Neuronal Cell Types Research Articles

Topological Sholl descriptors for neuronal clustering and classification.

Neuronal morphology is a fundamental factor influencing information processing within neurons and networks. Dendritic morphology in particular can widely vary among cell classes, brain regions, and animal species. Thus, accurate quantitative descriptions allowing classification of large sets of neurons is essential for their structural and functional characterization. Current robust and unbiased computational methods that characterize groups of neurons are scarce. In this work, we introduce a novel technique to study dendritic morphology, complementing and advancing many of the existing techniques. Our approach is to conceptualize the notion of a Sholl descriptor and associate, for each morphological feature, and to each neuron, a function of the radial distance from the soma, taking values in a metric space. Functional distances give rise to pseudo-metrics on sets of neurons which are then used to perform the two distinct tasks of clustering and classification. To illustrate the use of Sholl descriptors, four datasets were retrieved from the large public repository https://neuromorpho.org/ comprising neuronal reconstructions from different species and brain regions. Sholl descriptors were subsequently computed, and standard clustering methods enhanced with detection and metric learning algorithms were then used to objectively cluster and classify each dataset. Importantly, our descriptors outperformed conventional morphometric techniques (L-Measure metrics) in several of the tested datasets. Therefore, we offer a novel and effective approach to the analysis of diverse neuronal cell types, and provide a toolkit for researchers to cluster and classify neurons.

Abstract 209: Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice

Abstract Therapeutic options for glioblastoma multiforme (GBM), the most common and fatal brain tumor, have been limited over the course of recent decades, leaving radiation as one of the few effective therapies. While advancements in radiation therapy have improved life expectancy and patient outcome, exposure to radiotherapy causes normal tissue toxicity and damage to the central nervous system, resulting in cognitive impairment and negative patient side effects. In the hippocampus, the memory processing center of the brain, radiotherapy-induced elevated glutamate levels lead to over-excitation of N-methyl-D-aspartate receptors (NMDAR) and thus cell death. To investigate the NMDAR, we repurposed a NMDA subtype specific inhibitor (Ifenprodil) and treated non-tumor mice before radiation. We found that Ifenprodil rescues cell death and cognition by inhibiting the NR2B subunit of NMDA receptors and preventing over-excitement-induced excitotoxicity, despite presence of high glutamate levels. In order to relieve glioma patients of the negative side effects of radiotherapies, we aim to further investigate the critical role of NR2B inhibition in non-tumor and tumor-bearing mice to reduce excitotoxicity and prevent damage to the central nervous system, opening avenues for therapeutic targeting and drug development. To accomplish this, we designed promotor-specific plasmids, only activated when present in their designed neuronal cell types, to initiate CRISPR/Cas9 genome editing and knock out the NR2B subunit of NMDAR. With NR2B knocked out across 5 different cell types, we exposed hippocampal organotypic slice cultures, derived from non-tumor mice, to 2 Gy radiation and ran flow cytometry to reveal which neuronal NR2B subunits play critical roles in excitotoxic phenotypes. To further these findings, we utilized a recently developed in-utero-electroporation mouse model that produces glioblastomas in immunocompetent mice by inactivating select tumor-suppressor genes via CRISPR/Cas9 genome editing at embryonic day 14.5. By applying different CRISPR/Cas9 gRNAs into our tumor-bearing mice, we can not only explore the robust role of NR2B inhibition in tumor brains with varying expressions, but also shed light onto how NR2B inhibition protects normal brain while leaving tumors radiosensitive. In conclusion, this work seeks to further develop our understanding of radiotherapy-induced cytotoxicity in order to prevent cognitive impairment and negative side effects in patients, improving their quality of life. Ultimately, the results from our findings will lead to the advancement of targeted drug development and therapeutic options. Citation Format: Jesus Castro, Mariana Gaelzer, Scott Welford. Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 209.

