Altered firing output of VIP interneurons and early dysfunctions in CA1 hippocampal circuits in the 3xTg mouse model of Alzheimer’s disease

Reviewer #1 (Public Review):

Summary:

The work in the manuscript titled " Altered firing output of VIP interneurons and early dysfunctions in CA1 hippocampal circuits in the 3xTg mouse model of Alzheimer's disease" utilized patch-clamp techniques to explore the electrophysiological characteristics of VIP interneurons in the early stages of AD using the 3xTg mouse model. The study revealed that VIP interneurons exhibited prolonged action potentials and reduced firing rates. These changes could not be attributed to modifications in input signals or morphological transformations. The authors attributed aberrant VIP activity to the accumulation of beta-amyloid in those interneurons.

The decreased frequency of VIP inhibitory events was associated with no observed changes in excitatory drive to these interneurons. Consequently, heightened activity in the general population of CA1 interneurons was observed during a decision-making task and an object recognition test. In light of these findings, the authors concluded that the altered firing patterns of VIP interneurons may initiate early-stage dysfunction in hippocampal CA1 circuits, potentially influencing the progression of AD pathology.

Strengths:

Overall the work is novel and moves the field of Alzheimer's disease forward in a significant way. The manuscript reports a novel concept of aberrant activity in VIP interneurons during the early stages of AD thus contributing to dysfunctions of the CA1 microcircuit. This results in the enhancement of the inhibitory tone on the primary cells of CA1. Thus, the disinhibition by VIP interneurons of Principal Cells is dampened. The manuscript was skillfully composed, and the study was of strong scientific rigor featuring well-designed experiments. Necessary controls were present. Both sexes were included.

Limitations:

(1) The authors attributed aberrant circuit activity to the accumulation of "Abeta intracellularly" inside IS-3 cells. That is problematic. 6E10 antibody recognizes amyloid plaques in addition to Amyloid Precursor Protein (APP) as well as the C99 fragment. There are no plaques at the ages 3xTg mice were examined. Thus, the staining shown in Figure 1a is of APP/C99 inside neurons, not abeta accumulations in neurons. At the ages of 3-6 months, 3xTg starts producing abeta oligomers and potentially tau oligomers as well (Takeda et al., 2013 PMID: 23640054; Takeda et al., 2015 PMID: 26458742 and others). Emerging literature suggests that abeta and tau oligomers disrupt circuit function. Thus, a more likely explanation of abeta and tau oligomers disrupting the activity of VIP neurons is plausible.

(2) Authors suggest that their animals do not exhibit loss of synaptic connections and show Figure 3d in support of that suggestion. However, imaging with confocal microscopy of 70-micron thick sections would not allow the resolution of pre- and post-synaptic terminals. More sensitive measures such as electron microscopy or array tomography are the appropriate techniques to pursue. It is important for the authors to either remove that data from the manuscript or address the limitations of their technique in the discussion section. There is a possibility of loss of synaptic connections in their mouse model at the ages examined.

Reviewer #2 (Public Review):

Summary:

The submitted manuscript by Michaud and Francavilla et al., is a very interesting study describing early disruptions in the disinhibitory modulation exerted by VIP+ interneurons in CA1, in a triple transgenic model of Alzheimer's disease. They provide a comprehensive analysis at the cellular, synaptic, network, and behavioral level on how these changes correlate and might be related to behavioral impairments during these early stages of the disease.

Main findings:

- 3xTg mice show early Aß accumulation in VIP-positive interneurons.

- 3xTg mice show deficits in a spatially modified version of the novel object recognition test.

- 3xTg mice VIP cells present slower action potentials and diminished firing frequency upon current injection.

- 3xTg mice show diminished spontaneous IPSC frequency with slower kinetics in Oriens / Alveus interneurons.

- 3xTg mice show increased O/A interneuron activity during specific behavioral conditions.

- 3xTg mice show decreased pyramidal cell activity during specific behavioral conditions.

Strengths:

This study is very important for understanding the pathophysiology of Alzheimer´s disease and the crucial role of interneurons in the hippocampus in healthy and pathological conditions.

Weaknesses:

Although results nicely suggest that deficits in VIP physiological properties are related to the differences in network activity, there is no demonstration of causality.

Author Response

Public Reviews:

Reviewer #1

Strengths:

Overall, the work is novel and moves the field of Alzheimer's disease forward in a significant way. The manuscript reports a novel concept of aberrant activity in VIP interneurons during the early stages of AD thus contributing to dysfunctions of the CA1 microcircuit. This results in the enhancement of the inhibitory tone on the primary cells of CA1. Thus, the disinhibition by VIP interneurons of Principal Cells is dampened. The manuscript was skillfully composed, and the study was of strong scientific rigor featuring well-designed experiments. Necessary controls were present. Both sexes were included.

