Kainic acid seizure suppression by neuropeptide Y is not correlated to immediate early gene mRNA levels in rats

Neuroscience Letters 271 (1999) 21±24

Kainic acid seizure suppression by neuropeptide Y is not correlated to immediate early gene mRNA levels in rats Torsten M. Madsen a, b,*, David P.D. Woldbye c, Tom G. Bolwig a, Jens D. Mikkelsen b,1 a

Neuropsychiatry Laboratory, Department of Psychiatry, Rigshospitalet, Copenhagen, Denmark b H. Lundbeck A/S, Department of Neurobiology, DK±2500 Valby, Denmark c Department of Pharmacology, Copenhagen University, Copenhagen, Denmark Received 26 April 1999; received in revised form 28 May 1999; accepted 31 May 1999

Abstract Kainic acid induces seizures and a rapid induction of immediate early genes and neuronal death. Neuropeptide Y (NPY) is implicated in seizure inhibiting activity. In order to investigate the mechanisms by which NPY inhibits seizure activity, this study was carried out to measure the levels of mRNAs encoding three different immediate early genes, in regions of the hippocampus and relate their induction to the behaviour in the same animals. NPY inhibited both the time spent in seizures, and the number of generalized seizures. However, NPY did not inhibit the induction of c-fos, FosB or junB mRNA in any hippocampal region examined in the same animals, showing lack of correlation between immediate early gene induction and seizure activity q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: c-fos; fosB; junB; Seizure activity; In-situ hybridization; Image analysis

Systemic kainic acid (KA) injections in rats produce generalized convulsive seizures, a condition that resembles temporal lobe epilepsy and status epilepticus in man [2]. Days after the KA administration neuronal degeneration occurs in several areas of the brain, most notably in entorhinal cortex, amygdala and in the CA3 area and hilar interneurons of the hippocampus [3,14]. KA activates a polysynaptic pathway that involves both AMPA and kainate receptors [7]. KA acts on both types of receptors, leading to intraneuronal Ca 21 in¯ux, and excitatory synaptic activity [5], and KA also acts presynaptically on kainate receptors located on GABAergic interneurons, that causes a depression of inhibitory synaptic transmission [4]. Altogether, KA increases excitatory activity in the brain and induces a rapid transcriptional activation of several immediate early genes (IEGs) [13,16,17]. The protein products of these genes, such as Fos, FosB and JunB control the expression of genes containing an AP-1 binding site in the promotor region. However, the long-term consequences of an altered gene expression are poorly understood as is the identi®cation of genes that secondarily are affected by the * Corresponding author. Fax: 145-3545-6218. E-mail address: [email protected] (T.M. Madsen) 1 Present address: Zealand Pharmaceuticals A/S, Agern Alle 3, DK-2970 Hùrsholm, Denmark.

IEGs. The aim of this study was to identify the neurobiological background of events linked to seizures and neurotoxicity by studying the effect of seizure inhibition on IEG induction and behaviour. Neuropeptide Y (NPY), a 36 aminoacid peptide, is implicated in a number of physiological mechanisms in the CNS, including feeding, circadian rhythm and neuroendocrine regulation. When injected intracerebroventricularly shortly before KA administration, NPY signi®cantly inhibits the time spent in KA induced status epilepticus [17]. It has been shown that KA induces expression of NPY in both the hippocampus and in the piriform cortex [15,16], and because the NPY gene contains a promotor with an AP-1 binding site [6], this gene is likely to be one of those activated by binding complexes containing Jun-Fos dimers. Kainic acid induces a range of different seizure phenomena during its time of action. Initially, staring spells and wet dog shakes are observed, and later generalized seizures with clonic activity of forelimbs and neck occur. These episodes consist of rearing and loss of postural control resembling grade 4 and 5 seizures as de®ned by Racine et al. [12]. In this study we wanted to examine the effect of NPY on several aspects of KA induced generalized seizure behaviour. Also, given the inhibitory effect of NPY on seizures, we wanted to know if this inhibition is linked to decreased induction of the mRNAs of the immediate early genes.

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 50 7- 8

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T.M. Madsen et al. / Neuroscience Letters 271 (1999) 21±24

