Head Injury Treatment With Healthy and Advanced Dietary Supplements: A Pilot Randomized Controlled Trial of the Tolerability, Safety, and Efficacy of Branched Chain Amino Acids in the Treatment of Concussion in Adolescents and Young Adults.

Concussion is a common injury in the adolescent and young adult populations. Although branched chain amino acid (BCAA) supplementation has shown improvements in neurocognitive and sleep function in pre-clinical animal models of mild-to-moderate traumatic brain injury (TBI), to date, no studies have been performed evaluating the efficacy of BCAAs in concussed adolescents and young adults. The goal of this pilot trial was to determine the efficacy, tolerability, and safety of varied doses of oral BCAA supplementation in a group of concussed adolescents and young adults. The study was conducted as a pilot, double-blind, randomized controlled trial of participants ages 11-34 presenting with concussion to outpatient clinics (sports medicine and primary care), urgent care, and emergency departments of a tertiary care pediatric children's hospital and an urban tertiary care adult hospital, between June 24, 2014 and December 5, 2020. Participants were randomized to one of five study arms (placebo and 15 g, 30 g, 45 g, and 54 g BCAA treatment daily) and followed for 21 days after enrollment. Outcome measures included daily computerized neurocognitive tests (processing speed, the a priori primary outcome; and attention, visual learning, and working memory), symptom score, physical and cognitive activity, sleep/wake alterations, treatment compliance, and adverse events. In total, 42 participants were randomized, 38 of whom provided analyzable data. We found no difference in our primary outcome of processing speed between the arms; however, there was a significant reduction in total symptom score (decrease of 4.4 points on a 0-54 scale for every 500 g of study drug consumed, p value for trend = 0.0036, [uncorrected]) and return to physical activity (increase of 0.503 points on a 0-5 scale for every 500 g of study drug consumed, p value for trend = 0.005 [uncorrected]). There were no serious adverse events. Eight of 38 participants reported a mild (not interfering with daily activity) or moderate (limitation of daily activity) adverse event; there were no differences in adverse events by arm, with only two reported mild adverse events (both gastrointestinal) in the highest (45 g and 54 g) BCAA arms. Although limited by slow enrollment, small sample size, and missing data, this study provides the first demonstration of efficacy, as well as safety and tolerability, of BCAAs in concussed adolescents and young adults; specifically, a dose-response effect in reducing concussion symptoms and a return to baseline physical activity in those treated with higher total doses of BCAAs. These findings provide important preliminary data to inform a larger trial of BCAA therapy to expedite concussion recovery.

Temporal and structural sensitivities of major biomarkers for detecting neuropathology after traumatic brain injury in the mouse.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality, especially in teenagers to young adults. In recent decades, different biomarkers and/or staining protocols have been employed to evaluate the post-injury development of pathological structures, but they have produced many contradictory findings. Since correctly identifying the underlying neuroanatomical changes is critical to advancing TBI research, we compared three commonly used markers for their ability to detect TBI pathological structures: Fluoro-Jade C, the rabbit monoclonal antibody Y188 against amyloid precursor protein and the NeuroSilver kit were used to stain adjacent slices from naïve or injured mouse brains harvested at different time points from 30 min to 3 months after lateral fluid percussion injury. Although not all pathological structures were stained by all markers at all time points, we found damaged neurons and deformed dendrites in gray matter, punctate and perivascular structures in white matter, and axonal blebs and Wallerian degeneration in both gray and white matter. The present study demonstrates the temporal and structural sensitivities of the three biomarkers: each marker is highly effective for a set of pathological structures, each of which in turn emerges at a particular time point. Furthermore, the different biomarkers showed different abilities at detecting identical types of pathological structures. In contrast to previous studies that have used a single biomarker at a single time range, the present report strongly recommends that a combination of different biomarkers should be adopted and different time points need to be checked when assessing neuropathology after TBI.