Abstract 3650: Trace'n'Seq: Assessing neuronal infiltration and its impact in pancreatic ductal carcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor entity with dismal prognosis. PDAC is characterized by an extensive desmoplastic stroma and the tumor microenvironment (TME) has been recognized to play crucial roles in tumor development, therapy resistance, progression and metastasis. Although different cell types of the TME have been molecularly analyzed, tumor-infiltrating neurons could not be considered so far, because their cell bodies are located in adjacent ganglia of the peripheral nervous system and thus they are not part of the tumor mass (Demir, Thiel et al, Nature Cancer 2020; Demir, Thiel et al. Cancer Cell 2020). One of the unique features of PDAC is the strong infiltration by nerve fibers which correlates directly with tumor malignancy. In recent years it has been shown that either neurotransmitters secreted by neurons, or modulators of neuronal infiltration secreted by cancer cells can influence tumor growth. Here we use 3D light sheet fluorescence microscopy imaging (LSFM) of full tissue cleared PDAC specimen of tumors growing in genetically engineered mouse models or patient-derived xenograft models. Our data showthat these PDACs are innervated by a complex array of sensory and sympathetic neurons. To molecularly analyse neurons innervating the tumor mass, we have developed a novel approach we termed Trace’n’seq. This combines retrograde axonal tracing and FACS analysis with single cell RNA sequencing with the aim to identify, isolate and analyse hundreds of traced neurons infiltrating tumors or their respective healthy organs. Thereby, we have identified previously undescribed neuronal cell types and provide a landscape of sympathetic and sensory nerves innervating the healthy pancreas as well as PDAC. Our single cell transcriptomic analysis revealed signs of reprogramming of neuronal behaviour orchestrated by the tumor cells. These datasets combined with single cell profiling of PDAC cells and its associated stroma provide a first complete, unbiased insight into neurons infiltrating both healthy pancreas and PDAC. Based on a comprehensive interactome analysis of the interplay between tumor cells, stroma and the neurons, we have identified various candidate genes involved in tumor-nerve signalling. We are currently performing gain-and loss- of function approaches to dissect the role of individual factors. In summary, with Trace'n'seq, we have developed a novel technology pipeline to isolate tissue innervating neurons to investigate the role of neuronal infiltration in the development and progression of PDAC. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Julian Mochayedi, Rienk Offringa, Martin Sprick, Andreas Trumpp. Trace'n'Seq: Assessing neuronal infiltration and its impact in pancreatic ductal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3650.

Cell type specific cannabinoid CB1 receptor distribution across the human and non-human primate cortex

Alterations in cannabinoid CB1 receptor (CB1R) are implicated in various psychiatric disorders. CB1R participates in both depolarization induced suppression of inhibition (DSI) and depolarization induced suppression of excitation (DSE), suggesting its involvement in regulating excitatory and inhibitory (E/I) balance. Prior studies examining neuronal cell type specific CB1R distribution have been conducted near exclusively within rodents. Identification of these distribution patterns within the human and non-human primate cortex is essential to increase our insight into its function. Using co-labeling immunohistochemistry and fluorescent microscopy, we examined CB1R protein levels within excitatory and inhibitory boutons of male human and non-human primate prefrontal cortex and auditory cortices, regions involved in the behavioral effects of exogenous cannabinoid exposures. We found that CB1R was present in both bouton populations within all brain regions examined in both species. Significantly higher CB1R levels were found within inhibitory than within excitatory boutons across all regions in both species, although the cell type by brain region interactions differed between the two species. Our results support the importance of conducting more in-depth CB1R examinations to understand how cell type and brain region dependent differences contribute to regional E/I balance regulation, and how aberrations in CB1R distribution may contribute to pathology.

In-vivo egfp expression in the honeybee Apis mellifera induced by electroporation and viral expression vector.

In this study we describe egfp expression induced by two techniques: in vivo electroporation and viral transduction in several cell types of the adult honeybee brain. Non-neuronal and neuronal cell types were identified and the expression persisted at least during three days. Kenyon cells, optic lobe neurons and protocerebral lobe neurons were electroporated. Astrocyte-like glia cells, fibrous lamellar glia cells and cortex glia cells were identified. Viral transduction targeted one specific type of glia cells that could not be identified. EGFP positive cells types were rather variable after electroporation, and viral transduction resulted in more homogenous groups of positive cells. We propose that these techniques remain a good alternative to transgenic animals because they potentially target only somatic cells.

Quantitative relationship between cerebrovascular network and neuronal cell types in mice.

SUMMARYThe cerebrovasculature and its mural cells must meet brain regional energy demands, but how their spatial relationship with different neuronal cell types varies across the brain remains largely unknown. Here we apply brain-wide mapping methods to comprehensively define the quantitative relationships between the cerebrovasculature, capillary pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes in adult mice. Our results show high densities of vasculature with high fluid conductance and capillary pericytes in primary motor sensory cortices compared with association cortices that show significant positive and negative correlations with energy-demanding parvalbumin+ and vasomotor nNOS+ neurons, respectively. Thalamo-striatal areas that are connected to primary motor sensory cortices also show high densities of vasculature and pericytes, suggesting dense energy support for motor sensory processing areas. Our cellular-resolution resource offers opportunities to examine spatial relationships between the cerebrovascular network and neuronal cell composition in largely understudied subcortical areas.

