Brain Research 946 (2002) 239–246

www.elsevier.com / locate / bres

Research report

Regulation of norepinephrine transporter abundance by catecholamines

and desipramine in vivo
David Weinshenker a,b , *, Sylvia S. White c , Martin A. Javors e,f , Richard D. Palmiter a,b ,
Patricia Szot c,d
a
Howard Hughes Medical Institute, Box 357370, University of Washington, Seattle, WA 98195, USA
b
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
c
Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA 98195, USA
d
GRECC, Puget Sound Health Care System, Seattle, WA 98108, USA
e
Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78284, USA
f
Department of Psychiatry, University of Texas Health Science Center, San Antonio, TX 78284, USA

Accepted 18 April 2002

Abstract

The norepinephrine transporter (NET) regulates adrenoreceptor signaling by controlling the availability of synaptic norepinephrine
(NE), and it is a direct target for some classes of antidepressant drugs. NET levels are normal in dopamine b-hydroxylase knockout (Dbh
2 /2 ) mice that lack NE, demonstrating that the NET does not require endogenous NE for appropriate regulation under physiological
conditions. In contrast, tyrosine hydroxylase knockout (Th 2 /2 ) mice that lack both NE and dopamine (DA) have reduced levels of NET,
suggesting that it is down-regulated by a complete absence of catecholamines and not NE per se. Chronic treatment with the NET
inhibitor, desipramine (DMI), reduced NET levels in both control and Dbh 2 /2 mice, demonstrating that NE is not required for the
regulation of NET by antidepressant drugs. There are some qualitative and quantitative differences in the down-regulation of the NET by
catecholamine depletion and DMI treatment, suggesting that different mechanisms may be involved.
 2002 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Uptake and transporters

Keywords: Norepinephrine transporter; Desipramine; Knockout mouse; Dopamine b-hydroxylase; Nisoxetine; Antidepressant

1. Introduction To maintain homeostatic NE signaling, NET levels

Norepinephrine (NE) is an abundant neurotransmitter in
the peripheral and central nervous systems, where it is
involved in many physiological and behavioral processes,
including cardiovascular function, metabolism, embryonic
development, arousal, seizure susceptibility, maternal be-
havior, and responses to drugs of abuse [9,11,26–28]. The
norepinephrine transporter (NET) is a Na 1 / Cl 2 -dependent
transporter expressed by noradrenergic neurons, where it
modulates noradrenergic signaling by clearing secreted
norepinephrine via selective uptake (e.g. Ref. [1]).
*Corresponding author. Tel.: 11-206-543-6090; fax: 11-206-543-
0858.
E-mail address: dzw@genetics.washington.edu (D. Weinshenker).
would be expected to decrease when synaptic NE content
is low. The work of Lee et al. [19] supports this, as NET
levels decrease when NE is depleted with reserpine. These
results suggest that NE levels are sensed by the neuron and
NET abundance is then regulated accordingly. However,
because reserpine depletes dopamine (DA) and serotonin
(5-HT) as well as NE, it is unclear whether the depletion
of other monoamines contributes to the observed down-
regulation of the NET.
Among the drugs that inhibit the NET and block NE
reuptake are antidepressants such as DMI and reboxetine
[13]. Blockade of NE reuptake by antidepressant drugs
happens on the order of minutes to hours, while alleviation
of depressive symptoms requires weeks of chronic drug
administration (e.g. Ref. [21]). Therefore, it is thought that

