Molecular Brain Research, 15 (1992) 247-255 © 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00

247

BRESM 70476

Alterations in mRNA of enkephalin, dynorphin and thyrotropin releasing hormone during amygdala kindling: an in situ hybridization study J e f f r e y B. R o s e n ,

Christopher

J. C a i n , S u s a n R . B . W e i s s a n d R o b e r t

M. Post

Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892 (USA) (Accepted 28 April 1992)

Key words: Kindling; In situ hybridization; Enkephalin; Dynorphin; Thyrotropin-releasing hormone; RNA; Fos

The present study examined changes in mRNA expression of various neuropeptides at several stages of amygdala kindled seizures. 35S-labelled oligonucleotide probes for mRNA of enkephalin (ENK), dynorphin (DYN) and thyrotropin releasing hormone (TRH) were hybridized to brain sections of rats sacrificed 24 h after a stage 1 or stage 5 seizure, or 2 weeks after a stage 5 seizure. Changes in expression developed as kindling progressed, with long-lasting changes in ENK and transient changes in DYN and TRH. ENK mRNA levels increased in pyriform and entorhinal cortices at stage 1 and 5 and remained elevated in the pyriform two weeks after a stage 5 seizure. In contrast, DYN mRNA was decreased bilaterally in the dentate gyrus 24 h after a stage 5 seizure, but returned to control levels two weeks after a stage 5 seizure. TRH mRNA was dramatically increased 24 h after a stage 1 or stage 5 seizure. After a stage 1 seizure two patterns developed. One showed increases in the pyriform, entorhinal and perirbinal cortices ipsilateral to the stimulation. The other pattern displayed bilateral increases in the dentate gyrus with or without the unilateral increases the limbic cortices. Twenty-four hours after a stage 5 seizure, large bilateral increases were found in these areas, but these returned to baseline levels by two weeks after a stage 5 seizure. The data demonstrate a constellation of alterations in several peptide systems with distinct spatiotemporal patterns, particularly in regions known to be important in kindling and epilepsy, such as the dentate gyrus and pyriform and entorhinal cortices. The relationship of these neuropeptide mRNA changes to those previously found in c-fos mRNA expression during the development of kindling is discussed.

INTRODUCTION K i n d l i n g is the process of r e p e a t e d , i n t e r m i t t e n t a d m i n i s t r a t i o n of s u b c o n v u l s a n t electrical s t i m u l a t i o n , which e v e n t u a l l y i n d u c e s g e n e r a l i z e d seizures ~4. D u r ing the course of k i n d l e d seizure d e v e l o p m e n t , electrographic seizure activity initially is localized in limbic s t r u c t u r e s b u t e v e n t u a l l y spreads to o t h e r areas as seizures b e c o m e g e n e r a l i z e d 36. Behaviorally, k i n d l e d seizures evolve from b e h a v i o r a l arrest at the first several s t i m u l a t i o n s t h r o u g h orofacial m o v e m e n t s a n d forelimb clonus to m a j o r m o t o r seizures with r e a r i n g a n d falling 35. Finally, the a n i m a l r e m a i n s in the ' k i n dled state' w h e r e a single k i n d l i n g s t i m u l a t i o n can elicit a g e n e r a l i z e d seizure even after m o n t h s w i t h o u t stimulation or seizures. This evolution seems to develop in a stepwise m a n n e r which m a y involve specific n e u r o -

chemical c h a n g e s in distinct b r a i n regions at different stages of k i n d l i n g 2. A l t h o u g h the m o l e c u l a r a n d biochemical bases of k i n d l i n g are still u n c l e a r , n u m e r o u s studies have d e m o n s t r a t e d that n e u r o p e p t i d e s play a m o d u l a t o r y role in kindling. Exogenously a p p l i e d peptides, p e p t i d e a n a l o g u e s or a n t a g o n i s t s can either facilitate or suppress k i n d l e d seizures or k i n d l e d seizure d e v e l o p m e n t , d e p e n d i n g o n the p e p t i d e system b e i n g m a n i p u l a t e d . F o r instance, r e p e a t e d i n f u s i o n of e n k e p h a l i n ( E N K ) into the amygdala can i n d u c e k i n d l e d seizures 4, however, its complex effects may be either pro- or a n t i c o n v u l s a n t 12. D y n o r p h i n ( D Y N ) a d m i n i s t e r e d ' i n t r a c e r e brally has b e e n shown to suppress electroconvulsive shock (ECS), flurothyl a n d k i n d l e d seizures t'13'47. A n t i c o n v u l s a n t p r o p e r t i e s of t h y r o t r o p i n releasing h o r m o n e ( T R H ) have b e e n d e m o n s t r a t e d in various species a n d

Correspondence." J.B. Rosen, Biological Psychiatry Branch, National Institute of Mental Health, Building 10, Room 3N212, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: (1) (301) 402-0052.

