Kindled seizures induce a long-term increase in vasopressin mRNA

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 24 (1994) 20-26

Research Report

Kindled seizures induce a long-term increase in vasopressin m R N A R.S. Greenwood *, R.B. Meeker, A. Abdou, J.N. Hayward Departments of Neurology and Pediatrics and the Neurobiology Curriculum, CB 7025, 751 Burnett-Womack, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA (Accepted 7 December 1993)

Abstract

Neuroendocrine disturbances are among the significant problems associated with animal and human seizures. To investigate the mechanisms for these disturbances, we examined changes in the expression of vasopressin (VP) mRNA in the hypothalamic magnocellular neuroendocrine cells of rats after amygdala kindled seizures, a model for temporal lobe epilepsy. A prominent increase in VP mRNA was found in the supraoptic nucleus of kindled animals by one week after the last seizure which persisted for at least 4 months. The increase occurred bilaterally in the SON and remained unchanged despite the absence of further stimulation, seizures or change in body fluid homeostasis. Since the VP mRNA change after kindling correlated with the duration of afterdischarge but not the number of amygdala stimuli the change appears to be an effect of the seizure. This chronic increase in VP mRNA appears to reflect a change in neuroendocrine gene expression and may identify an important new mechanism of plasticity that contributes to the neuroendocrine disturbances accompanying epilepsy.

Key words: Kindling; Seizure; Vasopressin; Neuroendocrine; In situ hybridization; Hypothalamus; Supraoptic nucleus

I. Introduction

Neuroendocrine changes are one of the most significant abnormalities associated with naturally occurring seizures or seizures produced by electroconvulsive therapy (ECT). I m m e d i a t e elevations in blood levels of hormones follow seizures in humans and can be used as a reliable marker for the occurrence of some types of seizures [1,15,25,51]. Electroconvulsive seizures also produce acute changes in neuroendocrine function and these changes have been considered a potential source for the therapeutic effects of electroconvulsive therapy [23,32,47,53,54]. Chronic endocrine problems are also common in patients with intractable seizures [11,21,22] and may account for some of the psychological and social impact of seizures. In humans with epilepsy the pathologic events producing the seizures, the medications used to treat seizures as well as the seizures themselves have been considered potential sources for these endocrine abnormalities [4,14,21]. Unfortunately, the

* Corresponding author. Fax: (1) (919) 966-2922. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 3 ) E 0 2 3 0 - W

varying etiologies of human epilepsy, the differences in antiepileptic treatments, the psychosocial consequences of epilepsy, as well as the varying seizure types, frequency and severity in human epilepsy, have confounded the results of clinical studies and obscured the underlying mechanisms accounting for endocrine abnormalities [11]. To circumvent the complexities of human seizures the kindled seizure model, an animal model for partial seizures in humans, has been used to study seizure mechanisms [16]. In this model a stimulus that initially produces minimal behavioral change, with repetition, gives rise to more severe electrical and behavioral seizures [16]. The behaviors seen in the rat with electrical kindling in the amygdala and the hippocampus are analogous to the behaviors observed in humans with partial complex seizures [41]. The changes in brain excitability after kindling are p e r m a n e n t [16] and the seizures can be made to occur spontaneously [36]. Protein synthesis inhibitors block kindling, thus implying that an alteration in gene expression is involved. For these reasons, recent investigations have focused on changes in gene expression in the limbic system. Kindled seizures produce changes in immediate early genes [43,46], growth factors [2,3,13] and neuropeptides

R.S. Greenwood et al. /Molecular Brain Research 24 (1994) 20-26

[39,44,45] within the limbic system. The changes in the expression of these genes, however, are transient, lasting less than one week, so that they could not directly account for the permanent alterations in excitability. Long lasting changes in gene expression after kindling have also been reported, including a reduction of ligatin mRNA in the hippocampus and decreased type II calmodulin kinase mRNA [5,35]. The initiation of these gene expression changes are thought to invoNe, in part, glutamate [9,10] and other excitatory transmitters [27]. Since most studies have focused on limbic seizures, little is known about the effects of kindled seizures on brain regions outside the limbic system. We have previously reported that rats kindled to stage 5 seizures with amygdala stimulation display an enhanced release of vasopressin in response to a kindling stimulus and a modest but persistent elevation of plasma VP [17]. This elevation of plasma VP was not associated with overt seizure activity suggesting that kindling might chronically alter VP regulation in magnocellular neuroendocrine cells. In this study we show that kindling is associated with very long-term changes in the regulation of the expression of VP mRNA within magnocellular neuroendocrine cells of the supraoptic nucleus (SON) of the hypothalamus.

