VTechWorks Repository :: Browsing by Author "Fox, Michael A."

Browsing by Author "Fox, Michael A."

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3D electron microscopy and volume-based bouton sorting reveal the selectivity of inputs onto geniculate relay cell and interneuron dendrite segments
Maher, Erin E.; Briegel, Alex C.; Imtiaz, Shahrozia; Fox, Michael A.; Golino, Hudson; Erisir, Alev (Frontiers, 2023-03)
IntroductionThe visual signals evoked at the retinal ganglion cells are modified and modulated by various synaptic inputs that impinge on lateral geniculate nucleus cells before they are sent to the cortex. The selectivity of geniculate inputs for clustering or forming microcircuits on discrete dendritic segments of geniculate cell types may provide the structural basis for network properties of the geniculate circuitry and differential signal processing through the parallel pathways of vision. In our study, we aimed to reveal the patterns of input selectivity on morphologically discernable relay cell types and interneurons in the mouse lateral geniculate nucleus. MethodsWe used two sets of Scanning Blockface Electron Microscopy (SBEM) image stacks and Reconstruct software to manually reconstruct of terminal boutons and dendrite segments. First, using an unbiased terminal sampling (UTS) approach and statistical modeling, we identified the criteria for volume-based sorting of geniculate boutons into their putative origins. Geniculate terminal boutons that were sorted in retinal and non-retinal categories based on previously described mitochondrial morphology, could further be sorted into multiple subpopulations based on their bouton volume distributions. Terminals deemed non-retinal based on the morphological criteria consisted of five distinct subpopulations, including small-sized putative corticothalamic and cholinergic boutons, two medium-sized putative GABAergic inputs, and a large-sized bouton type that contains dark mitochondria. Retinal terminals also consisted of four distinct subpopulations. The cutoff criteria for these subpopulations were then applied to datasets of terminals that synapse on reconstructed dendrite segments of relay cells or interneurons. ResultsUsing a network analysis approach, we found an almost complete segregation of retinal and cortical terminals on putative X-type cell dendrite segments characterized by grape-like appendages and triads. On these cells, interneuron appendages intermingle with retinal and other medium size terminals to form triads within glomeruli. In contrast, a second, presumed Y-type cell displayed dendrodendritic puncta adherentia and received all terminal types without a selectivity for synapse location; these were not engaged in triads. Furthermore, the contribution of retinal and cortical synapses received by X-, Y- and interneuron dendrites differed such that over 60% of inputs to interneuron dendrites were from the retina, as opposed to 20% and 7% to X- and Y-type cells, respectively. ConclusionThe results underlie differences in network properties of synaptic inputs from distinct origins on geniculate cell types.
BDNF and Astrocyte TrkB.T1 Signaling as a Mechanism Underlying Astrocyte Synapse Interactions in Motor and Barrel Cortex
Pinkston, Beatriz T. Ceja (Virginia Tech, 2024-07-25)
Synapses are the fundamental units of communication in the brain, and their proper development and function are critical for cognitive processes and behavior. While the development of glutamatergic synapses has been extensively studied, the mechanisms underlying the formation of the tripartite synapse remain poorly understood. The tripartite synapse is a specialized structure consisting of the presynaptic terminal, the postsynaptic element, and a perisynaptic astrocyte process (PAP) that ensheathes the synaptic cleft. Increasing evidence demonstrates that PAPs are critical for synapse formation, stabilization, and plasticity. However, the mechanisms that govern the formation of tripartite synapses remain to be fully elucidated. This dissertation investigates the role of the astrocyte TrkB.T1 receptor, a truncated isoform of the canonical receptor for brain derived neurotrophic factor (BDNF), in mediating behavior and excitatory synapse development. Using an astrocyte-specific conditional TrkB.T1 knockout mouse model, we demonstrate that deletion of TrkB.T1 results in hyperactive locomotion, with increased voluntary running and perseverative motor behaviors. Through a combination of molecular and cellular approaches, we demonstrate that the behavioral abnormalities that result from TrkB.T1 deletion are accompanied by developmental reductions in glutamatergic synapses and astrocyte-synapse interactions in the motor and barrel cortex. Mechanistic studies using neuron-astrocyte co-cultures also reveal that loss of TrkB.T1 in astrocytes inhibits the formation of PAPs around glutamatergic synapses. Altogether, the insights presented herein present a novel astrocyte-mediated signaling mechanism that regulates excitatory synapse formation. These insights have important implications for understanding both neurodevelopmental and neuropsychiatric disorders involving synaptic dysfunction.
