GABAergic interneuronal loss and reduced inhibitory synaptic transmission in the hippocampal CA1 region after mild traumatic brain injury

Abstract

Patients that suffer mild traumatic brain injuries (mTBI) often develop cognitive impairments, including memory and learning deficits. The hippocampus shows a high susceptibility to mTBI-induced damage due to its anatomical localization and has been implicated in cognitive and neurological impairments after mTBI. However, it remains unknown whether mTBI cognitive impairments are a result of morphological and pathophysiological alterations occurring in the CA1 hippocampal region. We investigated whether mTBI induces morphological and pathophysiological alterations in the CA1 using the controlled cortical impact (CCI) model. Seven days after CCI, animals subjected to mTBI showed cognitive impairment in the passive avoidance test and deficits to long-term potentiation (LTP) of synaptic transmission. Deficiencies in inducing or maintaining LTP were likely due to an observed reduction in the activation of NMDA but not AMPA receptors. Significant reductions in the frequency and amplitude of spontaneous and miniature GABA_A-receptor mediated inhibitory postsynaptic currents (IPSCs) were also observed 7 days after CCI. Design-based stereology revealed that although the total number of neurons was unaltered, the number of GABAergic interneurons is significantly reduced in the CA1 region 7 days after CCI. Additionally, the surface expression of α1, ß2/3, and γ2 subunits of the GABA_A receptor were reduced, contributing to a reduced mIPSC frequency and amplitude, respectively. Together, these results suggest that mTBI causes a significant reduction in GABAergic inhibitory transmission and deficits to NMDA receptor mediated currents in the CA1, which may contribute to changes in hippocampal excitability and subsequent cognitive impairments after mTBI.

1. Introduction

Mild traumatic brain injury (mTBI) is a brain trauma that results in the disruption of brain function. mTBI is characterized by one or more of the following: a) loss of consciousness up to 30 min; b) posttraumatic amnesia up to 24 h; c) alterations in consciousness (i.e. dazed feeling, disorientation up to 24 h); d) transient neurological dysfunction such as a seizure and an intracranial lesion that does not require surgical intervention; and e) a Glasgow coma scale of 13–15 when performed at least 30 min after initial injury (Borg et al., 2004a,2004b; Carroll et al., 2004; Cassidy et al., 2004; Peloso et al., 2004). Occurring in more than 80% of all head trauma cases (Moore et al., 2006), mTBI patients commonly report acute memory and concentration problems, and other deficits in learning and memory, all of which can have devastating effects on patients and families (Gasquoine, 1997; Miotto et al., 2010; Moore et al., 2006; Rimel et al., 1981; Stuss et al., 1985).

Recent evidence from human and animal studies suggests that a single brain injury may cause acute changes in learning and cognition, though the mechanism by which this occurs remains poorly understood. Moreover, functional changes that occur after a mTBI may be exacerbated by multiple head injuries (Aungst et al., 2014; Prins et al., 2013; Shultz et al., 2012; Tavazzi et al., 2007; Vagnozzi et al., 2007, 2008). Indeed, compared to athletes that have experienced only a single brain injury, athletes experiencing three or more concussions display more severe symptoms, long-term cognitive deficits, and an increase in mood disorders, including depression (Guskiewicz et al., 2003, 2005, 2007). Thus, to understand why cognitive and neurological deficits are more severe after a repeated trauma, it is essential to first identify the functional and morphological deficits that take place after the initial injury in brain regions essential to learning and memory.

The hippocampus plays a major role in learning and memory, and is particularly susceptible to mTBI-related injury due to its anatomic location (Hicks et al., 1993; Kotapka et al., 1991; Umile et al., 2002). Following mTBI there is rarely overt morphological damage in the hippocampus (Bigler and Maxwell, 2012; Vos et al., 2012); stereological analysis of animals exposed to mTBI do not reveal a significant loss of hippocampal neurons (Almeida-Suhett et al., 2014a; Eakin and Miller, 2012). However, functional impairments to the hippocampus after mTBI may be associated with learning and memory deficits (McDonald et al., 2012). Indeed, studies suggest that up to 75% of patients suffering from mTBI may have functional abnormalities in the medial temporal lobe, including the hippocampus (Umile et al., 2002), and that alterations in hippocampal function and excitability after mild brain injury may be present in the absence of clear morphological damage (Eakin and Miller, 2012; Greer et al., 2012; Griesemer and Mautes, 2007; Reeves et al., 1995, 2000).

Changes in hippocampal excitability and memory deficits observed after TBI may result from reductions in GABAergic inhibitory synaptic transmission (Gupta et al., 2012; Mtchedlishvili et al., 2010; Pavlov et al., 2011; Raible et al., 2012; Witgen et al., 2005) or alterations in N-methyl-d-aspartate (NMDA) receptor function (Schwarzbach et al., 2006). Indeed, fluid percussion injury, which leads to mild to moderate injury, reduces NMDA receptor mediated currents (Schwarzbach et al., 2006), while severe TBI leads to reductions in GABA_A-mediated inhibitory synaptic transmission in dentate granule cells (DGCs) in rats (Mtchedlishvili et al., 2010; Pavlov et al., 2011) and mice (Witgen et al., 2005). Thus, while moderate to severe TBI is known to reduce GABAergic inhibition and impair NMDA receptor mediated currents, it remains unknown whether functional alterations to the GABAergic and glutamatergic systems occur in animals that receive a mild brain injury in the absence of overt morphological deficits. More specifically, it remains unknown how mTBI, using the controlled cortical impact (CCI) model, alters the activity of GABAergic inhibitory synaptic transmission and NMDA receptor function in the CA1 region of the hippocampus.

The purpose of the current study is to investigate the functional and morphological alterations in the CA1 hippocampal region that lead to acute cognitive deficits after mTBI. Our studies focus on the CA1 region as it is the major output within the hippocampal trisynaptic circuit and alterations in this region may have an impact in memory function (Daumas et al., 2005; Ji and Maren, 2008; Karasawa et al., 1994; Stubley-Weatherly et al., 1996; Vago et al., 2007; Zola-Morgan et al., 1986). We found that within 7 days of receiving a mild brain injury induced by a CCI, animals displayed significant cognitive deficits and an inability to induce long-term potentiation (LTP) of synaptic transmission. It is likely that the inability to induce LTP was a result of impaired activation of NMDA but not AMPA receptors. In addition, GABA_A receptor mediated inhibitory synaptic transmission was also impaired 7 days after CCI. Alterations in inhibitory synaptic transmission were due to a delayed loss of GABAergic interneurons and alterations in the surface expression of the α1, β2, or γ2 GABA_A receptor subunits, which comprise the majority of GABA_A receptors (Möhler, 2006).