248 epilepsy models. Intraventicular infusion of TRH and a TRH analogue (DN-1417) suppress kindling in rats, cats and baboons 38'39'4°. Other peptide systems also affect kindled and other types of seizures but have not been studied as extensively as ENK, DYN and TRH. The notion that endogenous levels of ENK, DYN and TRH may modulate the kindling process or kindled seizures is suggested by findings of alterations of endogenous levels of neuropeptides following kindled seizures. Met- and Leu-enkephalin immunoreactivity during and after kindling has been shown to be increased in the amygdala, hippocampus and substantia nigra and may persist for at least 21 days after generalized kindled seizures 19'46'49. Decreases in DYN A(1-8) immunoreactivity have been found in the hippocampus in rats 24 h after stage 5 amygdala-kindled seizures and 5 min after prepyriform cortex kindling 19'~2'23. However, DYN concentrations were not altered after partially kindled seizures 19'23 or 2 or 6 weeks after generalized kindled s e i z u r e s 23,22.

TRH levels have been found to be increased in the pyriform cortex 48 h after partially kindled (stage 2) and fully kindled seizures 2s and increased in the hippocampus, amygdala/pyriform cortex and ventral striarum 48 h after a generalized amygdala-kindled seizure 2~. Recently, molecular biological techniques have been employed to ask whether the mRNA levels for these peptides are also altered with kindling. Prepro-ENK mRNA has been shown to be elevated in various structures including the hippocampus, amygdala, nucleus accumbens and the entorhinal, frontal and occipital cortices 24 h after generalized amygdala-kindled seizures 3° or prepyriform cortex kindling 22. Partially kindled seizures (stage 2 or 3) also induce alterations of prepro-ENK mRNA levels in the hippocampus and entorhinal cortex 22'26. Prepro-DYN mRNA levels have been shown to decrease in the hippocampus 22'26'29'5° and increase in the striatum 5°. A preliminary report has suggested that prepro-TRH mRNA content appears de nouo in the dentate gyrus following generalized amygdala kindled seizures 2°. The results of the studies described above suggest that there is an integrated response of neuropeptide systems to kindled seizures that changes as kindling develops. Both the alterations in peptide levels and mRNA expression of these peptides seem to change as kindling evolves from focal afterdischarges and partial behavioral seizures to recruitment of extrafocal brain regions and generalized seizures. This evolution throughout kindling is reminiscent of the progressive changes seen in c-fos mRNA expression during the

development of kindling7'lt. c-los is a proto-oncogene which is transcribed rapidly following cellular activation and may be used as a marker to map activated pathways during seizures and other behaviors ~°'3v. Its protein product (Fos) acts as a transcription factor for other genes s. Little is known about the target genes which Fos may affect with kindling, although neuropeptides may be good candidates 9. In order to better understand the spatiotemporal relationship of peptide alteration during kindling and the possibility of peptides being target gene candidates for Fos, the present study utilized in situ hybridization techniques to anatomically analyze the changes in mRNA expression of ENK, DYN, and TRH at several stages during and after the development of amygdala kindling. MATERIALS AND METHODS

Animals and surgery Male Sprague-Dawley rats weighing approximately 300 g at the time of surgery were used. Rats were housed in individual cages. Food and water were freely available. Rats were anesthetized with 8% chloral hydrate (1 m l / 2 0 0 g) and a bipolar platinum electrode (Plastics One, Roanoke, VA), insulated except for the tip, was stereotaxically implanted into the left basolateral amygdala (3.3 m m posterior and 4.5 m m lateral to Bregma and 8.0 m m ventral from the skull surface). Four stainless steel screws were were used to anchor the electrode assembly to the skull. The electrode assembly was cemented in place with dental acrylic. One week following surgery, rats were either kindled with once daily stimulation with 800 ~zA of current (peak to peak intensity, 60 Hz, biphasic square waves) for 1 sec or were handled in the same m a n n e r but did not receive any current (sham controls). Fourteen rats received stimulation until a clear stage one behavioral seizure with accompanying afterdischarge was elicited. These rats were sacrificed 24 h after the last stimulation. Twenty-three rats were stimulated daily until each had one stage 5 seizure. Twelve of the rats were sacrificed 24 h after the last stimulation and the other 11 were sacrificed 2 weeks after the last stimulation. All rats were sacrificed by decapitation, the brain quickly removed and rapidly frozen in - 4 5 ° C isopentane, and stored at - 7 0 ° C . Fifteen p,m coronal sections at three levels of the brain were cut on a cryostat and thawed onto silanized or chrom-alum gelatinized microscope slides and stored with desiccant at - 7 0 ° C . Sections at the three coronal levels contained: (1) anterior caudate nucleus and pyriform, frontal and cingulate cortices (plate 16 in Paxinos and Watson34), (2) dorsal hippocampus, amygdala, hypothalamus, thalamus, posterior pyriform, retrosplenial and parietal cortices (plate 29), and (3) ventral hippocampus, substantia nigra, entorhinal, occipital and parietal and cortices (plate 39). Placement of the electrodes was verified at the time of slicing.

Oligonucleotide probes and labelling Oligonucleotide probes for T R H were a gift from W. Scott Young III (NIMH) or purchased from National Biosciences (Minnesota). T h e D Y N probe was a gift from from W. Scott Young III and Mark Smith (NIMH). The E N K probe was purchased from NEN. The oligonucleotide 5 ' - 3 ' sequences (base numbers in paranthesis) complementary to rat m R N A were: enkephalin(388-435) -ATC-TGC-ATC-CTT-CTT-CAT-GAA-ACC-GCC-ATA-CCTCTT-GGC-AAG-GAT-CTC; dynorphin(862-909)-GTT-GTC-CCACTF-AAG-C'Iq?-GGG-GCG-AAT-GCG-CCG-CAG-GAA-GCCCCC-ATA; thyrotropin-releasing hormone(319-366) -GTC-TTTTTC-CTC-CTC-CTC-CCT-T'Iq'-GCC-TGG-ATG-CTG-GCG-TTTTGT-GAT.

