Research report
Selective up-regulation of protein kinase Cϵ in granule cells after kainic acid-induced seizures in rat

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Abstract

Kainate-induced seizure activity causes persistent changes in the hippocampus that include synaptic reorganization and functional changes in the mossy fibers. Using in situ hybridization histochemistry, the expression of PKCα, PKCβ, PKCγ, PKCδ and PKCϵ mRNAs was investigated in the hippocampus of adult rats following seizures induced by a s.c. injection of kainic acid. In CA1 and CA3, we found a significant decrease in PKCγ mRNA 1 day after kainic acid which persisted for a 2nd day in CA1. None of the other PKC isoform mRNAs were altered in CA1 or CA3. In granule cells, a significant up-regulation specific to PKCϵ mRNA was observed. One week after kainic acid administration, a marked increase in PKCϵ immunoreactivity was found that persisted 2 months after kainic acid administration. PKCϵ immunoreactivity was found associated with mossy fibers projecting to the hilus of the dentate gyrus and to the stratum lucidum of the CA3 field and presumably with the newly sprouted mossy fibers projecting to the supragranular layer. These data provide the first evidence for a long-lasting increase of the PKCϵ in the axons of granule cells caused by kainate-induced seizures and suggest that PKCϵ may be involved in the functional and/or structural modifications of granule cells that occur after limbic seizures.

Introduction

After seizure activity in the hippocampus, there are a number of major changes that include changes in gene expression, alterations in synaptic efficacy, neurodegeneration and nerve sprouting [36]. One model of temporal lobe epilepsy involves systemic injection of kainic acid (KA) [23]which produces in rats a severe convulsive syndrome followed by neurodegeneration of pyramidal cells of the CA3 and CA1 fields and of the polymorphic cells of the hilus of the dentate gyrus 26, 39. As a consequence, a massive sprouting of the mossy fibers of granule cells occurs to form an aberrant supragranular band 24, 36. Recently, we and others have observed that GAP-43, a pre-synaptic protein and specific substrate of protein kinase C (PKC) [1], is rapidly and transiently up-regulated in the granule cells of hippocampus of rats treated with KA 8, 22and was found to be concentrated in the sprouted mossy fiber terminals, suggesting a role of this protein in the synaptic remodelling of these axons [7]. This change in GAP-43 levels has been shown to be dependent upon activation of NMDA receptors in the hippocampus 6, 22, 29. Similar changes in GAP-43 mRNA have been found after hippocampal kindling, suggesting that alterations in GAP-43 expression in granule cells is a common feature in animal models of epilepsy [16].

The events involved in mossy fiber sprouting and GAP-43 up-regulation during epileptogenesis are not known. A large number of molecular changes in hippocampal cells have been characterized after seizure activity [19]. PKC isoforms play a key role in intracellular signal transduction in the CNS and may be important in mediating some of the molecular and cellular changes after seizure activity. PKC is involved in the regulation of many neuronal functions. In the hippocampus, PKC has been shown to modulate the activity of many proteins, including sodium and calcium channels 5, 10, to alter neurotransmitter release [25], to phosphorylate a number of proteins, including GAP-43 1, 21, and modulate synaptogenesis and neurite outgrowth 9, 34. PKC can phosphorylate neurotransmitter receptors, including GABAA and glutamate receptors 20, 32. PKC may play a role in the initiation or maintenance of seizure activity itself: activators of PKC are proconvulsant [37]and a number of groups have reported increases in the levels or activity of PKC after induction of seizures by amygdala or hippocampal kindling 2, 13, 15, 18, 27, 28or by electroconvulsive and pentylentetrazole-induced seizures 11, 12.

Molecular studies have found that in mammalian brain PKC is a family of proteins consisting of three main groups: the classical Ca2+ and phorbol ester-sensitive PKC isoforms (α, β and γ), calcium-independent, phorbol ester-sensitive PKCs (δ, ϵ, η and θ) and calcium- and phorbol ester-insensitive PKCs (ζ and λ) [41]. Immunocytochemical and in situ hybridization studies have revealed that these PKC isoforms are differentially distributed in rat brain and may have distinctive substrate specificities 41, 42. Thus, each PKC subtype might subserve distinct neural functions in the brain. Some isoforms of PKC may be of particular importance during synaptic remodelling in granule cells after hippocampal injury. Kindling-induced seizures have been shown to produce increases in PKC in the hippocampus: Ono et al. [27]reported long-lasting increases in the γ and β isoforms in whole hippocampus after hippocampal kindling and Kamphuis et al. [18]demonstrated increases in the mRNA levels for the PKCϵ and PKCζ isoforms (but not PKCβ or PKCα mRNA) in several hippocampal subfields after hippocampal kindling. It is possible that changes in the levels or activity of PKC are associated with reactive synaptogenesis as well as with long-lasting molecular changes in granule cells. We have, therefore, chosen to examine the regulation of some PKC isoforms in granule cells of the hippocampus by KA-induced seizures activity. In the present study, we used oligonucleotide probes to detect α, β, γ, δ and ϵ PKC mRNAs using in situ hybridization. Since up-regulation of PKCϵ mRNA was found in the granule cells of rats treated with KA, we further analysed the hippocampal distribution of the PKCϵ protein using immunocytochemistry.

Section snippets

Animals treatment

Adult male Sprague-Dawley rats (250–300 g, Charles River, Italy) were housed at constant temperature (21±1°C) and relative humidity (60%) with a fixed 12-h light/dark cycle and free access to food and water. KA (12 mg/kg, Sigma) dissolved in phosphate buffer 0.1 M pH 7.4 was injected s.c. and the animals were observed for 3 h. Their behaviour was rated according to the scale described by Sperk et al. [39]. Only rats exhibiting full limbic seizures, including rearing, loss of postural control

Distribution of PKC isoenzymes mRNA in the brain of control rats

The distribution of the different PKC isoenzyme mRNAs in brain coronal sections of saline-treated rats is shown in Fig. 1.

PKCα mRNA showed high expression in the hippocampus with the maximal signal observed in the CA2 and CA3 pyramidal cells whereas a moderate signal was found in the CA1 region. Lower levels of PKCα mRNA were found in the granule cell layer of the dentate gyrus (Table 1). Moderate expression was found in the primary olfactory cortex and in the most superficial layer of the

Discussion

The pattern of distribution of the PKC isoenzymes mRNA analysed in this study agrees with previous in situ hybridization studies 18, 42. Except for PKCδ mRNA which is expressed almost exclusively in the thalamus, all the other isoenzyme mRNAs examined showed a prominent and unique distribution in the hippocampus. In granule cells, we found only moderate levels of PKCα and PKCβ mRNAs whilst these cells exhibited a high hybridization signal with PKCϵ and PKCγ oligonucleotide probes. PKCϵ was the

Acknowledgements

This study was supported by the National Research Council, CNR, Rome Convenzione Psicofarmacologia and Contract 96.00788.CT04.

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    Present address: Istituto di Clinica Neurologica, Università di Milano, via F. Sforza 35, 20122 Milan, Italy.

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