Reduced 5-HT1A- and GABAB Receptor Function in Dorsal Raphe Neurons Upon Chronic Fluoxetine...

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Reduced 5-HT1A- and GABAB receptor function in dorsal raphé neurons upon chronic fluoxetine treatment of socially stressed rats

Running head: Chronic fluoxetine treatment reduces GIRK activation

Cornelisse LN *†, Van der Harst JE ‡, Lodder JC *, Baarendse, PJJ †, Timmerman

AJ *, Mansvelder HD *, Spruijt BM ‡ and Brussaard AB *§

*Department of Experimental Neurophysiology & †Department of Functional

Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije

Universiteit (VU) and VU Medical Center (VUmc), De Boelelaan 1087, 1081 HV

Amsterdam. ‡ Department of Animals, Science and Society, Ethology and Animal

Welfare Group, Faculty of Veterinary Medicine, Universiteit Utrecht, Utrecht.

§ Corresponding author, Prof. Dr. A.B. Brussaard, Department of Experimental

Neurophysiology, CNCR, VU Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam,

the Netherlands, tel +31 20 5987098, e-mail: [email protected], fax +31 20 5987112

Number of figures: 5, Number of tables: 1, Number of pages: 28

Keywords: Whole-cell patch-clamp, social defeat, fluoxetine, GABAB receptor, 5-HT1A

receptor, GIRK current

Acknowledgements: The authors wish to acknowledge the excellent technical

assistance of Tessa Lodder, and comments on earlier drafts of the manuscript by Dr.

Matthijs Verhage, Dr. Witte Hoogendijk and Dr. Nail Burnashev. Financial support

was provided by the CNCR, NeuroBsik and Centre for Medical Systems Biology

(CMSB).

of 39 Articles in PresS. J Neurophysiol (April 25, 2007). doi:10.1152/jn.00109.2007

Copyright © 2007 by the American Physiological Society.

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Abstract

Autoinhibitory serotonin 1A receptors (5-HT1A) in dorsal raphé nucleus (DRN) have

been implicated in chronic depression and in actions of selective serotonin reuptake

inhibitors (SSRI). Due to experimental limitations, it was never studied at single cell

level whether changes in 5-HT1A receptor functionality occur in depression and during

SSRI treatment. Here we address this question in a social stress paradigm in rats

that mimics anhedonia, a core symptom of depression. We used whole-cell patch-

clamp recordings of 5-HT- and baclophen-induced G-protein-coupled inwardly

rectifying potassium (GIRK) currents as a measure of 5-HT1A- and GABAB receptor

functionality. 5-HT1A- and GABAB receptor-mediated GIRK-currents were not affected

in socially stressed rats, suggesting that there was no abnormal (auto)inhibition in the

DRN upon social stress. However, chronic fluoxetine treatment of socially stressed

rats restored anticipatory behavior and reduced the responsiveness of 5-HT1A

receptor-mediated GIRK currents. Since GABAB receptor-induced GIRK responses

were also suppressed, fluoxetine does not appear to desensitize 5-HT1A receptors

but rather one of the downstream components shared with GABAB receptors. This

fluoxetine effect on GIRK currents was also present in healthy animals and was

independent of the animal’s “depressed” state. Thus, our data show that symptoms

of depression after social stress are not paralleled by changes in 5-HT1A receptor

signaling in DRN neurons, but SSRI treatment can alleviate these behavioral

symptoms while acting strongly on the 5-HT1A receptor signaling pathway.

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Introduction

5-HT1A autoreceptors in the DRN have been implicated in mood disorders and

suicide although their direct role remains unclear. In the DRN of patients suffering

from major depression, both increased (Stockmeier et al. 1998) and reduced levels

(Arango et al. 2001) of 5-HT1A autoreceptors have been observed. Changes in 5-

HT1A autoreceptors may result from alterations in 5-HT1A receptor gene transcription

(Parsey et al. 2006), due to polymorphisms in the 5-HT1A receptor promoter region

(Albert and Lemonde 2004; Lemonde et al. 2003; Ou et al. 2003).

Moreover, SSRI treatment in patients may have long term effects on 5-HT1A

receptor levels in the DRN, since depressed patients treated with antidepressants

appear to have lower 5-HT1A receptors levels than antidepressant-naïve patients

(Parsey et al. 2006). Also in rodents desensitization of 5-HT1A autoreceptors as a

result of chronic SSRI administration has been observed (Blier et al. 1998; Czachura

and Rasmussen 2000; Elena Castro et al. 2003; Hensler 2002; Le Poul et al. 1995;

Pejchal et al. 2002; Shen et al. 2002; Subhash et al. 2000). Although it remains

unclear by which mechanism SSRI’s exert there antidepressant action, there is a

clear relation between reduced 5-HT1A receptor function and increased 5-HT levels

in DRN innervated forebrain areas after SSRI treatment in rodents. If SSRI’s are

acutely applied, locally enhanced 5-HT levels in the DRN inhibit serotonernergic

neurons through activation of the 5-HT1A autoreceptor. This results in decreased 5-

HT release in the projection areas (Artigas et al. 1996b). However, after chronic SSRI

treatment 5-HT1A receptors are desensitized and firing of DRN cells is restored

resulting in increased 5-HT levels in the frontal cortex (Artigas et al. 1996b; Bel and

Artigas 1993). Co-application of 5-HT1A receptor antagonists appears to prevent the

initial decrease of 5-HT release in the forebrain during SSRI treatment and may

accelerate antidepressant responses in patients (Artigas et al. 1996b; Blier et al.

1998).

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These studies suggest a role for DRN 5-HT1A autoreceptors in etology of

depression as well as in the antidepressant action of SSRI’s. However, in these

studies radioligand binding essays and in vivo recordings were used that do not allow

detection of changes in 5-HT1A receptor function at the cellular level. Moreover, the

experiments were performed in normal animals only and not in animal models for

depression.

Here we set out to quantify the 5-HT1A receptor function at the cellular level in

the DRN, using 1) control rats chronically treated with the SSRI fluoxetine, 2) a social

stress animal model for depression and 3) socially stressed rats chronically treated

with fluoxetine. Whole-cell patch-clamp recordings of 5-HT and baclophen (BAC)

induced GIRK currents were used as a measure of 5HT1A and GABAB receptor

functionality (Bayliss et al. 1997; Williams et al. 1988). With this approach we directly

tested if 5-HT1A-mediated inhibition is changed and whether this change is exclusive

for the 5-HT1A pathway or that also the GABAB pathway is affected. Thereby, we

discriminate between changes in receptor levels and changes downstream of the

receptor, since 5-HT1A- and GABAB receptors share the same intracellular pathway

through coupling to a pertussis toxin sensitive G-protein (Innis and Aghajanian 1987;

Innis et al. 1988; Williams et al. 1988).

