Local circuit allowing hypothalamic control of hippocampal area CA2 activity and consequences for CA1

  1. Vincent Robert
  2. Ludivine Therreau
  3. Arthur J.Y. Huang
  4. Roman Boehringer
  5. Denis Polygalov
  6. Thomas McHugh
  7. Vivien Chevaleyre
  8. Rebecca A. Piskorowski

  • Curated by eLife

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    Summary: 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 complementary methods to address this question including patch-clamp recordings and optogenetics. Overall the reviewers 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, and 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:

    1. The authors would need to show the effect of SuM stimulation 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/gamma rhythms in the CA1 area.

    This manuscript is under revision at eLife.

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Abstract

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. We found that SuM axon stimulation elicited mixed excitatory and inhibitory responses in area CA2 pyramidal neurons (PNs). We found that parvalbumin-expressing basket cells as 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 modulates 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.

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  1. eLife

    Reviewer #3:

    In this manuscript, Robert et al. demonstrated that medial SuM sends 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 an 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 (Li et al, 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.

    2. The authors claim that SuM-CA2 circuits recruit local PV basket cells to provide feedforward inhibition to CA2 pyramidal cells. While the data presented are supportive, they are not entirely convincing. Specifically, MOR agonist DAMGO is not specific to PV BCs. Though DAMGO has a preferential effect on PV cells over CCK cells, other interneuron types have been shown to be sensitive to DAMGO manipulation. Therefore, these results are subject to alternative interpretation that other types of CA2 local interneurons may be involved. To show whether PV BCs is the sole interneuron subtype involved, the authors may use a P/Q type calcium channel blocker, ω-agatoxin-TK, as P/Q Ca2+ channels are unique to PV BCs. In addition, chemogenetic inhibition of PV BCs was used, but light-evoked IPSCs are not completely blocked. The authors claimed this could be due to partial silencing of PV BCs. However, there is no evidence showing the efficacy of 10µM CNO application in suppressing CA2 PV basket cell activity. These data should be provided in order to draw such conclusions.

    3. CCK basket cells are known to excite PV basket cells (Lee et al 2011) 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/gamma rhythms in CA1? These data should be available in the tetrode recordings.

  2. eLife

    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 always be 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 (Dasgupta et al., 2017).

    3. Line 250: "BC transmission is insensitive to MOR activation (Glickfeld et al., 2008)."

    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?

    1. 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.
  3. eLife

    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 to answer the central question of the study. While the study is addressing an important question and provides novel data, the author's central claim about the role of feed-forward 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 feed-forward 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 (Gabernet et al., 2005, Pouille and Scanziani 2001). 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 and E-GABA would allow the authors to monitor excitatory and inhibitory events in a train and 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? Feed-forward and feed-back inhibition both could have an impact on the temporal precision of APs.

  4. eLife

    Summary: 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 complementary methods to address this question including patch-clamp recordings and optogenetics. Overall the reviewers 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, and 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:

    1. The authors would need to show the effect of SuM stimulation 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/gamma rhythms in the CA1 area.

    This manuscript is under revision at eLife.

  5. Version published to 10.1101/2020.09.18.303693 on bioRxiv