Aug 05, 2024
In vivo microdialysis for striatal DA release
- 1VHIR-CIBERNED-ASAP
- Vilalab Public
- Nuria
Protocol Citation: Ariadna Laguna, Miquel Vila 2024. In vivo microdialysis for striatal DA release. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8q1xv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 05, 2024
Last Modified: August 05, 2024
Protocol Integer ID: 104683
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
To assess local effects of nomifensine (DAT and NET inhibitor) on striatal DA
release in microdialysis experiments
Preparation of reagents
Preparation of reagents
Nomifensine (DAT and NET inhibitor) is dissolved in artificial cerebrospinal fluid (aCSF in mM: NaCl, 125; KCl, 2.5; CaCl2, 1.26 and MgCl2 1.18)
Concentrated solutions (1 mM; pH adjusted to 6.5–7 with NaHCO3 when necessary) are stored at -80ºC and working solutions are prepared daily by dilution in aCSF.
Mouse surgery
Mouse surgery
One concentric dialysis probe (Cuprophan membrane; 6000 Da molecular weight cut-off; 1.5 mm-long) is implanted in the striatum (AP, 0.5; ML, -1.7; DV, -4.5 in mm) of isoflurane-anesthetized mice
Microdialysis experiments are performed in freely-moving mice 24h after surgery.
Microdyalisis
Microdyalisis
Probes are perfused with aCSF at 1.5 μL/min
Following an initial 100-min stabilization period, 5 or 7 baseline samples are collected (20 min each) before local drug application
Nomifensine is administered by reverse dialysis at 10 and 50µM (uncorrected for membrane recovery) and then successive dialysate samples are collected
Dopamine levels determination
Dopamine levels determination
The concentration of DA in dialysate samples is determined by HPLC coupled to electrochemical detection (+0.7 V, Waters 2465), with 3-fmol detection limit.
The mobile phase containing 0.15 M NaH2PO4.H2O, 0.9 mM PICB8, 0.5 mM EDTA (pH 2.8 adjusted with orthophosphoric acid), and 10 % methanol is pumped at 1 ml/min (Waters 515 HPLC pump)
DA is separated on a 2.6 mm particle size C18 column (7.5 x 0.46 cm, Kinetex, Phenomenex) at 28°C.
Data representation
Data representation
Microdialysis data is expressed as femtomoles per fraction (uncorrected for recovery) and are shown as percentages of basal values (individual means of 5-7 pre-drug fractions).