A social stress paradigm in Wistar rats was used, consisting of one week of

daily social defeat encounters with a dominant intruder followed by approximately 3

months of social isolation. This paradigm induces symptoms resembling anhedonia

in depressed patients (Auriacombe et al. 1997), measured as the absence of

anticipatory behavior in response to sucrose rewards in these animals (Von Frijtag et

al. 2000; Von Frijtag et al. 2002). The anhedonia in rodents is a long-term effect that

correlates with impaired social memory and interactions (Rygula et al. 2005; Von

Frijtag et al. 2000), and is counteracted by imipramine (Von Frijtag et al. 2002) or

fluoxetine (Rygula et al. 2006) as also shown in this study. Using this animal model

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for depression we investigated the putative relationship between social stress, the

effects of chronic fluoxetine and 5-HT1A and GABAB receptor function in the DRN.

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Methods

Animals, housing and conditioning

Male Wistar rats (app. 8 wks old; HsdCpb:WU, Harlan, the Netherlands) were used in

experiments all approved by the Animal Ethical Committee of the Vrije Universiteit in

Amsterdam, which act in accordance to European law. The social stress procedure

has been described previously (Von Frijtag et al. 2001; Von Frijtag et al. 2000; Von

Frijtag et al. 2002). In short, the social defeat procedure consisted of daily resident-

intruder sessions on five consecutive days. Each defeat session consisted of a

forced introduction of an adult male rat (intruder) in the territory of a dominant male

rat (resident) and was divided in a pre- (5 min), fight- (5-10 min), and a post (5 min)-

phase. To minimize the risk of severe injuries residential male Long-Evans rats

(LE/CpbHsd, Harlan, UK) were selected for their fighting tactics. Only those rats that

terminated their attacks when the intruder showed submissive behaviors were

allowed to the defeat procedure. As part of the social stress paradigm, defeated

animals were housed individually in macrolon type III cages (30 cm long, 25 cm wide

and 15 cm high) following the first defeat session and control rats were housed

socially (two per cage). Three months later the chronic fluoxetine treatment started,

consisting of daily oral gavage with fluoxetine (10 mg/kg in tap water) or vehicle (tap

water) only, during a subsequent period of 21 days before the behavioral testing or

slice recordings started (fluoxetine HCl was a generous gift from Trifarma SpA

(Milan, Italy). Different animals were used for the behavioral assay and slice

recordings to avoid interference of experimental procedures. The behavioral assay

consisted of classical Pavlovian conditioning as previously described (Von Frijtag et

al. 2000); Van der Harst, 2005). A brief conditioning stimulus (CS, 2 x 60 W light-

input with 1 s interval followed by an auditory signal) was repeatedly paired with an

unconditioned stimulus (US, free access to 5% sucrose solution for 5 min, and of

which the consumption was measured). During a training period of 10 days, the

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offset of the CS and the onset of the US were progressively increased from 30 s to

10 min over 32 trials. Behavior was observed and analysed from videotape using the

computer program ‘The Observer’ (Noldus Information Technology, Wageningen, the

Netherlands). Behavioral activity displayed in the CS-US interval, reflected by the

frequency of behavioural transitions displayed in the time-interval between

announcement and presentation of the reward, was used as a parameter for

anticipation and reflects the level of reward-sensitivity (Von Frijtag et al. 2000; Von

Frijtag et al. 2002). Significant differences within groups between baseline and

posttraining testing was analyzed by paired t-test.

Slice recordings

Rats were anesthetized in a closed compartment with 4 ml Isoflurane (Abbot

Laboratories Ltd, Queenborough, Kent, U.K.) sprinkled on a tissue and decapitated

using a guillotine. Coronal midbrain slices (400 µm) were prepared in ice-cold slice

solution containing (in mM): 3.5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.2 KH2PO4, 212.5

Sucrose, 26 NaHCO3, 10 D-glucose, carboxygenated in 5% CO2 / 95% O2. Slices

were transferred to ACSF containing (in mM): 125 NaCl, 3 KCl, 1.2 NaH2PO4, 2.4

CaCl2, 1.3 MgSO4, 10 D-glucose, 25 NaHCO3, supplemented with 20 µM DNQX to

prevent overexcitation by glutamatergic inputs. Slices were kept at 37º C for 1 hour to

recover and were subsequently stored at room temperature. The recording chamber

was continuously perfused with ACSF, 20 µM DNQX and 20 µM bicuculline to block

AMPA and GABAA receptors. Cells were recorded in the whole-cell configuration in

voltage-clamp and current-clamp mode. To measure the I-V relation of GIRK currents

slow descending and ascending voltage ramps were applied between -60mV and -

120mV in figure 1-4, and between -60 and -100mV in figure 5. Patch pipettes had a

resistance of 2-2.5 MΩ. The internal solution contained (in mM) 5 NaCl, 1 MgCl2, 130

C6H11O7K (K-Gluconate), 10 HEPES, 2 Na2-ATP, 0.5 Tris-GTP, 0.02 EGTA. Series

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resistance was not compensated. Average access resistance at the end of the

recording was 9.6 MΩ (±0.3 SEM).

Large DRN neurons were filled through the recording pipette with 120 µM

Alexa 594 (Molecular Probes, Eugene, OR) dissolved in intracellular current clamp

medium. After fully loading with dye (15-30 min after break-in), these cells were

visualized with a Leica MP-RS 2 photon laser microscope (Leica, Mannheim,

Germany) and image projections (at 1 µm intervals) were obtained with Leica LCS

software and ImageJ (NIH), as described previously (de Rover et al. 2004).

The agents used in this study (5-HT, baclophen, DNQX, bicuculline) were

obtained from RBI (Natick, USA). Order of application in figure 2-5 was 5 µM 5-HT,

wash, 100 µM 5-HT or 1mM BAC. For this study we performed our recordings in the

absence of tryptophan loading (Liu et al. 2005). These conditions, which are

unfavorable for sustaining endogenous 5-HT synthesis, make it unlikely that

endogenous 5-HT secretion through spontaneous firing of 5-HT have contributed to

basal current responses in the absence of exogenous 5-HT application.