249 The oligonucleotides were labelled with [3sS]dATP (NEN) on the 3'-OH end with terminal transferase, CoC12 and 5xconcentrated potassium cacodylate/Tris buffer, pH 7.2 (all from Boehringer Mannheim). The reaction mixture for TRH and DYN was: 15/zl of 10.5 nmol/ml [35SldATP, specific activity of 1185 Ci/mmol; 1 ~1 of 5/J.M oligonucleotide; 1.5 ~1 of 5 mM COC12; 5 ~1 of 5 x concentrated potassium cacodylate/Tris buffer, pH 7.2; and 1.5/zl terminal transferase. The reaction mixture for ENK was the same except that different volumes of the reagents were used: 12/xl [3sS]dATP; 4/zl of 1.25 /xM oligonucleotide; 1.5 gl of 5 mM COC12; 5 /xl of 5 x concentrated potassium cacodylate/Tris buffer, pH 7.2; and 2.5/,1 of terminal transferase. The reaction was carried out at 37°C for 15 min. The reaction was terminated by being placed in ice for 10 min, then increased to room temperature, and adding 400/~1 of standard Tris/EDTA buffer and 2.5 /xl of yeast tRNA (20 mg/ml). The oligonucleotide was then extracted with conventional phenol : chloroform:isopentylalcohol methods and resuspended in 100 /,1 of standard Tris/EDTA buffer with 1 /zl of 1 M DTT. The probe was mixed to the concentration of 5 x 105 dpm per 20 /xl of hybridization buffer (Oncor, Gaithersburg, MD).

Data analysis The autoradiograms of the various peptide mRNAs were digitized using a Sierra Scientific CCD video camera with Image program (Wayne Rasband, NIMH) on an Apple Macintosh IIcx and then analyzed with the same program. Mean density was computed for anatomically circumscribed areas, regions or nuclei which may have differed between kindled and sham control rats. Only sections of kindled and control tissues that were hybridized and washed together, and then exposed to the same film were compared. The Image program was used to subtract background and measure the mean density of pixels within a circumscribed area. For each region analyzed, the area measured for kindled and control tissues was similar so that any differences reflected differences in density of the radioactive signal in comparable areas. Relative percent changes associated with kindling were computed for each measured area relative to the sham animals. At least five pairs of comparisons were made for each area analyzed. A group mean percent change was then calculated. Single sample t-tests were performed to test for significant percent increases (P < 0.05).

RESULTS

In situ hybridization In situ hybridization of the various neuropeptide mRNAs was performed according to a modified version of the Harper and Marselle technique iv. Tissue sections from sham controls were included in each assay for determining brain areas with alterations in levels of the peptide mRNAs. Sections were rinsed in 4% paraformaldehyde in 1 xphosphate-buffered saline and then rinsed in standard saline citrate buffer (2xSSC). The sections were then treated with 0.25% acetic anhydride for 10 min at room temperature followed by treatment with 0.1 M glycine (in 0.1 M Tris-HCl) for 30 min at room temperature. They were then rinsed in 2 x SSC followed by dehydration with increasing concentrations of ethanol. Twenty tzl of labelled probe-hybridization buffer were added to each tissue section, covered with a glass coverslip and incubated in a humidified box at 40°C overnight. The sections were rinsed twice in 50% formamide/2 x SSC at 40°C for 5 rain, then twice more for 10 min with constant agitation. The slides were finally rinsed twice for 1 min in 2×SSC, then again for 10 min and dehydrated with increasing concentrations of ethanol. They were then exposed to Kodak Min-R film for 7-14 days.

Kindling Rats kindled to stage 1 received 1 to 5 stimulations (mean _+ S.E.M. = 2.3 + 0.3). Rats kindled to stage 5 had a mean of 8.8 ___0.7 stimulations. T h e number of stimulations did not differ between the stage 5 rats sacrificed 24 h and 2 weeks after a stage 5 seizure. Electrode tips were found in the amygdala.

Expression of neuropeptide mRNA in normal and kindled rats A summary of the relative percent differences of the various peptides at the different stages, o f kindling is presented in Table I.

TABLE I

Significant alterations in mRNA expression following kindling Statistically significant scores (P < 0.05) are expressed as mean _+S.E.M. percent increases or decreases ( - ) of, mRNA levels in kindled rats compared to sham controls. Ipsi and contra denote the areas ipsilateral or contralateral to stimulation. NC indicates non-significant changes of less than 10%. ~,

Stage 1 (24 h)

Stage 5 (24 h)

1psi

Contra

Enkephalin Entorhinal cortex Pyriform cortex Retrosplenial cortex

33___ 4 NC NC

NC NC - 14-+ 5

Dynorphin Dentate gyrus

NC

NC

226+_82 153 ± 72 44 ± 31 *

NC NC NC

143 _+43 NC

164 _+52 NC

Thyrotropin releasing hormone Entorhinal cortex Pyriform cortex Perirhinal cortex Dentate gyrus long afterdischarge ( > 30 s) short afterdischarge ( < 30 s)

Stage 5 (2 weeks)

Ipsi

Contra

51_+ 12 24_+ 6 NC -23±

4

1psi

Contra

NC 23±8 NC

NC 25±7 NC

3

NC

NC

345_+ 65 380_+ 145 441 ± 59 123 -+ 26

NC NC NC NC

NC NC NC NC

28_+ 11 39± 10 NC -21±

245_+13 315 _+40 537 ± 71 147 -+ 20 , :

* Although the mean increase in TRH in the perirhinal was greater than 10%, it was not statistically significant.

