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Brain Research 946 (2002) 239–246

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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


would be expected to decrease when synaptic NE content
Norepinephrine (NE) is an abundant neurotransmitter in is low. The work of Lee et al. [19] supports this, as NET
the peripheral and central nervous systems, where it is levels decrease when NE is depleted with reserpine. These
involved in many physiological and behavioral processes, results suggest that NE levels are sensed by the neuron and
including cardiovascular function, metabolism, embryonic NET abundance is then regulated accordingly. However,
development, arousal, seizure susceptibility, maternal be- because reserpine depletes dopamine (DA) and serotonin
havior, and responses to drugs of abuse [9,11,26–28]. The (5-HT) as well as NE, it is unclear whether the depletion
norepinephrine transporter (NET) is a Na 1 / Cl 2 -dependent of other monoamines contributes to the observed down-
transporter expressed by noradrenergic neurons, where it regulation of the NET.
modulates noradrenergic signaling by clearing secreted Among the drugs that inhibit the NET and block NE
norepinephrine via selective uptake (e.g. Ref. [1]). reuptake are antidepressants such as DMI and reboxetine
[13]. Blockade of NE reuptake by antidepressant drugs
*Corresponding author. Tel.: 11-206-543-6090; fax: 11-206-543- happens on the order of minutes to hours, while alleviation
0858. of depressive symptoms requires weeks of chronic drug
E-mail address: dzw@genetics.washington.edu (D. Weinshenker). 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 Dbh 2 /2 genotype (data not shown). A subset of geno-
responsible for the efficacy of antidepressant drugs. One types was confirmed by PCR. Th 2 /2 mice were geno-
molecular change that follows the time course of clinical typed by PCR. Dbh 1 /2 and Th 1 /2 mice have normal
efficacy is down-regulation of the NET. For example, catecholamine levels and are indistinguishable from wild-
chronic, but not acute antidepressant treatment decreases type littermates for all previously tested phenotypes (e.g.
monoamine transporter density in rats [4,6]. However, it is Refs. [24,26,29,30]). Therefore, heterozygous (1 /2 ) litter-
not known whether the antidepressant-induced increase in mates were used as controls for all experiments in this
extracellular monoamines is required for this effect. study.
To determine if NE, the primary substrate for the NET, Experimental protocols were approved by the animal
is required for NET regulation under basal conditions or by care committee at the University of Washington and meet
chronic exposure to antidepressants in vivo, we measured the guidelines of the American Association for Accredita-
NET levels before and after chronic DMI administration in tion of Laboratory Animal Care.
dopamine b-hydroxylase (DBH) knockout (Dbh 2 /2 )
mice. These mice lack NE because DBH is essential for 2.2. Chronic DMI administration
the conversion of DA to NE. Because the NET is capable
of transporting DA [14,22] and Dbh 2 /2 mice have In the first experiment, DMI (10 mg / kg, 2.5 mg / ml)
elevated DA levels [30], NET levels were also determined was dissolved in 0.9% NaCl and administered i.p. once per
in tyrosine hydroxylase knockout (Th 2 /2 ) mice that lack day for 28 days. Mice were sacrificed on day 30 and brains
both NE and DA. 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
2. Material and methods dissolved in an aqueous solution containing 50% ethanol
and 0.9% NaCl, then adjusted to neutral pH (6.5–7) with 1
2.1. Animals M NaOH. The DMI solution was then loaded into osmotic
minipumps (28 days, 6.24 ml / day; Alza, Palo Alto, CA).