Data analysis

Current-voltage (I-V) curves were obtained by averaging the I-V responses during the

negative and positive slope of the voltage-clamp protocol in figure 1 C.

Conductances were calculated from the slope of the straight line fitted to the linear

part of the IV curves. 5-HT and BAC induced conductances shown in figure 2-5 were

corrected for basal conductance by subtraction of the basal conductance level at the

start of each experiment (Reanalysing the data by normalizing the induced GIRK

conductances to the basal conductance in each recording did not change the results;

not shown). Basal conductances were not different between the experimental groups

shown in figure 2-5 (figure 2: control + water 1.06±0.07 nS, control + fluoxetine

1.20±0.14 nS; figure 3: control 1.43±0.18 nS, social stress 1.65±0.16 nS; figure 4:

social stress + water 1.25±0.09 nS, social stress + fluoxetine 1.26±0.07 nS; figure 5:

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control + fluoxetine 1.73±0.54 nS, social stress + fluoxetine 1.65±0.61 nS) For

statistical analysis of the effects of different pharmaca within a given experimental

group (ie. comparison of the effect of 5 versus 100 µM 5-HT within the control for

instance) we used a two sample paired t-test assuming unequal variance (two tailed).

For comparison between experimental groups of the induced effect of 5-HT or BAC

at any given concentration and the conductance levels after wash we used a regular

ANOVA followed by a post hoc test (Boniferoni mode) for multiple comparisons. In

some instances, to increase statistical power for the data presented, we pooled the

data if experiments were conducted under identical experimental conditions; this was

done in particular for data shown in Fig. 2, where the control + fluoxetine data were

pooled with those also shown in Fig. 5. Data of Fig. 3 and 4 could not be pooled or

compared since the animal handling conditions were not identical, i.e. in Fig. 3

experimental animals were not treated orally either with water or fluoxetine, whereas

for data obtained for Fig. 4 they were. The significance found is indicated in the

figures with NS, *, ** or *** for not significant, p < 0.05, 0.01 and 0.005 respectively.

Error-bars show SEM. Not shown is the fact that all “wash conductances” were not

significantly different between experimental groups and that all 5-HT or BAC induced

conductances in figures 2-5 were significantly different from conductances before

application (at p < 0.01) unless mentioned otherwise explicitly (see figure 4 and figure

5, responses to 1 mM BAC). In addition, we used a multivariate ANOVA to test the

putative interaction between social defeat and SSRI treatment on GIRK current

activation by either 5-HT or BAC. Reanalysing the data by normalizing the induced

GIRK conductances to the basal conductance in each recording did not change the

results (not shown).

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Results

Assessing 5-HT1A receptor function in DRN neurons: GIRK currents

5-HT mediated auto-inhibition of serotonergic cells is extensively studied in rodents.

5-HT induces a hyperpolarizing current by activating the 5-HT1A receptor that is

coupled to a G-protein coupled potassium (GIRK) channel (Bayliss et al. 1997;

Katayama et al. 1997; Penington et al. 1993a; 1993b; Williams et al. 1988). Besides

5-HT1A receptors also GABAB receptors are coupled to GIRK channels in raphe

nucleus 5-HT cells (Bayliss et al. 1997; Innis and Aghajanian 1987; Innis et al. 1988;

Williams et al. 1988). This allows an accurate quantification on the cellular level of

the 5-HT1A receptor-mediated inhibition relative to the GABAB receptor-mediated

inhibition.

In whole-cell recordings from neurons in the ventral portion of the DRN we

distinguished between 5-HT-containing neurons and non-5-HT neurons based on

morphology and electrophysiological characteristics. The putative 5-HT neurons had

a morphology of magnocellular neurons (figure 1 A) and showed membrane potential

profiles in response to a series of current steps that were typical of 5-HT neurons

(figure 1 B, see (Li et al. 2001). In our experiments 89 % (n = 196) of the cells

displayed the characteristic electrophysiological profile of 5-HT neurons. The vast

majority of these cells fired spontaneously at the onset of recordings, unless slices

were exposed to tryptophan prior to recording (Liu et al. 2005) (n = 15, not shown). In

that case, DRN neurons secrete 5-HT within the nucleus, which hyperpolarizes DRN

neurons and reduce spontaneous activity (Johnson et al. 2002; Liu et al. 2005). In

the absence of the 5-HT precursor tryptophan, endogenous paracrine 5-HT release

is low or absent in DRN slices (Johnson et al. 2002), providing a good starting point

for quantitative measures of the exogenous 5-HT-induced GIRK responses. Current

responses were quantified by calculating the conductances from the measured IV

curves.

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Application of 5-HT induced a robust hyperpolarization of putative 5-HT

neurons (figure 1 C) but not of non-5-HT neurons (not shown). Subsequently, in the

same recording we switched to voltage-clamp and applied descending and

ascending voltage ramps, both in the absence and presence of various

concentrations of 5-HT (figure 1 C bottom traces). In the presence of 5-HT, the slope

of the current response during voltage ramps was much steeper, indicating that 5-HT

activated a GIRK conductance as reported previously (Bayliss et al. 1997; Katayama

et al. 1997; Penington et al. 1993a; 1993b; Williams et al. 1988) Subtraction of the

control current from the current during 5-HT application yielded the 5-HT activated

current with a reversal potential of -86 ± 7 mV (SD, figure 1 D, n = 38). This is in line

with the Nernst equilibrium potential for GIRK potassium currents calculated from the

intracellular and extracellular solutions used and corrected for the liquid junction

potential (-81 mV). Comparing conductances at different 5-HT concentrations

resulted in a dose-response relation with an EC50 of 2.7 µM 5-HT and 100 µM 5-HT

induced maximal GIRK conductance (figure 1 E).

