A Molecular Landscape of Mouse Hippocampal Neuromodulation

Smith, Stephen J.; Zastrow, Mark von

doi:10.3389/fncir.2022.836930

Cited by 3 publications

(1 citation statement)

References 113 publications

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“…This process is primarily based on the binding of a variety of secreted "modulatory" ligands to G protein-coupled receptors, which govern the operation of the ion channels affecting synaptic weights and membrane excitability. 20 The possibility to also apply our approach to studies on neuromodulation substantially widens the number of potentially interested researchers and opens yet unexplored avenues to implement the 3Rs principles.…”

Section: Introductionmentioning

confidence: 99%

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Merighi

Lossi²

2022

F1000Res

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Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants missing the Reelin gene (reln-/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing ex vivo cultured cerebellar slices one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs). Methods: We generated mouse hybrids (L7-GFPrelnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different reln genotypes and demonstrated that when the slices from reln-/- mutants were co-cultured with those from reln+/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion. Results: In images of the co-cultured slices from reln-/- mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of reln+/- animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from reln-/- mice were cultivated singularly or co-cultivated, supporting mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo. Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it for the possibility to study and manipulate ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective).

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Section: Introductionmentioning

confidence: 99%

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Merighi

Lossi²

2022

F1000Res

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Role of the G protein-coupled receptor kinase 2/3 N terminus in discriminating the endocytic effects of opioid agonist drugs

Li,

Inoue,

Manglik

et al. 2025

Molecular Pharmacology

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Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by GPCR kinases (GRKs) from the GRK2/3 subfamily. While these relatively 'downstream' steps in the agonist-selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished 'upstream' by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs, and that agonist-selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved PH domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist-selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist-selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and utilizes the NTD as an agonistselective sensor. Significance StatementThis study investigates how GPCR kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist-selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N-terminus is a key determinant of agonist discrimination.

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Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Merighi,

Lossi

2023

F1000Res

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Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler mouse phenotype. In reln(-/-) mutants, neurons are mispositioned in layered brain areas such as the cerebellar cortex. We demonstrate that in cultured cerebellar slices, one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs). Methods: We generated mouse hybrids (L7-GFP reln F1/) with GFP-tagged PNs, directly visible under fluorescence microscopy. We cultured singularly or in combination the slices from mice with different reln genotypes and used Voronoi tessellation and geographic information systems (GIS)-based spatial statistics to validate microscopic observations. Results: In co-cultured slices from reln(-/-) mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than in slices of reln(+/-) animals, thus indicating a rearrangement of the cortical architecture toward normality. The mean roundness factor, area disorder, and roundness factor homogeneity differed when slices from reln(-/-) mice were cultivated singularly or co-cultivated with slices from reln(+/-) mice. Analysis of Central Feature, Mean Center, Median Center, Directional Distribution, Standard Distance, Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, Global Moran’s I, Anselin Local Moran’s I, and Getis-Ord G* were fully supportive of Voronoi’s results giving further insight on the role of Reelin in cerebellar development. Our approach demonstrated mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo. Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it to study and manipulate ex vivo the activity of a bioactive protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the avoidance of severe surgery (3Rs perspective).

show abstract

A Molecular Landscape of Mouse Hippocampal Neuromodulation

Cited by 3 publications

References 113 publications

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Role of the G protein-coupled receptor kinase 2/3 N terminus in discriminating the endocytic effects of opioid agonist drugs

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

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