A possible inhibitory action of baclofen on hippocampus GABAergic neurones

Gen. Pharmac. Vol. 25, No. 2, pp. 297-301, 1994

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A Possible Inhibitory Action of Baclofen on Hippocampus GABAergic Neurones G. N. B A L E R I O , * M. E. O T E R O L O S A D A a n d M. C. R U B I O Instituto de Investigaciones Farmacol6gicas, CONICET, C6tedra de Farmacologia, Fac. de Farmacia y Bioquimica (UBA), Junin 956, 5 Piso, (1113% Buenos Aires, Argentina lFax 54-1-963-8593] (Received 8 July 1993)

Abstract--1.The function of the ~,-aminobutyricacid (GABA)ergicsystemin certain areas of the rat brain was investigated after baclofen treatment (30 mg/kg for 4 days). 2. Two h after the last dose of baclofen GAD activity was reduced in the hippocampus without changes in GABA levels. 24 h after baclofen, GAD activity was increased and the GABA content was decreased. 3. 48 h after the last dose both parameters returned to control values, 4. These results were not observed in any of the other areas investigated:frontal cerebral cortex, corpus striatum, olfactory bulbs, and medio basal hypothalamus. 5. In conclusion, the present study shows that baclofen 30 mg/kg for 4 days, induces an inhibitory action on hippocampus GABAergic neurones, which begins to disappear after 24 h free of drug. Key Words: Baclofen, GABA, hippocampus

INTRODUCTION 7-Aminobutyric acid (GABA) is the major inhibitory transmitter substance in the central nervous system and it is generally accepted that it acts on two types of receptors (Bowery, 1988). The GABA-A receptor, which is blocked by bicuculline, and activated by muscimol, is linked to the chloride channel and is responsible for postsynaptic inhibition. The GABA-B receptor is not blocked by bicuculline and is activated by baclofen (BAC). This drug, a lipophilic analogue of y-amino-butyric acid (GABA), is an effective antispastic drug (Bein, 1972; Fehr and Bein, 1974). Although its clinical use, the neuropharmacological actions of baclofen have not been well known. There has been some information available about the interaction of GABAergic system with another neurotransmitter in the brain and the role of B receptors mediated actions (Da Prada and Keller, 1976; Bowery et al. 1980; Otero Losada, 1987). It is clear that many GABA-B receptors are located on presynaptic nerve terminals and their activation inhibits transmitter release. There are several reports of inhibitory effects of BAC on the release of the classic neurotransmitters, noradrenaline (NA) (Bowery and Hudson, 1979), *To whom all correspondence should be addressed.

dopamine (DA) and serotonin (5-HT) (Curtis et al. 1974; Gray and Green, 1987). Earlier experiments indicate that B A C selectively inhibits excitatory transmission to the motoneurones, leaving the inhibitory transmission intact (Pierau and Zimmerman, 1973; Fukuda et al., 1977; Kato et al., 1978; Davies, 1981). On the other hand, BAC reduces the excitatory and the inhibitory potentials evoked in neurones of the hippocampal and cortical slices (Scholfield, 1983; Blaxter and Carlen, 1985; Inoue et al., 1985; Howe et al., 1987) as well as the inhibitory potencial in cultured hippocampal neurones (Harrison et al., 1988) and inhibits the release of GABA due to activation of presynaptic GABA-B autoreceptors localized on GABAergic terminals (Bonanno et aL, 1989). In the present study, we have attempted to investigate the effects of BAC on the release of endogenous GABA content and on the activity of its metabolic enzyme (GAD), from certain brain areas, as markers of GABAergic function.

MATERIALSANDMETHODS Male Wistar rats (150-180 g) were pretreated with BAC (30 mg/kg i.p. daily for 4 days) and were killed by decapitation 2 h, 24 h or 48 h after the last dose of the drug, The following areas were dissected: frontal

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G.N. BALERIOet al.

cerebral cortex (FC), corpus striatum (CS), olfactory bulbs (OB), medio basal hypothalamus (HYP) and hippocampus (HC). The corresponding control groups were pretreated with vehicle tween 80 (10%). Before and during the treatment the animals were allowed food and water ad libitum. G A B A concentration

GABA was measured by the method of Lindgren et al. (1982). In order to prevent postmortem increase

of GABA in the brain, the rats were injected with the G A D inhibitor 3-mercaptopropionic acid (dissolved in 0.01 N NaOH; l mmol/kg, i.p.) 2.5min before killing (Van der Heyden and Korf, 1978). Extraction o f GA BA

