Actigraphic evidence of persistent sleep disruption following repetitive mild traumatic brain injury in a gyrencephalic model.

We studied the effect of multimodal traumatic brain injuries on daily sleep/activity patterns and related histology. Gyrencephalic ferrets wore actigraphs and received military-relevant brain injuries including shockwaves, strong rotational impact, and variable stress, which were evaluated up to 6 months post injury. Sham and Baseline animals exhibited activity patterns occurring in distinct clusters of high activity, interspersed with periods of low activity. In the Injury and Injury + Stress groups, activity clusters diminished and overall activity patterns became significantly more dispersed at 4 weeks post injury with significant sleep fragmentation. Additionally, the Injury + Stress group exhibited a significant decrease in daytime high activity up to 4 months post injury. At 4 weeks post injury, the reactive astrocyte (GFAP) immunoreactivity was significantly greater in both the injury groups compared to Sham, but did not differ at 6 months post injury. The intensity of immunoreactivity of the astrocytic endfeet that surround blood vessels (visualized with aquaporin 4; AQP4), however, differed significantly from Sham at 4 weeks post injury (in both injured groups) and at 6 months (Injury + Stress only). As the distribution of AQP4 plays a key role in the glymphatic system, we suggest that glymphatic disruption occurs in ferrets after the injuries described here.

Evaluation of baseline behavioral tests in ferrets.

As the smallest mammal with a gyrencephalic cerebral cortex, ferrets are becoming increasingly important animal models to study neurological disorders. In order for them to be optimally used, typical behavioral measurements are highly desirable. To ascertain a baseline level of behavior, we conducted a battery of tests assessing motor, social, memory, headache, and aspects of depressive-like behavior. Adult male ferrets participated in open field, beam walk, sucrose preference, eye contact, light/dark box, socialization, and novel object recognition tests. The animals were assessed in three cohorts, which differed in age, with the youngest group being approximately 1 year younger than the oldest. Small, but significant, differences occurred between the youngest cohort and the older groups in several areas, suggesting that age may be an important factor when evaluating ferret behavior. Ferrets showed a high level of sociability in the eye contact tests and with novel animal preference. These experiments represent an important baseline of expected normative results that can provide a reference for normal ferret behavior and expected variability. The data reported here may serve as a reference for future intervention studies using the ferret. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Non-Communicable Neurological Disorders and Neuroinflammation.

Traumatic brain injury, stroke, and neurodegenerative diseases represent a major cause of morbidity and mortality in Africa, as in the rest of the world. Traumatic brain and spinal cord injuries specifically represent a leading cause of disability in the younger population. Stroke and neurodegenerative disorders predominantly target the elderly and are a major concern in Africa, since their rate of increase among the ageing is the fastest in the world. Neuroimmunology is usually not associated with non-communicable neurological disorders, as the role of neuroinflammation is not often considered when evaluating their cause and pathogenesis. However, substantial evidence indicates that neuroinflammation is extremely relevant in determining the consequences of non-communicable neurological disorders, both for its protective abilities as well as for its destructive capacity. We review here current knowledge on the contribution of neuroinflammation and neuroimmunology to the pathogenesis of traumatic injuries, stroke and neurodegenerative diseases, with a particular focus on problems that are already a major issue in Africa, like traumatic brain injury, and on emerging disorders such as dementias.

Expression of GFAP and Tau Following Blast Exposure in the Cerebral Cortex of Ferrets.

Blast exposures are a hallmark of contemporary military conflicts. We need improved preclinical models of blast traumatic brain injury for translation of pharmaceutical and therapeutic protocols. Compared with rodents, the ferret brain is larger, has substantial sulci, gyri, a higher white to gray matter ratio, and the hippocampus in a ventral position; these attributes facilitate comparison with the human brain. In this study, ferrets received compressed air shock waves and subsequent evaluation of glia and forms of tau following survival of up to 12 weeks. Immunohistochemistry and Western blot demonstrated altered distributions of astrogliosis and tau expression after blast exposure. Many aspects of the astrogliosis corresponded to human pathology: increased subpial reactivity, gliosis at gray-white matter interfaces, and extensive outlining of blood vessels. MRI analysis showed numerous hypointensities occurring in the 12-week survival animals, appearing to correspond to luminal expansions of blood vessels. Changes in forms of tau, including phosphorylated tau, and the isoforms 3R and 4R were noted using immunohistochemistry and Western blot in specific regions of the cerebral cortex. Of particular interest were the 3R and 4R isoforms, which modified their ratio after blast. Our data strongly support the ferret as an animal model with highly translational features to study blast injury.