Mild Traumatic Brain Injury Affects Orexin/Hypocretin Physiology Differently in Male and Female Mice.

Traumatic brain injury (TBI) is known to affect the physiology of neural circuits in several brain regions, which can contribute to behavioral changes after injury. Disordered sleep is a behavior that is often seen after TBI, but there is little research into how injury affects the circuitry that contributes to disrupted sleep regulation. Orexin/hypocretin neurons (hereafter referred to as orexin neurons) located in the lateral hypothalamus normally stabilize wakefulness in healthy animals and have been suggested as a source of dysregulated sleep behavior. Despite this, few studies have examined how TBI affects orexin neuron circuitry. Further, almost no animal studies of orexin neurons after TBI have included female animals. Here, we address these gaps by studying changes to orexin physiology using ex vivo acute brain slices and whole-cell patch clamp recording. We hypothesized that orexin neurons would have reduced afferent excitatory activity after injury. Ultimately, this hypothesis was supported but there were additional physiological changes that occurred that we did not originally hypothesize. We studied physiological properties in orexin neurons approximately 1 week after mild traumatic brain injury (mTBI) in 6-8-week-old male and female mice. mTBI was performed with a lateral fluid percussion injury between 1.4 and 1.6 atmospheres. Mild TBI increased the size of action potential afterhyperpolarization in orexin neurons from female mice, but not male mice and reduced the action potential threshold in male mice, but not in female mice. Mild TBI reduced afferent excitatory activity and increased afferent inhibitory activity onto orexin neurons. Alterations in afferent excitatory activity occurred in different parameters in male and female animals. The increased afferent inhibitory activity after injury is more pronounced in recordings from female animals. Our results indicate that mTBI changes the physiology of orexin neuron circuitry and that these changes are not the same in male and female animals.

Neuronal somatic plasmalemmal permeability and dendritic beading caused by head rotational traumatic brain injury in pigs-An exploratory study.

Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY+ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY+ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY+ and LY- cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.

Detection and verification of neurodegeneration after traumatic brain injury in the mouse: Immunohistochemical staining for amyloid precursor protein.

Previous studies of human traumatic brain injury (TBI) have shown diffuse axonal injury as varicosities or spheroids in white matter (WM) bundles when using immunoperoxidase-ABC staining with 22C11, a mouse monoclonal antibody against amyloid precursor protein (APP). These findings have been interpreted as TBI-induced axonal pathology. In a mouse model of TBI however, when we used immunofluorescent staining with 22C11, as opposed to immunoperoxidase staining, we did not observe varicosities or spheroids. To explore this discrepancy, we performed immunofluorescent staining with Y188, an APP knockout-validated rabbit monoclonal that shows baseline immunoreactivity in neurons and oligodendrocytes of non-injured mice, with some arranged-like varicosities. In gray matter after injury, Y188 intensely stained axonal blebs. In WM, we encountered large patches of heavily stained puncta, heterogeneous in size. Scattered axonal blebs were also identified among these Y188-stained puncta. To assess the neuronal origin of Y188 staining after TBI we made use of transgenic mice with fluorescently labeled neurons and axons. A close correlation was observed between Y188-stained axonal blebs and fluorescently labeled neuronal cell bodies/axons. By contrast, no correlation was observed between Y188-stained puncta and fluorescent axons in WM, suggesting that these puncta in WM did not originate from axons, and casting further doubt on the nature of previous reports with 22C11. As such, we strongly recommend Y188 as a biomarker for detecting damaged neurons and axons after TBI. With Y188, stained axonal blebs likely represent acute axonal truncations that may lead to death of the parent neurons. Y188-stained puncta in WM may indicate damaged oligodendrocytes, whose death and clearance can result in secondary demyelination and Wallerian degeneration of axons. We also provide evidence suggesting that 22C11-stained varicosities or spheroids previously reported in TBI patients might be showing damaged oligodendrocytes, due to a cross-reaction between the ABC kit and upregulated endogenous biotin.

