In the majority of experiments male C57BL/6J mice were used and purchased from Jackson Lab (Bar Harbor, Maine, USA). In a subset of experiments, male SERT knockout (KO) mice (backcrossed to C57BL/6J for >10 generations) were used. We maintain a breeding colony of SERT wild-type (+/+), heterozygote (+/−) and homozygote KO (−/−) (originally provided by Dr. Dennis Murphy (NIMH)). SERT KO mice were generated and maintained as described previously (Bengel et al., 1998
). Male mice weighing 25 to 30 g, were used for all experiments. All animal procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All measures were taken to limit the number of animals used and to minimize animal discomfort.
Mice were housed on a standard light cycle with lights on at 7:00 a.m. and off at 7:00 p.m. In mice, circulating corticosterone levels typically peak around the onset of the active period (Moore and Eichler, 1972
; Buijs et al., 1993
; La Fleur et al., 1999
; Li et al., 2006
), which would equate to ~6:00 – 7:00 p.m. in our studies. All experimental manipulations and data acquisition took place prior to this peak. Mice were swum between 9:00 a.m. and 10:30 a.m. Tail suspension tests (TST) were carried out between 9:00 a.m. and 12:00 noon, chronoamperometry recordings were made over the period from 10:00 a.m. and 6:00 p.m. and trunk blood was collected at approximately 6:00 p.m.
We selected repeated daily exposure to 10 min of swim as it is a known activator of the HPA axis and produces transient increases in extracellular 5-HT. For example, Boyce-Rustay and co-workers (2007)
showed that plasma corticosterone levels measured 30 min after either one 10 min swim or 14 days of 10 min/day swim were significantly elevated compared to non-swum control mice. Microdialysis studies revealed that exposure to one or two consecutive swims (2 hours apart) causes a rapid increase in ECF 5-HT in mouse hippocampus, peaking within a few min following swim and declining to baseline levels approximately 40 min later (Yoshitake et al., 2004
Animals were housed in groups of two or four. Half of the mice in each group received a 10-min swim, for 14 days, while the other half remained in the home cage, as previously described (Boyce-Rustay et al., 2007
). The swim tank consisted of a transparent Plexiglas cylinder (20 cm diameter) filled half way with water (24 ± 1 °C). Immediately following the swim, mice were towel dried before being returned to their home cage. In most instances separate cohorts of mice were used for the in vivo
electrochemistry, neurochemistry and behavioral studies. Quantitative autoradioradiography was carried out using brains from mice that were harvested immediately following chronoamperometric recordings. All experiments commenced 24 hr after the final swim.
In vivo electrochemistry
chronoamperometry was carried out according to the methods described in Daws and Toney (2007)
. We construct our own carbon fiber electrodes and a detailed description can be found in Williams et al., (2007)
. Recording electrode/micropipette assemblies were constructed using a single carbon-fiber (30 µm diameter; Specialty Materials Inc., Lowell, Massachusetts, USA), which was sealed inside fused silica tubing (Schott North America, Inc., Elmsford, New York, USA). We based our procedure for electrode construction on modifications of published methods (Gerhardt, 1995
; Perez et al., 2006
). Carbon fiber electrodes (30 µm tip diameter) were coated with Nafion® (5% solution, Aldrich Chemical Co, Milwaukee, WI), to prevent interference from anionic substances in extracellular fluid as previously described (Daws and Toney, 2007
). Electrodes were tested for sensitivity to the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA, 250 µM) and calibrated with accumulating concentrations of 5-HT (0–4 µM). Only electrodes displaying a selectivity ratio for 5-HT over 5-HIAA greater than 500:1 and a linear response (r2
≥ 0.9) to 5-HT were used.