The tissues were weighed and homogenized in 5 ml icecold 0.4 N HCIO4 containing Na2 SO3 (0.05%) and Na2 EDTA (0.1%). After centrifugation (10.000g, 10 min 4°C) the supernatants were adjusted to pH 3.0 using 5 N K 2 CO3 and centrifuged again 10.000 g, I0 min 4°C). The supernatants were passed through a column (75 mm length, 4.0 mm diameter) containing the strong cation exchange resin Dowex 50 W × 4 (200-400 mesh), which had previously been washed with 20 ml 0.1 M phosphate buffer, pH 6.5 containing 0.1% Na2 EDTA. The column was then treated as follows: 10ml water, 10ml 0.02 M citrate buffer, pH 4.0; 4 ml 0.05 M citrate buffer, pH 5.2; (fraction for GABA assay); 4 ml 0.05 M citrate buffer, pH 5.2 (fraction for tissue blank). Recovery was calculated by addition of a known amount of [3H]GABA to each sample at the beginning of the extraction procedure and by measuring the radioactivity in aliquots of the eluates obtained from the columns. Spectrofluorometric detection

The reaction mixture contained: 0.05 M sodium citrate buffer, pH 5.2, 0.3 ml; aliquot of the eluate from the column, 0.5 ml; 0.5 M sodium borate buffer, pH 9.3, 0.4ml; 2-mercaptoethanol (solution ! pl/ml of 95% ethanol), 0.1 ml; distilled water, 0.1 ml). The reaction was started by adding 30 #1 O-phthalaldehyde (1 mg/ml of 95% ethanol). Fluorescence was read within 30min at 335/455 nm wavelength, and was linear from 0.5 to 4/~g of GABA per test tube. Glutamate decarboxilase assay (GAD)

This was performed according to Albers and Brady (1959). Aliquots of 100#1 tissue homogenate were incubated in glass tubes for 60min at 37°C in the presence of: (concentration in mmol/I) potassium phosphate buffer (pH 6.4) 50, 2°mercaptoethanol 10, substrate DL-[I ~4C]glutamic acid (sp. act.

58 mCi/nmol) 100 (saturating concentration); o-penicillamine 10 nmol/l was added in the case of blanks. The final volume in each tube was 0.5 ml. In order to trap the L4CO2 formed, a plastic well was suspended from a rubber injection stopper containing folded paper soaked in 0.2 ml Protosol (New England Nuclear). The reaction was stopped by the injection of 0.5 ml 10% trichloroacetic acid and after 1 h of continuous shaking the plastic wells were removed, wiped with absorbent tissue, and placed in 5 ml of scintillation fluid for radioactivity counting. In this assay the cofactor pyridoxal-5-phosphate was exogenously added (1 mmol/l). Results are expressed as the mean + SEM and were analysed according to Student's t-test (Snedecor and Cochran, 1967). The following drugs were used: protosol (New England Nuclear; NEN), 7-2,3-[3H]aminobutiric acid (sp. act. 29.3Ci/mmol, NEN), O-phthaldehyde (Sigma), DL-[1--14C]glutamic acid (sp. act. 58 mCi/ mmol, Amersham), 3-mercaptopropionic acid (Sigma), D-penicillamine (Sigma). RESULTS In striatum, olfactory bulb, frontal cortex and hypothalamus, the chronic treatment with BAC failed to alter G A D activity [Fig.l(A)]. Similar result was obtained on GABA contents 2 h after last dose [Fig. I(B)]. The endogenous GABA levels or the G A D activity in this areas were not modified 24 h [Fig. 2(A, B)] and 48 h [Fig. 3(A, B)] after the last dose of BAC. However, 2 h after BAC in hippocampus the G A D activity [Fig. I(A)] was significantly decreased ( - 50%). Conversely, 24 h after the last dose of BAC the G A D activity [Fig. 2(A)] was increased and the GABA content [Fig. 2(B)] was decreased (37 and 35%, respectively). Finally, 48 h after BAC in hippocampus the G A D activity [Fig. 3(A)] and GABA endogenous levels [Fig. 3(B)] returned to control values. DISCUSSION The present study shows that the GABAergic system is affected by chronic treatment with BAC (30mg/kg/day, 4 days) only in the hippocampus (HC) and no differences could be detected in the other studied areas. After 2 h of this treatment decreased G A D activity in the HC; while GABA concentration was similar in both control and treated animals. This change in G A D activity should be compatible with a reduction in GABAergic neurone activity, mediated by an inhibitory action of BAC on presynaptic GABA-B receptors. These inhibitory receptors have been found

