University of Groningen Augmentation of the …GABA B receptors (Varga et al, 2002) as well as 5HT 2C and 5-HT 1A receptors (Serrats et al, 2005) have also been localized in the raphe,

University of Groningen

Augmentation of the neurochemical and behavioural effects of SSRIsRea, Kieran

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Augmentation of antidepressant effects of SSRIs by GABAB antagonists: mechanistic studies

205

Chapter 8

Augmentation of antidepressant effects of SSRIs by

GABAB antagonists: mechanistic studies

Chapter 8

206

Abstract

The slow onset of therapeutic effect of SSRIs has been linked to the gradual

desensitization of 5-HT autoreceptors during treatment. Recently, it has been reported

that 5-HT2C receptor antagonists augment the biochemical and behavioral effects of

SSRIs. This augmentation has been attributed to an attenuation of GABAB induced

feedback due to inhibition of GABA release by 5-HT2C antagonism. These

experiments also showed that local infusion of GABAB antagonists in hippocampus

augmented the effects of SSRIs in a similar fashion as 5-HT2C antagonists. In the

current study we present data on the augmentation of biochemical and behavioral

effects of SSRIs by GABAB antagonists. Using microdialysis coupled to HPLC, it

was observed that systemic administration of phaclofen and the GABAB antagonists,

CGP 463812 and SCH 50911, augmented the biochemical effects of citalopram on

central 5-HT levels in raphe, and ventral hippocampus in a bell-shaped manner.

While there was an augmentation, a bell-shaped profile of the combined

administration of phaclofen and citalopram was not observed in the prefrontal cortex.

Similar effects were observed in behavioural experiments involving the co-

administration of GABAB compounds with citalopram. The present data clearly

suggest that GABAB antagonists augment the efficacy of SSRIs. However, given

indications for bell-shaped dose dependency, this approach might be difficult to use

in the clinic.


207

Introduction

Serotonin-containing neurons are localized in the midbrain and brainstem raphe

nuclei and project to most regions of the brain, where they exert a wide array of

physiological functions. A number of conditions such as depression, anxiety and

various psychiatric illnesses are attributed, at least in part, to abnormalities of the

serotonergic system. Current popular antidepressant treatment involves the inhibition

of reuptake of monoamines such as noradrenaline and serotonin, or the manipulation

of specific target receptors which modulate monoamine levels. The administration of

selective serotonin reuptake inhibitors (SSRIs) dramatically increases the level of

extracellular serotonin in the brain. It is also known that with the administration of

SSRIs there is a certain amount of feedback to 5HT autoreceptors, and other 5HT

receptors located on various other neurons which also impact on the release and firing

of 5HT neurons. The firing and release of neurotransmitters from these serotonergic

neurons is under a fine control of a number of different neurotransmitter systems such

as serotonin itself (Barnes & Sharp, 1999), noradrenaline (Hjorth et al, 1995; Tao &

Hjorth, 1992; de Boer et al, 1996), dopamine (Ferre et al, 1993, 1994), histamine

(Schwarz et al, 1991; Barbera et al, 2002), glutamate (Ghersi et al, 2003; Pittaluga et

al, 1987), and many others, including GABA (Tao & Auerbach, 2000).

The inhibitory neurotransmitter GABA plays a critical role in the regulation

of firing of 5-HT neurons (Gallagher and Aghajanian, 1976; Abellan et al, 2000a;

Tao & Auerbach, 1996; Bowery et al, 1987; Chu et al, 1990; Mennini et al, 1986).

