J Neural Transm (1992) [Suppl] 35: 155-177
`©Springer-Verlag 1992
`
`Experimental absence seizures: potential role of y-hydroxybutyric
`acid and GABA8 receptors
`
`R. Bernasconi\ J. Lauber\ C. Marescaux2 , M. Vergnes3 , P. Martin1,
`V. Rubio\ T. Leonhardt\ N. Reymann1, and H. Bittiger1
`1 Research and Development Department, Pharmaceuticals Division, Ciba-Geigy,
`Basel, Switzerland
`2 Groupe de Recherche de Physiologie Nerveuse, Clinique Neurologique, Hospices
`Civils, and 3 Centre de Neurochimie du CNRS et de l'INSERM, Strasbourg, France
`
`Summary. We have investigated whether the pathogenesis of spontaneous
`generalized non-convulsive seizures in rats with genetic absence epilepsy is
`due to an increase in the brain levels of y-hydroxybutyric acid (GHB) or in
`the rate of its synthesis. Concentrations of GHB or of its precursor y(cid:173)
`butyrolactone (GBL) were measured with a new GC/MS technique which
`allows the simultaneous assessment of GHB and GBL. The rate of GHB
`synthesis was estimated from the increase in GHB levels after inhibition of
`its catabolism with valproate. The results of this study do not indicate
`significant differences in GHB or GBL levels, or in their rates of synthesis
`in rats showing spike-and-wave discharges (SWD) as compared to rats
`without SWD. Binding data indicate that GHB, but not GBL, has a selec(cid:173)
`tive, although weak affinity for GABAs receptors (IC50 = 150 µM). Similar
`IC50 values were observed in membranes prepared from rats showing SWD
`and from control rats. The average GHB brain levels of 2.12 ± 0.23nmol/g
`measured in the cortex and of 4.28 ± 0.90nmol/g in the thalamus are much
`lower than the concentrations necessary to occupy a major part of the
`GABAs receptors. It is unlikely that local accumulations of GHB reach
`concentrations 30-70-fold higher than the average brain levels. After injec(cid:173)
`tion of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain con(cid:173)
`centrations reach 240 ± 31 nmol/g (Snead, 1991) and GHB could thus
`stimulate the GABAs receptor.
`Like the selective and potent GABAs receptor agonist R( - )-baclofen,
`GHB causes a dose-related decrease in cerebellar cGMP. This decrease and
`the increase in SWD caused by R( - )-baclofen were completely blocked by
`the selective and potent GABAs receptor antagonist CGP 35348, whereas
`only the increase in the duration of SWD induced by GHB was totally
`antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited
`by GHB was only partially antagonized by CGP 35348.
`
`Ranbaxy Ex. 1014
`IPR Petition - USP 9,050,302
`
`

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`156
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`R. Bernasconi et al.
`
`These findings suggest that all effects of R( - )-baclofen are mediated by
`the GABAa receptor, whereas only the induction of SWD by GHB is
`dependent on GABAa receptor mediation, the decrease in cGMP being
`only partially so. Taken together with the observations of Marescaux et al.
`(1992), these results indicate that GABAa receptors are of primary import(cid:173)
`ance in experimental absence epilepsy and that GABAa receptor antag(cid:173)
`onists may represent a new class of anti-absence drugs.
`
`1. Introduction
`
`Primary generalized epilepsy of the absence type is a childhood-onset
`seizure disorder of unknown etiology characterized behaviourally by brief
`staring spells and arrest of motor activity, and electrically by generalized
`3 Hz spike-and-wave discharges (SWD) in the electroencephalogram (EEG)
`(Godschalk et al., 1976, 1977; Mirsky et al., 1986). Three Hz SWD are
`associated with enhanced GABA-mediated synaptic inhibition and absence
`epilepsy could conceivably represent generalized inhibitory seizures due to
`an excess, rather than to a deficit of GABA-mediated transmission (Fariello
`and Golden, 1987; Fromm and Kohli, 1972; Gloor and Fariello, 1988).
`Evidence for this premise is based on the fact that direct GABAA and
`GABAa receptor agonists, GABA uptake inhibitors and 4-aminobutyrate:
`2-oxoglutarate aminotransferase (EC 2.6.1.19; GABA-T) inhibitors aug(cid:173)
`ment the number and duration of discharges (King, 1979; Marescaux et al.,
`1984; Micheletti et al., 1985; Smith and Bierkamper, 1990; Snead, 1990;
`Vergnes et al., 1984). The GABA metabolite and/or putative neuro(cid:173)
`transmitter (Vayer et al., 1987) y-hydroxybutyric acid (GHB) or its
`lactonized prodrug y-butyrolactone (GBL), also induces 4-6Hz SWD
`accompanied by arrest of motor activity, with staring, facial myoclonus and
`vibrissal twitches, which mimic the events of absence seizures in rats (Snead
`et al., 1976; Snead, 1988). As the changes in EEG observed after admin(cid:173)
`istration of GHB are not followed by convulsions, GHB-induced seizures
`have been proposed as an animal model of petit mal epilepsy (Godschalk
`et al., 1976, 1977).