Single-cell transcriptomic classification of rabies-infected cortical neurons

Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear, and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets.

Biomarkers of Tuberculous Meningitis and Pediatric Human Immunodeficiency Virus on the African Continent.

Biomarkers in body fluids are helpful objective tools in diagnosis, prognosis and monitoring of (therapeutic) responses of many neurological diseases. Cerebrospinal fluid (CSF) biomarkers are part of the diagnostic toolbox for infectious neurological diseases. Tuberculous meningitis (TBM) and Human immunodeficiency virus (HIV), are important burdens of disease in Africa and can negatively affect brain health. Two thirds of the world's population of people living with HIV reside in sub-Saharan Africa and 25% of the global burden of tuberculosis (TB) is carried by the African continent. Neuroinflammation and damage of specific neuronal cell types are key constituents in the pathophysiology of these central nervous system (CNS) diseases, and important potential sources of circulating biomarkers. In this review, we summarize current research in the use of biomarkers in TBM and pediatric HIV as case demonstrations for high prevalence neurological diseases in Africa. Inflammatory molecules, primarily when detected in CSF, appear to have diagnostic value in these diseases, especially when measured as profiles. Brain injury molecules, such as S100, Neuron specific enolase and glial fibrillary acidic protein may have prognostic value in TBM, but more studies are needed. There is a need for more cost-economic and high sensitivity technologies to drive further biomarker discoveries and translate into healthcare improvements for these important healthcare problems in a globally fair way.

Adeno-Associated Viruses for Modeling Neurological Diseases in Animals: Achievements and Prospects.

Adeno-associated virus (AAV) vectors have become an attractive tool for efficient gene transfer into animal tissues. Extensively studied as the vehicles for therapeutic constructs in gene therapy, AAVs are also applied for creating animal models of human genetic disorders. Neurological disorders are challenging to model in laboratory animals by transgenesis or genome editing, at least partially due to the embryonic lethality and the timing of the disease onset. Therefore, gene transfer with AAV vectors provides a more flexible option for simulating genetic neurological disorders. Indeed, the design of the AAV expression construct allows the reproduction of various disease-causing mutations, and also drives neuron-specific expression. The natural and newly created AAV serotypes combined with various delivery routes enable differentially targeting neuronal cell types and brain areas in vivo. Moreover, the same viral vector can be used to reproduce the main features of the disorder in mice, rats, and large laboratory animals such as non-human primates. The current review demonstrates the general principles for the development and use of AAVs in modeling neurological diseases. The latest achievements in AAV-mediated modeling of the common (e.g., Alzheimer’s disease, Parkinson’s disease, ataxias, etc.) and ultra-rare disorders affecting the central nervous system are described. The use of AAVs to create multiple animal models of neurological disorders opens opportunities for studying their mechanisms, understanding the main pathological features, and testing therapeutic approaches.

Is it time for a moratorium on the use of benzalkonium chloride in eyedrops?

Is it time for a moratorium on the use of benzalkonium chloride in eyedrops?

Development of neurochemical labeling in the intermediolateral nucleus of cats' spinal cord.

NeuN is a neuron-specific nuclear protein expressed in most mature neuronal cell types, with some exceptions. These exceptions are known mainly for the brain but not for the spinal cord or the spinal visceral networks for which only scarce information is available. One of the most defined visceral structures in the spinal cord is the sympathetic intermediolateral nucleus located within the thoracolumbar segments. We investigated the NeuN staining in the intermediolateral nucleus and compared it with the staining for two neurochemical markers of visceral neurons: nitric oxide synthase and calcium-binding protein calretinin in adult cats and in kittens aged 0, 14, and 35 days. A clear NeuN-immunonegativity was obtained for intermediolateral neurons labeled for nitric oxide synthase for both adult cats and kittens. In contrast, a matched immunopositivity for the NeuN and calretinin was obtained, showing an age-dependent degree of this colocalization, which was high in newborn kittens, decreased on postnatal 14 and 35 days and persisted at a moderate level up to adulthood. Perhaps our data displayed a heterogeneity of the intermediolateral neurons.

