Adaptations in Dopamine Neurochemistry in SERT Knockout Mice by Microdialysis and
Chronoamperometry
X.A. Perez, T.A. Mathews, A.C. Chisnell, A.M. Andrews
Department of Chemistry, Penn State University, University Park, PA
Introduction
 The
presynaptic serotonin transporter (SERT) is the primary means by which
the concentration of 5-HT in the extracellular space is modulated and it is the
molecular target for the largest class of clinically relevant psychiatric drugs
(serotonin reuptake inhibitors; SRIs).
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Saline
 Mice with a targeted disruption of the SERT have been produced to
investigate transporter-mediated control of neurotransmission and behavior.
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SERT KO
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Surgery: Male mice were housed in groups of three to four per cage with food and water ad libitum (12-hr lightdark cycle) and weighed ~35g. The surgical procedure for implantation of a guide cannula for a CMA/7
microdialysis probe was as described previously with the following modifications. Mice were anesthetized with
2.5% Avertin (10g tribromoethylethanol diluted in 10mL t-amyl alcohol) administered in a volume of 16 ml/kg,
ip. A burr hole was drilled and the guide cannula was implanted into the striatum (A+0.6, L-1.8, V-2.5) of the
mouse brain.
Dialysis: The night before an experiment (1700h), mice were lightly anesthetized with 80mg/kg ketamine and
10mg/kg xylazine injected in a volume of 7ml/kg, ip, to remove the dummy probe and insert the dialysis
membrane (2mm length x 240mm diameter cuprophane, 10,000 MW cutoff). Mice were tethered to a dual
channel liquid swivel mounted on a weighted balance arm by a collar but otherwise had free movement in their
home cages containing bedding, food and water. Artificial cerebrospinal fluid (147mM NaCl, 3.5mM KCl, 1.0mM
CaCl2, 1.2 mM MgCl2, 1.0mM NaH2PO4, 2.5mM NaHCO3, pH 7.44) was dialyzed at a rate of 1.1 ml/min and
beginning at 700h, 6 baseline samples were collected at 20 minute intervals and analyzed immediately by online
HPLC-ED for DA at +220mV (ESA Coulochem II, Microdialysis Cell 5014B, 5 nA full scale). Following collection
of baseline samples, three different concentrations of DA ranging from 20-5.0 nM were perfused into the
microdialysis probes. Each different concentration was perfused for 1.66 hours using a programmable gradient
infusion pump. While artificial cerebrospinal fluid containing no DA was flowing through the implanted
microdialysis probe the night before the experiment, programmed concentrations of DA were allowed to bypass
the animal’s head and flow directly into the sample loop. The resulting samples were analyzed and compared
against a standard curve to determine the actual (true) Cin. Dopamine for no net flux infusion was diluted
immediately prior to analysis in artificial cerebrospinal fluid containing 57 mM ascorbate as an antioxidant.
Chronoamperometry: High-speed chronoamperometry was performed in the delayed-pulse mode. Changes in
current were recorded in response to a 1 Hz square wave potential step generated by an IVEC-10/FAST-12
system. A 100ms reductive pulse at 0.0V followed an oxidation potential consisting of a 100ms pulse at +0.55V.
The change in current was integrated over the last 80ms of each oxidative and reductive pulse. The potential was
then maintained at 0.0V for an additional 800ms (delayed-pulse mode) to prevent the fouling of the electrode.
Voltage at the carbon fiber working electrode was applied with respect to a sealed glass Ag/AgCl reference
electrode.
Synaptosome Preparation and Uptake experiments: Brains were extracted from wildtype, heterozygote
and homozygote SERT knockout mice, and bilateral striata and frontal cortex dissected out. Tissues were
prepared as a P2 pellet. The tissues were homogenized in 10 vols. of Tris-Sucrose Buffer (0.5mM Tris-HCl,
0,32mM Sucrose, pH 7.4). Homogenates were centrifuged at 4500 rpm for 10 min. The supernatant was
retained and centrifuged at 13,000 rpm for 10 min. The resulting pellet was resuspended in 40 Vols. of assay
buffer (124mM NaCl, 1.80mM KCl, 1.24mM KH2PO4, 1.40mM MgSO4, 2.50mM CaCl2, 26mM NaHCO3, 10mM
Glucose, saturated with 95% O2 / 5% CO2 gas mixture, pH7.4 with phosphoric acid). 25 L of each homogenized
solution were reserved for Lowry protein analysis and the striatal samples placed in 3ml vials for voltammetric
analysis.
Pre-calibrated microelectrodes and reference electrodes were placed in each synaptosomal solution. A
+0.55 V potential was applied and the current recorded until a stable baseline was obtained. serotonin (1.0M)
was added to each synaptosomal solution, and the change in current with respect to time was recorded and
compared for each of the three genotypes of SERT knockout mice. Some preparations were also challenged with
1.0M dopamine to determine dopamine uptake.