250

Enkephalin In both normal and kindled brain, E N K m R N A had a very distinct and heterogenous distribution. By far the most dense expression of E N K m R N A was in the dorsal and ventral striatum including the caudate putamen, nucleus accumbens, olfactory tubercle and hypothalamus. Prominent hybridization was also detectable in the entorhinal cortex, while a detectable but weaker signal was seen in the pyriform cortex. Weak labelling was seen in all neocortical areas with hybridization in distinct superficial and deep layers, while middle layers were devoid of signal. Very weak labelling was found in the hippocampus. Twenty-four hours after a stage 1 seizure, E N K m R N A expression was not changed in the caudateputamen, nucleus accumbens, olfactory tubercle or pyriform cortex (Fig. 1). Significant increases in the entorhinal cortex were observed ipsilateral to the amygdala stimulation (Table I). In sections that included the posteromedial amygdaloid Cortical nucleus, increases were also evident. Decreases contralateral to the stimulation in the retrosplenial cortex of 14% were also found. Hybridization in all other areas was weak and variable. Twenty-four hours after stage 5 seizures, significant bilateral elevations in pyriform cortex of 24 and 39% (ipsilateral and contralateral to stimulation, respectively) were found (Fig. 1). Bilateral increases of 51% ipsilateral and 28% contralateral to stimulation in the entorhinal cortex were also significant (Table I). The significant decrease in the retrospenial cortex found at stage 1 was not observable at stage 5. The bilateral elevation in the pyriform cortex (Fig. 1) cortex persisted 2 weeks following stage 5 seizures.

Dynorphin In normal and kindled rats substantial D Y N m R N A expression was seen in the granular cell layer of the dentate gyrus (Fig. 2). Less signal was detected in the striatum and CA1 of the hippocampus. Very light labelling was found in most cortical areas, however, it was too light to be analyzed. No changes in labelling were found 24 h after a stage 1 seizure (Fig. 2a). After a stage 5 seizure a 21-23% decrease in m R N A expression was seen bilaterally in the dentate gyrus (Fig. 2). Decreases were also observed in CA1 region of the hippocampus but the levels were too low to reliably analyze. Two weeks after a stage 5 seizure, these decreases became variable with some rats still showing slight decreases and others returning to baseline (Fig. 2), and as a group were not significantly different from sham controls. Detectable changes were not seen in other areas•

Prepro-ENK mRNA O • i¸ ~ i / : ~ ¸ ¸ • ~ i-i • •

•i

J Fig. 1. Comparison of prepro-enkephalin mRNA expression in pyriform cortex in sham and kindled rats 24 h after a stage 5 seizure. The boxes outline the area of the pyriform cortex that was analyzed. Significant bilateral increases in the kindled rat are shown. These increases were still detectable two weeks later. On more posterior brain sections, bilateral increases in the entorhinal cortex were also detectable 24 h after a stage 5 seizure (see Table I).

Thyrotropin-releasing hormone In the normal rat, T R H m R N A had a very limited distribution. A prominent signal is seen in the reticular thalamus nucleus. The paraventricular nucleus of the hypothalamus and cells in the ventro-lateral portion of the hypothalamus are the only other areas with sufficient signal to detect (Fig. 3). A very faint signal could be detected in the dentate gyrus and hippocampus with extended exposure times. Twenty-four hours following stage 1 kindling, two patterns of T R H m R N A hybridization were detectable in several areas which had no hybridization in the normal sham animals. In the first pattern, T R H m R N A was expressed in the granular layer of the dentate gyrus (Fig. 3), some with the increases in pyriform and entorhinal and cortices ipsilateral to the stimulation

251

Prepro-TRH mRNA

and others with only the bilateral dentate gyms increases. In the second pattern, the m R N A signalwas significantly elevated in the pyriform and entorhinal cortices (Fig. 3), and only unreliably increased in the perirhinal cortex ipsilateral to the stimulation, without any increase in the dentate gyrus. With both patterns, those sections that included the posterior cortical nucleus of the amygdala, T R H m R N A labelling was also seen. The two different patterns correlated with the length of afterdischarges. Those with afterdischarges longer than 30 seconds had significant bilateral increases in the dentate gyrus (pattern 2), whereas those rats with short afterdischarges of less than 30 s had elevated expression in the pyriform and entorhinal cortices without dentate gyrus increases (pattern 2; see Fig. 3 and Table I). Examination of the electrode placements did not distinguish between the two pat-

Prepro-DYN mRNA

Fig. 3. Comparison of prepro-thyrotropin releasing hormone mRNA expression in sham and kindled rats 24 h after a stage 1 seizure. Two patterns of mRNA expression related to different aflerdischarge durations were seen. In rats with short afterdischarges (less than 30 s), significant increases in the pyriform (PC) and entorhinal cortices (not shown) ipsilateral to the stimulation (right side of brain) were detectable, but no increase in the dentate gyrus was observed. With long afterdischarge durations (greater than 30 s), significant increases in the dentate gyms (DG) were found.