Translationally Relevant Magnetic Resonance Imaging Markers in a Ferret Model of Closed Head Injury.

Pre-clinical models of traumatic brain injury (TBI) have been the primary experimental tool for understanding the potential mechanisms and cellular alterations that follow brain injury, but the human relevance and translational value of these models are often called into question. Efforts to better recapitulate injury biomechanics and the use of non-rodent species with neuroanatomical similarities to humans may address these concerns and promise to advance experimental studies toward clinical impact. In addition to improving translational aspects of animal models, it is also advantageous to establish pre-clinical outcomes that can be directly compared with the same outcomes in humans. Non-invasive imaging and particularly MRI is promising for this purpose given that MRI is a primary tool for clinical diagnosis and at the same time increasingly available at the pre-clinical level. The objective of this study was to identify which commonly used radiologic markers of TBI outcomes can be found also in a translationally relevant pre-clinical model of TBI. The ferret was selected as a human relevant species for this study with folded cortical geometry and relatively high white matter content and the closed head injury model of engineered rotation and acceleration (CHIMERA) TBI model was selected for biomechanical similarities to human injury. A comprehensive battery of MRI protocols based on common data elements (CDEs) for human TBI was collected longitudinally for the identification of MRI markers and voxelwise analysis of T2, contrast enhancement and diffusion tensor MRI values. The most prominent MRI findings were consistent with focal hemorrhage and edema in the brain stem region following high severity injury as well as vascular and meningeal injury evident by contrast enhancement. While conventional MRI outcomes were not highly conspicuous in less severe cases, quantitative voxelwise analysis indicated diffusivity and anisotropy alterations in the acute and chronic periods after TBI. The main conclusions of this study support the translational relevance of closed head TBI models in intermediate species and identify brain stem and meningeal vulnerability. Additionally, the MRI findings highlight a subset of CDEs with promise to bridge pre-clinical studies with human TBI outcomes.

The effect of Zika virus infection in the ferret.

Although initial observations of infections with the Zika virus describe a mild illness, more recent reports show that infections by Zika result in neurotropism. In 2015, substantial congenital malformations were observed, with numerous infants born with microcephaly in Brazil. To study the underlying mechanism and effects of the disease, it is critical to find suitable animal models. Rodents lack an immune system parallel to humans and also have lissencephalic brains, which are likely to react differently to infections. As the smallest gyrencephalic mammal, ferrets may provide an important animal model to study the Zika virus, as their brains share many characteristics with humans. To evaluate the prospect of using ferrets to study Zika virus infection, we injected seven pregnant jills with the PR strain subcutaneously on gestational day 21, corresponding to the initiation of corticogenesis. These injections resulted in mixed effects. Two animals died of apparent infection, and all kits were resorbed in another animal that did not die. The other four animals remained pregnant until gestational day 40, when the kits were delivered by caesarian section. We evaluated the animals using CT, MRI, diffusion tensor imaging, and immunohistochemistry. The kits displayed a number of features compatible with an infection that impacted both the brain and skull. The outcomes, however, were variable and differed within and across litters, which ranged from the absence of observable abnormalities to prominent changes, suggesting differential vulnerability of kits to infection by the Zika virus or to subsequent mechanisms of neurodevelopmental disruption.

Viral infections of the central nervous system in Africa.

Viral infections are a major cause of human central nervous system infection, and may be associated with significant mortality, and long-term sequelae. In Africa, the lack of effective therapies, limited diagnostic and human resource facilities are especially in dire need. Most viruses that affect the central nervous system are opportunistic or accidental pathogens. Some of these viruses were initially considered harmless, however they have now evolved to penetrate the nervous system efficiently and exploit neuronal cell biology thus resulting in severe illness. A number of potentially lethal neurotropic viruses have been discovered in Africa and over the course of time shown their ability to spread wider afield involving other continents leaving a devastating impact in their trail. In this review we discuss key viruses involved in central nervous system disease and of major public health concern with respect to Africa. These arise from the families of Flaviviridae, Filoviridae, Retroviridae, Bunyaviridae, Rhabdoviridae and Herpesviridae. In terms of the number of cases affected by these viruses, HIV (Retroviridae) tops the list for morbidity, mortality and long term disability, while the Rift Valley Fever virus (Bunyaviridae) is at the bottom of the list. The most deadly are the Ebola and Marburg viruses (Filoviridae). This review describes their epidemiology and key neurological manifestations as regards the central nervous system such as meningoencephalitis and Guillain-Barré syndrome. The potential pathogenic mechanisms adopted by these viruses are debated and research perspectives suggested.