21

2. Materials and methods 2.1. Animals and kindling The care of the rats and all experiments were conducted in accordance with NIH and institutional guidelines. Under deep pentobarbital anesthesia 18 male Sprague-Dawley rats and 38 male Long-Evans rats weighing 250-300 g were implanted stereotaxically with bipolar electrodes (twisted, teflon-insulated platinum/iridium wire with bare tips separated by 500-750/xm) in the medial nucleus of the right amygdala (AP Bregma - 2 . 3 mm, ML 4.0 mm, depth 9 mm below the dura [34]). Location of the stimulating electrode in the amygdala was histologically confirmed in Giemsa counterstained sections. Following a one-week recovery, the rats received threshold testing for afterdischarge. All rats received threshold stimulation but the rats were then divided into matched pairs of control and experimental animals. All control animals, then, received threshold stimulation and some experienced at least one afterdischarge. The experimental group was kindled as previously described [17], receiving once or twice daily arnygdala stimulation (1 or 2 s trains of 60 hz, biphasic 0.45 ms pulses 400 /xA peak-to-peak). Behaviors accompanying the seizures were classified into five stages of increasing intensity (stages 1-5) [37]. Electrocorticographic recordings from the amygdala and the cortical surface were used for the determination of afterdischarge durations and seizure spread, respectively. After kindling, the rats were allowed stimulation-free intervals of 1, 7, 30 or 120 days in a controlled environment before sacrifice. Control animals for each of these intervals, except the 120 day interval, were implanted with electrodes but only sham-stimulated. Sham-stimulation consisted of attaching

Fig. 1. Photomicrographs of VP mRNA in situ hybridization in SON of kindled and control rats. These darkfield photomicrographs of autoradiograms illustrate the increased hybridization signal in the SON of kindled rats at 1 month (A) and 4 (C) months after kindling compared to that in the sham-stimulated control (B) and unstimulated control (D). Hybridization signal in the SON of kindled rats (A,C) was significantly greater than that in control rats (B,D).

22

R.S. Greenwood et al. ~Molecular Brain Research 24 (1994) 20-26

the animals by cable to allow recording the electrocorticogram but no stimulus was administered. The control animals at the 120 day interval were unimplanted. Hybridization of VP mRNA in the SON of unimplanted and implanted sham-stimulated animals was not different.

2.2. In situ hybridization At the end of the post-kindling interval each rat was perfused transcardially under deep pentobarbital anesthesia with a balanced salt solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After post-fixing overnight in the same solution, frozen 20 /zm coronal sections were cut from each brain on a Reichert cryostat and thaw mounted onto gelatin-chrome alum coated slides. Sections were processed immediately for in situ hybridization or stored at - 80°C. In situ hybridization for vasopressin mRNA was accomplished using a specific 30mer antisense oligonucleotide probe, 3' end-labeled with [125I]dCTP or [35S]dATP to a specific activity of 1-3 × 10 s cpm/txg DNA 129,56]. The vasopressin probe was complementary to bases 1785-1814 of exon C of the vasopressin gene [38] and was specific for vasopressin based on correlation with hybridization of two additional VP mRNA probes and VP immunocytochemistry. Sections were washed and permeabilized in 0.01 M PBS/0.3% Triton X-100 (2X30 min.) followed by prehybridization for 1 h at 45°C. After draining the prehybridization buffer, each section was covered with 100 tzl hybridization buffer containing labeled probe, coverslipped with parafilm and incubated at 45°C overnight. On the following day, the sections were washed sequentially in 2 x , 2X, 1 x , l x , 0.5xSSC at 45°C for 30 rain each. The slides were then dehydrated in ethanol (50%, 70%, 95%, 100%, 100%), dried with warm air and dipped in Kodak NBT-2 emulsion for autoradiography. Slides were developed in D19 after exposures of 2-8 days. The anatomical boundaries of the SON for silver grain density analysis were identified by Giemsa counterstain of the section. For silver grain density analysis, silver grain images under darkfield illumination (338 x magnification) were digitized and quantified using a Bioquant MegM Image Analysis system. The silver grain density was corrected for background by subtracting the grain density over the piriform cortex in the same section. Non-specific hybridization in the SON was assessed using randomer control probes, sense sequences, and probe sequences which are not expressed in the SON. These probes produced negligible hybridization.