Indeed both GABAA (Gao et al, 1993; Rodriguez-Pallares et al, 2001) and GABAB

receptors (Abellan et al, 2000b; Varga et al, 2002; Wirtshafter & Sheppard, 2001)

have been shown to be located in the raphe on serotonergic neurons. Interestingly,

GABAB receptors (Varga et al, 2002) as well as 5HT2C and 5-HT1A receptors (Serrats

et al, 2005) have also been localized in the raphe, on GABA interneurons which

synapse with serotonergic neurons. Previous experiments have reported an increase in

5HT levels due to SSRI administration, which can be further augmented by 5HT2C

and 5HT1A receptor antagonists (Cremers et al, 2004; Cremers et al, 2000; Hjorth et

al, 1997; Hjorth et al, 1996; Gartside et al, 1997a, b; Dawson et al, 2002; Bosker et al,

Chapter 8

208

1997). Also, the activation of GABAB receptors has been shown to hyperpolarize 5-

HT neurons (Innis & Aghajanian, 1987; Colmers & Williams, 1988; Innis et al, 1988).

It seems plausible that there may be a negative feedback loop in the raphe involving

5HT receptors located on GABAergic neurons and GABA receptors on serotonergic

neurons.

Emerging preclinical and clinical data implicate GABAergic dysfunction in

the pathophysiology of mood disorders such as anxiety and depression (Cryan &

Kaupmann, 2005). In a number of preclinical trials, GABAB antagonists were shown

to have antidepressant effects in the forced swim test (Slattery et al, 2005;

Mombereau et al, 2004a, b; Nakagawa et al, 1996; Borsini et al, 1986), learned

helplessness model (Nakagawa et al, 1996) and Morris water maze (Nakagawa &

Takashima, 1997). Similarly, the use of GABAB knockout mice in preclinical studies

have reinforced the involvement of these receptors in the etiology of anxiety and

depression (Mombereau et al, 2004; Kaupmann et al, 2003).

Following reports that there were 5-HT2C and 5-HT1A receptors localized on

GABAergic neurons in the raphe (Serrats et al, 2005; Serrats et al, 2003), and that

GABAB antagonists augment the SSRI-induced increase in extracellular 5-HT

(Cremers et al, 2006 in print), it was decided to examine the effects of SSRIs in the

presence of GABAB antagonists. Using microdialysis coupled to HPLC, 5-HT and

GABA levels from raphe nuclei, prefrontal cortex and hippocampus were monitored,

while animals were challenged with citalopram during systemic and local

administration of a number of GABAB antagonists. The effects of the combination

were also examined in the gerbil tail suspension test, to evaluate the effects in an

animal model of depression.


209

Materials and Methods

Animals

Neurochemical Studies

Male albino rats of a Wistar-derived strain (300-350 g, Harlan, Zeist, The

Netherlands) were used for the experiments. Animals were maintained on a standard

12-hour light/dark cycle at 24oC and allowed free access to food and water at all

times. After surgery, rats were housed individually in plastic cages (35 x 35 x 40 cm),

and had free access to food and water. Animals were kept on a 12 h light schedule

(light on 7:00 a.m.).

Behavioural Studies

Experimentally naïve male Mongolian gerbils (Morionsr unguiculatus, 60-80 g,

Charles River, Germany) were used for all behavioural studies. Gerbils (n = at least 8/

group) were housed four per cage, with food and water available ad libitum, in

temperature and humidity controlled holding rooms. The animals were allowed 4-7

days to acclimatise to housing conditions prior to testing. All testing were conducted

during the light phase of the light/dark cycle (lights on: 0600-18:00 h). Experiments

were carried out in accordance with the ethical rules of the Danish Committee on

Care and Use of Laboratory Animals.

Materials

Phaclofen, 2 hydroxysaclofen, CGP 46381, SCH 50911 and Citalopram

hydrobromide were kindly donated by Lundbeck A.S., Denmark. For subcutaneous

injections, drugs were dissolved in saline solution, while the drugs were dissolved in

Ringer solution for infusion regimes.