Chronic fluoxetine reduces 5-HT1A and GABAB induced GIRK-currents

It has been suggested that during treatment with SSRIs, 5-HT1A receptors on DRN

neurons desensitize (Elena Castro et al. 2003; Hensler 2002; Le Poul et al. 1995;

Pejchal et al. 2002; Shen et al. 2002; Subhash et al. 2000). To test whether

fluoxetine treatment would reduce 5-HT1A receptor-mediated inhibition, healthy rats

were treated for 21 days with daily oral administrations of fluoxetine. The 5-HT-

induced GIRK responses were probed with two 5-HT concentrations: 5 µM, close to

the EC50, and 100 µM. In line with data shown in figure 1 E, in DRN slices from

water-treated animals 100 µM 5-HT induced a significantly larger GIRK conductance

than 5 µM 5-HT (figure 2 A-B, E). In contrast, in fluoxetine-pretreated animals, paired

comparison of the GIRK responses induced by these two 5-HT concentrations

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showed that they were not significantly different (figure 2 A-B, E). Moreover, while

the response to 5µM 5-HT upon chronic fluoxetine was similar as in the water

treated group, the 100µM 5-HT response was significantly reduced, This implies that

5-HT1A receptor functionality is reduced after chronic fluoxetine treatment.

To examine whether the effect of fluoxetine treatment was unique to the 5-

HT1A receptor pathway, we also tested whether the GABAB receptor-induced GIRK

currents were affected. GIRK responses induced by a maximal dose of the GABAB

receptor agonist BAC (1 mM) were significantly reduced in 5-HT neurons after

fluoxetine treatment (figure 2 C-D, and F). This suggests that fluoxetine treatment

affects 5-HT1A receptor functionality downstream of the 5-HT1A receptor and at a level

of the signaling pathway that is shared with the GABAB receptor to the GIRK-

pathway.

Recently, it was claimed (Takahashi et al. 2006) that fluoxetine may suppress

basal GIRK conductance acutely and directly. To test whether the suppression of the

maximal GIRK current activation in our experiments was due to a chronic, rather than

to an acute or direct effect of fluoxetine, we measured basal conductance levels

before and after the acute application of 33 µM fluoxetine for 4 min. This

concentration corresponds to the theoretical maximal concentration of fluoxetine

when a dose of 10 mg/kg is used in vivo and is about 100 times higher than plasma

levels of fluoxetine measured after 10mg/kg injected i.p. or with osmotic minipumps

(Czachura and Rasmussen 2000). We found that acute exposure to fluoxetine

increased, rather than decreased, the normalized basal conductance (from 1 ± 0.15

nA to 1.33 ± 0.09 nA, n = 10, paired t-test, p < 0.002). We cannot exclude that this is

due to a fluoxetine-mediated increase in residual endogenously released,

extracellular 5-HT levels as a consequence of the acute block of 5-HT reuptake in the

brain slices. Furthermore, 5 µM 5-HT elicited normal GIRK responses in the

presence of fluoxetine (2.73 ± 0.38 fold increase, n = 6). Thus, in our recordings we

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find no evidence for a direct inhibition by fluoxetine of GIRK conductance level or of

5-HT receptor mediated GIRK currents.

Social defeat does not affect 5-HT1A receptor responses

To test whether 5-HT1A receptor function is affected in DRN neurons of animals that

exhibit symptoms of depression, we recorded 5-HT-induced GIRK currents in DRN 5-

HT neurons from rats that were subjected to a social stress paradigm (Von Frijtag et

al. 2000; Von Frijtag et al. 2002). These rats were compared to control rats that had

not received social defeat encounters nor isolation. It has previously been reported

that rats subjected to this social stress paradigm show anhedonia, a symptom of

depression, reflected by a suppression of in anticipatory behavior in expectation of a

reward. In the present experiments, socially stressed rats were also observed to

show a supressed anticipatory behavior as an indication for anhedonia (Table I).

When 5-HT-induced GIRK currents were recorded in DRN 5-HT neurons, no

differences were found between control and socially stressed animals. In both groups

GIRK currents were activated by 5-HT in a dose-dependent manner and to a similar

extent (figure 3 A-B and E). This suggests that the social stress paradigm does not

change 5-HT1A receptor function in DRN 5-HT neurons. Similarly, the maximal GIRK

conductance induced by the GABAB receptor agonist BAC was also not affected

upon social defeat stress (figure 3 C-D, and F). Thus, the induction of depression-like

symptoms in rats by social defeat stress does not affect 5-HT1A receptors or one of

the downstream components.

Rescue of anticipatory behavior after social stress by fluoxetine correlates with

reduced 5-HT1A- and GABAB receptor functionality

This insensitivity for reward (anhedonia), reflected by the impaired anticipatory

behavior in socially stressed rats was previously shown to be reversed by chronic

treatment of the antidepressant imipramine (Von Frijtag et al. 2002). We also

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observed relief from anhedonia-like symptoms in socially stressed rats after

treatment with fluoxetine for 21 days. In fluoxetine treated animals, anticipatory

behavior toward sucrose rewards was restored after three weeks of treatment

whereas in vehicle treated animals anticipatory behavior remained impaired (Table I).

No effect of social stress and/or fluoxetine treatment on the intake of 5% sucrose

consumption was observed (data not shown). To test whether the behavioral effect of

chronic fluoxetine treatment correlated with a cellular effect of fluoxetine in the DRN,

we examined GIRK currents induced by 5-HT and BAC in DRN 5-HT neurons of

animals subjected to the same social stress paradigm with or without subsequent

chronic fluoxetine treatment (figure 4 A-B, and E). In 5-HT neurons of socially-

stressed fluoxetine-treated animals, 100 µM 5-HT did not produce larger responses

than 5µM 5-HT (figure 4 B and E), similar to the effect seen of chronic fluoxetine

treatment in control animals (figure 2 B and E). Moreover the responsiveness to

either 5µM or 100µM 5-HT was significantly reduced when compared to socially

stressed animals not treated with fluoxetine (figure 4 E). Thus the 5-HT

responsiveness of DNR neurons in terms of 5-HT induced GIRK activation appeared

to be suppressed by fluoxetine-treatment in socially stressed animals, similar to the

apparent effect of chronic fluoxetine in control animals. However, the apparent

relative effect of fluoxetine upon social stress appeared to be larger than in control

animals (compare figure 4 E and figure 2 E respectively; see also below).

To test whether fluoxetine treatment reduced 5-HT1A receptor functionality

downstream of the 5-HT1A receptor, we also examined BAC-induced GIRK currents.