Dbh knockout (Dbh 2 /2 ) and Th knockout (Th 2 /2 ) Minipumps containing 50% ethanol, 50% water (pH 6.5–
mice, maintained on a 129 / SvEv and C57BL / 6J hybrid 7) were used as vehicle controls, and all pumps were
background, were developed and generated as described placed in a sterile 37 8C saline bath for 2 days before
[30,33] with the following modifications. Th 1 /2 males implantation. Mice were anesthetized with isoflurane and
were bred to Th 1 /2 females. To rescue the embryonic minipumps were implanted in the intraperitoneal cavity.
lethality caused by the lack of catecholamines in Th 2 /2 Surgery-associated fatality occurred in one Dbh 1 /2
mice, pregnant females were given L-DOPA (50 mg / kg, vehicle mouse, two Dbh 1 /2 DMI mice, and four Dbh
1.5 mg / ml) and vitamin C (2.5 mg / ml) both by daily 2 /2 DMI mice. Minipumps were removed 21 days later,
intraperitoneal (i.p.) injection and in the drinking water and orbital bleeds were carried out on a subset of mice for
until the birth of the litter, then discontinued. Therefore, DMI measurements. Blood was collected in heparinized
catecholamines were present in Th 2 /2 mice during but tubes, centrifuged, and plasma was collected and stored at
not after embryogenesis. Th 2 /2 animals die of aphagia 280 8C. Mice were sacrificed after a 2-day ‘washout’
and adipsia around the time of weaning because of a lack period. Trunk blood was collected from a subset of mice
of DA [32,33]. About a week before weaning, Th 2 /2 for DMI measurements and brains were frozen on dry ice
mice are distinguishable from their littermates because of and stored at 280 8C. To control for residual DMI in the
their reduced size, but otherwise appear healthy. Therefore, brains that was undetectable in the serum and could
postnatal day 15 (P15) Th 2 /2 mice and littermate interfere with [ 3 H]nisoxetine binding, mice with ‘vehicle’
controls were used for the [ 3 H]nisoxetine binding studies. minipumps received an i.p. injection of 30 mg / kg DMI (3
Because adrenoreceptor agonists and L-3,4-dihydrox- mg / ml) on day 21. Mice with DMI minipumps received an
yphenylserine (DOPS) were administered to pregnant Dbh i.p. injection of saline.
1 /2 mothers to rescue the embryonic lethality associated
with the lack of NE in knockout pups [29,30], catechol- 2.3. Measurement of DMI
amines were present in both Dbh 2 /2 and Th 2 /2
animals before but not after birth. Dbh 2 /2 mice were The measurement of DMI was carried out according to
reared in a specific pathogen-free facility and transferred to Benmansour et al. [6]) with minor modifications. Briefly,
a conventional facility for experiments, while Th 2 /2 25 ml of plasma for samples, calibrators, and controls were
mice were reared in a conventional facility. Both facilities aliquoted in polypropylene tubes along with 20 ml of a 10
had a 12-h light / dark cycle at the University of Washing- mg / ml solution of doxepin (internal standard). Next, 200
ton, and food and water were available ad libitum. ml of 5 N NaOH were added to each sample. After
Dbh 2 /2 mice were identified by the delayed growth vortexing, 3 ml of 5% isopropanol in hexane mixture were
and ptosis phenotype, which is 100% correlated with the added to the samples followed by gentle shaking for 10
D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246 241

min. Samples were then centrifuged at 12003g for 10 min of labeling in specific brain regions. Separate density
at 18 8C. Next, samples were placed in a methanol–dry ice measurements from three anatomically-matched sections
bath to freeze the aqueous layer. Then, the upper organic were made across all treatment groups for each brain
layers were decanted and mixed with 150 ml of 12 mM region that gave a consistent signal above background.