2. Experimental procedures

2.1. Animals

Experiments were performed on male Sprague–Dawley rats (Taconic Farms, Rockville, MD, USA), 5–6 weeks old, weighing 150–200 g at the start of the experiments. Animals were housed paired until the day of the surgery and then housed individually in an environmentally controlled room (20–23 °C, 12-h light/dark cycle, lights on at 6:00 AM), with food (Harlan Teklad Global Diet 2018, 18% protein rodent diet; Harlan Laboratories; Indianapolis, IN) and water ad libitum. Cages were cleaned weekly and animal handling was minimized to reduce animal stress (Prager et al., 2011). All animal experiments were conducted following the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council) and were in accordance with the guidelines and approved by the Uniformed Services University of the Health Sciences Institutional Animal Care and Use Committees (IACUC). All efforts were made to minimize the number of animals used and any pain or distress associated with these experiments.

2.2. Controlled cortical impact injury

A unilateral cortical contusion using the controlled cortical impact (CCI) model of traumatic brain injury was administered using a previously established protocol (Almeida-Suhett et al., 2014b; Lighthall, 1988). Briefly, animals were anesthetized with isoflurane (2.5%) and had their heads shaved and placed in the stereotaxic frame. Core body temperature of the animals was maintained at 36–37 °C using a heating pad and D.C. Temperature Control System (FHC, Model Number 40-90-8D, Bowdoin, ME). Without damaging the underlying dura mater, the skin was retracted, and a 4.0 mm craniotomy – 3.0 mm lateral to the midline and 4.0 mm posterior to the bregma over the left tempoparietal cortex – was performed. In these experiments, the contact velocity was set to 3.5 m/s with a dwell time of 200 ms and the amount of deformation was set to 2.0 mm using a 3.0 mm diameter impact tip. Following injury, the skullcap was replaced and fixed using bone wax (Ethicon, Sommerville, NJ) and the incision was closed with absorbable sutures (Stoelting, IL). The animals received subcutaneously buprenorphine (50 μL) for pain alleviation and Ringer's solution (5 mL) for rehydration after surgery. Sham-treated controls received the craniotomy, but no CCI injury.

2.3. Passive avoidance

One day prior to the CCI surgery, animals were submitted to the training session of the passive avoidance test, which provides a contextual measure of learning and memory (hippocampal dependent). During training, animals were placed into a lit chamber. After a delay of 60 s, the door to the other, still darkened chamber, opened. Each animal had up to 300 s to cross to the dark chamber. Once the animal crossed to the darkened chamber, the door closed, and a 0.8 mA shock was delivered through the grid floor for 1 s. The latency to cross to the darkened chamber was recorded. Testing trials were performed 7 days following CCI. Conditions were identical to the training session, except that the shock was not delivered if/when the animal crosses into the darkened chamber. Memory strength was observed as a longer latency to cross into the chamber in which it was previously shocked.

2.4. Electrophysiological experiments

Coronal slices containing the hippocampus were prepared from rats 1 and 7 days after surgery. The rats were anesthetized with isoflurane and then decapitated. Brain slices (400 μm-thick) were cut using a vibratome (series 1000; Technical Products International, St. Louis, MO), in ice-cold cutting solution consisting of (in mM): 115 sucrose, 70 NMDG, 1 KCl, 2 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, 30 NaHCO₃, 25 d-glucose. Slices were transferred to a holding chamber, maintained at 32 °C for 25 min and then at room temperature in a bath solution containing (in mM): 125 NaCl, 2.5 KCl, 2.0 CaCl₂, 2.0 MgCl₂, 25 NaHCO₃, 1.25 NaH₂PO₄, and 22 d-glucose. For field potential recordings, the bath/recording solution was the same as above, except for the concentration of MgCl₂ (1.5 mM). Recording solution was the same as the holding bath solution. All solutions were saturated with 95% O₂, 5% CO₂ to achieve a pH near 7.4. Field potential recordings were obtained in an interface-type chamber, maintained at 32–33 °C, with a flow rate of the ACSF at ~2 mL/min. For whole-cell recordings, slices were transferred to a submersion-type recording chamber (0.7 mL capacity), where they were continuously perfused with oxygenated ACSF (~ 3–4 mL/min). Neurons were visualized with an upright microscope (Zeiss Axioskop 2, Thronwood, NY) through a 40× water immersion objective, equipped with a CCD-100 camera (Dage-MTI, Michigan City, IN). All experiments were performed at 32 °C. Tight-seal (N1 GΩ) whole-cell recordings were obtained from the cell body of pyramidal-shaped neurons in the CA1 region. Patch electrodes were fabricated from borosilicate glass and had a resistance of 3.5–4.5 MΩ when filled with solution A containing (in mM): 135 Cs-gluconate, 10 MgCl₂, 0.1 CaCl₂, 1 EGTA, 10 Hepes, 2 Na-ATP, 0.2 Na₃GTP, pH 7.3 (285–290 mOsm) or solution B containing (in mM): 60 Cs-gluconate, 60 KCH₃SO₃, 10 KCl, 10 EGTA, 10 HEPES, 5 Mg-ATP, 0.3 NaGTP, pH 7.2 (280–290 mOsm/kg). Solution A was used to record AMPA receptor mediated excitatory postsynaptic currents (EPSCs) and NMDA currents; solution B was used to record spontaneous and miniature inhibitory postsynaptic currents (IPSCs). Neurons were voltage-clamped using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Inhibitory postsynaptic currents (IPSCs) were pharmacologically isolated and recorded at a −70 mV holding potential. Access resistance (5–24 MΩ) was regularly monitored during recordings, and cells were rejected if it changed by 15% during the experiment. Ionic currents and action potentials were amplified and filtered (1 kHz) using the Axopatch 200B amplifier (Axon Instruments, Foster City, CA) with a four-pole, low-pass Bessel filter, were digitally sampled (up to 2 kHz) using the pClamp 10.2 software (Molecular Devices, Sunnyvale, CA), and further analyzed using the Mini Analysis program (Synaptosoft Inc., Fort Lee, NJ) and Origin (OriginLab Corporation, Northampton, MA). The peak amplitude, 10–90% rise time, and decay time constant of EPSCs and IPSCs were analyzed off-line using pClamp 10.2 software and the Mini Analysis Program (Synaptosoft, Inc., Leonia, NJ, USA). Miniature IPSCs (mIPSCs) were analyzed off-line using the Mini Analysis Program (Synaptosoft, Fort Lee, NJ) and detected by manually setting the mIPSC threshold (~1.5 times the baseline noise amplitude) after visual inspection. Field potentials recorded from the CA1 were evoked by stimulation of the Schaffer Collaterals at 0.05 Hz. Recording glass pipettes were filled with ACSF and had a resistance of approximately 5 MΩ. Stimulation was applied with a bipolar concentric stimulating electrode made of tungsten (World Precision Instruments, Sarasota, FL). Signals were digitized using the pClamp 10.2 software (Molecular Devices, Union City, CA), analyzed using Clampfit 10.2 and final presentation was prepared using Origin (OriginLab Corporation, Northampton, MA).

