Regulation of Calcium Channel α1A Subunit Splice Variant mRNAs in Kainate-Induced Temporal Lobe Epilepsy

Neurobiology of Disease 6, 288–301 (1999) Article ID nbdi.1999.0248, available online at http://www.idealibrary.com on

Regulation of Calcium Channel ␣1A Subunit Splice Variant mRNAs in Kainate-Induced Temporal Lobe Epilepsy S. Vigues,1 M. Gastaldi,2 C. Chabret,1 A. Massacrier,2 P. Cau,2 and J. Valmier1,CA 1CNRS

UPR1142, Institut de Biologie, Blvd. Henri IV, 34060, Montpellier, Cedex, France. de Biologie Cellulaire, Faculte´ de Me´decine, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France 2Laboratoire

Received January 6, 1999, revised April 2, 1999, accepted for publication April 9, 1999

P/Q-type voltage-gated Ca2ⴙ channels (VGCC) regulate neurotransmitter release in the hippocampus and molecular alterations of their ␣1A pore-forming subunits are involved in various animal and human CNS diseases. We evaluated, using RT-PCR and in situ hybridization, the spatio-temporal activation of two ␣1A subunits splice variants (␣1A-a and ␣1A-b) in control and kainic acid (KA)-treated rats. Six hours after KA treatment, ␣1A-a and ␣1A-b mRNAs increased, decreased or remained unchanged with area specific patterns. These changes were evidenced in the hippocampus and the dentatus gyrus and absent in the cerebellum. The ␣1A mRNA upregulation lasted for at least 7 days after KA treatment. Altogether, these results indicate that ␣1A-a and ␣1A-b mRNAs following seizure onset exhibit a complex and specific spatio-temporal pattern. The long-lasting changes in ␣1A subunit mRNA contents suggests that VGCC may be involved in the mechanisms generating chronic focal hyperexcitability and/or cellular damage in temporal lobe epilepsy. r 1999 Academic Press

dling- or KA-related epileptiform seizures (Braun & Freed, 1990; De Sarro et al., 1990). High-voltage Ca2⫹ currents have been classified based on their electrophysiological and pharmacological properties into L-, N-, P/Q- and R-types (Tsien et al., 1988; Birnbaumer et al., 1994). These channels are composed of at least three subunits: a pore-forming ␣1 subunit and regulatory ␣2␦ and ␤ subunits. Molecular cloning has revealed at least six ␣1 gene products (␣1A–␣1F) expressed in the nervous system (Perez-reyes et al., 1994; Bech-Hansen et al., 1998; Strom et al., 1998). Although the identity of the channel formed after expression of ␣1E remains unclear (Soong et al., 1993; Zhang et al., 1993; Williams et al., 1994; Schneider et al., 1994; Bourinet et al., 1996), ␣1A and ␣1B are thought to encode respectively P/Q and N channels (Mori et al., 1991; Williams et al., 1992a; Sather et al., 1993; Zhang et al., 1993; Stea et al., 1994), and ␣1C, ␣1D and ␣1F are thought to be components of L channels (Hui et al., 1991; Singer et al., 1991; Snutch et al., 1991; Williams et al., 1992b; Bech-Hansen et al., 1998; Strom et al., 1998).

INTRODUCTION Voltage-gated Ca2⫹ channels (VGCC) regulate Ca2⫹ influx and neurotransmitter release and so are essential for controlling cell to cell communication and plasticity in the hippocampus. VGCC function is required for the induction of certain forms of hippocampal long-term potentiation (LTP), a model of memory formation (Nicoll & Malenka, 1995). VGCC are also implicated in hippocampus pathological processes such as epilepsy and ischemia (Heinemann et al., 1977; Cheung et al., 1986; Choi, 1995; Fletcher et al., 1996; Burgess et al., 1997). In the kindling model of epileptogenesis, enhancement of VGCC has been evidenced in different regions of the hippocampus (Vreugdenhil & Wadman, 1992; Faas et al., 1996) and Ca2⫹ entry blockers can prevent or reduce convulsions in kin-

Jean VALMIER, CNRS UPR1142, Institut de Biologie, Blvd Henri IV, 34060, MONTPELLIER, Cedex FRANCE. Te´l. (33).4.67.60.11.31; Fax. (33) 4.67.60.11.32; E-mail: [email protected]

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0969-9961/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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Calcium Channel mRNAs in Epilepsy

Most studies suggest that class A Ca2⫹ channels play a major role in the control of neurotransmitter release at the hippocampal synapses (Dunlap et al., 1995). From experiments performed on rabbit, rat, mouse and human ␣1A cDNAs, multiple isoforms are thought to be generated by alternative splicing in this single-copy gene from which at least ten variants are described (Mori et al., 1991; Starr et al., 1991; Fletcher et al., 1996; Ligon et al., 1998). They differ in their functional properties in expression systems, their cellular localization, their biochemical and pharmacological properties, their interaction with presynaptic protein and their involvement in animal and human genetic diseases (including epilepsy). Despite the fact that Ca2⫹ is essential for synaptic transmission and influencing cellular excitability, no data are presently available on the in vivo regulatory mechanisms that control the expression of the different ␣1A isoforms. As a first step to understanding the contribution of these different splice variants to physiological and pathological hippocampal processes, the present study investigated and compared, by PCR and in situ hybridization (ISH) analysis, the regulation of two ␣1A subunit isoforms (namely ␣1A-a and ␣1A-b), (Soong et al., 1994; Zamponi et al., 1996; Sutton et al., 1998) in control and KA-treated rats, a model of human temporal lobe epilepsy (Sperk, 1994).

