' connectivity between SuM and CA2 is mainly mediated by the regulation of PV+ basket cells that are involved in the feed forward inhibition of CA2 principal neurons. This study is also relevant in the context of neuropsychiatric disorders where PV+ IN density in the CA2 area is preferentially reduced.It would be good if some results and implications are further clarified for better understanding in the Discussion section:1. The results indicate that SuM recruits a feed forward inhibition onto CA2 PNs, which contributes to the shaping of CA2 AP firing. However, it is not entirely intuitive how the feed forward inhibition of CA2 PNs by SuM also reduces CA1 activity, as CA2 has also been known to recruit strong feed forward inhibition onto CA1. This would intuitively suggest that decrease in CA2 activity by photostimulation of SuM afferents will in turn decrease the feed forward inhibition by CA2 onto CA1, and thereby increase CA1 activity. However, the results suggest otherwise. Would this be suggestive of a stronger direct excitatory projection from CA2 to CA1 PNs that is more dominant than the feed forward inhibition of CA1 PNs by CA2? This may be a good point to further elaborate on in the Discussion section, so that the effect of SuM-CA2 connectivity on CA1 output becomes clearer.2. In the introduction section line 44, it is written that 'CA2 neurons do not undergo NMDA-mediated synaptic plasticity'. This may not be always the case; rather it may be better to rephrase 'NMDA-mediated' as 'high frequency stimulation-induced'. It has been shown previously that NK1 receptor activation by pharmacological application of substance P in hippocampal slices triggers a slow onset NMDA-dependent LTP in CA2 neurons by high frequency stimulation of CA3 afferents to CA2 .3. Line 250: "BC transmission is insensitive to MOR activation ."Was the Glickfeld study done in CA2 neurons? If not, it would be good to show that PV+ CA2 BCs are also sensitive to DAMGO and to what degree?The experiment shows that IPSC in PNs are inhibited by DAMGO that should have enhanced light induced EPSCs if PV+ BCs are responsible for feed forward inhibition. But it seems that has not been observed. What are direct EPSCs – electrical stimulation of CA3-CA2 synapses?4. Overall, the results seem to suggest that SuM stimulation would induce a net inhibition (?) of CA2 PNs by recruiting interneurons (INs). However, the role played by the direct glutamatergic connections from SuM to CA2 PNs is not entirely clear. Is it less prominent due to sparse SuM-PN projections compared to SuM-IN connections in the CA2 area? It may be good to elaborate on this a bit in the discussion.Reviewer #3:In this manuscript, Robert et al., demonstrated that medial SuM send glutamatergic projections to the hippocampal CA2 region, and stimulation of these projections exert mixed excitatory and inhibitory responses in CA2 pyramidal neurons. Furthermore, they showed that SuM-CA2 circuits recruit local PV basket cells to provide feedforward inhibition to CA2 pyramidal cells, which increases the precision of action potential firing in conditions of low and high cholinergic tone. Finally, they performed in vivo electrophysiology recording to show that stimulation of SuM-CA2 projections can influence CA1 activity. Overall, this is a well-designed study, and the quality of the data is high. The authors performed impressive amount of electrophysiology recording in acute slices and provided detailed information on how long-distance SuM projection neurons regulate CA2 pyramidal cell activity. These findings provide insights into how SuM activity directly acts on the local hippocampal circuit to modulate social memory encoding. However, there are some concerns that need to be addressed.eLife 2020) showing that lateral SuM neurons send dense projections to both CA2 and DG, and the SuM-DG projections release both glutamate and GABA to dentate granule cells. Based on the results from this study and the study from Li et al., does that mean medial SuM neurons are different from lateral SuM neurons in terms of the neurotransmitters they release? The authors need to clarify this point and provide additional ephys data to show that pyramidal cells do not receive direct GABAergic inputs upon stimulation of SuM-CA2 projections using high-chloride internal solution to reveal the IPSCs.1. The authors performed CAV-based retrograde tracing and demonstrated that medial SuM sends glutamatergic projections to CA2. These results are in contrast to a recent study  via a pertussin-toxin sensitive pathway. Is it possible that SuM-CA2 mediated excitation of PV basket cells includes a CCK intermediary? This point should be discussed.4. The in vivo recording data showed that SuM-CA2 circuit stimulation decreases the firing rate of CA1 pyramidal cells followed by increased firing rate in these cells. Then the authors performed slice recording and showed that the reduced firing rate of CA1 neurons in vivo is likely caused by increased inhibitory inputs onto CA1 pyramidal cells. Figure 7G-H seems to explain the reduced events in the first phase of the tetrode recordings, but not the rebound part. Is there some circuit component that is lost when making slices? Furthermore, what does SuM-CA2 circuit stimulation do to theta/γ rhythms in CA1? These data should be available in the tetrode recordings. The study describes the properties of inputs from the supramammillary nucleus (SuM) to the CA2 area of the hippocampus. Novel information is presented by the influence of the SuM input on the local hippocampal network in the CA2 and what the effect of this input is on network activity in the CA1. The authors use complimentary methods to address this question including patch-clamp recordings and optogenetics. Overall the reviewer found this study important, the experiments well-designed and the data of high quality. However, there are several key points raised by the reviewers to strengthen the data in order to fully support the authors' conclusions, addressing these will require additional experimental work. The list below summarizes the list of required experiments reviewers agreed would be necessary for having full confidence in the authors' conclusions. Reviewers' individual comments contain additional issues that would require the authors to make changes to the discussion, figure legends etc.1. The authors would need to show the effect of SuM stimulation on on synaptically triggered APs and not only on Aps evoked with a current step.We agree with the reviewers that establishing the role of SuM input in controlling synaptically-driven AP firing is most relevant. However, unlike other hippocampal pyramidal neurons, studying synaptically-evoked action potential firing in CA2 PNs is very difficult with optimal slice health and without blocking inhibitory transmission since CA2 PNs have low intrinsic excitability and receive strong inhibition as detailed below. Because of their low membrane resistance and very hyperpolarized membrane potential, CA2 PNs have an intrinsically low excitability which makes it difficult for any given input to cause AP firing in basal conditions. Furthermore, stimulation of CA3 axons evokes a very large feedforward inhibition that strongly prevents action potential firing. Likewise, distal dendritic inputs are extremely attenuated and also rarely lead to AP firing under physiological conditions. For these reasons we used current step injection-induced AP firing as a mean to induce spiking and test the effect of SuM input stimulation. Admittedly, this experimental design is somewhat artificial. We have previously discovered that CA2 PNs spontaneously depolarize and fire burst of action potentials under conditions of elevated cholinergic tone. It is known that the properties of these burst are strongly dependent on both excitatory and inhibitory synaptic transmission , and also the SuM is most likely to be active during theta rhythm, when cholinergic tone is