Cell-specific roles for CASK in the pathology of Optic Nerve Hypoplasia
Kerr, Alicia Marie (Virginia Tech, 2019-06-25)
Optic Nerve Hypoplasia (ONH) is the leading cause of childhood blindness in developed nations and its prevalence has been rising. Yet, we know little about the genetic, molecular, or cellular mechanisms underlying ONH. A previous study described ONH in a cohort of patients with mutations in CASK, an X-linked gene with established roles in neural development and synaptic function. I have demonstrated that heterozygous deletion of CASK in mice (Cask+/-) recapitulates many of the phenotypes observed in patients with CASK mutations, including ONH. This includes reduced optic nerve size, reduced numbers of retinal ganglion cells (RGCs), reduced RGC axonal diameter, and deficits in vision-related tasks. Further analysis on a homozygous partial loss of function variant (Caskfl/fl) also displayed ONH with reduced numbers of RGCs. In order to understand the mechanisms underlying CASK-associated ONH, I explored whether RGCs, the projection neurons of the retina and the cells whose axons comprise the optic nerve, generate CASK. Indeed, mRNA analysis revealed expression of CASK by a large cohort of RGCs. In order to assess whether loss of CASK from a majority of RGCs leads to ONH, I crossed a conditional allele of CASK (CASKfl/fl) with transgenic mice that express Cre Recombinase (Cre) in RGCs. Deletion of CASK from RGCs did not further alter ONH size nor RGC survival. These results demonstrate that loss of CASK signaling in this discrete neuronal populations is not sufficient to lead to further disruption in the assembly of the subcortical visual circuit, suggesting a non-cell autonomous mechanism for loss of CASK in ONH.
A cell–ECM mechanism for connecting the ipsilateral eye to the brain
Su, Jianmin; Sabbagh, Ubadah; Liang, Yanping; Olejníková, Lucie; Dixon, Karen G.; Russell, Ashley L.; Chen, Jiang; Pan, Yuchin Albert; Triplett, Jason W.; Fox, Michael A. (National Academy of Sciences, 2021-10-15)
Information about features in the visual world is parsed by circuits in the retina and is then transmitted to the brain by distinct subtypes of retinal ganglion cells (RGCs). Axons from RGC subtypes are stratified in retinorecipient brain nuclei, such as the superior colliculus (SC), to provide a segregated relay of parallel and feature-specific visual streams. Here, we sought to identify the molecular mechanisms that direct the stereotyped laminar targeting of these axons. We focused on ipsilateral-projecting subtypes of RGCs (ipsiRGCs) whose axons target a deep SC sublamina. We identified an extracellular glycoprotein, Nephronectin (NPNT), whose expression is restricted to this ipsiRGC-targeted sublamina. SC-derived NPNT and integrin receptors expressed by ipsiRGCs are both required for the targeting of ipsiRGC axons to the deep sublamina of SC. Thus, a cell–extracellular matrix (ECM) recognition mechanism specifies precise laminar targeting of ipsiRGC axons and the assembly of eye-specific parallel visual pathways.
Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria
Chavan, Vrushali; Willis, Jeffery; Walker, Sidney K.; Clark, Helen R.; Liu, Xinran; Fox, Michael A.; Srivastava, Sarika; Mukherjee, Konark (PLOS, 2015-04-30)
Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.
Collagen XIX is required for pheromone recognition and glutamatergic synapse formation in mouse accessory olfactory bulb
Amos, Chase; Fox, Michael A.; Su, Jianmin (Frontiers, 2023-04)
In mammals, the accessory olfactory bulb (AOB) receives input from vomeronasal sensory neurons (VSN) which detect pheromones, chemical cues released by animals to regulate the physiology or behaviors of other animals of the same species. Cytoarchitecturally, cells within the AOB are segregated into a glomerular layer (GL), mitral cell layer (MCL), and granule cell layer (GCL). While the cells and circuitry of these layers has been well studied, the molecular mechanism underlying the assembly of such circuitry in the mouse AOB remains unclear. With the goal of identifying synaptogenic mechanisms in AOB, our attention was drawn to Collagen XIX, a non-fibrillar collagen generated by neurons in the mammalian telencephalon that has previously been shown to regulate the assembly of synapses. Here, we used both a targeted mouse mutant that lacks Collagen XIX globally and a conditional allele allowing for cell-specific deletion of this collagen to test if the loss of Collagen XIX causes impaired synaptogenesis in the mouse AOB. These analyses not only revealed defects in excitatory synapse distribution in these Collagen XIX-deficient mutants, but also showed that these mutant mice exhibit altered behavioral responses to pheromones. Although this collagen has been demonstrated to play synaptogenic roles in the telencephalon, those roles are at perisomatic inhibitory synapses, results here are the first to demonstrate the function of this unconventional collagen in glutamatergic synapse formation.
