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, 28 (5), 1625-1644

Mild Traumatic Brain Injury Induces Structural and Functional Disconnection of Local Neocortical Inhibitory Networks via Parvalbumin Interneuron Diffuse Axonal Injury

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Mild Traumatic Brain Injury Induces Structural and Functional Disconnection of Local Neocortical Inhibitory Networks via Parvalbumin Interneuron Diffuse Axonal Injury

Michal Vascak et al. Cereb Cortex.

Abstract

Diffuse axonal injury (DAI) plays a major role in cortical network dysfunction posited to cause excitatory/inhibitory imbalance after mild traumatic brain injury (mTBI). Current thought holds that white matter (WM) is uniquely vulnerable to DAI. However, clinically diagnosed mTBI is not always associated with WM DAI. This suggests an undetected neocortical pathophysiology, implicating GABAergic interneurons. To evaluate this possibility, we used mild central fluid percussion injury to generate DAI in mice with Cre-driven tdTomato labeling of parvalbumin (PV) interneurons. We followed tdTomato+ profiles using confocal and electron microscopy, together with patch-clamp analysis to probe for DAI-mediated neocortical GABAergic interneuron disruption. Within 3 h post-mTBI tdTomato+ perisomatic axonal injury (PSAI) was found across somatosensory layers 2-6. The DAI marker amyloid precursor protein colocalized with GAD67 immunoreactivity within tdTomato+ PSAI, representing the majority of GABAergic interneuron DAI. At 24 h post-mTBI, we used phospho-c-Jun, a surrogate DAI marker, for retrograde assessments of sustaining somas. Via this approach, we estimated DAI occurs in ~9% of total tdTomato+ interneurons, representing ~14% of pan-neuronal DAI. Patch-clamp recordings of tdTomato+ interneurons revealed decreased inhibitory transmission. Overall, these data show that PV interneuron DAI is a consistent and significant feature of experimental mTBI with important implications for cortical network dysfunction.