elevated in the hippocampus. Therefore, we examined how the carbachol-induced bursting of CA2 PNs was altered by SuM input stimulation. We think that the results presented in Figure 7 are directly relevant to the reviewers’ request.We acknowledge that this experimental design does not allow for the contribution of specific inputs to AP bursting to be established. Thus, we performed additional experiments using electrical stimulation of axonal inputs in SR (CA3 axons) or SLM (EC axons) paired with SuM inputs stimulation. This new data is presented in Figure 2 and described in the text (pages 7-8). Unfortunately, for the vast majority of our slices, we were unable to evoke AP firing with reasonable electrical stimulation intensities, as is very typical for CA2 PNs. However, we were able to determine how SuM input summates with SR and SLM synaptic inputs, as well as determine how the summation ratio of these separate inputs is altered by SuM activity. Notably, we observed a reduction in PSP summation from both SR and SLM repeated stimulation when paired with SuM input activation, which is consistent with a shunting effect of the perisomatic-targeting basket cells recruited by SuM to drive inhibition in CA2 PNs.2. The change in the balance of EPCs and IPSCs in a train should be demonstrated in a single cell.We thank the reviewers for raising this point which is very important for the interpretation of the data with repeated SuM input stimulation presented in Figures 6, 7 and 8. In supplemental figure 4 we present IPSCs and EPSCs all recorded in the same CA2 PNs in the presence and absence of carbachol (described in pages 14-15). In short, we observed a consistent short-term depression of both SuM-driven monosynaptic EPSCs and di-synaptic IPSCs in CA2 PNs under basal conditions, with the resulting E/I ratio being stable over the train of SuM stimuli. Application of carbachol reduced initial amplitudes of both EPSCs and IPSCs while decreasing their short-term depression, resulting in a decrease of E/I ratio suggesting that the SuM inhibitory influence over CA2 PNs is even stronger and more sustained in conditions of elevated cholinergic tone.3. The properties of monosynaptic/disynaptic events should be compared and the lack of direct GABAergic input from the SuM demonstrated. The authors should quantify the delay time to light-evoked IPSCs to address whether the SuM-CA2 inputs are forming monosynaptic or disynaptic GABAergic connections to pyramidal neurons, as it is possible SuM neurons co-release glutamate and GABA to CA2. Given the importance of the mono vs. disynaptic innervation of different types of cells, the authors should go beyond the TTX experiments (as TTX would block a disynaptic EPSC) and also use 4-AP to recover the TTX blocked current to unequivocally prove that they inputs are monosynaptic.Nature 2020. The exclusive mono-synaptic glutamatergic nature of the SuM-CA2 transmission was demonstrated and directly compared to the SuM-DG synapse with measurements of EPSC vs IPSC onset latency and application of TTX with 4AP or NBQX + APV. We regret not summarizing more clearly in the text of this manuscript our previous findings and have added a description of these results to our manuscript (page 7).The reviewers are completely correct in asking for these experiments in order to confirm that indeed the SuM-CA2 inputs are entirely glutamatergic. All of these points are crucial to our investigation. In fact, we have already performed all of these requested experiments and analysis. These data were published as an extended data figure in Chen et al., 4. The preferential role of PV+ cells should be shown with a more selective pharmacological approach.We have taken the advice from reviewer #3 and performed a series of experiments using the P/Q voltage-gated calcium channel blocker ω-agatoxin TK. Block of these channels is predicted to only inhibit Gaba release from PV+ interneurons. These results of these experiments are in figure 5 and described page 12. Our experiments showed a near-complete block of SuM-driven IPSCs in CA2 PNs consistent with PV+ INs mediating this feedforward inhibition. However, the interpretation of these results is complicated by the fact that application of ω-agatoxin TK also reduced glutamatergic transmission at the SuM-CA2 synapse. If we assume that the SuM-IN synapse has a similar population of channels, then part of the reduction in inhibitory transmission may not be due to PV+ IN specificity, but by the reduction in glutamate release. While these results are not fully conclusive proof, we have still included them, as they further characterize the SuM input.As for the chemogenetic inhibition of PV+ INs, we took the reviewers advice and provided more direct evidence that 10 µM CNO is not sufficiently suppressing CA2 PV basket cell activity. We performed recordings of PV+ INs in area CA2 and measured changes in the intrinsic properties and action potential firing of these cells before and following CNO application. We did observe a small hyperpolarization of the membrane potential, however we still observed AP firing in several of the cells, indicating that this hyperpolarization is insufficient to fully silence PV+ INs. This is shown in supplemental figure 3 and described on page 11. GiDREADD can also act on pre-synaptic terminals, so it is not entirely conclusive if the PV+ cells that fire action potentials still release GABA. However, we feel that in combination with the incomplete expression of the GiDREADD in all PV+ interneurons, and the residual AP firing, that the 10 µM CNO is not entirely effective at suppressing PV+ IN activity in our experiments.We would like to mention that the expression of DOR receptors has been shown to be expressed in 35% of PV+ BCs in the hippocampus  and furthermore, we have shown that transmission exclusively from PV+ cells in area CA2 undergoes a DOR-dependent depression  that can be induced by application of DPDPE. We conclude that the effect of DPDPE on SuM transmission is compelling evidence that these inputs target PV+ cells.5. The authors should elaborate on how SuM stimulation influences theta/γ rhythms in the CA1 area.We have added a section to the discussion (pages 19-21) about theta/γ rhythms and the potential contribution of SuM input. We agree whole-heartedly with the reviewer about the fundamental importance of examining how SuM contributes to these brain activities in vivo. Please see our extended answer to reviewer 3 below.Reviewer #1:In this study Robert et al. describes the properties of long-range projections from the SuM to the CA2 area of the hippocampus. The authors identified direct excitatory and indirect inhibitory drive from SuM inputs on CA2 pyramidal neurons and showed that direct excitatory drive impinges on PV-positive basket cells. The overall effect of the input on CA2 activity was an increased precision of APs. The study also suggests that the input from the CA2 drives inhibition in the CA1 area. The study provides very interesting and new information about the cellular properties of SuM input in the CA2 area. This is an important question given the increasing importance of SuM inputs in social memory encoding. The study is timely, currently we have very limited data about the features and exact cellular profile of this input. The study is using elegant technical approaches o answer the central question of the study. While the study is addressing an important question and provides novel data, the authors central claim about the role of feedforward inhibition would need to be strengthened by the addition of experiments addressing how E-I balance changes in trains in individual neurons and how this can be linked to changes in the temporal precision of synaptically evoked APs.Action potentials are evoked with a current step. Since the study is focused on the network effects of feedforward inhibition, it would be useful to see how the properties of synaptically evoked action potentials