The electrochemical recording assembly consisted of a Nafion-coated, single carbon fiber electrode attached to a 4-barreled micropipette such that their tips were separated by ~200 µm. Depending on the experimental design (see details below), barrels were filled with combinations of either 5-HT (200 µM), histamine (200 µM), fluvoxamine (400 or 800 µM), corticosterone (2 mM) or vehicle phosphate buffered saline (PBS) +/− ethanol (EtOH) (6 mM). Serotonin and histamine were prepared in 0.1M PBS with 100 µM ascorbic acid added as an antioxidant and the pH adjusted to 7.4. The electrode-micropipette recording assembly was lowered into the CA3 region of the dorsal hippocampus (AP, −1.94 from bregma; ML, +2.0 from midline; DV, −2.0 from dura) (Franklin and Paxinos, 1997
) of anesthetized mice. We chose to study the CA3 region because this is a neural target for both stress (Watanabe et al., 1992
; Holmes & Wellman 2009
) and antidepressant drugs (de Montigny et al., 1990
), and because we have previously shown that 5-HT clearance in this region is mediated primarily by SERT (and not norepinephrine, dopamine or organic cation transporters) when exogenously applied 5-HT concentrations are 1 micromolar or less (Daws et al., 2005
; Daws and Toney, 2007
; Baganz et al., 2008
; Daws, 2009
For all experiments, mice were anesthetized by intraperitoneal (i.p.) injection (2 ml/kg body weight) of a mixture of chloralose (35 mg/kg) and urethane (350 mg/kg). A tube was inserted into the trachea to facilitate breathing and mice were then placed into a stereotaxic frame. Body temperature was maintained at 36–37 °C by a water circulated heating pad.
High-speed chronoamperometric recordings were made using the FAST-12 and FAST-16 systems (Quanteon, Nicholasville, Kentucky, USA) (Montañez et al., 2003
). Oxidation potentials consisted of 100 msec pulses of +0.55 volts. Each pulse was separated by a 900 msec interval during which the resting potential was maintained at 0.0 V. Voltage at the active electrode was applied with respect to a Ag/AgCl reference electrode positioned in the extracellular fluid of the ipsilateral superficial cortex. The oxidation and reduction currents were digitally integrated during the last 80 msec of each 100 msec voltage pulse.
At the conclusion of the experiment, an electrolytic lesion was made to mark the placement of the electrode tip. The brain was removed, rapidly frozen on dry ice and stored at −80°C until use. At this time brains were thawed to −15°C and sliced into 20 µm thick sections for histological verification of electrode localization. Only data from mice in which the electrode was confirmed to be in the CA3 region of the hippocampus were included in data analyses. An insufficient number of placements falling outside this region prevented assessment of drug effects on 5-HT clearance in other hippocampal regions.
The electrodes were not post-calibrated in these studies. We have carried out post-calibrations in the past and do find that electrodes decline in sensitivity to 5-HT over time; depending on the duration of the experiment this decline can range from 5 to 50%. Importantly, however, this decline occurs at a very similar rate, therefore the inter-electrode variability between experiments is very constant. Because of this we ensure that the timing of protocols and drug challenges is kept as similar as possible between experiments (but ensuring a randomized design to avoid order effects of drug administration). That is, when comparing 5-HT clearance rates between control and swum mice, in wild-type or SERT KO mice, this is done in a time matched manner. In addition, after establishing a stable baseline, (i.e. 3 consecutive 5-HT signals of the same amplitude, time course and clearance rate), we routinely switch out electrodes during the experiment if we find that signal amplitude (the best indicator of lost sensitivity) for a given amount of 5-HT declines by more than 20%. We find this occurs in <5% of experiments and with equivalent frequency across treatment groups. Combined, these strategies minimize potential confounds that may be introduced by loss of electrode sensitivity.
Drug Effects on Electrode Sensitivity for Serotonin
It is known that some compounds can interfere with the sensitivity of carbon fiber electrodes to detect neurotransmitter (Davidson et al., 2000
). Because of this we tested the effect of fluvoxamine, corticosterone and EtOH (vehicle for corticosterone) on electrode sensitivity for 5-HT. To do this, Nafion coated carbon fiber electrodes were calibrated to 5-HT (0.2 to 1.0 µM in 0.2 µM increments) in a beaker containing PBS according to standard protocol (Daws and Toney, 2007
). Each electrode was calibrated twice, the first time in the absence of drug or vehicle and the second time after the addition of drug or vehicle. Drug concentrations were based on those estimated to reach the recording electrode following pressure-ejection into brain. Based on our own estimates and that of others (Gerhardt and Palmer, 1987
; Daws et al., 2006
) the concentration of drug pressure-ejected a distance of ~200 µm from the recording electrode is diluted in the order of 10- to 200-fold by the time it reaches the recording site. Thus, in these experiments we tested drugs at both the low and high end of this range. The concentrations tested were 2 µM and 80 µM for fluvoxamine, 10 µM and 200 µM for corticosterone and 30 µM and 600 µM for EtOH. An equivalent volume of PBS was added as the control.
