Alzheimer's-linked axonal changes accompany elevated antidromic action potential failure rate in aged mice.

Alzheimer's disease (AD) affects both grey and white matter (WM), but considerably more is known about the former. Interestingly, WM disruption has been consistently observed and thoroughly described using imaging modalities, particularly MRI which has shown WM functional disconnections between the hippocampus and other brain regions during AD pathogenesis when early neurodegeneration and synapse loss are also evident. Nonetheless, high-resolution structural and functional analyses of WM during AD pathogenesis remain scarce. Given the importance of the myelinated axons in the WM for conveying information across brain regions, such studies will provide valuable information on the cellular drivers and consequences of WM disruption that contribute to the characteristic cognitive decline of AD. Here, we employed a multi-scale approach to investigate hippocampal WM disruption during AD pathogenesis and determine whether hippocampal WM changes accompany the well-documented grey matter losses. Our data indicate that ultrastructural myelin disruption is elevated in the alveus in human AD cases and increases with age in 5xFAD mice. Unreliable action potential propagation and changes to sodium channel expression at the node of Ranvier co-emerged with this deterioration. These findings provide important insight to the neurobiological substrates and functional consequences of decreased WM integrity and are consistent with the notion that hippocampal disconnection contributes to cognitive changes in AD.

Chronic, Reusable, Multiday Neuropixels Recordings during Free-Moving Operant Behavior.

Electrophysiological recording is a powerful technique to examine neuronal substrates underlying cognition and behavior. Neuropixels probes provide a unique capacity to capture neuronal activity across many brain areas with high spatiotemporal resolution. Neuropixels are also expensive and optimized for acute, head-fixed use, both of which limit the types of behaviors and manipulations that can be studied. Recent advances have addressed the cost issue by showing chronic implant, explant, and reuse of Neuropixels probes, but the methods were not optimized for use in free-moving behavior. There were specific needs for improvement in cabling/connection stability. Here, we extend that work to demonstrate chronic Neuropixels recording, explant, and reuse in a rat model during fully free-moving operant behavior. Similar to prior approaches, we house the probe and headstage within a 3D-printed housing that avoids direct fixation of the probe to the skull, enabling eventual explant. We demonstrate innovations to allow chronic headstage connection with protection against environmental factors and a more stable cabling setup to reduce the tension that can interrupt recording. We demonstrate this approach with rats performing two different behavioral tasks, in each case showing: (1) chronic single- or dual-probe recordings in free-moving rats in operant chambers and (2) reusability of Neuropixels 1.0 probes with continued good single-unit yield after retrieval and reimplant. We thus demonstrate the potential for Neuropixels recordings in a wider range of species and preparations.

The TgF344-AD rat: behavioral and proteomic changes associated with aging and protein expression in a transgenic rat model of Alzheimer's disease.

Animal models of Alzheimer's Disease (AD) are attractive tools for preclinical, prodromal drug testing. The TgF344-AD (Tg) rat exhibits cognitive deficits and 5 major hallmarks of AD. Here we show that spatial water maze (WMZ) memory deficits and proteomic differences in dorsal CA1 were present in young Tg rats. Aged learning-unimpaired (AU) and aged learning-impaired (AI) proteome associated changes were identified and differed by sex. Levels of phosphorylated tau, reactive astrocytes and microglia were significantly increased in aged Tg rats and correlated with the WMZ learning index (LI); in contrast, no significant correlation was present between amyloid plaques or insoluble Aß levels and LI. Neuroinflammatory markers were also significantly correlated with LI and increased in female Tg rats. The anti-inflammatory marker, triggering receptor expressed on myeloid cells-2 (TREM2), was significantly reduced in aged impaired Tg rats and correlated with LI. Identifying and understanding mechanisms that allow for healthy aging by overcoming genetic drivers for AD, and/or promoting drivers for successful aging, are important for developing successful therapeutics against AD.

Place cells are nonrandomly clustered by field location in CA1 hippocampus.

A challenge in both modern and historic neuroscience has been achieving an understanding of neuron circuits, and determining the computational and organizational principles that underlie these circuits. Deeper understanding of the organization of brain circuits and cell types, including in the hippocampus, is required for advances in behavioral and cognitive neuroscience, as well as for understanding principles governing brain development and evolution. In this manuscript, we pioneer a new method to analyze the spatial clustering of active neurons in the hippocampus. We use calcium imaging and a rewarded navigation task to record from 100 s of place cells in the CA1 of freely moving rats. We then use statistical techniques developed for and in widespread use in geographic mapping studies, global Moran's I, and local Moran's I to demonstrate that cells that code for similar spatial locations tend to form small spatial clusters. We present evidence that this clustering is not the result of artifacts from calcium imaging, and show that these clusters are primarily formed by cells that have place fields around previously rewarded locations. We go on to show that, although cells with similar place fields tend to form clusters, there is no obvious topographic mapping of environmental location onto the hippocampus, such as seen in the visual cortex. Insights into hippocampal organization, as in this study, can elucidate mechanisms underlying motivational behaviors, spatial navigation, and memory formation.