change. In the cortex and in the CA1 feed forward inhibition was shown to limit the temporal summation of excitatory inputs which lead to decrease in AP jitter . In order to map these dynamics APs should be evoked via synaptic stimulation and not through current injection.Please see our response to point #1 in the above section.The authors show recordings of monosynaptic EPSCs in pyramidal cells and interneurons. It would be important to know how inhibitory and excitatory PSCs change in a train. Recordings from single cells held at E-GLUT ad E-GABA would allow the authors to monitor excitatory and inhibitory events in a train ad map how their balance changes. Can the change in E-I balance explain the change in AP jitter?These experiments examining the changes in E/I balance with a train of inputs has been added to the manuscript . We thank the reviewer for this insight. In fact, the E-I balance does not change during a train of stimulation, staying between 0.5 and 0.6 . However, we do see a large change in the E/I ratio in high cholinergic tone , indicating that the inhibitory drive of the SuM input is increased under these conditions.What are the characteristics of the SuM-driven inhibitory currents? Does the latency and jitter of monosynaptic EPSCs and disynaptic IPSCs differ? If one is monosynaptic and the other is disynaptic one would expect significant differences in both of these parameters.These points are very critical to understanding the local SuM-CA2 circuitry. These experiments have been published as supplemental data for Chen et al., Nature 2020. We have also added a description of these results to our current manuscript (page 7).How do the authors exclude the contribution of feed-back inhibition? feedforward and feed-back inhibition both could have an impact on the temporal precision of APs.We thank the reviewer for raising this important point which we failed to address in our initial submission. In basal conditions, feedback inhibition is not at play because stimulation of the SuM input evokes only small amplitude EPSPs that remain subthreshold in CA2 PNs, as documented in Figures 1 and 2. Indeed, we never observed SuM-evoked AP firing from CA2 PNs in our recordings . However, when inducing action potential firing in CA2 PNs either by current injection  or CCh application , the contribution of feedback inhibition becomes very relevant. Indeed, it is likely that CA2 PNs recruit not only feedback inhibition  but also recurrent excitation , both of which could impact the temporal precision of APs. In the case of current step injection-induced firing, the contribution of feedback inhibition and recurrent excitation to reducing AP jitter can be examined with our pharmacology experiments: in the GABA receptors blocked condition , the effect of SuM input stimulation on both the AP latency and jitter is abolished, thereby indicating that the main factor imposing the initial AP delay and constraining AP jitter is SuM-driven feedforward inhibition. This is because, at least for the first AP evoked by current step injection in the single CA2 PN recorded from, it is reasonable to assume that other CA2 PNs were not actively firing thus removing the possibility of feedback inhibition and recurrent excitation from the equation. Furthermore, the small probability of PNs to be connected (1.4%) and the small size of CA2-CA2 EPSP (~0.5mV)  makes it very unlikely that recurrent excitation plays any role when a single PN is depolarized. The problem becomes much more complicated when considering the CCh-induced AP burst firing conditions, as many CA2 PNs likely burst fire somewhat independently of the single CA2 PN recorded in any given experiment. Therefore, feedback inhibition and recurrent excitation are to be expected in these conditions, thereby potentially contributing to the temporal precision of AP firing in the recorded CA2 PN. Unfortunately, typical pharmacology blocking GABA or glutamate receptors is not suitable here as the CCh-induced AP burst firing pattern of CA2 PNs relies on intact synaptic transmission , thus making the examination of the respective roles of feedforward versus feedback inhibition in these conditions difficult to probe experimentally. Nevertheless, we deem reasonable to interpret our data with SuM input stimulation during CCh-induced spontaneous AP burst firing as follows: by recruiting perisomatic-targeted feedforward inhibition on most CA2 PNs, SuM input stimulation might effectively enforce a restricted time window during which CA2 PNs can undergo or sustain AP burst firing, thereby limiting independent burst firing from individual CA2 PNs and instead shaping a synchronized output from many CA2 PNs subsequently driving feedforward inhibition over area CA1 as shown in Figures 7 and 8.Reviewer #2:The article brings to light the functional consequences of the activity of SuM afferents terminating at CA2 neurons in the hippocampus using a combination of a variety of methods like whole-cell voltage clamp and optogenetics. In addition, the authors provide evidence that modulation of the CA2 neurons by SuM afferents affects the activity pattern of CA1 neurons. Specifically, the study reveals that the 'functional' connectivity between SuM and CA2 is mainly mediated by the regulation of PV+ basket cells that are involved in the feed forward inhibition of CA2 principal neurons. This study is also relevant in the context of neuropsychiatric disorders where PV+ IN density in the CA2 area is preferentially reduced.It would be good if some results and implications are further clarified for better understanding in the Discussion section:1. The results indicate that SuM recruits a feed forward inhibition onto CA2 PNs, which contributes to the shaping of CA2 AP firing. However, it is not entirely intuitive how the feed forward inhibition of CA2 PNs by SuM also reduces CA1 activity, as CA2 has also been known to recruit strong feed forward inhibition onto CA1. This would intuitively suggest that decrease in CA2 activity by photostimulation of SuM afferents will in turn decrease the feed forward inhibition by CA2 onto CA1, and thereby increase CA1 activity. However, the results suggest otherwise. Would this be suggestive of a stronger direct excitatory projection from CA2 to CA1 PNs that is more dominant than the feed forward inhibition of CA1 PNs by CA2? This may be a good point to further elaborate on in the Discussion section, so that the effect of SuM-CA2 connectivity on CA1 output becomes clearer.The referee is correct in that the connection between CA2 and CA1 is predominantly excitatory. CA2 PNs make a powerful excitatory connection with CA1 PNs . CA2 PNs also drive feedforward inhibition onto CA1 PNs . Here we show that under conditions that resemble active exploration (high cholinergic tone) the SuM-CA2-CA1 elicits a reliable poly-synaptic IPSC in CA1 PNs , as well as an occasional poly-synaptic CA1 EPSC. Our data does not directly show that SuM feedforward inhibition of CA2 causes a drop in CA2-CA1 excitation underlying the decreased CA1 activity following SuM-CA2 stimulation. Instead, our data shows that SuM inhibition of CA2 yields synchronized feedforward inhibition from CA2 to CA1 which decreases CA1 activity. We appreciate the complexity of this idea and have done our best to elaborate in the discussion (page 19).2. In the introduction section line 44, it is written that 'CA2 neurons do not undergo NMDA-mediated synaptic plasticity'. This may not be always the case; rather it may be better to rephrase 'NMDA-mediated' as 'high frequency stimulation-induced'. It has been shown previously that NK1 receptor activation by pharmacological application of substance P in hippocampal slices triggers a slow onset NMDA-dependent LTP in CA2 neurons by high frequency stimulation of CA3 afferents to CA2 .We thank the reviewer for this clarification. In the text, “NMDA-mediated” has been replaced by “high frequency stimulation-induced” as suggested.3. Line 250: "BC transmission is insensitive to MOR activation ."Was the Glickfeld study done in CA2 neurons? If not, it would be good to show that PV+ CA2 BCs are also sensitive to DAMGO and to what degree?The experiment shows that IPSC in PNs are inhibited by DAMGO that should have enhanced light induced EPSCs if PV+ BCs are responsible for feed forward inhibition. But it seems that has not been observed. What are direct EPSCs – electrical stimulation of CA3-CA2 synapses?In the experiments where we applied DAMGO, the EPSCs were recorded in presence of GABAR blockers. Therefore, any change in GABA transmission would not affect the light induced EPSC.The Glickfeld study was performed in CA1 only. However, expression of MOR mRNA is very strong in area CA2 and a large fraction of PV+ INs are also positive for MOR throughout the hippocampus . We haven’t directly monitored the effect of DAMGO on PV+ transmission in area CA2. However, in an ongoing study, we stimulated CA3 inputs and monitors the electrically evoked IPSCs in CA2 pyramidal cells . We foun4. Overall, the results seem to suggest that SuM stimulation would induce a net inhibition (?) of CA2 PNs by recruiting interneurons (INs). However, the role played by the direct glutamatergic connections from SuM to CA2 PNs is not entirely clear. Is it less prominent due to sparse SuM-PN projections compared to SuM-IN connections in the CA2 area? It may be good to elaborate on this a bit in the discussion.We feel that the combined excitation and feedforward inhibition from the SuM to area CA2 is acting together to coordinate the timing of CA2 PN output. We have expanded this explanation in the discussion (page 18).Reviewer #3:In this manuscript, Robert et al. demonstrated that medial SuM send glutamatergic projections to the hippocampal CA2 region, and stimulation of these projections exert mixed excitatory and inhibitory responses in CA2 pyramidal neurons. Furthermore, they showed that SuM-CA2 circuits recruit local PV basket cells to provide feedforward inhibition to CA2 pyramidal cells, which increases the precision of action potential firing in conditions of low and high cholinergic tone. Finally, they performed in vivo electrophysiology recording to show that stimulation of SuM-CA2 projections can influence CA1 activity. Overall, this is a well-designed study, and the quality of the data is high. The authors performed impressive amount of electrophysiology recording in acute slices and provided detailed information on how long-distance SuM projection neurons regulate CA2 pyramidal cell activity. These findings provide insights into how SuM activity directly acts on the local hippocampal circuit to modulate social memory encoding. However, there are some concerns that need to be addressed.1. The authors performed CAV-based retrograde tracing and demonstrated that medial SuM sends glutamatergic projections to CA2. These results are in contrast to a recent study  showing that lateral SuM neurons send dense projections to both CA2 and DG, and the SuM-DG projections release both glutamate and GABA to dentate granule cells. Based on the results from this study and the study from Li et al., does that mean medial SuM neurons are different from lateral SuM neurons in terms of the neurotransmitters they release? The authors need to clarify this point and provide additional ephys data to show that pyramidal cells do not receive direct GABAergic inputs upon stimulation of SuM-CA2 projections using high-chloride internal solution to reveal the IPSCs.Nature 2020. As the reviewer mentions, in that publication we present multiple lines of evidence supporting the premise that the CA2-projecting and DG-projecting cells in the SuM are from different populations and use different neurotransmitter modalities. These results are supported by prior histological results by Soussi et al., EJN, 2010 that show the CA2-projecting SuMM fibers do not co-express GAD65 unlike the DG-projecting SUML fibers that express both VGLUT2 and GAD65.This is a very valid point, and as we have detailed above, the experiments directly comparing the SuM transmission into the DG and area CA2 have been published as a supplemental figure in Chen et al., eLife, 2020) the authors show that AAV5-DiO.eYFP injected into the SuM in a Vgat-cre mouse line results in fibers in area CA2. These results lack control data showing the specificity of the AAV5 viral vector in the vgat-cre line (co-labelling with Gad65/67…). In our experience, it is very easy to get non-specific expression of AA5 vectors in area CA2. In fact, we use this problem to our advantage in Dominguez et al., Cell Reports 2019.In the Li et al., paper .As the reviewer suggested, we used ω-agatoxin-TK to explore whether PV+ BCs are the sole population of interneurons. Consistent with PV+ cells being the primary population underlying the feedforward inhibition onto CA2 PNs, we saw a nearly complete block of light-evoked IPSCs. However, we also examined the direct excitatory SuM-CA2 glutamatergic synapse and found that application of ω-agatoxin-TK resulted in a 50% block of glutamatergic transmission. Thus, if we assume that the glutamatergic SuM-IN synapse is also reduced then the reduction of feedforward IPSC may not be because of the specificity of P/Q CaAs for the chemogenetic inhibition of PV BCs, we took the reviewers advice and provided more direct evidence that 10 µM CNO is not sufficiently suppressing CA2 PV basket cell activity. We performed recordings of PV+ INs in area CA2 and measured changes in the intrinsic properties and action potential firing of these cells before and following CNO application. We did observe a small hyperpolarization of the membrane potential, however in we still observed AP firing in several of the cells, indicating that this hyperpolarization is insufficient to fully silence PV+ INs. This is shown in supplemental figure 3 and described page 11. GiDREADD can also act on pre-synaptic terminals, so it is not entirely conclusive if the PV cells that fire action potentials still release GABA. However, we feel that in combination with the incomplete expression of the GiDREADD in all PV+ interneurons, and the residual AP firing, that the 10 µM CNO is not entirely effective at suppressing PV-BC activity in our experiments.3. CCK basket cells are known to excite PV basket cells  via a pertussin-toxin sensitive pathway. Is it possible that SuM-CA2 mediated excitation of PV basket cells includes a CCK intermediary? This point should be discussed.This is a very intriguing point, and we thank the reviewer for this suggestion. Lee et al. 2011 demonstrates that CCK application results in a depolarization of PV+ cells via a nonselective calcium conductance linked to intracellular calcium store release. The idea that CCK release would potentially enhance ability of SuM inputs to recruit PV+ cells is quite interesting and have added this to the discussion (page 19). Similarly, we have also mentioned the other intriguing finding that CCK cells actively inhibit PV+ basket cells (Dodek 2021), presenting two ways in which CCK cells may be acting indirectly to modulate SuM input to area CA2 (page 19).4. The in vivo recording data showed that SuM-CA2 circuit stimulation decreases the firing rate of CA1 pyramidal cells followed by increased firing rate in these cells. Then the authors performed slice recording and showed that the reduced firing rate of CA1 neurons in vivo is likely caused by increased inhibitory inputs onto CA1 pyramidal cells. Figure 7G-H seems to explain the reduced events in the first phase of the tetrode recordings, but not the rebound part. Is there some circuit component that is lost when making slices? Furthermore, what does SuM-CA2 circuit stimulation do to theta/γ rhythms in CA1? These data should be available in the tetrode recordings.This is a very interesting point, and a central flaw in acute slice physiology. There are definitely circuit components that are lost when making transverse