Total RNA was first treated with Dnase (Gibco, 1.5 U, 15 minutes, 37°C) to remove any residual genomic DNA. cDNA templates for amplification by Polymerase Chain Reaction (PCR) were synthesized by reverse transcription of 1 µg total RNAs using Moloney’s murine leukemia virus reverse transcriptase (Superscript II, Gibco) with random hexanucleotides primers (160 µM) and dNTP (300 µM) for 50 minutes at 42°C in the buffer (supplied by the manufacturer).

Amplification of ␣1A-a and ␣1A-b Subunit mRNAs PCR was carried out using synthetic oligonucleotide primers (Eurogentec, France) based on the rat calcium channel ␣1A subunit sequence (GenBank, sequence M64373). The specificity of the 4 primers was checked using the BLAST program. Sense primer I: 58-TCC AAA AAC CAG AGT GTG-38, nucleotide

5183-5200

Antisense primer II:

MATERIALS AND METHODS

58-TTG AAG TGA ACG GTG TTG-38, nucleotide

Animal Treatment

Antisense primer III:

Wistar adult rats (250–300 g) (Iffa Credo, L’Arbresle, France) were housed under standard laboratory conditions and maintained under a 12 h light/dark cycle with free access to food and water. Their care and use conformed to institutional policies and guidelines. Seizures were induced by a single intraperitoneal injection of KA (Sigma, 9 mg/kg dissolved in phosphate buffered saline), control animals received vehicle alone. The behavior of animals was continuously scored for at least 1 hour in order to evaluate the severity of KA seizures. Animals exhibiting a full limbic seizure syndrome were kept for experiments. Animals were anaesthetised by pentobarbital (50 mg/kg) and killed by decapitation. Brains were quickly removed, frozen in liquid nitrogen vapours and stored at ⫺80°C until use.

58-TAT TAC TCG CAA TAA ACT G-38, nucleotide

RNA Isolation and First cDNA Synthesis Total RNA was isolated from various brain regions by using the TRIzol protocol (Gibco) derived from Chomczinski & Sacchi (1987).

5517-5534

5412-5430

Antisense primer IV: 58-CAT GTG TCT CAG CAT CTG A-38, nucleotide

5412-5430

Primer I and II allowed us to amplify a 351 bp sequence, called ␣1A-EF, which was shared by ␣1A-a and ␣1A-b isoforms. ␣1A-EF encoded a polypeptide starting from the domain IV S6 (a sequence corresponding to that involved in the pore of Ca2⫹ channel) and going to the carboxyl terminal after the divalent ion binding domain (EF hand) of the Ca2⫹ binding proteins (figure 1). To confirm the presence of two isoforms within the PCR products, we subsequently cloned and sequenced these PCR products. As expected, sequence analysis of individual clones confirmed that the two isoforms ␣1A-a and ␣1A-b were present within the PCR products. Used in association with primer I, primer III and IV specifically amplify a 247 bp fragment of ␣1A-a and ␣1A-b respective sequences (figure 1). The difference between the ␣1A-a and ␣1A-b was the substitution of ten amino Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. EF-hand domain and primers positions. a- Schematization of the EF-hand domain located between 5347 and 5433 bp. ␣1A-a (grey) and ␣1A-b (black) specific regions are located between 5387 and 5433 bp. b- cDNA was synthesized by the reverse transcriptase in hippocampus and (1) a fragment of 351 bp was amplified by using I and II primers, (2) a 247 bp fragment was amplified by specific primer of ␣1A-a variant (I and III primers), (3) a 247 bp fragment was amplified by specific primer of ␣1A-b variant (I and IV). c- The PCR product was digested in a single restriction site AlwNI located at 5418 bp. The complete digestion generated 3 distinct bands at 351, 235 and 116 bp which correspond for the former: ␣1A-a and the two latter ␣1A-b PCR products.

acid residues among 32 amino acids between the 1795 and 1826 position. These data were in accordance with the presence of ␣1A-a and ␣1A-b in adult rat brain as previously described by Soong et al. (1994). The cDNA mixture was supplemented with 2.5 U Taq polymerase (Thermus aquaticus YT1 from Gibco), 25 pmoles of each primer, 5 µl of 10 ⫻ PCR buffer (100 mM Tris-HCl pH 8.4, 20 mM MgCl2, 500 mM KCl), 1 µl of dNTP mix (10 mM) and water to bring the final volume to 50 µl. The reaction mixture was overlaid with 50 µl mineral oil (Promega) and incubated in a thermal cycler (Eppendorf) for 30 cycles (1 min denaturation at 94°C, 1 min hybridization at 50°C, and 45 sec elongation at 72°C). After amplification, 10 µl of PCR product was run on an ethidium bromide-stained gel (1.5% agarose). These PCR conditions were used for all amplifications. PCR products are fragments of 351 or 247 nucleotides, they were sequenced (ACT gene) and the specificity was confirmed. Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Enzymatic Digestion The first PCR product (primers I and II) was digested with 2 µl of AlwNI enzyme (Amersham, 10 U/µl) and digestion buffer (10⫻, Amersham) in a final volume of 100 µl. Digestion product was precipitated with ethanol and was run on an ethidium bromide-stained gel (1% agarose). AlwNI enzyme was specific of the ␣1A-b isoform and cut the PCR product on a single position at 235 bp. Three bands are expected: 351 bp (␣1A-a) and 235 bp and 116 bp (␣1A-b isoform).