Learning-related changes in cellular activity within mouse dentate gyrus during trace eyeblink conditioning.

Because the dentate gyrus serves as the first site for information processing in the hippocampal trisynaptic circuit, it an important structure for the formation of associative memories. Previous findings in rabbit had recorded populations of cells within dentate gyrus that may bridge the temporal gap between stimuli to support memory formation during trace eyeblink conditioning, an associative learning task. However, this previous work was unable to identify the types of cells demonstrating this type of activity. To explore these changes further, we did in vivo single-neuron recording in conjunction with physiological determination of cell types to investigate the functional role of granule cells, mossy cells, and interneurons in dentate gyrus during learning. Tetrode recordings were performed in young-adult mice during training on trace eyeblink conditioning, a hippocampal-dependent temporal associative memory task. Conditioned mice were able to successfully learn the task, with male mice learning at a faster rate than female mice. In the conditioned group, granule cells tended to show an increase in firing rate during conditioned stimulus presentation while mossy cells showed a decrease in firing rate during the trace interval and the unconditioned stimulus. Interestingly, populations of interneurons demonstrated learning-related increases and decreases in activity that began at onset of the conditioned stimulus and persisted through the trace interval. The current study also found a significant increase in theta power during stimuli presentation in conditioned animals, and this change in theta decreased over time. Ultimately, these data suggest unique involvement of granule cells, mossy cells, and interneurons in dentate gyrus in the formation of a trace associative memory. This work expands our knowledge of dentate gyrus function, helping to discern how aging and disease might disrupt this process.

Sex-Dependent Effects of Chronic Microdrive Implantation on Acquisition of Trace Eyeblink Conditioning.

Neuroscience techniques, including in vivo recording, have allowed for a great expansion in knowledge; however, this technology may also affect the very phenomena researchers set out to investigate. Including both female and male mice in our associative learning experiments shed light on sex differences on the impact of chronic implantation of tetrodes on learning. While previous research showed intact female mice acquired trace eyeblink conditioning faster than male and ovariectomized females, implantation of chronic microdrive arrays showed sexually dimorphic effects on learning. Microdrive implanted male mice acquired the associative learning paradigm faster than both intact and ovariectomized females. These effects were not due to the weight of the drive alone, as there were no significant sex-differences in learning of animals that received "dummy drive" implants without tetrodes lowered into the brain. Tandem mass tag mass spectrometry and western blot analysis suggest that significant alterations in the MAPK pathway, acute inflammation, and brain derived neurotrophic factor may underlie these observed sex- and surgery-dependent effects on learning.

In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields.

Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3±5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.Significance statement:In vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3±5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.

Aging-Related Alterations to Persistent Firing in the Lateral Entorhinal Cortex Contribute to Deficits in Temporal Associative Memory.

With aging comes a myriad of different disorders, and cognitive decline is one of them. Studies have consistently shown a decline amongst aged subjects in their ability to acquire and maintain temporal associative memory. Defined as the memory of the association between two objects that are separated in time, temporal associative memory is dependent on neocortical structures such as the prefrontal cortex and temporal lobe structures. For this memory to be acquired, a mental trace of the first stimulus is necessary to bridge the temporal gap so the two stimuli can be properly associated. Persistent firing, the ability of the neuron to continue to fire action potentials even after the termination of a triggering stimulus, is one mechanism that is posited to support this mental trace. A recent study demonstrated a decline in persistent firing ability in pyramidal neurons of layer III of the lateral entorhinal cortex with aging, contributing to learning impairments in temporal associative memory acquisition. In this work, we explore the potential ways persistent firing in lateral entorhinal cortex (LEC) III supports temporal associative memory, and how aging may disrupt this mechanism within the temporal lobe system, resulting in impairment in this crucial behavior.

Diet-induced Alzheimer's-like syndrome in the rabbit.