NMDA (100 μM), a specific agonist to NMDA receptors was applied by pressure injection. Pressure application was performed with the help of a push–pull experimental arrangement (Pidoplichko and Dani, 2005), as utilized previously (Figueiredo et al., 2011b). Experiments were performed with similar ACSF composition, except MgCl₂ was reduced to 200 μM and 1 μM tetrototoxin (TTX) was also included. Pressure was applied to the pipette via a Picospritzer (General Valve Division, Parker Hannifin Corp., Fairfield, NJ), set at about 30 psi for 100 ms. A motorizer (Newport, Fountain Valley, CA) was coupled with the approach/withdrawal (push–pull) actuator of a micromanipulator (Burleigh PCS-5000 series; EXFO Photonic Solution Inc., Mississauga, Ontario, Canada). Motorizer movement and duration of application pulses were controlled with a Master-8 digital stimulator (AMPI; Jerusalem, Israel). Ionic currents were amplified and filtered (1 kHz) using an Axopatch 200B amplifier, with a four-pole low-pass Bessel filter, and were digitally sampled (up to 5 kHz). Currents were recorded using pClamp 10.2 software and further analyzed using OriginLab (Northampton, MA) and Mini60 software.

Drugs used were as follows: 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist; 50 μM d-2-amino-phosphonovalerate (AP-5), an N-methyl-d-aspartic acid (NMDA) receptor antagonist; 10 μM SCH50911, a GABA_B receptor antagonist, and 3 μM LY341495, a metabotropic group II/III glutamate receptor antagonist (all purchased from Tocris, Ellisville, MO). We also used 20 μM bicuculline methiodide, a GABA_A receptor antagonist and 1 μM tetrodo-toxin (TTX), a sodium channel blocker (purchased from Sigma-Aldrich, St. Louis, MO).

2.5. Biotinylation and Western blot

To identify alterations in the surface expression of GABA_A receptor subunits, we performed biotinylation to isolate and quantify proteins expressed on the cell membrane in intact brain slices as previously described (Goodkin et al., 2008; Grosshans et al., 2002; Holman and Henley, 2007; Gonzalez et al., 2013). Coronal slices containing the hippocampus were prepared as described for electrophysiology experiments. After a 1-hour recovery period in oxygenated ACSF, slices were incubated in ACSF containing 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) for 1 h on ice, followed by the addition of quench solution (provided in the Pierce Cell Surface Protein Isolation Kit, Cat No. 89881). The hippocampus was then dissected and tissue sections were transferred to small plastic tubes containing radioimmunoprecipitation assay (RIPA) buffer composed of (in mM) 50 Tris–HCl, pH 7.4, 150 NaCl, 2 EDTA, 50 NaF, 1 Na₃VO₄, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate, and a Protease Inhibitor Cocktail (Sigma-Aldrich, MO). Samples were sonicated and the homogenates were centrifuged at 14,000 g for 10 min at 4 °C. Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad, CA). Protein (1500 μg) was then mixed with 400 μL of UltraLink immobilized NeutrAvidin agarose beads (Pierce) for 1 h at room temperature. The beads were then washed 3 times with 500 μL wash buffer (provided in the kit). Samples were eluted in 400 μL of RIPA buffer containing Protease Inhibitor Cocktail supplemented with 50 mM dithiothreitol and mixed for 1 h at room temperature followed by centrifugation at 14,000 g for 10 min at 4 °C. Then, LDS 4x (Invitrogen) was added to protein samples. Biotinylated proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies anti-Alpha1 GABA-A receptor, anti-GABA-A receptor Beta 2,3 chain, anti-GABA-A receptor Gamma 2. The signal from the immunoreactive band was detected by using a gel imaging system (Fuji LAS-3000). Membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Millipore, Billerica, MA) and re-probed with anti-GLUT1 for loading control as anti-actin probes would not be detected in biotinylated sections (Wang and Quick, 2005) (see Table 1 for immunogen, source and details, and concentration information). Signal intensity was determined by densitometric scanning using ImageJ. When duplicate conditions were performed in one animal, the ratio values were averaged to obtain an animal average for that condition.

Table 1.

Primary and secondary antibodies used for Western blot and immunohistochemistry.

Antibody	Immunogen	Source, cat. #, clone, RRID	Species	Concentration used
Anti-GABA-A-R-Alpha1	Fusion protein amino acids 355–394 of human GABA-A-R-Alpha1	UC Davis/NIH NeuroMab Facility, clone: N95/35, AB_10697873	Mouse IgG, monoclonal	1:1000
GABA-A-R beta 2,3 subunit (bd17)	Purified GABA/benzodiazepine receptor from bovine cortex	Millipore, Cat# MAB341, AB_2314466	Mouse IgG, monoclonal	1:1000
Anti-GABA A Receptor gamma 2	Fusion protein of MBP with the amino acid sequence from the cytosolic loop of GABAA Receptor gamma2	Abcam, Cat# ab16213, AB_302324	Rabbit IgG, polyclonal	1:1000
Anti-GLUT1	KLH-conjugated linear peptide corresponding to the C-terminus of human GLUT1	Millipore, Cat #MABS132, Clone 5B12.3	Mouse IgG, monoclonal	1:1000
Anti-GAD67	Recombinent GAD67 protein	Millipore, Cat# MAB5406, Clone 1g10.2, AB_2278725	Mouse IgG, Monoclonal	1:1000
Cy3 AffiniPure Goat	Mouse IgG	Jackson ImmunoResearch, Cat# 115–165–166, AB_2338692	Goat, IgG, polyclonal	1:1000

2.6. Immunohistochemistry

2.6.1. Fixation and tissue processing

Seven (7) days after CCI, animals were deeply anesthetized using nembutal (75–100 mg/kg, i.p.) and transcardially perfused with phosphate buffered saline (PBS, 100 ml) followed by 4% paraformaldehyde (250 mL). Brains were removed and post-fixed in4% paraformaldehyde overnight at 4 °C, then transferred to a solution of 30% sucrose in PBS for 72 h, and frozen with dry ice before storage at −80 °C until sectioning. Sectioning was performed as previously described (Figueiredo et al., 2011a,2011b). A 1-in-5 series of sections containing the hippocampus was cut at 40 μm on a sliding microtome (Leica Microsystems SM2000R). One series of sections was mounted on slides (Superfrost Plus, Daigger, Vernon Hills, IL) for Nissl staining with cresyl violet.