We express our gratitude to the reviewer for their keen appreciation of our efforts and their enthusiasm for the outcomes of this research.

Limitations:

(1) The authors attributed aberrant circuit activity to the accumulation of "Abeta intracellularly" inside IS-3 cells. That is problematic. 6E10 antibody recognizes amyloid plaques in addition to Amyloid Precursor Protein (APP) as well as the C99 fragment. There are no plaques at the ages 3xTg mice were examined. Thus, the staining shown in Figure 1a is of APP/C99 inside neurons, not abeta accumulations in neurons. At the ages of 3-6 months, 3xTg starts producing abeta oligomers and potentially tau oligomers as well (Takeda et al., 2013 PMID: 23640054; Takeda et al., 2015 PMID: 26458742 and others). Emerging literature suggests that abeta and tau oligomers disrupt circuit function. Thus, a more likely explanation of abeta and tau oligomers disrupting the activity of VIP neurons is plausible.

The Reviewer correctly points out that 3xTg-AD mice typically do not exhibit plaques before 6 months of age, with limited amounts even up to 12 months, particularly in the hippocampus. To the best of our knowledge, the 6E10 antibody binds to an epitope in APP (682-687) that is also present in the Abeta (3-8) peptide. Consequently, 6E10 detects full-length APP, α-APP (soluble alpha-secretase-cleaved APP), and Abeta (LaFerla et al., 2007). Nonetheless, we concur with the Reviewer's observation that the detected signal includes Abeta oligomers and the C99 fragment, which is currently considered an early marker of AD pathology (Takasugi et al., 2023; Tanuma et al., 2023). Studies have demonstrated intracellular accumulation of C99 in 3-month-old 3xTg mice (Lauritzen et al., 2012), and its binding to the Kv7 potassium channel family, which results in inhibiting their activity (Manville and Abbott, 2021). If a similar mechanism operates in IS-3 cells, it could explain the changes in their firing properties observed in our study. Consequently, we will revise the manuscript to include this crucial information in both the Results and Discussion sections.

(2) Authors suggest that their animals do not exhibit loss of synaptic connections and show Figure 3d in support of that suggestion. However, imaging with confocal microscopy of 70micron thick sections would not allow the resolution of pre- and post-synaptic terminals. More sensitive measures such as electron microscopy or array tomography are the appropriate techniques to pursue. It is important for the authors to either remove that data from the manuscript or address the limitations of their technique in the discussion section. There is a possibility of loss of synaptic connections in their mouse model at the ages examined.

We appreciate the Reviewer’s perspective on the techniques used for imaging synaptic connections. While we acknowledge the limitations of confocal microscopy for resolving pre- and post-synaptic structures in thick sections, we respectfully disagree regarding the exclusive suitability of electron microscopy (EM). Our approach involved confocal 3D image acquisition using a 63x objective at 0.2 um lateral resolution and 0.25 Z-step, providing valuable quantitative insights into synaptic bouton density. Despite the challenges posed by thick sections, this method together with automatic analysis allows for careful quantification. Although EM offers unparalleled resolution, it presents challenges in quantification. We will ensure to include the important details regarding image acquisition and analysis in the revised manuscript.

Reviewer #2 (Public Review):

Summary:

The submitted manuscript by Michaud and Francavilla et al., is a very interesting study describing early disruptions in the disinhibitory modulation exerted by VIP+ interneurons in CA1, in a triple transgenic model of Alzheimer's disease. They provide a comprehensive analysis at the cellular, synaptic, network, and behavioral level on how these changes correlate and might be related to behavioral impairments during these early stages of the disease.

Main findings:

3xTg mice show early Aß accumulation in VIP-positive interneurons.

3xTg mice show deficits in a spatially modified version of the novel object recognition test. - 3xTg mice VIP cells present slower action potentials and diminished firing frequency upon current injection.

3xTg mice show diminished spontaneous IPSC frequency with slower kinetics in Oriens / Alveus interneurons.

3xTg mice show increased O/A interneuron activity during specific behavioral conditions.

3xTg mice show decreased pyramidal cell activity during specific behavioral conditions.

Strengths:

This study is very important for understanding the pathophysiology of Alzheimer´s disease and the crucial role of interneurons in the hippocampus in healthy and pathological conditions.

We are thankful to the reviewer for their insightful recognition of our efforts and their enthusiasm for the results of this research.

Weaknesses:

Although results nicely suggest that deficits in VIP physiological properties are related to the differences in network activity, there is no demonstration of causality.

RE: We completely agree with the reviewer's observation regarding the lack of demonstration of causality in our results. Investigating causality in the relationship between deficits in VIP physiological properties and differences in network activity is indeed a crucial aspect of this project. However, achieving this goal will require a significant amount of time and dedicated manipulations in a new mouse model (VIP-Cre-3xTg). We appreciate the importance of this line of investigation and consider it as a priority for our future research endeavors.