Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes

Journal of Neuroscience, Journal Year: 2023, Volume and Issue: 43(26), P. 4755 - 4774

Published: June 7, 2023

NMDA receptors (NMDARs) are ionotropic glutamate that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs regulated at several levels, including their externalization, internalization, lateral diffusion between the synaptic extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either smallest commercially available quantum dot 525 (QD525) or nanometer larger (and thus brighter) QD605 (referred as nanoGFP-QD525 nanoGFP-QD605, respectively). Targeting yellow protein-tagged GluN1 subunit rat hippocampal neurons, compared these two probes previously established probe, rabbit IgG together with secondary antiGFP-QD605). nanoGFP-based allowed faster NMDARs, several-fold increased median values coefficient ( D ). Using thresholded tdTomato-Homer1c signals mark regions, found nanoprobe-based sharply distances over 100 nm from edge, while for antiGFP-QD605 probe remained unchanged up 400 distance. nanoGFP-QD605 neurons expressing GFP-GluN2A, GFP-GluN2B, GFP-GluN3A subunits, detected subunit-dependent differences localization value, residence time, synaptic–extrasynaptic exchange rate. Finally, confirmed applicability study distribution by comparing data obtained nanoGFPs organic fluorophores, using universal point accumulation imaging nanoscale topography direct stochastic optical reconstruction microscopy. SIGNIFICANCE STATEMENT Our systematically mobility containing subunits expressed rodent anti-green protein (GFP) 605 (nanoGFP-QD605), well nanoGFP small fluorophores. comprehensive analysis showed method delineate region plays an important pools NMDARs. In addition, has optimal parameters studying because its high accuracy comparable microscopy longer scan time topography. developed approaches readily applicable any GFP-labeled membrane mammalian neurons.

Language: Английский

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Dynamic role of GlyT1 as glycine sink or source: pharmacological implications for the gain control of NMDA receptors

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 17, 2024

Abstract Glycine transporter 1 (GlyT1) mediates termination of inhibitory glycinergic receptors signaling in the spinal cord and brainstem, is also diffusely present forebrain. Here, it regulates ambient glycine concentration influencing ‘glycine’-site occupancy N -methyl-d-aspartate (NMDARs). GlyT1 a reversible with substantial, but not excessive, sodium-motive force for uphill transport. This study examines its potential role as source, either by reversed-uptake or heteroexchange. I explored how accumulation triggers release, facilitating activation NMDARs glutamate applied alone. Indeed, evokes no current “naive” oocytes coexpressing GluN1/GluN2A GlyT1, previously characterized cellular model, now using only source coagonist NMDAR activation. After uptake, however, large currents, blocked ALX-5407 potentiated sarcosine, specific inhibitor substrate respectively. These results suggest higher co-agonist site when functions A difference between these two glycine-release mechanisms occurs at hyperpolarized potentials, which induce an apparent voltage-dependent block whereas heteroexchange preserves potentials. Together, confirm GlyT1-mediated efflux positive regulator occupancy, demonstrate sarcosine effectiveness enhancing occupancy. Depending on actual mode transport, GlyT1-inhibitors may have distinct effects facilitation, be variation reversing hypofunction schizophrenia. Graphical

Language: Английский

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CA1i pyramidal neurons mediate the role of NMDA receptor subunit GluN3A in depressive behavior and D-serine anti-depression

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 28, 2024

Abstract Depression is a heterogeneous psychiatric disorder characterized by multiple symptom clusters. N-methyl-d-aspartic acid receptors (NMDARs), consisting of various subunit proteins GluN1-3, are known to be critical molecular bases for the occurrence and treatment depression. However, involvement NMDAR GluN3A in heterogeneity depressive symptoms antidepressant effects remains unclear. Here, we found that chronic social defeat stress (CSDS) induced range depression-related behaviors, including decreased interest, increased helplessness anxiety-like behavior, reduced mRNA protein expression hippocampal CA1 intermediate (CA1i) region. Additionally, knockout (KO) mice exhibited pronounced behavior. Increasing CA1i both models specifically reversed behavior but not interest Furthermore, lack activity pyramidal neurons during phenomenon also upregulating expression. Further bidirectional modulation neuron directly mimicked or CSDS-induced Finally, injection D-serine into rapidly improved CSDS while increasing neurons, whereas inhibition prevented effect D-serine. Our study elucidates role regulating its mechanisms, as well rapid D-serine, which deepen understanding complex pathophysiology depression develop potential clinical new target.

Language: Английский

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An increased copy number of glycine decarboxylase (GLDC) associated with psychosis reduces extracellular glycine and impairs NMDA receptor function

Molecular Psychiatry, Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 30, 2024

Language: Английский

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Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages

ACS Applied Materials & Interfaces, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 5, 2024

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest photolabile protecting groups (PPGs) photouncaging. growing need for detection of uncaging events has led to development reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags turn-off properties been drastically underexplored, although there are applications where they would be sought after. this work, a rhodamine-based tag is developed signal following One-photon photolysis experiments reveal ready loss red UV (365 nm) irradiation, while no significant change observed control absence PPG or irradiation around absorption maximum fluorophore (595 nm). two-photon explored hydrogel photolithography experiments. hydrogel-bound enables power-, dwell time-, wavelength-dependent construction intricate patterns gradients. Finally, prominent caged neurotransmitter (MNI-Glu) modified tag, resulting glutamate precursor named as GlutaTrace traceability successfully applied visualization distribution capillary microinjection selective excitation neurons mouse brain model.

Language: Английский

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