Determination of extracellular release of neurotensin in discrete rat brain regions utilizing in vivo microdialysis/electrospray mass spectrometry

Abstract

In vivo microdialysis was used together with structure-specific high sensitivity nano-flow capillary liquid chromatography/micro-electrospray mass spectrometry to quantify and compare extracellular neurotensin from discrete regions of the rat brain. Microdialysis probes were implanted in the hypothalamus or globus pallidus/ventral pallidum in unanesthetized freely moving animals. Utilizing this specific methodology, recovered basal levels of neurotensin were detectable in hypothalamus and globus pallidus/ventral pallidum. The basal level of neurotensin in these regions were slightly higher in hypothalamus (101±11 amol/10 μl, n=6) compared to those in the globus pallidus/ventral pallidum region (74±12 amol/10 μl, n=8) in samples collected for 30 min at a flow-rate of 0.4 μl/min 150–180 min after the microdialysis probe implantation. After a pulse of 1.0 μl of 100 mM KCl-containing artificial cerebrospinal fluid during the next 30-min sampling period (180–210 min), the recovered neurotensin increased in hypothalamus and globus pallidus/ventral pallidum by 544% (548±90 amol/10 μl) and 674% (499±99 amol/10 μl), respectively. The basal levels of endogenously released neurotensin in the hypothalamus and globus pallidus/ventral pallidum were lower in the present study compared to those previously reported in the rat brain using in vivo microdialysis and radioimmunoassays. Our data demonstrate the effectiveness of combining in vivo microdialysis and structure-specific micro-electrospray mass spectrometry for the quantitation of basal and stimulated in vivo levels of endogenous neurotensin (NT) in different brain areas.