2.6.2. GAD-67 immunohistochemistry

To estimate the total number of GABAergic interneurons, we labeled GAD-67 immunoreactive neurons as previously described (Stanley and Shetty, 2004; Shetty et al., 2009; Prager et al., 2014b; Figueiredo et al., 2011b). A 1-in-10 series of free-floating sections was collected from the cryoprotectant solution, washed three times for 5 min each in 0.1 M PBS, and incubated in a blocking solution containing 10% normal goat serum (Millipore Bioscience Research Reagents, Temecula, CA) and 0.5% Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with mouse anti-GAD-67 serum, 5% normal goat serum, 0.3% Triton X-100, and 1% bovine serum albumin overnight at 4 °C. After rinsing three times for 10 min each in 0.1% Triton X-100 in PBS, the sections were incubated with Cy3-conjugated goat anti-mouse antibody and 0.0001% 4,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich) in PBS for 1 h at room temperature. After a final rinse in PBS for 10 min, sections were mounted on slides, air-dried for at least 30 min, and coverslipped with ProLong Gold antifade reagent (Invitrogen, Carls-bad, CA).

2.6.3. Stereological quantification

Design-based stereology was performed to assess total neuronal loss from Nissl-stained sections and interneuronal loss from GAD-67-immunostained sections in the CA1 hippocampal region (Figueiredo et al., 2011b). Neurons were identified using previously established parameters. Nissl-stained neurons were distinguished from glial cells by their larger size and pale nuclei surrounded by darkly stained cytoplasm containing Nissl bodies (Figueiredo et al., 2011b; Prager et al., 2014b). Sections were viewed with a Zeiss (Oberkochen, Germany) Axioplan 2ie fluorescent microscope with a motorized stage, interfaced with a computer running StereoInvestigator 9.0 (MicroBrightField, Williston, VT). The CA1 region was identified on slide-mounted sections and delineated for each slide of each animal, under a 2.5× objective, based on the atlas of Paxinos and Watson (Paxinos and Watson, 2005). All sampling was done under a 63× oil immersion objective. The total number of Nissl-stained and GAD-67-immunostained neurons from the stratum pyramidale was estimated by using the optional fractionator probe, and, along with the coefficient of error (CE), were calculated by using StereoInvestigator 9.0 (MicroBrightField). The CE was calculated by the software according to the equations of Gundersen et al. (m = 1) (Gundersen et al., 1999) and Schmitz and Hof (second estimation) (Schmitz and Hof, 2000).

A 1-in-10 series of sections was analyzed (on average seven sections) for Nissl stained neurons and GABAergic interneurons immune-labeled for GAD-67 in the CA1. The counting frame was 50 × 50 μm, the counting grid was 100 × 100 μm, and the dissector height was 20 μm. Nuclei were counted when the top of the nucleus came into focus within the dissector, which was placed 2 μm below the section surface. Section thickness was measured at every fifth counting site, and the average mounted section thickness was 26.3 μm. An average of 257 neurons per side per rat was counted. Eight rats were analyzed per group, and the average CE was 0.08 for both the Gunderson et al. and Schmitz-Hof equations.

2.7. Statistical analysis

Statistical values are presented as mean ± standard error (SE). Results from ipsilateral and contralateral sides of sham-operated and traumatized animals were compared using one-way ANOVA followed by Bonferroni post-hoc test in the stereology and Western blot experiments. For passive avoidance experiments, two-way ANOVA followed by Tukey post-hoc test was used. For electrophysiology experiments, either one-way ANOVA followed by Bonferroni post-hoc test or independent t-tests were performed. Statistical significance for all statistical analysis was considered when P < 0.05. For pressure-applied experiments, six pressure-evoked currents mediated by NMDA receptors were collected at one location and additional six currents were collected in a separate location. All currents were averaged together. Sample sizes (n) refer to the number of rats, except for the electrophysiology results where “n” refers to the number of slices (extracellular recordings) or recorded cells (patch clamp recordings).

3. Results

3.1. mTBI induces memory deficits in the passive avoidance test

Rats were exposed to mild TBI and then tested for memory impairments in the passive avoidance context. Prior to surgery all animals were trained in the passive avoidance apparatus and their latency to cross to the dark chamber was recorded. There were no significant differences in latency to cross to the dark chamber between the sham (21.73 ± 3.7 s, n = 16) and CCI groups (20.05 ± 4.7 s, n = 16; P = 0.78) during the training session. However, CCI rats showed a significantly lower latency to cross to the darkened chamber (46.28 ± 7.8 s; n = 16) 7 days after CCI compared to sham rats (179 ± 22.48 s, n = 16; P < 0.001; Fig. 1).

Fig. 1.

Mild TBI leads to memory deficits in the passive avoidance test. A decreased latency to enter the darkened chamber was observed 7 days after CCI in rats that received an mTBI compared to sham animals. Bars show the mean ± SEM of latency time to cross to the darkened chamber. ***P < 0.001; n = 16 for each group.

3.2. Long-term potentiation in the CA1 is reduced 7 days after mTBI

Long-term potentiation (LTP) of synaptic transmission is a synaptic property that is considered the cellular mechanism underlying learning and memory processes (Malenka and Nicoll, 1999; Prager et al., 2014a; Teyler and DiScenna, 1987). The CA1 region of the hippocampus plays an important role in contextual memory acquisition and retrieval (Daumas et al., 2005; Hall et al., 2001; Ji and Maren, 2008). We examined whether the synaptic properties underlying memory formation were also impaired after mTBI and correlated with deficits in the passive avoidance 7 days after mTBI. Therefore, we investigated if the capacity of neuronal synapses in the CA1 to express LTP had been altered after mTBI. Potentiation of the evoked field potentials was measured by averaging the slope of the response from 50 to 60 min after high frequency stimulation (HFS), and expressing it as a percentage of the baseline response (Prager et al., 2014b). Compared to the percent change in control animals (146.9 ± 10.0%, from 0.33 ± 0.02 mV/ms at baseline to 0.49 ± 0.04 mV/ms at 50 to 60 min after HFS, n = 9), the percent change 7 days after CCI (102.4 ± 4.7%, from 0.26 mv/ms at baseline to 0.27 ± 0.03 mv/ms at 50 to 60 min after HFS, n = 10) was significantly lower (P = 0.001; Fig. 2).

Fig. 2.

Mild TBI impairs long-term potentiation in the CA1 hippocampal region. The plot shows the time course of the changes in the slope of the field potentials after high-frequency stimulation (HFS). The slope of three responses recorded in each min (stimulation at 0.05 Hz) was averaged, and each data point on the plot is the mean and standard error of those averages (n = 9 to 10 slices) (see sample sizes in the text). Traces over the plots are examples from a sham rat and a rat that received a CCI; the superimposed field potentials are a baseline response and a response at 50–60 min after HFS (each trace is the average of 20 sweeps). Potentiation of the response, measured at 50–60 min after HFS, was significantly lower, compared to the control group, 7 days after mTBI.