Maintenance of a Lateral Fluid Percussion Injury Device.

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and disability in children and young adults. TBI is caused by a sudden force applied to the head and, to better understand human TBI and its underlying mechanisms, experimental injury models are necessary. Lateral fluid percussion injury (LFPI) is a commonly used injury model due to similarities in the pathological changes found in human TBI compared to LFPI, including hemorrhages, vascular disruption, neurological deficits, and neuron loss. LFPI employs a pendulum and a fluid-filled cylinder, the latter having a moveable piston at one end, and a Luer lock connection to stiff, fluid-filled tubing at the other end. Preparation of the animal involves performing a craniectomy and attaching a Luer hub over the site. The next day, the tubing from the injury device is connected to the Luer hub on the animal's skull and the pendulum is raised to a specified height and released. The impact of the pendulum with the piston generates a pressure pulse which is transmitted to the intact dura mater of the animal via the tubing and produces the experimental TBI. Proper care and maintenance are essential for the LFPI device to function reliably, as the character and severity of the injury can vary greatly depending on the condition of the device. Here, we demonstrate how to properly clean, fill, and assemble the LFPI device, and ensure that it is adequately maintained for optimal results.

Dietary Supplementation With Branched Chain Amino Acids to Improve Sleep in Veterans With Traumatic Brain Injury: A Randomized Double-Blind Placebo-Controlled Pilot and Feasibility Trial.

Study Objectives: Traumatic brain injury (TBI) is associated with chronic sleep disturbances and cognitive impairment. Our prior preclinical work demonstrated dietary supplementation with branched chain amino acids (BCAA: leucine, isoleucine, and valine), precursors to de novo glutamate production, restored impairments in glutamate, orexin/hypocretin neurons, sleep, and memory in rodent models of TBI. This pilot study assessed the feasibility and preliminary efficacy of dietary supplementation with BCAA on sleep and cognition in Veterans with TBI. Methods: Thirty-two Veterans with TBI were prospectively enrolled in a randomized, double-blinded, placebo-controlled trial comparing BCAA (30 g, b.i.d. for 21-days) with one of two placebo arms (microcrystalline cellulose or rice protein, both 30 g, b.i.d. for 21-days). Pre- and post-intervention outcomes included sleep measures (questionnaires, daily sleep/study diaries, and wrist actigraphy), neuropsychological testing, and blood-based biomarkers related to BCAA consumption. Results: Six subjects withdrew from the study (2/group), leaving 26 remaining subjects who were highly adherent to the protocol (BCAA, 93%; rice protein, 96%; microcrystalline, 95%; actigraphy 87%). BCAA were well-tolerated with few side effects and no adverse events. BCAA significantly improved subjective insomnia symptoms and objective sleep latency and wake after sleep onset on actigraphy. Conclusion: Dietary supplementation with BCAA is a mechanism-based, promising intervention that shows feasibility, acceptability, and preliminary efficacy to treat insomnia and objective sleep disruption in Veterans with TBI. A larger scale randomized clinical trial is warranted to further evaluate the efficacy, dosing, and duration of BCAA effects on sleep and other related outcome measures in individuals with TBI. Clinical Trial Registration: [http://clinicaltrials.gov/], identifier [NCT03990909].

Endogenous Opioid Dynorphin Is a Potential Link between Traumatic Brain Injury, Chronic Pain, and Substance Use Disorder.

