Animals

Experiments were performed on adult male mice maintained in standard housing conditions on a 12 h light/dark cycle with food and water provided ad libitum. All protocols and procedures followed the guidelines approved by the University Laboratory Animal Resources at the University of California, Irvine and adhered to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All core and CCI-specific Common Data Elements (CDEs) defined by the Preclinical TBI Working Groups and the NIH/NINDS/DOD CDE Team (https://fitbir.nih.gov/content/preclinical-common-data-elements) can be found in Supplementary Data 2 and 3. For immunohistochemical cell characterization and seizure monitoring experiments, we used CD1 mice as recipients (Charles River Laboratories, cat no. 022). For all other experiments, we used C57BL/6J mice (Jax Stock No: 000664). The C57BL/6J strain was selected for behavior testing on the basis that it performs well in the behavioral paradigms used in our study and seizures are rare52. In contrast, the CD1 strain was selected for seizure testing on the basis that it is the most commonly used mouse strain for seizure monitoring after CCI injury43,44,52. However, this strain is a poor choice for fear memory studies64,65, so we did not perform behavior tests in these animals. Embryonic donor tissue was produced by crossing wild-type CD1 mice to homozygous β-actin EGFP mice (Jax Stock No: 006567) maintained on a CD1 background.

Randomization

Upon arrival, animals were habituated to their housing for up to 1 week before being coded and randomly assigned into uninjured (naive control), vehicle-injected (TBI) or MGE-injected (TBI-MGE) treatment groups. Brain injured mice and age-matched controls were housed together (2–5 animals per cage). The order of injury and cell transplantation (or vehicle injection) was also randomized. For all experiments, animals from each treatment group were run together (i.e., experiments were never staggered in time) and performed in a randomized order by an investigator blinded to treatment.

Blinding

All behavioral assays were conducted between 1 pm and 6 pm during the light phase of the light/dark cycle (lights off at 8 pm; lights on at 8 am). For all behavioral tests, including seizure assessments, mouse identities were coded and conducted by investigators who were blinded to animal treatment (MGE transplantation, vehicle injection or naive control). All behaviors were recorded using a video tracking system and analyzed using ANY-maze software or manually by investigators who were blind to the treatment of the animals. Immunostaining analyses were quantified by an investigator blinded to injury and/or treatment.

Exclusion

For behavior and EEG experiments, transplantations were only considered to be successful if GFP cells were evenly distributed in the recipient brain and confirmed as ≥6000 cells per mouse and ≥1000 μm from the injection site; these criteria were met in all MGE-grafted mice. One cohort of animals (n = 10 mice) was excluded from the y-maze analysis, because the animals were exposed to the first trial of the assay for only 5 min, rather than 10 min for all other cohorts. During the study, a total of four mice died unexpectedly, two from anesthesia overdose during surgery and two of unknown causes between the time of TBI and their use for experimentation. No additional animals were generated to replace these mice. All other brain-injured mice survived and remained otherwise healthy until the day of experimentation.

Brain injury

CCI injury was performed on adult male mice at P5519. Briefly, mice were anesthetized by 1.5% isoflurane inhalation and placed in a stereotaxic frame. The skull was exposed by midline incision, and a 4–5 mm craniotomy was made ~1 mm lateral to the sagittal suture and centered between bregma and lambda. The skull cap was removed without damage to the exposed underlying dura. The contusion device consisted of a computer-controlled, pneumatically driven impactor fitted with a beveled stainless-steel tip 3 mm in diameter (Precision Systems and Instrumentation; TBI-0310). Brain injury was delivered using this device to compress the cortex to a depth of 1.0 mm at a velocity of 3.5 m s−1 and 500 ms duration. The incision was sutured, without replacing the skull cap, and the animal was allowed to recover. A qualitative postoperative health assessment was performed daily for 7 d after TBI and periodically thereafter.

Tissue dissection and transplantation

Ventricular and subventricular layers of the MGE were harvested from E13.5 GFP + embryos. The time point at which the sperm plug was detected was considered E0.5. Embryonic MGE explants were dissected in Leibovitz L-15 medium, mechanically dissociated by repeated pipetting in L-15 medium and concentrated by centrifugation (3 min at 600 × g). Concentrated cell suspensions were front loaded into beveled glass micropipettes (50 μm tip diameter, Wiretol 5 μl, Drummond Scientific) and injected (3 × 104 cells per injection) into the hippocampus of adult brain injured mice 7 d after CCI injury. We chose 7 d post-TBI as the time for transplantation, because a one-week delay between lesion and transplantation has been widely reported as an optimal clinically-relevant therapeutic time-window for transplanting neural stem cells after neurotrauma59. Target coordinates were first verified in a series of preliminary dye injection studies into control and brain injured mice. Cell injections were made into stratum radiatum of the CA3 subfield at the following stereotaxic coordinates: anterior-posterior (AP) 1.75 mm, medial-lateral (ML) 2.3 mm, dorsal-ventral (DV) 1.7 mm. A second group of injections was made into area CA1: AP 1.75 mm, ML 1.75 mm and DV 0.75 mm. Brain injured controls were injected with an equal volume of L-15 media at each site (that is, two sites at the injury epicenter). Cell viability (78.3 ± 2.4%) and concentration were quantified using 0.5 μl of the cell suspension mixed with 24.5 μl of L-15 medium and 25 μl of Trypan Blue (Sigma)33.