Collagen-derived matricryptins promote inhibitory nerve terminal formation in the developing neocortex
Su, Jianmin; Chen, Jiang; Lippold, Kumiko; Monavarfeshani, Aboozar; Carrillo, Gabriela Lizana; Jenkins, Rchael; Fox, Michael A. (Rockefeller University Press, 2016-03-14)
Inhibitory synapses comprise only ∼20% of the total synapses in the mammalian brain but play essential roles in controlling neuronal activity. In fact, perturbing inhibitory synapses is associated with complex brain disorders, such as schizophrenia and epilepsy. Although many types of inhibitory synapses exist, these disorders have been strongly linked to defects in inhibitory synapses formed by Parvalbumin-expressing interneurons. Here, we discovered a novel role for an unconventional collagen—collagen XIX—in the formation of Parvalbumin+ inhibitory synapses. Loss of this collagen results not only in decreased inhibitory synapse number, but also in the acquisition of schizophrenia-related behaviors. Mechanistically, these studies reveal that a proteolytically released fragment of this collagen, termed a matricryptin, promotes the assembly of inhibitory nerve terminals through integrin receptors. Collectively, these studies not only identify roles for collagen-derived matricryptins in cortical circuit formation, but they also reveal a novel paracrine mechanism that regulates the assembly of these synapses.
Complement-dependent loss of inhibitory synapses on pyramidal neurons following Toxoplasma gondii infection
Carrillo, Gabriela L.; Su, Jianmin; Cawley, Mikel L.; Wei, Derek; Gill, Simran K.; Blader, Ira J.; Fox, Michael A. (Wiley, 2023-01-23)
The apicomplexan parasite Toxoplasma gondii has developed mechanisms to establish a central nervous system infection in virtually all warm-blooded animals. Acute T. gondii infection can cause neuroinflammation, encephalitis, and seizures. Meanwhile, studies in humans, nonhuman primates, and rodents have linked chronic T. gondii infection with altered behavior and increased risk for neuropsychiatric disorders, including schizophrenia. These observations and associations raise questions about how this parasitic infection may alter neural circuits. We previously demonstrated that T. gondii infection triggers the loss of inhibitory perisomatic synapses, a type of synapse whose dysfunction or loss has been linked to neurological and neuropsychiatric disorders. We showed that phagocytic cells (including microglia and infiltrating monocytes) contribute to the loss of these inhibitory synapses. Here, we show that these phagocytic cells specifically ensheath excitatory pyramidal neurons, leading to the preferential loss of perisomatic synapses on these neurons and not those on cortical interneurons. Moreover, we show that infection induces an increased expression of the complement C3 gene, including by populations of these excitatory neurons. Infecting C3-deficient mice with T. gondii revealed that C3 is required for the loss of perisomatic inhibitory synapses. Interestingly, loss of C1q did not prevent the loss of perisomatic synapses following infection. Together, these findings provide evidence that T. gondii induces changes in excitatory pyramidal neurons that trigger the selective removal of inhibitory perisomatic synapses and provide a role for a nonclassical complement pathway in the remodeling of inhibitory circuits in the infected brain.