INTRODUCTION: Although mouse models of Alzheimer's disease (AD) have increased our understanding of the molecular basis of the disease, none of those models represent late-onset Alzheimer's Disease which accounts for >90% of AD cases, and no therapeutics developed in the mouse (with the possible exceptions of aduhelm/aducanumab and gantenerumab) have succeeded in preventing or reversing the disease. This technology has allowed much progress in understanding the molecular basis of AD. To further enhance our understanding, we used wild-type rabbit (with a nearly identical amino acid sequence for amyloid as in humans) to model LOAD by stressing risk factors including age, hypercholesterolemia, and elevated blood glucose levels (BGLs), upon an ε3-like isoform of apolipoprotein. We report a combined behavioral, imaging, and metabolic study using rabbit as a non-transgenic model to examine effects of AD-related risk factors on cognition, intrinsic functional connectivity, and magnetic resonance-based biomarkers of neuropathology. METHODS: Aging rabbits were fed a diet enriched with either 2% cholesterol or 10% fat/30% fructose. Monthly tests of novel object recognition (NOR) and object location memory (OLM) were administered to track cognitive impairment. Trace eyeblink conditioning (EBC) was administered as a final test of cognitive impairment. Magnetic resonance imaging (MRI) was used to obtain resting state connectivity and quantitative parametric data (R2*). RESULTS: Experimental diets induced hypercholesterolemia or elevated BGL. Both experimental diets induced statistically significant impairment of OLM (but not NOR) and altered intrinsic functional connectivity. EBC was more impaired by fat/fructose diet than by cholesterol. Whole brain and regional R2* MRI values were elevated in both experimental diet groups relative to rabbits on the control diet. DISCUSSION: We propose that mechanisms underlying LOAD can be assessed by stressing risk factors for inducing AD and that dietary manipulations can be used to assess etiological differences in the pathologies and effectiveness of potential therapeutics against LOAD. In addition, non-invasive MRI in awake, non-anesthetized rabbits further increases the translational value of this non-transgenic model to study AD.

Brain activity studied with magnetic resonance imaging in awake rabbits.

We reviewed fMRI experiments from our previous work in conscious rabbits, an experimental preparation that is advantageous for measuring brain activation that is free of anesthetic modulation and which can address questions in a variety of areas in sensory, cognitive, and pharmacological neuroscience research. Rabbits do not struggle or move for several hours while sitting with their heads restrained inside the horizontal bore of a magnet. This greatly reduces movement artifacts in magnetic resonance (MR) images in comparison to other experimental animals such as rodents, cats, and monkeys. We have been able to acquire high-resolution anatomic as well as functional images that are free of movement artifacts during several hours of restraint. Results from conscious rabbit fMRI studies with whisker stimulation are provided to illustrate the feasibility of this conscious animal model for functional MRI and the reproducibility of data gained with it.

Variability in sub-threshold signaling linked to Alzheimer's disease emerges with age and amyloid plaque deposition in mouse ventral CA1 pyramidal neurons.

The hippocampus is vulnerable to deterioration in Alzheimer's disease (AD). It is, however, a heterogeneous structure, which may contribute to the differential volumetric changes along its septotemporal axis during AD progression. Here, we investigated amyloid plaque deposition along the dorsoventral axis in two strains of transgenic AD (ADTg) mouse models. We also used patch-clamp physiology in these mice to probe for functional consequences of AD pathogenesis in ventral hippocampus, which we found bears significantly higher plaque burden in the aged ADTg group compared to corresponding dorsal regions. Despite dorsoventral differences in amyloid load, ventral CA1 pyramidal neurons of aged ADTg mice exhibited subthreshold physiological changes similar to those previously reported in dorsal neurons, indicative of an HCN channelopathy, but lacked exacerbated suprathreshold accommodation. Additionally, HCN channel function could be rescued by pharmacological manipulation of the endoplasmic reticulum. These observations suggest that an AD-linked HCN channelopathy emerges in both dorsal and ventral CA1 pyramidal neurons, but that the former encounter an additional integrative obstacle in the form of reduced intrinsic excitability.

Intact Female Mice Acquire Trace Eyeblink Conditioning Faster than Male and Ovariectomized Female Mice.

Female subjects have been widely excluded from past neuroscience work because of a number of biases, including the notion that cycling sex hormones increase variability. However, it is necessary to conduct behavioral research in mice that includes both sexes as mice are typically used for developing and evaluating future therapeutics. Understanding sex differences in learning is fundamental for the development of targeted therapies for numerous neurologic and neurodegenerative disorders, including Alzheimer's disease, which is more prevalent in females than males. This study set out to confirm the role of sex and necessity of circulating ovarian hormones in the acquisition of the temporal associative memory task trace eyeblink conditioning (tEBC) in C57BL/6J mice. We present evidence that sex and ovarian hormones are important factors in learning. Specifically, intact female mice learn significantly faster than both male and ovariectomized (ovx) female mice. Data from pseudoconditioned control mice indicate that sex differences are because of differences in learned associations, not sensitization or spontaneous blink rate. This study strengthens the idea that ovarian hormones such as estrogen and progesterone significantly influence learning and memory and that further research is needed to determine the underlying mechanisms behind their effects. Overall, our findings emphasize the necessity of including both sexes in future behavioral studies.