Image Analysis of PCR Gels After digestion, the relative intensity of the bands was analysed using Image Tool 1.23, a free image analysis program available in the department of dental

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Calcium Channel mRNAs in Epilepsy

diagnostic Science at the University of Texas Health Science Center, San Antonio, Texas (ftp://maxrad6. uthscsa.edu).

In situ Hybridization Frozen brain saggital sections (12 µm) were cut using a cryostat (Microm, France), collected onto silanized slides (Sigma) and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature (RT) for 30 min, then rinsed in the same buffer. Slides were dehydrated in graded ethanols, then stored at ⫺80°C until use. cRNA probes were designed from sequences described by Sutton et al., (1998). They corresponded to specific 38 region of each Ca2⫹ channel mRNA subunit. Each cRNA probe was obtained by a 3 step process: i) a RT-PCR from rat cerebral cortex total RNAs produced both ␣1A-a and ␣1A-b sequences. ii) four specific nested PCR with extended primers produced ␣1A-a and ␣1A-b sense and antisense templates with T7 promoters at their 58 extremities. iii) In vitro transcription from these templates produced specific sense and antisense cRNA probes. Primers used for these synthesis were as follows: i) First RT-PCR: ComR3:

58-CAA GGT GGA GTT GAA GTG AAC G-38

ComD2:

58-AAC CAG AGT GTG GCA ACG AG-38

This amplification produced a 358 bp fragment ii) Specific PCR: 7aD

58-CTG CGG CCG CAT TCA CTA TAA-38

T74aR

58-GTT TAA TAC GAC TCA CTA TAG GAG CCT CTT GCA AGC AAC CCT AT-38

These two primers were used for the amplification of the antisense ␣1A-a template (129 bp) 7aDT7

58-GTT TAA TAC GAC TCA CTA TAG GCT GCG GCC GCA TTC ACT ATA A-38

4aR

58-AGC CTC TTG CAA GCA ACC CTA T-38

These two primers were used for the amplification of the sense ␣1A-a template (129 bp) 1bR

58-CCT CTT GTA AGC CAC TCT GGC-38

T74bD

58-TGT TAA TAC GAC TCA CTA TAG GACATG TAT CAG ATG CTG AGA C-38

These primers were used for the amplification of the sense ␣1A-b template (104 bp) T71bR

58-GTT TAA TAC GAC TCA CTA TAG GCC TCT TGT AAG CCA CTC TGG C-38

3bD

58-GGA CAT GTA TCA GAT GCT GAG ACA C-38

These primers were used for the amplification of the antisense ␣1A-b template (106 bp) Each spliced variant-amplified template was purified on Nick-column (Pharmacia). The quantity was estimated using a 2% agarose gel electrophoresis and the amplifiate was resuspended in DEPC-treated water. Antisense and sense cRNA probes were produced by in vitro transcription using T7 RNA polymerase (Promega) and were labelled with digoxigenin-11-UTP (Boehringer Mannheim) using 100 ng of the template in a final volume of 100 µl with ATP-CTP-GTP mixture (0.5 mM each), UTP (0.3 mM), digoxigenin-11-UTP (0.2 mM), DTT (1 mM), transcription buffer, T7 RNA polymerase (40 U) and RNasin (200 U, Promega). Transcription was carried out at 37°C for 1 h 30 and template DNA was digested with RNase-free DNase RQ1 (Promega, 1 U) at 37°C for 15 min. The probes were then extracted with phenol/chloroform, ethanol precipitated and dissolved in DEPC-treated water. Unincorporated nucleotides were removed by filtration through a 1 ml Sephadex G50 column (Pharmacia). Probe amount was quantified by a spectrophotometric UV determination, and their integrity checked by electrophoresis on a 2% agarose gel. Labelling efficiency of sense and antisense probes was estimated using serial dilutions of the labelled probes spotted and fixed onto nylon membranes (Hybond, Amersham). Dots were imaged using a TV camera connected to an Apple Macintosh IICi fitted with a digitization card (Perceptics Pipeline, Graftek, France). The luminance of each dot was measured using Image 1.49 program (NIH) and transformed into optical density (OD) values. The OD of dots from Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