`Because of the structural resemblance of GHB to GABA, GHB has also
`been described as a "GABA agonist" (Meldrum, 1981), suggesting that the
`epileptiform discharges caused by GHB may be due to its GABAergic
`activity. In agreement with this hypothesis, Pericic et al. (1978) have shown
`that GHB, like GABAA agonists, does not alter GABA levels, but pro(cid:173)
`duces a marked and dose-related reduction in the rate of GABA synthesis,
`indicating strong interactions between GHB and GABA-mediated inhibit(cid:173)
`ion. In contrast to the action of muscimol, this effect is not secondary to
`a direct effect of GHB on GABAA receptors (Enna and Snyder, 1975).
`Thus, GHB modulates GABA neurotransmission and induces absence-like
`seizures by way of a mechanism which is not mediated through GABAA
`receptors.
`
`

`
`Experimental absence seizures
`
`157
`
`Since exogeneous GHB is capable of inducing absence seizures, the
`question naturally arises whether GHB-mediated mechanisms might play a
`role in the genesis of petit mal epilepsy. One possibility of testing the GHB
`hypothesis of petit mal epilepsy is to assess biochemical parameters related
`to GHB activity in the brain (e.g. GHB levels, its rate of synthesis, GHB
`binding or second messengers) in animals with absence seizures as com(cid:173)
`pared to non-epileptic animals.
`Recently, a genetic model of spontaneous generalized non-convulsive
`seizures has been described (Vergnes et al., 1982), which satisfies most
`of the criteria proposed for a useful animal model of petit mal epilepsy
`(Mirsky et al., 1986). Spontaneous and recurrent SWD were originally seen
`in the EEG of some Wistar rats (Vergnes et al., 1982). By successive
`inbreeding of such rats, a strain in which spontaneous SWD can be recorded
`in 100% of the animals has been selected and named the Genetic Absence
`Epilepsy Rats from Strasbourg (GAERS) (Vergnes et al., 1987). Con(cid:173)
`currently, another strain of rats was selected which never displayed SWD
`(controls). Both the electrographic characteristics and pharmacological
`response of these SWD are reminiscent of petit mal epilepsy in man
`(Vergnes et al., 1982; Micheletti et al., 1985). The GAERS strain thus
`affords a reproducible and pharmacologically specific model for the study
`of biochemical mechanisms involved in spontaneous generalized non(cid:173)
`convulsive seizures (Engel et al., 1990).
`The aim of the present study was to examine the involvement of GHB
`and GBL in such seizures by measuring the endogenous concentration of
`both in hippocampus, thalamus and frontal cortex in GAERS and to com(cid:173)
`pare them with the levels in seizure-free rats. The increase in GHB induced
`by valproate, an index of its rate of synthesis, was also examined in both
`strains. Levels of GHB and GBL were assessed by a new capillary gas
`chromatography-mass spectrometry method with selected-ion monitoring
`(GC-MS) which allows simultaneous measurement of GHB and GBL with
`the necessary sensitivity. As the SWD induced by GHB are antagonized by
`the selective GABA8 receptor antagonist CGP 35348 (Marescaux et al.,
`1992), the interactions of GHB and of its prodrug GBL with 12 neuro(cid:173)
`transmitter receptors and neuromodulator binding sites, in particular those
`controlling GABA-mediated inhibition, were evaluated. In addition, we
`assessed the effects of GBL and of the selective GABA8 receptor agonist
`R( - )-baclofen either alone or in combination with CGP 35348, on cGMP
`levels and we used this paradigm to study the potential interactions between
`GHB and GABA8 receptors in vivo.
`
`2. Material and methods
`
`Animals
`
`Experiments for the development of the GC-MS procedure and for the assessment of
`cGMP were conducted on male Tif: RAIF (SPF) rats (Tierfarm Sisseln, Switzerland)
`
`

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`158
`
`R. Bernasconi et al.