Immunostaining

Mice were transcardially perfused with 4% paraformaldehyde (v/v) and free-floating vibratome sections (50 μm) were processed using standard immunostaining procedures33. Primary antibodies and dilutions are provided in Supplementary Table 2. All antibodies have been previously used for immunostaining analysis in brain. For secondary antibodies (1:1000, Invitrogen), we used Alexa 488–conjugated goat antibody to chicken IgG (cat. no. A11039); Alexa 546–conjugated goat antibody to mouse IgG (cat. no. A11030); Alexa 594–conjugated goat antibody to mouse IgG (cat. no. A11005), goat antibody to rabbit IgG (cat. no. A11012), goat antibody to guinea pig IgG (cat. no. A11076) and donkey antibody to goat IgG (cat. no. A11058); and Alexa 647–conjugated goat antibody to rabbit IgG (cat. no. A21244). Sections were then mounted on charged slides (Superfrost plus, Fisher Scientific) with Fluoromount-G that contained DAPI. Confocal images were obtained with an Olympus FV3000 laser scanning microscope. Epifluorescent images were obtained using a Leica DM6 microscope. Brightness and contrast were adjusted manually using Adobe Photoshop, as needed.

Cell quantification

Fluorescently labeled sections (50 μm) were imaged using a Leica DM6 microscope with a ×10 or ×20 objective or Olympus FV3000 confocal microscope with a ×20 or ×40 objective and counted using FIJI (ImageJ)66. All cells that expressed GFP and/or a subtype marker were counted in every sixth coronal section through the entire brain (that is, 300 μm apart). All sections containing grafted cells were analyzed per animal and the values averaged to obtain a mean cell density (cells/mm2). For quantification of GFAP and IBA1 immunostaining, measurements were analyzed at three different locations and the percentage of area above fluorescence threshold was applied using ImageJ according to a previous protocol67. The same settings were used for all sections.

Volumetric analysis

Coronal brain sections (50 μm thick, 300 μm apart) that comprised the entire rostral to caudal extent of the lesion were stained with cresyl violet for histological structure identification. Sections were imaged at ×10 magnification using a Leica DM6 microscope. Eight sections were analyzed per animal, and the % ipsilateral cortex remaining was calculated by outlining the borders of neocortex in both hemispheres using FIJI (ImageJ) and calculating the ratio of ipsilateral to contralateral cortex volume19.

Electrophysiology

Coronal brain slices (300 μm thick) were prepared from recipient mice 45–60 DAT. Slices were submerged in the recording chamber and continuously perfused with oxygenated artificial cerebrospinal fluid (32–34 °C) containing 124 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 4 -H 2 O, 2 mM MgSO 4 -7 H 2 O, 26 mM NaHCO 3 , 10 mM dextrose and 2 mM CaCl 2 (pH 7.2–7.4, 300–305 mOsm kg−1). In MGE-grafted animals, recordings were only performed in slices in which GFP+ could be visually identified. Whole-cell patch-clamp recordings were performed at x40 using an upright, fixed-stage microscope (Olympus BX50WI) equipped with infrared differential interference contrast and epifluorescence optics. For current-clamp recordings and voltage-clamp recordings of EPSCs, patch pipettes (2–4 MΩ) were filled with an internal solution containing 140 mM potassium gluconate, 1 mM NaCl, 5 mM EGTA, 10 mM HEPES, 1 mM MgCl 2 , 1 mM CaCl 2 , 3 mM KOH, 2 mM ATP (pH 7.25, 295 mOsm kg−1). For voltage-clamp recordings of IPSCs, patch pipettes (2–4 MΩ) were filled with an internal solution containing 140 mM CsCl, 11 mM EGTA, 10 mM HEPES, 1 mM MgCl 2 , 2 mM NaATP, 0.5 and mM NaGTP (pH 7.2, 293 mOsm kg−1). Recordings were obtained with a Multiclamp 700B amplifier, filtered at 4 kHz, and recorded to pClamp 10.7 software (Clampfit; Molecular Devices). For current-clamp experiments, cells were held at −70 mV, and electrophysiological properties were measured in response to a series of long (1000 ms) hyperpolarizing and depolarizing current-injections (10 pA steps; range: −80 pA to 400 pA). Voltage-clamp recordings were examined at a holding potential of −70 mV. GABAergic currents were measured in the presence of 1 mM kynurenic acid (Sigma-Aldrich, K3375). Series resistance was typically <15 MΩ and was monitored throughout the recordings. Data were only used for analysis if the series resistance remained <20 MΩ and changed by ≤20% during the recordings. Recordings were not corrected for a liquid junction potential. Resting membrane potentials were measured immediately after breakthrough by temporarily removing the voltage clamp and monitoring voltage. Data analysis was performed using pClamp 10.7 (Clampfit, Molecular Devices), MiniAnalysis 6.0 (Synaptosoft), Microsoft Excel and Sigmaplot 13.1 programs. A 2-min sample recording per cell was used for measuring post-synaptic currents. Events characterized by a typical fast rising phase and exponential decay phase were manually detected using MiniAnalysis. The threshold for event detection was currents with amplitudes greater than three times the root mean square noise level.