3.3. Deficits to NMDA but not AMPA receptor mediated excitatory postsynaptic currents after mTBI

Following mTBI, mechanisms of learning and memory were impaired; this was associated with the cognitive deficits observed in the passive avoidance test and an inability to induce and maintain LTP. Impairments in learning and memory processes may be due to deficits in excitatory synaptic transmission, including deficits in NMDA and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor activation, both of which play essential roles in memory formation (Bassani et al., 2013; Bliss and Collingridge, 2013; Collingridge, 1987; Collingridge et al., 2013; Henley and Wilkinson, 2013; Myhrer, 2003; Riedel et al., 2003; Staubli et al., 1994; Stern and Alberini, 2013). To determine whether excitatory synaptic transmission was altered after mTBI, we pressure-applied NMDA (100 μM; 100 ms; 30 psi; Fig. 3 bottom), while recording from principal neurons at a holding potential of −70 mV in the presence of CNQX, bicuculline, SCH50911, LY341495, and TTX. For these experiments, bath application of ACSF containing MgCl₂ was reduced to 200 μM and the mean amplitude was examined. Six pressure evoked currents mediated via NMDA receptors were collected at one location from a pipette (2.5 μM in diameter at tip) positioned about 50 μM off the patch-clamped neurons; an additional six pressure evoked currents were collected at a second location from the same cell. Approximately 128 current amplitudes were averaged from sham animals (averaged 9 amplitudes per cell) and 121 amplitudes were averaged from CCI animals (averaged 7 amplitudes per cell). The mean amplitude of NMDA receptor mediated currents in sham animals was 160 ± 8 pA (n = 14; Fig. 3). 7 days after CCI the mean amplitude of NMDA receptor mediated currents (110 ± 6 pA; n = 15) was significantly reduced (P < 0.001), suggesting that NMDA receptor mediated currents are reduced after TBI (Fig. 3).

Fig. 3.

N-methyl-d-aspartate receptor mediated currents are reduced 7 days after mild TBI. Recordings were obtained in the presence of TTX, CNQX, bicuculine, SCH50911, LY341495, at a holding potential of −70 mV. MgCl₂ was reduced to 200 μM. Top: average amplitude of pressure-application evoked NMDA receptor-mediated currents for CA1 neurons from sham (160 ± 8 pA; n = 14) and CCI (110 ± 6 pA; n = 15) animals. Bottom: example of NMDA receptor mediated currents evoked by pressure application of NMDA (100 μM) [arrowhead; 100 ms; 30 psi]. ***P < 0.001.

AMPA receptors are the major mediators of fast excitatory synaptic transmission throughout the brain and also play an important role in memory, learning, and cognition (Bassani et al., 2013; Henley and Wilkinson, 2013). To determine whether there are alterations in tonic glutamatergic activity in the CA1 after mTBI, we recorded AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) from principal neurons in the presence of d-APV, bicuculline, SCH50911, LY341495, at a holding potential of −70 mV. The mean frequency of AMPA receptor mediated sEPSCs did not differ significantly between control rats (0.55 ± 0.12 Hz; n = 6; Fig. 4A) and CCI rats (0.50 ± 0.08 Hz; n = 7; P > 0.05; Fig. 4B). Similarly, the amplitude of AMPA receptor mediated sEPSCs did not differ significantly in the CCI rats (18.7 ± 0.8 pA; n = 7) compared to the sham rats (19.7 ± 2.1 pA; n = 6; P > 0.05; Fig. 4). These results suggest that there is no significant difference in AMPA receptor mediated sEPSCs after mTBI.

Fig. 4.

Mild TBI has no significant effect on the frequency and amplitude of AMPA receptor mediated sEPSCs in the CA1 7-days after CCI. sEPSCs were recorded from pyramidal-shaped neurons in the presence of D-APV, bicuculine, SCH50911, LY341495, at a holding potential of −70 mV. Representative examples of recordings obtained in the CA1 are shown in (A) and (B) for sham and CCI 7-day animals, respectively. (C) Group data demonstrate that there is no significant difference in the frequency, amplitude, rise time, or decay time constant of sEPSCs from CCI animals relative to sham animals.

3.4. Alterations in GABA_A receptor mediated spontaneous and miniature IPSCs

To determine whether mTBI impaired GABAergic inhibitory synaptic transmission in the CA1, we recorded GABA_A receptor mediated spontaneous sIPSCs from principal neurons in the presence of CNQX, D-AP5, SCH50911, and LY 3414953 at a holding potential of −70 mV, 1 and 7 days after CCI. Sham animals did not differ in the frequency and amplitude of sIPSCs at either 1- or 7-days after surgery and so we averaged together the amplitude and frequency of all sham animals (data not shown). One day after the surgery the mean frequency of sIPSCs in the CCI group (3.6 ± 0.7 Hz; n = 15) did not differ significantly from sham controls (3.2 ± 0.9 Hz; n = 15; P > 0.05); no difference in the mean amplitude of sIPSCs were observed between sham (254 ± 8 pA; n = 15) and CCI rats (258 ± 10 pA; n = 15; P > 0.05). However, 7 days after CCI the mean frequency of GABA_A receptor mediated sIPSCs recorded from CCI rats (2.47 ± 0.5 Hz; n = 15) was reduced by 35 ± 4% when compared to sham controls (3.8 ± 1.1 Hz; n = 15; P < 0.05; Fig. 5). In addition, the mean amplitude of GABA_A receptor mediated sIPSCs was reduced by 32 ± 5% in CCI rats (192 ± 7 pA; n = 15; P < 0.05) when compared to sham controls (282 ± 8 pA; n = 15). There were no significant differences in the rise and the decay time constant of the GABA_A receptor mediated currents in CCI rats and sham controls 1 and 7 days after CCI (Fig. 5).

Fig. 5.

Mild TBI causes a significant decrease in the frequency and amplitude of GABA_A receptor mediated sIPSCs in the CA1 7-days after CCI. sIPSCs were recorded from pyramidal-shaped neurons in the presence of CNQX, D-AP5, SCH50911, and LY 3414953, at a holding potential of −70 mV. Representative examples of recordings obtained in the CA1 are shown in (A) and (B) for Sham and CCI 7-day animals, respectively. (C) Group data showing the change in the percentage frequency and amplitude of sIPSCs from CCI animals relative to sham animals. The frequency and amplitude, but not the rise time and the decay time constant of the sIPSCs were significantly reduced in the CCI group compared to the sham controls. *P < 0.05; n = 15 for each group.