292 serially diluted sense and antisense probes was compared. The dilution of sense and antisense probes used in ISH experiments was adjusted so that both probes gave the same dot labelling intensity. ISH was performed according to Gastaldi et al. (1995). Tissue sections were briefly rehydrated in PB (0.1 M pH 7.4 containing 0.1 M glycin), incubated in 1⫻ standard saline citrate (SSC), then in proteinase K solution (Boehringer Mannheim, 0.2 µg ml⫺1 in 1⫻ SSC), 10 min at room temperature (RT) and fixed with 4% paraformaldehyde in PB for 15 min. Slides were subsequently rinsed twice in 4⫻ SSC (for 15 min) and prehybridized at RT in 4⫻ SSC, 1⫻ Denhardt for 10 min, then 4⫻ SSC, 1⫻ Denhardt, 43% formamide for 60 min. Slides were dehydrated in graded alcohols, then allowed to dry at least 30 min before hybridization. Ca2⫹ channel probes were diluted at a final concentration of 80 ng/ml into hybridization buffer (4⫻ SSC, 43% deionised formamide, 1⫻ Denhardt solution, 100 mM phosphate buffer pH 7.4, 0.9% sarcosyl, 1% dextran sulfate, 145 µg ml-1 yeast tRNA, 250 µg ml-1 sheared salmon sperm DNA). Twenty µl of the hybridization mixture was applied to the slides which were covered with Parafilm coverslips and hybridized overnight at 40°C in an humid chamber. Parafilm coverslips were removed by immersion in 4⫻ SSC. Tissue sections were washed in 4⫻ SSC (15 min, 40°C), then in 1⫻ SSC (30 min at RT), and finally in 0.1⫻ SSC (2 ⫻ 5 min at RT). The sections were treated for 5 min at RT with a solution of RNase A (Boehringer Mannheim, 10 µg ml-1 in 300 mM NaCl, 10 mM Tris-HCl pH 7.5, containing 5 mM EDTA), and fixed with 1% paraformaldehyde in PB for 30 min. Then tissue sections were washed for 30 min in PB containing 0.1 M glycin. Detection of digoxigenin-labelled probes was performed according to the Boehringer Mannheim protocol. After a brief wash in buffer 1 (150 mM NaCl, 100 mM Tris-HCl pH 7.5), slides were incubated for 30 min in buffer 2 (buffer 1 containing 10% normal sheep serum and 0.3% Triton X100) then 15 min in buffer 1. Slides were then incubated at RT for 2 h with polyclonal sheep antidigoxigenin Fab fragments (diluted 1:1000 in buffer 2), conjugated with calf intestinal alkaline phosphatase (Boehringer Mannheim). Slides were washed 3 times in buffer 1 for 5 min and in buffer 3 (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 5 min, and colour development was achieved by an overnight incubation in NBT and BCIP (Gibco). The reaction was stopped in 10 mM Tris-HCl pH 8.0, 1 mM EDTA. Slides were mounted in Mowiol (Calbiochem) and coverslipped. Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Vigues et al.

ISH Controls In each experiment, the following three ISH controls were prepared: (1) digestion with RNase A before hybridization: sections were incubated for 30 min at RT in a solution of RNase A (Boehringer Mannheim, 10 µg.ml⫺1 in 300 mM NaCl, 10 mM Tris-HCl buffer pH 7.5, containing 5 mM EDTA), before proteinase K digestion; (2) hybridization using sense probes diluted at the concentrations giving the same labelling signal intensity in dots as antisense probes; (3) hybridization without probes (control of the immunocytochemical detection of digoxigenin).

ISH Signal Intensity Measurements Slides with sense and antisense probes from two control and KA-treated animals and for each period (24 h, n ⫽ 3; 7 days, n ⫽ 2) were observed using a Zeiss photomicroscope connected to DXC101P TV camera (Sony). The video signal was digitized (256 gray levels) using the Perceptics Pipeline card (Graftek, France) fitted into an Apple Macintosh IIci computer. Pictures were captured using image 1.44 program (NIH), averaged and corrected for non-homogeneous illumination using a blank image. Two slides were analysed for each brain area, for each sense and antisense probe and for each experimental condition. When sectioning tissues, serial sections were obtained in order to test the labelling of identical cells with the two splice variant cRNA probes. For each brain area, three to twelve fields were selected from each hybridized slide using a drawing tool. We can therefore assume that, for a given brain region, the same section surface area of neuronal cell bodies was analyzed in control and KA-treated animals. CA4 neuronal cell bodies from KA-treated animals (24 h) were also extracted by grey level tresholding in order to discard unlabelled neuropil and their luminance was recorded. The luminance of neuronal cell bodies was measured, the mean luminance for a given experiment was computed and then transformed into OD values, corresponding to the ISH signal intensity. When control and KA-treated rats were compared, results were expressed as relative changes in mean labelling intensity values in the two data sets, the OD values measured in the control group being used as reference: (Kainate OD minus Control OD) divided by Control OD. Mean and SD were computed and statistical analysis was performed using Student’s t-test, after checking the equality of variances.

