On the day of live cell calcium imaging, 50 µg Fluo-4 AM cell-permeant dye (Invitrogen) was diluted in 10 µl of 20% pluronic F-127 in DMSO. Fluo-4 AM was diluted 1:1000 in BrainPhys medium and added to coverglass chamber wells after rinsing cells once in HBSS. Samples were incubated at 37°C for 30 min. After incubation, Fluo-4 AM medium was removed, washed once in HBSS and fresh BrainPhys without phenol red was added. Samples were imaged directly on the Zeiss Axio Observer.Z1 system. Time-lapse series were acquired at 50 ms exposure using a 488 nm LED at 200 ms intervals for 1–1.5 min duration. After imaging baseline activity in BrainPhys, 1 μM DAMGO (MOR agonist, 10 mM stock in water; Abcam) was added directly to chamber wells and imaged again. The competitive opioid receptor antagonist naloxone hydrochloride was then added at 10 μM (10 mM stock in water; Abcam). EMLO-dissociated cultures, spinal sensory, and spinal motor neuron samples were imaged in succession to control for time of Fluo-4 AM cell loading. Time-lapse series were quantified in ImageJ.
Damgo
Manufactured by Abcam
Sourced in United Kingdom
DAMGO is a synthetic peptide that acts as a selective agonist for the mu-opioid receptor (MOR). It is commonly used in pharmacological research to study the effects and mechanisms of MOR activation.
Automatically generated - may contain errors
5 protocols using damgo
1
Live-cell Calcium Imaging of Neuronal Responses
2
Sexually Distinct Effects of LC Microdosing
3
Modulation of Opioid Receptor Signaling
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We purchased the following MOP‐targeting ligands: [D‐Ala 2 , NMe‐Phe 4 ‐, Gly‐ol 5 ] (DAMGO) (Abcam), and morphine hydrochloride (Takeda Pharmaceutical Company). Brain‐derived neurotrophic factor (BDNF) and all‐trans retinoic acid (ATRA) were purchased from FUJIFILM Wako. We altered the medium every 24 h for prolonged treatment with ATRA or BDNF or prolonged stimulation with a MOP ligand.
4
Investigating IPSC Modulation by Drugs
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Spontaneous inhibitory postsynaptic currents (sIPSC) were recorded in the absence and presence of a variety of drugs including bicuculline, morphine, DAMGO, DPDPE (Abcam, Cambridge, UK), PP2, PP3 and SL327 (all from Tocris, Bristol, UK). All drug solutions were prepared as required on the day of use from frozen stock solutions and were diluted in the extracellular solution. Control and drug containing solutions were applied to the recording chamber using a peristaltic pump (Scientifica, East Sussex, UK). DAMGO and morphine were applied at ascending concentrations to establish the concentration-response relationships. Each concentration was applied to slices for 450 s to establish steady state; with up to seven morphine concentrations per neuron, there was a maximum of 53 min of exposure.
5
Intra-LC Microinfusion of DAMGO and Clonidine
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Drugs used for intra-LC microinfusion were DAMGO ([D-Ala2, N-MePhe4, Gly-ol]enkephalin; Abcam, Cambridge, MA), a synthetic opioid peptide with high MOR specificity and clonidine HCl (Sigma, St Louis, MO). These compounds were aliquoted and concentrated using a Speed Vac and stored at -20 o C before experimental procedures. On the day of the experiments, drugs were dissolved in artificial cerebrospinal fluid (ACSF). The doses that were microinfused into the LC were DAMGO (10 pg in 200 nl) and clonidine (50 ng in 200 nl). The dose of DAMGO is one that has been demonstrated to produce sexually distinct effects on LC activity and on cognitive endpoints when microinfused into the LC (Guajardo et al, 2017) . The dose of clonidine has been demonstrated to produce a long lasting cessation of LC spontaneous discharge (Page et al, 1993) .
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