Traumatic brain injury (TBI) is a serious public health problem associated with numerous physical and neuropsychiatric comorbidities. Chronic pain is prevalent and interferes with post-injury functioning and quality of life, whereas substance use disorder (SUD) is the third most common neuropsychiatric diagnosis after TBI. Neither of these conditions has a clear mechanistic explanation based on the known pathophysiology of TBI. Dynorphin is an endogenous opioid neuropeptide that is significantly dysregulated after TBI. Both dynorphin and its primary receptor, the ĸ-opioid receptor (KOR), are implicated in the neuropathology of chronic pain and SUD. Here, we review the known roles of dynorphin and KORs in chronic pain and SUDs. We synthesize this information with our current understanding of TBI and highlight potential mechanistic parallels between and across conditions that suggest a role for dynorphin in long-term sequelae after TBI. In pain studies, dynorphin/KOR activation has either antinociceptive or pro-nociceptive effects, and there are similarities between the signaling pathways influenced by dynorphin and those underlying development of chronic pain. Moreover, the dynorphin/KOR system is considered a key regulator of the negative affective state that characterizes drug withdrawal and protracted abstinence in SUD, and molecular and neurochemical changes observed during the development of SUD are mirrored by the pathophysiology of TBI. We conclude by proposing hypotheses and directions for future research aimed at elucidating the potential role of dynorphin/KOR in chronic pain and/or SUD after TBI.

Blocking Cross-Species Secondary Binding When Performing Double Immunostaining With Mouse and Rat Primary Antibodies.

Immunostaining is a powerful technique and widely used to identify molecules in tissues and cells, although critical steps are necessary to block cross-reaction. Here we focused on an overlooked cross immunoreactivity issue where a secondary antibody (secondary) cross-reacts with a primary antibody (primary) from a different species. We first confirmed the previously reported cross-species binding of goat anti-mouse secondary to rat primary. This was accomplished by staining with a rat primary against glial fibrillary acidic protein (GFAP) and visualizing with goat (or donkey) anti-mouse secondary. We then further revealed the converse cross-species binding by staining with a mouse primary against neuronal nuclear protein (NeuN) and visualizing with anti-rat secondaries. We speculate that mouse and rat primaries share antigenicity, enabling either secondary to recognize either primary. To block this cross-species binding in double staining experiments, we compared three protocols using mouse anti-NeuN and rat anti-GFAP, two primaries whose antigens have non-overlapping distributions in brain tissues. Simultaneous staining resulted in cross-species astrocytic staining (anti-mouse secondary to rat anti-GFAP primary) but no cross-species neuronal staining (anti-rat secondary to mouse anti-NeuN primary). Cross-species astrocytic staining was missing after sequential same-species staining with mouse anti-NeuN primary, followed by rat anti-GFAP. However, cross-species astrocytic staining could not be diminished after sequential same-species staining with rat anti-GFAP primary, followed by mouse anti-NeuN. We thus hypothesize that a competition exists between anti-mouse and anti-rat secondaries in their binding to both primaries. Single staining for NeuN or GFAP visualized with dual secondaries at different dilution ratio supported this hypothesis.

Variability in atlas registration of optical intrinsic signal imaging and its effect on functional connectivity analysis.

To compare neuroimaging data between subjects, images from individual sessions need to be aligned to a common reference or "atlas." Atlas registration of optical intrinsic signal imaging of mice, for example, is commonly performed using affine transforms with parameters determined by manual selection of canonical skull landmarks. Errors introduced by such procedures have not previously been investigated. We quantify the variability that arises from this process and consequent errors from misalignment that affect interpretation of functional neuroimaging data. We propose an improved method, using separately acquired high-resolution images and demonstrate improvements in variability and alignment using this method.

Wavelength censoring for spectroscopy in optical functional neuroimaging.

