Sustained Hippocampal Synaptic Pathophysiology Following Single and Repeated Closed-Head Concussive Impacts

Abstract

Traumatic brain injury (TBI), and related diseases such as chronic traumatic encephalopathy (CTE) and Alzheimer's (AD), are of increasing concern in part due to enhanced awareness of their long-term neurological effects on memory and behavior. Repeated concussions, vs. single concussions, have been shown to result in worsened and sustained symptoms including impaired cognition and histopathology. To assess and compare the persistent effects of single or repeated concussive impacts on mediators of memory encoding such as synaptic transmission, plasticity, and cellular Ca²⁺ signaling, a closed-head controlled cortical impact (CCI) approach was used which closely replicates the mode of injury in clinical cases. Adult male rats received a sham procedure, a single impact, or three successive impacts at 48-hour intervals. After 30 days, hippocampal slices were prepared for electrophysiological recordings and 2-photon Ca²⁺ imaging, or fixed and immunostained for pathogenic phospho-tau species. In both concussion groups, hippocampal circuits showed hyper-excitable synaptic responsivity upon Schaffer collateral stimulation compared to sham animals, indicating sustained defects in hippocampal circuitry. This was not accompanied by sustained LTP deficits, but resting Ca²⁺ levels and voltage-gated Ca²⁺ signals were elevated in both concussion groups, while ryanodine receptor-evoked Ca²⁺ responses decreased with repeat concussions. Furthermore, pathogenic phospho-tau staining was progressively elevated in both concussion groups, with spreading beyond the hemisphere of injury, consistent with CTE. Thus, single and repeated concussions lead to a persistent upregulation of excitatory hippocampal synapses, possibly through changes in postsynaptic Ca²⁺ signaling/regulation, which may contribute to histopathology and detrimental long-term cognitive symptoms.

Keywords: CTE; Ca2+; LTP; concussion; hippocampus; synaptic transmission; tau.

Figure 1

Single or repeated traumatic brain injury (TBI) results in persistent hyperexcitability at CA3-CA1 synapses. Rats subjected to a single TBI (sTBI), or repeated TBI (rTBI), exhibited persistent upregulation at CA3-CA1 synapses, as revealed using extracellular field recordings (fEPSPs). (A1) fEPSP Input-output curves obtained from CA1 hippocampal field recordings using Schaffer collateral stimulation, 30 days after sham surgery, sTBI or rTBI, demonstrating increasing field potential (fEPSP) slopes resulting from increasing stimulus intensity ranging from 100 μA to 1,000 μA. fEPSPs from both the sTBI and rTBI groups showed a steeper slope at most stimulus intensities compared to the sham treatment group, with no significant difference between the sTBI and rTBI groups. (A2) Representative input-output fEPSP traces taken from sham, sTBI, and rTBI rats. (B1) sTBI and rTBI did not affect the paired-pulse ratio of fEPSPs, compared to sham, indicating that there was no significant difference in the presynaptic release probability of glutamate between groups. (B2) Representative traces showing paired fEPSPs evoked at a 50 ms interstimulus interval. All recordings were on the hippocampal side ipsilateral to the TBI impact site (over the right sensorimotor cortex). ^#p < 0.05 for sham vs. sTBI. *p < 0.05.for sham vs. rTBI. Error bars represent the standard error of the mean.

Figure 2

Acute induction of hippocampal synaptic plasticity is unaffected by single or repeated TBI. (A) High-frequency stimulation (HFS) of CA3-CA1 Schaffer collaterals resulted in short-term post-tetanic synaptic potentiation (PTP), lasting for several minutes. Graph shows averaged time course of PTP in sham, single TBI (sTBI), and repeated TBI (rTBI) groups. (B) High-frequency stimulation (HFS) of CA3-CA1 Schaffer collaterals also resulted in long-term potentiation (LTP) lasting for 1 h. Graph shows averaged time course of early (E-LTP) and late-phase LTP (L-LTP) in sham, sTBI, and rTBI groups. Time of HFS indicated by the arrow. (C) Bar graph shows the percentage change in post-HFS response relative to baseline as PTP (0–2 min after HFS), E-LTP (15–20 min after HFS), and L-LTP (50–60 min after HFS). HFS consisted of two 100 Hz pulse trains of 1 s duration each, with a 10 s interval between pulse trains. There was no significant effect of sTBI or rTBI on the magnitude of PTP, E-LTP, or L-LTP. Superimposed trendlines represent mean data for sham (blue), sTBI (orange), and rTBI (red). All recordings were on the hippocampal side ipsilateral to the TBI impact site (over the right sensorimotor cortex). Error bars represent the standard error of the mean.

