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Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

easy-reproducible-low-density-primary-culture-using-frozen-stock

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

University of Manitoba

Health Sciences Centre

Gunma University

Abnormal architecture of dendritic spines is associated with neurodevelopmental and neurodegenerative diseases. The 5-HT(2A) receptor is a potential therapeutic target for mental illnesses and it is functionally and genetically associated with many types of psychiatric disorders. It has been reported that 5-HT(2A) receptor activation alters spine architecture. Although actin cytoskeleton has a key role in the regulation of spine architecture, it is not clarified whether 5-HT(2A)+ receptor activation affect the actin cytoskeleton in dendritic spines. In the present study, we examined the effect of 5-HT(2A) receptor activation on the actin cytoskeleton in dendritic spines of mature hippocampal neurons in low-density culture. Immunocytochemical analysis showed that 15 min exposure of 5-HT(2A) receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) significantly decreased the cluster densities of drebrin (control, 37.0±6.9 per 100 μm, DOI, 12.5±2.9) and F-actin (control, 18.3±4.9; DOI, 7.7±2.1) at dendritic spines without any detectable changes in the cluster densities of synapsin I and PSD-95. At the same time period DOI exposure did not affect spine architecture (spine density: control, 38.3±5.1 per 100 μm; DOI, 25.6±3.5; spine length: control, 1.99±0.18; DOI, 2.00±0.29; spine width: control, 0.72±0.06; DOI, 0.77±0.11). Thus, it is indicated that decrease of drebrin and F-actin can occur at the dendritic spines without morphological changes. Together our data suggest that 5-HT(2A) receptors activation is involved in the regulation of distribution of cytoskeleton in the dendritic spines.

Selective reduction of drebrin and actin in dendritic spines of hippocampal neurons by activation of 5-HT(2A) receptors.

Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.

Spikar, a novel drebrin-binding protein, regulates the formation and stabilization of dendritic spines.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be useful for developing novel methods of treatment for various neurological diseases. However, the detailed process of functional maturation of hiPSC-derived neurons (hiPS neurons) remains poorly understood. This study analyzes development of hiPS neurons, focusing specifically on early developmental stages through 48 hr after cell seeding; development was compared with that of primary cultured neurons derived from the rat hippocampus. At 5 hr after cell seeding, neurite formation occurs in a similar manner in both neuronal populations. However, very few neurons with axonal polarization were observed in the hiPS neurons even after 48 hr, indicating that hiPS neurons differentiate more slowly than rat neurons. We further investigated the elongation speed of axons and found that hiPS neuronal axons were slower. In addition, we characterized the growth cones. The localization patterns of skeletal proteins F-actin, microtubule, and drebrin were similar to those of rat neurons, and actin depolymerization by cytochalasin D induced similar changes in cytoskeletal distribution in the growth cones between hiPS neurons and rat neurons. These results indicate that, during the very early developmental stage, hiPS neurons develop comparably to rat hippocampal neurons with regard to axonal differentiation, but the growth of axons is slower.

Early-stage development of human induced pluripotent stem cell-derived neurons.

LRRTMs are postsynaptic cell adhesion proteins that have region-restricted expression in the brain. To determine their role in the molecular organization of synapses in vivo, we studied synapse development and plasticity in hippocampal neuronal circuits in mice lacking both  and . We found that LRRTM1 and LRRTM2 regulate the density and morphological integrity of excitatory synapses on CA1 pyramidal neurons in the developing brain but are not essential for these roles in the mature circuit. Further, they are required for long-term-potentiation in the CA3-CA1 pathway and the dentate gyrus, and for enduring fear memory in both the developing and mature brain. Our data show that LRRTM1 and LRRTM2 regulate synapse development and function in a cell-type and developmental-stage-specific manner, and thereby contribute to the fine-tuning of hippocampal circuit connectivity and plasticity.

Distinct but overlapping roles of LRRTM1 and LRRTM2 in developing and mature hippocampal circuits.

Alzheimer's disease (AD)-like cognitive impairment, a kind of Neuro-COVID syndrome, is a reported complication of SARS-CoV-2 infection. However, the specific mechanisms remain largely unknown. Here, we integrated single-nucleus RNA-sequencing data to explore the potential shared genes and pathways that may lead to cognitive dysfunction in AD and COVID-19. We also constructed ingenuity AD-high-risk scores based on AD-high-risk genes from transcriptomic, proteomic, and Genome-Wide Association Studies (GWAS) data to identify disease-associated cell subtypes and potential targets in COVID-19 patients. We demonstrated that the primary disturbed cell populations were astrocytes and neurons between the above two dis-eases that exhibit cognitive impairment. We identified significant relationships between COVID-19 and AD involving synaptic dysfunction, neuronal damage, and neuroinflammation. Our findings may provide new insight for future studies to identify novel targets for preventive and therapeutic interventions in COVID-19 patients.

Reiko T. Roppongi

Department of Physiology and Pathophysiology

University of Manitoba

Low-Density Primary Hippocampal Neuron Culture

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Reiko T. Roppongi

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University of Manitoba

Low-Density Primary Hippocampal Neuron Culture

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Department of Physiology and Pathophysiology