terns. Twenty-four hours after a stage 5 seizure, T R H m R N A was expressed bilaterally in the dentate gyrus and pyriform, entorhinal and perirhinal cortices (Fig. 4). Two weeks after a stage 5 seizure, m R N A expression was at pre-kindling levels where no detectable signal was evident in the dentate gyrus or pyriform, entorhinal or perirhinal cortices (Fig. 4). Fig. 2. Comparison of prepro-dynorphin mRNA expression in dentate gyrus in sham and kindled rats 24 h after a stage 5 seizure. The boxes outline the area of the dentate gyrus that was analyzed. Significant bilateral decreases in the kindled rat are shown. These decreases returned to control levels within 2 weeks.

DISCUSSION As shown in this experiment, the response in different neuropeptide systems to amygdala kindling, as

252 Prepro-TRH m R N A

PR PC

PR PC

PR PC

Fig. 4. Comparison of prepro-thyrotropinreleasing hormone mRNA expression in sham and kindled rats 24 h after a stage 5 seizure. Large increases were seen in dentate gyrus(DG), and pyriform(PC) and perirhinal (PR) cortices. On more posterior brain sections, bilateral increases in the entorhinal cortex were also detectable. These increases returned to control levelswithin 2 weeks.

measured by m R N A levels, varies with anatomical localization, stage of kindling development and time following a seizure. Some alterations are seen after only a single stage 1 seizure (ENK and TRH), whereas others are evident only after a generalized stage 5 seizure (DYN). Some are transient (DYN and T R H ) and others are more persistent (ENK). All are regionally specific. Although changes in neuropeptide systems following kindled seizures may play a modulatory role in the kindling process, these alterations could also reflect aftereffects of convulsions. Similar increases in ENK and T R H immunoreactivity a n d / o r mRNAs and decreases in measures of DYN have been found following repeated ECS, although repeated ECS does not kindle 18'41'5t. Whether neuropeptide changes play a role in epileptogenesis or are aftereffects of seizures cannot be distinguished in the present study.

E N K m R N A is widely distributed in the brain t6, however, alterations in levels were seen in only a few cortical areas. Substantial increases were seen in the entorhinal cortex after stage 1 seizures, and only ipsilateral to the stimulation. Small but consistent decreases were also found in the retrosplenial cortex (posterior cingulate cortex) contralateral to the stimulation. As with T R H mRNA, there was a progressive recruitment in the entorhinal cortex as kindling developed. Twenty-four hours after a stage 5 seizure, increases in the entorhinal cortex became bilateral. Also, after a stage 5 seizure, bilateral increases were seen in the pyriform cortex. The decreases in the retrosplenial cortex became variable 24 h after a stage 5 seizure and were not significant. However, the elevation in the pyriform cortex was persistent and still robust two weeks following a stage 5 seizure, although the increases in the entorhinal cortex contralateral to the stimulation returned to normal. The increases in the entorhinal cortex were similar to those found by Naranjo et al. 3° with northern blots, while changes in the retrosplenial cortex have not been previously reported. The increase in hybridization in the pyriform cortex after a stage 5 seizure but not following stage 1 seizure has also been shown by Shinoda et al. 44. In contrast to our and Shinoda et al.'s finding of no change in the hippocampus using in situ hybridization techniques, others using isolated m R N A have found increases in the hippocampus with kindling. Both Naranjo et al. 3° and Lee et al. 22 reported increases in ENK m R N A in the hippocampus 24 h following kindling and levels returning to normal after 42 and 90 days. However, in both of these studies, hippocampal tissue from several rats were pooled, possibly in order to get sufficient levels of ENK m R N A to make the necessary measurements. Because the basal and postkindling levels of ENK m R N A are so low in the hippocampus, it may not be possible to get a sufficiently large enough signal in 15-/zm-thick sections to measure reliably. Nevertheless, the changes in the entorhinal, pyriform and retrosplenial cortices were consistent and large enough to be detected using the in situ hybridization technique. Met-Enkephalin immunoreactivity has also been shown to increase after kindling in hippocampus, amygdala, nucleus accumbens and entorhinal cortex 19'30'49. Increases in the amygdala have been detected 21 days after stage 5 kindled seizures 46. The effects of exogenously applied enkephalins and opiate agonists are primarily anticonvulsant on kindled seizures, particularly during postictal and interictal phases and have been suggested to play a role in limiting seizures 5'48, however, repeated intracranial in-