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Studies of the Colocalization of Type ␣1A-a and ␣1A-b mRNAs Serial sections alternatively hybridized with type ␣1A-b and type ␣1A-a cRNA probes were used for colocalization studies. Fields from CA1 and CA3 areas, and from Dentate gyrus region (including CA4 neurons) from control animals were digitized as described above. The two grey level pictures were thresholded and binarized, the picture corresponding to type ␣1A-a mRNAs was inverted. Using identical neurons present in serial sections, the two pictures were superimposed using PHOTOSHOP 4.0 program (Adobe, San Jose´, CA, USA) in order to visualize in grey the cells expressing both type ␣1A-a and ␣1A-b mRNAs. The surface area (in pixel units) of black cells (expressing type ␣1A-b mRNAs only), of white cells (expressing type ␣1A-a mRNAs only) and of grey cells (expressing both ␣1A-a and ␣1A-b mRNAs) was measured using IMAGE 1.44 program. The colocalization was expressed as the percentage of grey pixels related to black ⫹ grey or white ⫹ grey pixels of pictures. The same protocol was applied to CA4 area from serial sections hybridized with type ␣1A-a and type ␣1A-b cRNA probes and collected from rats 24 hours after KA administration.

conserved region, the second pair of primers (I and III) was designed to specifically amplify the ␣1A-a splice variant while the third pair (I and IV) allows us to specifically amplify the ␣1A-b splice variant (see Methods). In figure 1b, lane 1 shows the presence of a 351 bp of the ␣1A-EF amplified sequence. The two splices variants of the ␣1A: ␣1A-a (lane 2), ␣1A-b (lane 3) produced a 247 bp fragment. All fragments were sequenced and the specificity of each PCR was checked by comparing the sequence of the amplification product with those of previously characterized ␣1A-a and ␣1A-b subclones (data not shown). Both isoforms were found to be present in the hippocampus, in the cerebellum and the neocortex of control adult rats. We have evaluated the relative quantity of each splice variant using the restriction site AlwNI specific for the ␣1A-b isoform. When using primer I and II (see Methods), both splice variants were amplified. This AlwNI restriction site is localized at position 235 and cuts the 351 bp ␣1A PCR product in two fragments of 235 and 116 bp (figure 1a and c). The quantitative analysis of the electrophoretic pattern electrophoresis showed that the ratio ␣1A-b/(␣1A-a ⫹ ␣1A-b) is higher in the neocortex and in the hippocampus regions than in cerebellum (Table 1).

␣1Aa and bmRNA Changes in Hippocampus, Cerebellum and Neocortex Following KA-induced Limbic Seizures

RESULTS Relative ␣1A-a and ␣1A-b mRNA Expression in Hippocampus, Cerebellum and Neocortex of Control Adult Rat In order to examine the pattern of expression of the ␣1A-a and ␣1A-b splice variants in several brain regions, we carried out PCR amplification with primers able to specifically amplify ␣1A-EF as well as ␣1A-a and ␣1A-b splice variants. The first pair of primers (I and II) was designed to amplify the ␣1A Ca2⫹ channel in a highly-

The same PCR strategies were used to follow the temporal pattern of the relative expression of the two ␣1A isoforms in KA-treated adult rats. No changes were observed 3 hours after KA administration but by 6 hours after the drug administration, ␣1A-a and ␣1A-b isoforms were increased (Table 1). Quantitative analysis of the relative amount of ␣1A-b versus ␣1A-a ⫹ ␣1A-b isoforms showed a decrease, 24 hours after KA, in comparison with control ␣1A-b/(␣1A-a ⫹ ␣1A-b) ⫽ 0.49 ⫾

TABLE 1 ␣1A⫺b /(␣1A⫺a ⫹ ␣1A⫺b ) Ratio in Different Brain Regions

Hippocampus Cortex Cerebellum

Control

3 hours

6 hours

24 hours

7 days

0.73 ⫾ 0.04 n⫽3 0.75 ⫾ 0.07 n ⫽ 14 0.59 ⫾ 0.04 n⫽2

0.68 ⫾ 0.05 n⫽3 0.80 ⫾ 0.06 n⫽3 0.65 ⫾ 0.04 n⫽3

0.54 ⫾ 0.07 n⫽3 0.51 ⫾ 0.02 n⫽3 0.69 ⫾ 0.07 n⫽2

0.49 ⫾ 0.09 n⫽5 0.58 ⫾ 0.05 n⫽4 0.65 ⫾ 0.09 n⫽3

0.52 ⫾ 0.09 n⫽5 0.45 ⫾ 0.07 n⫽8 0.63 ⫾ 0.03 n⫽2

Six hours, 24 hours and 7 days after kainate treatment, ␣1A⫺b /(␣1A⫺b ⫹ ␣1A⫺a ) ratios were decreased in hippocampus and cortex whereas it was unchanged in cerebellum.

Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

294 0.09 and 0.73 ⫾ 0.04 respectively in KA-treated (n ⫽ 5) and control (n ⫽ 3 rats) (Table 1). These modifications were present in hippocampus as well as in cortex (Table 1). By contrast, no changes in the relative amount of these two isoforms were observed in the cerebellum. To test the persistence of the changes induced by epilepsy and separate them from the immediate effects of seizures, KA-treated rat hippocampus were analyzed up to 7 days after KA injection (Table 1). In KA-treated rats, the ␣1A-b/(␣1A-a ⫹ ␣1A-b) ratio decreased in hippocampus (0.52 ⫾ 0.09; n ⫽ 5) and (0.45 ⫾ 0.07; n ⫽ 8) in the neocortex when compared to control values (0.73 ⫾ 0.04; n ⫽ 3; 0.75 ⫾ 0.07; n ⫽ 14 respectively). In the cerebellum the ratio was not significantly modified.