Chapter 8

210

Neurochemical Procedures

Surgery

Microdialysis of extracellular serotonin levels was performed using I-shaped

microdialysis probes with a polyacrylonitrile/ sodium methyl sulfonate copolymer

dialysis fibre (Brainlink, Groningen, the Netherlands). The dialysis probe was

stereotactically implanted under the following conditions; isofluorane 2%: N2O 300

mL/ min: O2 300 mL/ min. Microdialysis probes were implanted at AP: -0.53, ML:

+0.48, VD: -0.80 for hippocampus (4 mm dialysing membrane), AP: +0.33, ML

+0.08, VD: -0.60 for medial prefrontal cortex (3 mm dialysing membrane) and Intra

Aural: +0.12, ML: +0.14, VD: -0.90 (4 mm dialysing membrane) at a 10o angle for

raphe nuclei; according to coordinates from bregma (Paxinos et al, 1985). The

microdialysis probes were permanently fixed to the skull using stainless steel screws

and methylacrylic cement. Animals were allowed to recover 18-24 hours before

microdialysis experiments commenced.

Microdialysis Experiments

Rats were allowed to recover for at least 24 h. Probes were perfused with artificial

cerebrospinal fluid containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.0

mM MgCl2, at a flow-rate of 1.5 µl / min by TSE Univentor 802 syringe pump

(Technical and Scientific Equipment (TSE), Bad Homburg, Germany). Microdialysis

samples were collected every 15 minutes in HPLC vials containing 32.5 µL of 0.02

M acetic acid for serotonin and 7.5 µL for GABA analysis. The collected samples

were stored in a freezer at -80 oC.

5-HT analysis

Serotonin concentrations were determined using HPLC coupled with electrochemical

detection. 20µL of microdialysate fractions were injected via an autoinjector (Gilson

223 XL, The Netherlands) onto a 100 ± 2.0 mm C18 Hypersil 3 µm column (Bester,

Amstelveen, the Netherlands) and separated with a mobile phase consisting of 4.1 g/L

sodium acetate, 500 mg/L Na2-EDTA, 50 mg/L heptane sulphonic acid, 4.5 %


211

methanol v/v, and 30 µL/L of triethylamine, pH 4.75 at a flow rate of 0.4 mL/min

(Shimadzu LC-10 AD). 5-HT was detected amperometrically at a glassy carbon

electrode at 500 mV vs Ag/AgCl (Antec Leyden, Leiden, Netherlands). The detection

limit was 0.5 fmol 5-HT per 20 µL sample (signal-to-noise ratio 3).

GABA analysis

GABA concentrations in the dialysates were determined off-line by pre-column

derivatization with o-phtaldialdehyde/mercaptoethanol reagent, and separation by

reverse-phase HPLC on a Supercosil LC-18-DB column as previously described (Rea

et al, 2005). Samples were derivatized as follows, based on the derivitisation by

Lindroth and Mopper (1979). 100 mg o-phtaldialdehyde was dissolved in 2 mL

methanol and added to 200 mL 0.5 mol/L NaHCO3 (pH adjusted to 9.5 with NaOH)

containing 20 µL 2-mercaptoethanol. The reagent was freshly prepared daily.

30 µL microdialysate samples were derivatized with 50 µL o-phtaldialdehyde/

mercaptoethanol reagent, mixed, and allowed to react for two minutes. 50 µL of the

reaction mixture was then injected by a Gilson 231 XL sampling injector (Gilson,

Villiers Le Bel, France) onto the HPLC apparatus. The mobile phase consisted of

30% methanol, 70mM di-sodium hydrogen phosphate, 400µM EDTA and 0.15%

tetrahydrofuran, and orthophosphoric acid was added dropwise until a pH of 5.25 was

obtained. Fluorescent detection was performed off-column using a JASCO FP-1520

detector (excitation λ = 350nm, emission λ = 450nm).

Behavioural Procedures

Tail Suspension Test

The tail suspension procedure was a modified version of that validated for NMRI

mice by Steru and colleagues (Porsolt et al, 1987; Steru et al, 1987). Animals were

transported a short distance from the holding facility to the testing room and left

undisturbed for at least 30 mins. Subjects were randomly allocated to treatment

conditions and tested in counterbalanced order. Thirty minutes after injection, gerbils

were suspended by the tail with the use of adhesive tape with one end of the tape

Chapter 8

212

wrapped around the animal’s tail (approx 2 cm from the tip of the tail) and the other

pushed through a hook which was connected to a PC. During a 6 min test session,

immobility time was recorded via an automated system. A reduction in immobility is

considered to reflect an antidepressant-like response.