3. Results In kindled rats VP m R N A labeling in the SON was consistently greater than in sham-stimulated rats (Fig. 1). By 1 week after the last stimulation, labeling of VP m R N A in the SON of rats kindled to stage 5 seizures (Fig. 1A) was clearly greater than VP m R N A labeling in the SON of the sham-stimulated animals (Fig. 1B). The increased VP m R N A in the SON was also observed in 15 out of 16 matched pairs of rats at one month post-kindling. Similar increases were seen in the magnocellular portions of the paraventricular nucleus (Fig. 2). Comparisons at even longer times after the last stimulation or seizure revealed that this increase in VP m R N A labeling was still seen in the kindled animals at 4 months after the last kindled seizure (Fig. 1(2). The effect was not strain specific since similar changes were found in both Sprague-Dawley and L o n g - E v a n s rats. The time course of the VP m R N A content in the right and left SON of the experimental rats relative to the matched control rats is illustrated in Fig. 3. No significant change in VP m R N A could be detected 24 h after the last stage 5 seizure. The increase in VP m R N A labeling in sections from kindled animals was, however, significantly greater than in sections from control animals one week, one month and 4 months after the last stimulation (Fig. 3). At each of these time points both the right and left SON showed increases in VP m R N A after kindling although the electrodes were implanted in the right amygdala. Some control animals manifested a single AD at the time of threshold testing. The VP m R N A labeling in control animals that experienced a single AD at the time of threshold testing was not significantly different from the VP m R N A labeling in control animals that did not experience AD at the time of threshold testing (VP

Fig. 2. Photomicrographs of VP mRNA in situ hybridization in paraventricular nucleus (PVN) of kindled and control rats. These dark field photomicrographs of autoradiograms illustrate the increased hybridization signal in the PVN of a kindled rat at 4 months after the last seizure (A) compared to hybridization in the PVN of a control (B). As in the SON the hybridization signal in the PVN of the kindled rat was significantly greater over magnocellular neurons.

R.S. Greenwood et aL /Molecular Brain Research 24 (1994) 20-26

23

Table 1 Comparison of body weight, hematocrit and osmolality in experimental and control animals

400

300

Measurement

Experimental

Control

Body weight (g) Before kindling After kindling

320 323

317 320

Hematocrit % O ..~ 200

Osmolality ( m O s m / k g )

.E

Values are mean_+ S.E.M. * P > 0.05, t-test.

+6* + 8.3 *

+ 6.5 + 6.6

40.3 + 0.5 *

42.1 + 0.5

283.6 + 1.2 *

283.3 + 0.8

100

0

Rt. Lt. 1 Day

Rt. Lt. 1 Week

Rt. Lt. 1 Month

Rt. Lt. 4 IM[os.

Fig. 3. Time course of the increase in VP m R N A in situ hybridization in kindled animals. In this graph, m e a n values for the hybridization signal in the S O N of the kindled rats in the right amygdala and left amygdala, are expressed as the percent of the control animals at 4 different times after the last amygdala stimulation. Percentages ( E / C × 100) were used to normalize values from individual time points which were obtained from different in situ hybridization runs with variable absolute grain densities. Control animals at the 4 m o n t h time point were unimplanted controls. Probe hybridization in both the right and the left S O N was significantly greater in kindled rats at 1 week (n = 10 pairs), 1 month (n = 16 pairs) and 4 months (n = 5 pairs) after the last kindled seizure (** P < 0.01; * P < 0.05, paired t-test). In contrast, no significant difference in hybridization was found in animals 1 day (n = 5 pairs) after the last stimulation. The m e a n absolute digitized grain densities in the S O N for control animals was 0.126 + 0.01 (11.8%) p i x e l s / ~ m 2.