Sensitivity was determined as the slope of the calibration curve. As expected under control conditions, the electrode’s sensitivity for 5-HT declined after the first calibration by ~30% (69 ± 15%, n=3, slope of second calibration as a percent of first). Neither corticosterone nor EtOH, at either concentration, changed the sensitivity of the Nafion-coated carbon fiber electrode for 5-HT compared to the PBS control. Expressed as a percent of the control condition the sensitivity of the electrode following corticosterone was 100 ± 26% (n=5, 10 µM and 200 µM pooled) and following EtOH, 115 ± 14% (n=6, 30 µM and 600 µM pooled). There was a non-significant trend for fluvoxamine (2.0 µM) to reduce the sensitivity of the electrode for 5-HT (PBS 100 ± 23% n=3, vs fluvoxamine 68 ± 15%, n=6). The higher fluvoxamine concentration (80 µM) reduced the sensitivity of the electrode for 5-HT (15 ± 3%, n=3). It is important to note however, that we do not find any evidence for fluvoxamine interfering with electrode sensitivity for 5-HT under our in vivo
recording scenario. For example, 5-HT signals remain reproducible following intrahippocampal application of fluvoxamine (200 µM barrel concentration) to SERT knockout out mice (Montañez et al., 2003
). Together, these results support the view that drugs are diluted much in excess of 10-fold, and perhaps even more than 200-fold, by the time they diffuse the 200 µm from the micropipette to the recording electrode. None of the conditions significantly changed the selectivity of the electrodes for 5-HT over 5-HIAA. The implication is that data presented in the current study are not confounded by drugs interfering with the sensitivity or selectivity of the carbon fiber electrode for 5-HT.
We first assessed basal 5-HT clearance in the CA3 region of the hippocampus of mice exposed to repeated swim (10 min/day for 14 days) or not exposed to swim. Serotonin (2.5, 5.0, 10.0, or 20.0 pmol) was locally pressure ejected into the hippocampus to obtain 5-HT signals with increasing amplitude (µM). Current induced by oxidation of 5-HT at the carbon fiber recording electrode was measured as a function of time and converted to micromolar units based on a calibration factor determined prior to the experiment in vivo
(for details see Daws and Toney, 2007
Effect of Local Fluvoxamine on 5-HT Clearance in Hippocampus
Exogenous 5-HT was applied into the CA3 region of the hippocampus by pressure-ejection (5–25 psi for 0.25–3.0 sec). Advantages of this approach are that clearance can be measured without an associated ‘release’ component and that measurements can be made in vivo with excellent temporal (msec) resolution. The amount of 5-HT pressure ejected was adjusted so that baseline peak signal amplitudes did not exceed 1.0 µM. By keeping signal amplitudes in this range we can maintain the sensitivity of the electrode for 5-HT for several hours, and importantly, provide conditions that favor SERT mediated 5-HT uptake.
Once reproducible 5-HT electrochemical signals were obtained, fluvoxamine or PBS vehicle was applied locally into the CA3 region of hippocampus 2 min before the next application of 5-HT. Different pmol amounts of fluvoxamine were delivered by varying the barrel concentration of drug (400 or 800 µM) as well as the volume ejected. This drug application protocol was chosen to cause minimal disturbance to the baseline electrochemical signal and to allow sufficient time for drugs to diffuse to the recording site. Three signal parameters were analyzed; the peak signal amplitude, the T80 time course parameter, defined as the time for the signal to decline by 80% of the peak signal amplitude and the Tc, a measure of clearance rate, defined as the slope of the most linear portion of the descending limb of the signal, between T20 and T60 (the time for the signal to decline by 20% to 60% of the peak signal amplitude).
