Anexo III Transcripción de las entrevistas

Data from studies of GABAa receptor subunit mRNA distribution and levels of expression in several animal models of epilepsy is summarised in Table 1.2. The majority o f these studies utilised the in situ hybridisation technique although use of Northern hybridisation and PCR techniques have also been reported (Pratt et al 1993). Changes in mRNA expression associated with increased seizure activity at the acute stage was most commonly reported, with fewer studies addressing the question of whether long-term changes in subunit mRNA expression are associated with increased seizure susceptibility. Notable changes in subunit mRNA at the chronic stage included an increase in a3 mRNA levels in the dentate gyrus and a decrease in y2l mRNA throughout the hippocampus (21-27%) (possibly coinciding with an increase in y2g mRNA) in rats kindled via the Schaffer collaterals (Kamphuis et al 1995). In addition, increased seizure susceptibility in the pilocarpine model of epilepsy was associated with increased a5 mRNA in the dentate gyrus and reduced levels o f a2 and a5 mRNA in the CA in chronic studies (Rice et al 1996).

Interpretation of observed changes in GABAa subunit mRNA expression at the various temporal stages chosen is speculative at this time but it has been proposed that changes at intermediate time stages represents a reaction to the occurrence of seizure activity, possibly compensatory (Kamphuis et al 1995). Changes at the acute stages, on the other hand, may be more relevant for the pathogenesis of seizures and altered mRNA levels at the chronic time points could reflect pathologic or adaptive changes in the hippocampus underlying, or in response to, increased seizure susceptibility.

Parallel changes in receptor binding and mRNA expression have led some others to propose that no epilepsy-associated alteration occurs in the pharmacological profile of the GABAa receptor (Clark et al 1994; Kamphuis et al 1995).

A number of the studies included in Table 1.2 evaluated subunit mRNA expression at the cellular level (Kokaia et al 1994; Friedman et al 1994; Rice et al 1996) and confirmed changes in expression per surviving neurone, however, other studies

Model / Ref Region Acute Intermediate Chronic

subunits studied <12 hours 12-48 hours >48 hours

Amygdala kindling 1 DG T a4 p ip 3 NA NA

a l , a2, a4, p i, p3. CA3

y2 CAl Ta2

Schaffer kindling 2 DG T a la 2 a 4 p i Tp2p3y2y2L iy lL Ta3y2

a l , a2, a3, a4. CA3 Ta2 Ty2y2L ij2 L

P1,P2, p 3 ,y l,y 2 CAl Ta4 Taly2y2L •IjIl

Hippocampal 3 DG J<aip3y2 Taly2

kindling a l , P3, y2 CAl >Ly2 T a l

Electroconvulsive 4 CBM T a lp 2

Shock HIP

a l , P2 CTX

Kainate (i.p.) 5 DG NA tGluR2/3 NA

a l , GluR2, GluR3 CA3/4 i a l , GluR2/3

Pilocarpine (i.p.) 6 DG NA NA Ta5

a l , a2, a5, p2, y2 CA \La2a5

Hippocampal Slice 7 HIP NA NA

(+ bicuculline)

a2, a5, y2

Hippocampal Slice 8 DG ]<al NA NA

(Mg^-free) CA3 l 'a i

a l , a l CAl l 'a i

Hippocampal 9 HIP NA i a l NA

Culture (low Mg"^

a l , a l

Table 1.2 Data from studies of GABAa receptor subunit mRNA in animal

models of epilepsy.

Key: T = increased expression; 'I = decreased expression; o = no change. Unless otherwise indicated, mRNA expression may be assumed to be unchanged for all subunits examined. Abbreviations: DG - dentate gyrus; CA - Ammon's Horn; CBM - cerebellum; HEP - hippocampus; CTX - cortex; NA - not assessed at this stage. References: 1 (Clark et al

1994); 2 (Kamphuis et al 1995); 3 (Kokaia et al 1994); 4 (Pratt et al 1993); 5 (Friedman et al 1994); 6 (Rice et al 1996); 7 (Gerfinmoser et al 1995); 8 (Vick et al 1996); 9 (Blair et al 1995).

reporting decreased mRNA expression without correction for neuronal density must be interpreted with caution.

Studies incorporating targeted disruption of selected subunits o f the GABAa receptor

have revealed interesting results, possibly o f some relevance for TLE. These studies have included treatment of adult animals with antisense oligonucleotides to a portion of the mRNA encoding the subunit of interest and gene knockout strategies inhibiting expression of a particular subunit from early development.

Jakoi et al (1995) examined seizure activity in cultured hippocampal neurones administered an antisense oligonucleotide to the a2 subunit of the GABAaR. Synchronous epileptiform bursting was observed in antisense-treated cultures exposed to a low magnesium medium. Seizure activity was not observed in control cultures treated with a missense oligonucleotide however, administration of bicuculline was found to mimic the effects o f the a2 antisense treatment.

Karle et al (1995) reported a 43% loss in ^H-flunitrazepam binding to hippocampal membrane preparations following a 5 day continuous infusion of an antisense oligonucleotide into the rat hippocampus, targeting the y2 subunit o f the GABAa receptor compared to untreated controls. Scatchard analysis revealed a 27% decrease in Bmax and a 15% increase in Kj (decrease in affmity) in treated animals. Interestingly, a significant reduction in total binding sites was also detected in rats infused with a mismatch oligonucleotide as a control, in comparison with untreated animals thus prompting caution in the interpretation o f these results. Binding changes for ^^S-TBPS and ^H-muscimol mimicked the findings for flumazenil possibly suggesting a complete loss of all GABAa receptors containing the y2 subunit. Also reported in the antisense- treated animals was a marked loss o f neurones upon histological examination and the authors propose that neuronal death may be due to diminished GABAergic inhibition induced by a decrease in functional GABAa receptors.

Romanics et al (1997) reported the results of a study o f mice devoid of the p3 subunit following gene targeting of this subunit. GABAa receptor density was found to be

halved in p3-deficient mice as assessed by ^H-muscimol and ^H-Ro 15-4513 binding. The majority o f the animals died as neonates, many with cleft palate. Surviving mice demonstrated grossly abnormal brain morphology, hypersensitivity and neurological impairment as well as Sequent myoclonus and occasional epileptic seizures. Electrophysiological studies of dorsal root ganglion cells in the spinal cord of the knockout animals, revealed approximately 80% decrease in GABAa chloride current maximal amplitude in comparison with control mice.

Blair and colleagues (1995) reported the results of slot blot hybridisation studies of hippocampal neonatal cultures exposed to three hours of magnesium-free or control media. The development o f long lasting recurrent seizures in the cultures maintained in magnesium-free medium was associated with a 39% decrease in GABAa a2 mRNA and no change in a l mRNA in comparison with control cultures.