Hilar mossy cells control structural and functional organization in the dentate gyrus

  1. Corwin R. Butler
  2. Connor R. Squellati
  3. Alyssa B. Danis
  4. Arielle Isakharov
  5. Gary L. Westbrook
  6. Eric Schnell

  • Curated by eLife

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    eLife Assessment

    This important study demonstrates how ablation or silencing of hilar mossy cells in the mouse influences the primary location where the mossy cells project, the inner molecular layer of the dentate gyrus. The anatomical findings are convincing and include altered adult-born granule cells and the shrinkage of the inner molecular layer following mossy cell ablation. However, the mechanisms and their functional significance are unclear, so more of these types of experiments/analyses would strengthen the study, especially the support for the broader conclusions.

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Abstract

Hilar mossy cells in the dentate gyrus project widely throughout the hippocampus, broadly contributing to circuit function. Their loss in disease is associated with local functional and structural rearrangements, including retrograde granule cell axon sprouting, aberrant neurogenesis, and disinhibition. To examine how mossy cell loss contributes to these circuit rearrangements, we ablated or silenced hilar mossy cells using viral approaches in transgenic (Crlr-Cre) mice. Both mossy cell ablation and silencing dramatically altered dentate gyrus structure and function, as assessed using immunohistochemical, viral labeling, electrophysiology, and anatomical methods. Both manipulations accelerated the maturation of adult-born neurons, but did not alter neuroblast proliferation or cause granule cell axon sprouting. However, mossy cell ablation, but not silencing, caused collapse of the inner molecular layer accompanied by proximal translocation of middle molecular layer inputs. In both cases, granule cell activity measured by cFos labeling and seizure susceptibility were unchanged after mossy cell loss, indicating functional compensation for the altered network organization. Our results highlight how mossy cells influence dentate gyrus organization and adult neurogenesis but also demonstrate the resilience of the hippocampal circuit to structural or functional perturbations.

Article activity feed

  1. eLife

    eLife Assessment

    This important study demonstrates how ablation or silencing of hilar mossy cells in the mouse influences the primary location where the mossy cells project, the inner molecular layer of the dentate gyrus. The anatomical findings are convincing and include altered adult-born granule cells and the shrinkage of the inner molecular layer following mossy cell ablation. However, the mechanisms and their functional significance are unclear, so more of these types of experiments/analyses would strengthen the study, especially the support for the broader conclusions.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    This study provides valuable evidence that hilar mossy cells play important roles in maintaining the structural organization of the dentate gyrus and regulating the maturation of adult-born granule cells. The evidence for the structural reorganization and for the accelerated dendritic maturation of adult-born granule cells is convincing: it rests on converging anatomical, viral tract-tracing, retroviral birth-dating, and electrophysiological measurements, with appropriate controls for viral spread, off-target CA3 expression, and axonal degeneration. Support for the study's broader interpretive claim - that the dentate circuit functionally compensates for mossy cell loss - is incomplete. That claim rests on two null results obtained under baseline conditions (home-cage cFos and PTZ seizure metrics) in small cohorts, without behavioral assessment and without a stimulus-driven activity readout, and the manuscript does not engage with published work showing that mossy cells regulate neural stem cell activation and are required for stimulus-evoked neurogenic and behavioral responses.

    Strengths:

    (1) The study is technically rigorous and employs multiple complementary approaches, including selective genetic manipulations, viral tracing, immunohistochemistry, retroviral labeling of adult-born neurons, electrophysiology, and anatomical analyses. The comparison between complete mossy cell ablation and chronic synaptic silencing is particularly powerful, allowing the authors to examine the significant role of mossy cells in structural and functional organization in the dentate gyrus.

    (2) One of the most notable findings is the identification of a previously unrecognized collapse of the inner molecular layer following extensive mossy cell ablation. This observation substantially expands current understanding of dentate gyrus structural plasticity. The demonstration that adult-born granule cells undergo accelerated dendritic maturation after both mossy cell loss and silencing also provides important insight into how mossy cells regulate adult neurogenesis.

