Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo

Abstract

The combination of microdialysis and a highly sensitive radioimmunoassay was developed in order to monitor the in vivo extracellular levels of endogenous opioid peptides from discrete regions of the rat brain. The radioimmunoassay cross-reacts 100% with peptides with alpha N-acetyl Tyr.Gly.Gly.Phe-Met or -Leu at the N terminus and thus recognizes all known endogenous opioid peptide fragments following acetylation of the sample. The assay was conducted on solid phase with antibody bound via protein A to 96-well plates and provided a limit of detection of approximately 0.2 fmol. A variety of dialysis membranes were evaluated with respect to their efficiency in recovering opioid peptides in vitro. Custom-made probes (4 mm active length) manufactured from polyacrylonitrile membranes and commercially available polycarbonate membrane probes proved most suitable with relative recoveries for [Met]- and [Leu]enkephalin in the range 6–10% at a flow rate of 2.7 μl/min. Probes implanted in the globus pallidus/ventral pallidum of halothane/N 2O anaesthetized rats recovered approximately 1.5 fmol of immunoreactive opioid material per 30-min sample in the absence of peptidase inhibitors. The majority of this immunoreactivity co-eluted with [Met]- and [Leu]enkephalin on reverse-phase high-performance liquid chromatography. A 2-min pulse of 100mM K +-containing artificial cerebrospinal fluid in the perfusion medium during a 30-min sampling period increased the recovered immunoreactive material to 43.9fmol± 12.4 S.E.M. A second stimulation 3 h later also resulted in elevated levels with an S2:S1 ratio of 0.64 ± 0.03. The second stimulation was completely blocked by perfusion of a 10 mM EGTA-containing medium, basal release on average remaining unaffected. Repeated K + stimulations (50 mM) at 1-h intervals resulted in progressively diminishing elevations in opioid peptide release. Incorporation of veratridine (50μM) in the perfusion medium for 2 min of a 30-min collection period increased the release to 16.8fmol± 0.5 with an S2:S1 ratio of 0,62 ± 0.17 after a 3-h interval. The second stimulation was prevented by simultaneous perfusion of a 2 μM tetrodotoxin-containing medium, basal release remaining unaffected. The results demonstrate the potential of this technique for studying the regulation of opioid peptide release and metabolism from discrete regions of the rat brain in vivo.