hippocampal slices. We have added two paragraphs to the Discussion section detailing the potential role of SuM-CA2 input to theta and γ oscillations (pages 19-21).The in vivo data in the paper was based on short stimulation pulses in resting animals where theta and γ are minimal. Thus, further examination of theta and γ requires further distinct experiments. As this manuscript elucidates the local circuitry underlying the SuM input in area CA2, and the overall effect of SuM activity on action potential firing in this region, further examination of network oscillations is outside the scope of this work.However, we still went through all of our data to try to determine if there is a large apparent effect of SuM activity in theta / γ. Because of the current Covid-19 crisis, we have not been able to continue with the majority of our experimental work for this project, so we can only present preliminary data from two animals with acceptable trials, tetrode locations (in CA1), fiber placement over the area CA2, and properly targeted virus in the SuM. We present in As visible in the wavelet spectragrams across the whole trial, we do not see any obvious change in theta or γ power when the laser is on. We also present  the norm


In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca2+ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca2+ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca2+ signals. Because the abnormal astrocytic Ca2+ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP3RII), we reanalyzed these pathological events using IP3RII-deficient mice and found that all abnormal Ca2+ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca2+ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca2+ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca Thus, reactive astrocytes have received much attention, and researchers are continuing intense investigations to understand the nature and roles of these cells. The term reactive astrocyte includes a variety of astrocyte states. Researchers are attempting to identify simpler ways to describe these astrocytes, such as neurotoxic A1 astrocytes and neuroprotective A2 astrocytes . Ax. AxGFAP 2+ signals mediated by IP3RII in AxD astrocytes were found to be associated with the molecular pathogenesis of AxD, it remains unclear whether these are cell-autonomous actions of astrocytes or whether they involve the communication with other cells, such as neurons, microglia, and blood vessels. Recently, in iPS cell-derived astrocytes from AxD human patients, aberrant Ca2+ signals were also observed [2+ propagation, which was due to impaired extracellular release of ATP. In this experimental system, only iPS cell-derived astrocytes are present, and there is no communication with other types of cells. Therefore, aberrant Ca2+ signaling in AxD astrocytes occurs as a cell-autonomous action of astrocytes, suggesting that GFAP mutations would be translated into abnormal Ca2+ signals in astrocytes, leading to the development of AxD. However, the molecular mechanisms and cascades leading from aberrant Ca2+ signals to AxD remain unclear. By focusing on Ca2+ abnormalities in astrocytes, future research on AxD is expected to make significant progress.Although aberrant Caobserved . AxD iPS2+ signals”. However, Ca2+ signal shows more complex spatiotemporal variations than we thought, and the mechanisms by which these occur are different. For example, Ca2+ signals have three different patterns in striatal astrocytes, defined as global Ca2+ signals, local Ca2+ signals, and Ca2+ microdomains [2+ signals are well observed in the somata of astrocytes and are mainly due to Ca2+ release from IP3RII, followed by Ca2+ entry via store-operated Ca2+ entry mechanisms. In addition, Ca2+ shut-off mechanisms mediated by the endoplasmic reticulum Ca2+ pump, the plasma membrane Ca2+ pump, and the Na+/Ca2+ exchanger also affect Ca2+ signals there. On the contrary, Ca2+ microdomains are mainly observed in the fine processes of astrocytes and are rather produced by Ca2+ entry via Ca2+ entry channels such as TRPA1 [2+ signals. In addition, the word “aberrant Ca2+ signal” means not only an abnormal increase but also an abnormal decrease in Ca2+ signals. However, other than the fact that differences in the spatiotemporal pattern of Ca2+ signals in astrocytes have a significant effect on astrocyte function, research in this field has not progressed, and the actual situation is not well understood. Therefore, in future studies, we should investigate and categorize the aberrant Ca2+ signals in astrocytes from the point of view of temporal and spatial differences as well as of qualitative differences, so to classify them more precisely.In this manuscript, we have simply described astrocyte dysfunctions in terms of “aberrant astrocyte Caodomains . Global as TRPA1 . Of cour2+ signals, and very importantly, aberrant Ca2+ signaling was found to be a cause of these diseases. As shown in 2+ signals are induced in various diseases differ (2+ signals affect the development and progression of various diseases also markedly differ (2+ signaling or that abnormal astrocytic Ca2+ signals may be a common underlying dysfunction in neurodegenerative diseases, because inhibition of these abnormal Ca2+ signals by deletion of IP3RII dramatically reduced all three disease states (2+ signaling, especially abnormal Ca2+ signaling, is a fascinating subject that may reveal common principles of neurodegenerative diseases and directly lead to the development of high-impact therapeutic strategies common to many brain diseases. In the future, it will be necessary to clarify the actual Ca2+ abnormalities at the molecular level by comprehensive analyses, such as single-cell RNA sequencing and proteome analysis, and to analyze the mechanisms responsible for Ca2+ signaling abnormalities (2+ signaling abnormalities (2+ signaling in astrocytes, the nature of the “abnormality” in each disease is different. The degree to which Ca2+ signals are abnormal in astrocytes also needs to be carefully studied.As shown in the above examples, astrocytes show common characteristics in three very different diseases, i.e., they appeared reactive and exhibited abnormal Cae states B. Thus,


Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium (ICaL) current and slowed ICaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K+ current reserve, even at end-stage disease.Dysfunctional sarcoplasmic reticulum Ca This balance has important implications for arrhythmia mechanisms in the heart, and these can be clearly illustrated by considering differences across mammalian species.Dysfunctional sarcoplasmic reticulum (SR) Caopathies , 2002 toopathies . However2+ that fuels cardiac contraction is obtained from L-type Ca2+ current (ICaL)-mediated Ca2+ influx  plateau, which itself results from a relatively delicate balance of inward and outward currents in large mammals   have suggested that the dominant mechanisms of arrhythmia in these mice are focal activity (perhaps originating from DADs in the His-Purkinje network), and APD alternans driven by aberrant SR Ca2+ handling  . This pr release , and we  in mice . In vivohandling . Togethein vivo and cellular arrhythmogenesis in a conditional SR Ca2+ ATPase type 2 (SERCA2) knockout mouse (KO). These mice progress to contractile failure in 7–10 weeks and display remarkable adaptations to the rapid loss of cardiac SR Ca2+ reuptake . In combination, these adaptations constitute a genetic model of extreme loss of ICaL control in the mouse, and present a unique opportunity to directly dissect the role of sarcolemmal versus intracellular mechanisms in murine arrhythmia.To interrogate this hypothesis, we have examined reuptake . Most prICaL and Na+-Ca2+ exchange, both of which contribute to marked prolongation of the AP and are ordinarily interpreted as strongly proarrhythmic changes. In combination, these data strongly suggest that SR Ca2+ release is required for cellular arrhythmogenesis