calmodulin system in neuronal hyperexcitability

The International Journal of Biochemistry & Cell Biology 33 (2001) 439–455 www.elsevier.com/locate/ijbcb

Review

The Ca2 + /calmodulin system in neuronal hyperexcitability Carme Sola` a,*, Sonia Barro´n a, Josep M. Tusell b, Joan Serratosa a a Department of Pharmacology and Toxicology, Institut d’In6estigacions Biome`diques de Barcelona-Consell Superior d’In6estigacions Cientı´fiques (CSIC), Institut d’In6estigacions Biome`diques August Pi i Sunyer (IDIBAPS), c/Rossello´ 161, 6th Floor, 08036 Barcelona, Spain b Department of Neurochemistry, Institut d’In6estigacions Biome`diques de Barcelona-Consell Superior d’In6estigacions Cientı´fiques (CSIC), Institut d’In6estigacions Biome`diques August Pi i Sunyer (IDIBAPS), c/Rossello´ 161, 6th Floor, 08036 Barcelona, Spain

Received 5 November 2000; accepted 17 January 2001

Abstract Calmodulin (CaM) is a major Ca2 + -binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2 + concentration. Calmodulin modulates numerous Ca2 + -dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2 + /CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2 + /CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2 + concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2 + levels described in different pathological situations. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Calmodulin; Calmodulin-binding protein; Convulsion; Central nervous system; c-fos expression

Abbre6iations: CaM, calmodulin; CaMBP, calmodulin binding protein; CaMKII, Ca2 + /calmodulin dependent protein kinase II; CaN, calcineurin; GABA, g-aminobutyric acid; HCH, hexachlorocyclohexane; KA, kainic acid; NMDA, N-methyl-D-aspartate. * Corresponding author. Tel.: + 34-93-3638328; fax: + 34-93-3638301. E-mail address: [email protected] (C. Sola`). 1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 0 3 0 - 9

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Contents 1. 1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Calmodulin and seizure activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Anticonvulsant action of Ca2 + and CaM antagonists . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. CaM levels after seizure activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Calmodulin and the induction of c-fos expression by convulsant agents. . . . . . . . . . . . . . . . . . . 3.1. CaM and the induction of c-fos expression in cell cultures. . . . . . . . . . . . . . . . . . . . . . . 3.2. CaM and induction of c-fos expression in experimental animals . . . . . . . . . . . . . . . . . . .

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4. Expression of calmodulin in neuronal hyperexcitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Modifications in the expression of CaM after g-HCH seizures . . . . . . . . . . . . . . . . . . . . 4.2. Modifications in the expression of CaM after KA seizures. . . . . . . . . . . . . . . . . . . . . . .

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5. Expression of calmodulin binding proteins in neuronal hyperexcitability . . . . . . . . . . . . . . . . . .

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6. Concluding comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Calmodulin (CaM) is a Ca2 + -binding protein present in all eucaryotic cells, which has a major role as Ca2 + mediator in non-muscle and smooth muscle cells [1,2]. It is a small, acidic, heat-stable, monomeric protein with a molecular weight of about 17 000. The molecule has four similar domains, each containing one Ca2 + -binding site. The binding of Ca2 + alters the conformation of CaM, and the resulting CaM-Ca2 + complex can interact with target proteins and modulate their activity (Fig. 1). Numerous CaM-binding proteins (CaMBPs) have been described in relation to a wide range of cellular functions, from structural roles to the regulation of gene expression. CaM is also important in the regulation of cell proliferation [3]. CaM is highly abundant in the mammalian central nervous system [4,5], and it is specially enriched in sites involved in neurotransmission, such as postsynaptic membranes [6,7], postsynaptic densities [8–10] and synaptic vesicles [11]. It is widely distributed in the brain, where it modulates the action of numerous Ca2 + -dependent enzymes, including adenylate cyclase, Ca2 + -ATPase, cyclic nucleotide phosphodiesterase and certain protein