monobasic potassium phosphate, pH 2.5, for the back Across three atlas-matched sections from each animal,
extraction of the drugs. The samples were shaken, cen- three readings were taken from the dorsal raphe (DR; plate
trifuged, placed in the methanol–dry ice bath, then the 67 of Ref. [12]), and three readings were taken from the
upper organic layer was poured off and discarded. The left and right sides of the locus coeruleus (LC; plate 75),
aqueous fraction was placed in a fume hood for 45 min to dorsal and ventral hippocampus (HP; dorsal plate 44,
allow any residual isopropanol / hexane to evaporate, then ventral plate 55), the lateral habenular nucleus of the
100 ml of sample was injected into the HPLC system. The thalamus (plate 46), and the bed nucleus of stria terminalis
HPLC system included a Beckman model 110B pump, a (BNST; plate 29). Density was similar between the dorsal
Waters model 717 sample injector, a Waters 2487 UV and ventral hippocampus, so dorsal hippocampal measure-
detector for HPLC, and a Spherisorb S5 CN column (5 ments were used in the analysis. Therefore, for each
mm). animal, the mean6S.E.M. for the DR were an average of
Samples were analyzed by UV detection at a fixed three density readings, and an average of six density
wavelength of 214 nm. The mobile phase contained 70% readings for the LC, HP, THAL and BNST. For each brain
acetonitrile, 13% methanol, and 17% of a solution of 10 region, the size and shape of the area where density was
mM KH 2 PO 4 (pH 6.7). The flow rate was 2.0 ml / min. measured was identical for all sections. In cases where a
DMI concentrations were quantified by comparing samples brain region was damaged or missing because of tissue
of unknown concentration against the linear regression of a loss during slide mounting, the animal was excluded from
calibrator concentration curve. The detection limit for DMI the analysis for that brain region. Non-specific binding was
was 5 ng / ml at a signal-to-noise ratio of five. Recovery determined in sections incubated with 1 mM mazindol and
was 85% and the coefficient of variation for within-day subtracted from the value of density reading.
variability among identical samples was less than 6%. [ 3 H]Nisoxetine binding was quantified by an independent
observer blind to genotype and drug treatment.
2.4. Measurement of NET mRNA by in situ
hybridization 2.6. Statistics

A cDNA NET clone containing the entire nucleotide Data were analyzed by Student’s t-test or Wilcoxon–
sequence for the human NET was kindly provided by Dr S. Mann–Whitney U-test when comparing two groups, and
Amara (Vollum Institute, Oregon Health Sciences Uni- ANOVA when comparing more than two groups.
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 3. Results
labeling of the riboprobe was performed as described [25].
The probe was applied to tissue at a concentration of 3.1. Dbh 2 /2 mice have normal NET levels
0.7310 6 cpm / 50 ml. Hyperfilm (Amersham, Arlington
Heights, IL) was exposed to slides containing tissue NET levels were measured in adult Dbh 1 /2 and Dbh
hybridized with [ 35 S]NET riboprobe for 7 days and 2 /2 mice by [ 3 H]nisoxetine binding. No differences
quantified as previously described by an independent between genotypes were detected in any of the five brain
observer blind to genotype and drug treatment. regions measured (Fig. 1A), demonstrating that NET levels
do not change in response to an absence of NE in mice.
2.5. Measurement of NET binding sites
3.2. Th 2 /2 mice have reduced NET levels
NET binding using [ 3 H]nisoxetine was performed on
18-mm mouse brain sections as described [4]. Incubation Because DBH converts DA to NE, Dbh 2 /2 mice
buffer (400 ml / slide) contained [ 3 H]nisoxetine at |3 nM. produce DA from ‘noradrenergic’ terminals by default.