and tissue-level triggered events in the mouse.At end-stage life we found SERCA2 KO mice are remarkably resistant to arrhythmia, both at the cellular level and in intact conscious animals. This resistance exists in the face of large increases in both All experiments were performed in accordance with the Norwegian Animal Welfare Act, which conforms to NIH guidelines .The SERCA2 knock out mouse (KO) has previously been described and studied in detail as a model of contractile failure . BrieflyTelemetry transmitters  were used to record electrocardiograms in freely moving animals. Implantation was performed 6 weeks after tamoxifen injection, and as previously described . IntrapePremature ventricular complexes (PVCs) were manually identified, and episodes of ventricular tachycardia (VT) were defined as 4 or more PVCs occurring in sequence. Runs of VT were defined as non-sustained (NSVT) if they lasted less than 20-s, and as sustained (SVT) if longer.2, 0.4 NaH2PO4 (pH 7.4). Each heart was then retrograde-perfused under constant flow conditions (3 ml/min) with warmed (37°C) isolation buffer for 4 min, and then with the same buffer supplemented with 400 U/ml collagenase Type II  and 0.015 mmol/L Ca2+. After 10 min of enzyme perfusion, hearts were cut down and the left ventricle was removed, diced, and triturated in collagenase-free isolation buffer including 1% BSA and 0.02 U/L deoxyribonuclease I (Worthington), again at 37°C. The resulting cell suspension was filtered (200 μm nylon mesh) and sedimented, the cell pellet was washed in isolation buffer supplemented with 1% BSA, and Ca2+ was progressively reintroduced . The Ca2+-tolerant cardiomyocytes were stored at room temperature until use, which occurred within 8 h of isolation.Cardiomyocytes were isolated from ventricles of FF and KO mice similarly to our prior studies . Briefly+ currents in voltage clamp, where the internal solution contained , and external (in mM): NaCl 140, HEPES 5, KCl 5.4, MgCl2⋅6H2O 0.5, Glucose 5.5, NaH2PO4H2O 0.4, CaCl2 1. For voltage clamp recordings of the transient outward potassium current (Ito), the internal solution contained (in mM) 110 K-Asp, 20 KCl, 0.5 MgCl2, 4 K2-ATP, 5 EGTA, 5 HEPES, and 10 D-Glucose - pH 7.2 with KOH, with a Na+ and Ca2+ free external solution (mM): 140 CholineCl, 1 CdCl2, 0.5 MgCl2, 5 HEPES, 5.5 Glucose, 5.4 KCl, pH 7.2 with KOH. For ICa,L the internal solution was (mM) 130 CsCl, 0.33 MgCl2, 4 Mg-ATP, 0.06 EGTA, 10 HEPES and 20 tetraethylammonium chloride - pH 7.2 with CsOH, and the bathing solution included (mM) 135 NaCl, 20 CsCl, 1 MgCl2, 10 glucose, 10 HEPES, 1 CaCl2, and 4 4-AP (pH 7.4).Single rod-shaped cardiomyocytes with clear striations were patch-clamped in whole-cell configuration using Axoclamp 2A and 2B amplifiers. Borosilicate patch-pipettes had resistances of 2–3 MOhms with the corresponding internal and external solutions. We used identical solutions for current clamp AP recordings and end-pulse KICaL was elicited by a 200-ms depolarizing voltage step (INa). Current amplitude was measured as the difference between peak inward current and steady-state current at the end of the test pulse. Ito (peak IK) was elicited by 300-ms steps to test potentials between −40 and +60 mV in 10-mV increments from a holding potential of −70 mV  was calculated as the mean of the final 20-ms of 500-ms test-pulse steps between −100 and +60 mV, again at 10 mV increments from a –70 mV holding potential. All currents were normalized to capacitance, which was calculated as the integral of the transient current response to a 10-mV hyperpolarizing step (150 ms) from a holding potential of −70 mV.Cells were superfused with external solution in all experiments, and all current measurements were made during step-pulse protocols at 1 Hz. Following ten 50-ms conditioning pulses  to 0 mV from a holding potential of −70 mV, age step . Test pof −70 mV . Peak IKAction potential waveforms were also recorded at 1 Hz in all experimental conditions, and at least 20 sequential beats were allowed to achieve steady state. In experiments assessing EAD and DAD susceptibility, Iso-containing superfusate (1 μM) was applied after 30-s of baseline recording, and maintained for at least 3 min further.The computational models used here are modifications of those published by IK, IKp, ICaL, and AP durations at 20, 50, 70, and 90% repolarization were all tested by RMANOVA where step potential was repeated for current measurements, and treatment conditions  were repeated for AP recordings.Steady state electrocardiographic parameters were assessed by repeated measures ANOVA (RMANOVA: Genotype × Epi) with repeated measures for Epi. ECG arrhythmia outcomes (PVC and NSVT frequency) were non-normally distributed, and interrogated by Kruskal-Wallis rank sum test after being separated into baseline and Epi recordings. Similarly, skewed data for EAD frequency were assessed via the Kruskal-Wallis test, and the Fisher exact test was used to determine differences in EAD incidence. Peak post hoc. In the event that the sphericity assumption was not valid, RMANOVA was replaced by the Mann-Whitney-Wilcoxon test. Significant effects were defined at p < 0.05, and p-values are explicitly stated for all marginal results (0.05 ≤ p ≤ 0.1). All statistical analyses were performed either with SigmaPlot , or R software .For all RMANOVAs, the Holm-Sidak test was used to identify pairwise effects p < 0.01, p < 0.0001), particularly during epinephrine challenge . In fact, during Epi challenge we observed reduced frequency of PVCs (p < 0.05) and a tendency for reduced NSVT frequency in the KO group  VT (50 and 230% longer APD70 in KO myocytes (both p < 0.01). Panels B and C show that this effect is due to modest potentiation of peak ICaL , and dramatically slowed ICaL inactivation , which together result in a 200% increase in Ca2+ influx, measured as the ICaL integral during the square-pulse voltage clamp protocol (p < 0.001). Exacerbating this gain in ICaL was a moderate reduction of end-pulse potassium current (IKp) at positive potentials (p < 0.05). While we did not attempt to experimentally dissect the different components of this current, we used a computational model to assess the ability of those components to contribute to the observed changes in AP morphology. These analyses are described in further detail below.ICaL in slowing early repolarization either by acutely changing superfusate Ca2+ concentration, or applying ICaL blockade by Nifedipine . 50 and APD70 is removed when Ca2+ is excluded from the bath, and slightly exaggerated when it is increased to 1.8 mM (both p < 0.05). These alterations in extracellular [Ca2+] did not have statistically discernible effects on the kinetics of early repolarization in FF myocytes (p > 0.1). Because field-screening effects and non LCC-specific Ca fluxes2+ (such as Na+/Ca2+ exchange) will be altered by modulation of extracellular Ca2+, we also used Nifedipine to pharmacologically antagonize ICaL . This suggests that inward ICaL is the major contributor to the small phase 2 plateau in KO cells. Finally, to determine whether maneuvers that ordinarily modulate Ca2+-dependent ICaL inactivation (via SR Ca2+ release) are ineffective in KO mice, we also assessed the frequency-dependence of peak ICaL and ICaL inactivation , further indicating the loss of SR-dependent ICaL control in KO animals.In ize ICaL . As for tivation . In movilic Ca2+ . In KO a50 and APD70 are lost by 90% (APD90) repolarization. The dominant inward currents modulating this late phase of repolarization in the mouse are INaCa and recovering INa  to shape terminal repolarization. While we have not directly assessed the balance of these currents during late repolarization in KO myocytes, the near complete absence of triggered Ca2+ release  for at least 3 min while pacing at 1 Hz in current clamp, and defined EADs as any upward deflection in the voltage signal exceeding 3 mV. p > 0.1), rather EAD frequency tended to be higher  