kinases [2] (Table 1). CaM is involved in the synthesis of neurotransmitters, such as noradrenaline and serotonin [12,13]. It also participates in synaptic function and neurotransmitter release, playing a role in the Ca2 + -stimulated phosphorylation of synaptic proteins [14]. In addition, CaM controls the function of the cytoskeleton, where it regulates the assembly and disassembly of microtubules [15]. CaM is also involved in the Ca2 + -mediated regulation of gene expression. Among the different genes that are regulated by changes in intracellular Ca2 + , the immediate-early genes are the most rapidly induced. They often encode transcription factors, such as c-fos and c-jun. Such genes may function as third messengers in an intracellular cascade linking extracellular stimuli to long-term adaptative processes (Fig. 2). CaM plays a role in a signal transduction pathway leading to c-fos gene activation in response to certain stimuli [16,17]. In addition, CaM is also directly or indirectly involved in the regulation of transcription factors controlling the expression of certain groups of genes [18–20]. The 148-amino acid sequence of CaM is highly conserved through evolution, and is encoded by multiple genes in vertebrates. However, the

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Fig. 1. The Ca2 + /CaM signalling system. Schematic diagram of intracellular Ca2 + signal processing by CaM. (A) CaM has four Ca2 + binding sites with reasonably high affinity (Ka  10 − 6 M − 1). In the resting state of the cell (Ca2 + concentration of  10 − 7 M), very few CaM molecules have Ca2 + bound. (B) In response to stimulation, influx of Ca2 + raises its cellular concentration to  10 − 5 M. The binding of 1–4 Ca2 + modifies the conformation of CaM. (C) The resulting Ca2 + -CaM complex can specifically interact with target proteins (CaMBPs) and modulate their activity. More than 40 different proteins and enzymes are modulated by CaM in a Ca2 + -dependent manner.

protein produced by the different genes within a vertebrate organism is always identical. The presence of different genes encoding exactly the same Table 1 CaM functions in the central nervous system Cyclic nucleotide metabolism Ca2+ pumps Ion transport Protein phosphorylation/dephosphorylation Cytoskeletal function Neurotransmitter synthesis Synaptic function and neurotransmitter release Others (nuclear functions, …)

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Fig. 2. CaM regulation of early gene expression. c-fos activation in response to certain stimuli is modulated by CaM, linking extracellular stimuli to long-term adaptative processes. ROCC, receptor-operated Ca2 + channel; VSCC, voltage-sensitive Ca2 + channel.

protein, all of which have a high basal expression, reflects the importance of this protein in the normal function of the cells. Three genes encoding CaM have been isolated from the rat brain [21– 23], namely CaM I, CaM II and CaM III. The coding regions of the three genes are identical, but differences in their 3% and 5% non-coding regions suggest a differential regulation of the genes. CaM I produces two transcripts of different sizes, 1.7 and 4.0 kb, CaM II a single transcript of 1.4 kb and CaM III two transcripts of 2.3 and 1.0 kb. The two mRNA species for CaM I and CaM III genes are the result of alternative polyadenylation signals. cDNAs representing the transcripts for the three CaM genes have been obtained from the mouse, although only the gene that is equivalent to rat CaM II has been isolated to date [24– 27]. cDNAs representing the transcripts for the three CaM genes have also been obtained from hu-

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mans, and the genes equivalent to rat CaM I, CaM II and CaM III have been isolated [28– 31]. All the CaM genes are widely expressed throughout the rodent brain [32– 35], although a different pattern of expression of the three genes has been observed during development [36]. A different intracellular distribution of the mRNAs for the three CaM genes has also been suggested [37]. In addition, different modifications in the expression of CaM genes have been observed in several experimental situations [34,38–46]. Nevertheless, the mechanisms involved in the regulation of the different CaM genes remain unknown. The transduction of a Ca2 + signal by CaM requires the interaction of the complex Ca2 + CaM with specific proteins. Among the different cerebral CaMBPs, the Ca2 + /CaM dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN; phosphatase 2B, PP-2B) are especially important because they are abundant and they are involved in numerous neural functions. CaMKII constitutes between 0.25 and 2% of the total protein of the brain, and it is especially enriched in the hippocampus, where it accounts for 1–2% of total protein. The kinase phosphorylates and regulates a large number of substrates. CaN is the only CaM-dependent phosphatase known, and it constitutes 1% of total protein. Although CaN has a narrow substrate specificity, it can trigger a protein phosphatase cascade involving the dephosphorylation of numerous substrates (for review see [47–50]). Depending on the way Ca2 + enters the neuron, it interacts with different Ca2 + -binding proteins leading to the activation of specific intracellular signalling pathways, which are responsible for the cellular response to the initial Ca2 + signal. Here we present an overview of the different studies we have performed on the role of CaM as mediator of Ca2 + signal in the central nervous system in situations of neuronal hyperexcitability induced by convulsant agents. We performed in vitro and in vivo studies using different convulsant agents and we used several experimental approaches to determine the involvement of CaM in the cellular response to these agents (Fig. 3).

