Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior

Abstract

The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET(+/-) ), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET(+/-) mouse establishes an activated state of existing surface NET proteins. The NET(+/-) mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET(+/-) mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET(+/-) mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders.

Keywords: Anxiety; Morris water maze; depression; genetic; heterozygote; knockout; light-dark box; norepinephrine; tail suspension; transporter.

Figure 1

NET expression and activity in synaptosomes from NET^+/+ versus NET^+/− mice. Synaptosomes were subjected to surface biotinylation (sulfo-NHS-SS biotin) followed by immunoisolation. Total and surface NET and TH levels were analyzed on Western blot (a,b). In the same sample, [³H]NE transport was measured in a ten minute transport assay at 37°C using 150 nM [³H]NE. Data are expressed as the mean percent ± SEM of NET^+/+ results for each assay and brain region. *p < 0.05, **p < 0.01, ***p < 0.001, N=3 (b). Saturation kinetics of [³H]NE transport. Synaptosomes were incubated in 10 nM-1 μM [³H]NE for 10 min at 37°C (c). TH, tyrosine hydroxylase; CTX, cortex; HPC, hippocampus.

Figure 2

Specificity of NET-mediated transport in brain synaptosomes. Saturation kinetics of [³H]NE transport in the presence of either 1 μM atomoxetine (ATE) or 1 μM atomoxetine plus 1 μM citalopram (CIT). Synaptosomes were incubated in 10 nM-1 μM [³H]NE for 10 min at 37°C (N=3) (a). [³H]NE transport in the presence of 1 μM desipramine or 1 μM decynium-22 (D-22) followed by a 10 min incubation in 150 nM [³H]NE at 37°C. ***p < 0.001, N=4 (b). Saturation kinetics of of [³H]NE transport in the presence or absence of Na⁺. Synaptosomes were incubated in 10 nM-1 μM [³H]NE for 10 min at 37°C (c). [³H]NE transport in normal, Na⁺-free, and normal Na⁺ following Na⁺-free conditions. Synaptosomes were incubated in 150 nM [³H]NE for 10 min at 37°C. **p < 0.01, ***p < 0.001, N=4 (d).

Figure 3

Anxiety/depression phenotypes in NET^+/+ versus NET^+/− mice. Open field exploration during a 1 h trial (a,b) Time spent in the center versus periphery of the open field. Data are expressed as mean time in s ± SEM for each area of the field. **p < 0.01, N=14-20 (a). Distance traveled in the open field displayed in 5 min blocks (N=14-20) (b). Time spent in the light vs. dark in a 5 min light-dark test. Data are expressed as mean time in s ± SEM for each compartment. **p < 0.01, N=21-29 (c). Immobility during a 7 min-duration tail suspension test. N=14-20 (d).

Figure 4

Performance in the Morris water maze. For the acquisition and reversal phases, each mouse underwent four trials per day and the mean per mouse for a measure was used to calculate the mean ± SEM for the NET^+/+ or NET^+/− group on that day (a,c,e). Latency to find the platform during the acquisition (a) and reversal (c) phases of the task. *p < 0.05, N=8-20. Probe trials were performed once for 60 seconds on the days following the acquisition (b) and reversal (d) phases and are expressed as percent time spent in the target quadrant versus other quadrants ± SEM. Swim speed during the reversal phase of mice shown in c (e).