Thus, we have compared the behaviour of individual animals with the level of c-fos, fosB and junB mRNA in areas of the hippocampus in animals with and without inhibition of NPY. A total of 18 male Wistar rats (Mùllegaard, L1. Skensved, DK), weighing 250±280 g at the beginning of the study were anaesthetized with hypnorm/dormicum (Janssen/Roche (3 ml/kg, s.c.)) and a stainless steel i.c.v. cannula (22 gauge) was placed in the right ventricle 2 mm lateral to bregma, and secured with dental cement. All animals were allowed 1 week of recovery before the experiment. The study was performed as two separate experiments: In the ®rst experiment, six animals (n ˆ 3 in each treatment group) were used, and rated for behaviour. In the second experiment, 12 animals were used (six in each treatment group) that were both rated and used for mRNA measurement. The two experiments were pooled for behavioural analysis. In the NPY treated group, animals were injected i.c.v. with vehicle or 6 nmol of NPY (Bachem, Switzerland) in 10 ml of saline and 1% bovine serum albumin (Sigma, St. Louis, MO). Five minutes after the i.c.v. injection, the rats were injected with KA (Sigma, St. Louis, MO; 10 mg/kg, s.c.). The rats were coded and placed in individual observation cages, and then rated for appearance of seizures over the next 120 min by observers blind to the treatment. Two seizure ratings were used. One counting the time spent in seizures, including only seizure activity (clonic movements) that last at least 15 s [17]. As this excluded analysis of seizures of short duration, the other rating counted the number of grade 4 seizures consisting of rearing, clonic movement of the forelimbs, and grade 5 seizures with rearing, and loss of balance [12]. At the end of the observation period, animals were killed by decapitation, and the brains removed and frozen on powdered dry ice. Brains were sectioned in 12 m thick serial sections at the level of the hippocampus, thawed onto gelatine coated slides, and kept at 2808C until further use. The placement of the i.c.v. cannula was con®rmed visually in each animal by observing the cannula track ending in the lateral ventricle. The sections were removed from the freezer, dried and ®xed in 4% paraformaldehyde for 5 min. Then, after washing twice for 5 min in phosphate buffered saline (PBS), they were placed in PBS with triethanolamine and acetic anhydride for 10 min, delipidated in chloroform, and rehydrated in a series of graded alcohols. DNA oligoprobes complementary to the IEG mRNA sequences (c-fos: nucleotides 133±180, fosB: nucleotides 1206±50, junB: nucleotides 2438±82. GenBank accession numbers: X06769, X14897, X54686, respectively) were 35S-ATP (Amersham) endlabelled using TdT enzyme (Promega) and extracted using the phenol/chloroform method. Sections were hybridized (1:25 £ 106 cpm/slide) in a hybridization buffer consisting of 25% v/v deionized formamide, 4£ SSC, 1£ Denhardt's solution, 0.5 mg/ml salmon sperm DNA, and 0.25 mg/ml yeast tRNA. The sections were incubated in sealed cham-

bers at 378C overnight. After three brief rinses in 1£ SSC at room temperature, the sections were washed for 4 £ 15 min in SSC at 558C, air dried in a stream of cold air and apposed to hyper®lm b-max (Amersham) for 5±7 days together with 14 C standards (Amersham). The grey values over the dentate gyrus, the CA1 and CA3 areas of the hippocampus were measured using an image analysis program (ImagePro plus, Media Cybernetics, Silver Spring, MD). The regions of interest were delineated manually. A background signal measured over the white matter in the corpus callossum was subtracted from the value of each section. The ®nal value for each animal was the average of values from six measurements. The values of intensity were calibrated using 14C-standards. Treatment with KA induced characteristic seizure activity as earlier reported [8,15,16]. Following an initial silent period, the animals began to display wet dog shakes. Later, but prior to the onset of seizures, gazing and salivation appeared. Injection of 6 nmol NPY i.c.v. signi®cantly reduced generalized KA induced seizures. At the end of the 120-min observation period, the NPY injected group had spent signi®cantly less time in seizure than the vehicle injected controls (Fig. 1a). Also, the NPY treated group had experienced signi®cantly fewer grade 4 and 5 seizures (Fig. 1b), although these were still present. Also, the NPY treated group showed a longer latency to both the ®rst long lasting ($15 s) (Fig. 1c) and the ®rst grade 4/5 seizure (data not shown). Notably, a great variability in the NPY group was observed despite that all cannulas were correctly placed. Thus, in the NPY treated group, one-third (three) of the animals did not show any seizure activity at all. Two animals in the NPY treated group had seizures lasting more than 5 min. In the vehicle treated group, one animal had seizures lasting a total of 6 min, whereas all the other animals in the group had seizures lasting more than 30 min. In the vehicle injected group, the seizure activity was fatal for two animals within the observation period, and none in the NPY treated group. c-Fos mRNA was robustly induced in many areas of the

Fig. 1. (a) Minutes spent in seizure within the observation period; (b) number of grade IV and V seizures within the observation period; (c) time to ®rst long lasting (.15 s) seizure. White columns: vehicle treated animals; hatched columns: 6 nmol NPY i.c.v. Asterisk denotes statistical signi®cance (P , 0:05), Mann± Whitney U test. Values ˆ means. Error bars: SEM.