2. Calmodulin and seizure activity Convulsant agents induce seizures in the experimental animals through their interaction with different kinds of receptors. Activation of Nmethyl-D-aspartate (NMDA) receptors and their associated Ca2 + channels has been reported to induce seizures [51]. In contrast, competitive and non-competitive antagonists of NMDA receptors are anticonvulsant [52–54]. g-Aminobutyric acid A (GABAA) receptor antagonists also induce seizures, which can be inhibited by dihydropyridine Ca2 + antagonists [55,56]. These facts suggest that Ca2 + may play a critical role in seizure etiology. We suggested that Ca2 + mobi-

Fig. 3. The Ca2 + /CaM system and neuronal hyperexcitability. Schematic representation of different approaches considered to study the involvement of the Ca2 + /CaM signalling system in the seizure activity induced by convulsant agents. NMDA, KA, or BayK and g-HCH are used as convulsant agents acting at the NMDA receptor (R), the AMPA/KA-R or the L-type voltage-sensitive Ca2 + channel (VSCC), respectively. The VSCC antagonist nifedipine, the central nervous system depressant d-HCH and the CaM antagonist W-7 are used as potential anticonvulsant agents.

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Table 2 Anticonvulsant activity of d–HCH, nifedipine and W-7a Anticonvulsant agents

d-HCH (up to 300 mg/kg) Nifedipine (up to 20 mg/kg) W-7 (up to 75 mg/kg)

Convulsant agents g-HCH

BayK-8644

KA

NMDA

b

b

c

c

b

b

b

d

b

b

c

e

a Each anticonvulsant agent was injected i.p. 30 min before administration of convulsants. Presence and absence of convulsions (loss of righting reflex) was noted during a 30 min observation period after convulsant drug administration. b Positive anticonvulsant activity. c No anticonvulsant activity. d 25% protection at 20 mg/kg. e No dose-response relationship.

lization might be involved in the convulsant action of g-hexachlorocyclohexane (HCH) [57], whose mechanism of action has been associated with its interaction with the GABAA receptor. In addition we described the involvement of Ca2 + and CaM in the seizure activity induced by different convulsant agents [57– 59].

2.1. Anticon6ulsant action of Ca 2 + and CaM antagonists Different convulsant agents — the GABAergic antagonist g-HCH, the Ca2 + channel agonist BayK-8644 and two agonists of the excitatory amino acid receptors, kainic acid (KA) and NMDA- are able to induce seizures in experimental mice. d-HCH, a strong depressant of the central nervous system, the voltage-sensitive Ca2 + channel antagonist nifedipine and/or the CaM antagonist W-7 can selectively inhibit these seizures [57,59] (Table 2). Thus, d-HCH protects from convulsions induced by g-HCH and BayK8644, but it fails to antagonize the seizures induced by KA and NMDA. Nifedipine antagonizes the seizures induced by g-HCH, BayK-8644 and KA and partially protects against the convulsions induced by NMDA. W-7 antagonizes the convulsions induced by g-HCH and BayK-8644 and partially protects against the convulsions induced by NMDA, but it has no effect on the convulsions induced by KA. These results show how the mechanism of action of different

convulsant agents is associated with Ca2 + mobilization and in some cases with CaM. However, the effect of the anticonvulsants used was different depending on the convulsant agent considered, which permits relationships to be established between the mechanisms of action of the different convulsant agents. Thus, although g-HCH acts theoretically on the GABAA receptor, the response it gives to the different anticonvulsants suggests that other mechanisms are probably involved in its mechanism of action. In fact, g-HCH and BayK-8644 gave similar responses to the different anticonvulsant agents tested, suggesting that g-HCH has a direct or indirect action on the Ca2 + channels. The two glutamate agonists, NMDA and KA, partially differed in their pattern of response to the anticonvulsants tested, showing that their convulsant action is associated with Ca2 + mobilization but that the pathways involved are different.