Non-specific binding was defined in the presence of 1 mM Central DA tissue levels are |10% higher in Dbh 2 /2
mazindol. Slides were apposed to LKB 3 H-Ultrafilm (LKB mice, although DA release has not been measured [30]. In
Instruments, Gaithersburg, MD) for 4 weeks. Each sheet of addition, the affinity of the NET for DA is at least as high
film contained brain sections from each genotype and as that for NE [14,22]. To determine whether NET levels
treatment group. Autoradiograms were analyzed using in Dbh 2 /2 mice are normal due to uptake of ectopic DA,
MicroComputer Imaging Device Systems (MCID; Imaging we examined NET levels in tyrosine hydroxylase knockout
Research Inc, Ontario, Canada). Either relative optical (Th 2 /2 ) mice that lack both NE and DA [33]. Because
density or 3 H-standards were used to quantitate the amount 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 levels in most terminal fields, but not in the region
experiment was done on P15 animals, a time when Th containing cell bodies and dendrites of noradrenergic
2 /2 mice show no obvious signs of malaise [32,33]. We neurons. To control for the effects of age on NET levels,
found that Th 2 /2 mice had less [ 3 H]nisoxetine binding we measured [ 3 H]nisoxetine binding in P15 Dbh 1 /2 and
across all brain regions than Th 1 /2 controls F(4, 44)5 Dbh 2 /2 mice and found no difference across all brain
2.97, P50.0296. This difference was significant in the regions F(4, 68)50.5648, P50.689. Young Dbh 2 /2
dorsal raphe nucleus (DR), the bed nucleus of the stria mice appeared to have slightly lower NET levels in some
terminalis (BNST) and the thalamus (THAL) but not in the brains regions compared to littermate controls, and when
locus coeruleus (LC) or hippocampus (HP) (Fig. 1B–D). individual brain regions were compared between genotypes
This result suggests that the ectopic DA produced by the a significant difference was found only in the THAL (Fig.
Dbh 2 /2 mice is responsible for maintaining normal NET 1E).
D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246 243

3.3. NE is not required for the reduction of NET levels Benmansour et al. [6]. We administered DMI at 20, 30, or
by chronic DMI 40 mg / kg per day for 21 days via an osmotic mini-pump
and measured serum DMI concentrations after a 2-day
To determine whether NE is required for the down- washout period. We chose 30 mg / kg per day for our
regulation of NET by antidepressant drugs, we measured subsequent experiments because it produced serum DMI
NET levels in Dbh 1 /2 and Dbh 2 /2 mice treated concentrations (61–488 ng / ml, n56) on day 21 similar to
chronically with DMI using a daily injection paradigm (10 that seen clinically (125–600 ng / ml; [15]). Following the
mg / kg / d, i.p.) that has been shown to reduce 2-day washout period, NET levels were measured by
[ 3 H]nisoxetine binding to the NET in rats [4]. Because [ 3 H]nisoxetine binding to brain sections. This paradigm
DMI failed to reduce NET levels in mice of either resulted in a significant reduction (17–42%, depending on
genotype (data not shown), we considered the possibility brain region) in NET levels in Dbh 1 /2 mice compared
that this regimen did not maintain sufficient drug con- to control animals implanted with vehicle-containing
centrations in the brain and switched to a modified version minipumps across all brain regions measured, F(4, 44)5
of chronic antidepressant drug administration described by 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. depletes DA and 5-HT in addition to NE, MAO inhibitors
A similar effect of DMI (13–48% reduction) was observed affect the metabolism of all monoamines, and NET levels
in Dbh 2 /2 mice with this paradigm, F(4, 36)54.36, were measured with [ 3 H]desipramine (DMI), which has
P50.006 (Fig. 2C–E). NET levels in vehicle-treated, binding sites in addition to the NET [3,18,19]. We clarified
F(4,48)50.177, P50.95, and DMI-treated, F(4, 32)5 these results using a specific genetic ablation of NE and
1.728, P50.168, Dbh 1 /2 and Dbh 2 /2 mice were substituting the more NET-selective reuptake inhibitor
comparable (Fig. 2E). We controlled for the possibility that [ 3 H]nisoxetine for [ 3 H]DMI. NET levels in adult Dbh
occupancy of the antidepressant binding site of the NET by 2 /2 mice that lack NE are indistinguishable from Dbh
residual DMI after the 2-day washout period was respon- 1 /2 controls, suggesting that NE is not required for the
sible for the observed reduction in [ 3 H]nisoxetine binding maintenance of normal NET levels. Compared to controls,
in two ways. First, DMI was undetectable in the serum Dbh 2 /2 mice had significantly lower levels of
following the 2-day washout period (data not shown). [ 3 H]nisoxetine binding in the thalamus, at P15, although
Second, the vehicle-treated animals in Fig. 2E were given no differences were detected in other brain regions.