Optical neuromonitoring provides insight into neurovascular physiology and brain structure and function. These methods rely on spectroscopy to relate light absorption changes to variation of concentrations of physiologic chromophores such as oxy- and deoxyhemoglobin. In clinical or preclinical practice, data quality can vary significantly across wavelengths. In such situations, standard spectroscopic methods may perform poorly, resulting in data loss and limiting field-of-view. To address this issue, and thereby improve the robustness of optical neuromonitoring, we develop, in this manuscript, novel methods to perform spectroscopy even when data quality exhibits wavelength-dependent spatial variation. We sought to understand the impact of spatial, wavelength-based censoring on the physiologic accuracy and utility of hemoglobin spectroscopy. The principles of our analysis are quite general, but to make the methodology tangible we focused on optical intrinsic signal imaging of resting-state functional connectivity in mice. Starting with spectroscopy using four sources, all possible subset spectroscopy matrices were assessed theoretically, using simulated data, and using experimental data. These results were compared against the use of the full spectroscopy matrix to determine which subsets yielded robust results. Our results demonstrated that accurate calculation of changes in hemoglobin concentrations and the resulting functional connectivity network maps was possible even with censoring of some wavelengths. Additionally, we found that the use of changes in total hemoglobin (rather than oxy- or deoxyhemoglobin) yielded results more robust to experimental noise and allowed for the preservation of more data. This new and rigorous image processing method should improve the fidelity of clinical and preclinical functional neuroimaging studies.

Traumatic Brain Injury Diminishes Feedforward Activation of Parvalbumin-Expressing Interneurons in the Dentate Gyrus.

Traumatic brain injury (TBI) is associated with aberrant network hyperexcitability in the dentate gyrus (DG). GABAAergic parvalbumin-expressing interneurons (PV-INs) in the DG regulate network excitability with strong, perisomatic inhibition, although the posttraumatic effects on PV-IN function after TBI are not well understood. In this study, we investigated physiological alterations in PV-INs one week after mild lateral fluid percussion injury (LFPI) in mice. PV-IN cell loss was observed in the dentate hilus after LFPI, with surviving PV-INs showing no change in intrinsic membrane properties. Whole-cell voltage clamp recordings in PV-INs revealed alterations in both EPSCs and IPSCs (EPSCs/IPSCs). Evoked EPSCs (eEPSCs) in PV-INs from perforant path electrical stimulation were diminished after injury but could be recovered with application of a GABAA-receptor antagonist. Furthermore, current-clamp recordings using minimal perforant path stimulation demonstrated a decrease in evoked PV-IN action potentials (APs) after LFPI, which could be restored by blocking GABAAergic inhibition. Together, these findings suggest that injury alters synaptic input onto PV-INs, resulting in a net inhibitory effect that reduces feedforward PV-IN activation in the DG. Decreased PV-IN activation suggests a potential mechanism of DG network hyperexcitability contributing to hippocampal dysfunction after TBI.

Unsupervised Machine Learning Reveals Novel Traumatic Brain Injury Patient Phenotypes with Distinct Acute Injury Profiles and Long-Term Outcomes.

The heterogeneity of traumatic brain injury (TBI) remains a core challenge for the success of interventional clinical trials. Data-driven approaches for patient stratification may help to identify TBI patient phenotypes during the acute injury period as well as facilitate targeted trial patient enrollment and analysis of treatment efficacy. In this study, we implemented an unsupervised machine learning approach to identify TBI subpopulations at injury baseline using data from 1213 TBI patients who participated in the Citicoline Brain Injury Treatment Trial (COBRIT) Trial. A wrapper framework utilizing generalized low-rank models automatically selected relevant clinical features that were subsequently used to cluster patients using a partitioning around medoids clustering algorithm. Using this approach, we identified three patient phenotypes with unique clinical injury profiles based on a subset of acute injury features. Phenotype-specific differences in long-term functional outcome trajectories were respectively observed at 3 and 6 months after injury. In comparison, when patients were grouped by baseline Glasgow Coma Scale (GCS), no differences in baseline clinical feature profiles or long-term outcomes were observed. To test phenotype reproducibility in an external validation data set, we used a K-nearest neighbors algorithm to classify subjects in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Pilot data set into corresponding phenotypes, then measured the Gower's dissimilarities between TRACK-TBI and COBRIT subjects in each phenotype. No significant differences were found between trial subjects within two phenotypes, suggesting that these phenotypes may be generalizable within a broad range of TBI severity. Further, Extended Glasgow Outcome Scale (GOS-E) outcomes in the TRACK-TBI data set similarly demonstrated phenotype-specific differences in long-term outcomes. Our results suggest that unsupervised machine learning is a promising and effective approach for discovery of novel injury subpopulations over the conventional GCS-based method, and may improve patient selection in future TBI clinical trials.

