Persistent working memory dysfunction following traumatic brain injury: evidence for a time-dependent mechanism

Abstract

The prefrontal cortex is highly vulnerable to traumatic brain injury (TBI) resulting in the dysfunction of many high-level cognitive and executive functions such as planning, information processing speed, language, memory, attention, and perception. All of these processes require some degree of working memory. Interestingly, in many cases, post-injury working memory deficits can arise in the absence of overt damage to the prefrontal cortex. Recently, excess GABA-mediated inhibition of prefrontal neuronal activity has been identified as a contributor to working memory dysfunction within the first month following cortical impact injury of rats. However, it has not been examined if these working memory deficits persist, and if so, whether they remain amenable to treatment by GABA antagonism. Our findings show that working memory dysfunction, assessed using both the delay match-to-place and delayed alternation T-maze tasks, following lateral cortical impact injury persists for at least 16 weeks post-injury. These deficits were found to be no longer the direct result of excess GABA-mediated inhibition of medial prefrontal cortex neuronal activity. Golgi staining of prelimbic pyramidal neurons revealed that TBI causes a significant shortening of layers V/VI basal dendrite arbors by 4 months post-injury, as well as an increase in the density of both basal and apical spines in these neurons. These changes were not observed in animals 14 days post-injury, a time point at which administration of GABA receptor antagonists improves working memory function. Taken together, the present findings, along with previously published reports, suggest that temporal considerations must be taken into account when designing mechanism-based therapies to improve working memory function in TBI patients.

Figure 1. Four month post-TBI rats display working memory deficits in the delay match-to-place task

A) Schematic depiction of the delay match-to-place testing paradigm. Each location-match pair of trials is carried out with the hidden platform (filled circle) in a novel location within the water tank (large, open circle). A 5-second delay is used between the location and match trials. Rats were tested using at least five different platform positions, with each location-match pair separated by a 4 min resting period. B) Summary data for performance in the delay match-to-place task. Data are presented as the mean ± SEM. ǂ, group main effect by two-way ANOVA. *, significant difference between match trials for sham and injured groups.

Figure 2. Four month post-TBI rats display working memory deficits in the water version of the delayed alternation task

A) Summary data for the performance of sham-operated and injured animals in the delayed alternation T-maze task. The percent correct alternations for each animal was calculated as the average of two days of testing. Data are presented as the mean ± SEM. ǂ, group main effect by repeated measures two-way ANOVA. *, significant difference between between sham and injured groups. B) Intra-mPFC infusion of 1.0 μg/side muscimol impairs performance in the delayed alternation task. Data are presented as the mean ± SEM. ǂ, interaction between group and condition by repeated measures two-way ANOVA. *, significant difference between vehicle and muscimol groups.

Figure 3. Working memory deficits are not alleviated by GABA_A antagonism in 4 month post-TBI animals

A) Summary data for performance in the delay match-to-place task for injured animals i.p. injected with 0.5 mg/kg bicuculline or vehicle. B) Representative Deep Purple stained gel illustrating the equality of loading for the samples used in C). C) Representative western blots and summary data for GAD67 immunoreactivity within the mPFC for sham-operated and 4 month post-TBI animals. Data are presented as the mean ± SEM.

Figure 4. Dendritic arbors of layer II/III pyramidal neurons are not altered as a result of TBI

A) Computer assisted traces of layer II/III pyramidal neurons from sham and 14 day and 4 month post-injury rats. B) Sholl analysis revealed no significant difference in either basilar or apical dendritic arbors from 14 day post-injury animals compared to sham-operated controls. C) Similarly, no significant changes in arborization were detected in basal or apical dendrites from layer II/III pyramidal neurons at the 4 month post-injury time point. Data are presented as the mean ± SEM.

Figure 5. Layer V/VI pyramidal neurons from 4 month injured animals have reduced basal dendrite lengths

A) Computer assisted traces of layer V/VI pyramidal neurons from sham and 14 day and 4 month post-injury rats. B) No significant difference in dendrite arbors was detected for either the basal or apical dendrites between the sham and 14 day injured groups. C) Although not significant by two-way repeated measures ANOVA (p=0.053), a trend towards reduced basal dendrite arborization was found in the layer V/VI pyramidal neurons of 4 month post-injury rats. D) The total number and cumulative length of basal dendrites for layer V/VI pyramidal neurons. Data are presented as the mean ± SEM. ǂ, group main effect by repeated measures two-way ANOVA. *, significant difference in dendrite number between sham and 4 month post-TBI animals.

Figure 6. Layer V/VI pyramidal neurons from 4 month injured animals have increased spine densities

A) Representative photomicrographs of 3^rd order layer V/VI apical dendrites from sham and 4 month post-injury rats. Scale bar = 10μm. B) Quantification of layer II/III spine density (spines/10 μm) from 2^nd order basal and 3^rd order apical dendrites from sham and 4 month post-injury rats. C) The spine densities of both 2^nd order basal and 3^rd order apical dendrites from layer V/VI pyramidal neurons were found to be significantly increased (group main effect by repeated measures two-way ANOVA) in 4 month post-injured animals compared to age-matched sham controls.