Quantitative microdialysis of neuropeptide Y

The feasibility of using the difference method of quantitative microdialysis to measure neuropeptide Y (NPY) was evaluated in vitro and in vivo. The accuracy of this method was tested in vitro under steady-state conditions for 3 test solutions containing known concentrations of NPY. The estimated concentrations of NPY were 1.2 ± 0.6, 3.7 ± 0.9, and 15.1 ± 0.7 pg/μl (mean ± SEM) in agreement with the actual concentrations of NPY in the test solutions which were 1.1 ± 0.8, 4.6 ± 0.6, and 14.6 ± 0.5 pg/μl (mean ± SEM of solution samples), respectively. The responsiveness of the estimated NPY_ext measure to changes in the external concentration of NPY was also evaluated in vitro. An accurate estimate of NPY_ext was obtained within the first sampling period (within 15 min) after a 2-3-fold increase in the test solution concentration of NPY and within 2-3 sampling periods (15-45 min) in response to a 2-3-fold decrease in the test solution concentration of NPY. In vivo, the estimated basal concentration of NPY in dialysis samples from probes in the medial basal hypothalamus of anesthetized female rats (n = 4) was 4.0 ± 1.6 pg/μl and increased to 9.5 ± 0.3 pg/μl during K⁺ stimulation. Relative recovery was 22% in vivo under steady-state conditions and ranged from 14% to 30% during dynamic conditions. These results demonstrate that the difference method of quantitative microdialysis accurately estimates picomolar concentrations of NPY in vitro, and is sufficiently sensitive to detect basal and increasing concentrations of NPY in vivo.