Aol Z n" E I

0

25

35

45

55

65

75

85

95

105 115 125 135 145

AD Duration (sec)

B mRNA labeling, Controls with AD 0.122 ± 0.011 pixels//xm2: Controls without A D 0.149 + 0.015 pixels//~m2: t-test, T = -1.68, P > 0.05). Control animals at 1 week and 1 month consistently had less VP mRNA labeling than kindled animals. No detectable disturbances of water balance regulation accompanied the increase in VP mRNA since body weight, hematocrit, and plasma osmolality in the control and experimental animals were not significantly different (Table 1). Other investigators have shown that afterdischarge (AD) duration accompanying amygdala kindling may be an important predictor of the expression of the immediate early gene, c-los, in the limbic system [8]. We analyzed the relationship between AD duration and VP mRNA labeling to determine if the magnitude of VP mRNA increase with kindling was related to AD duration. Linear regression analysis (Fig. 4A) revealed a significant positive relationship (r = 0.645, P < 0.007) between mean duration of the last three ADs and VP mRNA labeling. No correlation was found between VP

~2 < Z

rr E

1

i|

0

0

i

~

i

L

5

10

15

20

t •

25

i

i

30

35

40

Number of Stimuli

Fig. 4. Plot of the average afterdischarge (AD) duration of the last three stage 5 seizures (A) or the n u m b e r of kindling stimuli to reach stage 5 seizures (B) versus the increase in VP m R N A labeling in kindled animals. In A the relative increase in VP m R N A (pixel ratio) is expressed as the ratio of the grain density in the S O N of the experimental animals minus that in the matched-control animal divided by the grain density in the matched-control animal ( E C/C). A significant positive correlation between A D duration and the VP pixel ratio was found (correlation coef. = 0.645; P < 0.007) but the correlation between the n u m b e r of kindling stimuli and the pixel ratio was not significant (correlation coef. = 0.096; P > 0.1).

24

R.S. Greenwood et al. / Molecular Brain Research 24 (1994) 20-26

mRNA content and the number of electrical stimulations to reach stage 5 seizures (Fig. 4B).

4. Discussion

These findings provide the first indication that long-term changes in neuroendocrine gene expression can be induced by kindled seizures. By one week after the last seizure, VP mRNA labeling in the kindled animals was increased by nearly 75% in comparison to that in the sham-stimulated animals. This increase in steady-state levels of VP mRNA persisted for 4 months without further stimulation or overt seizure activity, demonstrating a clear long-term plastic change in a neuroendocrine gene product. The increase in VP mRNA is large, approximately that seen in response to moderate water deprivation [29,42] but, in contrast, does not appear to be secondary to substantial changes in water balance regulation. No changes in body weight regulation, hematocrit, or plasma osmolality were observed in the present study or in our previous study [17]. These result do not exclude the possibility that the response of the vasopressinergic system to extreme perturbations of fluid balance could be altered. Our earlier studies did reveal that after kindling, vasopressin release occured with the same amygdala kindling stimulus that failed to elicit significant vasopressin release at the start of kindling [17]. The large increase in VP mRNA and the modest elevation in resting plasma VP noted in our previous study, cautions against over interpretation of the results in this study. The increase in VP mRNA not only could reflect increased transcription but also could reflect reduced message turnover. The delay in the increase in VP mRNA in the SON after kindled seizures was a remarkable finding in this study. Although very significant and long-lasting VP mRNA was apparent in nearly all animals studied 1 week or longer after the last stimulation or kindled seizure, no increase was seen at one day after the last kindled seizure. This result implies either that the time course of VP mRNA up-regulation requires more than one day or that some intervening process must occur before up-regulation of VP mRNA takes place. The first of these possibilities seems unlikely since studies of VP mRNA changes after physiologic stimuli, like dehydration, indicate a rapid rise in VP mRNA readily detectable by in situ hybridization [29]. In so far as the second possibility is concerned, other investigators studying kindling have reported similar time-delayed changes after kindling. A delay has been described in the positive transfer effect of kindling [26]. The positive transfer effect is observed after kindling in a primary limbic site and refers to the more rapid kindling rate at a secondary site, a brain site distant to the primary