Detection and Distinction of Mild Brain Injury Effects in a Ferret Model Using Diffusion Tensor MRI (DTI) and DTI-Driven Tensor-Based Morphometry (D-TBM).

Mild traumatic brain injury (mTBI) is highly prevalent but lacks both research tools with adequate sensitivity to detect cellular alterations that accompany mild injury and pre-clinical models that are able to robustly mimic hallmark features of human TBI. To address these related challenges, high-resolution diffusion tensor MRI (DTI) analysis was performed in a model of mild TBI in the ferret - a species that, unlike rodents, share with humans a gyrencephalic cortex and high white matter (WM) volume. A set of DTI image analysis tools were optimized and implemented to explore key features of DTI alterations in ex vivo adult male ferret brains (n = 26), evaluated 1 day to 16 weeks after mild controlled cortical impact (CCI). Using template-based ROI analysis, lesion overlay mapping and DTI-driven tensor-based morphometry (D-TBM) significant differences in DTI and morphometric values were found and their dependence on time after injury evaluated. These observations were also qualitatively compared with immunohistochemistry staining of neurons, astrocytes, and microglia in the same tissue. Focal DTI abnormalities including reduced cortical diffusivity were apparent in 12/13 injured brains with greatest lesion extent found acutely following CCI by ROI overlay maps and reduced WM FA in the chronic period was observed near to the CCI site (ANOVA for FA in focal WM: time after CCI p = 0.046, brain hemisphere p = 0.0012) often in regions without other prominent MRI abnormalities. Global abnormalities were also detected, especially for WM regions, which demonstrated reduced diffusivity (ANOVA for Trace: time after CCI p = 0.007) and atrophy that appeared to become more extensive and bilateral with longer time after injury (ANOVA for D-TBM Log of the Jacobian values: time after CCI p = 0.007). The findings of this study extend earlier work in rodent models especially by evaluation of focal WM abnormalities that are not influenced by partial volume effects in the ferret. There is also substantial overlap between DTI and morphometric findings in this model and those from human studies of mTBI implying that the combination of DTI tools with a human-similar model system can provide an advantageous and informative approach for mTBI research.

Emerging Viral Infections in Sub-Saharan Africa and the Developing Nervous System: A Mini Review.

The global public health concern is heightened over the increasing number of emerging viruses, i.e., newly discovered or previously known that have expanded into new geographical zones. These viruses challenge the health-care systems in sub-Saharan Africa (SSA) countries from which several of them have originated and been transmitted by insects worldwide. Some of these viruses are neuroinvasive, but have been relatively neglected by neuroscientists. They may provide experiments by nature to give a time window for exposure to a new virus within sizeable, previously non-infected human populations, which, for instance, enables studies on potential long-term or late-onset effects on the developing nervous system. Here, we briefly summarize studies on the developing brain by West Nile, Zika, and Chikungunya viruses, which are mosquito-borne and have spread worldwide out of SSA. They can all be neuroinvasive, but their effects vary from malformations caused by prenatal infections to cognitive disturbances following perinatal or later infections. We also highlight Ebola virus, which can leave surviving children with psychiatric disturbances and cause persistent infections in the non-human primate brain. Greater awareness within the neuroscience community is needed to emphasize the menace evoked by these emerging viruses to the developing brain. In particular, frontline neuroscience research should include neuropediatric follow-up studies in the field on long-term or late-onset cognitive and behavior disturbances or neuropsychiatric disorders. Studies on pathogenetic mechanisms for viral-induced perturbations of brain maturation should be extended to the vulnerable periods when neurocircuit formations are at peaks during infancy and early childhood.

Distribution and Cellular Localization of KCC2 in the Ferret Neocortex.