2.2. CaM le6els after seizure acti6ity Once established that different convulsant agents need CaM for the induction of convulsions, such as g-HCH and BayK-8644, it is interesting to know what happens to CaM levels. Although these convulsant agents do not induce significant modifications in the levels of CaM in brain homogenate nor in cytosolic or synaptosomal fractions, they induce a 1.7-fold increase in nuclear CaM levels (Table 3) [58]. In addition, the

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administration of the anticonvulsant agents dHCH or nifedipine prior to g-HCH decrease CaM levels to control values. The effect of BayK-8644 is reversed by nifedipine. The increase observed in nuclear CaM levels in the brain after inducing seizures with g-HCH and BayK-8644 is probably a consequence of the increased Ca2 + influx through the L-type voltage-sensitive Ca2 + channels, as convulsions induced by these agents are inhibited by nifedipine. This increase appears to be necessary for the development of the seizures induced by g-HCH and BayK-8644, since these seizures are inhibited by W-7.

3. Calmodulin and the induction of c-fos expression by convulsant agents The immediate early genes, such as c-fos, which are rapidly activated by different stimuli, have been proposed to act as third messengers in the cascade linking external stimuli to long-term cellular responses. Different stimuli, such as an increase in the intracellular Ca2 + concentration, result in c-fos activation, and several signal transduction pathways can mediate such activation [16,60,61]. c-Fos activation has been proposed as a marker of neuronal injury, because it is induced when alterations in the normal function of the Table 3 CaM concentration in neuronal nucleia Treatments

Control g-HCH d-HCH-g-HCH Nifedipine-BayK-8644 BayK-8644 Nifedipine-g-HCH

CaM concentration (mg/mg protein) 0.57 0.99b 0.61 0.29b 0.95b 0.41

a CaM concentration was measured by using the activation of cyclic AMP phosphodiesterase as a test [58]. The values given are the mean of three separate samples of nuclei. Each sample of nuclei corresponds to 5 pooled animals. S.E.M. was B10% in all cases. CaM concentration is expressed in micrograms of CaM per milligram of protein. b PB0.01, significantly different from control. Student’s ttest.

Fig. 4. Effect of W-7 on c-fos-induced immunoreactivity in cell cultures, shown as the percentage of c-fos-immunoreactive neurons in control culture (C) and in cells maintained in contact with g-HCH (g; 10 mM, 10 min), BayK-8644 (BayK; 1 mM, 2 h), KA (7.5 mM, 2 h), and NMDA (10 mM, 2 h). W-7 (10 mM, 6 h) was added to the cultures alone or before convulsants. Data are mean 9S.D. (bars) values of at least three separate duplicate experiments. *P B0.05 by Student’s t-test compared with control values.

brain occur [62]. Convulsant agents induce c-fos expression. Here we show the involvement of CaM in the signal transduction pathways involved in c-fos induction by several convulsant agents, whose mechanism of action is associated with Ca2 + influx through different pathways.

3.1. CaM and the induction of c-fos expression in cell cultures In primary cortical cultures, which are constituted by a mixture of neurons and glial cells, 30% of neurons display basal c-fos nuclear immunostaining [17]. Neurons form clumps surrounded by astrocytes, but only the neurons express c-fos protein. The addition of different convulsant agents to the culture, such as g-HCH, BayK-8644, KA, and NMDA, results in an increase in c-fos immunostaining (Figs. 4 and 5). Pretreatment of cells with W-7 blocks this increase in g-HCH-, BayK-8644- and KA-treated cells (Figs. 4 and 5), while W-7 has no significant effect on NMDAtreated cells (Figs. 4 and 5). These alterations of c-fos immunostaining detected are correlated to

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alterations of c-fos mRNA levels [17]. These results show the involvement of CaM in the increased c-fos expression induced by g-HCH, BayK-8644 and KA in cell cultures.