`
`weighing 240-280g. Other experiments on cGMP levels were performed on male
`Tif: MAGf (SPF) mice, 23-27 g body weight, 5-8 weeks of age (Tierfarm Sisseln,
`Switzerland). The animals were kept in an air-conditioned room at 21°C, with a 12 hour
`light-dark cycle and were sacrificed between 8:30 and 10:00 a.m. to avoid circadian
`variations of the different biochemical parameters measured.
`
`Rats with spontaneous absence-like seizures
`
`Male Wistar rats (350-400 g) from the breeding colony at the Centre de Neurochimie,
`C.N.R.S., Strasbourg were used in this study. They were chosen from the 9th genera(cid:173)
`tion of a strain with spontaneous generalized non-convulsive seizures, in which bilateral
`SWD (frequency = 7-9c/sec, amplitude = 300-1,000µV, mean duration = 6.0 ±
`3.4sec with a variance between 0.5 and 40sec, occurrence = 1/min) are observed in
`awake but inactive animals. Controls were also from the 9th generation of a strain
`which never displayed SWD. Epileptic and non-epileptic rats were of the same age.
`They were sacrificed after an acclimatization period of 15 days in Basel.
`
`Chemicals for GC and GC-MS
`
`(DPTMDS, Cat. No 43340), hexa(cid:173)
`1,3-Diphenyl-l,l,3,3-tetramethyldisilazane
`methylsilazane (HMDS, Cat No 52619), acetonitrile, and acetic acid anhydride were
`purchased from Fluka. The internal standard for GHB and GBL, GBL-2,2,3,3,4,4-d6
`(GBL-d6) was from Merck, Sharp & Dohme Ltd, Pointe Claire, Quebec, Canada. The
`stationary phase CP-51 wax was from Chrompack International (Middleburg, The
`Netherlands). All other chemicals and reagents were of analytical-reagent grade and
`were used without purification.
`
`Drugs
`
`GHB (sodium salt) and GBL were purchased from Fluka. [2,3-3H]GHB, potassium salt
`(spec. act. lOOCi/mmol) was prepared by the CEA (Gif-sur-Yvette, France). Valproate
`sodium was synthesized in our laboratories by Dr. H. Allgeier. Drugs were dissolved in
`saline 0.9% such that the volume of injection was 1 ml/kg and were used on the same
`day, if necessary the pH was adjusted to pH = 5 with NaOH lN. We only used
`subanaesthetic (200- 400 mg/kg) doses of GBL, which produce EEG and behavioural
`changes corresponding to stage 1 and 2 of Snead (1988). Doses larger than 400mg/kg
`i.p. are associated with a burst suppression pattern described as stage 3 by Snead
`(1988) .
`
`Sample preparation for GC-MS-analysis
`
`Rats were killed by fast focused microwave irradiation of the head (Piischner GmbH,
`Schwanewede, F.R.G; l.6sec, 7.5kW). The brains were rapidly removed, cooled on
`dry ice and dissected immediately into different brain areas according to the method of
`Glowinski and Iversen (1966). The brain structures were divided into two equal parts
`(left and right). One part of the samples was homogenized for lOrnin at room tempera-
`
`

`
`Experimental absence seizures
`
`159
`
`ture in a ground-glass homogenizer with 2 ml acetonirile containing 20 ng of the the
`internal standard GBL-d6 . Since GHB does not undergo lactonisation under these
`conditions (Vayer et al., 1988; Snead et al., 1989), any GHB present would not be
`lactonized and thus not extracted into the acetonitrile. Therefore, the values obtained
`represent only GBL. The contralateral brain structures were extracted for 10 min at
`room temperature in the ground-glass homogenizer with a solution of 5% acetic
`anhydride in acetonitrile containing 80 ng of GBL-d6• This procedure lactonizes all the
`GHB present in the sample, such that the value obtained represents GHB plus GBL.
`Hence, by subtracting the value obtained from the pure acetonitrile extract, it is
`possible to determine the concentration of GHB. The acetonitrile solutions were
`allowed to stand for 1 hr at room temperature and were centrifuged at 10,000 g for 1 hr
`at 4°C. Owing to the selectivity of the GC-MS method, prior purification of these
`solutions of GBL in acetonitrile or acetic anhydride/acetonitrile is not necessary.
`
`GC-MS assay for GHB and GBL
`
`The GC-MS analyses were carried out on a Finnigan 4500 mass spectrometer interfaced
`with an Incas data-processing system and coupled to a Carlo Erba gas chromatograph
`model 5160, Mega series equipped with the Ciba-Geigy injector model 1988 and the A
`200S autosampler. The injector developed at Ciba-Geigy (Lauber-Injector) can be
`variously operated for split/splitless injection mode, for cold quasi on column injection
`mode or for hot quasi on column injection mode. All three injection techniques were
`automated by the autosampler A 200S from Carlo Erba Instruments, Milan, Italy for
`Europe and Leap Technology, Chapel Hill, NC, for USA.