Complete loss of the X-linked gene CASK causes severe cerebellar degeneration
Patel, Paras A.; Hegert, Julia; Cristian, Ingrid; Kerr, Alicia; LaConte, Leslie E. W.; Fox, Michael A.; Srivastava, Sarika; Mukherjee, Konark (BMJ, 2022-02-11)
Background Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes developmental delay in girls, while in boys, loss of the only allele of these genes leads to epileptic encephalopathy. The mechanism for these disorders remains unknown. CASK-linked cerebellar hypoplasia is presumed to result from defects in Tbr1-reelin-mediated neuronal migration. Method Here we report clinical and histopathological analyses of a deceased 2-month-old boy with a CASK-null mutation. We next generated a mouse line where CASK is completely deleted (hemizygous and homozygous) from postmigratory neurons in the cerebellum. Result The CASK-null human brain was smaller in size but exhibited normal lamination without defective neuronal differentiation, migration or axonal guidance. The hypoplastic cerebellum instead displayed astrogliosis and microgliosis, which are markers for neuronal loss. We therefore hypothesise that CASK loss-induced cerebellar hypoplasia is the result of early neurodegeneration. Data from the murine model confirmed that in CASK loss, a small cerebellum results from postdevelopmental degeneration of cerebellar granule neurons. Furthermore, at least in the cerebellum, functional loss from CASK deletion is secondary to degeneration of granule cells and not due to an acute molecular functional loss of CASK. Intriguingly, female mice with heterozygous deletion of CASK in the cerebellum do not display neurodegeneration. Conclusion We suggest that X-linked neurodevelopmental disorders like CASK mutation and Rett syndrome are pathologically neurodegenerative; random X-chromosome inactivation in heterozygous mutant girls, however, results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.
Contributions of VLDLR and LRP8 in the establishment of retinogeniculate projections
Su, Jianmin; Klemm, Michael A.; Josephson, Anne M.; Fox, Michael A. (BioMed Central, 2013-06-13)
Background: Retinal ganglion cells (RGCs), the output neurons of the retina, project to over 20 distinct brain nuclei, including the lateral geniculate nucleus (LGN), a thalamic region comprised of three functionally distinct subnuclei: the ventral LGN (vLGN), the dorsal LGN (dLGN) and the intergeniculate leaflet (IGL). We previously identified reelin, an extracellular glycoprotein, as a critical factor that directs class-specific targeting of these subnuclei. Reelin is known to bind to two receptors: very-low-density lipoprotein receptor (VLDLR) and low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2). Here we examined the roles of these canonical reelin receptors in retinogeniculate targeting. Results: To assess the roles of VLDLR and LRP8 in retinogeniculate targeting, we used intraocular injections of fluorescently conjugated cholera toxin B subunit (CTB) to label all RGC axons in vivo. Retinogeniculate projections in mutant mice lacking either VLDLR or LRP8 appeared similar to controls; however, deletion of both receptors resulted in dramatic defects in the pattern of retinal innervation in LGN. Surprisingly, defects in vldlr(-/-); lrp8(-/-) double mutant mice were remarkably different than those observed in mice lacking reelin. First, we failed to observe retinal axons exiting the medial border of the vLGN and IGL to invade distant regions of non-retino-recipient thalamus. Second, an ectopic region of binocular innervation emerged in the dorsomedial pole of vldlr(-/-); lrp8(-/-) mutant dLGN. Analysis of retinal projection development, retinal terminal sizes and LGN cytoarchitecture in vldlr(-/-); lrp8(-/-) mutants, all suggest that a subset of retinal axons destined for the IGL are misrouted to the dorsomedial pole of dLGN in the absence of VLDLR and LRP8. Such mistargeting is likely the result of abnormal migration of IGL neurons into the dorsomedial pole of dLGN in vldlr(-/-); lrp8(-/-) mutants. Conclusions: In contrast to our expectations, the development of both the LGN and retinogeniculate projections appeared dramatically different in mutants lacking either reelin or both canonical reelin receptors. These results suggest that there are reelin-independent functions of VLDLR and LRP8 in LGN development, and VLDLR- and LRP8-independent functions of reelin in class-specific axonal targeting.
The Cx43 Carboxyl-Terminal Mimetic Peptide Î±CT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner
Strauss, Randy E. (Virginia Tech, 2022-01-20)
The Cx43 CT mimetic peptide, Î±CT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a TRITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, Î±CT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, Î±CT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1) , and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of Î±CT1, Î±CT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of Î±CT1 in the treatment of vascular edema.