Expression of results and statistics

The neurochemical data are expressed as area under the curve (AUC). The average

concentration of five stable baseline samples was set at 100%. Statistic analysis was

performed using one way ANOVA followed by Dunnet’s t-test for parametric data.

For the Tail Suspension Test, dose-response studies were analysed by one

factor ANOVA followed by Dunnett’s t-tests, or two factor ANOVA followed by

Student Newmann Keuls post hoc test.


213

Results

Baseline levels in dialysates from hippocampus (n = 94), prefrontal cortex (n = 31)

and raphe nuclei (n = 24) samples were determined as 7.71 ± 0.67 fmol/ sample, 5.72

± 0.82 fmol/ sample and 37.84 ± 2.51 fmol/ sample for 5-HT; and 318.78 ± 26.77 and

337.08 ± 17.18 fmol/ sample GABA for hippocampus, and raphe nuclei respectively.

Basal dialysates were not corrected for in vitro recovery. The average concentration

of five stable baseline samples was set at 100%, and all data were corrected

correspondingly. The area under the curve for all treatments was then determined and

statistics performed on these results.

The effect of citalopram during GABAB antagonist challenges on 5-HT in ventral

hippocampus and raphe nuclei

The systemic administration of citalopram (3.0 mg/kg) increased basal output by

600% in ventral hippocampus. The systemic co-administration of 3.0 mg/kg

citalopram with varying doses of the GABAB antagonist phaclofen and 2-hydroxy

saclofen (2.0 mg/kg) (Fig. 1) augmented the increase in extracellular 5-HT (F(5, 42)

= 5.676, p < 0.001). Post hoc analysis determined that the systemic application of 2.0

mg/kg phaclofen in combination with citalopram significantly augmented the

extracellular increase in 5-HT in hippocampus (p = 0.03), while the other doses of

phaclofen had no significant effect on the output of 5-HT. 2-hydroxysaclofen was

also shown to augment the effect of citalopram on extracellular 5-HT (p = 0.018).

Chapter 8

214

Figure 1 The effect of varying doses of phaclofen, and 2-hydroxy saclofen, on the

citalopram-induced increase in extracellular 5-HT, in hippocampal dialysates. Results

are expressed as mean ± SEM (n = 5-12). * represents a significant difference as

compared to citalopram + vehicle controls (p < 0.05).

The effects of the acute systemic administration of citalopram on extracellular 5-HT

in hippocampal dialysates was also augmented when co-administrated with

CGP46381 (F(3, 33) = 9.823, p < 0.001) (Fig. 2). Post hoc analysis determined that

the effect of citalopram was significantly augmented by the systemic co-

administration of 0.5 mg/kg, and 2.0 mg/kg CGP 46381 (p = 0.001, and 0.012

respectively) while the augmentation was not apparent at higher concentrations (10.0

mg/kg) of CGP46381 (p = 0.961).

Fig. 1Hippocampus

Veh/ Veh

Cit + Veh

Cit + 0.2 Phac

Cit + 2.0 Phac

Cit + 10.0 Phac

Cit + 20.0 Phac

Cit + 2-OH Sac

0

20000

40000

60000

80000

100000

120000*

*

% AUC (5-HT)


215

Figure 2 The effect of varying doses of CGP 46381, on the citalopram-induced

increase in extracellular 5-HT, in hippocampal dialysates. Results are expressed as

mean ± SEM (n = 5-12). * represents a significant difference as compared to

citalopram + vehicle controls (p < 0.05).

Similarly, the effect of citalopram on 5-HT was augmented in a bell-shaped dose

response by systemic SCH50911 administration (F(3, 33) = 2.835, p = 0.055) (Fig.