in the FF (9.3 ± 4.7%) than KO group (2.1 ± 1.8%). In FF cells, EADs exhibited signature properties identified in previous work  to result from subcellular Ca2+ waves. Thus, it is very likely that EADs in FF myocytes were carried almost entirely by non-equilibrium reactivation of the fast component of the sodium current (INa) (p < 0.001), were much larger in amplitude , and lasted much longer than EADs in the FF myocytes . While these EADs initiated later than those in FF cells, because spontaneous SR Ca2+ release in KO cells is virtually negligible  . In contgligible , it is vtivation , the −40 mammals .ICaL deactivation and limits the opportunity for Ca2+-dependent ICaL inactivation, which is prominent in larger species. Combining this with the known redundancy and different kinetic characteristics among the various K+ currents active in this early phase of repolarization, it becomes much more difficult to predict or account for how changes in ICaL regulation can contribute to modulating early repolarization. To interrogate these dynamics, we employed a published computational model of the mouse cardiomyocyte, which is specifically parameterized to incorporate the reduced SR Ca2+ handling and altered Na+ balance in failing KO myocytes [16]. This model already incorporates the increased Cav1.2 and NCX1 expression in KO cells, which leads to exaggerated peak ICaL and INaCa. However, it does not fully capture the pronounced slowing of ICaL inactivation we have observed in KO myocytes at 7 weeks of age, and incorporating this characteristic was our first alteration. ICaL model in square-pulse voltage-clamp protocols identical to our experiments  during early repolarization in both the FF and KO models, and had relatively little impact on the trajectory of early repolarization  is minimally expressed in mice, and that currents carried by Kv1.5 and Kv2.1 make important contributions (IKslow), also commonly referred to as the ultrarapidly activating delayed rectifier (IKur) K+ current. IKslow is a dominant contributor to the end-pulse K+ current in mice. It combines with the steady state K+ current  and measured end pulse IK with the KO model, we also had to employ the approach of IKslow into Kv1.5- and Kv2.1- specific components. However, unlike IKslow,2, as shown in IKslow enough to permit ICaL modulation of intermediate repolarization, while also matching end-pulse IK and permitting stable overall repolarization, as observed in our experiments.As mentioned above, KO myocytes also exhibit a reduction in the measurable total ibutions . Togethechannels , and IK1kinetics . To simuICaL , SERCA (50–60% reduction in the Km for cytosolic Ca2+ binding), the Na+-K+ ATPase (25–30% reduction in the Km for cytosolic Na+ binding), and IKur (15–20% increase in whole cell conductance)  are increased similarly to the experimental recordings  . The stecordings . These d mammals . Howeverals  Shifting the burden of contractile calcium flux away from the SR and toward the sarcolemma elicits a pronounced, positive, and prolonged T-wave that is not normally observable in mice. While these effects are substantial, they are not sufficient to destabilize cardiac activation or repolarization. (2) The major cellular effect underlying these global changes is a very large increase in ICaL-mediated Ca2+ influx, due largely to slowed current inactivation, and which results in a pronounced delay of early repolarization creating an unusual AP plateau around 0 mV. (3) This slowing of early repolarization is difficult to capture in models due to the rapid kinetics of the large measurable peak IK, which is broadly thought to be carried by Ito,f and IKur/IKslow,1. The manner in which K+ current activation interacts with ICaL inactivation to shape early repolarization of the murine AP in this way requires further review. (4) The overall shift in the balance of membrane currents in SERCA2-KO away from a mouse-like phenotype and toward a large mammalian phenotype recruits EAD dynamics that are closer to those in large mammals. However, this shift is not sufficient to increase EAD frequency or incidence, and initiation of infrequent EADs in KO myocytes still likely relies upon INa reactivation, which rarely drives EADs in large mammalian myocytes.The ability for SR Ca30 years , and is ICaL in mice and rats. Early rat studies of agonist dihydropiridines (Bay K 8644), which both potentiate and markedly prolong ICaL, noted similar overall changes and included additional ST segment elevation due to fusion of the QRS complex and T-wave (ICaL is Timothy syndrome (formerly type 8 long QT syndrome). While mice have been generated for major mutations known to cause this channelopathy, and brief reports suggest QT prolongation and abnormal ECG characteristics . 2+ transport but achieve sufficient contractile activation through very high gain EC coupling  and QT intervals, they do not present an overt arrhythmia phenotype (90), EADs, and an overt susceptibility to arrhythmia  in KO myocytes while constraining IK1 to the measured inward end-pulse current below EK (2+ release and resulting inward INaCa are major modulators of this phase of the murine AP. This suggests that more detailed accounting for components of IK that are active and available between −80 and −40 mV (particularly IK1), is likely necessary for properly capturing the interaction among INaCa, recovering INa and IK in determining the trajectory of terminal repolarization in the mouse.Additionally, we had limited ability to reconcile the more rapid terminal repolarization (APDbelow EK  and othebelow EK  have sho2+ transport resulting from cardiac specific SERCA2 knock out are clearly not sufficient to destabilize myocyte repolarization or global electrophysiology in the mouse. Furthermore, those shifts only alter EAD dynamics in a relatively subtle fashion. In sum, our data suggest that, in the mouse, the destabilizing influence of extreme changes to sarcolemmal Ca2+ influx and extrusion can be readily outweighed by the stabilizing influence of an absent SR Ca2+ store and dramatic reduction in spontaneous SR Ca2+ release. Of course, this characteristic relies on the very strong overall repolarization present in the mouse, and should not be interpreted to extend to humans or indeed any larger mammals exhibiting an AP plateau.The major shifts in sarcolemmal CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Norwegian National Committee for Animal Welfare.AGE collected and analyzed data, performed computational analyses, conceived and designed aspects of the study, and wrote the manuscript. HM collected and analyzed data, conceived and designed aspects of the study, and reviewed the manuscript. MKS collected and analyzed data, and reviewed the manuscript. DBL collected and analyzed data. IS conceived and designed aspects of the study, and reviewed the manuscript. SR and WEL collected and analyzed data, conceived and designed aspects of the study, reviewed the manuscript, and contributed to funding the study. OMS conceived and designed aspects of the study, reviewed the manuscript, and contributed to funding the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inhibitors of mutant IDH enzymes and their 2-HG production have been approved by the FDA to be used in patients. However, 60% of IDH mutant AML patients do not respond to these inhibitors or develop mechanisms of resistance, leading to relapse. Among these mechanisms, some produce a 2-HG rebound. Alternative therapies exploiting the 2-HG-dependent molecular effects could therefore be of clinical interest. In this study, we demonstrate that 2-HG specifically activates vitamin D receptor (VDR) in IDH mutant AML cells leading to increased sensitivity to the combination of vitamin D (or VDR agonist) and all-Relapses and resistance to therapeutic agents are major barriers in the treatment of acute myeloid leukemia (AML) patients. These unfavorable outcomes emphasize the need for new strategies targeting