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3.2. CaM and induction of c-fos expression in experimental animals Experimental mice showing seizures after the administration of g-HCH, BayK-8644, KA or NMDA present an induction of c-fos mRNA expression in the brain showing specific patterns (Fig. 6). Hippocampus and cerebral cortex are the brain areas mainly showing c-fos induction. The dentate gyrus shows the highest levels of c-fos expression, with the exception of BayK-8644 treatment, which induces a strong expression in the cerebral cortex. c-fos expression induced by g-HCH or BayK-8644 is blocked by the previous administration of W-7 (Fig. 6). Mice treated with W-7 before KA or NMDA do not elicit convulsions, but they show hyperexcitability and tremors. In these cases, c-fos expression is inhibited in the cerebral cortex but is still present in the hippocampus (Fig. 6). Therefore, CaM has a role in the induction of c-fos expression by g-HCH or BayK-8644. In the case of KA and NMDA the partial inhibition of c-fos expression observed could be either due to an indirect effect of W-7 through suppression of convulsions or to the fact that other signal transduction pathways are also involved. 4. Expression of calmodulin in neuronal hyperexcitability

Fig. 5. Microscopic images of c-fos immunoreactivity in cell cultures. (A) Basal c-fos immunostaining in control cells. B –N, c-fos immunostaining after different treatments. (B) W-7; (C) g-HCH; (D) W-7 and g-HCH; (E) BayK-8644; (F) W-7 and BayK-8644; (G) KA; (H) W-7 and KA; (I) NMDA; (J) W-7 and NMDA. Doses and schedule of administration were as in figure 4. Bar =30 mm.

In the mammalian central nervous system, three CaM genes coexist to produce exactly the same protein. All CaM mRNAs are mainly localized in gray matter structures, showing a preferential neuronal localization [35]. In general, CaM mRNAs are preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II mRNA also in striatum. Nevertheless, CaM mRNAs show a different pattern of cellular distribution in the hippocampus and the cerebellar cortex. CaM immunoreactivity is widely distributed in the mouse brain, mainly in gray matter structures. A strong immunoreactivity is observed in neuronal cells throughout the brain, cerebral cortex, striatum, hippocampus, septum, thalamus, cerebellum, brainstem nuclei. At the subcellular level, CaM immunostaining is localized in the

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Fig. 6. c-fos mRNA expression in mouse brain after treatments with convulsants or with W-7 30 min before convulsants. (A) Control animals; (B) W-7 (75 mg/kg); (C) g-HCH (60 mg/kg); (D) W-7 before g-HCH; (E) BayK-8644 (6 mg/kg); (F) W-7 before BayK-8644; (G) KA (60 mg/kg); (H) W-7 before KA; (I) NMDA (80 mg/kg); (J) W-7 before NMDA. CA1, CA2 and CA3, fields of Ammon’s horn; DG, dentate gyrus; RS, retrosplenial cortex; Pir, piriform cortex.

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Fig. 7. Effect of different treatments on CaM I and CaM II mRNA expression. All animals, at least three for each group, were killed 24 h after administrations. In ordinate, the levels of each CaM mRNA transcript relative to that seen in control group. (A) Effect on CaM I transcripts. (B) Effect on CaM II transcript. In abscise, C are control animals (olive oil treated); g, animals treated with g-HCH (30 mg/kg); d, animals treated with d-HCH (100 mg/kg); and d-g, animals treated with d-HCH prior to g-HCH administration. The gels were quantified by a spot scanner and normalized using an actin probe as reference. The values are the mean of at least four experiments. Standard deviations were in all cases less than 15% of the mean. Statistical analysis performed using the Student’s t-test revealed that the differences between values of control animals and all the other treatment groups are significant at P B 0.001.

nucleus, where it is specially strong, as well as in the cytoplasm and the cellular processes. As explained above, Ca2 + mobilization plays a role in the development of convulsions in experimental animals. In addition, CaM levels are modified after seizures elicited by specific convulsant agents and CaM is involved in the c-fos activation induced by some convulsant agents. These results suggest an active role of the Ca2 + CaM system in the response of the central nervous system to hyperexcitability. As mentioned in the Section 1, CaM genes are differentially regulated in the brain during development. In addition, the expression of one or another CaM gene is modified in the adult rat brain in several experimental conditions [38– 40]. We shall now report modifications in the expression of CaM genes in the mouse brain after inducing neuronal hyperexcitability.