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 02 )02889-5
240 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246
adaptive changes that follow chronic reuptake blockade are
responsible for the efficacy of antidepressant drugs. One
molecular change that follows the time course of clinical
efficacy is down-regulation of the NET. For example,
chronic, but not acute antidepressant treatment decreases
monoamine transporter density in rats [4,6]. However, it is
not known whether the antidepressant-induced increase in
extracellular monoamines is required for this effect.
To determine if NE, the primary substrate for the NET,
is required for NET regulation under basal conditions or by
chronic exposure to antidepressants in vivo, we measured
NET levels before and after chronic DMI administration in
dopamine b-hydroxylase (DBH) knockout (Dbh 2 /2 )
mice. These mice lack NE because DBH is essential for
the conversion of DA to NE. Because the NET is capable
of transporting DA [14,22] and Dbh 2 /2 mice have
elevated DA levels [30], NET levels were also determined
in tyrosine hydroxylase knockout (Th 2 /2 ) mice that lack
both NE and DA.
2. Material and methods
2.1. Animals
Dbh knockout (Dbh 2 /2 ) and Th knockout (Th 2 /2 )
mice, maintained on a 129 / SvEv and C57BL / 6J hybrid
background, were developed and generated as described
[30,33] with the following modifications. Th 1 /2 males
were bred to Th 1 /2 females. To rescue the embryonic
lethality caused by the lack of catecholamines in Th 2 /2
mice, pregnant females were given L-DOPA (50 mg / kg,
1.5 mg / ml) and vitamin C (2.5 mg / ml) both by daily
intraperitoneal (i.p.) injection and in the drinking water
until the birth of the litter, then discontinued. Therefore,
catecholamines were present in Th 2 /2 mice during but
not after embryogenesis. Th 2 /2 animals die of aphagia
and adipsia around the time of weaning because of a lack
of DA [32,33]. About a week before weaning, Th 2 /2
mice are distinguishable from their littermates because of
their reduced size, but otherwise appear healthy. Therefore,
postnatal day 15 (P15) Th 2 /2 mice and littermate
controls were used for the [ 3 H]nisoxetine binding studies.
Because adrenoreceptor agonists and L-3,4-dihydrox-
yphenylserine (DOPS) were administered to pregnant Dbh
1 /2 mothers to rescue the embryonic lethality associated
with the lack of NE in knockout pups [29,30], catechol-
amines were present in both Dbh 2 /2 and Th 2 /2
animals before but not after birth. Dbh 2 /2 mice were
reared in a specific pathogen-free facility and transferred to
a conventional facility for experiments, while Th 2 /2
mice were reared in a conventional facility. Both facilities
had a 12-h light / dark cycle at the University of Washing-
ton, and food and water were available ad libitum.
Dbh 2 /2 mice were identified by the delayed growth
and ptosis phenotype, which is 100% correlated with the
Dbh 2 /2 genotype (data not shown). A subset of geno-
types was confirmed by PCR. Th 2 /2 mice were geno-
typed by PCR. Dbh 1 /2 and Th 1 /2 mice have normal
catecholamine levels and are indistinguishable from wild-
type littermates for all previously tested phenotypes (e.g.
Refs. [24,26,29,30]). Therefore, heterozygous (1 /2 ) litter-
mates were used as controls for all experiments in this
study.
Experimental protocols were approved by the animal
care committee at the University of Washington and meet
the guidelines of the American Association for Accredita-
tion of Laboratory Animal Care.
2.2. Chronic DMI administration
In the first experiment, DMI (10 mg / kg, 2.5 mg / ml)
was dissolved in 0.9% NaCl and administered i.p. once per
day for 28 days. Mice were sacrificed on day 30 and brains
were frozen on dry ice and stored at 280 8C. In the second
experiment, DMI (96, 144, or 192 mg / ml, corresponding
to 20, 30, or 40 mg / kg per day for a 30 g mouse) was
dissolved in an aqueous solution containing 50% ethanol
and 0.9% NaCl, then adjusted to neutral pH (6.5–7) with 1
M NaOH. The DMI solution was then loaded into osmotic
minipumps (28 days, 6.24 ml / day; Alza, Palo Alto, CA).
Minipumps containing 50% ethanol, 50% water (pH 6.5–
7) were used as vehicle controls, and all pumps were
placed in a sterile 37 8C saline bath for 2 days before
implantation. Mice were anesthetized with isoflurane and
minipumps were implanted in the intraperitoneal cavity.
Surgery-associated fatality occurred in one Dbh 1 /2
vehicle mouse, two Dbh 1 /2 DMI mice, and four Dbh
2 /2 DMI mice. Minipumps were removed 21 days later,
and orbital bleeds were carried out on a subset of mice for
DMI measurements. Blood was collected in heparinized
tubes, centrifuged, and plasma was collected and stored at
280 8C. Mice were sacrificed after a 2-day ‘washout’
period. Trunk blood was collected from a subset of mice
for DMI measurements and brains were frozen on dry ice
and stored at 280 8C. To control for residual DMI in the
brains that was undetectable in the serum and could
interfere with [ 3 H]nisoxetine binding, mice with ‘vehicle’
minipumps received an i.p. injection of 30 mg / kg DMI (3
mg / ml) on day 21. Mice with DMI minipumps received an
i.p. injection of saline.
2.3. Measurement of DMI
The measurement of DMI was carried out according to
Benmansour et al. [6]) with minor modifications. Briefly,
25 ml of plasma for samples, calibrators, and controls were
aliquoted in polypropylene tubes along with 20 ml of a 10
mg / ml solution of doxepin (internal standard). Next, 200
