VIP/CR IS-3 cells survive but accumulate intracellular Aβ in VIP-Tg mice.

a. Confocal image of the CA1 hippocampal area showing intracellular accumulation of APP/Aβ (6E10) in cell bodies of the pyramidal layer in a 3-month-old VIP-Tg mouse (left, scale bar: 100 μm; inset shows the area indicated with a white box) and summary box plot showing the cell number expressing the Aβ intracellularly in 3-month vs. 6-month-old mice (right; n = 8 slices from 3 animals per group). b. Summary bar graphs showing the average number of VIP+ cells expressing Aβ intracellularly per slice (left), and representative confocal images of the VIP-IN labelled with eGFP and showing intracellular accumulation of Aβ in a 6-month-old VIP-Tg mouse. Scale bar: 10 μm c. Representative confocal images of the CA1 hippocampal area in VIP-nonTg (top) and VIP-Tg (bottom) mice showing eGFP expression in VIP INs (left), CR expression (middle) and an overlay of both markers (right). Scale bar: 100 μm. d. Summary plots showing no changes in the number of VIP-INs (left), CR-INs (middle) and VIP/CR co-expressing IS-3 cells (right) in VIP-Tg mice.

I-S3 cells exhibit wider spikes and lower firing rate in VIP-Tg mice.

a. Representative voltage responses (top, middle) showing the firing pattern of IS-3 cells in VIP-nonTg (left, black) and VIP-Tg (right, blue) mice in response to depolarizing and hyperpolarizing current injections (bottom). b. Representative voltage traces in VIP-nonTg (black) and VIP-Tg (blue) mice superimposed for the first (top) and last (bottom) AP showing the longer AP duration in VIP-Tg mice. c. Summary plots showing the amplitude (left), half-width (middle left), depolarization rate (middle), repolarization rate (middle right) and area (right) of the first AP in IS-3 cells. *p<0.05. d. Summary plots showing changes in the AP amplitude (left), half-width (middle) and area (right) during the train of APs in IS-3 cells. ****p<0.0001. e. Summary plots showing no changes in the AP depolarization rate (left) but slower repolarization rate (right) during the train of APs in IS-3 cells. ****p<0.0001. f. Summary plot showing changes in the number of AP during the train of APs in relation to the injected current in IS-3 cells. The shaded areas show the SEM. *p<0.05.

Altered inhibition of CA1 interneurons in Tg mice.

a. Representative traces for sIPSCs in O/A INs from nonTg (top, black) and Tg (bottom, blue) mice. b. Summary plots showing the sIPSCs amplitude (left) and frequency (right) in nonTg (black) and Tg (blue) groups. Insets show data for animal-wise comparison. **p<0.01. c. Summary plots showing the sIPSC rise time left), decay time constant (middle), and charge transfer (right) in O/A INs of nonTg and Tg animals. *p<0.05, **p<0.01, ***p<0.001. d. Representative confocal images showing immunoreactivity for VGAT (left) and VGAT+CR (right) in the CA1 O/A of the nonTg (top, scale bar: 20 μm) and Tg (bottom, scale bar: 5 μm) mice. e. Summary plots showing no changes in the density of VGAT-expressing axonal boutons in the O/A.

Enhanced activation of CA1 interneurons during decision making and object exploration.

a. Schematic of cannula implantation and the experimental setup for wireless fiberphotometry calcium imaging in freely behaving mice (top). b. Representative confocal image of the CA1 hippocampal area showing the cannula track and GCaMPF6f expression in the CA1 parvalbumin-expressing (PV-INs) or somatostatin-expressing (SOM-INs), with a bar graph summarizing the fraction of PV-INs and SOM-INs contributing to the photometry signal. White arrowheads depict examples of cell bodies expressing GCaMPF6f. Scale bars: top left, 100 μm; bottom left, 50 μm. c. Summary plots showing the mean speed, mobility rate, total distance traveled and the number of rearing episodes during the open field exploration in nonTg (black) and Tg (blue) animals. d. Representative individual (top) and average (bottom) traces obtained from oriens/alveus interneurons (O/A INs) in nonTg (black) and Tg (blue) mice during walking in the open field arena. e. Summary plots showing the average and peak Z-scored values of calcium transients in O/A INs in nonTg (black) and Tg (blue) mice during walking. f. Schematic of the behavioral paradigm in T-maze: correct-choice animals visit an alternative to the previously visited arm during the 2nd trial. The decision zone (DZ) is illustrated with the shaded area. g. Bar graph showing the percentage of alternation in T-maze test in nonTg and Tg animals. h. Summary plots showing the average and peak Z-scored values of calcium transients in O/A INs of nonTg (black) and Tg (blue) mice during exploration in the stem vs. DZ. Only mice making the correct choice (those that visited alternative to the previously visited arm) were included in this analysis. *p<0.05. i. Schematic of the arena used to examine the object-related modulation of neuronal activity, with summary plot showing the number of entries inside the object exploration zone, the average duration of object exploration episodes, and the mean Z-scored values of calcium transients recorded in interneurons during object exploration periods. *p<0.05.