drug-resistant cells. As IDH mutations are present in the preleukemic stem cells and systematically conserved at relapse, targeting IDH mutant cells could be essential to achieve a long-term remission in the IDH mutant AML subgroup. Here, using a panel of human AML cell lines and primary AML patient specimens harboring IDH mutations, we showed that the production of an oncometabolite (R)-2-HG by IDH mutant enzymes induces vitamin D receptor-related transcriptional changes, priming these AML cells to differentiate with pharmacological doses of ATRA and/or VD. This activation occurs in a CEBPα-dependent manner. Accordingly, our findings illuminate potent and cooperative effects of IDH mutations and the vitamin D receptor pathway on differentiation in AML, revealing a novel therapeutic approach easily transferable/immediately applicable to this subgroup of AML patients. MUT) AML cells to differentiation therapy using all-trans retinoic acid (ATRA) − species at m/z 147.0299 and 145.0142, respectively. Two-HG and α-KG, were quantified by isotope dilution using the corresponding 13C-labeled homologues as internal standards.Metabolites LC-HRMS quantification of 2-HG and α-KG was performed using an Ultimate 3000 chromatographic system coupled with an exactive mass spectrometer  fitted with an electrospray source. Chromatographic separation was performed on a Hypercarb column  maintained at 30 °C. Mobile phase A consisted of an aqueous buffer of 5 mM of ammonium formate at pH 2.7, while mobile phase B was made of acetonitrile with 0.1% formic acid. Chromatographic elution was achieved at a flow rate of 300 μL/min. After injection of 10 μL of sample, elution started with an isocratic step of 0.5 min at 95% A, followed by a linear gradient from 95% to 5% of phase A in 4.5 min. The chromatographic system was then rinsed for 1 min at 95% B, and the run ended with an equilibration step of 2 min. The column effluent was directly introduced into the electrospray source of the mass spectrometer, and analyses were performed in the negative ion mode. Source parameters were as follows: capillary voltage, −2.5 kV; capillary temperature, 350 °C; sheath gas and auxiliary gas pressures set at 35 and 10 arbitrary units, respectively. The detection was performed from p < 0.05, ** is p < 0.01, *** is p < 0.005 and **** is p < 0.001. Combination indexes were calculated using Compusyn software .GraphPad Prism Software version 9  was used for the statistical analysis. The results were expressed as a mean ± SEM. Statistical analyses were performed using two-tailed  Student’s t test with Welch’s correction The significance is represented by stars in which * is R132H or IDH1WT  (R132H HL60 cells compared to wild-type isoforms (WT AML cells treated with exogenous 2-HG and in patients harboring IDH mutations (R312H AML blasts from a patient-derived xenografted (PDX) model in NSG mice, we confirmed this phenotype in vivo. Of note, the Late VD/VDR activation pathway gene signature was strongly correlated to gene signatures of IDH1MUT patients generated from two independent public cohorts in the PDX IDH1 R132H  ar IDH1WT  [4]. We A and VD . Both AT, CEBPε) . Using gisoforms . Particuisoforms  is furthutations . Using sH1 R132H D. This cH1 R132H . FinallyTaken together, transcriptomic analysis of cell lines, primary patient cells and PDX with IDH mutations showed that VD/VDR pathway is activated in this subgroup of AML patients. Furthermore, this VD/VDR gene signature is enhanced in patients who are relapsing from IDH inhibitor treatments, suggesting that Vitamin D-based therapies could represent a promising alternative for these patients.WT or IDH1R132H. Consistently, we observed an increased expression in VDR, RARα (ATRA receptor/transcription factor) and RXRα (VDR and RARα co-receptor/transcription factor) in IDH1R132H  and primary patient cells  with IDHWT or IDHMUT. We first observed that the combination therapy induced a higher differentiation potentiation as shown with an increased CD11b expression in IDH1R132H and IDH2R172K HL60 cells compared to IDHWT HL60 cells , CD11b responders (n = 4), CD15 responders (n = 2) and the non-responders (n = 4)  A. Next, fication  , that leads to MYC/CEBPα inhibition. However, most IDHMUT AML cells upregulate MYC which counteracts the effect of 2-HG on proliferation, CEBPα expression and metabolic consequences [MUT AML biology [MUT context. Markedly, Glass et al. showed a differential CpG methylation in RXR promoter specifically in IDH2MUT AML cells [MUT differentiation and almost completely abrogated differentiation in IDHWT treated with exogenous 2-HG. Of note, high serum 2-HG and low serum VD levels are associated with shorter overall survival [MUT AML patients. Although randomized clinical trials are still lacking, epidemiological and preclinical data suggest that VD-based combinations could be a promising therapeutic strategy by activating VD signaling pathway. In this context, better designs of combinations would circumvent potential limitations of VD alone such as high risk of hypercalcemia.Numerous studies have investigated the potential benefit of VD-induced differentiation through interaction with VDR in different cancers including AML, without encouraging results . Indeed,sitivity . Additiositivity  and in Hsitivity , for whisitivity . Of notesitivity . In thesequences ,37. Inde biology ,13. In t biology ,38. Note biology . CEBPα s biology . TherefoML cells . Overallsurvival ,41,42, tMUT cells, the use of a hypomethylating agent would be relevant in this subgroup of patients. Surprisingly, as opposed to first studies  could help determine if patients with IDH mutations are more likely to respond. If so, further studies measuring the expression of VDR and VDR target genes, as well as 2-HG level, differentiation and impact on the stem cell compartment specifically in IDHMUT AML cells following these combinations would be of particular interest and help determine potential biomarkers of response.Finally, hypomethylating agents such as 5-azacytidine (AZA) or decitabine induce an increase in expression of VDR and its target genes in breast cancer cells  and AMLa, 2014; ), AML ana, 2014; ,45,46. Ma, 2014; . In a seMUT primary cells. This experiment could also enable to consider the potential toxicity of ATRA/VD association on normal hematopoietic stem cells. Indeed, both ATRA and VD are known to induce side effect including differentiation syndrome and hypercalcemia, respectively [MUT inhibitors and treatment interruption is rarely considered [MUT patients and their response to ATRA/VD combination therapy are needed to consider 2-HG as a biomarker of response to this treatment. Response to IDHMUT inhibitors is not associated with 2-HG levels since non-responding patients display a reduction in 2-HG amount after treatment [MUT inhibitors [There are several limitations to our study. Before clinical application of ATRA/VD combination therapy, these results first need to be confirmed in preclinical models. In this context, the efficacy of this doublet therapy to enhance differentiation and anti-leukemic activity should be evaluated in vivo using mice xenografted with IDHectively ,49. Todansidered . Moreovensidered ,51. Howereatment ,30. In thibitors ,9,10. ThMUT subgroup, especially at relapse post-IDHm inhibitor. Moreover, VD could enhance ATRA effect in AML patients with IDH mutation through a 2-HG/CEBPα/VDR axis. Thus, our study describes an alternative approach with approved differentiation drugs ATRA and VD with a highly efficient and safety profile, already in the clinics. This strategy could be specifically used for non-eligible AML patients to IDH mutant inhibitor therapies or AML patients developing resistance to newly FDA-approved IDH mutant-specific inhibitors. This is timely and clinically relevant and forms the basis of novel doublet and triplet therapies in IDH-mutated AML patients.In conclusion, these data demonstrate that the VDR pathway is increased in IDH