Cognitive aging is associated with redistribution of synaptic weights in the hippocampus.

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others' lifespans outlast their "mindspans."

Detection of memory- and learning-related brain connectivity changes following trace eyeblink-conditioning using resting-state functional magnetic resonance imaging in the awake rabbit.

Animal imaging studies have the potential to further establish resting-state fMRI (rs-fMRI) and enable its validation for clinical use. The rabbit subjects used in this work are an ideal model system for studying learning and behavior and are also an excellent established test subject for awake scanning given their natural tolerance for restraint. We found that analysis of rs-fMRI conducted on a cohort of rabbits undergoing eyeblink conditioning can reveal functional brain connectivity changes associated with learning, and that rs-fMRI can be used to capture differences between subjects with different levels of cognitive performance. rs-fMRI sessions were conducted on a cohort of rabbits before and after trace eyeblink conditioning. MRI results were analyzed using independent component analysis (ICA) and network analysis. Behavioral data were collected with standard methods using an infrared reflective sensor aimed at the cornea to detect blinks. Behavioral results were analyzed, and a median split was used to create two groups of rabbits based on their performance. The cohort of rabbits undergoing eyeblink conditioning exhibited increased functional connectivity in the cingulate cortex, retrosplenial cortex, and thalamus consistent with brain reorganization associated with increased learning. Differences in the striatum and right cerebellum were also identified between rabbits in the top or bottom halves of the group as measured by the behavioral assay. Thus, rs-fMRI can provide not only a tool to detect and monitor functional brain changes associated with learning, but also to discriminate between different levels of cognitive performance.

Aß oligomer induced cognitive impairment and evaluation of ACU193-MNS-based MRI in rabbit.

INTRODUCTION: Amyloid-beta oligomers (AßOs) accumulate in Alzheimer's disease and may instigate neuronal pathology and cognitive impairment. We examined the ability of a new probe for molecular magnetic resonance imaging (MRI) to detect AßOs in vivo, and we tested the behavioral impact of AßOs injected in rabbits, a species with an amino acid sequence that is nearly identical to the human sequence. METHODS: Intracerebroventricular (ICV) injection with stabilized AßOs was performed. Rabbits were probed for AßO accumulation using ACUMNS (an AßO-selective antibody [ACU193] coupled to magnetic nanostructures). Immunohistochemistry was used to verify AßO presence. Cognitive impairment was evaluated using object location and object recognition memory tests and trace eyeblink conditioning. RESULTS: AßOs in the entorhinal cortex of ICV-injected animals were detected by MRI and confirmed by immunohistochemistry. Injections of AßOs also impaired hippocampal-dependent, but not hippocampal-independent, tasks and the area fraction of bound ACUMNs correlated with the behavioral impairment. DISCUSSION: Accumulation of AßOs can be visualized in vivo by MRI of ACUMNS and the cognitive impairment induced by the AßOs can be followed longitudinally with the novel location memory test.

Persistent firing in LEC III neurons is differentially modulated by learning and aging.

Whether and how persistent firing in lateral entorhinal cortex layer III (LEC III) supports temporal associative learning is still unknown. In this study, persistent firing was evoked in vitro from LEC III neurons from young and aged rats that were behaviorally naive or trained on trace eyeblink conditioning. Persistent firing ability from neurons from behaviorally naive aged rats was lower compared to neurons from young rats. Neurons from learning impaired aged animals also exhibited reduced persistent firing capacity, which may contribute to aging-related learning impairments. Successful acquisition of the trace eyeblink task, however, increased persistent firing ability in both young and aged rats. These changes in persistent firing ability are due to changes to the afterdepolarization, which may in turn be modulated by the postburst afterhyperpolarization. Together, these data indicate that successful learning increases persistent firing ability and decreases in persistent firing ability contribute to learning impairments in aging.

Contingency awareness, aging, and the parietal lobe.