`For GBL analysis, the temperature-controlled "cold on column" mode was chosen.
`The temperature was kept at 20°C. GC analyses were performed with a 50m x 0.3mm
`glass capillary column pretreated and coated with CP 51 Wax at a film thickness of
`1 µm according to Grob (1986), with a 25 m retention gap. The GC oven programme
`started at 70°C, increased at a rate of 7.5°C per minute to 220°C and was kept for
`10 min at this temperature. The temperature of the GC-MS interface and the ion source
`were kept constant at 250°C and 100°C, respectively. Hydrogen was used as carrier
`gas at a pressure of 80 kPa. The mass spectra were obtained in the total ion current
`(TIC) mode. The following mass spectrometric conditions were used: positive chemical
`ionization with methane as reactant gas at an ion source pressure of 45 kPa measured
`with an uncalibrated thermocouple gauge. The filament current was kept at 200 µA,
`and the electron energy at 70 e V. The mass spectrometer was scanned from m/z 50 to
`250 daltons in 1 sec intervals. Multiple Ion Detection was used for sensitive, selective
`simultaneous mass specific detection of the GBL-d0 and GBL-d6 at m/z 87 and 93.
`These base peaks were used for the quantitative assessment of GHB and GBL in brain
`structures (Fig. lB). Under these conditions the retention time for GBL and the
`internal standard GBL-d6 was 8: 15 min (Fig. lA). Every sample was injected twice.
`
`Measurement of GHB rate of synthesis
`
`The time-dependent accumulation of GHB and GBL following a dose of 400 mg/kg
`valproate was determined from 0 to 240min at fixed intervals. GHB and GBL levels
`were determined by the GC-MS method previously described. Turnover rates were
`estimated by measuring the accumulation of GHB and GBL in the linear part of the
`curves obtained.
`
`

`
`160
`
`R. Bernasconi et al.: Experimental absence seizures
`
`Receptor binding assays
`
`.To demonstrate the selectivity of the interactions, GHB and GBL were tested in
`a battery of 12 assays including GABAA, GABA8 , benzodiazepine, ai, a2 and ~­
`adrenoceptors, muscarinic cholinergic, 5HTi, histamine Hl, adenosine Al, opiate µ
`and substance P receptors. Methods for receptor-binding assays used in the present
`investigation are documented in table 3. All assays were validated using appropriate
`reference standards. When testing the affinity of GHB for GABA8 receptors in epilep(cid:173)
`tic as compared to non-epileptic conrol rats, we used the potent and selective tritiated
`GABA8 receptor agonist 3-aminopropylphosphinic acid, [3H]CGP 27492 (15.0Ci/
`mmol, Ciba-Geigy Horsham, UK) as described by Bittiger et al. (1990).
`
`cGMP determination
`
`cGMP assays were performed using a radioimmunoassay kit with [3H]cGMP obtained
`from Arnersham (Amersham, Buckinghamshire, UK). Groups of 8mice or rats were
`injected i.p. with test compounds or saline and sacrificed by fast focused microwave
`irradiation of the head (for mice: 3sec, 2.8kW, operating power; 2,450MHz, 54cm-2 ;
`Medical Engineering Consultants, Lexington, MA) to prevent post mortem changes in
`levels of cGMP. Each cerebellum was dissected and homogenized by ultrasonication in
`lml 0.05M tris buffer with 4mM EDTA, pH 7.5 (to prevent enzymatic degradation of
`cGMP), followed by heating 800 µl of the solution for 3 minutes at 120°C in a glycerine
`bath to coagulate protein. Homogenized samples were then centrifuged for 5 min at
`40,000 x g in the cold. cGMP levels in 100 µl aliquots of the supernatants were assayed
`in duplicate with the radioimmunoassay kit. The procedure involved incubating
`[3H]cGMP, antiserum and sample at 4°C for 1.5 to 18 hr. The antibody-cGMP com(cid:173)
`plex was pellected by the addition of chilled ammonium sulfate (60% saturated) and
`centrifugation. Pellets were resuspended in water, the suspension added to a scintilla(cid:173)
`tion cocktail, and radioactivity measured. Control experiments were carried out with an
`acetylated [125I]cGMP RIA kit of Advanced Magnetic (Cambridge, MA).
`
`Analysis of data
`
`Results are expressed as means ± standard deviation for 6 to 10 animals per group.