a single bolus injection of 30 mg / kg DMI on day 21 when Because central NE concentrations have not reached adult
the mini-pumps were removed to mimic the amount of levels in rodents at P15 [8,10], NET regulation may be
DMI that the drug-treated animals would have to clear more sensitive to changes in catecholamine levels in
during the washout period. We conclude that the reduction younger mice. In contrast, the complete lack of catechol-
of [ 3 H]nisoxetine binding observed in DMI-treated animals amines in P15 Th 2 /2 mice reduced NET density
was not due to residual DMI. significantly in several brain regions. This suggests that the
In situ hybridization revealed similar NET mRNA levels DA produced in Dbh 2 /2 mice was sufficient to maintain
in the LC in all groups (Dbh 1 /2 saline 0.11460.008 normal NET levels and indicate that NET levels are
mCi / g, Dbh 1 /2 DMI 0.10860.011, Dbh 2 /2 saline regulated by substrate transport in general and not by a
0.09960.01, Dbh 2 /2 DMI 0.10260.006), demonstrating mechanism that specifically senses synaptic NE concen-
that the reduction in [ 3 H]nisoxetine binding by chronic tration. The reduction in NET levels observed by Lee et al.
DMI was not a due to a reduction in NET mRNA. [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
4. Discussion aspect of Dbh 2 /2 mice, as the NET has similar affinity
for NE and DA, and substantial amounts of DA are taken
4.1. Overview up by the NET in the prefrontal cortex (e.g. Refs.
[7,20,31]). Therefore, extracellular DA levels likely con-
Although the regulation of NET levels by NE and tribute to NET abundance in wild-type animals in regions
antidepressants has been examined both in vitro and in of the brain where DA is abundant and under conditions of
vivo, the mechanisms remain poorly understood. We have diminished NE.
used mice with a genetic NE deficiency to show that in Substrate transport by the serotonin transporter (SERT)
vivo, NET levels appear to be modulated by endogenous prevents its phosphorylation and internalization by protein
catecholamines and an antidepressant drug. The absence of kinase C (PKC) in vitro, and NET internalization is also
all catecholamines reduces NET levels in terminal regions, regulated by PKC-dependent phosphorylation [2,23].
but a loss of only NE has no effect, suggesting that Given the structural and functional similarities between the
substrate transport is important for NET regulation. Reduc- NET and SERT, it is tempting to speculate that the
tion of the NET by DMI occurs in both terminal regions reduction of [ 3 H]nisoxetine binding in Th 2 /2 mice is
and cell body / dendritic regions and does not require NE, due to increased phosphorylation and subsequent internali-
indicating that a mechanism independent of substrate zation of the NET when catecholamines are absent. One
transport may be involved. caveat to this interpretation is that NET levels are not
significantly reduced in the LC of Th 2 /2 mice. This
4.2. Regulation of the NET by catecholamines suggests that something present in the cell bodies or
dendrites, but not terminal fields, of noradrenergic neurons
Because the NET is a key regulator of synaptic NE can support normal NET expression in the absence of
levels, it seems likely that the transporter would be substrate transport. This could involve an unidentified
upregulated in the presence of high NE and down-reg- substrate, regulation by cotransmitters found in the LC
ulated in the presence of low NE to maintain neuro- such as galanin and NPY, or differences in PKC-dependent
transmitter homeostasis. Indeed, reserpine, which depletes phosphorylation / dephosphorylation.