kindling site [37]. The positive transfer effect is not fully developed at 24 h after the last kindled seizure although after a two week interval a fully-developed kindled convulsion occurs with one stimulus [26]. Most investigators believe that this transfer effect involves propagation of the seizure activity by way of brainstem structures [6]. Hernandez and Gallager [20] have also noted a delay after kindling in the appearance of reduced GABA sensitivity in dorsal raphe neurons of midbrain slices from kindled animals. In this well characterized model, these investigators noted that the subsensitivity to GABA was not present at 24 h after the last kindled seizure in dorsal raphe neurons from animals that had several stage 5 kindled seizures but was present at 120 h and 4 weeks after the last kindled seizure [20]. A similar mechanism could occur in the SON since GABA inhibition is the major inhibitory neurotransmitter in the SON. Loss of inhibition could, therefore, account for the observed delay in the VP mRNA. Our data indicate that kindling is an important prerequisite to the increase in VP mRNA. The magnitude of the VP mRNA was correlated with the duration of stage 5 AD but not the number of amygdala stimuli. Since control animals did receive threshold stimulation and manifested a single AD, the increase in VP mRNA does not occur simply with stimulation as has been reported with other peptides. Additional studies will be needed to determine if the increase in VP mRNA occurs at an earlier kindling stage. The lack of correlation of the number of stimuli with VP mRNA and the absence of significant asymmetry in VP mRNA increases in the right and left SONs strongly argue that direct stimulation of the SON by current spread or unilateral pathways from the stimulated amygdala do not mediate the VP mRNA increases. The symmetry of the increase in VP mRNA after stage 5 kindling is consistent with other studies of changes after kindling. Decrease in hippocampal ligatin [35], reorganization of mossy fiber synapses in the dentate gyrus [49], immediate early gene expression changes [8] and thyrotropin releasing hormone and proenkephalin changes [39] after amygdala kindling are all bilateral and symmetric. The susceptibility of VP magnocellular cells to the effects of kindling may be due to properties they have in common with ceils of the limbic system; properties thought to be important for the establishment of kindling [10,28]. These properties include a relatively dense innervation from glutamate presynaptic terminals [30, 31,52], a full complement of ionotropic glutamate receptor subtypes (NMDA, AMPA and kainate) [24], presence of low voltage-activated calcium channels [48], bursting patterns of activity resembling hippocampal pyramidal cell responses during seizure spread [18,19] and the induction of c-los during cell activation [40]. Studies which have examined glutamate receptor den-

R.S. Greenwood et al. /Molecular Brain Research 24 (1994) 20-26

sities in the hippocampus after kindling have found sustained increases in glutamate binding but not in the first 24 h after the last kindled seizure [33,55]. These results parallel our observation of a delayed increase in VP mRNA after the last kindled seizure. A modification in glutamate receptors, therefore, may be another potential mechanism accounting for the delayed change in VP mRNA. We do not yet know how far-reaching these kindling-induced changes are, but the presence of extensive limbic-neuroendocrine pathways [12] suggests that similar changes to those observed in VP SON neurons will be found in other neuroendocrine systems. From the current studies, however, we may conclude that at least some neuroendocrine cells exhibit pathologic plasticity at the level of neuropeptide gene regulation in response to kindled seizures. These observations raise a multitude of questions which remain to be answered. Further study of this novel process in the neuroendocrine system promises to contribute greatly to our understanding of pathologic plasticity associated with epilepsy.

Acknowledgments Supported by NIH Grants NS30923 and NS13411 and the Ministry of Higher Education of Egypt. The authors thank Lisa Rietz and Rhonda Baldwin for their technical assistance.

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