Similar to the effect of fluoxetine-treatment in healthy rats, upon social stress

conditioning, the BAC-induced GIRK currents were significantly reduced in 5-HT

neurons after fluoxetine treatment (figure 4 C-D, and F). This suggests that in social

defeated animals a reduced functionality of the 5-HT1A receptor coupling to GIRK

induced by fluoxetine treatment is expressed downstream of the 5-HT1A receptor and

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most likely at a level of the signaling pathway that is shared with the GABAB receptor

to the GIRK-pathway. Again the relative effect of fluoxetine upon social defeat

appeared larger than in control animals (compare figure 4 F and figure 2 F

respectively).

Fluoxetine acts on 5-HT1A receptor functionality independent of social

conditions

Fluoxetine appears to have mood-elevating effects predominantly in depressive

patients. In healthy human subjects, no significant effects of fluoxetine on mood and

other psychological variables were found (Gelfin et al. 1998). As indicated above, in

our experiments, fluoxetine treatment appeared to affect 5-HT- and GABAB GIRK

activation in DRN 5-HT neurons of both control and socially stressed rats, but

possibly to a different extent. We directly addressed the hypothesis that fluoxetine

treatment has a stronger impact on 5-HT1A receptor responses in DRN 5-HT neurons

of animals upon social stress compared to control conditioning. To this end, we

tested 5-HT-induced GIRK activation in fluoxetine-treated, socially stressed rats and

in fluoxetine-treated, control rats. Recordings of DRN 5-HT neurons of these animals

showed that, while 5 µM 5-HT induced GIRK responses were significantly different

from basal levels, the 5-HT-induced GIRK conductance at 5 µM and 100 µM were not

different in either group (figure 5 A, B). Thus, no significant differences were found in

the relative effect of fluoxetine between the control and socially stressed groups

(figure 5 E). Similarly, BAC-induced GIRK currents were similarly suppressed in both

groups (figure 5 F), indicating that also in these experiments fluoxetine acted

downstream of the 5-HT1A receptor. The BAC-induced GIRK conductance change

was also not different from 100 µM 5-HT responses, either in control and in socially

stressed animals (figure 5 E and F). These data show that fluoxetine acts on DRN 5-

HT neurons independent of whether animals show symptoms of depression.

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Subsequent multivariate ANOVA revealed no interaction between social

defeat and fluoxetine treatment. Simple contrast (K matrix) testing, for all data pooled

in this study, showed no significant effect of social stress (compared to controls, p =

0.563 and p = 0.154). Basal conductances were not different between social stress

and control animals. In contrast, chronic treatment with fluoxetine produced robust

overall effects (p = 0.006 and p = 0.001 respectively for 5 and 100 µM 5-HT, with no

effects on basal responses). Additional multivariate analysis of possible interaction

between social stress and chronic SSRI treatment revealed no significance. This

confirms that DRN 5-HT neurons in socially stressed animals are not more sensitive

to fluoxetine treatment than 5-HT neurons of control animals.

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Discussion

In this study, we addressed the question of whether 5-HT1A receptor function in

dorsal raphé nucleus 5-HT neurons is affected in animals that show symptoms of

depression and upon fluoxetine treatment. Rats exposed to social stress exhibited

behavioral symptoms of depression that could be relieved by fluoxetine treatment. In

socially stressed animals, neither 5-HT1A - nor GABAB receptor-induced GIRK

currents in DRN 5-HT neurons were different in amplitude from GIRK currents in

healthy animals. However, when these animals were treated with fluoxetine for 21

days, both 5-HT1A- and GABAB receptor mediated GIRK responses were markedly

reduced. This implies that fluoxetine acts on downstream components shared by

both receptor-types. We found that fluoxetine treatment also affects both 5-HT1A- and

GABAB receptor functionality in 5-HT neurons of healthy animals. When directly

tested, the effects of fluoxetine treatment on 5-HT1A- and GABAB induced GIRK

currents were not different in healthy and socially stressed animals, indicating that

the action of fluoxetine is independent of the animal’s “depressed” state.

5-HT1A receptor function in the DRN and depression

Previous studies on depressed patients suggest either a positive or an inverse

correlation between chronic depression and 5-HT1A receptor levels in the brain. In

postmortem brains of patients suffering from major depression, increased

(Stockmeier et al. 1998) and decreased (Arango et al. 2001) levels of 5-HT1A

receptors were reported. In patients with bipolar or late-life depression, reduced

levels of 5-HT1A receptors were found (Drevets et al. 2000; Drevets et al. 1999;

Meltzer et al. 2004). In socially stressed rats that show behavioral symptoms of

depression, we found no changes in levels or functionality of 5-HT1A-receptors at the

level of DRN neurons. Our results suggest that in DRN neither receptor levels, nor

downstream components, such as G-proteins or GIRK channel activity, are affected

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after severe social stress. This suggests that the behavioral symptoms of depression

observed in these animals, such as a loss of reward-sensitivity, an impaired social

memory and social interaction behavior that resemble anhedonia and motivational

deficits in humans (Von Frijtag et al. 2001; Von Frijtag et al. 2000; Von Frijtag et al.

2002), are brought about by other mechanisms than an alteration in 5-HT1A-receptor

signaling in DRN 5-HT neurons. These results appear in line with findings in

monkeys that show symptoms of behavioral depression. In these animals, 5-HT1A

receptor binding at the level of the raphé nucleus (Shively et al. 2006) were not

affected.

Chronic treatment with the SSRI fluoxetine did affect 5-HT1A receptor

functionality in DRN 5-HT neurons, both in healthy and socially stressed animals.

Fluoxetine treatment also restored anticipatory responses in the latter group (Table

I), just as the antidepressant imipramine reverses the symptoms of anhedonia after

social stress (Von Frijtag et al. 2001). Previous studies have shown that fluoxetine

treatment affects 5-HT1A receptor-stimulated G-protein-binding in the DRN in healthy

rats (Elena Castro et al. 2003; Hensler 2002; Pejchal et al. 2002; Shen et al. 2002).