ml of 5 N NaOH were added to each sample. After
vortexing, 3 ml of 5% isopropanol in hexane mixture were
added to the samples followed by gentle shaking for 10
 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246
min. Samples were then centrifuged at 12003g for 10 min
at 18 8C. Next, samples were placed in a methanol–dry ice
bath to freeze the aqueous layer. Then, the upper organic
layers were decanted and mixed with 150 ml of 12 mM
monobasic potassium phosphate, pH 2.5, for the back
extraction of the drugs. The samples were shaken, cen-
trifuged, placed in the methanol–dry ice bath, then the
upper organic layer was poured off and discarded. The
aqueous fraction was placed in a fume hood for 45 min to
allow any residual isopropanol / hexane to evaporate, then
100 ml of sample was injected into the HPLC system. The
HPLC system included a Beckman model 110B pump, a
Waters model 717 sample injector, a Waters 2487 UV
detector for HPLC, and a Spherisorb S5 CN column (5
mm).
Samples were analyzed by UV detection at a fixed
wavelength of 214 nm. The mobile phase contained 70%
acetonitrile, 13% methanol, and 17% of a solution of 10
mM KH 2 PO 4 (pH 6.7). The flow rate was 2.0 ml / min.
DMI concentrations were quantified by comparing samples
of unknown concentration against the linear regression of a
calibrator concentration curve. The detection limit for DMI
was 5 ng / ml at a signal-to-noise ratio of five. Recovery
was 85% and the coefficient of variation for within-day
variability among identical samples was less than 6%.
2.4. Measurement of NET mRNA by in situ
hybridization
A cDNA NET clone containing the entire nucleotide
sequence for the human NET was kindly provided by Dr S.
Amara (Vollum Institute, Oregon Health Sciences Uni-
versity, Portland, OR). The NET riboprobe was a 226-bp
fragment (nucleotides 1–226) which was subcloned into
the Sma I site of pBluescript SK1. Transcription and
labeling of the riboprobe was performed as described [25].
The probe was applied to tissue at a concentration of
0.7310 6 cpm / 50 ml. Hyperfilm (Amersham, Arlington
Heights, IL) was exposed to slides containing tissue
hybridized with [ 35 S]NET riboprobe for 7 days and
quantified as previously described by an independent
observer blind to genotype and drug treatment.
2.5. Measurement of NET binding sites
NET binding using [ 3 H]nisoxetine was performed on
18-mm mouse brain sections as described [4]. Incubation
buffer (400 ml / slide) contained [ 3 H]nisoxetine at |3 nM.
Non-specific binding was defined in the presence of 1 mM
mazindol. Slides were apposed to LKB 3 H-Ultrafilm (LKB
Instruments, Gaithersburg, MD) for 4 weeks. Each sheet of
film contained brain sections from each genotype and
treatment group. Autoradiograms were analyzed using
MicroComputer Imaging Device Systems (MCID; Imaging
Research Inc, Ontario, Canada). Either relative optical
density or 3 H-standards were used to quantitate the amount
241
of labeling in specific brain regions. Separate density
measurements from three anatomically-matched sections
were made across all treatment groups for each brain
region that gave a consistent signal above background.
Across three atlas-matched sections from each animal,
three readings were taken from the dorsal raphe (DR; plate
67 of Ref. [12]), and three readings were taken from the
left and right sides of the locus coeruleus (LC; plate 75),
dorsal and ventral hippocampus (HP; dorsal plate 44,
ventral plate 55), the lateral habenular nucleus of the
thalamus (plate 46), and the bed nucleus of stria terminalis
(BNST; plate 29). Density was similar between the dorsal
and ventral hippocampus, so dorsal hippocampal measure-
ments were used in the analysis. Therefore, for each
animal, the mean6S.E.M. for the DR were an average of
three density readings, and an average of six density
readings for the LC, HP, THAL and BNST. For each brain
region, the size and shape of the area where density was
measured was identical for all sections. In cases where a
brain region was damaged or missing because of tissue
loss during slide mounting, the animal was excluded from
the analysis for that brain region. Non-specific binding was
determined in sections incubated with 1 mM mazindol and
subtracted from the value of density reading.
[ 3 H]Nisoxetine binding was quantified by an independent
observer blind to genotype and drug treatment.
2.6. Statistics
Data were analyzed by Student’s t-test or Wilcoxon–
Mann–Whitney U-test when comparing two groups, and
ANOVA when comparing more than two groups.
3. Results
3.1. Dbh 2 /2 mice have normal NET levels
NET levels were measured in adult Dbh 1 /2 and Dbh
2 /2 mice by [ 3 H]nisoxetine binding. No differences
between genotypes were detected in any of the five brain
regions measured (Fig. 1A), demonstrating that NET levels
do not change in response to an absence of NE in mice.
3.2. Th 2 /2 mice have reduced NET levels
Because DBH converts DA to NE, Dbh 2 /2 mice
produce DA from ‘noradrenergic’ terminals by default.
Central DA tissue levels are |10% higher in Dbh 2 /2
mice, although DA release has not been measured [30]. In
addition, the affinity of the NET for DA is at least as high
as that for NE [14,22]. To determine whether NET levels
in Dbh 2 /2 mice are normal due to uptake of ectopic DA,
we examined NET levels in tyrosine hydroxylase knockout
(Th 2 /2 ) mice that lack both NE and DA [33]. Because
Th 2 /2 mice that survive embryogenesis die from
242 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246