Figure 3

TBI results in enhanced voltage-gated Ca²⁺ channel activity in hippocampal CA1 pyramidal neurons. (A) Left, bar graph summarizing peak Ca²⁺ effects of voltage-gated Ca²⁺ channel (VGCC) activation (using a depolarizing current injection in CA1 pyramidal neurons) 30 days after either a sham procedure, single TBI (sTBI), or repeated TBI (rTBI). Peak VGCC Ca²⁺ effects were larger in both the sTBI (gray bar) and rTBI (black bar) groups relative to sham (white bar). The number of recorded cells per group is in each bar. Right, action potential train in response to a depolarizing current injection in a CA1 pyramidal neuron (action potential characteristics were unchanged by either sTBI or rTBI, see Table 1). (B) Sample traces illustrating Ca²⁺ effects of VGCC activation in individual sham (left), sTBI (middle) or rTBI (right) CA1 pyramidal neurons. Calibration bars illustrate the % Ca²⁺ change over baseline. (C) Fluorescent (monochrome) and pseudocolored images of fura-2 loaded CA1 pyramidal neurons. Pseudocolored images illustrate relative Ca²⁺ responses to VGCC activation, from sham (left), sTBI (middle) and rTBI (right) groups. *p < 0.05. All recordings were on the hippocampal side ipsilateral to the TBI impact site (over the right sensorimotor cortex). Error bars represent the standard error of the mean.

Figure 4

TBI results in increased resting Ca²⁺ and blunted RyR-evoked Ca²⁺ release in hippocampal CA1 pyramidal neurons. (A) Left, bar graph summarizing averaged resting Ca²⁺-based fluorescence (F₀) in CA1 pyramidal neurons 30 days after either a sham procedure (white bar), single TBI (sTBI, gray bar), or repeated TBI (rTBI, black bar). The decreased resting fluorescence (F₀) values of both TBI groups compared to the sham group are indicative of increased resting Ca²⁺. Right, a bar graph illustrating that the averaged RyR-Ca²⁺ response to caffeine (20 mM) was decreased in the rTBI group (black bar) but not the sTBI group (gray bar), relative to sham (white bar). The number of recorded cells per group is in each bar. (B) Sample traces illustrating RyR-Ca²⁺ effects of caffeine (20 mM), in individual sham (left), sTBI (middle), and rTBI (right) CA1 pyramidal neurons. Calibration bars illustrate the % Ca²⁺ change over baseline. (C) Fluorescent (monochrome) and pseudocolored images of fura-2 loaded CA1 pyramidal neurons. Pseudocolored images illustrate relative RyR-Ca²⁺ effects of caffeine, from sham (left), sTBI (middle), and rTBI (right) groups. *p < 0.05. All recordings were on the hippocampal side ipsilateral to the TBI impact site (over the right sensorimotor cortex). Error bars represent the standard error of the mean.

Figure 5

Spontaneous synaptic events and contribution of RyR activation are unaffected by repeated TBI (rTBI). (A) The frequency of baseline spontaneous excitatory postsynaptic potentials (sEPSPs; gray bars) measured in hippocampal CA1 pyramidal neurons was not affected by rTBI at 30 days post-injury. sTBI neurons were not tested. In both the sham and rTBI groups, RyR activation by caffeine (20 mM; black bars) increased the frequency of sEPSPs similarly. (B) Baseline sEPSP amplitude (gray bars) was also unaffected by rTBI, but RyR activation by caffeine (20 mM; black bars) resulted in a significant increase in sEPSP amplitude in the rTBI group. (C) Representative traces illustrating effects of bath application of caffeine (20 mM) on the frequency and amplitude of sEPSPs during a whole-cell current-clamp recording in a CA1 pyramidal neuron from the rTBI group. **p < 0.001, *p < 0.05. All recordings were on the hippocampal side ipsilateral to the TBI impact site (over the right sensorimotor cortex). Error bars represent the standard error of the mean.

Figure 6

Hippocampal and cortical phosphorylated tau is elevated after single (sTBI) or repeated TBI (rTBI). (A–C) Representative confocal images (63×, multi-plane) show immunolabeling of phosphorylated tau (pTau S262, green) within the CA1 region of the hippocampus (left) and Layer V of the cortex (right) ipsilateral to injury in sham (A), sTBI (B), and rTBI (C) animals. (D) Bar graphs show a significant increase in hippocampal phosphorylated tau burden in the rTBI group (black) relative to the sTBI (gray) and sham groups (white; n = 16 slices/4 rats for each treatment condition). (E) Bar graphs show a significant increase in cortical phosphorylated tau burden in the rTBI group (black) relative to the sTBI (gray) and sham groups (white; n = 16 slices/4 rats for each treatment condition). Representative images were obtained on a Leica Microsystems SP8 confocal microscope. The injury was over the right sensorimotor cortex. *p ≤ 0.05 and **p ≤ 0.01. Error bars represent the standard error of the mean.

Figure 7

Schematic of effects of TBI on hippocampal synaptic transmission and Ca²⁺ handling. Thirty days after single (sTBI) or repeated TBI (rTBI), we observed potentiation of evoked fEPSPs, indicating synaptic potentiation, possibly resulting from postsynaptic effects including increased AMPA receptor effects. VGCC function was also increased in the sTBI and rTBI groups, and this, combined with increased synaptic transmission, may play a role in the increased basal cytosolic Ca²⁺ observed in both TBI groups. RyR-mediated Ca²⁺ release was decreased, but only in the rTBI group, possibly due to depletion of endoplasmic reticulum (ER) Ca²⁺ stores.