4.1. Modifications in the expression of CaM after k-HCH seizures The administration of g-HCH to experimental

rats induces neuronal excitation resulting in seizures. These effects occur in the absence of neuronal death. The expression of CaM I mRNAs (two transcripts of 4.0 and 1.7 kb), which are expressed in brain and other tissues, and CaM II mRNA, mainly expressed in the brain, is modified in the rat brain as a consequence of g-HCH administration [41]. The expression of CaM II mRNA is drastically reduced in the cerebral cortex of g-HCH-treated animals (Fig. 7), while the 4.0 kb CaM I mRNA decreases to a lesser extent (Fig. 7). Moreover, the central nervous system depressant d-HCH induces a decrease in CaM I mRNAs expression (Fig. 7). When d-HCH is administered prior to g-HCH, the level of the 4.0 kb CaM I mRNA is lower than when g-HCH is administered alone (Fig. 7). These results show that the expression of CaM genes is modified in response to changes in neuronal activity. CaM I mRNAs show a decrease after the administration of the convulsant agent g-HCH and the central nervous system depressant d-HCH. In contrast, CaM II mRNA expression is decreased after the administration of the convulsant agent, but is

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slightly increased after the administration of dHCH. The differential effect of d-HCH in the expression of the CaM gene selectively expressed in the brain may be related to the opposite effect of this agent in the neuronal excitability in relation to the other agents tested. These results suggest a differential regulation of CaM I and CaM II gene expression in the rat brain.

actly the same protein. However, as the effect induced in CaM I mRNAs is larger than the effect induced in CaM II mRNA, a resultant increase in the expression of CaM may be expected. These results clearly suggest a differential regulation of the three CaM genes in the mouse brain.

4.2. Modifications in the expression of CaM after KA seizures

5. Expression of calmodulin binding proteins in neuronal hyperexcitability

The expression of CaM I, CaM II and CaM III mRNAs and CaM immunoreactivity are modified in the mouse brain after the administration of a convulsant dose of KA, a neurotoxic agent widely used in experimental models of neuronal damage [44]. We center our interest in the hippocampus, the main target of the neurotoxic action of KA, and the results described hereafter correspond to it. The expression of the three CaM genes is modified after KA induced seizures, although each gene gives a different response. Thus, CaM I mRNAs expression is rapidly and transiently increased in KA-treated animals, while there is a transient decrease in CaM II mRNA (Fig. 8). CaM III mRNA is mainly unaffected in the mouse hippocampus, with the exception of the dentate gyrus, where a rapid and transient decrease occurs (Fig. 8). CaM immunoreactivity, determined 24 h after KA administration, increases in the pyramidal cell layer of Ammon’s horn of KA-treated mice. A large number of CaM-immunoreactive glial cells are detected throughout the hippocampus of KA-treated mice (Fig. 9). The effects above described are always associated with no neuronal damage, as tested by histological stainings. KA-induced seizures result in both an increase and a decrease in the expression of CaM I and CaM II genes, respectively, while the presence of CaM is apparently increased, at least in the hippocampus. It is difficult to establish a correlation between the effect of KA-induced seizures on the levels of CaM mRNAs and the effect on the protein level, because the individual contribution of each CaM gene to the total CaM level cannot be discriminated, as the three genes encode ex-

The transduction of a Ca2 + signal via CaM requires the interaction of the Ca2 + -CaM complex with a series of effector proteins, or CaMBPs, which are specifically modulated by it. Among the different CaMBPs, the kinase CaMKII and the phosphatase CaN are especially important in the central nervous system. They are highly concentrated in the brain and are involved in numerous cellular functions. As these enzymes share common substrates, a balance between their phosphorylation-dephosphorylation activities has been suggested to constitute a relevant mechanism in the control of protein function at the postranslational level. Among the multiple isoforms described for CaMKII and CaN, we center the interest on the most abundant isoforms in the brain, CaMKIIa, CaMKIIb and CaN Aa isoforms. Experimental mice administered with a convulsant dose of KA show a transient decrease in CaMKIIa, CaMKIIb and CaN Aa subunit mRNAs in the hippocampus (Figs. 10 and 11) [63]. In contrast, no alterations in CaMKIIa or CaN A immunoreactivity are observed after KA-treatment. The modifications observed could be due to the neuronal hyperexcitability induced by KA rather than neuronal degeneration, because no areas of neuronal loss were observed. These results suggest that CaMKII and CaN mRNAs are down-regulated in neuronal cells in response to the hyperexcitability induced by KA. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage. A decrease in the expression of CaMKII mRNAs, protein or enzymatic activity is also observed in