A. Serotonin is synthesized inside serotonin neurons where it is stored in
synaptic vesicles and later released into the synapse.
B. Once serotonin is in the synapse, it can be taken up by the serotonin
transporter back into serotonin neurons. It can also interact with pre and
post-synaptic receptors. The interaction of serotonin with serotonin
receptors on dopamine neurons elicits the release of dopamine.
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C o n c e n t r a t io n ( n M )
Materials and Methods
Figure 1. Schematic of Serotonin-influenced Dopamine
Neurotransmission
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Figure 4. Administration of 5 mg/kg of methamphetamine to
SERT KO mice
METH has a significant effect to increase extracellular DA at all time
points.
Effect of METH was different on each genotype with respect to time
DA release was significantly differ between genotypes at 60, 80 and
100 min.
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(+/-)
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Figure 7. DA Uptake in SERT KO Mice
Uptake of 1M DA into striatal synaptosomes from Wildtype
(+/+), SERT (+/-), and SERT (-/-) mice.
Dopamine clearance was significantly reduced in a gene dosedependent manner (438±46, n=10; 318±32, n=12; and 305±16,
n=11 pmol/mg protein/min; respectively. **p<0.01 vs wildtype
mice).
Preincubation with paroxetine (1.0M, 30 min) significantly
reduced dopamine uptake in wildtype mice, 211±24 (n=7) pmol/mg
protein/min (†p<0.001 vs preincubation of wildtype synaptosomes
in asssay buffer).
Paroxetine had a modest but not significant effect in SERT (+/-)
and SERT (-/-) mice (225±13, n=7; 314±36, n=5 pmol/mg
protein/min, respectively).
S E R T(+ /-)
S E R T(-/-)
Conclusions
Microdialysis in SERT KO versus wildtype mice show no difference in
dopamine or DOPAC extracellular levels in striatum.
S tria tu m
Zero net flux was employed to determine if neuroadaptive changes were
evident in the DA system; however, zero net flux revealed no statistically
significant changes in recovery or “corrected” extracellular DA levels.
Figure 1. Basal Uncorrected DA and DOPAC levels in SERT KO
Basal levels in SERT KO mice across the genotypes is not statistically significant.
DA levels were 3.5±0.5 (n=22), 2.9±0.4 (n=17) and 3.7±0.5 nM (n=19) in
wildtype, SERT(+/-) and SERT (-/-), respectively.
DOPAC levels were not statistically significant across the genotypes. DOPAC is a
metabolite of DA that is metabolized via the monoamine oxidase enzyme
Although, administration of METH releases a substantial amount of
DA in both genotypes this response is blunted in the SERT KO. The
SERT KO mice might have a diminished responses due to
neuroadaptations in the DA system.
Results from our chronoamperometry experiments indicate that DA
uptake rates decrease in a gene dose-dependent manner. ~40% of the
DA uptake observed in striatum of SERT KO mice might be due to
promiscuous SERT activity.
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Future chronoamperometry experiments will further investigate
whether these decreases in dopamine uptake rates are completely due
to decreased SERT expression.
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nM or % R ecovery
 Since our long-term goal is to characterize neuroadaptation resulting from
decreases in SERT expression, in the present study, we investigate possible
compensatory changes in the dopamine system of SERT knockout mice.
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 In addition, our chronoamperometry studies in these mice has shown a 50%
decrease in the uptake rate of 5-HT in synaptosomes from heterozygote
knockout mice compared to wildtype SERT knockout mice in striatum and
frontal cortex. No uptake was observed in synaptosomes from homozygote
SERT knockout mice.
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Time (min)
 Our previous studies using microdialysis and the method of no-net-flux show
a 6-fold and 3-fold increase in the concentration of extracellular 5-HT in
striatum of homozygote and heterozygote SERT knockout mice as compared to
wildtype mice, respectively.
pmol/mg protein/min
Percent of Control
 Chronic decreases in 5-HT uptake are believed to eventually result in
prolonged increases in extracellular 5-HT. In turn, this fundamental adjustment
in serotonergic signaling is thought to lead to adaptive responses, some of
which underlie the efficacy of 5-HT reuptake inhibiting drugs.
Paroxetine
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Figure 3. DA Zero Net Flux
DA zero net flux was employed in SERT mice to determine if there
were neuroadaptive changes in this system unable to be detected
using conventional microdialysis techniques.
DA zero net flux showed no change in “true” extracellular DA levels
(6.61 vs. 6.09 nM, WT and (-/-) respectively).
Future dialysis experiments will further examine DA at lower dose
of METH and the DA-5- HT interaction with amphetamine.
Figure 4. DA Uptake by Chronoamperometry
Representative DA electrochemical signal from SERT knockout mice striatal
synaptosomes after the addition of 1 M DA. Only oxidation signals are
illustrated.
Acknowledgements
X.A. Perez and T.A. Mathews are supported through Penn State Chemistry Travel
Award.
This project was supported by a grant from NIH (RO1 MH64756-01).