`Dunnet's multiple comparison two-tailed test (Winer, 1971) was used to assess the
`significance of differences between several groups and Student's t-test for paired
`groups. Means ± SEM were considered to be statistically different when p < 0.05.
`
`3. Results
`
`Quantification, linearity, recovery and reproducibility
`
`The GC characteristics and mass spectra of GBL are shown in Fig. lA and
`lB. The yield for the extraction of GBL using brain homogenates spiked
`with pure [2,33H]GHB was 100% (N = 8). Total recovery of the method,
`extraction plus derivatization, as estimated by adding different quantities of
`GHB (sodium salt) to brain extracts, was 100 ± 7.7%. The conversion of
`
`

`
`460
`
`480
`
`500
`
`520
`
`540
`
`560
`
`580
`
`TIME
`
`100 PCl/CH4
`
`93
`
`B
`
`50
`
`M/Z 60
`
`I
`
`80
`
`1/io
`
`I
`
`128
`
`140
`
`160
`
`188
`
`Fig. 1. Gas chromatography and mass spectra of GBL and GBL-d6 . These lactones
`were analyzed by extracting them from brain tissue with acetonitrile or with the
`combination acetonitrile and 5% acetic anhydride as described in "Materials and
`methods". 2 µl was injected into GC/MS system. GC conditions as described in
`"Materials and methods". A Mass spectrum of GBL-d0 m/z = 87 and GBL-d6 m/z = 93
`used as internal standard for quantification. Ionization conditions are positive ion
`chemical ionization with methane as reagent gas. Time = retention time in sec. The
`number at the top of each peak represents the retention time of the cortesponding
`lactone. RIC Reconstructed ion current. B GBL-do m/z = 87 and GBL-d6 m/z = 93 as
`internal standard selected ion mass chromatograms from brain extract. Ordinate =
`relative intensity in %
`
`

`
`162
`
`R . Bernasconi et al.
`
`0
`
`l<l
`
`(.!)
`
`.,,
`...
`....
`.,,
`«i
`"'
`<-! = N
`
`..l
`l<l
`(.!)
`
`:3 .. It:
`Q) = ... .. Q)
`
`:Ci
`.2!'
`
`Cl.
`
`2
`
`0
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`[ng/l GHBI
`
`Fig. 2. Calibration curve GHB versus GBL-d6 for the determination of GHB. Test
`samples containing various amounts of GHB and constant amounts of internal standard
`GBL-d6 were derivatized to GBL-d0 using conditions described in "Materials and
`methods" and injected into the gas chromatograph. Every point is the average of two
`determinations. Data are expressed as peak height ratio GHB-do: GBL-d6
`
`GHB to GBL under the conditions of acidification used was also quantita(cid:173)
`tive. Standard curves were obtained by derivatizing quantities ranging from
`5 to 100 ng GHB with 40 GBL-d6 . The calibration curves of GHB/internal
`standard peak area versus the GHB/internal standard concentration ratio
`showed a linear response in the range studied. The regression coefficient for
`the calibration curves was r ~ 0.99 (Fig. 2).
`The sensitivity of the assay is high, as the quantities of GHB-d6 injected
`to get the fragmentograms of Fig. lB are about 125 pg, and the setting
`of the electron multiplier is very low. In the total reproducibility assay
`(extraction, derivatization and GC-MS measurement), the quantities of
`GHB plus GBL measured from a pool of cortices were 2.36 ± 0.09 nmol/g,
`which corresponded to a coefficient of variation of 3.88% (N = 40). This
`variation coefficient is low because the internal standard is the deuterated
`derivative and GBL and GBL-d6 are eluted at the same time (Fig. lA). The
`mean cortical concentrations of GHB in all control samples (N = 57) was
`2.12 ± 0.23 nmol/g, GBL was also found to be present in all brain structures
`investigated. The cortical concentration was 0.370 ± 0.025 nmol/g (N = 57).
`This is about 15% of the concentration of GHB in this brain area.
`
`Extraction with organic solvents
`
`A study of the most favorable conditions for the isolation and extraction of
`GBL and GHB from brain tissues was carried out with several organic
`
`

`
`Experimental absence seizures
`
`163
`
`Table 1. Regional distribution of GHB and GBL in brain of rats
`
`Brairr areas
`
`GBL levels nmol/g wet wt
`
`GHB levels nmol/g wet wt
`
`2.12 ± 0.23
`0.37 ± 0.02 (57)
`(57)
`Cortex
`4.67 ± 0.25**
`0.65 ± 0.06 (12)
`(12)
`Striatum
`4.49 ± 0.91 **
`0.39 ± 0.04 (38)
`(38)
`Hippocampus
`4.25 ± 0.48**
`(28)
`N.D.