Deficiency in Parkinson's Disease risk gene CD38 as it relates to glial function: dysregulation of astrocyte genes and bioenergetics as a result of CD38 deficiency
Hernandez, Raymundo Daniel (Virginia Tech, 2024-01-12)
Parkinson's disease (PD) is the second most prevalent age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of dopaminergic neurons within the substantia nigra of the basal ganglia (BG). Motor coordination becomes severely affected over the course of the disease, causing patients to experience tremors at rest, bradykinesia, and body rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and treatment options are limited for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to monogenic variations. Evidence strongly indicates that the majority of PD cases are idiopathic and likely driven due to gene by environmental interactions. Reflective of this idea, recent research efforts have turned to genome-wide association studies (GWAS) to provide indications of gene variations, that while not causative of PD, incur increased risk within patient populations. GWAS findings play a particularly crucial role in neurodegenerative interventions, as early identification of patient risk may allow for preventative therapeutics to delay disease onset or reduce symptom severity. Amongst the many gene variants identified as incurring increased PD risk, single-nucleotide polymorphisms (SNPs) in the loci for CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immunological response and a second as an ectoenzyme that modulates bioenergetic functions. The particular functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede pathological indications. In the brain, CD38 expression is most enriched in astrocytes in BG regions, including substantia nigra, midbrain, and striatum. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/-, and Cd38-/- mice by magnetic-activated cell sorting (MACS) to acquire astrocyte isolates. Numerous differentially expressed genes (DEGs) were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and bioenergetic functions. GO analysis further suggested mitochondrial dysfunction in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered bioenergetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk.
The development, cytoarchitecture, and circuitry of the ventral lateral geniculate nucleus
Sabbagh, Ubadah (Virginia Tech, 2021-05-28)
In the visual system, retinal axons convey visual information from the outside world to dozens of distinct retinorecipient brain regions. In rodents, two major areas that are densely innervated by this retinal input are the dorsal lateral geniculate nucleus (dLGN) and ventral lateral geniculate nucleus (vLGN), both of which reside in the thalamus. The dLGN is well-studied and known to be important for classical image‐forming vision. The vLGN, on the other hand, is associated with non‐image‐forming vision and its neurochemistry, cytoarchitecture, and retinothalamic connectivity all remain unresolved, raising fundamental questions of its role within the visual system. Here, we sought to shed light on these important questions by studying the cellular and extracellular landscape of the vLGN and map its connectivity with the retina. Using bulk RNA sequencing and proteomics, we identified extracellular matrix proteins that form two molecularly distinct types of perineuronal nets in two major laminae of vLGN: the retinorecipient external vLGN (vLGNe) and the non‐retinorecipient internal vLGN. Using in situ hybridization, immunohistochemistry, electrophysiology, and genetic reporter lines, we found that vLGNe and vLGNi are also composed of diverse subtypes of neurons. In vLGNe, we discovered at least six transcriptionally distinct subtypes of inhibitory neurons that are distributed into distinct adjacent sublaminae. Using trans‐synaptic viral tracing and ex vivo electrophysiology, we found that cells in each these sublaminae receive direct inputs from retina. Lastly, by genetically removing visual input, we found that the organization of these sublaminae is dramatically disrupted, suggesting a crucial role for sensory input in the cytoarchitectural maintenance of the vLGN. Taken together, these results not only identify novel subtypes of vLGN cells, but they also point to new means of organizing visual information into parallel pathways – by anatomically creating distinct sensory channels. This subtype-specific organization may be key to understanding how the vLGN receives, processes, and transmits light‐derived signals in the subcortical visual system.
Developmental remodeling of relay cells in the dorsal lateral geniculate nucleus in the absence of retinal input
El-Danaf, Rana N.; Krahe, Thomas E.; Dilger, Emily K.; Bickford, Martha E.; Fox, Michael A.; Guido, William (BMC, 2015)
Background: The dorsal lateral geniculate nucleus (dLGN) of the mouse has been an important experimental model for understanding thalamic circuit development. The developmental remodeling of retinal projections has been the primary focus, however much less is known about the maturation of their synaptic targets, the relay cells of the dLGN. Here we examined the growth and maturation of relay cells during the first few weeks of life and addressed whether early retinal innervation affects their development. To accomplish this we utilized the math5 null (math5−/−) mouse, a mutant lacking retinal ganglion cells and central projections. Results: The absence of retinogeniculate axon innervation led to an overall shrinkage of dLGN and disrupted the pattern of dendritic growth among developing relay cells. 3-D reconstructions of biocytin filled neurons from math5−/− mice showed that in the absence of retinal input relay cells undergo a period of exuberant dendritic growth and branching, followed by branch elimination and an overall attenuation in dendritic field size. However, math5−/− relay cells retained a sufficient degree of complexity and class specificity, as well as their basic membrane properties and spike firing characteristics. Conclusions: Retinal innervation plays an important trophic role in dLGN development. Additional support perhaps arising from non-retinal innervation and signaling is likely to contribute to the stabilization of their dendritic form and function.