It is important to note that the concentration of drug reaching the recording electrode is approximately 200 fold less than the concentration of drug within the barrel (Daws et al., 2006
). Thus, barrel concentrations were determined to achieve concentrations of drug reaching the recording site in a range that would yield pharmacologically and physiologically relevant concentrations.
Effect of Local Corticosterone on 5-HT Clearance in Hippocampus
These experiments were carried out as described for fluvoxamine, with two notable exceptions. To favor detection of OCT3 mediated 5-HT clearance, 5-HT signals in a range of 2–4 µM were obtained. Second, the barrel concentration of corticosterone was 2 mM. We have previously shown that a corticosterone barrel concentration of 100 µM does not affect 5-HT clearance in C57BL/6J mice, but does impede 5-HT clearance in SERT deficient mice where OCT3 expression is elevated (Baganz et al., 2008
). Therefore, we used this relatively high concentration of corticosterone to increase the likelihood of detecting an ability of corticosterone (an OCT3 blocker) to inhibit 5-HT clearance in C57BL/6J mice exposed to repeated swim, compared to control mice. The vehicle for corticosterone was PBS containing 6 mM EtOH. This would yield a concentration of approximately 0.03 mM EtOH at the recording site, a concentration that does not influence 5-HT clearance when signal amplitudes are kept below 1 µM (Daws et al., 2006
To directly assess the function of OCT3, we measured clearance of the high-affinity substrate, histamine (200 µM barrel concentration and delivering 140 pmol), in the CA3 region of the hippocampus of mice exposed to 0 or 14 days of swim using chronoamperometry. These experiments were carried out in the same way as described above for 5-HT clearance with the following exceptions. The sensitivity for carbon fiber electrodes is much lower for histamine than 5-HT. Therefore, to increase the surface area of the electrode we created micro-fractures in the carbon fiber by applying a potential of +1.50 V for 100 msec and stepping back to a resting potential of −1.50 V for 100 msec for a period of 10 min prior to coating the electrode with Nafion, calibrating in vitro and recording in vivo. Histamine is oxidized at a higher potential than 5-HT, and so for in vivo recordings 100 msec pulses of +1.0V were applied (compared to 0.55V for 5-HT recordings). Each pulse was separated by a 900 msec interval during which the resting potential was maintained at 0.0 V.
A separate cohort of animals was exposed to repeated swim, or not, as described above and 24 hr following the final swim, mice were sacrificed by rapid decapitation; brains were rapidly removed, frozen on dry ice, and stored at −80°C until use. Coronal, 20 µm brain sections were cut in a cryostat and mounted onto gelatin-coated slides.
To determine SERT expression level, we used quantitative autoradiography to measure binding of the SERT-specific radioligand, [3
H]-cyanoimipramine (CN-IMI) (80–85 Ci/mmol: American Radiolabeled Chemicals, St. Louis, Missouri, USA) in hippocampus of whole brain sections, as described by Hensler et al. (1994)
. Slide-mounted sections were incubated with CN-IMI (1 nM) in a buffer of 50 mM Tris, pH 7.4, and 120 nM NaCl at 4°C for 24 hr. Nonspecific binding was defined in the presence of 1 µM fluvoxamine. Sections were then washed in cold buffer (4°C) and dipped in cold distilled water. At the end of the autoradiographic assay, sections were dried on a slide warmer at 60°C and apposed to Kodak Biomax MR film (Amersham, Piscataway, New Jersey, USA) for 4 weeks to generate autoradiograms. Analysis of the digitized autoradiograms was performed using the image analysis program NIH Image, version 1.47 (NIH, Bethesda, Maryland, USA), and optical densities of brain images were converted to femtomoles/mg protein (fmol/mgpt). The concentration of [3
H]-CN-IMI used is approximately 8 times its KD
value (Kovachich et al., 1988
) so the values obtained approximate Bmax values.