Altered activation of CA1 pyramidal cells during cognitive tasks.

a. Representative confocal image of the CA1 hippocampal area showing the cannula track and GCaMPF6f expression in the CA1 PCs. Scale bar: 100 μm. b. Representative calcium traces and summary plots showing the peak amplitude (left) and frequency (right) of calcium transients recorded in the CA1 PCs in nonTg (black) and Tg (blue) mice when animals were in their homecage. Insets show summary boxplots for animal-wise mean comparison. c. Representative average traces obtained from PCs in nonTg (black) and Tg (blue) mice during walking. d. Summary plots showing the peak and area under the curve (AUC) for Z-scored values of calcium transients in PCs of nonTg (black) and Tg (blue) mice during walking. e. Representative average traces obtained from PCs in nonTg (black) and Tg (blue) mice during exploration in the D-zone of T-maze. f. Summary plot demonstrating the area under the curve (AUC) for Z-scored values of calcium transients in PCs of nonTg (black) and Tg (blue) mice in the D-Zone. Only mice making the correct choice (those that visited alternative to the previously visited arm) were included in this analysis. *p<0.05. g. Representative average traces obtained from PCs in nonTg (black) and Tg (blue) mice during object exploration. h. Summary plots showing the area under the curve (left, AUC) and the root mean square (RMS) for Z-scored values of calcium transients in PCs of nonTg (black) and Tg (blue) mice during object exploration. *p<0.05.

Summary of statistical analysis conducted throughout the study.

Notes:

1) The reported n corresponds to the number of animals, cells (underlined) or slices (italic).

2) Animals of both sexes with equal distribution were used throughout the study.

Early deficits in the object-place test of VIP-Tg mice.

a. Schematic for the object-place test (OPT): Day 1 – open field maze (OFM) exploration; Day 2 – Sampling of two different objects; Day 3 – OPT, in which one of the objects has been replaced by a new one and the second was displaced. In this test, mice show preference for a novel object (Francavilla et al., 2020). b. Summary plots showing the recognition index (RI) on day 2 (left) and on day 3 (right) for VIP-nonTg and VIP-Tg mice. ***p<0.001. c. Summary plots showing the total object exploration time on day 3 (left) and the total horizontal activity (right) for VIP-nonTg and VIP-Tg mice. Note a significant decrease of RI in VIP-Tg mice despite unaltered activity and total exploration time.

Unaltered synaptic drive to IS-3 cells in VIP-Tg mice.

a. Representative traces for spontaneous inhibitory (sIPSCs, left) and excitatory (sEPSCs, right) postsynaptic currents in IS-3 cells from VIP-nonTg (top, black) and VIP-Tg (bottom, blue) mice. b. Summary plots showing the cumulative probability for sIPSCs amplitude (left) and frequency (middle left) as well as the sEPSCs amplitude (middle right) and frequency (right) in IS-3 cells from VIP-nonTg (black) and VIP-Tg (blue) groups. c. Summary plots showing the animal-wise data for sIPSCs amplitude (left) and frequency (middle left) as well as the sEPSCs amplitude (middle right) and frequency (right) in IS-3 cells.

Unaltered morphological properties of IS-3 cells in VIP-Tg mice.

a. Anatomical reconstructions of IS-3 cells filled with biocytin from VIP-nonTg (left) and VIP-Tg (right) animals. Scale bar: 50 μm. b-e. Summary plots showing the soma area (b), the total dendritic surface (c), the dendritic length (d), and the number of dendritic branching points (e) of IS-3 cells for VIP-nonTg and VIP-Tg groups. f. Sholl analysis of the dendritic length (left), number of intersections (middle) and number of dendritic nodes (right) in relation to the distance from the soma in IS-3 cells for VIP-nonTg and VIP-Tg groups.

Excitatory drive to O/A INs remains unchanged in Tg mice.

a. Representative traces for sEPSCs in O/A INs from nonTg (top, black) and Tg (bottom, blue) mice. b. Summary distribution plots showing the sEPSCs amplitude (left) and frequency (right). Insets show summary data for animal-wise comparison. c. Summary boxplots showing the rise time (left), decay time constant (middle), and charge transfer (right) for sEPSCs.

Unaltered activity of CA1 interneurons in homecage and open field.

a. Representative calcium traces from CA1 O/A INs in nonTg (black) and Tg (blue) mice recorded when animals were in their homecage. b. Summary plots showing the peak amplitude (left) and frequency (right) of calcium transients in O/A INs in non-Tg (black) and Tg (blue) animals in their homecage. Insets show boxplots for animal-wise mean comparison. c. Representative movement trajectories during OFM for non-Tg (black) and Tg mice (blue). d. Summary plots showing the animal-wise comparison for the mean Z-scored values of calcium transients during periods of locomotion, immobility and rearing in the OFM.