To determine whether the reduction of GABA_A receptor mediated sIPSCs was associated with a decreased responsiveness of postsynaptic GABA_A receptors or reduced presynaptic GABA release, we recorded action potential-independent, miniature IPSCs (mIPSCs) 1 and 7 days after CCI. Action potential-independent mIPSCs were recorded at a holding potential of −70 mV in the presence of CNQX, D-AP5, SCH50911, LY 3414953, and TTX. The frequency and amplitude of GABA_A receptor mediated mIPSCs recorded from sham animals did not differ at either 1- or 7-days after surgery and so we averaged together the amplitude and frequency of all sham animals (data not shown). The frequency and amplitude of GABA_A receptor mediated mIPSCs recorded from CCI animals (mean frequency = 1.9 ± 0.4 Hz; mean amplitude = 64 ± 3; n = 16) did not differ from those recorded from sham animals (mean frequency = 1.7 ± 0.6; mean amplitude = 65 ± 4; n = 16; P > 0.05) 1 day after the surgery. However, we found a 29 ± 3% reduction in frequency (1.34 ± 0.5 Hz; n = 15; P < 0.05) and a 25 ± 3% reduction in the amplitude (48 ± 7 pA; n = 15; P < 0.01) of mIPSCs recorded 7 days after the surgery from CCI rats (Fig. 6).

Fig. 6.

Mild CCI causes a significant decrease in the frequency and amplitude of mIPSCs in the CA1 7-days after CCI. mIPSCs were recorded from pyramidal-shaped neurons in the presence of CNQX, D-AP5, SCH50911, LY 3414953, and TTX at a holding potential of −70 mV. Representative examples of recordings obtained in the CA1 are shown in (A) and (B) for sham and CCI 7 day animals, respectively. (C) Group data showing the change in the percentage frequency and amplitude of mIPSCs from CCI animals relative to sham animals. The frequency and amplitude, but not the rise time and the decay time constant of the sIPSCs were significantly reduced in the CCI group compared to the sham controls. The recorded currents were blocked by the GABA_A receptor antagonist bicuculline (data not shown). *P < 0.05; **P < 0.01; n = 15 for each group.

3.5. Reduced surface expression of GABA_A receptor subunits in the hippocampus

Because we found a decrease in both the amplitude and frequency of GABA_A receptor-mediated IPSCs, we next examined whether reduced surface expression of the GABA_A receptor contributed to the reduced amplitude of the GABA_A receptor mediated mIPSC. To determine whether the surface expression of GABA_A receptor subunits was reduced after CCI, we examined the GABA_A subunits α1, ß2/3, and γ2 from the CA1, subunits that constitute the majority of GABA_A receptors in the brain and are highly expressed in the CA1 (Möhler, 2006). Membrane proteins were isolated by biotinylation assay and levels of specific subunits were quantified by Western blot. After densiometric analysis, Western blot membranes were stripped and re-probed for GLUT1. It has been previously demonstrated that expression of GLUT1 is unaltered following TBI (Hamlin et al., 2001) and therefore is used as a loading control. Seven days after the surgery the surface expression of all three GABA_A subunits examined were reduced in CCI animals compared to sham controls. Ipsilateral to the side of injury, a 67.3% (P < 0.001) reduction of the α1 subunit, a 37.4% (P < 0.001; Fig. 7A) reduction of the ß2/3 subunit, and a 61.7% (P < 0.001; Fig. 7B) reduction of the γ2 subunit were found, whereas a 56.8% (P < 0.001; Fig. 7C) reduction of the α1 subunit, a 44.8% (P < 0.001) reduction of the ß2/3 subunit, and a 54.4% (P < 0.001) reduction of the γ2 subunit was observed in the contralateral hippocampus, indicating that the reduced amplitude in the GABA_A receptor-mediated sIPSCs may be due to a reduction in the number of GABA_A receptors expressed on the surface of the postsynaptic cell membrane (Fig. 7).

Fig. 7.

Surface expression of α1, β2/3, and γ2 GABA_A receptor subunits is reduced in the hippocampus of CCI animals 7 days after mild CCI. Western blot for subunits of (A) α1, (B) β2/3, and (C) γ2 subunits, respectively was performed using biotinylated proteins isolated from the ipsilateral and contralateral sides of sham (open bars) and CCI 7-day animals (black bars). Group data showing the mean ± SE of the ratio between each subunit and GLUT1 optical densities. Top panel: representative Western blot for α1 (A), ß2/3 (B), and γ2 (C) subunits of GABA_A receptors, respectively. Bottom panel: representative Western blot for GLUT1 to ensure equal loading. ***P < 0.001; n = 4 for each group.

3.6. Neuropathology of neurons and interneurons in the CA1 hippocampal region after CCI

To ensure that the injury severity was mild and did not produce any overt morphological damage to the hippocampus, Nissl staining was performed on coronal brain slices and design-based stereology was used to count neurons in the CA1 region (Fig. 8). Similar to our previous results indicating that there were no significant decreases in neurons from ipsilateral and contralateral brain regions (Almeida-Suhett et al., 2014a,2014b), we found that at 24 h (644,113 ± 33,635; P = 0.95; Fig. 8B) and 7 days (626,370 ± 30,858; P = 0.99; Fig. 8C) after CCI, there was no significant decrease in the total number of CA1 neurons compared to controls (630,215 ± 23,603; Fig. 8D).

Fig. 8.

Mild CCI does not produce any significant neuronal loss in the CA1 hippocampal region. Representative photomicrograph of Nissl stained brain sections that contain the CA1 hippocampal region from control animals (A), CCI animals 24 h after injury (B) and CCI animals 7 days after injury (C). (D) Group data showing the mean and standard error off the stereologically estimated total number of neurons in the CA1 1- and 7-days after CCI compared with sham animals. n = 8 for each group.

We next examined whether reductions in the frequency of GABA_A receptor mediated mIPSCs was due to a loss of GABAergic interneurons. The loss of GABAergic interneurons in the hippocampus has been previously associated with memory and learning deficits (Andrews-Zwilling et al., 2010; Leung et al., 2012; Murray et al., 2011) and may be an underlying cause of cognitive impairment following mTBI. Moreover, because CA1 excitability and function are tightly regulated by local interneurons (Antonucci et al., 2012; Harvey and Svoboda, 2007; Hausser et al., 2000), we investigated whether a specific loss of GABAergic interneurons occurs in the CA1 region after CCI. Design-based stereology revealed that there was no significant reduction in the total number of interneurons 1 day after CCI either ipsilateral (13,165 ± 615.3; P = 0.64; n = 8) or contralateral (14,262 ± 621.6; P = 0.998; n = 8) to the site of injury when compared to sham controls (ipsilateral — 14,367 ± 97.78; contralateral — 14,587 ± 281.5; Fig. 9A and B). GAD67-positive cells from 1- and 7-day sham control groups did not display any significant differences and were therefore averaged together (data not shown). However, the total number of GAD67-positive cells was reduced by 23.1% ipsilateral (11,058 ± 559.3; P = 0.026; n = 8) and by 14.96% contralateral (11,974 ± 572.1; P = 0.012; n = 8) to the lesion seven days after CCI when compared to sham animals (ipsilateral — 14,367 ± 97.8; contralateral — 14,587 ± 281.5; Fig. 9A and B). At 24 h after CCI, we did not observe a significant decrease in the ratio of interneurons to neurons (ipsilateral — 0.042 ± 0.001; contralateral — 0.041 ± 0.001; P = 0.8731), but the ratio of GABAergic interneurons to the total number of neurons was significantly reduced from the controls at 7 days (ipsilateral — 0.035 ± 0.003; contralateral — 0.034 ± 0.002; P = 0.035) compared to controls (ipsilateral — 0.045 ± 0.001; contralateral — 0.047 ± 0.002; Fig. 9C). Importantly, our data indicate that the ratio of GABAergic interneurons to the total number of neurons in the CA1 was altered after CCI and further suggests that a mild brain injury selectively led to the loss of GABAergic interneurons.