Cellular Expression of ␣1A Isoforms in Control Hippocampus Rat Brain None of the control slides displayed any ISH signal: slides hybridized without probes, or with sense probes or sections digested by RNAse A before the hybridization step (data not shown). The ␣1A-b isoform was present in DG and CA regions with a signal intensity higher in pyramidal cell layer than in DG cells (figure 2 a–d). The ␣1A-a variant exhibited a weak labelling signal from CA4 to CA1 areas and even weaker in the DG area (figure 3 a–d). By contrast, in the cerebellum, the ␣1A-a variant was the predominant isoform in the Purkinje cell layer (data not shown). At the cellular level, it was evident that ␣1A-a and ␣1A-b variants were present in all types of neurons (including pyramidal cells, interneurons and granule cells) in the hippocampus although differentially expressed. Colocalization of the two isoforms in the same neuron was a common pattern of expression (figure 4). For example, 46% of ␣1A-a containing neurons expressed ␣1A-b mRNA and 56% of ␣1A-b containing neurons expressed ␣1A-a mRNAs in the CA3 region. No glial cells were labelled, for example, astrocytes and oligodendrocytes in the Corpus callosum (see figures 2a and 3a).

Cellular Expression of ␣1A Isoforms in KA-treated Hippocampus Rat Brain The pattern of expression of the ␣1A-a and ␣1A-b splice variants was analyzed, by ISH, in hippocampus 24 hours (n ⫽ 3) and 3–7 days (n ⫽ 3) after KA administration and compared with corresponding control animals. 24 hours after KA administration, Nissl staining Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Vigues et al.

failed to detect significant neuronal loss whatever the region studied and an increase in the amounts of both isoform mRNAs was detected in the DG. Hybridization signal for ␣1A-b increased in CA and DG areas (figure 3) but decreased for ␣1A-a in CA1–CA4 areas (figure 2). Densitometric analyses confirmed qualitative data concerning KA-induced changes in the expression of type ␣1A-a mRNAs: an increase in the hybridization signal in DG granule cells (⫹35% related to control animals) and a decrease in CA1 (⫺21%) and CA4 (⫺16%) areas with no changes in CA3 areas. Densitometric analyses also confirmed qualitative data concerning KA-induced changes in the expression of type ␣1A-b mRNAs: an increase in the hybridization signal in DG granule cells (⫹96% related to control animals), in CA1 (⫹60%) and in CA3 (⫹32%) areas. The ISH signal intensity was similar in CA4 area from control and KA-treated rats. No changes in the two isoforms expression were observed in cerebellum in KA-treated in comparison with control rats (data not shown). 3–7 days after KA administration, variable hippocampal neuronal cell loss was apparent with Nissl staining in CA1, CA3 and CA4 areas (data not shown). ISH signal intensity for ␣1A-a and ␣1A-b was increased (figure 5) and was due to an increase in the ISH signal in remaining neurons, as shown by densitometric measurements of several individual neurons. For example in CA4 area, neurons are dispersed and can be analyzed as single units: ␣1A-b ISH signal intensity of the whole area was increased (⫹28% related to control animals) which corresponded to an increase in the signal intensity (⫹18% related to individual CA4 neurons from control animals) and of mRNA content in these remaining neurons. It is noteworthy that no changes were observed in ISH signal intensity for ␣1A-a and ␣1A-b in the cerebellum (data not shown).

DISCUSSION In this study, we report that KA-treated rats exhibited complex and specific spatiotemporal patterns of Ca2⫹ channel ␣1A-a and ␣1A-b subunit mRNA changes. As soon as 6 hours after KA administration, both isoforms are differentially regulated with noticeable brain regional and cellular distribution. While no changes were evidenced in cerebellum, neuronal ␣1A-a and ␣1A-b mRNAs increased, decreased or were unchanged in the hippocampus and the dentatus gyrus. Moreover both splice variants are regulated during the long-term course of the disease (at least 7 days after KA treatment): an upregulation of ␣1A-a and ␣1A-b mRNA

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FIG. 2. ISH detection of mRNAs encoding ␣1A-b isoform in control rat (left panel, a to d) and in rat, 24 hours after kainate administration (right panel, e to h). Antisense digoxigenin-labelled cRNA probe a and e: whole hippocampus area, b and f: dentate gyrus (DG) and CA4 area, c and g: CA3 area, d and h: CA1 area (a and e: scale bar: 300 µm; other pictures: scale bar: 75 µm). ␣1A-b mRNAs were detected in all neurons from DG and from Ammon horn in control rat. 24 hours after kainate administration, the hybridization signal increased in DG granule cells (e, f), in CA3 (g) as well as in CA1 areas (h). Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 3. ISH detection of mRNAs encoding ␣1A-a isoform in control rat (left panel, a to d) and in rat, 24 hours after kainate administration (right panel, e to h). Antisense digoxigenin-labelled cRNA probe a and e: whole hippocampus area, b and f: dentate gyrus (DG) and CA4 area, c and g: CA3 area, d and h: CA1 area (a and e: scale bar: 300 µm; other pictures: scale bar: 75 µm). ␣1A-a mRNAs were easily detected in all neurons from Ammon horn in control rat, while DG granule cells appeared slightly labelled (b). 24 hours after kainate administration, the hybridization signal increased in DG granule cells (e, f), and decreased in CA4 (f), CA3 (g) and in CA1 areas (h).