Enhancing Membrane Repair in Neuronal Cell Types as a Potential Therapeutic for Neurodegenerative Diseases

Cell membrane repair is an essential cellular process to repair damage to the membrane and maintain the integrity of the cell membrane. Impaired membrane resealing kinetics can potentially lead to neurodegenerative diseases. Enhancing membrane repair via therapeutic intervention could minimize neurodegeneration in neuronal disease states. Recombinant human MG53 (rhMG53) and poloxamers have been an effective therapeutic to enhance membrane repair in muscle and non‐muscle cell types. Here, we aimed to assess if the membrane repair response is active and the efficacy of enhancing membrane resealing in neuronal cells. To assess if neuronal cells have an active membrane repair response, mouse neuroblastoma N2a cells were transfected with GFP or GFP‐MG53. MG53 localization was tracked following multiphoton infrared laser injury. N2a cells were transfected with GFP or GFP‐MG53, and subjected to multiphoton infrared laser injury to investigate if MG53 expression enhances membrane resealing following damage. An infrared laser was used to damage the cell membrane of N2a cells and primary mouse dorsal root ganglion neurons (DRG) in the presence of FM4‐64 dye. To test the efficacy of therapeutics, N2as and DRGs were treated with 1μM rhMG53 or 100μM Poloxamer 188 (P188) and challenged with the multiphoton laser injury. Lastly, the therapeutic potential of rhMG53 was tested in vivo with a sciatic nerve crush injury model. C57Bl/6 mice sciatic nerves were mechanically crushed for 10 seconds and then injected with 1μL rhMG53 (1mg/mL) or saline. The sciatic nerves were harvested and immunostained with an axonal regeneration marker, SCG10. SCG10 staining intensity was measured to calculate the regeneration index. Overall, we observed MG53 trafficking to the injury site which indicates there is an activate membrane repair mechanism within neuronal cells. Therapeutic levels of rhMG53 and P188 increased membrane resealing in both N2as and DRGs. Lastly, rhMG53‐treated sciatic nerves displayed a two‐fold increase in regenerative capacity following sciatic nerve crush injury compared to the saline vehicle control. These results illustrate an active membrane repair process in neuronal cells and potential for therapeutic intervention in the context of neuronal diseases, specifically neurodegenerative diseases.

Matrisome Expression in the Dorsal Root Ganglion

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides mechanical support to cells and tissues. The ECM also plays a role in cell signaling and morphology. It comprises various proteins which are called the matrisome. It consists of a core matrisome and matrisome-associated proteins. Recently, the ECM was identified as the most regulated pathway in animal pain models. However, mechanistic insights into the role of ECM in pain are lacking. Therefore, we will examine matrisome gene expression in the peripheral nervous system, in particular in murine dorsal root ganglia (DRG). This study's goal is to investigate the cellular distribution for matrisome gene expression in the DRG. Single-cell RNA sequencing (scRNAseq) was performed on 11 pooled 18-week-old male wild-type mice L3-L5 DRGs to analyze matrisome gene expression. Samples were sequenced by 10X Genomics and analyzed with the Seurat 4.0 package in Rstudio. The DRG consist of different cell types from various neuronal cells, supporting cells, vascular cells, pericytes and immunological cells. The core matrisome (glycoproteins (83% expressed), collagens (98%) and proteoglycans (92%)) is highly expressed in the DRG (Fig1A). Collagens and proteoglycans are mainly expressed by vascular cell types, Schwann, and satellite glial cells, while glycoproteins are expressed by neuronal, vascular cell types and pericytes (Fig1B). Matrisome associated proteins had a lower coverage but were still widely expressed by DRG cells: secreted factors (68%), ECM-affiliated proteins (75%), and ECM-regulators (62%). Neuronal cell types are the dominant cell type for expression of the matrisome-associated proteins. DRG neurons express both core matrisome as well as matrisome-associated genes, supporting the idea that the matrisome may contribute to pain signaling. Identifying the cellular distribution of the matrisome genes provides a framework to study the role of the ECM in peripheral neuronal tissue and its influence on pain. Grant support from Rachel Miller: R01AR077019 Anne-Marie Malfait: R01AR064251, R01AR060364, P30AR079206 Delfien Syx:12Q5920N Fransiska Malfait: 1842318N FWO project: G041519N.