Fig. 9.

Delayed loss of GABAergic interneurons in the CA1 region 7 days after mild CCI. (A) Representative photomicrographs of GAD-67 immunohistochemically stained GABAergic interneurons in the CA1 of sham (left), 1-day CCI (middle), and 7-day CCI (right) animals. Total magnification is 630×; scale bar, 50 μm. (B) Group data showing the mean and standard error of the stereologically estimated total number of GAD-67-positive cells in the CA1 1- and 7-days after CCI compared with sham. (C) Group data showing the mean and standard error of the ratio of GABAergic interneurons to the total number of neurons in the CA1 1- and 7-days after CCI compared to sham. *P < 0.05; n = 8 for each group.

4. Discussion

Mild traumatic brain injury can lead to cognitive and neuropsychiatric impairments in humans despite the absence of clear structural damage. We show, for the first time, that a single, mild brain injury leads to functional deficits in GABAergic and glutamatergic synaptic transmission in the CA1 hippocampal region, which may contribute to the cognitive deficits observed after a mild TBI. In addition, a cellular mechanism underlying learning and memory, LTP of synaptic transmission, is also impaired after mTBI and may be the result of decreased activation of NMDA but not AMPA receptors. We also observed significantly reduced GABAergic inhibitory synaptic transmission; reductions in mIPSC frequency and amplitude were likely due to the loss of GABAergic interneurons and reductions in the surface expression of the α1, ß2/3, γ2 subunits that make up the GABA_A receptor, respectively.

While in most cases the cognitive, emotional, and behavioral impairments that are observed in patients during an acute period after injury improve, a significant minority of patients (ranging between 1% and 20%; Katz and DeLuca, 1992; Dikmen et al., 2001; Arciniegas et al., 2005) will have impairments that persist well after the initial injury. Memory deficits are often observed early and may persist beyond one year after brain injury (Bohnen et al., 1994; Humayun et al., 1989; Miotto et al., 2010; Rimel et al., 1981; Stulemeijer et al., 2010; Stuss et al., 1985; van der Naalt et al., 1999). Indeed, mTBI is associated with the loss of memories acquired prior to the onset of mTBI (i.e., retrograde amnesia) (Cantu, 2001; Hunkin et al., 1995), but patients suffering from mTBI may also report learning difficulties (Draper and Ponsford, 2008; Miotto et al., 2010). Similar observations have been made in animals, where exposure to a mild brain injury is associated with the loss of previously acquired memories (Lyeth et al., 1990; Whiting and Hamm, 2008) and difficulties in learning new tasks (Darwish et al., 2012; Henninger et al., 2005). Our results suggest that a mild injury that does not induce clear morphological damage to the hippocampus still disrupts memory processes, including the consolidation and retrieval of memories; when animals receive mTBI 1-day after passive avoidance training, they are unable to recall the aversive stimulus they received 7 days later. Our study is in line with groups that have found, though with different TBI models that were more severe than that used in our model, that impairments in passive avoidance testing can be observed as early as the 5th day following surgery (Hogg et al., 1998) and last between 25 and 30 days after injury (Milman et al., 2005; Zhao et al., 2012). The animals may not be able to retrieve this memory because mechanisms associated with the consolidation of the memory may be impaired (Anagnostaras et al., 2001; Brun et al., 2001; Sutherland et al., 2001, 2010). In addition to an inability to recall aversive memories, learning is also impaired in both animals and humans that have sustained an mTBI (Draper and Ponsford, 2008; Miotto et al., 2010). We found that 7 days after mTBI, we were unable to induce or maintain LTP. Thus, it is likely that deficits in synaptic plasticity and impaired synaptic transmission may lead to learning and memory impairments.

Following mTBI, functional deficiencies have been observed in both humans (Christodoulou et al., 2001; Inglese et al., 2005; McAllister et al., 1999) and animals (Cohen et al., 2007; Eakin and Miller, 2012; Greer et al., 2012; Witgen et al., 2005), which may contribute to deficits in learning. We demonstrate that LTP, a cellular model for learning and memory (Malenka and Nicoll, 1999; McGaugh, 2000; Shors and Matzel, 1997), is impaired in the CA1 region after mTBI. Indeed, our results are in agreement with others that the functional deficiencies associated with impaired LTP may be an underlying cause of memory impairment observed after head injury (D'Ambrosio et al., 1998; Miyazaki et al., 1992; Reeves et al., 1995; Sanders et al., 2000; Schwarzbach et al., 2006; Sick et al., 1998; Zhang et al., 2011).

The inability to induce or maintain LTP was not an artifact of decreased synaptic activation observed in slices obtained from CCI animals; rather the inability to induce LTP may be due to impaired synaptic activation of postsynaptic glutamate receptors (Malenka and Nicoll, 1999). AMPA receptors, for example, provide the majority of excitatory current under basal conditions. However, NMDA receptors, which exhibit profound voltage dependence and contribute little to basal postsynaptic responses, are critical in triggering LTP (Malenka and Nicoll, 1999; Shors and Matzel, 1997). Following mTBI, we did not observe any significant differences in the slope of baseline field potentials evoked by stimulating the Schaffer collaterals; nor did we observe any significant difference in AMPA receptor mediated sEPSCs, suggesting that basal excitatory synaptic transmission was unaltered 7 days after mTBI. However, and in agreement with Schwarzbach et al. (2006), we observed significant decreases in pressure-evoked activation of NMDA receptor mediated currents 7 days after a mild brain injury. Decreases in currents mediated by NMDA receptors may have reduced the influx and intracellular accumulation of Ca²⁺ needed to trigger LTP (Malenka and Nicoll, 1999) 7 days after injury and subsequently contributed to the cognitive deficits observed after mTBI.

Deficits in GABAergic inhibitory synaptic transmission may also constitute an underlying mechanism for impaired memory formation and consolidation (Collinson et al., 2002; Izquierdo and Medina, 1991; Sharma and Kulkarni, 1990; Yonkov and Georgiev, 1985; Zarrindast et al., 2002). GABAergic interneurons are essential in modulating synaptic plasticity and for synchronizing activity (known as theta rhythm) in the CA1 region, both of which are important for proper memory function (Antonucci et al., 2012; Harvey and Svoboda, 2007; Hausser et al., 2000). Administration of bicuculline, a GABA_A receptor antagonist, exacerbates memory impairments induced by TBI (O'Dell et al., 2000), whereas administration of drugs that enhance GABAergic neurotransmission improves behavioral and cognitive outcomes of rodents after TBI (Dash et al., 2010; O'Dell et al., 2000).