    Weaknesses:

    (1) The functional significance of the observed structural remodeling remains incompletely addressed. Mossy cells have been strongly implicated in pattern separation, spatial information, and emotional behavior, yet no behavioral analyses were conducted. Consequently, it remains unclear whether the dramatic anatomical changes observed following mossy cell ablation translate into meaningful behavioral alterations.

    (2) The conclusion that the dentate gyrus exhibits remarkable homeostatic compensation is reasonable but remains indirect. Although cFos expression and PTZ-induced seizure susceptibility are unchanged despite altered E:I balance, the mechanisms responsible for maintaining network stability are not investigated. Additional analyses of inhibitory circuit remodeling or compensatory synaptic adaptations would strengthen this conclusion.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors examine how hilar mossy cells (MCs) influence adult-born dentate granule cell (abDGC) maturation and dentate gyrus (DG) structural integrity. Using both MC ablation and chronic functional silencing, they find that lacking MC inputs accelerates early abDGC maturation without altering mature cellular or intrinsic properties. MC silencing specifically decreased inner molecular layer (IML) spine density, whereas MC ablation led to IML collapse and an increased E/I ratio. However, neither intervention altered overall network excitability (measured via c-Fos and seizure induction) or seizure thresholds. These results advance our understanding of DG circuit plasticity during neurodegeneration.

    Strengths:

    (1) The side-by-side comparison of ablation vs. silencing provides a clear distinction between structural synapse loss and functional inactivation.

    (2) The multi-level analysis spanning structural anatomy, single-cell physiology, and network-level assays yields a rich, comprehensive dataset.

    Weaknesses:

    (1) Measuring composite E/I ratios without parsing isolated EPSCs and IPSCs limits direct evaluation of MC-driven excitatory inputs. Furthermore, electrical stimulation in the IML likely recruits local interneuron axons directly alongside MC fibers, complicating the attribution of these responses solely to feed-forward MC circuits.

    (2) The dramatic structural reorganization and IML collapse observed following MC ablation make it difficult to attribute changes in the E/I ratio purely to functional synaptic remodeling rather than physical circuit distortion.

    (3) Layer boundary shifts following MC ablation complicate the interpretation of site-specific spine density (Figure 4); without accounting for IML collapse, classifying spine loss purely by traditional layer boundaries rather than proximal vs. distal dendrites may obscure local structural changes.

    (4) The convulsive dosing protocol used for the seizure threshold test lacks the sensitivity required to reveal subtle changes in excitability.

  4. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This study provides valuable evidence that hilar mossy cells play important roles in maintaining the structural organization of the dentate gyrus and regulating the maturation of adult-born granule cells. The evidence for the structural reorganization and for the accelerated dendritic maturation of adult-born granule cells is convincing: it rests on converging anatomical, viral tract-tracing, retroviral birth-dating, and electrophysiological measurements, with appropriate controls for viral spread, off-target CA3 expression, and axonal degeneration. Support for the study's broader interpretive claim - that the dentate circuit functionally compensates for mossy cell loss - is incomplete. That claim rests on two null results obtained under baseline conditions (home-cage cFos and PTZ seizure metrics) in small cohorts, without behavioral assessment and without a stimulus-driven activity readout, and the manuscript does not engage with published work showing that mossy cells regulate neural stem cell activation and are required for stimulus-evoked neurogenic and behavioral responses.

    Thank you and we agree with all points. We specifically focused on the structural aspects of dentate rearrangement, the impact of mossy cell loss/silencing on dentate neurogenesis, and dentate function at the circuit level. Although our home-cage cFos and PTZ susceptibility assays are limited in terms of their sensitivity, these assays were chosen to address the role of mossy cells in controlling overall dentate activity levels and seizure susceptibility, and our data demonstrate no dramatic changes in overall activity levels or increased/decreased seizure susceptibility.

    Strengths:

    (1) The study is technically rigorous and employs multiple complementary approaches, including selective genetic manipulations, viral tracing, immunohistochemistry, retroviral labeling of adult-born neurons, electrophysiology, and anatomical analyses. The comparison between complete mossy cell ablation and chronic synaptic silencing is particularly powerful, allowing the authors to examine the significant role of mossy cells in structural and functional organization in the dentate gyrus.