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results showing that ␣1A-a mRNAs are present in cerebellar purkinje cells where P-type Ca2⫹ channel is present and ␣1A-b is predominant in hippocampus pyramidal neurons where Q-type Ca2⫹ channel regulates neurotransmitter release. In addition, many native P/Q-type ␻-Aga-IVA-sensitive VGCC display variable gating characteristics and different pharmacological sensitivity to ␻-Aga-IVA contributing to the difficulties to classify in situ ␻-Aga-IVA-sensitive VGCC subtypes at cellular level (Mintz et al., 1992; Hilaire et al., 1996). The demonstration of a colocalization of this two splices variants in different proportions from one neuron to another may be a contributing factor of some in situ heterogeneous ␻-Aga-IVA-sensitive channel properties.

␣1A Splice Variant Expressions in KA-treated Hippocampus

FIG. 4. In situ co-localization of ␣1A-a and ␣1A-b in (a) CA4, (b) CA1 and CA2 regions in adult hippocampus. White-labelled cells possess both ␣1A-a and ␣1A-b mRNAs.

contents were observed in the hippocampus of the epileptic rat while no changes were observed for both variants in the cerebellum. Therefore these complex and specific spatio-temporal patterns most likely represent important molecular characteristics of both the epileptogenesis and the long-lasting adaptative changes associated with KA-induced epilepsy, a model of human temporal lobe epilepsy.

␣1Aa Splice Variant Expressions in Normal Hippocampus ␣1A-a and ␣1A-b variants have been recently identified that result from alternative splicing (Soong et al., 1994; Zamponi et al., 1996) and are though to generate two different in situ ␻-Aga-IVA-sensitive Ca2⫹ currents, i.e. P- and Q-type Ca2⫹ channels respectively (Sutton et al., 1998). Present morphological analysis confirmed these

Seizure activity has been shown to alter gene expressions i.e. genes encoding transcription factors, neuropeptides, neurotrophic factors, enzymes for GABA synthesis, glutamate receptors or voltage dependent channels (Nedivi et al., 1993; Sperk, 1994; Khrestchatisky et al., 1995). Such an alteration of splicing during seizure activity has been reported for the flip and flop variants of the AMPA-type glutamate receptor subunits and the isoforms II and III of the Na channels in KA-induced epilepsy (Pollard et al., 1993; Kamphuis et al., 1994; Gastaldi et al., 1997). To our knowledge, it is the first time that modification of VGCC isoforms would be reported in KA-induced epilepsy. Interestingly, alterations in ␣1A isoform expression have been recently reported in genetic model of epilepsy in mice (Ophoff et al., 1996; Doyle et al., 1997). No difference in the pattern of expression of the two isoforms was observed three hours after seizures and six hours was necessary to observe altered gene expression. This new pattern of expression with a global increase of each variant as well as a decrease of the ratio ␣1A-b/(␣1A-a ⫹ ␣1A-b) was seen in the hippocampus and cortex and absent in the cerebellum. At the cellular level, the increase in ␣1A variant mRNAs was only localized in neuronal cells as shown with ISH. Shortterm changes in neuronal VGCC after seizures had also been reported, at the protein level, using the patch-clamp technique (Faas et al., 1996). 24 hours after the last seizure induced by kindling, mean current amplitudes of the high-VGCC in CA1 and DG hippocampal neurons significantly increased (from 25% to 80%) with no or subtle differences in biophysical properties depending of the preparation used (Faas et al., 1996). Such enhancement of Ca2⫹ conductance Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 5. The hippocampus 7 days after kainate administration. a to d: a neuronal cell loss is observed in CA4, CA3 and in CA1 areas. Nissl stain: a: whole hippocampus area: ISH (in situ hybridization) detection of mRNAs encoding ␣1A-b isoform (Antisense digoxigenin-labelled cRNA probe): b: dentate gyrus (DG) and CA4 area, c: CA3 area, d: CA1 area (a: scale bar: 300 µm; other pictures: scale bar: 75 µm)

were supposed to be due to (i) an increase in single channel conductance or mean open time (ii) increase in channel densities or recruitability (iii) changes in sensitivity to intracellular modulating factors (including Ca2⫹). While no pharmacological dissection of the different VGCC had been done by these authors, the present study indicates that an increase in P/Q-type ␣1A subunit density may explain in part this Ca2⫹ current enhancement. Moreover, qualitative differences in biophysical properties of the high-VGCC after kindling have been reported. Since ␣1A-a and ␣1A-b variants differ by their biophysical properties in expression systems, changes in the ratio of these two isoforms may contribute to the observed differences in the characteristics of Ca2⫹ expressed by control and 9epileptic: neurons (Faas et al., 1996). The present results also evidenced a long-lasting upregulation of ␣1A mRNA, in KA-treated rats. Westenbroek et al. (1998) found, using immunochemical method, no change in the expression level of any of the VGCC classes in neurons, 2–3 weeks after KA injection. However, only large changes are easily detected with this technic. Previous ISH study could not evidence Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

change at the RNA level of ␣1A subunits, three/six weeks after the last kindling-induced seizures (Hendriksen et al., 1997). The authors did not analyze, as we did, ␣1A splice variants separately but as a whole, and ex vivo data, using the patch-clamp technique, showed a long-term increase in Ca2⫹ currents, several weeks after kindling-induced generalized seizures (Vreugdenhil & Wadman, 1992).