KCC2 (a brain-specific potassium-chloride cotransporter) affects development of the cerebral cortex, including aspects of neuronal migration and cellular maturation and differentiation. KCC2 also modulates chloride homeostasis by influencing the switch of GABA from depolarizing in young neurons to hyperpolarizing in mature neurons. We describe the expression pattern, regional distribution, and cellular colocalization of KCC2 in the ferret cortex in normal kits and those treated with methylazoxymethanol acetate (MAM). We earlier developed a model of impaired cortical development by injecting MAM during mid-cortical gestation, which briefly interferes with neuronal production and additionally results in increased levels of KCC2 at P0. Using immunohistochemistry, we show a shift in KCC2 expression during development, being high in the subplate at P0, repositioning into a subtle laminar pattern in the neocortex at P7-P14, and becoming homogeneous at P35. KCC2 colocalizes with neuronal markers in the developing and mature cerebral cortex of normal ferrets and those treated with MAM, but shows a differential pattern of expression at different ages and locates in distinct cellular compartments during development. Subcellular localization shows that KCC2 predominantly situates in the membrane fraction of neocortical samples. These findings reveal that KCC2 colocalizes differentially with neurons and its expression pattern alters during development.

Progression of histopathological and behavioral abnormalities following mild traumatic brain injury in the male ferret.

White matter damage is an important consequence of traumatic brain injury (TBI) in humans. Unlike rodents, ferrets have a substantial amount of white matter and a gyrencephalic brain; therefore, they may represent an ideal small mammal model to study human-pertinent consequences of TBI. Here we report immunohistochemical and behavioral results after a controlled cortical impact (CCI) injury to the sensorimotor cortex of adult male ferrets. We assessed inflammation in the neocortex and white matter, and behavior at 1 day post injury and 1, 4, and 16 weeks post injury (WPI). CCI in the ferret produced inflammation that originated in the neocortex near the site of the injury and progressed deep into the white matter with time. The density of microglia and astrocytes increased in the neocortex near the injury, peaking at 4WPI and remaining elevated at 16WPI. Microglial morphology in the neocortex was significantly altered in the first 4 weeks, but showed a return toward normal at 16 weeks. Clusters of microglial cells in the white matter persisted until 16WPI. We assessed motor and cognitive behavior using the open field, novel object recognition, T-maze, and gait tests. A transient deficit in memory occurred at 4WPI, with a reduction of rearing and motor ability at 12 and 16WPI. Behavioral impairments coincide with features of the inflammatory changes in the neocortex revealed by immunohistochemistry. The ferret represents an important animal model to explore ongoing damage in the white matter and cerebral cortex after TBI.

Diffusion MRI and the detection of alterations following traumatic brain injury.

This article provides a review of brain tissue alterations that may be detectable using diffusion magnetic resonance imaging MRI (dMRI) approaches and an overview and perspective on the modern dMRI toolkits for characterizing alterations that follow traumatic brain injury (TBI). Noninvasive imaging is a cornerstone of clinical treatment of TBI and has become increasingly used for preclinical and basic research studies. In particular, quantitative MRI methods have the potential to distinguish and evaluate the complex collection of neurobiological responses to TBI arising from pathology, neuroprotection, and recovery. dMRI provides unique information about the physical environment in tissue and can be used to probe physiological, architectural, and microstructural features. Although well-established approaches such as diffusion tensor imaging are known to be highly sensitive to changes in the tissue environment, more advanced dMRI techniques have been developed that may offer increased specificity or new information for describing abnormalities. These tools are promising, but incompletely understood in the context of TBI. Furthermore, model dependencies and relative limitations may impact the implementation of these approaches and the interpretation of abnormalities in their metrics. The objective of this paper is to present a basic review and comparison across dMRI methods as they pertain to the detection of the most commonly observed tissue and cellular alterations following TBI.

Establishing the ferret as a gyrencephalic animal model of traumatic brain injury: Optimization of controlled cortical impact procedures.