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Fig. 2. In-situ hybridization. Three 12 m sections from a kainate treated brain hybridized with a c-fos probe (A), a FosB probe (B) or junB (C). Small arrow in (A) points to the CA1 area, large arrow point to dentate gyrus. Scale bar, 2 mm (A). Same magni®cation in all sections.

brain. Of interest for the present study, high levels of c-fos mRNA were observed in the hippocampus and piriform cortex (Fig. 2). Similarly, junB mRNA was present in high levels in the hippocampus. FosB hybridization gave a lower signal, and only small amounts of fosB mRNA were observed outside the dentate gyrus (Fig. 2). NPY treatment prior to the KA injection did not signi®cantly change the induction of mRNA for any of the IEGs measured in the regions measured (Fig. 3). No correlation between the time spent in seizures or the number of grade 4/ 5 seizures and the amount of IEG induction was observed. However, one animal that did not display any seizure activity due to inhibition by NPY, also had very low levels of IEG mRNA. Notably, animals displaying the same behaviour did not have similar IEG responses. This study corroborates our previous observations showing that NPY inhibits generalized seizures produced by KA [17]. The aim of the present study was to determine if NPY

Fig. 3. Densitometric analysis of mRNA levels. (a) c-fos; (b) fosB; (c) junB; measurements from four to six animals in each group. White columns: DG from saline treated animals; upward hatched column: DG from NPY treated animals; downward hatched column: CA1 from saline treated animals; cross-hatched: CA1 from NPY treated animals. Error bars: SEM.

inhibits not only the time spent in seizures, but also to see if NPY prevents severe seizures like grade 4 and 5 seizures of the Racine scale. NPY signi®cantly inhibited both kinds of seizures within the 120 min of the observation period. Further, it shows that NPY delays the time to onset of both the ®rst long-lasting seizure and grade 4/5 seizure. This inhibition was not correlated to inhibition of IEG induction in the hippocampus, since NPY produced no change in the pattern of IEG induction after kainic acid administration. It is well known that c-fos is induced in brains of animals with seizures [10]. Other IEGs, such as FosB and JunB have also been reported to be linked to the development and propagation of KA induced seizures [1,9]. Though activated acutely by the excitation the roles of the IEG is important long after the seizure has occurred since it takes time before the protein product exert an intracellular function. Dimers of jun/fos proteins form the AP-1 binding complex that binds to regulatory sequences. In this study the full limbic seizures that were expected from the KA treatment were blocked by i.c.v. injection of NPY, but not accompanied by lower expression of either c-fos, fosB or junB mRNA. This result is in apparent disagreement with previous results from our group, showing a reduction of Fos protein after KA [17]. The previous study was based on relatively few animals, and no attempt to quantify the signals were done. Alternatively, this may be explained by the choice of timepoint, and also the fact that in the present study, quanti®cation was performed on the mRNA level. Furthermore, in one animal which did not experience any seizure activity of the kind measured in this study, low levels of IEG mRNAs were also present. This variability of IEG response both in intensity and anatomical distribu-

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tion after KA has also been observed by others [16]. This could indicate that the IEGs are induced at the appearance of the ®rst seizure, and that unless seizures are completely absent, there will be a high IEG response, which is not correlated to the either length nor the severity of the seizure. This is supported by the lack of correlation between the level of mRNA in the hippocampus, and the severity of seizure, and although NPY signi®cantly reduces seizure activity, it is not capable of attenuating the level of IEG mRNA. Previous reports [8,16] divides KA induced seizures in phases, involving an increasing number of regions. In particular, Willuoghby et al. [16] note that the initial step after a KA challenge is a near maximal induction of Fos protein in limbic regions arising within minutes after i.v. KA administration, which is not correlated to motor seizure activity. This initial response is followed by a later seizure related induction in other forebrain areas. Assuming that this pattern of induction holds true for the other IEGs measured in this study, this indicates that NPY is not capable of modulating the initial IEG induction occurring in the limbic regions, prior to the appearance of the ®rst seizure. Consequently, when NPY exerts its seizure inhibiting effect, the IEG mRNA is already present in the hippocampus in high levels, and no inhibition is possible. Also, it may be that NPY exerts its anticonvulsant effect through a pathway that is different from the induction pathway of the IEGs. Blockade of Ca 21 channels in cortical cells, which have little effect on spontaneous synaptic activity can suppress expression of c-fos, junB and FosB mRNA [11], indicating that neuronal excitation and IEG induction are regulated through distinct cellular effects. T. Halborg and P.Mùller Carstensen are thanked for skilful technical assistance. This work was supported by grants from the Danish Medical Research Council, the Ivan Nielsen Foundation and the Strategic Drug Research Centre. [1] Beer, J., Mielke, K., Zipp, M., Zimmermann, M. and Herdegen, T., Expression of c-jun, junB, c-fos, fra-1 and fra-2 mRNA in the rat brain following seizure activity and axotomy. Brain Res., 794 (1998) 255±266. [2] Ben-Ari, Y., Limbic seizure and brain damage produced by kainic acid and relevance to human temporal lobe epilepsy. Neuroscience, 14 (1985) 375±403. [3] Buckmaster, P.S. and Dudek, F.E., Neuron loss, granule cell axon reorganization, and functional changes in the

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