3). The lower dose (2.0 mg/kg), and the higher dose (30.0 mg/kg) had no significant

effect (p = 0.939, and 0.894 respectively) on the citalopram-induced increase in 5-

HT, while a dose of 10.0 mg/kg resulted in a significant augmentation of the response

(p = 0.023).

Fig. 2Hippocampus

Veh/ Veh

Cit + Veh

Cit + 0.5 CGP

Cit + 2.0 CGP

Cit + 10.0 CGP

0

20000

40000

60000

80000

100000

120000

* *

% AUC (5-HT)

Chapter 8

216

Figure 3 The effect of varying doses of SCH 50911, on the citalopram-induced

increase in extracellular 5-HT, in hippocampal dialysates. Results are expressed as

mean ± SEM (n = 5-12). * represents a significant difference as compared to

citalopram + vehicle controls (p < 0.05).

The effect of citalopram on 5-HT during phaclofen co-administration was also

examined in raphe nuclei (F(2, 15) = 3.173, p = 0.075) (Fig. 4). It was determined

that the systemic administration of 2.0 mg/kg phaclofen significantly augmented the

citalopram effect (p = 0.011), while 10.0 mg/kg phaclofen displayed no significant

difference from the effect seen with citalopram administration alone (p = 0.676). The

administration of phaclofen, 2-hydroxy saclofen, CGP 46381, or SCH 50911 alone

had no significant effect on basal 5-HT levels as compared to vehicle treated animals

(data not shown).

Fig. 3Hippocampus

Veh/ Veh

Cit + Veh

Cit + 2.0 SCH

Cit + 10.0 SCH

Cit + 30.0 SCH

0

20000

40000

60000

80000

100000

120000

*

% AUC (5-HT)


217

Figure 4 The effect of systemic co-administration of 2.0 mg/kg, and 20.0 mg/kg

phaclofen, on the citalopram-induced increase in extracellular 5-HT, in dialysates

from the raphe nuclei. Results are expressed as mean ± SEM (n = 5-8). * represents a

significant difference as compared to citalopram + vehicle controls (p < 0.05).

The local infusion of phaclofen (50µM) (p = 0.017) and CGP 46381 (1µM) (p =

0.002) also augmented the increase in hippocampal 5-HT as compared to the

systemic administration of citalopram (F(2, 26) = 23.008, p < 0.001) (Fig. 5).

Fig. 4Raphe Nucleus

Veh/ Veh

Cit + Veh

Cit + 2.0 PHAC

Cit + 20.0 PHAC

0

20000

40000

60000

80000

100000

*

% AUC (5-HT)

Chapter 8

218

Figure 5 The effect of local infusion of 50µM phaclofen, and 1.0 µM CGP 46381 on

the citalopram-induced increase in extracellular 5-HT, in hippocampal dialysates.

Results are expressed as mean ± SEM (n = 5-12). * represents a significant difference

as compared to citalopram controls (p < 0.05).

Neither the combination of 2.0 mg/kg or 10.0 mg/kg phaclofen with citalopram

resulted in a significant change in GABA levels (Fig. 6) as compared to citalopram

alone in hippocampus (F(2, 19) = 0.907, p = 0.422) or raphe (F(2, 12) = 0.856, p =

0.454).

Fig. 5Hippocampus

Veh/ Veh

Cit + Veh Inf

Cit + CGP Inf

Cit + PHAC Inf

0

20000

40000

60000

80000

100000

120000

140000

*

*

% AUC (5-HT)


219


phaclofen, with 3.0 mg/kg citalopram on extracellular GABA in dialysates from the

hippocampus, and raphe nuclei. Results are expressed as mean ± SEM (n = 4-7).

