Comparison of regulation of K+- and d-amphetamine-induced noradrenaline efflux in NK1-/- and NK1+/+ mice

Yan T, 1Hunt SP and Stanford SC. Depts Pharmacology and 1Anatomy & Developmental Biology, University College London, London, UK

In our previous studies, anaesthetised NK1-/- mice (in which expression of the substance P‑preferring (NK1) receptor is disrupted), display ed 2 ~4 fold greater noradrenaline (NA) efflux than the wildtype (NK1+/+). This is possibly due to impaired regulation of NA release by presynaptic α 2-adrenoceptors (Herpfer et al., 2005). Here, in vivo microdialysis was used to investigate differences in K+-evoked and d-amphetamine (d-AMP)-induced NA efflux (considered to be impulse-independent) in the frontal cortex of freely-moving NK1-/- and NK1+/+ mice. Further, we tested our proposal that α2-adrenoceptor function is impaired in the NK1-/- phenotype. Microdialysis probes were implanted in male mice (29~36g; 129/Sv X C57BL/6 crossed with an outbred MF1 strain) under halothane anaesthesia. The next day, the probes were perfused with Ringer’s solution (1.5 μl min-1). Samples were collected every 20 min and their NA content measured by HPLC-ECD. Data were analysed by split-plot ANOVA.

In Experiment 1, NK1+/+ and NK1-/- mice (n=11~13) were given either 2 x 40-min pulses of 80 mM K+, via the probe, or a pulse of 80 mM K+ followed by a pulse of Ca2+-free Ringer’s solution. The first K+ challenge augmented NA efflux by 6.0 ± 1.0 fmol 20 min-1 (n=11~13) in both phenotypes. The response to the second K+ pulse was smaller in NK1+/+ but not NK1-/- mice. Removal of Ca2+ attenuated the NA response in NK1-/- mice, only ( F1,20=6.0; P=0.02). However, in NK1+/+ mice, the NA response to 80 mM K+ was blunted when the Ca2+-free medium contained EGTA (4 mM; F1,4=20.8; P=0.01). In Experiment 2 , different batches of mice (n=12~14) were infused with Ringer’s solution containing d-AMP (10  μM and then 100 μM). The effect of pretreatment with the α2-adrenoceptor antagonist, atipamezole (10 mg kg-1, i.p), was investigated, also. 10 μM d-AMP transiently increased NA efflux in both phenotypes, but only in NK1+/+ mice was there a greater response at 100  μM ( 3.0 ± 0.8 and 4.9 ± 1.5 fmol 20 min-1, respectively). At both concentrations of d-AMP, the NA response was greater in NK1+/+ mice. Moreover , only in NK1+/+ mice, was the response to 10 μM d-AMP increased by atipamezole ( F1,26=4.5; P=0.04) .

The findings indicate that basal NA efflux rests on Ca2+-dependent release in both phenotypes. However, NK1-/- mice showed abnormal NA responses in both experiments. The sustained response to a second K+ pulse suggests that they have a larger releasable pool of NA and/or more efficient excitation-release coupling, but they are more vulnerable to Ca2+ depletion. The lack of an increase in NA efflux at the higher concentration of d-AMP and the smaller NA response in NK1-/- mice could be due to greater competition between d-AMP and extracellular NA at the uptake transporter. The failure of atipamezole to increase the NA response to d-AMP in NK1-/- mice supports our proposal that their α2- adrenoceptor function is impaired (Herpfer et al., 2005). In conclusion, NK1-/- mice display abnormal K+- and d-AMP-induced NA efflux, possibly arising from impaired α2-adrenoceptor function, a larger NA release pool and/or greater sensitivity to Ca2+.

Herpfer, I. et al. (2005) Neuropharmacology, 48, 706-19.

This work was funded in part by SM Yan and JP Wen