Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs

Abstract

In the rTg4510 mouse model, expression of the mutant human tau variant P301L leads to development of neurofibrillary tangles (NFTs), neuronal death, and memory impairment, reminiscent of the pathology observed in human tauopathies. In the present study, we examined the effects of mutant tau expression on the electrophysiology and morphology of individual neurons using whole-cell patch-clamp recordings and biocytin filling of pyramidal cells in cortical slices prepared from rTg4510 (TG) and wild-type (WT) littermate mice. Among the TG cells, 42% contained a clear Thioflavin-S positive inclusion in the soma and were categorized as NFT positive (NFT+), while 58% had no discernable inclusion and were categorized as NFT negative (NFT-). The resting membrane potential (V(r)) was significantly depolarized (+8 mV) in TG cells, and as a consequence, evoked repetitive action potential (AP) firing rates were also significantly increased. Further, single APs were significantly shorter in duration in TG cells and the depolarizing voltage deflection or "sag" evoked by hyperpolarization was significantly greater in amplitude. In addition to these functional electrophysiological changes, TG cells exhibited significant morphological alterations, including loss or significant atrophy of the apical tuft, reduced dendritic complexity and length, and reduced spine density. Importantly, NFT- and NFT+ TG cells were indistinguishable with regard to both morphological and electrophysiological properties. Our observations show that expression of mutated tau results in significant structural and functional changes in neurons, but that these changes occur independent of mature NFT formation.

Figure 1. Confocal images of representative streptavidin-Alexa 546-labeled layer 3 pyramidal cells from WT (left) and TG (center and right) mice

Top row: 10× confocal images of the cells with Thioflavin-S staining showing a high density of NFTs in the cortex of TG mice. Middle row: Somata of cells shown in top row at 40×. Imaging the Thioflavin-S staining in the soma allows for the classification of the TG cells into one of 2 groups based on the presence or absence of a NFT, respectively named NFT− cells (center column) and NFT+ cells (right column). Bottom row: 3-D reconstruction of cells imaged at high resolution (xy and xz projections on the left and right side of each panel, respectively). Scale bars: top row = 40 µm; middle row = 5 µm.

Figure 2. Membrane voltage responses to injected current from representative WT, NFT− and NFT+ cells

A) Top: Cells from which recordings shown in panels A1 and 3 were obtained. Panels 1–3: Membrane voltage responses evoked by 2 s current steps in cells with different V_r (1), and in cells matched by V_r (2), or by a 10 s depolarizing current ramp from 0 to 200 pA (3). WT cells fired at a lower frequency than either NFT− or NFT+ cells while the latter two groups did not differ (1). Arrowheads indicate a pronounced “sag” in TG (but not WT) cells. None of the groups differed significantly in firing frequency when matched by Vr (2). Scale bars: 1 and 2 = 20 mV, 500 ms; 3 = 20 mV, 2 s. B) Mean frequency-current plots for all WT and TG cells (left, p < 0.05 at each current step) and for subgroups of WT and TG cells that are matched by Vr (right).

Figure 3. Reconstructions of representative cells from WT and TG mice showing morphological diversity

Fully reconstructed frontal cortical pyramidal cells of WT (top), NFT− (middle) and NFT+ (bottom). Dashed lines indicate the pial surface. Arrowheads indicate severe apical tuft atrophy. Scale bar = 200 µm.

Figure 4. Significant alterations in the length and complexity of apical and basal dendritic arbors of TG cells

Proximal, middle and distal thirds of the dendritic arbors were examined. Complexity, as reflected by the number of bifurcation nodes (top), and total length (bottom) are plotted for the apical (left) and basal (right) dendritic arbors of WT and TG cells. Note the overall decrease in complexity and length from the proximal to the distal third of the basal dendritic arbor. In the apical arbors, a decrease in complexity was observed in the middle and distal thirds, as well as a dramatic decrease (−66%) in length in the distal third. #p = 0.062; *p < 0.05; ***p < 0.002.

Figure 5. Significant reduction in dendritic spine density in TG cells

Left panel, top: confocal images of apical dendritic tufts from the cells shown in Figure 1. bottom: higher magnification view of the boxed areas shown above. Right panel, significant reduction in spine density were observed in both the apical (top) and basal (bottom) dendritic arbors in the TG cells as compared to WT. There was no difference between the NFT− and NFT+ cells within the TG group. Scale bars: top left = 10 µm; bottom left = 5 µm. **p ≤ 0.02.