The effect of citalopram during phaclofen challenges on 5-HT in prefrontal cortex

The systemic administration of phaclofen at 2.0 mg/kg (p = 0.010) and 20.0 mg/kg (p

= 0.001) significantly augmented the citalopram-induced increase in 5-HT (F(2, 29) =

8.661, p = 0.001). Contrary to results obtained in the ventral hippocampus, the

administration of phaclofen with citalopram in the prefrontal cortex did not display

the same bell-shaped dose dependency (Figure 7). Again, the administration of

phaclofen alone did not significantly alter basal 5-HT levels as compared to vehicle

administration (data not shown).

Fig. 6 Raphe and Hippocampus

RN - Cit + Veh

RN - Cit + 2.0 PHAC

RN - Cit + 10.0 PHAC

HC - Cit + Veh

HC - Cit + 2.0 PHAC

HC - Cit + 10.0 PHAC

0

20000

40000

% AUC (GABA)

Chapter 8

220


phaclofen, on the citalopram-induced increase in extracellular 5-HT, in dialysates

from the prefrontal cortex. Results are expressed as mean ± SEM (n = 5-8). *

represents a significant difference as compared to citalopram + vehicle controls (p <

0.05).

The effect of the co-administration of phaclofen with citalopram in the Gerbil Tail

Suspension Test

A dose response curve was first constructed (Fig. 8) to determine which

concentration of citalopram should be used in the study to allow for the detection of

an antidepressant effect. While the oral doses of 1.0, 2.0, 4.0 and 16.0 mg/kg

citalopram were significantly different from vehicle dose (F(5, 47) = 10.004, p <

0.01), it was decided to use the 0.5 mg/kg citalopram dose. Despite the fact that it did

not produce a significant decrease in antidepressant response (p = 0.445), there was

Fig. 7Prefrontal cortex

Veh/ Veh

Cit + Veh

Cit + 2.0 PHAC

Cit + 20.0 PHAC

0

10000

20000

30000

40000

50000

60000

70000

80000 **

% AUC (5-HT)


221

an inclination towards a decrease in immobility, and hence towards an antidepressant-

like response. This dose allowed for an easier detection of a potentiation in the

antidepressant effect of citalopram.

Figure 8 The effect of various orally administered doses of citalopram on immobility

in the gerbil tail suspension test. Results are expressed as mean ± SEM (n = 8). *

represents a significant difference as compared to vehicle controls (p < 0.05).

Fig. 8

Vehicle

0.5 mg/kg p.o.

1.0 mg/kg p.o.

2.0 mg/kg p.o.

4.0 mg/kg p.o.

16.0 mg/kg p.o.

0

50

100

150

200

250

**

*

*

Immobility time (sec)

Chapter 8

222

Using this citalopram dose (0.5 mg/kg p.o.), a study was performed investigating the

effect of oral phaclofen administration on immobility (2.5, 5.0 and 10.0 mg/kg).

There was an overall effect of citalopram treated groups as compared to vehicle

treated groups (F(1, 63) = 17.084, p < 0.001), while the administration of phaclofen

had no significant effect at any of the oral doses tested (2.5, 5.0 and 10.0 mg/kg) as

compared to vehicle (F(3, 63) = 0.807, p = 0.496). There was no significant decrease

in immobility with the combination of the two treatments as compared to their

respective controls (F(3, 63) = 1.742, p = 0.169), except when 2.5 mg/kg phaclofen

was co-administered with 0.5 mg/kg citalopram (p = 0.005) (Fig. 9).

Figure 9 The effect oral phaclofen administration alone, and in combination with 0.5

mg/kg citalopram, on immobility in the gerbil tail suspension test. Results are

expressed as mean ± SEM (n = 8). * represents a significant difference as compared

to control (p < 0.05).