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other situations of neuronal hyperexcitability, such as kindling [64,65], recurrent limbic seizures induced by a electrolytic lesion [66] and status epilepticus [67,68]. In fact, increases in neuronal activity are usually associated with a decrease in either the expression or the activity of the kinase, while decreases in neuronal activity are associated with an increase in these parameters

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[64– 66,69,70]. In the case of CaN, such a relationship can not be established because the existing data are controversial [71–73]. However, Moriwaki et al. [74] show that the CaN inhibitor FK506 has no effect on the seizure activity induced by KA, but it protects against the neuronal dysfunction induced by this neurotoxic agent.

Fig. 8. Time course of CaM mRNAs expression in the hippocampus after KA treatment. Data represent percentage of modification of the hybridization signal in several areas of the hippocampus of KA-treated mice killed at different times after treatment (5, 24 h, and 2, 4 and 8 days) versus the signal in the equivalent controls (C). Measurements of the hybridization signal were obtained by densitometric analysis in the pyramidal cell layer from different fields of Ammon’s horn (CA1, CA2 and CA3) and the granule cell layer of the dentate gyrus (DG) (n, three to five mice per group). Error bars indicate S.E.M. Significant modifications, *P B 0.05, Student-Newman– Keuls multiple comparison test.

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Fig. 9. CaM immunoreactivity in the hippocampus of control and KA-treated mice (24 h). (A, B) Cresyl violet staining of coronal 50-mm vibratome sections of a control (A) and a KA-treated mouse (B), showing that there are no areas of neuronal loss. (C) CaM immunoreactivity in a control hippocampus, where immunostaining is mainly localized in the pyramidal cell layer of Ammon’s horn and the granule cell layer of the dentate gyrus. (D) CaM immunostaining in a KA-treated mouse showing both an increased immunoreactivity in the pyamidal cell layer (arrows) and the presence of numerous immunoreactive glial cells (arrowheads). (E, F) Higher magnification of a portion of the images shown in Ca and D showing CaM immunoreactivity in the pyramidal cells of the CA1 field (arrows) and the cerebral cortex (small arrows) of a control (E) and a KA-treated mouse (F), as well as the presence of immunoreactive glial cells in the KA-treated animal (arrowheads). (G) Cellular localization of CaM immunostaining in the pyramidal cells of the CA1 field in a 5-mm paraffin section from a KA-treated mouse. Immunohistochemical studies were performed in a total of seven control and seven KA-treated mice. CA1, CA2, and CA3, fields CA1-CA3 of Ammon’s horn; DG, dentate gyrus; G, granule cell layer of the dentate gyrus; Py, pyramidal cell layer of Ammon’s horn. Scale bars = 500 mm (A– D), 100 mm (E, F), 20 mm (G).

6. Concluding comments In summary, different evidences show the implication of the Ca2 + /CaM signalling system in neuronal hyperexcitability induced by convulsant agents. Firstly, Ca2 + mobilization is involved in

the development of neuronal excitability and seizures induced by different convulsant agents (g-HCH, BayK-8644, NMDA and KA). Accordingly, seizures induced by these agents can be inhibited by the voltage-sensitive Ca2 + channel antagonist nifedipine. Moreover, the CaM antag-

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onist W-7 is able to inhibit the seizures induced by most of these convulsants, at least in part, implicating the Ca2 + -CaM signalling system in the development of the seizures. In addition, at least

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seizures induced by g-HCH and BayK-8644 are associated with an increase in CaM levels. Secondly, at the molecular level, CaM is differentially involved in the transduction of Ca2 +

Fig. 10. CaMKII mRNAs expression in the hippocampus after KA administration. Autoradiographic images showing CaMKIIa (A, C, E and G) and CaMKIIb (B, D, F and H) mRNAs hybridization signal in consecutive coronal sections from control (A and B) and KA-treated mice killed 5 h (C and D), 24 h (E and F) and 8 d (G and H) after treatment. KA-treatment induced a decrease in the signal for CaMKII mRNAs both in the pyramidal cell layer of Ammon’s horn and the granule cell layer of the dentate gyrus. Abbreviations, CA1, CA2 and CA3, CA1-3 fields of Ammon’s horn; DG, dentate gyrus; G, granule cell layer of the dentate gyrus; Py, pyramidal cell layer of Ammon’s horn.