    (2) One of the most notable findings is the identification of a previously unrecognized collapse of the inner molecular layer following extensive mossy cell ablation. This observation substantially expands current understanding of dentate gyrus structural plasticity. The demonstration that adult-born granule cells undergo accelerated dendritic maturation after both mossy cell loss and silencing also provides important insight into how mossy cells regulate adult neurogenesis.

    We were also surprised by the inner molecular layer (IML) collapse, as disease models that produce mossy cell loss often involve granule cell axon (mossy fiber) sprouting (and maintained IML thickness) rather than IML collapse. It is unclear whether axon sprouting, reduced degrees of mossy cell loss, or other signaling pathways drive the differences between our selective ablation and translational disease models. We agree that the differential effects of mossy cell ablation and silencing on adult neurogenesis and proximal spine formation highlight the remarkable plasticity in this circuit and provide insights into both the functional and structural circuit roles of mossy cell inputs.

    Weaknesses:

    (1) The functional significance of the observed structural remodeling remains incompletely addressed. Mossy cells have been strongly implicated in pattern separation, spatial information, and emotional behavior, yet no behavioral analyses were conducted. Consequently, it remains unclear whether the dramatic anatomical changes observed following mossy cell ablation translate into meaningful behavioral alterations.

    Our functional assays were primarily focused on the circuit (synaptic) level, with additional assessment of how mossy cell manipulations affected overall dentate activity levels (as reflected by cFos expression). Our limited behavioral analysis focused on seizures, based on prior foundational work on the roles of mossy cells in seizures/epilepsy. We tested the hypothesis that seizure susceptibility might be markedly changed in the near absence of mossy cells, using a “threshold” dose of PTZ that is just above that required to produce seizures, and which can produce dramatically enhanced seizures in hyperexcitable mice. Alternative seizure assays (dose-response curves, continuous monitoring, different seizure-inducing protocols), measurements of granule cell activity in response to environmental contingencies, and the assessments of the response of the dentate stem cell pool to neurogenesis-enhancing stimuli might absolutely produce further insights into how functional mossy cell inputs control stimulus-related dentate activation and/or neurogenesis. Our resubmitted manuscript will clarify that the preserved basal level of dentate gyrus activity after mossy cell loss does not preclude altered activity-dependent activation in other settings or behavioral/learning changes. This could be uncovered with additional behavioral testing or seizure modeling, and is something that we expect to address in future studies.

    (2) The conclusion that the dentate gyrus exhibits remarkable homeostatic compensation is reasonable but remains indirect. Although cFos expression and PTZ-induced seizure susceptibility are unchanged despite altered E:I balance, the mechanisms responsible for maintaining network stability are not investigated. Additional analyses of inhibitory circuit remodeling or compensatory synaptic adaptations would strengthen this conclusion.

    We believe that there are many potential mechanisms that could explain how the nearly complete loss of a major population of dentate neurons is not accompanied by dramatic changes in overall activity levels. Although a fully comprehensive functional assessment of dentate circuit elements is prohibitive, we will undertake what we believe to be the highest-yield analyses in this regard. We propose to stain tissue for inhibitory circuit markers such as VGAT and PV, to determine whether mossy cell loss alters the density or localization of inhibitory synapses as well as circuit elements involved in feed-forward inhibition. We also plan to perform additional electrophysiological experiments to directly assay whether changes in feed-forward inhibition, overall synaptic inhibition (sIPSCs) and/or tonic inhibition might accompany functional mossy cell loss. We will incorporate the outcomes from these additional assays into a revised manuscript. This will shed light on whether inhibitory circuit remodeling also contributes to compensation after mossy cell loss, and hopefully provide additional insights relevant to translational disease models that involve mossy cell loss.