253 jection of Met-enkephalin can induce kindled seizures 4. The progressive increases in ENK mRNA in the entorhinal cortex found in this study suggest that enkephalin may play a role in kindled seizures during their development. The initial decreases of ENK mRNA in the retrosplenial cortex intimate that enkephalin may play a role in cortical structures as kindling proceeds from a localized event at stage 1 to a generalized seizure during stage 5. Moreover, because the pyriform cortex has been suggested to be a primary generator of kindled seizure activity24, the evolving and persistent alterations in the synthesis of enkephalin in the pyriform cortex imply a role in both the development and maintenance of kindled seizures 3. Levels of DYN mRNA are greatest in the dentate gyrus, although moderate to low levels are also found in the striatum, amygdala, hypothalamus and cortex 28. Changes after stage 5 kindled seizures were found only in the dentate gyrus and possibly in the CA1 region of the hippocampus. This agrees with the results of others 22'26'29'5° and is probably not due to cell loss 29. This also is consistent with decreases found in DYN immunoreactivity in the dentate gyrus following kindling 19'22'23. Our finding of normal levels two weeks after the last stage 5 seizure agrees with the return of DYN mRNA to normal levels 8 days following stage 3 perforant path kindled seizures 26 and the return of immunoreactivity to normal after two weeks 23. We did not find increases in the striatum as were reported by Xie et al. 5° following prepyriform cortex kindling. However, the rates of kindling were different: ours was once daily, whereas Xie et al. 5° stimulated twice daily, and our stimulation was focused in the amygdala while Xie et al. kindled in the prepyriform cortex. Injections of DYN into the ventricles or substantia nigra have been shown to suppress ECS, fluorthyl and kindled seizures 1'13'47. Speculatively, decreases in DYN mRNA and DYN immunoreactivity in the hippocampus following kindling may play a role in releasing excitatory processes from inhibition6 thus allowing the hippocampus to be more susceptible to seizure activity. The most striking alteration in any of the mRNAs studied was with TRH. In normal rats, in the sections taken in this experiment, TRH mRNA expression was spatially very limited with detectable lex~els only in the reticular thalamic nucleus and hypothalamus42. However, following either a stage 1 or stage 5 amygdala kindled seizure, large de novo levels in TRH mRNA were seen in the dentate gyrus and pyriform, entorhinal and perirhinal cortices. There was a progressive recruitment of these increases as the kindling proceeded. At stage 1, two patterns of TRH mRNA induction were seen. A unilateral pattern associated

with short afterdischarges was revealed in the posteromedial cortical amygdaloid nucleus, and the pyriform, entorhinal and perirhinal cortices ipsilateral to the stimulation. A bilateral pattern of increases in TRH mRNA in the dentate gyrus was associated with long afterdischarges. The distinct dentate gyrus versus cortical patterns of TRH mRNA elevation as a function of afterdischarge duration parallel similar distinct patterns observed with c-los mRNA at stage 1 kindled seizures, which also differed as a function of afterdischarge duration 7. Shin et al. 43 have also shown that with angular bundle kindling c-los mRNA expression is increased in the dentate gyrus only if the electrographic afterdischarge durations are longer than 30 s. Whether there is a direct relationship between the c-fos and TRH expression, as their spatiotemporal distribution with kindling would suggest, is under investigation and is discussed further below. After a stage 5 seizure, TRH mRNA increases were expressed both bilaterally in the pyriform and entorhinal cortices, and bilaterally in the dentate gyrus. These increases were nevertheless transient and by 2 weeks after a seizure they had dissipated. These data parallel the transient increases in TRH peptide levels reported after kindled seizures 21'25,33. Several studies have demonstrated that administration of TRH or a TRH analogue (DN-1417) may suppress kindled seizures 33'38'39'40.TRH may thus play a role as an endogenous anticonvulsant in a few select brain regions (i.e., target regions of TRH-containing efferents of the entate gyrus and pyriform, entorhinal and perirhinal cortices). In the present study, the brain regions in which the greatest alterations in neuropeptide mRNA levels were found correspond to the areas where c-fos mRNA increases were found following kindled seizures TM. clos is a proto-oncogene located in neurons that is rapidly transcribed following stimulation (such as seizures) and may be used as a marker for neuronal activation~°'37. Functionally, the Fos protein and other related gene products may act as transcription factors for the expression of neuropeptides, including ENK, DYN and TRH 15"31'32'45. Comparing the c-los mRNA distribution following kindled seizures described by Clark et al. 7 and the peptide mRNA patterns in the present study, the similarities between the c-fos and TRH patterns in the limbic cortices and dentate gyrus are striking. Both have negligible basal levels, but are dramatically increased in specific and discrete anatomical areas following a kindled seizure. Also, these increases demonstrate similar spatial and temporal patterns as kindling develops. Two nearly identical patterns are seen for

