Browsing by Subject "Glutamatergic"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research
    (Springer Nature, 2014-09-16) Ray, Balmiki; Chopra, Nipun; Long, Justin M.; Lahiri, Debomoy K.; Psychiatry, School of Medicine
    Background: Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. Results: This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. Conclusion: We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.
  • Thumbnail Image
    Item
    Spinophilin-dependent regulation of GluN2B-containing NMDAR-dependent calcium influx, GluN2B surface expression, and cleaved caspase expression
    (Wiley, 2023) Salek, Asma B.; Claeboe, Emily T.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of Science
    N-methyl-d-aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor conductance and surface localization, two alterations that can modulate overall channel activity. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates the amount of calcium entering the cell and subsequent activation of calcium-dependent processes. The dendritic spine enriched protein, spinophilin, is the major synaptic protein phosphatase 1 (PP1) targeting protein. Depending on the substrate, spinophilin can act as either a PP1 targeting protein, to permit substrate dephosphorylation, or a PP1 inhibitory protein, to enhance substrate phosphorylation. Spinophilin limits NMDAR function in a PP1-dependent manner. Specifically, we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284 on GluN2B. However, how spinophilin modifies NMDAR function is unclear. Herein, we utilize a Neuro2A cell line to detail that Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs. Moreover, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression, an effect that is independent of Ser-1284 phosphorylation. In hippocampal neurons isolated from spinophilin knockout animals, there is an increase in cleaved caspase-3 levels, a marker of calcium-associated apoptosis, compared with wildtype mice. Taken together, our data demonstrate that spinophilin regulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin enhances neuronal cleaved caspase-3 expression.