Mechanisms underlying the inability to induce area CA1 LTP in the mouse after traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a significant health issue that often causes enduring cognitive deficits, in particular memory dysfunction. The hippocampus, a structure crucial in learning and memory, is frequently damaged during TBI. Since long-term potentiation (LTP) is the leading cellular model underlying learning and memory, this study was undertaken to examine how injury affects area CA1 LTP in mice using lateral fluid percussion injury (FPI). Brain slices derived from FPI animals demonstrated an inability to induce LTP in area CA1 7 days postinjury. However, area CA1 long-term depression could be induced in neurons 7 days postinjury, demonstrating that some forms of synaptic plasticity can still be elicited. Using a multi-disciplined approach, potential mechanisms underlying the inability to induce and maintain area CA1 LTP were investigated. This study demonstrates that injury leads to significantly smaller N-methyl-D-aspartate potentials and glutamate-induced excitatory currents, increased dendritic spine size, and decreased expression of alpha-calcium calmodulin kinase II. These findings may underlie the injury-induced lack of LTP and thus, contribute to cognitive impairments often associated with TBI. Furthermore, these results provide attractive sites for potential therapeutic intervention directed toward alleviating the devastating consequences of human TBI.

FIGURE 1

Inability to induce area CA1 LTP but not LTD following lateral FPI in mice. A: High frequency tetanic stimulation (HFS, 2 × 100 Hz trains) denoted by arrows resulted in facilitation of the evoked field excitatory postsynaptic potential (fEPSP) in hippocampal slices from sham (filled circles, n = 4) but not in slices from brain injured (open circles, n = 7) mice. B: Low frequency stimulation (LFS, 1 Hz, 10 min) resulted in LTD of the fEPSP in slices derived from both injured (open circles, n = 4) and sham (filled circles, n = 4) animals. Inset: Paired pulse facilitation is observed in slices from FPI and sham mice (n = 3 FPI, n = 6 sham P > 0.05). Scale bar: 0.5 mV, 5 ms. C: Histogram demonstrating that LTP can be induced in slices from sham and naïve animals using either the standard HFS or theta burst stimulation (TBS); however, with both protocols, LTP cannot be induced in slices from FPI animals. LTP can be induced and maintained in area CA1 in the hippocampus contralateral to injury (n = 4), but ipsilateral area CA1 LTP cannot be rescued by varying the stimulation protocol, altering extracellular Ca²⁺ and Mg²⁺ concentrations (n = 4), or bath application of BMI (n = 4). All FPI treatment groups were significantly different from the sham slices exhibiting LTP (P < 0.05, denoted by *). Furthermore, no rescue was observed when statistically comparing BMI and altered aCSF treatment groups to the FPI LTP treatment group (P > 0.05). D: LTD induced by a LFS (denoted by LFS and a line) in slices from FPI animals does not make subsequent LTP induction by HFS (2 × 100 Hz trains, denoted by arrows) possible. Responses were followed for an additional 30 min post-tetanic stimulation, no potentiation (even a return to baseline) was observed (n = 6).

FIGURE 2

Injury diminishes isolated NMDA receptor potentials and EPSCs following injury. A (sham), B (FPI): Trace of fEPSP, isolated NMDA potential, recording after addition of APV to ensure the waveform is NMDA dependent. Scale bar: 0.5 mV, 10 ms. C: Histogram showing the quantification of the reduction in the peak amplitude of the NMDA potential recorded in slices from FPI mice compared to sham animals (n = 11 and 7 for FPI and sham, respectively, P < 0.05, denoted by *). D (sham), E (FPI): Glutamatergic currents were recorded by focal application of glutamate (100 μM, 50 ms, 40 psi), the NMDA component was isolated with the addition of CNQX to the bath, and at the end of each experiment, APV was perfused to ensure the current is NMDA-dependent. Scale bar: 15 pA, 100 ms. F: The histogram shows FPI causes a significant reduction in the peak amplitude of the NMDA current. G: The histogram is created by subtracting the isolated NMDA-mediated current from the entire glutamate-evoked current to determine the AMPA-mediated current. This shows FPI also causes a significant reduction in the peak amplitude of the AMPA currents. *denotes significance, P < 0.05.

FIGURE 3

Increased dendritic spine size in slices from injured mice. A: CA1 pyramidal neuron and its spiny dendrites from an FPI-injured mouse loaded with Lucifer yellow. pcl, pyramidal cell layer; sl, stratum lucidum; so, stratum oriens. Scale bar: 10 μm. B: Higher magnification of area highlighted by box in section A showing fourth order basal dendrite segment spines. Scale bar: 1 μm. C: Higher magnification of area highlighted by box in section A showing second order apical dendrite segment spines. Scale bar: 1 μm. D: Neurolucida drawing of the spiny segment shown in B (noted by asterisk *). Scale bar: 1 μm. E: Comparison of the distribution of dendritic spine size of sham (black) and FPI (gray) apical (E) and basal (F) CA1 pyramidal cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FIGURE 4

Decreased α-CaMKII expression following FPI. A: Western blot analysis demonstrating a decrease in α-CaMKII protein expression from tissue regionally dissected from area CA1 (n = 3, each independent sample from tissue pooled from five sham or FPI mice). The blot was reprobed with β-actin to ensure proper loading of the samples. B: Histogram depicting a significant reduction of α-CaMKII protein expression in tissue from FPI mice, which is specific to area CA1.