In transgenic mice lacking 5-HT transporter protein, similar adaptive changes in 5-

HT1A receptor signaling were found in DRN (Fabre et al. 2000; Gobbi et al. 2001;

Holmes et al. 2003; Li et al. 2000; Mannoury la Cour et al. 2001; Mannoury la Cour et

al. 2004). Recently, it was confirmed that fluoxetine treatment can alleviate

behavioral deficits evoked by chronic social stress in rats (Rygula et al. 2006). How

reduced 5-HT1A receptor functionality in the DRN contributes to the suppression of

depressive symptoms in socially stressed animals is not clear. However, previous

studies show a clear relation between 5-HT1A receptor mediated autoinhibition at the

DRN level and 5-HT levels in the projection areas. 5-HT levels in DRN innervated

regions and the activity of serotonergic neurons are both reduced after acute

application of SSRI’s (Artigas et al. 1996a; Artigas et al. 1996b; Czachura and

Rasmussen 2000). This is attributed to the inhibiting action of 5-HT1A receptors that

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are activated by the increased concentration of 5-HT in the DRN after blocking 5-HT

reuptake. Long-term treatment with SSRI’s produces a robust increase in

extracellular 5-HT levels in the projection area’s and restores firing activity of

serotonergic cells, which is consistent with the desensitization of 5-HT1A receptors in

the DRN (Bel and Artigas 1993; Ferrer and Artigas 1994; Czachura and Rasmussen

2000). The similar time course of 5-HT levels in frontal cortex and the antidepressant

response by SSRI’s suggests that these are related. This idea is supported by the

fact that coaplication of SSRI’s with a 5-HT1AR antagonist like pindolol prevents the

initial reduction of 5-HT in forebrain projection areas and accelerates antidepressant

action (Artigas et al. 1996b). Our findings therefore suggests that the restoration of

hedonic behavior in socially stressed animals after treatment with fluoxetine might

possibly be due to augmented serotonergic output of DRN as a result of reduced 5-

HT1A receptor functionality in DRN.

Action of fluoxetine at the cellular level

The observed reduction of 5-HT-induced GIRK currents after chronic SSRI treatment

may result from desensitization of 5-HT1A receptors or may be due to desensitization

of a component downstream of the receptor. The fluoxetine-induced reduction in 5-

HT1A receptor functionality is paralleled by a reduction in GABAB receptor-mediated

GIRK activation after treatment with fluoxetine, both in healthy as well as in socially

stressed animals. Although a parallel effect of fluoxetine on both receptors can not be

excluded it is more likely that fluoxetine treatment affects 5-HT1A receptor signaling in

DRN 5-HT neurons downstream of the receptors, since both 5-HT1A and GABAB

receptors couple to the same type of GIRK channel through Gi-proteins in these

neurons (Innis and Aghajanian 1987; Innis et al. 1988; Williams et al. 1988).

Radioligand binding assays suggest that chronic fluoxetine treatment reduces the

coupling of 5-HT1A-receptors and G-proteins without changing 5-HT1A-receptor levels

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(Elena Castro et al. 2003; Hensler 2002; Pejchal et al. 2002; Shen et al. 2002). In

transgenic mice in which 5-HT reuptake is prevented by knocking out the 5-HT

transporter (5-HTT null mice), functionality of 5-HT1A- as well as GABAB receptors is

strongly reduced in DRN 5-HT cells (Mannoury la Cour et al. 2004), supporting the

idea that both receptor types partly share a common signal transduction pathway in

5-HT neurons.

Thus far, the best supported candidate mechanism by which fluoxetine

treatment leads to inhibition of GIRK channel activity is through uncoupling of Gi-

proteins from their receptors (Elena Castro et al. 2003; Hensler 2002; Pejchal et al.

2002; Shen et al. 2002). Alternative mechanisms could be through PKC-dependent

phosphorylation of GIRK channels (Mao et al. 2004), depletion of PIP2, an

endogenous co-factor necessary for GIRK channel activity (Cho et al. 2005; Lei et al.

2003) or through a glucocorticoid-mediated modulation of GIRK responsiveness

(Fairchild et al. 2003). Recently, the background potassium channel TREK-1 was

proposed as a target for antidepressant action in DRN neurons ((Heurteaux et al.

2006)). In this model, fluoxetine acts as antidepressant through direct inhibition of

TREK-1 channels. However, in our experiments, we could not detect a direct

inhibition of background whole-cell potassium currents by fluoxetine in DRN 5-HT

neurons that would support this model.

GABAB receptors and the action of SSRI’s

There is an emerging body of data purporting a role for GABAB receptors as potential

targets for antidepressant actions (Mombereau et al. 2004a; Mombereau et al. 2005;

Petty 1995; Slattery et al. 2005). Although previous studies reported no change in

GABAB receptor levels (Cross et al. 1988) or functionality (Monteleone et al. 1990) in

depressed patients, more recent studies showed antidepressant effects of GABAB

antagonists. Transgenic mice lacking either GABAB1 or GABAB2 receptor subunits

show enhanced resistance against symptoms of depression in the forced swim test

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(Mombereau et al. 2005; Mombereau et al. 2004b; Slattery et al. 2005). We show

that chronic SSRI treatment suppresses GABAB receptor functionality of DRN

neurons, confirming a possible role for GABAB receptors as target for

antidepressants. Since local GABAergic interneurons in the DRN are activated by 5-

HT through 5-HT2A/C receptors (Liu et al. 2000), GABA signaling through GABAB

receptors is likely to provide an additional negative feedback loop of 5-HT neuron

activity. Therefore, reduction of GABAB receptor functionality may have a similar

disinhibiting effect of 5-HT neuron activity as decreased 5-HT1A receptor functionality.

This model may further suggest that co-application of GABAB antagonists might

speed up the antidepressant action of SSRI’s by blocking GABAB receptor activity.

Significance from a clinical perspective

The prevalent view on the cellular mechanisms underlying the origin of depression in

humans and the cellular mechanisms of SSRI treatment is that they both reside in

the serotonergic system. Our results suggest that these mechanisms could diverge.

On the one hand, severe social stress can induce behavioral symptoms of

depression, such as anhedonia, without affecting 5-HT1A receptor functionality in 5-

HT neurons. On the other hand, fluoxetine treatment can alleviate these symptoms of

depression by reducing 5-HT1A receptor functionality in DRN 5-HT neurons. Thus,

although the possibility remains that stress hormones affects components in the

serotonergic cell system other than GIRK currents (Abrams et al. 2005; Lowry et al.

2000), it becomes more and more likely that non-aminergic pathways located in

target areas of the DRN may also be involved (Berton and Nestler 2006). Also in the

latter model, antidepressants are thought to act primarily on the serotonergic system

(Duman 2002; Duman et al. 1999). Cause and therapy of depression may therefore

diverge. Using animal models, such as social stress in rodents, to identify the

neuronal mechanisms underlying both cause and therapy of mood disorders is

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indispensable to eventually get from palliative to curative treatments of these

disorders in the future.