After a severe TBI, deficits in GABAergic activity have been observed (Mtchedlishvili et al., 2010; Pavlov et al., 2011; Witgen et al., 2005) and may contribute to impaired cognition. Indeed, between one month and six months after a severe brain injury induced by the lateral fluid perfusion model, there was a significant decrease in the frequency of GABA_A receptor mediated mIPSCs in the dentate gyrus, but no changes in tonic inhibition or to the subunit expression of the GABA_A receptor. The decrease in inhibitory synaptic transmission is paralleled by a loss of parvalbumin-immunopositive neurons in the dentate gyrus (Pavlov et al., 2011). By comparison, 90 days after a severe CCI, there is a significant decrease in GABA_A receptor mediated mIPSC frequency in the dentate gyrus, but an increase in tonic inhibition (Mtchedlishvili et al., 2010). Importantly, the results from Pavlov et al. (2011) and Mtchedlishvili et al. (2010) differ from our study in two important ways. First, those two studies used animals that were considerably older (11–13 weeks at the start of experiments) compared to our study, which used animals that were 5–6 weeks at the start of the experiments. Second, Pavlov et al. (2011) and Mtchedlishvili et al. (2010) focused on the long-term changes occurring in the dentate gyrus at least 90 days after CCI/FPI, while we demonstrate, for the first time, that following a single mild brain injury, the significant decrease in GABA_A receptor mediated inhibitory synaptic transmission occurs within 7 days of injury in the CA1 hippocampal region. We found significant reductions in both the frequency and amplitude of GABA_A receptor mediated mIPSCs 7 days after CCI. The decrease in the frequency of mIPSCs was likely due to a selective loss of GABAergic inhibitory interneurons, whereas the decrease in the amplitude of mIPSCs was associated with reduced surface expression of the α1, ß2/3, and γ2 subunits of the GABA_A receptor, subunits that make up the majority of GABA_A receptors in the CA1 (Möhler, 2006). One other known study has found, using the fluid percussion injury model, reduced expression of GABA_A receptor subunits in the dentate gyrus and CA3 region as early as 6 hours after injury (Drexel et al., 2015). Importantly, Drexel et al. reported that the downregulation in GABA_A receptor subunit mRNA recovered within 4 months of injury. Our data are the first to demonstrate significant decreases in the surface expression of different GABA_A receptor subunits in the CA1 hippocampal region after a single, mild injury. However, we cannot exclude the possibility that in our model, the protein expression of the GABA_A subunits may recover to basal levels over time.

Our model also demonstrates that this injury is mild as there was no overt neuronal loss in the CA1 region (Almeida-Suhett et al., 2014a); rather, we found only a selective loss of GABAergic interneurons, as indicated by the loss of GAD-67 immunoreactive neurons and a decrease in the ratio of interneurons to neurons. While it remains unknown why there was a selective loss of GABAergic interneurons, this loss may be the result of a gradual upregulation of d-serine, a positive NMDA receptor modulator (Liu et al., 2009; Mothet et al., 2000; Wolosker, 2007) that selectively binds to GABAergic interneurons and may contribute to the death of GABAergic interneurons. Alternatively, excitotoxicity induced by NMDA receptor hyperactivation (for a review see Obrenovitch and Urenjak, 1997) on GABAergic interneurons or the loss of functional inhibitory synapses, which could lead to posttraumatic epileptic conditions (Stief et al., 2007), could contribute to their selective death. It is also possible that the delayed loss of GAD67 immunoreactivity may not reflect the actual death of GABAergic interneurons but rather may involve a prolonged suppression of GAD67 expression. It is known that the expression of GAD67 can be differentially regulated in response to diverse stimuli (Hill et al., 2010; Kobori and Dash, 2006; Perreault et al., 2012; Sangha et al., 2012). Thus, a sustained downregulation of GAD67 could underlie pathophysiological alteration that occurs in response to mTBI. Regardless of the cause, the observed reduction in GAD67 immunoreactivity has a major implication to the pathophysiology of mTBI. This finding reveals the possibility that loss of GABAergic inhibition may result in a state of neuronal hyper-excitability that may contribute the associated cognitive deficit.

While advances in an understanding of the functional alterations occurring in the CA1 and other brain regions improves patient care, no specific pharmacological therapy for TBI is currently available that would improve neurological, behavioral, and cognitive outcomes (Beauchamp et al., 2008). Indeed, treatments directed at AMPA receptors may be ineffective, as no significant differences in the binding of extracellular excitatory amino acids to AMPA receptors following brain injury have been observed (McIntosh, 1993; Miller et al., 1990). NMDA receptor antagonists may be effective within hours of a TBI to reduce overstimulation, but may be ineffective or counter-effective 7 days after TBI (Beauchamp et al., 2008). On the contrary, stimulation of NMDA receptors within 24 to 48 h post-injury has been found to attenuate neurological deficits and restore cognitive performance (Biegon et al., 2004; Yaka et al., 2007). Finally, because of the loss of GABAergic interneurons and reduced surface expression of GABA_A receptor subunits, both of which contribute to reduced inhibitory synaptic transmission, GABA_A receptor agonists may be ineffective in enhancing inhibitory synaptic transmission and contributing to enhanced cognitive outcomes following mTBI. Thus, the effects of a mild brain injury may lead to lasting behavioral deficits unless treatments are designed to target specific pathophysiological pathways to improve functional outcomes, including preventing the loss of GABAergic inhibition and increase NMDA receptor mediated excitatory synaptic transmission.

5. Conclusions

The results from this study demonstrate that a single mild brain injury leads to cognitive deficits including impaired mechanisms associated with learning (LTP of synaptic transmission). Our data confirm that there is no significant difference in AMPA receptor mediated excitatory synaptic transmission after mTBI, but that within 7 days of mTBI, there is a significant decrease in NMDA receptor mediated currents. Moreover, we found significant deficits in GABA_A receptor mediated inhibitory synaptic transmission, which was due to a significant loss of GABAergic interneurons and reduced surface expression of GABA_A receptors. These findings, therefore, suggest that the learning and memory deficits observed in mTBI victims, may be due to damage to the GABAergic system, but may also be due to deficits in NMDA receptor mediated excitatory synaptic transmission. Our results also provide promising sites for the development of therapeutic interventions directed at enhancing NMDA activity and preventing the loss of GABA_A receptor activity, so that cognitive deficits following a mild brain injury may be ameliorated and patients can recover fully.