Fig. 1. NET levels in catecholamine-deficient mice. NET levels were quantitated by autoradiography of [ 3 H]nisoxetine binding to brain sections. LC, locus
coeruleus; THAL, thalamus; HP, hippocampus; BNST, bed nucleus of the stria terminalis; DR, dorsal raphe. (A) Quantitation of [ 3 H]nisoxetine binding
from all brain regions examined of adult Dbh 1 /2 (n57) and Dbh 2 /2 (n57) mice. Units are relative optical density (ROD). (B) Representative section
at the level of the dorsal raphe are shown from a P15 Th 1 /2 mouse and (C) Th 2 /2 mouse. (D) Quantitation of [ 3 H]nisoxetine binding from all brain
regions examined of P15 Th 1 /2 (n57) and Th 2 /2 (n57) mice. Units are mCi of 3 H per g tissue. (E) Quantitation of [ 3 H]nisoxetine binding from all
brain regions examined of P15 Dbh 1 /2 (n510) and Dbh 2 /2 (n510) mice. Units are mCi of 3 H per g tissue. *P,0.05, **P,0.01, ***P,0.001
compared to 1 /2 control.

aphagia and adipsia around the time of weaning, this
experiment was done on P15 animals, a time when Th
2 /2 mice show no obvious signs of malaise [32,33]. We
found that Th 2 /2 mice had less [ 3 H]nisoxetine binding
across all brain regions than Th 1 /2 controls F(4, 44)5
2.97, P50.0296. This difference was significant in the
dorsal raphe nucleus (DR), the bed nucleus of the stria
terminalis (BNST) and the thalamus (THAL) but not in the
locus coeruleus (LC) or hippocampus (HP) (Fig. 1B–D).
This result suggests that the ectopic DA produced by the
Dbh 2 /2 mice is responsible for maintaining normal NET
levels in most terminal fields, but not in the region
containing cell bodies and dendrites of noradrenergic
neurons. To control for the effects of age on NET levels,
we measured [ 3 H]nisoxetine binding in P15 Dbh 1 /2 and
Dbh 2 /2 mice and found no difference across all brain
regions F(4, 68)50.5648, P50.689. Young Dbh 2 /2
mice appeared to have slightly lower NET levels in some
brains regions compared to littermate controls, and when
individual brain regions were compared between genotypes
a significant difference was found only in the THAL (Fig.
1E).
 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246 243

3.3. NE is not required for the reduction of NET levels
by chronic DMI
To determine whether NE is required for the down-
regulation of NET by antidepressant drugs, we measured
NET levels in Dbh 1 /2 and Dbh 2 /2 mice treated
chronically with DMI using a daily injection paradigm (10
mg / kg / d, i.p.) that has been shown to reduce
[ 3 H]nisoxetine binding to the NET in rats [4]. Because
DMI failed to reduce NET levels in mice of either
genotype (data not shown), we considered the possibility
that this regimen did not maintain sufficient drug con-
centrations in the brain and switched to a modified version
of chronic antidepressant drug administration described by
Benmansour et al. [6]. We administered DMI at 20, 30, or
40 mg / kg per day for 21 days via an osmotic mini-pump
and measured serum DMI concentrations after a 2-day
washout period. We chose 30 mg / kg per day for our
subsequent experiments because it produced serum DMI
concentrations (61–488 ng / ml, n56) on day 21 similar to
that seen clinically (125–600 ng / ml; [15]). Following the
2-day washout period, NET levels were measured by
[ 3 H]nisoxetine binding to brain sections. This paradigm
resulted in a significant reduction (17–42%, depending on
brain region) in NET levels in Dbh 1 /2 mice compared
to control animals implanted with vehicle-containing
minipumps across all brain regions measured, F(4, 44)5
11.3, P50.0001 (Fig. 2A,B,E). Individually, this reduction