Behavior testing

All mice were individually habituated to handling for 2–5 min on 5 consecutive days before testing. Handling took place in the holding room where the mice lived. Mice were then tested in two separate groups. Group 1 was evaluated in the open field test (35 DAT), novel object location (36-37 DAT), Y-maze (44 DAT), social approach (51 DAT), elevated plus maze (58 DAT), fear conditioning (65-67 DAT) and forced swim test (72 DAT). Group 2 was evaluated in contextual fear conditioning at 37–41 DAT. The investigator was not visible to the animals during training or testing.

Object location memory

This task consisted of three phases: habituation, exposure and testing according to a previous protocol49,66. On day 1, animals were habituated individually to the open field arena. Mice were placed in the center of a 40 L × 40 W × 35 H cm open field arena with a vertical marking strip for 10 min under dim overhead lighting conditions (45 lux). For the training session (day 2), two identical objects were placed in the open field, 1 cm from the back wall and mice were placed in the center of the opposite wall. Animals were allowed to explore each object for 10 min. The arena and objects were cleaned with 70% (v/v) EtOH (OLM) or 1% acetic acid (ORM) between trials. A retention test was performed 24 h after the training session (day 3). For OLM, one object was placed in a different location. The objects used were Falcon 50 mL conical centrifuge tubes (Fisher, Cat no. 14-432-22) filled with beach sand. A mouse was scored as exploring an object when its head was oriented toward the object within a distance of 1 cm or when the nose was touching the object. The relative exploration time was recorded and expressed by a discrimination index (DI = [t novel – t familiar ]/[t novel + t familiar ] × 100) where t represents time. Mean exploration times were calculated and the discrimination indexes between treatment groups were compared. To diminish bias, animals from each treatment group were evaluated on the same day in the same arena, and the location of the novel object was counterbalanced across animals.

Y-maze

The y-maze (Panlab, model no. LE847) consisted of three identical enclosed arms (30 L × 6 W × 15 H cm) set at an angle of 120° to each other, with visual cues located above and outside the maze, but not within it. The orientation of the maze and start arm both remained constant, but the other and novel arms were counterbalanced across animals. The test consisted of two trials separated by 90 min. In trial 1 (exposure), mice were first placed at the end of the start arm and allowed to explore the maze for 10 min with one of the arms closed. Mice were returned to their home cage located away from the test apparatus for 90 min. In trial 2 (test), mice were again placed in the start arm and allowed to explore all three arms for 5 min. The floor of the maze was cleaned with 70% EtOH (v/v) between trials. Behavior was videotaped and time spent in each arm was quantified by ANY-maze software. The number and sequence of arms entered were recorded at a later date by an investigator blind to animal treatment or arm identities. Percent alternation was calculated as the number of alternations (entries into three different arms consecutively) divided by the total possible alternations (i.e., the number of arms entries minus 2) and multiplied by 100.

Social approach

Animals were tested in a rectangular three-chambered box with Methacrylate floor and transparent walls (Panlab, model no. LE894). Each chamber was 42 L × 20 W × 22 H cm. The assay consisted of three 10 min phases spaced 30 min apart, two habituation phases and a test phase. Mice were first placed into the center chamber and allowed to explore for 10 min with the doorways into the two side chambers closed. Thirty min later, mice were placed into the center chamber and allowed to explore all three chambers for 10 min along with empty grid enclosures in each side chamber (Panlab, model no. LE894A; 8 × 18 cm, 3 mm bars spaced 7.4 mm apart). Then, 30 min later, an unfamiliar mouse (age-matched CD1 male that had previously been habituated to placement in the small cage) was enclosed in one of the grid enclosures and placed in a side chamber. The grid enclosures allowed nose contact between the bars, but prevented fighting, and were attached to the bottom of the assay with double sided tape. An unfamiliar object (T25 tissue culture flask) was placed in the other enclosure. Mice were placed into the center chamber and allowed to explore the entire social test box for 10 min. Time spent in each chamber was measured using ANY-maze software. The chambers were cleaned with 70% EtOH (v/v) between trials. To diminish bias, animals from each treatment group were evaluated on the same day in the same arena, and the location of the unfamiliar mouse and object were counterbalanced across animals.