`Hypothalamus
`4.28 ± 0.90**
`(20)
`N.D.
`Thalamus
`0.33 ± 0.02 (6)
`2.33 ± 0.16 N.S. (6)
`Cerebellum
`Each value represents the mean ± SEM for (N) rats. ND = not determined.
`Interregional significancies were estimated relative to cortical GHB level by the
`Student's t-test for paired groups. ** p < 0.01
`
`solvents by adding GBL and GHB before the homogenisation of the
`irradiated tissue and analysis with the GC-MS method. Best recovery
`(>95%) was obtained with acetonitrile. The acetonitrile extracts yield
`much cleaner chromatograms than do extracts prepared from other organic
`solvents (ethanol, methanol, chloroform, dioxane and tetrahydrofurane).
`
`Assay of GHB and GBL in different brain structures
`
`The amounts of GHB and GBL in 6 regions of the rat brain are indicated
`in Table 1. The structures richest in GHB are striatum, hypothalamus,
`hippocampus and thalamus, whereas cortex and cerebellum had a relatively
`
`B
`
`Striatum
`
`•
`
`Cortex
`
`GHB
`nmol/g
`
`8
`
`6
`
`4
`
`2
`
`0
`
`0
`
`30
`
`60
`
`120
`
`240 min
`
`Fig. 3. Time course of GHB accumulation in cortex and striatum of rats treated with
`valproate (400mg/kg i.p.). Results are the mean ± S.E.M. for six animals. Statistical
`significance was calculated by Dunnett's test: *p < 0.05, **p < 0.01 when compared to
`the control group at t = 0. The initial rate of GHB synthesis in the cortex and striatum
`were 3.84nmol/g/h and 7.78nmol/g/h, respectively
`
`

`
`164
`
`R. Bernasconi et al.
`
`low content of GHB. Statistically significant differences (p < 0.01; Student's
`t test for paired group) were observed between the cortex ( = 100%) versus
`the following areas: striatum, 220%; hippocampus 210%; hypothalamus
`200% and thalamus 200%. In general, the distribution pattern for GBL
`seems to follow that of GHB.
`
`Time course of GHB accumulation in rats treated with valproate
`
`Rats were treated with valproate (400mg/kg i.p.), killed by microwave
`irradiation 0, 30, 60, 120 and 240 minutes later; GBL and GHB levels were
`determined in cortex and striatum. In these two regions, valproate induced
`a rapid and strong increase of GHB and GBL levels (about 180%) for 30
`minutes; then the content of the two GABA metabolites decreased slightly
`until a plateau was reached (Fig. 3). This rapid accumulation of GHB
`observed 30 minutes after enzymatic inhibition of its metabolization was
`used to determine the rate of GHB synthesis by calculating the difference
`between GHB content 30 minutes after treatment with valproate and the
`control level. In the different regions investigated, the accumulation of
`GBL caused by valproate was of the same oi:der of magnitude as for GHB
`(180%); but these increases never reached the level of significance (results
`not shown).
`
`GHB and GBL content and rate of synthesis in rats with SWD
`compared to controls
`
`The concentrations of endogenous GHB and of its prodrug GBL were
`measured in hippocampus, thalamus and frontal cortex in GAERS and
`compared to those observ.ed in rats from the selected control group (Table
`2, Fig. 4). Levels of GHB in GAERS were never different from those
`observed in control animals. Cortical and hippocampal GBL concentrations
`were also similar in both strains (results not shown).
`The rate of GHB synthesis was assessed in GAERS as well as in control
`rats by reference to the valproate-induced accumulation of GHB (Table 2
`and Fig. 4). The increases in GHB concentrations in GAERS were not
`different from control rats (Table 2, Fig. 4). The same is true of the
`valproate-induced increases in cortical and hippocampal GBL content in
`rats with SWD and in those without SWD (results not shown).
`
`Selectivity of interactions of GHB with GABAB receptors
`
`GHB interacted with the GABAa receptors with an IC50 of 1.5 x 10- 4 M.
`This value was obtained in three different experiments using [3H]baclofen
`as radioligand and membranes prepared from cerebral cortices according to
`
`

`
`Experimental absence seizures
`
`165
`
`D Control
`
`•
`
`Rats with SWD
`
`D Control+
`
`Valproic A.
`
`Rats with SWD
`+ Valproic A.