Brain segmentation, spatial censoring, and averaging techniques for optical functional connectivity imaging in mice.

Resting-state functional connectivity analysis using optical neuroimaging holds the potential to be a powerful bridge between mouse models of disease and clinical neurologic monitoring. However, analysis techniques specific to optical methods are rudimentary, and algorithms from magnetic resonance imaging are not always applicable to optics. We have developed visual processing tools to increase data quality, improve brain segmentation, and average across sessions with better field-of-view. We demonstrate improved performance using resting-state optical intrinsic signal from normal mice. The proposed methods increase the amount of usable data from neuroimaging studies, improve image fidelity, and should be translatable to human optical neuroimaging systems.

Neuronal network dysfunction precedes storage and neurodegeneration in a lysosomal storage disorder.

Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage-sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs before notable lysosomal storage and neuronal loss in 2 CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding - hippocampal sharp-wave ripples. This discovery, which is also seen in Alzheimer's disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describe new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system and highlight the need for new therapeutic interventions targeting early circuit defects.

Memory Deficit in an Object Location Task after Mild Traumatic Brain Injury Is Associated with Impaired Early Object Exploration and Both Are Restored by Branched Chain Amino Acid Dietary Therapy.

The relation between traumatic brain injury (TBI) and memory dysfunction is well established, yet imprecise. Here, we investigate whether mild TBI causes a specific deficit in spatial episodic memory. Fifty-eight (29 TBI, 29 sham) mice were run in a spatial recognition task. To determine which phase of memory might be affected in our task, we assessed rodent performance at three different delay times (3 min, 1 h, and 24 h). We found that sham and TBI mice performed equally well at 3 min, but TBI mice had significantly impaired spatial recognition memory after a delay time of 1 h. Neither sham nor injured mice remembered the test object locations after a 24-h delay. In addition, the TBI-specific impairment was accompanied by a decrease in exploratory behavior during the first 3 mins of the initial exposure to the test objects. These memory and exploratory behavioral deficits were linked as branched-chain amino acid (BCAA) dietary therapy restored both memory performance and normal exploratory behavior. Our findings 1) support the use of BCAA therapy as a potential treatment for mild TBI and 2) suggest that poor memory performance post-TBI is associated with a deficit in exploratory behavior that is likely to underlie the encoding needed for memory formation.

Dietary therapy restores glutamatergic input to orexin/hypocretin neurons after traumatic brain injury in mice.

Study Objectives: In previous work, dietary branched-chain amino acid (BCAA) supplementation, precursors to de novo glutamate and γ-aminobutyric acid (GABA) synthesis, restored impaired sleep-wake regulation and orexin neuronal activity following traumatic brain injury (TBI) in mice. TBI was speculated to reduce orexin neuronal activity through decreased regional excitatory (glutamate) and/or increased inhibitory (GABA) input. Therefore, we hypothesized that TBI would decrease synaptic glutamate and/or increase synaptic GABA in nerve terminals contacting orexin neurons, and BCAA supplementation would restore TBI-induced changes in synaptic glutamate and/or GABA. Methods: Brain tissue was processed for orexin pre-embed diaminobenzidine labeling and glutamate or GABA postembed immunogold labeling. The density of glutamate and GABA immunogold within presynaptic nerve terminals contacting orexin-positive lateral hypothalamic neurons was quantified using electron microscopy in three groups of mice (n = 8 per group): Sham/noninjured controls, TBI without BCAA supplementation, and TBI with BCAA supplementation (given for 5 days, 48 hr post-TBI). Glutamate and GABA were also quantified within the cortical penumbral region (layer VIb) adjacent to the TBI lesion. Results: In the hypothalamus and cortex, TBI decreased relative glutamate density in presynaptic terminals making axodendritic contacts. However, BCAA supplementation only restored relative glutamate density within presynaptic terminals contacting orexin-positive hypothalamic neurons. BCAA supplementation did not change relative glutamate density in presynaptic terminals making axosomatic contacts, or relative GABA density in presynaptic terminals making axosomatic or axodendritic contacts, within either the hypothalamus or cortex. Conclusions: These results suggest TBI compromises orexin neuron function via decreased glutamate density and highlight BCAA supplementation as a potential therapy to restore glutamate density to orexin neurons.