BACKGROUND: Although rodent TBI studies provide valuable information regarding the effects of injury and recovery, an animal model with neuroanatomical characteristics closer to humans may provide a more meaningful basis for clinical translation. The ferret has a high white/gray matter ratio, gyrencephalic neocortex, and ventral hippocampal location. Furthermore, ferrets are amenable to behavioral training, have a body size compatible with pre-clinical MRI, and are cost-effective. NEW METHODS: We optimized the surgical procedure for controlled cortical impact (CCI) using 9 adult male ferrets. We used subject-specific brain/skull morphometric data from anatomical MRIs to overcome across-subject variability for lesion placement. We also reflected the temporalis muscle, closed the craniotomy, and used antibiotics. We then gathered MRI, behavioral, and immunohistochemical data from 6 additional animals using the optimized surgical protocol: 1 control, 3 mild, and 1 severely injured animals (surviving one week) and 1 moderately injured animal surviving sixteen weeks. RESULTS: The optimized surgical protocol resulted in consistent injury placement. Astrocytic reactivity increased with injury severity showing progressively greater numbers of astrocytes within the white matter. The density and morphological changes of microglia amplified with injury severity or time after injury. Motor and cognitive impairments scaled with injury severity. COMPARISON WITH EXISTING METHOD(S): The optimized surgical methods differ from those used in the rodent, and are integral to success using a ferret model. CONCLUSIONS: We optimized ferret CCI surgery for consistent injury placement. The ferret is an excellent animal model to investigate pathophysiological and behavioral changes associated with TBI.

Analysis of the effects of noise, DWI sampling, and value of assumed parameters in diffusion MRI models.

PURPOSE: This study was a systematic evaluation across different and prominent diffusion MRI models to better understand the ways in which scalar metrics are influenced by experimental factors, including experimental design (diffusion-weighted imaging [DWI] sampling) and noise. METHODS: Four diffusion MRI models-diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), mean apparent propagator MRI (MAP-MRI), and neurite orientation dispersion and density imaging (NODDI)-were evaluated by comparing maps and histogram values of the scalar metrics generated using DWI datasets obtained in fixed mouse brain with different noise levels and DWI sampling complexity. Additionally, models were fit with different input parameters or constraints to examine the consequences of model fitting procedures. RESULTS: Experimental factors affected all models and metrics to varying degrees. Model complexity influenced sensitivity to DWI sampling and noise, especially for metrics reporting non-Gaussian information. DKI metrics were highly susceptible to noise and experimental design. The influence of fixed parameter selection for the NODDI model was found to be considerable, as was the impact of initial tensor fitting in the MAP-MRI model. CONCLUSION: Across DTI, DKI, MAP-MRI, and NODDI, a wide range of dependence on experimental factors was observed that elucidate principles and practical implications for advanced diffusion MRI. Magn Reson Med 78:1767-1780, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Quantitative MRI and DTI Abnormalities During the Acute Period Following CCI in the Ferret.

During the acute time period following traumatic brain injury (TBI), noninvasive brain imaging tools such as magnetic resonance imaging (MRI) can provide important information about the clinical and pathological features of the injury and may help predict long-term outcomes. In addition to standard imaging approaches, several quantitative MRI techniques including relaxometry and diffusion MRI have been identified as promising reporters of cellular alterations after TBI and may provide greater sensitivity and specificity for identifying brain abnormalities especially in mild TBI. However, for these imaging tools to be useful, it is crucial to define their relationship with the neurophysiological response to brain injury. Recently, a model of controlled cortical impact (CCI) has been developed in the ferret which has many advantages compared with rodent models (e.g., gyrencephalic cortex and high white matter volume). The objective of this study was to evaluate quantitative MRI metrics in the ferret CCI model, including T2 values and diffusion tensor imaging (DTI) metrics, during the acute time period. Longitudinal quantitative comparisons of in vivo MRI and DTI metrics were evaluated to identify abnormalities and characterize their spatial patterns in the ferret brain. Ex vivo MRI and DTI maps were then compared with histological staining for glial and neuronal abnormalities. The main findings of this article describe T2, diffusivity, and anisotropy markers of tissue change during the acute time period following mild TBI, and ex vivo analyses suggest that MRI and DTI markers are sensitive to subtle cellular alterations in this model. This was confirmed by comparison with immunohistochemistry, also showing altered markers in regions of MRI and DTI change.

Prenatal carbon monoxide impairs migration of interneurons into the cerebral cortex.