`
`GHB
`nmol/g
`
`15
`
`10
`
`5
`
`0
`
`Front. Cortex
`
`Thalamus
`
`Hippocampus
`
`Fig. 4. GHB levels and GHB rate of synthesis in rats with SWD as compared to "non(cid:173)
`epileptic" control rats. Animals treated with valproate were sacrificed 30min later.
`Results are the mean ± S.E.M. for groups of ten rats. **p < 0.01 when compared to
`the respective control group (Dunnett's test)
`
`Table 2. Kinetic parameters for the synthesis of GHB in rats with SWD as compared to
`controls
`
`Brain areas
`
`Cortex GAERS
`Cortex control
`Hippoc.GAERS
`Hippoc.control
`Thalam.GAERS
`Thalam.control
`
`Control GHB
`content
`nmol/g
`
`GHB content
`after valproate
`nmol/g
`
`Initial rate of
`GHB synthesis
`nmol/g/h
`
`Turnover
`time
`h
`
`2.02 ± 0.83
`3.06 ± 0.84
`8.34 ± 1.15
`6.67 ± 0.91
`5.69 ± 0.71
`6.25 ± 1.05
`
`8.90 ± 1.14**
`8.31±1.54**
`16.17 ± 3.34**
`16.30 ± 2.86**
`13.38 ± 1.46**
`12.47 ± 1.83**
`
`13.76
`10.50
`15.68
`19.24
`15.38
`12.44
`
`0.15
`0.29
`0.53
`0.35
`0.37
`0.50
`
`Wistar rats from the colony of Strasbourg (GAERS) were treated with valproate, killed
`30 min later and GHB levels were determined in dissected brain regions. Control GHB
`concentrations were determined in animals receiving saline. All values are means
`± S.E.M. for 10 animals per group and refer to wet weight.
`Statistical significance of difference was calculated by Dunnet's test: ** p < 0.01
`
`Bernasconi et al. (1986). Interactions with other receptors (including
`GABA A and central benzodiazepine receptors) were absent at a concentra(cid:173)
`tion of 100 µM (Table 3). GBL did not interact with the 12 receptors listed
`in Table 3, including GABA8 receptors, at a concentration of 100 µM. Thus,
`the interaction of GHB with GABA8 receptors appears to be selective.
`For the measurement of the interaction of GHB with GABA8 receptors
`in GAERS as compared to control rats, the potent and highly selective
`
`

`
`166
`
`R. Bernasconi et al.
`
`Table 3. Inhibition of binding by y-butyrolactone (GBL) and by y-hydroxybutyric acid
`(GHB) in 12 receptor binding assays
`
`Putative receptor
`
`Radio ligand
`
`Inhibition of
`binding
`(% at
`10-4 M)
`GHB GBL
`
`Method
`
`a 1-Adrenergic
`Clz-Adrenergic
`~-Adrenergic
`5-HT1
`Histamine1
`Muscarinic
`Mu-opiate
`GABA A
`GAB AB
`Benzodiazepine
`Adenosine Al
`Substance P
`
`[3H]prazosin
`[3H]clonidine
`[3H]DHA
`[3H]5-HT
`[3H]doxepine
`[3H]QNB
`[3H]naloxone
`[3H]muscimol
`[3H]baclofen
`[3H]ftunitrazepam
`[3H]CHA
`[3H]substance P
`
`0
`0
`0
`0
`5*
`0
`0
`0
`44**
`0
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`Greengrass and Bremner (1979)
`Tanaka and Starke (1980)
`Bylund et al. (1976)
`Nelson et al. (1978)
`Tran et al. (1981)
`Yamamura et al. (1974)
`Bradbury et al. (1976)
`Beaumont et al. (1978)
`Bernasconi et al. (1986)
`Speth et al. (1978)
`Patel et al. (1982)
`Bittiger et al. (1982)
`
`The receptor binding assays were performed essentially as described in the references.
`Abbreviations: DHA dihydro-alprenolol; 5-HT serotonin; QNB quinuclidinyl
`benzylate; CHA cyclohexyl-adenosine. * =55% at 10-3 M; ** IC50 = 1.5 x 10-4 M
`obtained from 3 inhibition curves
`
`Table 4. Interactions of y-hydroxybutyric acid with GABAB
`receptors in rats with SWD and in rats from the selected
`control group
`IC50 in µM
`
`Brain structures
`
`Control rats
`
`Rats with SWD
`
`Cortex
`Cerebellum
`Thalamus
`
`152.5
`138.0
`166.7
`
`132.2
`168.4
`157.2
`
`Membranes were prepared from male Wistar rats from the
`breeding colony at the Centre de Neurochirnie, C.N.R.S.,
`Strasbourg according
`to Bittiger et al.