Western Blot Analysis
Brains were removed from mice sacrificed by rapid decapitation and hippocampi were dissected out on ice and stored at −80°C until use. Samples were suspended in 500 µL of ice cold PBS (0.1 M, pH 7.4) and pulsed in centrifuge. The supernatant was removed, and the pellets were then homogenized in 500 µL of homogenizing buffer (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25mM sucrose, 1.5mM MgCl2
, 50mM NaCl, pH=7.2, aprotinin, leupeptin, pepstatin, phenylmethanesulfonylfluoride (PMSF)) using glass Potter-Elvehjem tissue grinders. Total protein concentrations of each sample were measured by Bradford Protein Assay (Bradford, 1976
). Samples were solubilized in a buffer containing bromophenyl blue, heated for 5 minutes at 95°C, and separated by electrophoresis using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was then transferred to a polyvinylidine difluoride (PVDF) membrane (Immobilon-P, Millipore, Bedford, MA, USA). The membrane was blocked in 5% nonfat dry milk in 1X TBS-Tween at room temperature for 1 hour, washed twice with 1X TBS-Tween, and incubated overnight at 4°C with polyclonal OCT3 antibody (Alpha Diagnostics International, San Antonio, Texas, USA; Catalogue # OCT31-A). The primary antibody was then removed, and the membrane was washed 3 times and incubated for 1 hour at 4°C with the secondary ECL donkey anti-rabbit IgG horseradish peroxidase-linked antibody (1:2500) (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Bound antibody was detected on x-ray film using enhanced chemiluminescence reagents (GE Healthcare, Little Chalfont, Buckinghamsire, UK). Note that affinity purified rabbit polyclonal OCT3 primary antibodies used here were generated against a 19 amino acid C-terminal cytoplasmic domain of mouse OCT3. This peptide has no significant sequence homology with other mouse OCTs. Together with our previously published immunohistochemical data using the same antibody and controlling for non-specific staining (Baganz et al., 2008
), the antibody used in these studies appears to be highly specific for OCT3.
Mice were anesthetized with isoflurane (3%) and bilateral adrenalectomy performed according to Castro (1974)
. Sham-operated animals were surgically incised to visually identify intact adrenal glands. Incisions were sutured and animals were allowed to recover from surgery for 1 week prior to exposure to repeated forced swim. Drinking water was replaced with 0.45% saline for ADX mice.
Corticosterone Radioimmunoassay (RIA)
Immediately following chronoamperometry recordings in ADX or sham operated mice (i.e. ~30 hr after the last swim), the animals were sacrificed, and trunk blood was collected for analysis of corticosterone content. Blood was centrifuged (6000 min−1
for 15 min) with 300 µL ethylenediaminetetraacetic acid (EDTA, anticoagulant) and plasma was separated according to methods described in Li et al., 1999
(and Dr. Li at Univ Kansas, Lawrence, Kansas – personal communication). Plasma corticosterone concentration was measured using a radioimmunoassay kit purchased from MP Biomedicals, LLC (Diagnostic Division, 29525 Fountain Pkwy. Solon, Ohio 44139, Cat No. 07120102) as described by the manufacturer.
Tail Suspension Test
A separate cohort of mice was used to assess the effect of the SSRIs fluvoxamine and fluoxetine, as well as the selective norepinephrine uptake inhibitor, desipramine (DMI) on behavior. Mice were exposed to 0 or 14 days of swim as described. Twenty-four hr after the final swim, mice were administered fluvoxamine (30 mg/kg), fluoxetine (15 mg/kg), desipramine (DMI) (15 mg/kg), dissolved in 0.9% v/v saline, injected intraperitoneally at 10 ml/kg body weight), or saline control. Thirty min following injection, the TST was carried out as previously described (Steru et al., 1985
). Mice were secured and suspended by the distal end of the tail to a flat metallic surface for 6 min. The presence or absence of immobility, defined as the absence of movement other than passive swaying, was sampled every 5 s over 6-min by a trained observer who remained blind to treatment (Boyce-Rustay et al., 2006
Data are presented as the mean and standard error of the mean and analyzed with ANOVA followed by Bonferroni or Newman-Keuls post-hoc comparisons or t-tests for independent samples. The threshold for statistical significance was set at p < 0.05
Serotonin, 5HIAA, histamine, corticosterone, desipramine, fluvoxamine, urethane and chloralose were purchased from Sigma (Houston, Texas, USA). Fluoxetine was purchased from LKT Laboratories (St. Paul, Minnesota, USA).