Diminished Dentate Gyrus Filtering of Cortical Input Leads to Enhanced Area Ca3 Excitability after Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) disrupts hippocampal function and can lead to long-lasting episodic memory impairments. The encoding of episodic memories relies on spatial information processing within the hippocampus. As the primary entry point for spatial information into the hippocampus, the dentate gyrus is thought to function as a physiological gate, or filter, of afferent excitation before reaching downstream area Cornu Ammonis (CA3). Although injury has previously been shown to alter dentate gyrus network excitability, it is unknown whether mTBI affects dentate gyrus output to area CA3. In this study, we assessed hippocampal function, specifically the interaction between the dentate gyrus and CA3, using behavioral and electrophysiological techniques in ex vivo brain slices 1 week following mild lateral fluid percussion injury (LFPI). Behaviorally, LFPI mice were found to be impaired in an object-place recognition task, indicating that spatial information processing in the hippocampus is disrupted. Extracellular recordings and voltage-sensitive dye imaging demonstrated that perforant path activation leads to the aberrant spread of excitation from the dentate gyrus into area CA3 along the mossy fiber pathway. These results suggest that after mTBI, the dentate gyrus has a diminished capacity to regulate cortical input into the hippocampus, leading to increased CA3 network excitability. The loss of the dentate filtering efficacy reveals a potential mechanism by which hippocampal-dependent spatial information processing is disrupted, and may contribute to memory dysfunction after mTBI.

A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse.

The hippocampus plays a critical role in learning and memory and higher cognitive functions, and its dysfunction has been implicated in various neuropathological disorders. Electrophysiological recording undertaken in live brain slices is one of the most powerful tools for investigating hippocampal cellular and network activities. The plane for cutting the slices determines which afferent and/or efferent connections are best preserved, and there are three commonly used slices: hippocampal-entorhinal cortex (HEC), coronal and transverse. All three slices have been widely used for studying the major afferent hippocampal pathways including the perforant path (PP), the mossy fibers (MFs) and the Schaffer collaterals (SCs). Surprisingly, there has never been a systematic investigation of the anatomical and functional consequences of slicing at a particular angle. In the present study, we focused on how well fiber pathways are preserved from the entorhinal cortex (EC) to the hippocampus, and within the hippocampus, in slices generated by sectioning at different angles. The postmortem neural tract tracer 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label afferent fibers to hippocampal principal neurons in fixed slices or whole brains. Laser scanning confocal microscopy was adopted for imaging DiI-labeled axons and terminals. We demonstrated that PP fibers were well preserved in HEC slices, MFs in both HEC and transverse slices and SCs in all three types of slices. Correspondingly, field excitatory postsynaptic potentials (fEPSPs) could be consistently evoked in HEC slices when stimulating PP fibers and recorded in stratum lacunosum-moleculare (sl-m) of area CA1, and when stimulating the dentate granule cell layer (gcl) and recording in stratum lucidum (sl) of area CA3. The MF evoked fEPSPs could not be recorded in CA3 from coronal slices. In contrast to our DiI-tracing data demonstrating severely truncated PP fibers in coronal slices, fEPSPs could still be recorded in CA1 sl-m in this plane, suggesting that an additional afferent fiber pathway other than PP might be involved. The present study increases our understanding of which hippocampal pathways are best preserved in the three most common brain slice preparations, and will help investigators determine the appropriate slices to use for physiological studies depending on the subregion of interest.