Figures

Figure 1.
Figure 1.
Characterization of PV-Cre;Ai9 mouse and RFP antibody for enhanced visualization of tdTomato expression. AH, Representative maximal intensity projections from sham-injured PV-Cre;Ai9 mice that express tdTomato (tdTom) in PV+ interneurons. All images are oriented perpendicular to the SCWM (E). (A) Overview image of a coronal section showing broad overlap between native tdTomato expression and endogenous PV immunoreactivity with S1 delineated. BG, Higher-magnification images showing colocalization of tdTomato with PV (BD) and immunoreaction using antibodies against RFP (that recognize tdTomato; EG) within layer 5 neocortex. (BD, H) Typical profiles of tdTomato+/PV+ perisomatic innervation of a layer 5 pyramidal neuron (asterisks). (H) Zoomed image of box in D, showing an ascending axonal projection (arrowheads) juxtaposed by tdTomato+ presynaptic terminals with characteristic “basket” morphology. Note the fine diameter of the axon (~0.5 µm), which could be followed for ~30 µm distal to the soma of origin. EG, Colocalization of tdTomato with RFP immunoreactivity, which was visualized using secondary antibodies conjugated to Alexa Fluor 488 (F). Parallel anti-RFP immunoreaction in a C57BL/6 J (WT) tissue section (I), the background strain of PV-Cre and Ai9 mice, did not reveal evidence of non-specific binding. (J) Summary data (mean with 95% CI error bars and cell counts) from quantitative colocalization analysis of expression specificity between Cre-driven tdTomato and endogenous PV (n = 6 FOV from 3 animals) and native tdTomato signal with RFP immunoreactivity (n = 4 FOV from 2 animals) within neocortical layers 2–6.
Figure 2.
Figure 2.
Repertoire of tdTomato+ neuropathology following mTBI. Representative images of tdTomato+ morphological changes at 3 h (A–G) and 24 h (HK) post-mTBI. (AE) Within 3 h post-mTBI, tdTomato+ axonal swellings, occurring near the soma of origin, were observed across S1 layers 2–6. (F) A representative tdTomato+ interneuron showing a perisomatic axonal swelling (arrow, oriented toward pia) and the distal disconnected axonal segment with lobular/varicose morphology (arrowheads) that can be traced to the initial branch point (outline). While the majority of tdTomato+ perisomatic axonal swellings were oriented toward the pia (AF), occasionally profiles with descending and/or lateral trajectories were found (G). (HK) At 24 h post-mTBI, perisomatic axonal swellings were difficult to identify; however, widespread tdTomato+ axonal debris was readily observable. (IK) Zoomed images corresponding to insets in H, reflecting progressive tdTomato+ anterograde changes (arrowheads) likely associated with distal disconnected axonal arbor (F) degeneration. (K) Layer 5 pyramidal neuron silhouettes (asterisks) showing evidence of tdTomato+ axonal terminal degeneration, ranging from punctate (left arrowhead) to varicose (right arrowheads) profiles.
Figure 3.
Figure 3.
Ultrastructural analysis of tdTomato+ PSAI identified via confocal microscopy. The same tdTomato+ profile was followed from the light (A,B) to electron (CE) microscopy level at 3 h post-mTBI. (A, B) Confocal images capturing native tdTomato signal. (A) Overview image of S1BF with inset in layer 5/6 corresponding to tdTomato+ neuron in B, showing PSAI (arrow, oriented toward pia) and related disconnected distal segment (arrowhead). Employing the same RFP antibodies used for photostabilizing allowed use to follow the same tdTomato+ neuron from confocal (B) to electron (CE) microscopy level. (C) The tdTomato+ neuron was identified based on morphology, including the perisomatic axonal swelling (arrow) and disconnected distal axonal segment (arrowhead), as well as other fiduciary markers including an adjacent tdTomato+ neuron (left), nearby capillaries (c), and a non-tdTomato expressing neuron adjacent to the tdTomato+ disconnected distal axonal segment (outline, upper right). (DE) Ultrastructural analysis of the tdTomato+ distal disconnected segment (D) showed disorganized cytoskeleton consistent with the onset of Wallerian degeneration and the perisomatic swelling (E) laden with organelles and vesicles, indicating a focal site of impaired axonal transport.
Figure 4.
Figure 4.
GABAergic markers accumulate in APP+/tdTomato+ perisomatic axonal swellings. Representative images at 3 h post-mTBI showing GABAergic markers (A,E,I) with respect to APP immunoreactivity (B, F, J), which accumulates at focal sites of impaired axonal transport (arrows). Colocalization of tdTomato+ PSAI with PV (AD), VGAT (EH), and GAD67 (IL) immunoreactivity confirms GABAergic interneuron axonal injury. (A) Normal uninjured intact (int) axonal profile (wide arrowheads) juxtaposed by PV+ interneuron PSAI (arrow). (B) APP is not detected within the intact axonal profile, while robust APP immunoreactivity colocalizes with tdTomato+/PV+ interneuron PSAI (C, D). Within sites of tdTomato+ PSAI (C,G,K), the immunoreactive profiles of VGAT (E) and GAD67 (I) are similar to APP+ axonal swellings (F and J, respectively). Qualitatively, the GAD67+ axonal swelling profile (I, L) has a better signal-to-noise than VGAT (E, H). Note non-GABAergic APP+ axonal swellings have opposite trajectories.
Figure 5.
Figure 5.