254

c-los

and TRH after a stage 1 seizure, which correlate with afterdischarge duration. Finally, whereas c-los mRNA expression is maximal at 15-30 min after stimulation and Fos protein levels are greatest 1-4 h following a seizure 27, the TRH message does not appear until more than 1 h after stimulation, peaks 6 to 12 h later and is still present 24 h after a seizure 2°, suggesting that Fos may act as a transcription factor for TRH in the dentate gyrus and limbic cortices with kindled seizures. The potential connection between Fos and ENK or DYN does not seem as apparent because of the relatively high basal levels of ENK in the entorhihal cortex and DYN in the dentate gyrus, where basal levels of Fos are very low, and ENK is not increased in the dentate gyrus where c-los mRNA levels are dramatically elevated following kindled seizures 7. In conclusion, in situ hybridization methods can be used to assess alterations in mRNA levels of many gene products associated with kindling. The levels of the mRNAs coding for the three peptides ENK, DYN and TRH have been shown to change both spatially and temporally as kindling proceeds from focal to generalized seizure events. It is possible that some peptides may have excitatory, proconvulsant actions (e.g., ENK), while others such as TRH, may be anticonvulsant. Thus, the contribution of the different proand anti-convulsant forces of the peptides may change both temporally as kindled seizures develop and spatially as more areas are recruited into the seizure activity. Understanding of the functional role of different peptide systems in the evolution and maintenance of kindling may assist in the elucidation of the role of neuropeptides in human epilepsy. REFERENCES 1 Bonhaus, D.W., Rigsbee, C.C. and McNamara, J.O., Intranigral dynorphin-l-13 suppresses kindled seizures by a naloxone insensitive mechanism, Brain Res., 405 (1987) 358-363. 2 Burchfiel, J.L. and Applegate, C.D., Stepwise progression of kindling: Perspectives from the kindling antagonism model, Neurosci. Biobehav. Rev., 13 (1989) 289-299. 3 Cain, D.P,, Excitatory neurotransmitters in kindling: excitatory amino acid, cholinergic, and opiate mechanisms, Neurosci. Biobehay. Rev., 13 (1989) 269-276. 4 Cain, D.P, and Corcoran, M.E., Intracerebral Beta-endorphin, met-enkephalin and morphine: kindling of seizures and handling-induced potentiation of epileptiform effects, Life Sci., 34 (1984) 2535-2542. 5 Caldecott-Hazard, S. and Engel, J., Limbic postictal events: anatomical substrates and opioid receptor involvement, ~Prog. Neuropsychopharmacol. Biol. Psychiat. , 11(1987)389-418. 6 Chavkin, C., Neumaier, J.F. and Swearengen, E., Opioid receptor mechanisms in the rat hippocampus, NIDA Res. Monogr., 82 (1988) 94-117. 7 Clark, M., Post, R.M., Weiss, S.R.B., Cain, C.J. and Nakajima, T., Regional expression of c-los mRNA in rat brain during the evolution of amygdala kindled seizures, Mol. Brain Res., 11 (1991) 55-64.

8 Curran, T. and Morgan, J.I., Memories of los, BioEssays, 7 (1987) 255-258. 9 Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 10 Dragunow, M. and Faull, R., The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods, 29 (1989) 261-265. 11 Dragunow, M., Robertson, H.A. and Robertson, G.S., Amygdala kindling and c-los protein(s), Exp. Neurol., 102 (1988) 261-263. 12 Frenk, H., Pro- and anticonvulsant actions of morphine and the endogenous opioids: involvement and interactions of multiple opiate and non-opiate systems, Brain Res. Rev., 6 (1983) 197-210. 13 Garant, D.S. and Gale, K., Infusion of opiates into substantia nigra protects against maximal electroshock seizures in rats, Z Pharmacol. Exp. Ther., 234 (1985) 45-48. 14 Goddard, L.S., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 15 Goodman, R.H., Regulation of neuropeptide gene expression, Annu. Rev. Neurosci., 13 (1990) 111-127. 16 Harlan, R.E., Shivers, B.D., Romano, G.J., Howells, R.D. and Pfaff, D.W., Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization, J. Comp. Neurol., 258 (1987) 159-184. 17 Harper, M.E. and Marselle, L.M., RNA detection and localization in cells and tissue sections by in situ hybridization of 35Slabeled RNA probes, Methods Enzymol., 151 (1987) 539-551. 18 Hong, J.S., Yoshikawa, K., Kanamatsu, T., McGinty, J.F., Mitchell, C.L. and Sabol, S.L., Repeated electroconvulsive shocks alter the biosynthesis of enkephalin and concentration of dynorphin in the rat brain, Neuropeptides, 5 (1985) 557-560. 19 Iadarola, M.J., Shin, C., McNamara, J.O. and Yang, H.-Y.T., Changes in dynorphin, enkephalin and cholecystokinin content of hippocampus and substantia nigra after amygdala kindling, Brain Res., 365 (1986) 185-191. 20 Kubek, M.J., Knoblach, S.M., Fuson, K.S. and Aydelotte, M.R., Amygdaloid kindling induces TRH mRNA in specific limbic foci as determined by in situ hybridization histochemistry (ISHH), Soc. Neurosci. Abstr., 16 (1990) 1266. 21 Kubek, M.J., Low, W.C., Sattin, A., Morzorati, S.L., Meyerhoff, J.L. and Larsen, S.H., Role of TRH in seizure modulation, Ann. N.Y. Acad. Sci., 553 (1989) 286-303. 22 Lee, P.H.K., Zhao, D., Xie, C.W., McGinty, J.F., Mitchell, C.L. and Hong, J.S., Changes of proenkephalin and prodynorphin mRNAs and related peptides in rat brain during the development of deep prepyriform cortex kindling, Mol. Brain Res., 6 (1989) 263-273. 23 McGinty, J.F., Kanamatsu, T., Obie, J., Dyer, R., Mitchell, C. and Hong, J.S., Amygdaloid kindling increases enkephalin-like immunoreactivity but decreases dynophin A-like immunoreactivity in rat hippocampus, Neurosci. Lett., 71 (1986) 31-36. 24 Mclntyre, D.C. and Racine, R.J., Kindling mechanisms: current progress on an experimental epilepsy model, Prog. NeurobioL, 27 (1986) 1-12. 25 Meyerhoff, J.L., Bates, V.E. and Kubek, M.J., Elevated TRH levels in pyriform cortex after partial and fully generalized kindled seizures, Brain Res., 525 (1990) 144-148. 26 Moneta, M.E. and Hollt, V., Perforant path kindling induces differential alterations in the mRNA levels coding for prodynorphin and proenkephalin in the rat hippocampus, Neurosci. Len., 110 (1990) 273-278. 27 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 28 Morris, B.J., Haarmann, S.J, Kempter, B., Hollt, V. and Herz, A., Localization of prodynorphin messenger RNA in rat brain by in-situ hybridization using a synthetic oligonucleotide probe, Neurosci. Lett., 69 (1986) 104-108. 29 Morris, B.J., Moneta, M.E., ten Bruggencate, G. and Hollt, V., Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling, Neurosci. Lett., 80 (1987) 298-302.