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Fig. 11. CaN mRNAs expression in the hippocampus after KA administration. Autoradiographic images showing CaN Aa mRNA hybridization signal in consecutive coronal sections from a control (A) and KA-treated mice killed 5 h (B), 24 h (C) and 8 d (D) after treatment. KA-treatment induced a decrease in the signal for CaN Aa mRNA in both the pyramidal cell layer of Ammon’s horn and the granule cell layer of the dentate gyrus. Abbreviations, CA1, CA2 and CA3, CA1-3 fields of Ammon’s horn; DG, dentate gyrus; G, granule cell layer of the dentate gyrus; Py, pyramidal cell layer of Ammon’s horn.

signals leading to an increase in c-fos expression after inducing convulsions with a GABAA receptor antagonist (g-HCH), a L-type voltage-sensitive Ca2 + channel agonist (BayK-8644), or a NMDA or a non-NMDA receptor agonist (NMDA or KA). In some cases (g-HCH and BayK-8644) the induction of c-fos expression is mediated by a Ca2 + /CaM dependent pathway (W-7 sensitive), whereas in other cases (NMDA and KA) it is mediated, at least in part, by a CaM independent pathway (W-7 insensitive). These differences agree with the hypothesis that differences in the transduction of Ca2 + signals are due to the presence of spatially different sites of Ca2 + entry into neurons, resulting in the activation of different enzymes located at different sites in the cell [16]. Thus, although CaM appears to be necessary for the development of seizures induced by the different convulsant agents tested, the induction of c-fos by these convulsant agents does not necessarily depend on CaM. Thirdly, CaM gene expression is modified after g-HCH- and KA-induced seizures. The expression of each CaM gene is specifically modified after the seizures induced by these convulsant agents, which is in accordance with the presence of a differential regulation of CaM genes. In the brain, a differential response of CaM genes to several stimuli has been observed in various experimental situations [34,39,41, 44,46]. These results are in favour of a very precise control of CaM levels through the different contribution of each CaM gene, and show how the same stimulus can induce, inhibit or even have no effect on the expression of each CaM gene. Finally, a transient decrease in the expression of CaMKII and CaN mRNAs is observed in the hippocampus after inducing convulsions with KA. A decrease in CaMKII expression has been reported in several experimental situations associated with an increase in neuronal activity, while an increase in the expression of the kinase has been associated with a decrease in neuronal activity [67–71]. This relationship has not been established for CaN. The expression of these Ca2 + /CaM-binding proteins appears to be down-

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regulated in response to the increased neuronal excitability and intracellular Ca2 + levels induced by KA. However, no significant changes at the protein level are observed in neuronal cells, suggesting that the neuronal insult induced with KA and the transient effect at the mRNA level were not strong enough to reflect significant changes at the protein level. In conclusion, the Ca2 + /CaM signalling system is involved in the development of seizures induced by different convulsant agents. However, processes occurring in association with seizures, such as c-fos gene expression, are not always sensitive to CaM, but depend on the convulsant agent. In addition, at least some of the convulsant agents considered modify CaM levels and CaM gene expression after inducing seizures. Alterations in the expression of the effector proteins CaMKII and CaN are observed after KA-induced convulsions. Therefore, the Ca2 + /CaM signalling system plays a role in the neuronal hyperexcitability associated to the seizure activity induced by different convulsant agents, being involved either directly in the development of seizures or in different processes associated to the neuronal dysfunction induced in parallel by the convulsant agents. Given the relevance of the Ca2 + /CaM signalling system in the processing of a Ca2 + signal and taking into account that alterations in the homeostasis of Ca2 + are very often associated with the development of neuronal damage, the study of the Ca2 + /CaM signalling system both in physiological and pathological conditions is a subject of interest. Alterations in this system, especially in CaMKII or CaN levels or activity, have been reported in different situations of neuronal damage, such as ischemia, neurotoxic agents, hypoxia/hypoglycemia or in neurodegenerative diseases.

Acknowledgements This work was partially supported by grants SAF 98-0067 from CICYT (Comisio´ n de Investigacio´ n Cientı´fica y Te´ cnica) and Fundacio´ la Marato´ de TV3 010/97.

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