(Pro)renin Receptor Knockdown in Tyrosine Hydroxylase neurons in the PVN Protects Against the Development of HFD‐Induced Hyperglycemia and Increases Energy Expenditure in Mice

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin‐angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy‐expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)‐induced metabolic physiology is not well understood. We recently showed that neuron‐specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)‐containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD‐induced hyperglycemia. To test this hypothesis, we injected both male and female TdTomato‐PRR‐LoxP mice with adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre) to ablate PRR in the THPVN neurons (THPVNPRRKO). The Tdtomato‐LoxP mice received the same virus were used as control. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following 6‐week HFD (60% calories from fat). Mouse metabolic parameters were measured over 48 hours in Sable Promethion metabolic cages with ad libitum access to HFD. In male mice, PRR‐LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, P= 0.0059) at the end of the six week HFD treatment. Interestingly, PRR ablation in the THPVN did not improve FBG and glucose handling in female mice. Metabolic cage measurements revealed that THPVN PRRKO mice had elevated oxygen consumption (VO2, ml/minute/g body weight) (0.047 ± 0.009 vs. 0.043 ± 0.0008 P= 0.027) and carbon dioxide emission (VCO2, ml/minute/g body weight) (0.035 ± 0.0005 vs. 0.032 ± 0.0005, P=0.0125) versus control HFD‐fed mice over 48 hours, indicating enhanced metabolic rate. The energy expenditure was significantly higher (0.0125 ± 0.00017 vs 0.0116 ± 0.00014, kCal/Hr/g body weight P=0.0026) in the THPVN PRRKO mice compared with their control counterparts. Locomotor activity over 48 hours was similar between the two groups (59.94 ± 19.6 vs. 103.5 ± 42.42 total meters traveled, P=0.204), and no significant difference in food intake (0.254 ± 0.011 vs. 0.274 ± 0.012 g, P=0.090) or body weight (42.7 ± 3.65 vs. 43.6 ± 1.41 g, P=0.842) was observed. This data suggests that PRR knockout in THPVN neurons protects against glucose dysregulation and elevates energy expenditure in mice following HFD. We conclude that PRR THPVN neurons play a key regulatory role in glucose and metabolic regulation.

Neuronal and Non‐neuronal Cell Type Mapping in the Mouse Brain

The brain contains multiple neuronal and non‐neuronal cell types with distinct spatial relationship that serve as a basic building block in the brain. I previously developed high resolution mapping methods to visualize and to quantify these individual brain cell types across the entire mouse brain. In this talk, I will present my lab’s effort to understand spatial arrangement of several major cell types in the mouse brain across lifetime. I previously applied my mapping methods to examine GABAergic neuronal cell types, c‐Fos‐labeled cells representing functional network activation, and long‐range anatomical connectivity of oxytocin neurons. Moreover, I used the method to trace the complete network of cerebrovasculature and pericytes that showed striking correlation with cortical parvalbumin and neuronal nitric oxide synthase (nNOS) positive neurons. Recently, my lab established a new online anatomical atlas for the adult mouse brain that combines the segmentation of the Allen Mouse Brian Atlas and Paxinos Mouse Brain Atlas, and developed new atlas framework for early postnatal brains. Using these resources, I have mapped developmental trajectories of oxytocin receptor‐expressing neurons during early postnatal development, unveiling spatial temporal patterns of the brain cell type. In summary, high resolution anatomical mapping of cell types helps to understand overall anatomical and functional organization of the brain.

Brain Penetrant Humanized scFv Antibody Block of P2 × 4 Receptor for Treatment of Chronic Pain

Our collaborative efforts are focused on validation of promising humanized single chain Fragment variable (hscFv) antibodies targeting a unique amino acid peptide sequence of P2 × 4 receptor (P2 × 4R). Data indicate single dose post-treatment with our newly engineered hP2 × 4 scFv antibody restores mechanical and cold pain-related behaviors to baseline in our trigeminal nerve compression model (FRICT-ION). The reversal to naïve baseline persists until experiment end at 10 weeks. Anxiety-like behaviors that develop in mice with chronic pain do not develop in hscFv antibody treated mice. We are characterizing the efficacy of lead P2 × 4R hscFv antibodies on neuronal cell-type specific activation patterns in medullary spinal V nucleus slices from Fos-TRAP(targeted recombination in active populations) mice and cultured trigeminal ganglia (TG) neurons from FRICT-ION mice. Engineered scFv antibodies feature binding affinity and activity similar to or better than monoclonal antibodies because they are much smaller in size. Thus, they are brain/nervous tissue penetrant due to their small size and have promising biotherapeutic applications for both the nervous and immune systems, now recognized as interactive in chronic pain. The ribosome display method utilized to generate the scFvs is a powerful cell-free technology widely used to select single-chain antibody fragments against the target of choice. This method is inexpensive, rapid, and was used to quickly develop repertoires of high-affinity antibodies targeting P2 × 4. In our hands the scFvs developed have high affinity in the picomolar range, superior stability and solubility. The patent was filed and published. Moving forward we currently continue evaluating these humanized P2 × 4R scFv antibodies that have strong nanomolar binding affinity, reduced self-immunogenicity, and relatively inexpensive large-scale production toward new drug application status based on the successful reversal of pain related measures and reduction of neuronal activation. This project opens new avenues for development of highly effective non-opioid biologic therapeutic interventions for chronic pain. Grant support from DoD grant W81XWH2010930 (KNW, SRAA); VA Merit grant BX002695 (KNW); NIH NIDCR R21 DE028096; NIH UG3 NS123958.