Fig. 9

Veh/ Veh

Veh + 0.5 Cit

2.5 Phac + 0.5 Cit

5.0 Phac + 0.5 Cit

10.0 Phac + 0.5 Cit

0

50

100

150

200

250

*

Immobility time (sec)


223

Discussion

Recently, it has been reported that 5-HT2C antagonists are capable of augmenting the

biochemical and behavioral effects of SSRIs (Cremers et al, 2004). There is also

evidence supporting the localization of 5-HT2C and 5-HT1A receptors on GABAergic

neurons (Serrats et al, 2003; Eberle-Wang et al, 1997), and that these serotonergic

receptors activate local GABA inhibitory circuits (Liu et al, 2000) which impact on

GABAB receptors located on raphe serotonergic neurons (Serrats et al, 2003; Abellan

et al, 2000; Varga et al, 2002; Wirtshafter & Sheppard, 2001). Activation of GABAB

receptors has also been shown to inhibit 5-HT levels in terminal areas (Tao &

Auerbach, 2000). While investigating the mechanisms involved in the augmentation

of the citalopram-induced increase in 5-HT by 5-HT2C antagonists, we observed an

increase in hippocampal 5-HT when phaclofen was locally infused prior to systemic

citalopram administration. However, to date no GABAB receptors have been

localized to serotonergic neuron terminals. The increase in serotonin was similar in

magnitude to the augmentation seen with co-administration of 5-HT1A (Hjorth et al,

1997) and 5-HT2C (Cremers et al, 2004) antagonists with citalopram. This

augmentation was also demonstrated when the GABAB antagonist 2-hydroxy

saclofen was systemically co-administered with citalopram. Using microdialysis

coupled to HPLC, we examined the effect of citalopram on hippocampal 5-HT when

systemically challenged with a series of doses of phaclofen. Interestingly we

observed that the combination strategy did not display a simple dose response curve

but a bell-shaped response. Similarly, this bell-shaped dose response curve was

observed in hippocampus when citalopram was challenged with the more selective

GABAB antagonists, CGP43681, and SCH50911. This effect was again seen in the

raphe when phaclofen was co-administered with citalopram. However, when we

examined GABA levels, there was no significant attenuation in extracellular GABA

levels in raphe or hippocampal dialysate samples as compared to citalopram controls.

In this study we show that the local infusion of phaclofen augments the citalopram-

induced increase in 5-HT in hippocampus. In an earlier study (Rea et al, 2005), we

showed that the local infusions of GABAB agonists and antagonists decreased, and

Chapter 8

224

increased GABA levels respectively. However, the infusion of these compounds had

no effect on 5-HT levels at the concentrations infused (data not shown). It is possible

that the local infusion of these GABAB compounds influences the GABA tonus of the

serotonergic neuron, and facilitates a further augmentation of the effects of SSRIs

without directly affecting the basal release of 5-HT.

Interestingly, the bell-shaped effect on 5-HT with the combination of an SSRI

with a GABAB antagonist was not apparent in the prefrontal cortex. An augmentation

in the citalopram-induced increase in 5-HT was still observed with the co-

administration of 20.0 mg/kg phaclofen s.c. This may be due to a difference in

GABA receptor density or function (Amantea et al, 2004), or possibly due to the

interaction of phaclofen on GABAB receptors on cortical neurons other than

serotonergic or GABAergic neurons (Santiago et al, 1993).

We also show evidence for a bell-shaped dose response phenomenon in an

animal model for depression. It was determined in the gerbil tail suspension test that

the administration of a dose of 2.0 mg/kg phaclofen with citalopram was significantly

different to the amount of time spent immobile as compared to citalopram alone. This

effect was not seen when citalopram was co-administered with higher (5.0 or 10.0

mg/kg) doses. The tail suspension test is classically used for the discrimination of

antidepressant compounds. While SCH50911 showed a tendency for a bell shaped

response in combination with citalopram, the results were not significant (data not

shown). The compound CGP43681 showed little or no response alone, or in

combination with citalopram (data not shown). The doses of citalopram used in this

study were lower than those of the microdialysis experiments, as the lower doses of

citalopram sufficiently decreased immobility (Varty et al, 2003), as compared to

controls, and it would still be possible to observe a shift in the dose response curve, or

a further augmentation of the immobility response. Although a bell-shaped dose

response change in behaviour was observed with the combination studies, it is

possible that a more pronounced effect would be observed at higher doses of

citalopram in combination with the GABAB antagonists.