Quantitative analysis of GABAergic DAI at 3 h post-mTBI. (A) Representative low-magnification (×10 objective) maximum intensity projection of S1 layers 2–6, with corresponding XZ (bottom) and YZ (right) planes through the center of a GAD67+/APP+ axonal swelling, which can be readily identified visually (arrows) and also using automated image analysis (cyan outlines). Note the scattered distribution of APP+ axonal swellings across the XY plane of S1 layers 2–6 and the sparse distribution of the APP+/GAD67+ subpopulation. XZ and YZ planes highlight GAD67 colocalization with APP is not an artifact of flattening z-stacks. (B) Representative higher-magnification (×20 objective) single optical slices through the center of visually identified GAD67+/APP+ axonal swellings. Top and middle panels show tdTomato+ and tdTomato– PSAI (yellow arrow), respectively. Bottom panel shows an isolated/remote tdTomato– axonal swelling (yellow arrowheads). (C,D) Summary of quantitative data reported as mean percentage with 95% CI error bars. (C) GAD67+ accumulations are a positive predictor of APP+ axonal swellings. The proportion of GAD67+ profiles with areas >10 µm2 that colocalized with APP+ axonal swellings (0.819, 95% CI: 0.746–0.874) was significantly greater (X2 = 426, ***P < 0.0001) than the proportion of GAD67+ profiles with areas <10 µm2 (0.078, 95% CI: 0.061–0.98). (D) The majority of GABAergic DAI is represented by tdTomato+ PSAI. The proportion of tdTomato+ PSAI (0.779, 95% CI: 0.708–836) was significantly greater (X2 = 53.9, ***P < 0.0001) than the proportion of tdTomato– axonal injury (0.040, 95% CI 0.007–0.195). Statistics: Chi-squared test used to determine significance. Counts obtained from n = 15 sections from 5 mice.
Figure 6.
Figure 6.
tdTomato+ interneuron PSAI is associated with retrograde nuclear expression of p-c-Jun within 3 h post-mTBI. (A) Representative images of 2 distinct tdTomato+ somas within a field of p-c-Jun+ nuclei in S1 layer 5. “TOP,” tdTomato+/p-c-Jun+ soma (green arrowhead) contiguous with PSAI (green arrow). “BOTTOM,” tdTomato+/p-c-Jun– soma (yellow arrowhead) with an intact axon (yellow arrows). (B) Summary data reported as mean percentage with 95% CI error bars. The proportion of tdTomato+/p-c-Jun+ somas with perisomatic swellings (PSAI: 0.636, 95% CI: 0.430–80.3) was significantly greater (***P < 0.0001; Fisher's exact test) than the proportion with intact profiles (0.016, 95% CI: 0.008–0.031%). Counts obtained from single sections taken from n = 5 mice.
Figure 7.
Figure 7.
Anterograde axonal degeneration and retrograde p-c-Jun nuclear expression progresses rapidly following mTBI. (AC) Representative maximum intensity projections showing mTBI-induced changes in tdTomato+ profiles and p-c-Jun immunoreactivity across S1 layers 2–6. (A) Sham-injury did not induce any tdTomato+ neuropathology and p-c-Jun immunoreactivity was virtually absent. (B) Within 3 h post-mTBI tdTomato+ axonal debris (black/white arrowheads) is seen near a tdTomato+/p-c-Jun+ neuronal soma (green arrowhead). (C) At 24 h post-mTBI the density of tdTomato+ axonal debris and p-c-Jun+ nuclei increase drastically. (DG) Quantitative data summarized using median, IQR, and min/max values. The density of tdTomato+ axonal debris (D), tdTomato+/p-c-Jun+ somas (E), and total p-c-Jun+ nuclei (F) were significantly different across experimental groups (X22 = 8.61, *P = 0.0135; X22 = 10.5, **P = 0.0053; and X22 = 10.7, **P = 0.0048, respectively). Post hoc analyses revealed significant increases in at 24 h post-mTBI compared with sham (D, tdTomato+ axonal debris: *P = 0.0381; E, tdTomato+/p-c-Jun+ somas: **P = 0.0034; F, total p-c-Jun+ neurons: **P = 0.0031). (G) tdTomato+ soma density did not change overtime (P = 0.4401). H,I, Overall burden of injury, estimated as percentage of tdTomato+/p-c-Jun+ somas, with respect to total tdTomato+ somas (H) significantly increased overtime (X2 = 5.33, *P = 0.0209), while there was no change with respect to total p-c-Jun+ nuclei (P = 0.3865). Statistics: Significant differences were determined using Kruskal–Wallis test followed by post hoc pair-wise comparisons using Dunn's method with control (sham) for joint ranking (DG) and Wilcoxon test (H, I). Each data point (statistical unit) corresponds to a single section per animal (n). The total counts per group (N) are denoted underneath each graph.
Figure 8.
Figure 8.
sIPSC is reduced in tdTomato+ fast-spiking interneurons 24 h post-mTBI. (A,B) Representative traces of whole-cell patch-clamp recordings made in S1BF layer 5 showing fast-spiking action potentials characteristic of PV+ interneurons (A), and sIPSCs from sham-injury (black) and mTBI (gold) mice (B). (C) A representative biocytin-filled tdTomato+ interneuron from a mTBI animal revealing an ascending intact axon that can be continuously traced distally through and past the initial branch point (arrowhead). (D) Post hoc confocal image showing a biocytin-filled tdTomato+ interneuron (top left, cyan arrowhead) within a field of diffuse p-c-Jun immunoreactivity (green). Note the tdTomato+/p-c-Jun+ PSAI neuron (bottom right, magenta arrowhead). (EG) sIPSC recordings summarized using median, IQR, and min/max values. At 24 h post-mTBI, there was a significant decrease in frequency (E, X2 = 17.2, ***P < 0.0001), amplitude (F, X2 = 8.56, **P = 0.0034), and event area/charge transfer (G, X2 = 8.17, **P = 0.0043; G) of sIPSC compared with shams. Statistics: Individual tdTomato+ interneurons were considered as the statistical unit (Sham: n = 18 from 9 mice; mTBI: n = 28 from 12 mice). Significant differences determined using Wilcoxon test.

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