P480. Single-Nucleus Profiling Reveals Diverse Neuronal Composition of Human Mediodorsal Thalamus

Multiple lines of evidence implicate the thalamus in the pathophysiology of severe mental illness, and reduced connectivity between the mediodorsal thalamus (MDT) and prefrontal cortex is a consistent and early finding in psychotic patients. Prior work indicated a close relationship between cell type and projection properties of thalamic neurons. Studies of human MDT are severely limited by the lack of knowledge about its neuronal cell type composition. We previously found unappreciated diversity of cell types in mouse MDT. Given major conservation across species, similar or greater cell type diversity is expected for human MDT.

A comparative transcriptomic analysis of glucagon-like peptide-1 receptor- and glucose-dependent insulinotropic polypeptide receptor-expressing cells in the hypothalamus

ObjectiveThe hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and humans. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r-expressing hypothalamic cells using single-cell RNA sequencing. MethodsUsing Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1rEYFP+ or GiprEYFP+ cells were FACS-purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1rEYFP+ and GiprEYFP + populations were made. ResultsA total of 14,091 Glp1rEYFP+ and GiprEYFP+ cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1rEYFP+ and GiprEYFP+ hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of GiprEYFP+ cells were non-neuronal, whereas the Glp1rEYFP+ population was evenly split between neuronal and non-neuronal cell types. Both Glp1rEYFP+ and GiprEYFP+ oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1rEYFP+ and GiprEYFP+ populations, Glp1rEYFP+ mural cells are largely smooth muscle cells, while the majority of GiprEYFP+ mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1rEYFP+ and GiprEYFP+ neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus. ConclusionsWe have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr- and Glp1r-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance.

What Makes Organoids Good Models of Human Neurogenesis?

Human neurogenesis occurs mainly in embryonic, fetal, and neonatal stages and generates tremendously diverse neural cell types that constitute the human nervous system. Studies on human neurogenesis have been limited due to a lack of access to human embryonic and fetal tissues. Brain organoids derived from human pluripotent stem cells not only recapitulate major developmental processes during neurogenesis, but also exhibit human-specific features, thus providing an unprecedented opportunity to study human neurodevelopment. First, three-dimensional brain organoids resemble early human neurogenesis with diverse stem cell pools, including the presence of primate-enriched outer radial glia cells. Second, brain organoids recapitulate human neurogenesis at the cellular level, generating diverse neuronal cell types and forming stratified cortical layers. Third, brain organoids also capture gliogenesis with the presence of human-specific astrocytes. Fourth, combined with genome-editing technologies, brain organoids are promising models for investigating functions of human-specific genes at different stages of human neurogenesis. Finally, human organoids derived from patient iPSCs can recapitulate specific disease phenotypes, providing unique models for studying developmental brain disorders of genetic and environmental causes, and for mechanistic studies and drug screening. The aim of this review is to illustrate why brain organoids are good models to study various steps of human neurogenesis, with a focus on corticogenesis. We also discuss limitations of current brain organoid models and future improvements.

Role of Extracellular Vesicles in Glia-Neuron Intercellular Communication.

Cross talk between glia and neurons is crucial for a variety of biological functions, ranging from nervous system development, axonal conduction, synaptic transmission, neural circuit maturation, to homeostasis maintenance. Extracellular vesicles (EVs), which were initially described as cellular debris and were devoid of biological function, are now recognized as key components in cell-cell communication and play a critical role in glia-neuron communication. EVs transport the proteins, lipids, and nucleic acid cargo in intercellular communication, which alters target cells structurally and functionally. A better understanding of the roles of EVs in glia-neuron communication, both in physiological and pathological conditions, can aid in the discovery of novel therapeutic targets and the development of new biomarkers. This review aims to demonstrate that different types of glia and neuronal cells secrete various types of EVs, resulting in specific functions in intercellular communications.