`(1990). The
`radioreceptor assay was performed with [3H]CGP 27492 as
`radioligand according to Bittiger et al. (1990)
`
`GABAa radioligand, [3H]CGP 27492, was used (Bittiger et al., 1988, 1990).
`The IC50 ranged from 1.38 x 10-4 M in the cerebellum to 1.66 x 10- 4 M in
`the thalamus and were similar in GAERS and in control rats (Table 4) and
`not different from the IC50 values obtained with [3H]baclofen as radioligand
`and membranes prepared from cerebral cortices (Table 4).
`
`

`
`cGMP
`pmol/mg
`protein
`
`3
`
`2
`
`Experimental absence seizures
`
`A
`
`cGMP
`pmol/mg
`
`167
`
`B
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`R-(-)-baclofen
`
`3
`
`10 mg/kg i.p.
`
`0.1 -+--.-----.----.----..----------..--
`
`0
`
`30
`
`60
`
`120
`Postinjection time
`
`240 min
`
`Fig. 5. Cerebellar cGMP concentrations in mice exposed to R( - )-baclofen. A Dose(cid:173)
`dependent decrease in cGMP content. Animals (n = 8) were administered R( - )(cid:173)
`baclofen and killed 60rnin later, controls received 0.9% saliµe. cGMP levels were
`determined by radioimmunoassay and expressed as mean ± S.E.M. **p < 0.01,
`***p < 0.001 (Dunnett's test). B Time course of cGMP levels in the cerebellum follow(cid:173)
`ing administration of R(-)-baclofen (6mg/kg, i.p.). Each value represents the mean±
`S.E.M. of 8 mice. Controls received 0.9% saline and were killed 30 min later
`
`Effects of GHB and the GABA8 receptor agonist R(-)-baclofen on cerebellar
`cGMP content
`
`The GABAs receptor agonist R( - )-baclofen dose-dependently decreased
`cerebellar cGMP levels (Fig. SA). The threshold dose 60min after injection
`was between 1 and 3mg/kg i.p. (56% of control at 3mg/kg) and the con(cid:173)
`tent of cGMP after 6 mg/kg R( - )-baclofen was 28% of control value and
`decreased to 20% at 10 mg/kg. Figure SB shows the time-course for the
`decrease of cerebellar cGMP observed after injection of 6 mg/kg of the
`agonist. The onset of the decrease of cGMP content caused by R( - )(cid:173)
`baclofen was very rapid; 30 min after administration of R( - )-balcofen
`cGMP levels were 38% of controls and decreased further to 28% 60min
`after drug treatment. Then, levels of the second messenger increased again
`and reached 59% of control values 2 hours after administration of R( - )(cid:173)
`baclofen. After 4 hours cerebellar cGMP concentrations were normalized
`and ataxia had disappeared in mice. This suggests that the behavioural
`effects induced by R( - )-baclofen correlate with the decrease in cGMP.
`GBL decreased cGMP levels in a dose-dependent manner (Fig. 6A).
`While lOOmg/kg GBL i.p. did not alter cerebellar cGMP levels significantly
`45 min after administration of the drug (7 4 % ) , the reductions by 200 mg/kg
`GBL i.p. (42%) and 400mg/kg GBL i.p. (24%) were statistically significant
`(p < 0.01). The time course of the effect of GBL on levels of cerebel(cid:173)
`lar cGMP is shown in Fig. 6B. After intra peritoneal administration of
`
`

`
`168
`
`cGMP
`pmol/mg
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`R. Bernasconi et al.
`
`A
`
`cGMP
`pmol/mg
`
`B
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`GBL
`
`0
`
`100
`
`200
`
`400 mg/kg i.p.
`
`0
`
`20
`Postinjection
`
`time
`
`40
`
`60
`
`Fig. 6. Effect of GBL on cGMP content in the cerebellum of mice. A Decrease in
`cGMP in function of the doses of GBL. The drug was administered 45 min before
`microwave irradiation. Each value represents the mean± S.E.M. of 8 animals. Statis(cid:173)
`tical significance w.as calculated by Dunnett's test. B Time course of cGMP concentra(cid:173)
`tions in the cerebellum of mice following injection of 200 mg/kg GBL. Each point
`represents the mean ± S.E.M. of 8 animals. *p < 0.05, **p < 0.01
`
`200 mg/kg of GBL, there was a rapid and marked decrease in the con(cid:173)
`tent of cGMP, the maximal effect being achiev