    Reviewer #2 (Public review):

    Summary:

    The authors examine how hilar mossy cells (MCs) influence adult-born dentate granule cell (abDGC) maturation and dentate gyrus (DG) structural integrity. Using both MC ablation and chronic functional silencing, they find that lacking MC inputs accelerates early abDGC maturation without altering mature cellular or intrinsic properties. MC silencing specifically decreased inner molecular layer (IML) spine density, whereas MC ablation led to IML collapse and an increased E/I ratio. However, neither intervention altered overall network excitability (measured via c-Fos and seizure induction) or seizure thresholds. These results advance our understanding of DG circuit plasticity during neurodegeneration.

    Strengths:

    (1) The side-by-side comparison of ablation vs. silencing provides a clear distinction between structural synapse loss and functional inactivation.

    (2) The multi-level analysis spanning structural anatomy, single-cell physiology, and network-level assays yields a rich, comprehensive dataset.

    Thank you for these positive assessments of our study.

    Weaknesses:

    (1) Measuring composite E/I ratios without parsing isolated EPSCs and IPSCs limits direct evaluation of MC-driven excitatory inputs. Furthermore, electrical stimulation in the IML likely recruits local interneuron axons directly alongside MC fibers, complicating the attribution of these responses solely to feed-forward MC circuits.

    We fully expect electrical stimulation of the proximal molecular layer to directly recruit local interneuron axons in addition to feed-forward inhibition. Thus, our experimental design did not distinguish between directly stimulated and feed-forward inhibitory circuits, and we were only able to conclude that mossy cell loss caused circuit rearrangement without clearly attributing the differences specifically to feed-forward mechanisms. To provide additional insights into the underlying changes, we plan to examine both inhibitory circuit structure (using immunohistochemistry) and function using assays designed to distinguish between directly stimulated vs. feed-forward inhibitory mechanisms, which will be incorporated into the revised manuscript.

    (2) The dramatic structural reorganization and IML collapse observed following MC ablation make it difficult to attribute changes in the E/I ratio purely to functional synaptic remodeling rather than physical circuit distortion.

    We actually consider physical circuit remodeling after MC ablation to be the primary explanation for the E/I ratio changes, in that the proximal translocation of MEC synapses following mossy cell ablation allows them to be electrically stimulated in the proximal molecular layer. Thus, the altered E/I ratio of proximal synapses after MC ablation largely represents the fact that we are stimulating proximal MEC inputs rather than mossy cell inputs (which are now absent). Our MML terminal stain (VGlut2) and MEC viral labeling support this interpretation, which we will clarify in the results and discussion of this data.

    (3) Layer boundary shifts following MC ablation complicate the interpretation of site-specific spine density (Figure 4); without accounting for IML collapse, classifying spine loss purely by traditional layer boundaries rather than proximal vs. distal dendrites may obscure local structural changes.

    We initially kept the classic nomenclature (IML vs OML) to avoid confusion for readers, and defined “IML” vs “OML” spines based on proximity to the inner and outer edges of the molecular layer (the innermost and outermost 40 µm; see Methods). Thus, in the setting of IML collapse after mossy cell ablation, these spines almost certainly occurred in regions innervated by the MEC (formerly “MML”). To avoid obscuring this aspect of the data, we will clarify this in the Results and Figure 4, making the proximal vs. distal designations clear.

    (4) The convulsive dosing protocol used for the seizure threshold test lacks the sensitivity required to reveal subtle changes in excitability.

    Our PTZ dose (40 mg/kg i.p.) is just above a dose (30 mg/kg i.p.) that almost never causes seizures in healthy mice in our hands, making it potentially able to detect seizure resistance. This 40 mg/kg dose causes short, limited seizures with a relatively consistent latency, and in other (unrelated) experiments, mice with genetic hyperexcitability have dramatically increased seizure duration and accelerated seizure onset (and sometimes mortality) at this dose, indicating that it is sensitive to at least some forms of increased seizure susceptibility. That stated, we agree that this single-dose PTZ protocol could miss subtle changes in dentate excitability or seizure susceptibility. These could be unmasked by a more detailed dose-response analysis or by other induced seizure assays; we will clarify this limitation in our manuscript.

  5. Version published to 10.64898/2026.07.15.738260 on bioRxiv