Regulation of ␣1A Splice Variants in KA-induced Epilepsy Until recently, very little had been known about the signalling mechanisms involved in regulating neuronal VGCC. Using rat hippocampal culture, recent evidence has indicated that glutamate can induce an increase in VGCC currents by stimulating earlyresponse c-fos gene transcriptions (Cavalie et al., 1994). Interestingly, in KA-induced epileptic activity, c-fos mRNAs increase preceeded the increase in ␣1A-b variant mRNAs and major modification of the latter were found in dentate gyrus and in CA1 area of the hippocampus, areas in which NMDA-type glutamate

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receptors are particularly abundant (Popovici et al., 1990). It is possible that the induction of early-response genes by Ca2⫹ entry through NMDA receptor and the regulation of ␣1A variant mRNAs could be initiated by a common Ca2⫹-dependent mechanism. In addition, prolonged in vivo decreased in corticosteroid actions have also been reported to increase VGCC, especially ␣1A, and NMDA receptor subunit mRNA in CA1 hippocampal neurons resulting in increase in cell firing and in neuronal death, particularly when concomitant excitatory challenges are present (Sapolsky et al., 1988; Eliott & Sapolsky, 1993; Nair et al., 1998). This corticosteroid-receptor effect required protein synthesis. In KA-treated rats, glucocorticoid receptor levels were decreased in hippocampus, both in early (3–24 hours) and late (7 days) stages (Lowy et al., 1992). It is tempting to speculate that corticosteroids might regulate coordinate expression of those mRNAs that play a critical role in Ca2⫹ permeability, synaptic transmission and neuronal death in CA1 neurons.

Physiopathological Significance The early- and long-lasting changes in ␣1A subunit mRNA contents in KA-treated rats suggest that VGCC may be involved in the mechanisms generating chronic hyperexitability and/or cellular damage in the epileptic focus. Epileptogenesis is though to be due to an imbalance between excitation (mainly glutamatergic) and inhibition (mainly GABAergic) (Heinemann et al., 1977). The Ca2⫹ influx involved in neurotransmitter release in the hippocampus is most prominantly mediated by the P/Q-type subunit (Dunlap et al., 1995), which is upregulated in the CA1/CA3 and DG areas during the early stages of the KA-induced epileptic activity (Sperk, 1994). Assuming that the changes in mRNA levels are translated into similar changes of the functional protein levels, this ␣1A mRNA upregulation might increase neurotransmitter release as previously reported in this model and consequently change neuronal activity and excitability. At 6 hours following KA treatment, mRNA levels of the ␣1A mRNA splice variants are increased in the hippocampus. These changes precede any lesion as the first signs of neuronal death are observed 12 hours following treatment (Pollard et al., 1993). Changes in ␣1A mRNA levels have been also evidenced during the early stages of the kindling where no neuronal death is present (Hendriksen et al., 1997). Therefore, it is likely that the early changes in ␣1A mRNA expression are a direct consequence of the paroxysmal activity occuring

during the status epilepticus. Conversely, long-lasting changes in these splice variants may be related to neuronal damage since there is also precedent for Ca2⫹ involvement in neuronal death (Choi, 1995; Orrenius & Nicotera, 1994). Enhancement in Ca2⫹ influx and intracellular free Ca2⫹ concentrations cause not only an enhancement of neurotransmitters release but also modulation of Ca2⫹-dependent enzymes involved in signal transduction, gene transcription and sometimes cell death or survival (Dolmetsch et al., 1998; Meldolesi, 1998). Correlative evidence between modifications in neuronal VGCC and neurodegeneration in ischemic injury, seizure activity and ageing have been reported (Cheung et al., 1986; Choi, 1995; Thibault & Landfield, 1996). Moreover, mutations in ␣1A subunit are implicated in cerebellar Purkinje cell degeneration (Burgess et al., 1997). Interestingly, KA-treated rats showed a long-term enhancement of ␣1A variant mRNAs in hippocampus when neuronal damage was present, increase not found in KA-treated rat with nonlesionned hippocampus (data not shown). So, these changes could be linked to the damage and independent of the epileptogenic process. The short- and long-lasting regulations of these two ␣1A splice variants, with unique functional and pharmacological properties, may represent components of a chain of genomic events which result in cellular strategies for protecting neurons against brain injury after seizure insults. VGCC blockers have been proposed as a therapeutic strategy in patients with epilepsy and some clinicallyused antiepileptic drugs modulated VGCC behaviours (Schachter et al., 1995). While substantial safety concerns would have to be addressed before any P/Qtype VGCC inhibition strategy could be deployed, the present study provides additional data indicating that modulating pore-forming ␣1A subunits might benefit patients with intractable temporal lobe epilepsy.

ACKNOWLEDGMENTS We thank S. Valentin and A. Roig for technical assistance. This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM).

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