Fig. 2. NET levels in vehicle and DMI-treated adult Dbh 1 /2 and Dbh 2 /2 mice. DMI (30 mg / kg per day) was administered for 21 days via osmotic
minipumps. NET levels were quantitated by autoradiography of [ 3 H]nisoxetine binding to brain sections. (A) Representative section at the level of the locus
coeruleus from a vehicle-treated Dbh 1 /2 mouse, (B) DMI-treated Dbh 1 /2 mouse, (C) vehicle-treated Dbh 2 /2 mouse, and (D) DMI-treated Dbh
2 /2 mouse. (E) Quantitation of [ 3 H]nisoxetine binding from all brain regions examined. Vehicle-treated mice received a one-time 30 mg / kg dose of DMI
(see Material and methods). Units are mCi of 3 H per g tissue. LC, locus coeruleus (Dbh 1 /2 vehicle n59, Dbh 1 /2 DMI n58, Dbh 2 /2 vehicle n510,
Dbh 2 /2 vehicle n55); DR, dorsal raphe (Dbh 1 /2 vehicle n59, Dbh 1 /2 DMI n58, Dbh 2 /2 vehicle n59, Dbh 2 /2 vehicle n56); HP,
hippocampus (Dbh 1 /2 vehicle n59, Dbh 1 /2 DMI n58, Dbh 2 /2 vehicle n510, Dbh 2 /2 vehicle n56); THAL, thalamus (Dbh 1 /2 vehicle n59,
Dbh 1 /2 DMI n58, Dbh 2 /2 vehicle n510, Dbh 2 /2 vehicle n56); BNST, bed nucleus of the stria terminalis (Dbh 1 /2 vehicle n58, Dbh 1 /2
DMI n55, Dbh 2 /2 vehicle n57, Dbh 2 /2 vehicle n56). *P,0.05, **P,0.01, ***P,0.001 compared to vehicle-treated control for each genotype.
244 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246
was significant in the LC, HP, DR, BNST, and the THAL.
A similar effect of DMI (13–48% reduction) was observed
in Dbh 2 /2 mice with this paradigm, F(4, 36)54.36,
P50.006 (Fig. 2C–E). NET levels in vehicle-treated,
F(4,48)50.177, P50.95, and DMI-treated, F(4, 32)5
1.728, P50.168, Dbh 1 /2 and Dbh 2 /2 mice were
comparable (Fig. 2E). We controlled for the possibility that
occupancy of the antidepressant binding site of the NET by
residual DMI after the 2-day washout period was respon-
sible for the observed reduction in [ 3 H]nisoxetine binding
in two ways. First, DMI was undetectable in the serum
following the 2-day washout period (data not shown).
Second, the vehicle-treated animals in Fig. 2E were given
a single bolus injection of 30 mg / kg DMI on day 21 when
the mini-pumps were removed to mimic the amount of
DMI that the drug-treated animals would have to clear
during the washout period. We conclude that the reduction
of [ 3 H]nisoxetine binding observed in DMI-treated animals
was not due to residual DMI.
In situ hybridization revealed similar NET mRNA levels
in the LC in all groups (Dbh 1 /2 saline 0.11460.008
mCi / g, Dbh 1 /2 DMI 0.10860.011, Dbh 2 /2 saline
0.09960.01, Dbh 2 /2 DMI 0.10260.006), demonstrating
that the reduction in [ 3 H]nisoxetine binding by chronic
DMI was not a due to a reduction in NET mRNA.
4. Discussion
4.1. Overview
Although the regulation of NET levels by NE and
antidepressants has been examined both in vitro and in
vivo, the mechanisms remain poorly understood. We have
used mice with a genetic NE deficiency to show that in
vivo, NET levels appear to be modulated by endogenous
catecholamines and an antidepressant drug. The absence of
all catecholamines reduces NET levels in terminal regions,
but a loss of only NE has no effect, suggesting that
substrate transport is important for NET regulation. Reduc-
tion of the NET by DMI occurs in both terminal regions
and cell body / dendritic regions and does not require NE,
indicating that a mechanism independent of substrate
transport may be involved.
4.2. Regulation of the NET by catecholamines
Because the NET is a key regulator of synaptic NE
levels, it seems likely that the transporter would be
upregulated in the presence of high NE and down-reg-
ulated in the presence of low NE to maintain neuro-
transmitter homeostasis. Indeed, reserpine, which depletes
NE, decreased [ 3 H]DMI binding sites and treatment with
the monoamine oxidase (MAO) inhibitors increased
[ 3 H]DMI binding sites [19]. However, these experiments
are somewhat difficult to interpret because reserpine
depletes DA and 5-HT in addition to NE, MAO inhibitors
affect the metabolism of all monoamines, and NET levels
were measured with [ 3 H]desipramine (DMI), which has
binding sites in addition to the NET [3,18,19]. We clarified
these results using a specific genetic ablation of NE and
substituting the more NET-selective reuptake inhibitor
[ 3 H]nisoxetine for [ 3 H]DMI. NET levels in adult Dbh
2 /2 mice that lack NE are indistinguishable from Dbh
1 /2 controls, suggesting that NE is not required for the
maintenance of normal NET levels. Compared to controls,
Dbh 2 /2 mice had significantly lower levels of
[ 3 H]nisoxetine binding in the thalamus, at P15, although