Digitally Augmented Neurorehabilitation: Potential for Treatment and Tele-Assessment
Mann, Jessie Elizabeth (Virginia Tech, 2021-03-10)
Neurorehabilitation, a relatively new domain of clinical intervention has, from its outset, been a rapidly evolving practice, with ongoing advancements in neuroimaging and neuroscience leading to new insights into how the brain grows and recovers from insult. The field of neurorehabilitation is tasked with translating this research into maximally effective treatments. This document addresses how digitally augmented neurorehabilitation, has, and can help meet, these translational needs and clinical imperatives. The first chapter is a review of the literature on the use of avatars in neurorehabilitation and their potential to promote neurological repair and plasticity. The second explores the use of a wearable remote control device for the promotion of enjoyability and intensity in the pediatric neurorehabilitation context. The third chapter pilot tests a video-based assessment methodology and explores the telehealth potential of such an assessment methodology and the final chapter demonstrates how such an assessment methodology can be implemented in pediatric neurorehabilitation in a case study on the treatment of Kernicterus. Collectively these works provide an overview of a selection of digitally augmented neurorehabilitation techniques and tools and preliminary data on how these approaches might be implemented in the field of pediatric neurorehabilitation.
Diverse GABAergic neurons organize into subtype-specific sublaminae in the ventral lateral geniculate nucleus
Sabbagh, Ubadah; Govindaiah, Gubbi; Somaiya, Rachana D.; Ha, Ryan V.; Wei, Jessica C.; Guido, William; Fox, Michael A. (Wiley, 2020-05-19)
In the visual system, retinal axons convey visual information from the outside world to dozens of distinct retinorecipient brain regions and organize that information at several levels, including either at the level of retinal afferents, cytoarchitecture of intrinsic retinorecipient neurons, or a combination of the two. Two major retinorecipient nuclei which are densely innervated by retinal axons are the dorsal lateral geniculate nucleus, which is important for classical image-forming vision, and ventral LGN (vLGN), which is associated with non-image-forming vision. The neurochemistry, cytoarchitecture, and retinothalamic connectivity in vLGN remain unresolved, raising fundamental questions of how it receives and processes visual information. To shed light on these important questions, used in situ hybridization, immunohistochemistry, and genetic reporter lines to identify and characterize novel neuronal cell types in mouse vLGN. Not only were a high percentage of these cells GABAergic, we discovered transcriptomically distinct GABAergic cell types reside in the two major laminae of vLGN, the retinorecipient, external vLGN (vLGNe) and the non-retinorecipient, internal vLGN (vLGNi). Furthermore, within vLGNe, we identified transcriptionally distinct subtypes of GABAergic cells that are distributed into four adjacent sublaminae. Using trans-synaptic viral tracing and in vitro electrophysiology, we found cells in each these vLGNe sublaminae receive monosynaptic inputs from retina. These results not only identify novel subtypes of GABAergic cells in vLGN, they suggest the subtype-specific laminar distribution of retinorecipient cells in vLGNe may be important for receiving, processing, and transmitting light-derived signals in parallel channels of the subcortical visual system.