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Figure 1 . Morphological and biophysical identification of 5-HT neurons. A: 2-photon

laser scanning microscopy of a serotonergic cell. Serotonergic cells displayed a

current clamp profile shown in B. C: Currents in response to a voltage clamp protocol

(-60 mV to -120 mV) before, during, and after application of 5-HT (5 µM). Membrane

potential changes after wash-in and wash-out of 5-HT were monitored in current

clamp. D: I-V plots of the currents as induced in C. The conductance was calculated

from the slope of the linear part of the I-V. E: Dose response curve of GIRK

conductance induction by 5-HT (EC 50 = 2.66 µM).

Figure 2 . Chronic application of fluoxetine affects both 5-HT and GABAB mediated

GIRK responses. A-D: Example traces of 5-HT- and BAC induced currents in control

animals not treated (water) (A, C) and treated with fluoxetine (B, D). E: 5-HT-

activated GIRK conductances in control animals not treated (5µM 5-HT 2.3±0.3,

n=19; wash 0.2±0.1, n=19; 100µM 5-HT 4.9±0.5, n=15; wash 0.8±0.4, n=11) and

treated with fluoxetine (5µM 5-HT 2.6±0.2, n=42; wash 0.4±0.1, n=35; 100µM 5-HT

3.3±0.4, n=30; wash 1.3±0.2, n=24). F: BAC-affected GIRK conductances in control

animals not treated (wash 0.6±0.3, n=7; 1mM BAC 5.1±0.7, n=7) and treated with

fluoxetine (wash 0.8±0.2, n=16; 1mM BAC 2.2±0.3, n=16). All induced effects, with

the exception of the BAC effect in unconditioned animals treated with fluoxetine,

were significantly different from their controls (p<0.05). The 100 µM 5-HT effect was

significantly different from the 5 µM 5-HT (p<0.01) in the control animals-not

fluoxetine treated group but not in the fluxetine-treated group. The BAC effect in the

fluoxetine treated group was significantly different from 100 µM 5-HT effect (p<0.05).

Figure 3 . Social stress does not affect 5-HT induced GIRK responses. A-D: GIRK

currents in response to 5-HT and BAC in control (A, C) and socially stressed animals

(B, D). E: 5-HT-activated GIRK conductances in control (5µM 5-HT 3.6±0.5, n=13;

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wash 0,3±0.1, n=12; 100µM 5-HT 6.3±0.8, n=10; wash 0.6±0.2, n=9) and socially

stressed animals (5µM 5-HT 3.3±0.3, n=19; wash 1,1±0.4, n=19; 100µM 5-HT

6.0±0.8, n=11; wash 2.0±0.5, n=10). F: BAC-activated GIRK conductances in control

(wash 0.3±0.1, n=9; 1mM BAC 2.9±0.6, n=9) and socially stressed animals (wash

1.5±0.6, n=10; 1mM BAC 4.6±1.2, n=10). All induced effects were significantly

different from their control (p<0.05) but not from the induced effects in the other

experimental group. The 100 µM 5-HT effect was significantly different from the 5 µM

5-HT effect (*=p<0.05, **=p<0.01) but not from the 1 mM BAC effect, within each

experimental group.

Figure 4. Chronic application of fluoxetine to socially stressed animals leads to

reduction of both 5-HT and GABAB mediated GIRK responses. A-D: Example traces

of 5-HT- and BAC induced currents in socially stressed animals not treated (A, C) or

treated with fluoxetine (B, D). E: 5-HT-activated GIRK conductances in socially

stressed animals not treated (5µM 5-HT 4.7±0.6, n=19; wash 0,3±0.2, n=19; 100µM

5-HT 6.3±0.7, n=11; wash 0.9±0.3, n=10) and treated with fluoxetine (5µM 5-HT

2.7±0.2, n=31; wash 0.3±0.1, n=31; 100µM 5-HT 2.7±0.5, n=15; wash 1.4±0.5, n=8).

F: BAC-activated GIRK conductances in socially stressed animals treated not treated

(wash 0.1±0.1, n=4; 1mM BAC 4.1±0.3, n=4) and treated with fluoxetine (wash

0.2±0.1, n=14; 1mM BAC 0.6±0.2, n=13). All induced effects, with the exception of

the BAC effect in socially stressed animals treated with fluoxetine, were significantly

different from their control (p<0.05) and from the induced effects in the other

experimental group (**<0.01, ***<0.005). The 100 µM 5-HT effect was significantly

different from the 5 µM 5-HT effect (p<0.05) in the social stress-not treated with

fluoxetine- group, but not in the social stress -treated with fluoxetine- group. The BAC

effect is significantly different from 100 µM 5-HT effect (p<0.05) in both groups.

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Figure 5 . No interaction between effect of chronic fluoxetine treatment and social

defeat conditioning. A-D: Example traces of 5-HT- and BAC induced currents in

control animals (A, C) and socially stressed animals (B, D), both treated with

fluoxetine (10mg/kg) for 21 days. E: 5-HT-activated GIRK conductances in control

animals treated with fluoxetine (5µM 5-HT 2.4±0.3, n=22; wash 0.5±0.1, n=15;

100µM 5-HT 2.5±0.4, n=13; wash 1.1±0.4, n=9) and socially stressed animals treated

with fluoxetine (5µM 5-HT 2.1±0.2, n=19; wash 0,5±0.1, n=18; 100µM 5-HT 2.1±0.2,

n=12; wash 1.2±0.2, n=7) . F: BAC-activated GIRK conductances in control animals

treated with fluoxetine (wash 0.3±0.4, n=5; 1mM BAC 1.4±0.4, n=5) and socially

stressed animals treated with fluoxetine (wash 0.4±0.2, n=5; 1mM BAC 1.5±0.4,

n=5). All 5-HT induced effects were significantly different from their control (p<0.05)

but not from the induced effects in the other experimental group. The BAC effect was

not significantly different from its control or from the induced effect in the other group.

Tabel I. Relief from anhedonia-symptoms upon chronic fluoxetine treatment in

socially stressed animals. Mean number of transition between behavioural elements

(± SEM), displayed in the interval (10 min) between the announcement (light+sound)

and the arrival of the reward (sucrose 5%, 5 min), reflecting baseline activity at trial 0,

and anticipatory activity (post-training) in expectation of the reward at trial 32.