Elevated plus maze

The elevated plus maze apparatus (Panlab, model no. LE842) was comprised of two open arms (6 W × 29.5 L × 1.8 H cm) and two enclosed arms (6 W × 29.5 L × 40 H cm), elevated 65 cm above the floor. Mice were placed in the center platform always facing the same open arm. Test duration was 10 min under standard dimmed lighting conditions (45 lux). All data were collected and analyzed automatically using ANY-maze software.

Contextual fear conditioning

Mice were subjected to either a 3 d or 5 d contextual fear conditioning assay. On the training day (day 1), mice were placed into context A of a fear conditioning chamber (Med Associates, model no. MED-VFC-OPTO-M) for 366 s, and a single shock (0.45 mA, duration) was delivered at 180 s, 242 s, and 304 s. On days 2–5, mice were tested for freezing behavior in two different contexts (A and B). All testing sessions were 300 s in duration. Context A was identical to the training conditioning, except that no shocks were presented, and consisted of a stainless steel grid floor, bright white light illumination in the chamber, 70% EtOH odor, lights dimmed to 20 lux in the test room and animals were transported in a transparent plastic container. Context B consisted of the same chamber as context A with some of the visual cues from context A intact (e.g., rectangular box, stainless steel grid floor) but other cues were derived from a distinct context B (i.e., dim light illumination in the chamber, 1% acetic acid odor, red lab tape under the grid floor, black plexiglass triangle insert overhead, bright white lights in the test room and animals were transported in a cardboard box). Animals from each treatment group were evaluated on the same day in the same chamber, and the order of testing was counterbalanced across animals. On days 4 and 5, Compound 21 (1 mg kg−1) was delivered i.p. to mice 30–60 min before testing by an investigator who was not involved in performing the behavior assay. Immediately after each session, mice were placed in a transport cage, walked back to the holding room and placed in a holding cage until experiments on all of the animals from the home cage were performed. Behavioral performance was recorded by digital video camera (sample rate, 30 fps). Freezing responses and duration were quantified using Video Freeze software (Med Associates) with motion threshold set at 18 au and minimum freeze duration set to 30 frames (1 s).

Forced swim test

The mouse forced swim test was conducted identical to the method described previously33. Mice were placed individually into a glass cylinder (height = 40 cm, diameter = 15 cm) containing 22 cm of water (22–23 °C) for 6 min. The total duration of immobility was recorded during the last 4 min of the 6 min testing period. A mouse was considered to be immobile when it floated in an upright position and made only minimal movements to keep its head above water. Trials were video recorded and scored offline by an investigator blind to the experimental outcome of each animal.

Video-EEG

EEG recordings were obtained using a time-locked video-EEG monitoring system (Pinnacle Technologies). Each mouse was anesthetized by 1.5% isoflurane inhalation so that there was no limb-withdrawal response to a noxious foot pinch. Sterile, stainless steel bone screw recording electrodes were placed epidurally through burr holes in the skull (one electrode on either side of the sagittal suture, overlying the hippocampus and ≈1mm from the midline) using surface head-mount EEG hardware (Pinnacle Technologies). Electrodes were cemented in place with a fast-acting adhesive and dental acrylic. Two wires were laid on the shoulder muscles for electromyographic recording. Mice were allowed to recover for at least 7 d before experiments were initiated. Electrographic seizures were defined as high-frequency, high-voltage synchronized polyspike or paroxysmal sharp waves with amplitude more than two-fold greater than background that lasted ≥15 s. Electrographic EEG seizures were analyzed by an investigator blinded to the treatment condition of the mice using Sirenia Seizure software (Pinnacle) and confirmed by offline review of behavioral video recordings. Behaviors were scored according to a modified Racine rating scale in CCI injured mice33,42,43. Experimental mice were monitored for 7–20 d (24 h/d). To diminish bias, brain injured animals receiving vehicle or MGE grafts were monitored together at the same time in the same room.

Statistical analysis

All statistical analyses were performed with GraphPad Prism 8 software. Data were compared by two-tailed t-test, nonparametric Fisher exact test, one-way ANOVA for multiple comparisons or two-way repeated-measures ANOVA. A Bonferroni post hoc test was performed when appropriate. No data were excluded from analysis. Sample sizes were determined based on a priori power analyses and previous MGE transplantation studies33. Data are expressed as mean ± s.e.m., and significance was set at P < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.