Contingency awareness is thought to rely on an intact medial temporal lobe and also appears to be a function of age, as older subjects tend to be less aware. The current investigation used functional magnetic resonance imaging, transcranial direct current stimulation, and eyeblink classical conditioning to study brain processes related to contingency awareness as a function of age. Older adults were significantly less aware of the relationship between the tone-airpuff pairings than younger adults. Greater right parietal functional magnetic resonance imaging activation was associated with higher levels of contingency awareness for younger and older subjects. Cathodal transcranial direct current stimulation over the right parietal lobe led to lower levels of awareness in younger subjects without disrupting conditioned responses. Older adults exhibited hyperactivations in the parietal and medial temporal lobes, despite showing no conditioning deficits. These findings strongly support the idea that the parietal cortex serves as a substrate for contingency awareness and that age-related disruption of this region is sufficient to impair awareness, which may be a manifestation of some form of naturally occurring age-related neglect.

Intrinsic Excitability Increase in Cerebellar Purkinje Cells after Delay Eye-Blink Conditioning in Mice.

Cerebellar-based learning is thought to rely on synaptic plasticity, particularly at synaptic inputs to Purkinje cells. Recently, however, other complementary mechanisms have been identified. Intrinsic plasticity is one such mechanism, and depends in part on the downregulation of calcium-dependent SK-type K+ channels, which contribute to a medium-slow afterhyperpolarization (AHP) after spike bursts, regulating membrane excitability. In the hippocampus, intrinsic plasticity plays a role in trace eye-blink conditioning; however, corresponding excitability changes in the cerebellum in associative learning, such as in trace or delay eye-blink conditioning, are less well studied. Whole-cell patch-clamp recordings were obtained from Purkinje cells in cerebellar slices prepared from male mice ∼48 h after they learned a delay eye-blink conditioning task. Over a period of repeated training sessions, mice received either paired trials of a tone coterminating with a periorbital shock (conditioning) or trials in which these stimuli were randomly presented in an unpaired manner (pseudoconditioning). Purkinje cells from conditioned mice show a significantly reduced AHP after trains of parallel fiber stimuli and after climbing fiber evoked complex spikes. The number of spikelets in the complex spike waveform is increased after conditioning. Moreover, we find that SK-dependent intrinsic plasticity is occluded in conditioned, but not pseudoconditioned mice. These findings show that excitability is enhanced in Purkinje cells after delay eye-blink conditioning, and point toward a downregulation of SK channels as a potential underlying mechanism. The observation that this learning effect lasts at least up to 2 d after training shows that intrinsic plasticity regulates excitability in the long term.SIGNIFICANCE STATEMENT Plasticity of membrane excitability ("intrinsic plasticity") has been observed in invertebrate and vertebrate neurons, coinduced with synaptic plasticity or in isolation. Although the cellular phenomenon per se is well established, it remains unclear what role intrinsic plasticity plays in learning and if it even persists long enough to serve functions in engram physiology beyond aiding synaptic plasticity. Here, we demonstrate that cerebellar Purkinje cells upregulate excitability in delay eye-blink conditioning, a form of motor learning. This plasticity is observed 48 h after training and alters synaptically evoked spike firing and integrative properties of these neurons. These findings show that intrinsic plasticity enhances the spike firing output of Purkinje cells and persists over the course of days.

Conditioned Contextual Freezing is A Neurobehavioral Biomarker of Axonal Injury Indicated by Reduced Fractional Anisotropy in A Mouse Model of Blast-Induced Mild Traumatic Brain Injury.

Mild traumatic brain injury (TBI) is an important public health problem generated by closed head injury. This study is focused on the impact of blast-induced mild TBI on auditory trace and delay fear conditioning, models of declarative and non-declarative memory, respectively, and the correlation of conditioned freezing and fractional anisotropy, a measure of axonal state. A supersonic helium pressure wave was generated by a shock tube to blast 8-week-old male mice on Day 1 for 1.4 msec with an incident pressure of 16 psi, corresponding to a reflected pressure of 56.9 psi at the mouse head. On Day 3, the mice were subjected to auditory trace- or delay-fear conditioning. On Day 4, contextual freezing in the trained context, and precue and cued freezing in a novel context were determined. After cardiac perfusion on Day 5, ex vivo images were obtained with diffusion tensor imaging at 14.1 Tesla. We observed that delay fear conditioning prevented or reversed the decrease in fractional anisotropy in both the medial and lateral corpus callosum suggesting axonal stabilization of potentially behavioral therapeutic significance. Moderately strong and statistically significant Pearson correlations were found between fractional anisotropy and contextual freezing in the medial and lateral corpus callosum of blasted and sham-blasted delay- or trace-fear conditioned mice. Thus, contextual freezing is a neurobehavioral biomarker for axonal injury in mild TBI and is a reliable and high-throughput behavioral assay for the evaluation of potential therapeutics to treat mild TBI.