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Figure 1 . Morphological and biophysical identification of 5-HT neurons. A: 2-photon laser scanning microscopy of a serotonergic cell. Serotonergic cells displayed a current clamp profile shown in B. C: Currents in response to a voltage clamp protocol (-60 mV to -120 mV) before, during, and after application of 5-HT (5 M). Membrane potential changes after wash-in and wash-out of 5-HT were monitored in current clamp. D: I-V plots of the

currents as induced in C. The conductance was calculated from the slope of the linear part of the I-V. E: Dose response curve of GIRK conductance induction by 5-HT (EC 50 = 2.66

M).

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Figure 2 . Chronic application of fluoxetine affects both 5-HT and GABAB mediated GIRK responses. A-D: Example traces of 5-HT- and BAC induced currents in control animals not

treated (water) (A, C) and treated with fluoxetine (B, D). E: 5-HT-activated GIRK conductances in control animals not treated (5 M 5-HT 2.3±0.3, n=19; wash 0.2±0.1, n=19; 100 M 5-HT 4.9±0.5, n=15; wash 0.8±0.4, n=11) and treated with fluoxetine (5 M 5-HT 2.6±0.2, n=42; wash 0.4±0.1, n=35; 100 M 5-HT 3.3±0.4, n=30; wash

1.3±0.2, n=24). F: BAC-affected GIRK conductances in control animals not treated (wash 0.6±0.3, n=7; 1mM BAC 5.1±0.7, n=7) and treated with fluoxetine (wash 0.8±0.2, n=16;

1mM BAC 2.2±0.3, n=16). All induced effects, with the exception of the BAC effect in unconditioned animals treated with fluoxetine, were significantly different from their

controls (p<0.05). The 100 M 5-HT effect was significantly different from the 5 M 5-HT (p<0.01) in the control animals-not fluoxetine treated group but not in the fluxetine-treated group. The BAC effect in the fluoxetine treated group was significantly different

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from 100 M 5-HT effect (p<0.05).

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Figure 3 . Social stress does not affect 5-HT induced GIRK responses. A-D: GIRK currents in response to 5-HT and BAC in control (A, C) and socially stressed animals (B, D). E: 5-HT-activated GIRK conductances in control (5 M 5-HT 3.6±0.5, n=13; wash 0,3±0.1, n=12; 100 M 5-HT 6.3±0.8, n=10; wash 0.6±0.2, n=9) and socially stressed animals (5 M 5-HT 3.3±0.3, n=19; wash 1,1±0.4, n=19; 100 M 5-HT 6.0±0.8, n=11; wash

2.0±0.5, n=10). F: BAC-activated GIRK conductances in control (wash 0.3±0.1, n=9; 1mM BAC 2.9±0.6, n=9) and socially stressed animals (wash 1.5±0.6, n=10; 1mM BAC 4.6±1.2, n=10). All induced effects were significantly different from their control

(p<0.05) but not from the induced effects in the other experimental group. The 100 M5-HT effect was significantly different from the 5 M 5-HT effect (*=p<0.05,

**=p<0.01) but not from the 1 mM BAC effect, within each experimental group.

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Figure 4. Chronic application of fluoxetine to socially stressed animals leads to reduction of both 5-HT and GABAB mediated GIRK responses. A-D: Example traces of 5-HT- and BAC induced currents in socially stressed animals not treated (A, C) or treated with fluoxetine (B, D). E: 5-HT-activated GIRK conductances in socially stressed animals not treated (5M 5-HT 4.7±0.6, n=19; wash 0,3±0.2, n=19; 100 M 5-HT 6.3±0.7, n=11; wash 0.9±0.3,

n=10) and treated with fluoxetine (5 M 5-HT 2.7±0.2, n=31; wash 0.3±0.1, n=31; 100 M 5-HT 2.7±0.5, n=15; wash 1.4±0.5, n=8). F: BAC-activated GIRK conductances in

socially stressed animals treated not treated (wash 0.1±0.1, n=4; 1mM BAC 4.1±0.3, n=4) and treated with fluoxetine (wash 0.2±0.1, n=14; 1mM BAC 0.6±0.2, n=13). All

induced effects, with the exception of the BAC effect in socially stressed animals treated with fluoxetine, were significantly different from their control (p<0.05) and from the

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induced effects in the other experimental group (**<0.01, ***<0.005). The 100 M 5-HT effect was significantly different from the 5 M 5-HT effect (p<0.05) in the social

stress-not treated with fluoxetine- group, but not in the social stress -treated with fluoxetine- group. The BAC effect is significantly different from 100 M 5-HT effect

(p<0.05) in both groups

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Figure 5 . No interaction between effect of chronic fluoxetine treatment and social defeat conditioning. A-D: Example traces of 5-HT- and BAC induced currents in control animals

(A, C) and socially stressed animals (B, D), both treated with fluoxetine (10mg/kg) for 21 days. E: 5-HT-activated GIRK conductances in control animals treated with fluoxetine (5 M 5-HT 2.4±0.3, n=22; wash 0.5±0.1, n=15; 100 M 5-HT 2.5±0.4, n=13; wash

1.1±0.4, n=9) and socially stressed animals treated with fluoxetine (5 M 5-HT 2.1±0.2, n=19; wash 0,5±0.1, n=18; 100 M 5-HT 2.1±0.2, n=12; wash 1.2±0.2, n=7) . F: BAC-activated GIRK conductances in control animals treated with fluoxetine (wash 0.3±0.4,

n=5; 1mM BAC 1.4±0.4, n=5) and socially stressed animals treated with fluoxetine (wash 0.4±0.2, n=5; 1mM BAC 1.5±0.4, n=5). All 5-HT induced effects were significantly different from their control (p<0.05) but not from the induced effects in the other

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experimental group. The BAC effect was not significantly different from its control or from the induced effect in the other group

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Baseline Post-training p-value

social defeat 16.9 ±1.09 16.0 ±0.82 0.4 (n = 9)

social defeat+ fluoxetine

17.0 ±1.01 21.1 ±0.92 0.02 (n = 10)

Table I

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Reduced 5-HT1A- and GABAB Receptor Function in Dorsal Raphe Neurons Upon Chronic Fluoxetine...

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Transcript of Reduced 5-HT1A- and GABAB Receptor Function in Dorsal Raphe Neurons Upon Chronic Fluoxetine...