255 30 Naranjo, J.R., Iadarola, M.J. and Costa, E., Changes in the dynamic state of brain proenkephalin-derived peptides during amygdaloid kindling, J. Neurosci. Res., 16 (1986) 75-87. 31 Naranjo, J.R., Mellstrom, B., Achaval, M. and Sassone-Corsi, P., Molecular pathways of pain: fos/jun-mediated activation of a noncanonical AP-1 site in the prodynorphin gene, Neuron, 6 (1991) 607-617. 32 Noguchi, K., Kowalski, K., Traub, R., Solodkin, A., Iadarola, M. and Ruda, M., Dynorphin expression and fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons, Mol. Brain Res., 10 (1991) 227-233. 33 Owaga, N., Kajita, S., Sato, M. and Mori, A., Seizures and thyrotropin-releasing hormone (TRH) neural system in the rat brain, F. Psychiat. Neurol. Jpn., 39 (1985) 309-312. 34 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. 35 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 36 Racine, R.J., Modification of seizure activity by electrical stimulation: I. Afterdischarge threshold, Electroencephalogr. Clin. NeurophysioL, 32 (1972) 269-279. 37 Sagar, S.M., Sharp, T. and Curran, T., Expression of c-fos protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 38 Sakai, S., Baba, H., Sato, M. and Wada, J.A., Effect of DN-1417 on photsensitivity and cortically kindled seizure in senegalese baboons, Papio papio, Epilepsia, 32 (1991) 16-21. 39 Sato, M. and Morimoto, K., Anti-epileptic effects of TRH-T and DN-1417, Kurume Med. J., 30 (1983) $57-$64. 40 Sato, M., Morimoto, K. and Wada, J.A., Antiepileptic effects of thyrotropin-releasing hormone and its new derivative, DN-1417, examined in feline amygdaloid kindling preparation, Epilepsia, 25 (1984) 537-544. 41 Sattin, A., Hill, T.G., Meyerhoff, J.L., Norton, J.A. and Kubek, M.J., The prolonged increase in thyrotropin-releasing hormone in rat limbic forebrain regions following electrocouvulsive shock, Regul. Pept., 19 (1987) 13-22.

42 Segerson, T.P., Hoefler, H., Childers, H., Wolfe, H.J., Wu, P., Jackson, I.M.D. and Lechan, R.M., Localization of thyrotropinreleasing hormone prohormone messenger ribonucleic acid in rat brain by in situ hybridization, Endocrinology, 121 (1987) 98-107. 43 Shin, C., McNamara, J.O., Morgan, J.I., Curran, T. and Cohen, D.R., Induction of c-los mRNA expression by afterdischarge in the hippocampus of naive and kindled rats, J. Neurochem., 55 (1990) 1050-1055. 44 Shinoda, H., Nadi, N.S. and Schwartz, J.P., Alterations in somatostatin and proenkephalin mRNA in response to a single amygdaloid stimulation versus kindling, Mol. Brain Res., 11 (1991) 221-226. 45 Sonnenberg, J i . , Rauscher III, F.J., Morgan, J.I. and Curran, T., Regulation of proenkephalin by los and jun, Science, 246 (1989) 1622-1625. 46 Talavera, E., Omana-Zapata, I., Asai, M. and Condes-Lara, M., Regional brain IR-Met-, IR-Leu-enkephalin concentrations during progess and full electrical amygdaloid kindling, Brain Res., 485 (1989) 141-148. 47 Tortella, F.C. and Holaday, J.W., Dynorphin A (1-13): in vivo opioid antagonist actions and non-opoiod anticonvulsant effects in the rat flurothyl test, NIDA Res. Monogr., 75 (1986) 539-542. 48 Tortella, F.C., Long, J.B. and Holaday, J.W., Endogenous opioid systems: physiolgogical role in the self-stimulation of seizures, Brain Res., 332 (1985) 174-178. 49 Wanscher, B., Kragh, J., Barry, D.I., Bolwig, T. and Zimmer, J., Increased somatostatin and enkephalin-like immunoreactivity in the rat hippocampus following hippocampal kindling, Neurosci. Lett., 118 (1990) 33-36. 50 Xie, C.W., Lee, P.H.K., Douglass, J., Crain, B. and Hong, J.S., Deep prepyriform cortex kindling differentially alters the levels of prodynorphin mRNA in rat hippocampus and striatum, Brain Res., 495 (1989) 156-160. 51 Xie, C.W., Lee, P.H.K., Takeuchi, K., Owyang, V., Li, S.J., Douglass, J. and Hong, J.S., Single or repeated electroconvulsive shocks alter the levels of prodynorphin and proenkephalin mRNAs in rat brain, Mol. Brain Res., 6 (1989) 11-19.