Browsing by Author "Pan, Yuchin Albert"
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- A cell–ECM mechanism for connecting the ipsilateral eye to the brainSu, 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.
- Cre-Dependent Anterograde Transsynaptic Labeling and Functional Imaging in Zebrafish Using VSV With Reduced CytotoxicityKler, Stanislav; Ma, Manxiu; Narayan, Sujatha; Ahrens, Misha Benjamin; Pan, Yuchin Albert (Frontiers, 2021-10-06)The small size and translucency of larval zebrafish (Danio rerio) have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory (vglut2a positive) and inhibitory (gad1b positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5–8 days post fertilization (2–5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.
- Deficiency in the endocytic adaptor proteins PHETA1/2 impairs renal and craniofacial developmentAtes, Kristin M.; Wang, Tong; Moreland, Trevor; Veeranan-Karmegam, Rajalakshmi; Ma, Manxiu; Jeter, Chelsi; Anand, Priya; Wenzel, Wolfgang; Kim, Hyung-Goo; Wolfe, Lynne A.; Stephen, Joshi; Adams, David R.; Markello, Thomas; Tifft, Cynthia J.; Settlage, Robert E.; Gahl, William A.; Gonsalvez, Graydon B.; Malicdan, May Christine; Flanagan-Steet, Heather; Pan, Yuchin Albert (2020-05)A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, phetal and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient. This article has an associated First Person interview with the first author of the paper.
- The development, cytoarchitecture, and circuitry of the ventral lateral geniculate nucleusSabbagh, 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.
- Effects of constitutive and acute Connexin 36 deficiency on brain-wide susceptibility to PTZ-induced neuronal hyperactivityBrunal-Brown, Alyssa Alexandra (Virginia Tech, 2020-10-30)Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: a) does Cx36 deficiency affect seizure susceptibility, b) does seizure-like activity affect Cx36 expression patterns, and c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol (PTZ; a GABA(A) receptor antagonist) induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within. 30 minutes and one-hour exposure to 20 mM PTZ significantly reduced the expression of Cx36. This Cx36 reduction persists after one-hour of recovery but recovered after 3-6 hours. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.
- Effects of Constitutive and Acute Connexin 36 Deficiency on Brain-Wide Susceptibility to PTZ-Induced Neuronal HyperactivityBrunal, Alyssa A.; Clark, Kareem C.; Ma, Manxiu; Woods, Ian G.; Pan, Yuchin Albert (Frontiers, 2021-01-11)Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: (a) does Cx36 deficiency affect seizure susceptibility, (b) does seizure-like activity affect Cx36 expression patterns, and (c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol [PTZ; a GABA(A) receptor antagonist] induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored.We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within 30min. This Cx36 reduction persists after 1-h of recovery but recovered after 3–6 h. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes regionspecific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.
- The Effects of Lead Toxicity on Thyroid Hormone Physiology in the Developing Brains of Xenopus laevis TadpolesDahora, 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.
- Investigation into the molecular mechanisms underlying circadian rhythm disruption and human cancerJanoski, Jesse Ryan (Virginia Tech, 2023-08-22)
- Structural Neural Connectivity Analysis in Zebrafish With Restricted Anterograde Transneuronal Viral Labeling and Quantitative Brain MappingMa, Manxiu; Kler, Stanislav; Pan, Yuchin Albert (2020-01-23)The unique combination of small size, translucency, and powerful genetic tools makes larval zebrafish a uniquely useful vertebrate system to investigate normal and pathological brain structure and function. While functional connectivity can now be assessed by optical imaging (via fluorescent calcium or voltage reporters) at the whole-brain scale, it remains challenging to systematically determine structural connections and identify connectivity changes during development or disease. To address this, we developed Tracer with Restricted Anterograde Spread (TRAS), a novel vesicular stomatitis virus (VSV)-based neural circuit labeling approach. TRAS makes use of replication-incompetent VSV (VSV Delta G) and a helper virus (lentivirus) to enable anterograde transneuronal spread between efferent axons and their direct postsynaptic targets but restricts further spread to downstream areas. We integrated TRAS with the Z-Brain zebrafish 3D atlas for quantitative connectivity analysis and identified targets of the retinal and habenular efferent projections, in patterns consistent with previous reports. We compared retinofugal connectivity patterns between wild-type and down syndrome cell adhesion molecule-like 1 (dscaml1) mutant zebrafish and revealed differences in topographical distribution. These results demonstrate the utility of TRAS for quantitative structural connectivity analysis that would be valuable for detecting novel efferent targets and mapping connectivity changes underlying neurological or behavioral deficits.
- Targeting NMDA Receptors to Tune Corticothalamic Circuit FunctionChen, Yang (Virginia Tech, 2023-02-09)The somatosensory corticothalamic (CT) circuit processes ascending sensory signals, and disruption to the balance of excitation and inhibition (E/I) within CT circuitry leads to absence seizures, sleep disorders, and attention deficits. E/I balance may be restored by independently modulating excitatory CT input to the ventral posteromedial (VPM) thalamus and inhibitory input to the VPM through the CT-thalamic reticular nucleus (nRT)-VPM pathway. This work revealed novel N-methyl-D-aspartate receptor (NMDAR) nucleus-specific and frequency-dependent functional diversity in the somatosensory CT circuit. Specifically, these findings illustrate the different effects of NMDAR negative modulation in the nRT and the VPM, which offers a method to preferentially decrease high frequency excitatory CT input to the VPM while having no significant effect on nRT activity. These results demonstrate the potential of utilizing NMDAR selective modulators to decrease overall excitation within the somatosensory CT circuit. Further investigation is required to elucidate the precise mechanisms underlying this phenomenon, including where NMDARs are localized at CT synapses and the effect of positive NMDAR modulators on nRT and VPM activity.