Culturing primary neurons from rat hippocampus and cortex

Sahu, Madhusmita Priyadarshini; Nikkilä, Outi; Lågas, Seija Anneli; Kolehmainen, S.; Ċastrén, Eero

doi:10.1101/491118

Cited by 21 publications

(22 citation statements)

References 18 publications

(20 reference statements)

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“…Primary cultures of cortical and hippocampal cells from rat embryos (E18) or P0 mice (for AP2M.het and wt) were prepared as described in the literature (25) , and maintained in NeuroBasal medium (Thermo Fisher Scientific) supplemented with 1% B-27 supplement (Thermo Fisher Scientific), 1% penicillin/streptomycin, and 1% L-glutamine at 37°C (25) .…”

Section: Cell Culture and Silencing Of Ap2m Expressionmentioning

confidence: 99%

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2

Fred

Laukkanen

Brunello

et al. 2019

Journal of Biological Chemistry

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Antidepressant drugs activate TRKB (tropomyosin-related kinase B), however it remains unclear whether these compounds employ a common mechanism for achieving this effect. We found by using mass spectrometry that the interaction of several proteins with TRKB was disrupted in the hippocampus of fluoxetine-treated animals (single intraperitoneal injection), including members of the AP-2 complex (adaptor protein complex-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking mu subunit of the AP-2 complex (AP2M) was disrupted by both acute and repeated fluoxetine treatment.However, while the coupling between full length TRKB and AP2M was disrupted by fluoxetine, the interaction between AP2M and the TRKB C-terminal peptide was resistant to this drug, indicating that the binding site targeted by fluoxetine must lie outside of the TRKB:AP2M interface. In addition to fluoxetine, other pharmacologically diverse antidepressants imipramine, rolipram, phenelzine, ketamine, and the ketamine metabolite 2R,6R-hydroxynorketamine (RR-HNK) also decreased the interaction between TRKB:AP2M in vitro , as measured by ELISA. Silencing the expression of AP2M in MG87.TRKB cell line led to increased surface positioning of TRKB and to a higher response to BDNF (brain-derived neurotrophic factor), observed as the levels of active TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB in vivo , as well as an enhanced cell surface expression of the receptor in cultured hippocampal neurons.Taken together, our data suggests that disruption of the TRKB:AP2M interaction is an effect shared by several antidepressants with diverse chemical structures and canonical modes of action.

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Section: Cell Culture and Silencing Of Ap2m Expressionmentioning

confidence: 99%

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2

Fred

Laukkanen

Brunello

et al. 2019

Journal of Biological Chemistry

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“…Primary cultures of hippocampal cells from E17-E18 rat embryos were prepared as detailed in literature (Sahu et al, 2019). Briefly, hippocampus of E18 rat embryos were dissected and suspended cells were seeded on poly-L-lysine coated 24-well plates (250,000 cells/well, 1.9cm 2 ) in Neurobasal medium supplemented with B27, 1% penicillin/streptomycin, 1% L-glutamine.…”

Section: Cell Culturementioning

confidence: 99%

Nitric Oxide Synthase inhibition counteracts the stress-induced DNA methyltransferase 3b expression in the hippocampus of rats

Maciel

Sales

Casarotto

et al. 2020

Preprint

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It has been postulated that activation of NMDA receptors (NMDAr) and nitric oxide (NO) production in the hippocampus is involved in the behavioral consequences of stress. Stress triggers NMDAr-induced calcium influx in limbic areas, such as the hippocampus, which in turn activates neuronal NO synthase (nNOS). Inhibition of nNOS or NMDAr activity can prevent stress-induced effects in animal models, but the molecular mechanisms behind this effect are still unclear. In this study, cultured hippocampal neurons treated with NMDA or dexamethasone showed increased of DNA methyltransferase 3b (DNMT3b) mRNA expression, which was blocked by pre-treatment with nNOS inhibitor nω-propyl-L-arginine (NPA). In rats submitted to the Learned Helplessness paradigm (LH), we observed that inescapable stress increased of DNMT3b mRNA expression at 1h and 24h in the hippocampus. The NOS inhibitors 7-NI and aminoguanidine (AMG) decreased the number of escape failures in LH, and counteracted the changes in hippocampal DNMT3b mRNA induced in this behavioral paradigm. Altogether, our data suggest that NO produced in response to NMDAr activation following stress upregulates DNMT3b in the hippocampus.

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“…Hippocampal cells from rat embryo (E18; DIV16-18) were cultivated onto glass coverslips coated with poly-L-lysine at 200 000 cells/well (Sahu et al 2019) . The cultures were maintained at 37ºC in serum-free Neurobasal medium (supplemented with 2% B27, 1% L-glutamine, and 1% ampicillin) and transfected by calcium phosphate co-precipitation method (Xia et al 1996) to overexpress GFP-tagged TRKB constructs from mouse sequence (TRKB.WT or TRKB.R427A/Y433F).…”

Section: Cell Cultures and Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

Cannarozzo

Fred

Girych

et al. 2019

Preprint

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Cholesterol is an essential constituent of cell membranes. Recently, the discovery of cholesterol recognition amino acid consensus (CRAC) on proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon recognition of the conserved CARC motif in the TRKB, we compared the effect of point mutations in CARC on structural changes in the TMR of mouse TRKB. The alignment of wild-type and mutant TMR indicates small morphological changes across the 6 mutations analyzed (Y433F, Y433C, Y433A, V437K, R427A, and the double mutation R427A/Y433F), as demonstrated by the root-mean-squared deviation values for the superimposed structures. A molecular dynamics simulation with the mouse TRKB TMR sequence indicated that cholesterol interaction with the TRKB CARC motif is reduced by the R427A/Y433F mutation. Experimental data assayed by fluorescence recovery after photobleaching indicated a reduction in brain-derived neurotrophic factor-induced mobility of TRKB.R427A/Y433F in the spine of cultured hippocampal neurons. Therefore, CARC/CRAC motifs may have a role in the function of the RTK family TMR.

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Culturing primary neurons from rat hippocampus and cortex

Cited by 21 publications

References 18 publications

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2

Nitric Oxide Synthase inhibition counteracts the stress-induced DNA methyltransferase 3b expression in the hippocampus of rats

Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

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