no differences were detected in other brain regions.
Because central NE concentrations have not reached adult
levels in rodents at P15 [8,10], NET regulation may be
more sensitive to changes in catecholamine levels in
younger mice. In contrast, the complete lack of catechol-
amines in P15 Th 2 /2 mice reduced NET density
significantly in several brain regions. This suggests that the
DA produced in Dbh 2 /2 mice was sufficient to maintain
normal NET levels and indicate that NET levels are
regulated by substrate transport in general and not by a
mechanism that specifically senses synaptic NE concen-
tration. The reduction in NET levels observed by Lee et al.
[19] probably resulted from the depletion of all NET
substrates by reserpine and not the lack of NE per se. The
regulation of the NET by DA is probably not a unique
aspect of Dbh 2 /2 mice, as the NET has similar affinity
for NE and DA, and substantial amounts of DA are taken
up by the NET in the prefrontal cortex (e.g. Refs.
[7,20,31]). Therefore, extracellular DA levels likely con-
tribute to NET abundance in wild-type animals in regions
of the brain where DA is abundant and under conditions of
diminished NE.
Substrate transport by the serotonin transporter (SERT)
prevents its phosphorylation and internalization by protein
kinase C (PKC) in vitro, and NET internalization is also
regulated by PKC-dependent phosphorylation [2,23].
Given the structural and functional similarities between the
NET and SERT, it is tempting to speculate that the
reduction of [ 3 H]nisoxetine binding in Th 2 /2 mice is
due to increased phosphorylation and subsequent internali-
zation of the NET when catecholamines are absent. One
caveat to this interpretation is that NET levels are not
significantly reduced in the LC of Th 2 /2 mice. This
suggests that something present in the cell bodies or
dendrites, but not terminal fields, of noradrenergic neurons
can support normal NET expression in the absence of
substrate transport. This could involve an unidentified
substrate, regulation by cotransmitters found in the LC
such as galanin and NPY, or differences in PKC-dependent
phosphorylation / dephosphorylation.
Interestingly, dopamine transporter (DAT) levels are
normal in dopamine-deficient mice [16]. Because the DAT
is also capable of transporting NE, albeit at |10-fold lower
affinity [14], examination of DAT levels in Th 2 /2 mice
 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246
will be required to determine whether DAT is also
regulated by catecholamine transport in vivo.
4.3. Regulation of the NET by DMI
Daily i.p. DMI administration (10 mg / kg per day)
reduced NET levels in some regions of the rat brain [4].
Our failure to observe that in mice is probably related to
differences in DMI metabolism and / or excretion between
the two species. We switched to a 21-day minipump
paradigm using a dose that mimics serum antidepressant
concentrations in humans because this regimen resulted in
a more consistent and robust decrease in serotonin trans-
porter levels in rats than what is observed with daily
injections of serotonin reuptake inhibitors [6]. This tech-
nique of chronically administering DMI significantly re-
duced NET levels in all brain regions measured in Dbh
1 /2 mice. Similar reductions were seen in Dbh 2 /2
mice, demonstrating that NE is not required for the NET
down-regulation observed. The failure of the reduction in
NET density to reach significance in some brain regions of
Dbh 2 /2 mice is likely a result of the smaller sample size
for the Dbh 2 /2 DMI group due to surgery-related
fatality, as there were no genotype differences in NET
levels under any treatment condition.
It is not clear whether our [ 3 H]nisoxetine-binding
paradigm measures all NET molecules or only those at the
plasma membrane. If it measures all of the NET, regard-
less of cellular location, then the effect of DMI on
functional NET activity may be even greater than that
reported, because some of the binding sites may be
internalized and unavailable for NE uptake. Reversal of the
DMI-induced down-regulation during the 2-day washout
period may have also resulted in an overestimate of plasma
membrane NET. Because of the required washout period,
we also cannot rule out the possibility that the reduction in
NET density we observe is a relatively acute drug with-
drawal-related effect and does not reflect the state of the
chronically-treated animal.
How does DMI reduce NET levels while catecholamines
maintain them? Catecholamines and DMI bind to different
regions of the NET [14], and catecholamines support