Prenatal exposure to carbon monoxide (CO) disrupts brain development, however little is known about effects on neocortical maturation. We exposed pregnant mice to CO from embryonic day 7 (E7) until birth. To study the effect of CO on neuronal migration into the neocortex we injected BrdU during corticogenesis and observed misplaced BrdU+ cells. The majority of cells not in their proper layer colocalized with GAD65/67, suggesting impairment of interneuron migration; interneuron subtypes were also affected. We subsequently followed interneuron migration from E15 organotypic cultures of mouse neocortex exposed to CO; the leading process length of migrating neurons diminished. To examine an underlying mechanism, we assessed the effects of CO on the cellular cascade mediating the cytoskeletal protein vasodilator-stimulated phosphoprotein (VASP). CO exposure resulted in decreased cGMP and in a downstream target, phosphorylated VASP. Organotypic cultures grown in the presence of the phosphodiesterase inhibitor IBMX resulted in a recovery of the leading processes. These data support the idea that CO acts as a signaling molecule and impairs function and neuronal migration by acting through the CO/NO-cGMP pathway. In addition, treated mice demonstrated functional impairment in behavioral tests.

Transplanted Neural Progenitor Cells from Distinct Sources Migrate Differentially in an Organotypic Model of Brain Injury.

Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs) obtained from rats to repair damaged neocortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0-3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ) for excitatory cells, ganglionic eminence (GE) for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ) or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically derived NPCs from the VZ/SVZ migrated less extensively. In the injured neocortex, transplanted cells moved predominantly into the wounded area. NPCs derived from the GE tended to be immunoreactive for GABAergic markers while those derived from the neocortex were more strongly immunoreactive for other neuronal markers such as MAP2, TUJ1, or Milli-Mark. Cells transplanted in vitro acquired the electrophysiological characteristics of neurons, including action potential generation and reception of spontaneous synaptic activity. This suggests that transplanted cells differentiate into neurons capable of functionally integrating with the host tissue. Together, our data suggest that transplantation of neural progenitor cells holds great potential as an emerging therapeutic intervention for restoring function lost to brain damage.

Fine-tuning of neurogenesis is essential for the evolutionary expansion of the cerebral cortex.

We used several animal models to study global and regional cortical surface expansion: The lissencephalic mouse, gyrencephalic normal ferrets, in which the parietal cortex expands more than the temporal cortex, and moderately lissencephalic ferrets, showing a similar degree of temporal and parietal expansion. We found that overall cortical surface expansion is achieved when specific events occur prior to surpragranular layer formation. (1) The subventricular zone (SVZ) shows substantial growth, (2) the inner SVZ contains an increased number of outer radial glia and intermediate progenitor cells expressing Pax6, and (3) the outer SVZ contains a progenitor cell composition similar to the combined VZ and inner SVZ. A greater parietal expansion is also achieved by eliminating the latero-dorsal neurogenic gradient, so that neurogenesis displays a similar developmental degree between parietal and temporal regions. In contrast, mice or lissencephalic ferrets show more advanced neurogenesis in the temporal region. In conclusion, we propose that global and regional cortical surface expansion rely on similar strategies consisting in altering the timing of neurogenic events prior to the surpragranular layer formation, so that more progenitor cells, and ultimately more neurons, are produced. This hypothesis is supported by findings from a ferret model of lissencephaly obtained by transiently blocking neurogenesis during the formation of layer IV.

Culturing layer-specific neocortical neurons as a cell replacement therapy following traumatic brain injury.

Neurophysiological changes resulting from traumatic brain injury (TBI) can result in adverse changes in behavior including mood instability and cognitive dysfunction. Cell death following TBI likely contributes to these altered behaviors and remains an elusive but attractive target for therapies aiming at functional recovery. Previously we demonstrated that neural progenitor cells derived from embryonic rats can be transplanted into donor neonatal rat brain slices and, over the course of 2 weeks in culture, mature into neurons that express neuronal immunohistochemical markers and develop electrophysiological profiles consistent with excitatory and inhibitory interneurons. Here we examine the potential of generating electrophysiologically mature neurons with a layer-specific phenotype as a next step in developing a therapy designed to rebuild a damaged cortical column with the functionally appropriate neuronal subtypes. Preliminary results suggest that neurons derived from passaged neurospheres and grown in dissociated cell culture develop GABAergic and presumed glutamatergic phenotypes and that the percentage of GABAergic cells increases as a function of passage. After 2 weeks in culture, the neurons have a mix of immature and mature neuronal electrophysiological profiles and receive synaptic inputs from surrounding neurons. Subsets of cells expressing neuron specific markers also express layer-specific markers such as Cux1, ER81, and RORß. Future studies will investigate the potential of transplanting layer-specific neurons generated and isolated in vitro into the neocortex of neonatal brain slices and their potential to maintain their phenotype and integrate into the host tissue.