The Effects of Aging on EGFR/pSTAT3-Dependent Gliovascular Structural Plasticity
Mills, William A. III (Virginia Tech, 2021-05-28)
Astrocytes comprise the most abundant cell population in human brain (1). First described by Virchow as being 'glue' of the brain (2), modern research has truly extended our knowledge and understanding regarding the vast array of roles these cells execute under normal physiological conditions. Examples include neurotransmitter reuptake at the synapse (3), the regulation of blood flow at capillaries to meet neuronal energy demand (4), and maintenance/repair of the blood-brain barrier (BBB) (5), which is comprised, in part, of tight junction proteins such zonula-occludens-1 (ZO1) (6) and Claudin-5 (7). Underlying the execution of these processes is the morphological and spatial arrangement of astrocytes between neurons and endothelial cells comprising blood vessels, where comprehensively speaking, these cells form what is known as the gliovascular unit (8). Astrocytes extend large processes called endfeet that intimately associate with and enwrap up to 99% of the cerebrovascular surface (9). Disruptions to this association can occur in the form of retracted endfeet, and this has been characterized in several disease states such as major depressive disorder (10-12), ischemia (13-15), and normal biological aging (16-18). Disruption can also take the form of cellular/protein aggregate intercalation, which our lab previously characterized in a human-derived glioma model (19) and vascular amyloidosis human Amyloid Precursor Protein J20 (hAPPJ20) animal model (20). In both models, focal astrocyte-vascular disruptions coincided with perturbations to astrocyte control of blood flow, with deficits in BBB integrity present in the glioma model as well. These findings lead to the preliminary work in this dissertation where we aimed to extend BBB findings in the glioma model to the hAPPJ20 vascular amyloidosis model. Immunohistochemical analysis in two-year old hAPPJ20 animal arterioles revealed that indeed in locations of vascular amyloid buildup and endfoot separation, there was a significant reduction in a tight junction protein critical for BBB maintenance, ZO1. This reduction in ZO1 expression was accompanied by extravasation of 70kDa FITC and the ~1kDa Cadaverine, suggesting that BBB integrity was compromised. These findings led to the objective of this dissertation, which was to determine if focal ablation of an astrocyte is sufficient to disrupt BBB integrity. By utilizing the in vivo 2Phatal single-cell apoptosis induction method (21), we found that 1) focal loss of astrocyte-vascular coverage does not result in barrier deficits, but rather induces a plasticity response whereby surrounding astrocytes extend processes to reinnervate vascular vacancies no longer occupied by previously ablated astrocytes. 2) Replacement astrocytes are capable of inducing vasocontractile responses in blood vessels, and that 3) aging significantly attenuates the kinetics of this process. We then tested the hypothesis that focal loss of astrocyte-vascular coverage leads to a gliovascular structural plasticity response, in part, through the phosphorylation of signal transducer and activator of transcription 3 (STAT3) by Janus Kinase 2 (JAK2). This dissertation found that 4), this was indeed the case, and finally, 5) we determined that gliovascular structural plasticity occurs after reperfusion post-focal photothrombotic stroke. Together, the work presented in this dissertation sheds light on a novel plasticity response whereby astrocytes maintain continual cerebrovascular coverage and therefore physiological control. Future studies should aim to determine if 1) astrocytes also replace the synaptic contacts with neighboring neurons once held by a previous astrocyte, and 2) what therapeutic opportunity gliovascular structural plasticity may present regarding BBB repair following stroke.
The Effects of Lead Toxicity on Thyroid Hormone Physiology in the Developing Brains of Xenopus laevis Tadpoles
Dahora, Lara Iza (Virginia Tech, 2023-07-17)
This dissertation focuses on the effects of lead (Pb) on the expression of thyroid hormone distributor proteins and how that affects the developing brain in Xenopus laevis tadpoles. Previous work has shown that Pb has the ability to dysregulate thyroid hormone (TH)-signaling in vertebrates and that Pb can impair brain development. This dissertation reports results for a series of Pb-treatment experiments conducted in Xenopus laevis tadpoles. The first primary hypothesis of this dissertation is that Pb impairs TH-dependent mechanisms of brain development. The second primary hypothesis of this dissertation is that Pb-induced impairments of brain development happen via dysregulation of thyroid hormone distributor proteins (THDPs) transthyretin (TTR) and β-trace. Analyses of the effects of Pb on overall body growth showed dose-dependent decreases in body length with increasing concentrations. Evaluation of the effect of Pb on tectal size and cell death in the developing brain yielded bimodal changes that depended upon Pb concentration in both features. Furthermore, Pb impaired TH-induced changes in brain development, including neurogenesis and brain volume. Pb abolished the T4-mediated increase in proliferating cell nuclear antigen (PCNA) expression, while having only marginal effects on neuronal regeneration related protein (NREP) and Krueppel-like factor 9 (klf9). Analyses of the effects of Pb on TTR and β-trace expression yielded results demonstrating a significant decrease in expression of both proteins in response to Pb-treatment. Contrary to prior studies in the literature, I demonstrate here that TTR is present in the brains of Xenopus. While electroporation of TTR morpholino did result in fewer TTR puncta, electroporation with morpholinos for TTR and β-trace knock down did not mimic the effects of Pb on neurogenesis. However, overexpression of these proteins in the choroid plexus (CP) of these animals was sufficient to produce an increase in neurogenesis. Finally, overexpression of these proteins was sufficient to ameliorate the effects of Pb-treatment on neurogenesis. The results affirm both the primary and secondary hypotheses, illustrating that Pb does, indeed, impair TH-mediated mechanisms of brain development and that these impairments are mitigated by dysregulation of TTR and β-trace.