Baclofen

in central including

neurones dopamine

tryptamine

utilizing

several

and GABAergic

central

system

299

transmitters,

(Bowery et al., 1980) 5-hydroxy-

(5-HT, Gray and Green,

1987) and chole-

cystokinin (Conzelmann et al., 1986). However, in the case of GABAergic neurones themselves, the acceptance of presynaptic inhibitory autoreceptors has been poor. In several studies, muscimol was found to decrease the K-evoked release of [3H]-GABA from brain slices or synaptosomes suggesting the presence of GABA-A

autoreceptors

on GABAergic

nerve

terminals (Mitchell and Martin, 1978; Brennan er al., 1981; Kuriyama et al., 1984). However, in similar experiments, other authors have found that BAC, but not muscimol, decreased GABA release, suggesting

800

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the presence of GABA-B autoreceptors (Anderson and Mitchell, 1985; Limberger et al., 1986; Pittaluga et al., 1987; Waldmeier et al., 1988). On the other hand, BAC reduces the excitatory

2

potentials evoked in neurones and cortical slices (Scholfield,

and

of the 1983;

Blaxter and Carlen, 1985; Inoue et al., 1985, Howe et al., 1987) as well as the inhibitory potential in cultured

hippocampal

neurones

(Harrison

et al.,

1988).

(A)

1200

FC

OB

HC

HYP

FC

400

200

2 0

cs

Fig. 2. Effect of baclofen (30 mg/kg i.p. daily for 4 days) 24 h after the last dose on: (A) GAD activity which was measured under saturating conditions (substrate glutamic acid 100 mmol/l). Results are expressed in pmol/g tissue/h. Mean values + SEM of 7-10 experiments are shown. Control 0 and Baclofen 0 *p < 0.05 compared with control. (B) GABA concentration. Mean values f SEM of 8-9 animals per group are shown. CS: corpus striatum, OB: olfactory bulbs, HC: hippocampus, HYP: hypothalamus, FC: frontal cerebral cortex. Control 0 and Baclofen 0 *p < 0.05 compared with control.

studies

indicate

the

presence

of

two

distinct types of GABA-B recephippocampus. The postsynaptic

GABA-B receptors mediating polarization, are coupled to

membrane hyperK+ channels and

blocked by the GABA-B antagonist phaclofen, whereas the presynaptic GABA-B receptors responsible for the depression of synaptic potentials, appear to be coupled to different ionic channels

(B)

3 =, .3 _

FC

600

Recent

HYP

HYP

W

pharmacologically tors in the rat HC

HC

2

.3 -

k 0 M $y

the inhibitory hippocampal

OB

900

and are insensitive

to phaclofen

(Dutar

and Nicoll,

1988). To determine if our effect was transient, we evaluated the BAC actions 24 h after the last dose of BAC.

”

CS

OB

HC

HYP

FC

Fig. I. Effect of baclofen (30 mg/kg i.p. daily for 4 days) 2 h after the last dose on: (A) GAD activity which was measured under saturating conditions (substrate glutamic acid 100 mmol/l). Results are expressed in pmol/g tissue/h. Mean values k SEM of 7-10 experiments are shown. Control q and Baclofen IJ. *p i 0.04 compared with control. (B) GABA concentration. Mean values + SEM of 8-9 animals per group are shown. CS: corpus striatum, OB: olfactory bulbs, HC: hippocampus, HYP: hypothalamus, FC: frontal cerebral cortex. Control 0 and Baclofen IJ.

Now GAD activity and GABA levels were modified in the HC. But when we measured these parameters 48 h after the last dose, both had returned to control values in the HC without changes in any of the other areas investigated. We can conclude that after 24 h of drug suppression there exists a compensatory mechanism which increases the GABAergic activity, returning to the basal steady state 48 h after treatment. Our study shows that these changes in the GABAergic system are different according to the area of the brain.

G. N. BALERIO et al.

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Fig. 3. Effect of baclofen (30 mg/kg i.p. daily for 4 days 48 h after the last dose on: (A) G A D activity which was measured under saturating conditions (substrate glutamic acid 100 mmol/1). Results are expressed in #mol/g tissue/h. Mean values + SEM of 7-10 experiments are shown. Control [] and Baclofen [] (B) GABA concentration. Mean values + SEM of 8-9 animals per group are shown. CS: corpus striatum, OB: olfactory bulbs, HC: hippocampus, HYP: hypothalamus, FC: frontal cerebral cortex. Control [] and Baclofen [:3.

T h e s e c h a n g e s m a y be exerted directly u p o n the G A B A e r g i c s y s t e m by B A C b u t an indirect action due to c h a n g e s in the activity o f o t h e r n e u r o n a l systems w h i c h in t u r n affects G A B A e r g i c f u n c t i o n can n o t be ruled out.

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