NE, decreased [ 3 H]DMI binding sites and treatment with Interestingly, dopamine transporter (DAT) levels are
the monoamine oxidase (MAO) inhibitors increased normal in dopamine-deficient mice [16]. Because the DAT
[ 3 H]DMI binding sites [19]. However, these experiments is also capable of transporting NE, albeit at |10-fold lower
are somewhat difficult to interpret because reserpine affinity [14], examination of DAT levels in Th 2 /2 mice
D. Weinshenker et al. / Brain Research 946 (2002) 239 – 246 245

will be required to determine whether DAT is also DMI treatment in non-neuronal cells ectopically expressing
regulated by catecholamine transport in vivo. the NET [34,35]. These results indicate that DMI binding
to the NET can alter NET abundance independent of
4.3. Regulation of the NET by DMI substrate transport.

Daily i.p. DMI administration (10 mg / kg per day) 4.4. NET regulation and depression
reduced NET levels in some regions of the rat brain [4].
Our failure to observe that in mice is probably related to Long-term regulation of the NET may be an important
differences in DMI metabolism and / or excretion between aspect of the pathophysiology of depressive illness and
the two species. We switched to a 21-day minipump antidepressant drug efficacy. The NET is a critical reg-
paradigm using a dose that mimics serum antidepressant ulator of noradrenergic transmission, and there is a striking
concentrations in humans because this regimen resulted in similarity in the time-course of downregulation of NET by
a more consistent and robust decrease in serotonin trans- antidepressants and their clinical efficacy. In addition,
porter levels in rats than what is observed with daily reduced levels of the NET have been found in the locus
injections of serotonin reuptake inhibitors [6]. This tech- coeruleus of depressed patients, perhaps as an adaptation
nique of chronically administering DMI significantly re- of the depressed brain to low NE levels that mimics the
duced NET levels in all brain regions measured in Dbh effect of antidepressant drugs [17]. Understanding how the
1 /2 mice. Similar reductions were seen in Dbh 2 /2 NET is regulated may be a key factor in determining the
mice, demonstrating that NE is not required for the NET molecular cognates of depressive illness and the design of
down-regulation observed. The failure of the reduction in improved antidepressant drugs. Regulation of the NET
NET density to reach significance in some brain regions of may also be important for the effects of drugs of abuse that
Dbh 2 /2 mice is likely a result of the smaller sample size bind to the NET such as amphetamine and cocaine. We
for the Dbh 2 /2 DMI group due to surgery-related have demonstrated that the sustained transport of endogen-
fatality, as there were no genotype differences in NET ous catecholamines and transport blockade by DMI are
levels under any treatment condition. important aspects of NET regulation in vivo.
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- Acknowledgements
less of cellular location, then the effect of DMI on
functional NET activity may be even greater than that We thank Sumitomo Pharmaceuticals for the generous
reported, because some of the binding sites may be donation of L-3,4 dihydroxyphenylserine (DOPS), R.
internalized and unavailable for NE uptake. Reversal of the Steiner for use of his anesthesia equipment, N. Rust and N.
DMI-induced down-regulation during the 2-day washout Miller for maintaining the Dbh mouse colony, M. Hunsley
period may have also resulted in an overestimate of plasma for help with statistical tests, and D. Kim and M. Szczypka
membrane NET. Because of the required washout period, for critical reading of this manuscript. This work was
we also cannot rule out the possibility that the reduction in supported by the Howard Hughes Medical Institute (D.W.
NET density we observe is a relatively acute drug with- and R.D.P.), the National Alliance for Research on Schizo-
drawal-related effect and does not reflect the state of the phrenia and Depression and the Department of Veterans
chronically-treated animal. Affairs (P.S. and S.S.W.).
How does DMI reduce NET levels while catecholamines
maintain them? Catecholamines and DMI bind to different
regions of the NET [14], and catecholamines support References
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may tip the balance in favor of the phosphorylated state, sites not related to noradrenaline neurons, J. Neurochem. 52 (1989)
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