transport while DMI prevents it. Substrate transport by the
SERT limits PKC-mediated phosphorylation and SERT
internalization in vitro, an effect that is abolished by
antidepressant treatment [23]. Because both the SERT and
NET are found in complexes with protein phosphatase 2A
(PP2A), it appears that the plasma membrane localization
of these transporters is regulated by phosphorylation state
[5]. Blockade of substrate transport by reuptake inhibitors
may tip the balance in favor of the phosphorylated state,
which stimulates the removal of functional transporters
from the plasma membrane. However, we observed a
reduction in NET in the LC in DMI-treated animals but not
Th 2 /2 mice that presumably lack substrate transport.
NET down-regulation is also observed after long-term
245
DMI treatment in non-neuronal cells ectopically expressing
the NET [34,35]. These results indicate that DMI binding
to the NET can alter NET abundance independent of
substrate transport.
4.4. NET regulation and depression
Long-term regulation of the NET may be an important
aspect of the pathophysiology of depressive illness and
antidepressant drug efficacy. The NET is a critical reg-
ulator of noradrenergic transmission, and there is a striking
similarity in the time-course of downregulation of NET by
antidepressants and their clinical efficacy. In addition,
reduced levels of the NET have been found in the locus
coeruleus of depressed patients, perhaps as an adaptation
of the depressed brain to low NE levels that mimics the
effect of antidepressant drugs [17]. Understanding how the
NET is regulated may be a key factor in determining the
molecular cognates of depressive illness and the design of
improved antidepressant drugs. Regulation of the NET
may also be important for the effects of drugs of abuse that
bind to the NET such as amphetamine and cocaine. We
have demonstrated that the sustained transport of endogen-
ous catecholamines and transport blockade by DMI are
important aspects of NET regulation in vivo.
Acknowledgements
We thank Sumitomo Pharmaceuticals for the generous
donation of L-3,4 dihydroxyphenylserine (DOPS), R.
Steiner for use of his anesthesia equipment, N. Rust and N.
Miller for maintaining the Dbh mouse colony, M. Hunsley
for help with statistical tests, and D. Kim and M. Szczypka
for critical reading of this manuscript. This work was
supported by the Howard Hughes Medical Institute (D.W.
and R.D.P.), the National Alliance for Research on Schizo-
phrenia and Depression and the Department of Veterans
Affairs (P.S. and S.S.W.).
References
[1] S.G. Amara, M.J. Kuhar, Neurotransmitter transporters: recent
progress, Annu. Rev. Neurosci. 16 (1993) 73–93.
[2] S. Apparsundaram, S. Schroeter, E. Giovanetti, R.D. Blakely, Acute
regulation of norepinephrine transport: II. PKC-modulated surface
expression of human norepinephrine transporter proteins, J. Phar-
macol. Exp. Ther. 287 (1998) 744–751.
[3] I.T. Backstrom, S.B. Ross, J.O. Marcusson, [ 3 H]Desipramine bind-
ing to rat brain tissue: binding to both noradrenaline uptake sites and
sites not related to noradrenaline neurons, J. Neurochem. 52 (1989)
1099–1106.
[4] M.E. Bauer, S.M. Tejani-Butt, Effects of repeated administration of
desipramine or electroconvulsive shock on norepinephrine uptake
sites measured by [ 3 H]nisoxetine autoradiography, Brain Res. 582
(1992) 208–214.
[5] A.L. Bauman, S. Apparsundaram, S. Ramamoorthy, B.E. Wadzinski,
246 D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246
R.A. Vaughan, R.D. Blakely, Cocaine and antidepressant-sensitive
biogenic amine transporters exist in regulated complexes with
protein phosphatase 2A, J. Neurosci. 20 (2000) 7571–7578.
[6] S. Benmansour, M. Cecchi, D.A. Morilak, G.A. Gerhardt, M.A.
Javors, G.G. Gould, A. Frazer, Effects of chronic antidepressant
treatments on serotonin transporter function, density, and mRNA
level, J. Neurosci. 19 (1999) 10494–10501.
[7] E. Carboni, G.L. Tanda, R. Frau, G. Di Chiara, Blockade of the
noradrenaline carrier increases extracellular dopamine concentra-
tions in the prefrontal cortex: evidence that dopamine is taken up in
vivo by noradrenergic terminals, J. Neurochem. 55 (1990) 1067–
1070.
[8] J.C. Chen, G. Turiak, J. Galler, L. Volicer, Postnatal changes of brain
monoamine levels in prenatally malnourished and control rats, Int. J.
Dev. Neurosci. 115 (1997) 257–263.
[9] L. Darracq, G. Blanc, J. Glowinski, J.P. Tassin, Importance of the
noradrenaline-dopamine coupling in the locomotor activating effects
of D-amphetamine, J. Neurosci. 18 (1998) 2729–2739.