Similarly, in agreement with the current data, no significant antidepressant

effects were observed in the tail suspension test when GABAB antagonists were


225

administered alone (Cryan et al, 2004; Cryan et al, 2005; Mombereau et al, 2004). It

is of interest however, that administration of GABAB antagonists were found to have

antidepressant-like qualities in a number of other animal models of depression

including the forced swim test (Mombereau et al, 2004; Nakagawa et al, 1996;

Slattery et al, 2005; Borsini et al, 1986) and learned helplessness models (Nakagawa

et al, 1996; Nakagawa et al, 1997). It is also interesting that when co-administered

with the GABAB agonist, baclofen, the effects of antidepressants are blocked in

certain animal models of depression (Nakagawa et al, 1996; Nakagawa et al, 1997).

This however may be due to the sedative nature of baclofen. While there appears to

be a role for GABAB receptors in mediating an antidepressant response in many of

these animal models, the mechanism behind how the GABAB receptor is mediating

its effect is unclear.

It is possible that in the presence of an SSRI, the resulting surplus of serotonin

activates 5-HT1A and/or 5-HT2C receptors localized on GABAergic cells. Indeed, this

was shown to be the case with 5-HT2C antagonism significantly impacting on GABA

release in the presence of citalopram (Cremers et al, 2006 in print). It seems plausible

that the administration of citalopram may increase the release of GABA, which in

turn could activate GABAB receptors located on serotonergic neurons. The resulting

decrease in serotonergic firing would reduce the amount of serotonin being released

from serotonergic terminals. The supposition is that by co-administering a 5-HT2C or

5-HT1A antagonist, or GABAB antagonist, we are preventing the effect of GABA on

serotonergic firing either indirectly by preventing GABA release, or directly by

GABAB receptor antagonism. This would lead to an increase in 5-HT release due to

disinhibition, and antagonism of effects of GABA on serotonergic neurons,

respectively. Despite the fact that these proposed augmentations of GABA effects

were not observed in the present study, it is possible that these effects are being

mediated at sites in other brain areas outside the vicinity of the microdialysis probe. It

is likely that, when co-administered with citalopram, the homeostatic balance

between the GABA and serotonergic neurons becomes compromised, and the effects

of the GABAB antagonist become more pronounced. It should be noted however, that

with GABAB antagonist administration, there will be antagonism at both presynaptic

Chapter 8

226

GABAB receptors on GABAergic neurons and postsynaptic GABAB receptors on

serotonergic neurons. It has been implied that some GABAB agonists and antagonists

express a preference for either the pre- or post-synaptic receptor due to differences in

the heterodimer composition of the receptors (Phelan, 1999; Yamada et al, 1999;

Pozza et al, 1999).

The GABAB receptor has recently been shown to be an allosterically

modulated G-protein receptor (Urwyler et al, 2001) and although the compounds

used in this study have not been tested for allosteric modulating activity, a possible

explanation for the bell-shaped dose response phenomenon is that in excess amounts,

GABAB antagonists bind to other allosteric sites on the GABAB receptor resulting in

conformational changes which prevents the binding of the antagonist. A recent theory

is that the GABAB heterodimer undergoes a conformational change (Venus Fly Trap

model) when bound by endogenous GABA which allows the allosteric modulation of

response by affecting the stability of the GABA-GABAB receptor complex (Pin et al,

2004).

The current data suggest that the GABAergic and 5-HTergic systems are

involved in regulating the levels of their respective systems, and perhaps by

manipulating certain receptors we can provide novel therapeutic targets. However,

given indications for bell-shaped dose dependency, the manipulation of GABAB

receptors may compromise its use in the clinic.


227

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Documents

University of Groningen Augmentation of the …GABA B receptors (Varga et al, 2002) as well as 5HT 2C and 5-HT 1A receptors (Serrats et al, 2005) have also been localized in the raphe,