Evaluation of biofeedback components for the management of acute stress in healthcare
Kennedy-Metz, Lauren Rose (Virginia Tech, 2018-11-27)
Medical error is the third leading cause of death in the United States, with surgery being a critical area for improvement. Of particular interest for this dissertation is understanding and mitigating the impact of acute stress experienced by surgeons. Previous research demonstrates the detrimental effects mismanaged acute stress can have on cognitive performance integral in optimal surgical practice. Biofeedback consists of objectively monitoring signs of stress, presenting physicians with their own physiological output in real time. Introducing appropriate, targeted coping mechanisms when they are most needed may facilitate behavioral adjustments in the face of acute stress. The goal of this dissertation research was to evaluate the potential benefit of biofeedback and coping instructions, measured by reduced perceived and physiological stress, and improved task performance. In the first study, college students participated in a first-person shooter videogame while receiving visual coping instructions. Instructions that were presented at moments of elevated stress improved downstream physiology compared to randomly administered instruction, and the presence of coping instructions was more beneficial than their absence at highly stressful times. In the second study, I adapted and validated a computer-based task to focus on components of workload experienced by physicians. This study yielded one high-stress and one-low stress version of a more demographic-appropriate task. In the final study, medical students and residents completed this task. The independent variables tested included a visual biofeedback interface, intermittent auditory coping instructions, and/or brief training on stress management and emotional intelligence. Results from this study showed that despite high cognitive workload experienced by participants receiving both biofeedback and coping instructions, performance across stress levels was indistinguishable, and physiological indicators of stress immediately following discrete coping instructions was reflective of decreased stress. Taken together, the results of these studies validate the generation of a new lab-based task to induce stress among healthcare providers, and the physiological and performance benefits associated with physiologically-based coping instructions. Future work should investigate how these concepts can be tailored towards surgical workflow with feedback modality in mind, extended to teams, and/or scaled up to higher levels of fidelity to better capture the work environment.
Exposure of Rats to Environmental Tobacco Smoke during Cerebellar Development Alters Behavior and Perturbs Mitochondrial Energetics
Fuller, Brian F.; Fox, Michael A. (National Institute of Environmental Health Sciences, 2012-12)
Background: Environmental tobacco smoke (ETS) exposure is linked to developmental deficits and disorders with known cerebellar involvement. However, direct biological effects and underlying neurochemical mechanisms remain unclear. Objectives: We sought to identify and evaluate underlying neurochemical change in the rat cerebellum with ETS exposure during critical period development. Methods: We exposed rats to daily ETS (300, 100, and 0 μg/m3 total suspended particulate) from postnatal day 8 (PD8) to PD23 and then assayed the response at the behavioral, neuroproteomic, and cellular levels. Results: Postnatal ETS exposure induced heightened locomotor response in a novel environment on par initially with amphetamine stimulation. The cerebellar mitochondrial subproteome was significantly perturbed in the ETS-exposed rats. Findings revealed a dose-dependent up-regulation of aerobic processes through the modification and increased translocation of Hk1 to the mitochondrion with corresponding heightened ATP synthase expression. ETS exposure also induced a dose-dependent increase in total Dnm1l mitochondrial fission factor; although more active membrane-bound Dnm1l was found at the lower dose. Dnm1l activation was associated with greater mitochondrial staining, particularly in the molecular layer, which was independent of stress-induced Bcl-2 family dynamics. Further, electron microscopy associated Dnm1l-mediated mitochondrial fission with increased biogenesis, rather than fragmentation. Conclusions: The critical postnatal period of cerebellar development is vulnerable to the effects of ETS exposure, resulting in altered behavior. The biological effect of ETS is underlain in part by a Dnm1l-mediated mitochondrial energetic response at a time of normally tight control. These findings represent a novel mechanism by which environmental exposure can impact neurodevelopment and function.

Browsing by Author "Fox, Michael A."

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