[10] M. Elias, T. Deacon, V.S. Caviness Jr., The development of
neocortical noradrenergic innervation in the mouse: a quantitative
radioenzymatic analysis, Brain Res. 255 (1982) 652–656.
[11] R.S. Feldman, J.S. Meyer, L.F. Quenzer (Eds.), Principles of
Neuropsychopharmacology, Sinauer, Sunderland, MA, 1992, pp.
324–344.
[12] K.B.J. Franklin, G. Paxinos, The Mouse Brain Atlas in Stereotaxic
Coordinates, Academic Press, San Diego, CA, 1997.
[13] A. Frazer, Norepinephrine involvement in antidepressant action, J.
Clin. Psychiatry 61 (Suppl. 10) (2000) 25–30.
[14] B. Giros, Y.M. Wang, S. Suter, S.B. McLeskey, C. Pifl, M.G. Caron,
Delineation of discrete domains for substrate, cocaine, and tricyclic
antidepressant interactions using chimeric dopamine-norepinephrine
transporters, J. Biol. Chem. 269 (1994) 15985–15988.
[15] C.M. Kaye, R.E. Haddock, P.F. Langley, G. Mellows, T.C. Tasker,
B.D. Zussman, W.H. Greb, A review of the metabolism and
pharmacokinetics of paroxetine in man, Acta Psychiatr. Scand. 350
(Suppl.) (1989) 60–75.
[16] D.S. Kim, M.S. Szczypka, R.D. Palmiter, Dopamine-deficient mice
are hypersensitive to dopamine receptor agonists, J. Neurosci. 20
(2000) 4405–4413.
[17] V. Klimek, C. Stockmeier, J. Overholser, H.Y. Meltzer, S. Kalka, G.
Dilley, G.A. Ordway, Reduced levels of norepinephrine transporters
in the locus coeruleus in major depression, J. Neurosci. 17 (1997)
8451–8458.
[18] P.M. Laduron, M. Robbyns, A. Schotte, [ 3 H]Desipramine and
[ 3 H]imipramine binding are not associated with noradrenaline and
serotonin uptake in the brain, Eur. J. Pharmacol. 78 (1982) 491–
493.
[19] C.M. Lee, J.A. Javitch, S.H. Snyder, Recognition sites for norepi-
nephrine uptake: regulation by neurotransmitter, Science 220 (1983)
626–629.
[20] J.A. Moron, A. Brockington, R.A. Wise, B.A. Rocha, B.T. Hope,
Dopamine uptake through the norepinephrine transporter in brain
regions with low levels of the dopamine transporter: evidence from
knock-out mouse lines, J. Neurosci. 22 (2002) 389–395.
[21] R.F. Prien, J.H. Kocsis, Psychopharmacology. A Fourth Generation
of Progress, in: F.E. Bloom, D.J. Kupfer (Eds.), Raven, New York,
1995, pp. 1067–1079.
[22] M. Raiteri, R. Del Carmine, A. Bertollini, G. Levi, Effect of
sympathomimetic amines on the synaptosomal transport of norad-
renaline, dopamine and 5-hydroxytryptamine, Eur. J. Pharmacol. 41
(1977) 133–143.
[23] S. Ramamoorthy, R.D. Blakely, Phosphorylation and sequestration
of serotonin transporters differentially modulated by psycho-
stimulants, Science 285 (1999) 763–766.
[24] M.S. Szczypka, M.A. Rainey, D.S. Kim, W.A. Alaynick, B.T.
Marck, A.M. Matsumoto, R.D. Palmiter, Feeding behavior in
dopamine-deficient mice, Proc. Natl. Acad. Sci. USA 96 (1999)
12138–12143.
[25] P. Szot, S.S. White, R.C. Veith, Effect of pentylenetetrazol on the
expression of tyrosine hydroxylase mRNA and norepinephrine and
dopamine transporter mRNA, Brain Res. Mol. Brain Res. 44 (1997)
46–54.
[26] P. Szot, D. Weinshenker, S.S. White, C.A. Robbins, N.C. Rust, P.A.
Schwartzkroin, R.D. Palmiter, Norepinephrine-deficient mice have
increased susceptibility to seizure-inducing stimuli, J. Neurosci. 19
(1999) 10985–10992.
[27] S.A. Thomas, R.D. Palmiter, Impaired maternal behavior in mice
lacking norepinephrine and epinephrine, Cell 91 (1997) 583–592.
[28] S.A. Thomas, R.D. Palmiter, Thermoregulatory and metabolic
phenotypes of mice lacking noradrenaline and adrenaline, Nature
387 (1997) 94–97.
[29] S.A. Thomas, A.M. Matsumoto, R.D. Palmiter, Noradrenaline is
essential for mouse fetal development, Nature 374 (1995) 643–646.
[30] S.A. Thomas, B.T. Marck, R.D. Palmiter, A.M. Matsumoto, Resto-
ration of norepinephrine and reversal of phenotypes in mice lacking
dopamine beta-hydroxylase, J. Neurochem. 70 (1998) 2468–2476.
[31] B.K. Yamamoto, S. Novotney, Regulation of extracellular dopamine
by the norepinephrine transporter, J. Neurochem. 71 (1998) 274–
280.
[32] Q.Y. Zhou, R.D. Palmiter, Dopamine-deficient mice are severely
hypoactive, adipsic, and aphagic, Cell 83 (1995) 1197–1209.
[33] Q.Y. Zhou, C.J. Quaife, R.D. Palmiter, Targeted disruption of the
tyrosine hydroxylase gene reveals that catecholamines are required
for mouse fetal development, Nature 374 (1995) 640–643.
[34] M.Y. Zhu, G.A. Ordway, Down-regulation of norepinephrine trans-
porters on PC12 cells by transporter inhibitors, J. Neurochem. 68
(1997) 134–141.
[35] M.Y. Zhu, R.D. Blakely, S. Apparsundaram, G.A. Ordway, Down-
regulation of the human norepinephrine transporter in intact 293-
hNET cells exposed to desipramine, J. Neurochem. 70 (1998)
1547–1555.