Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure sensitive Mongolian gerbils

Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure sensitive Mongolian gerbils

Brain Research, 401 (1987) 353-358 353 Elsevier BRE 21974 Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure sensiti...

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Brain Research, 401 (1987) 353-358

353

Elsevier BRE 21974

Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure sensitive Mongolian gerbils Randall J. Lee ~, J.-S. Hong 2, J.F. McGinty 3 and Peter Lomax 1 lDepartment of Pharmacology, UCLA School of Medicine and the Brain Research Institute, Los Angeles. ( A 90024 (U.S.A.), 2Laborato
(Accepted 2 September 1986) Key words: Seizure; Gerbil; Enkephalin; Dynorphin

Radioimmunochemistry (RIA) and immunocytochemistry (ICC) were used to measure proenkephalin and prodynorphin peptidcs in the brain of a genetic model of epilepsy, the seizure-sensitive (SS) Mongolian gerbil. Brain levels of both [Metq- or [LeuS]-enkephalin (ME-LI) and dynorphin A~_s and dynorphin A 1 17(DN-LI) like immunoreactivity were increased in the hippocampal region of the SS gerbil. However, ME-LI and DN-LI did not follow the same patterns. ME-LI was significantly increased in the SS gerbils (postseizure) compared to SR gerbils while ME-LI in SS (preseizure) gerbils was not significantly different from SR gerbils. DN-LI was significantly increased in the hippocampal region of both SS (preseizure) and SS (postseizure) gerbils compared to SR gerbils. These resuits strongly imply differences in the regulation of proenkephalin and prodynorphin metabolism in the Mongolian gerbil. The differences in metabolic regulation may signal fundamentally different roles of these opioid peptides in the modulation of seizure activity in this animal. A variety of neuropeptides has been shown to modulate epileptic phenomena ~'2. Of particular interest are the opioid peptides in the hippocampal formation which are believed to play an important role in the regulation of neuronal excitability. Several studies have demonstrated changes in brain enkephalin and dynorphin levels following electroconvulsive shock (ECS)7'I°; amygdaloid kindlingg'J9'2124; and intracerebral kainic acid 8"11'12"j7. We have reported previously that, one day after cessation of repeated ECS, [MetS]-enkephalin-like immunoreactivity (ME-LI) was increased in certain regions of the limbic system and the basal ganglia of the rat brain, including the perforant pathway of the hippocampal formation 7m. The level of dynorphin AI_ 8 or dynorphin Al_17 also increased in most limbic-basal ganglia regions after seizures induced by repeated ECS >. However, in the hippocampal mossy fibers, dynorphin-like immunoreactivity (DN-LI) was de° pleted. These results demonstrated that the same seizure conditions can differentially alter the concentra-

tions of peptides derived from two opioid families m. Subsequent measurements of opioid peptides within 3-12 h of kainic acid administration has revealed a depletion of M E - L I and D N - L I in hippocampal mossy fibers followed by a rebound increase above normal in later postseizure (24-72 h) periods 11. To investigate further the role of proenkephalin and prodynorphin peptides in seizure activity, a genetic model of epilepsy, the seizure-sensitive Mongolian gerbil, was used. This model of epilepsy offers the advantage of a seizure-free strain for comparison. Therefore, proenkephalin and prodynorphin opioid peptides can be compared in epileptic and non-epileptic gerbils and the metabolic responsiveness of the opioid peptides can be measured by radioimmunoassay ( R I A ) and immunocytochemistry (ICC) prior to and after a seizure. Adult Mongolian gerbils, weighing 65-100 g, of either sex from the U C L A seizure-sensitive (SS) (WJL/UC) or seizure-resistant (SR) strain were used for all experiments 15. Gerbils were housed individu-

Correspondence: P. Lomax, Department of Pharmacology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.

354 ally in metal cage pans with screen lids at an ambient temperature of 19-21 °C on a 24 h continuous light cycle. Each animal was tested a minimum of 3 times by the triple handling technique to determine scores for severity of seizures 3. The rating for seizures was as

TABLE I

described by Loskota et al) 5. Briefly, seizures were classified: grade 0, normal exploratory behavior during the entire testing period; grade 1, a brief pause in normal activity accompanied by vibrissae twitches and retraction of the pinnae; grade 2, motor arrest with twitching of the vibrissae and retraction of the pinnae; grade 3, motor arrest with myoclonic jerks; grade 4, clonic-tonic seizures; grade 5, clonic-tonic seizures with body rollover; grade 6, seizures progressing to death (status epilepticus). These behavioral seizures correlate well with E E G epileptic activity 16,20.

Hippocampus Hypothalamus Brainstem Midbrain

Only SS gerbils with consistent stage 4 or 5 seizures were selected. SR gerbils never demonstrated seizure activity (stage 0), R I A and ICC were performed on tissues from comparable SR and SS gerbils. SS gerbils formed 2 groups: those which had not experienced a seizure (preseizure) within 72 h prior to sacrifice and those sacrificed 5 min after the ictal phase of a seizure (postseizure). Following decapitation, brains were quickly removed, dissected according to the method of Glowinski and Iversen 5 and stored at - 7 0 °C. The tissue levels of [Met5]-enkephalin-like immunoreactivity (ME-LI) and dynorphin Al_s-like immunoreactivity (DN-LI) were determined by R I A as described previously ~':°. Briefly, tissue was homogenized in acetic acid (2 M) and immersed in boiling water for 5 rain. Following centrifugation at 25,000 g for 20 rain, the supernatant was lyophilized. The residue was then reconstituted in R I A buffer solutions and atiquots were used for R I A . The specificity of the antiserum against ME has been described in a previous report 6. A n antiserum against dynorphin Al_ 8, which does not cross-react with ME or [LeuS]-enkephalim but cross-reacts to a small degree with dynorphin AI_13 (0.02%) and dynorphin AI_17 (0.01%), was used in R I A for determining the brain levels of DNEl. Gerbils were perfused with a 4% paraformaldehyde solution and brains were prepared for immunocytochemistry as described previously:. Three serial

[MerS]-enkephalin irnmunoreactivity in the gerbil brain

Number of animals (n) = 6. Values are in ng/g wet wt. Brain region

Seizure resistant

Seizure-sensitive Preseizure

Postseizure

10.1 _+ 1.0 770 _+37.7 123_+2.3 164 _+ 19

13.3 ± 1,9 741 + 52 126 ± 8.1 182 _+32

19.7 L 2.6 ~ 854 +_50.7 118+_5.1 206 _+ 16

*P < 0.005 (Student's t-test) compared to seizure resistant. 50/~m frozen sections were cut at 400 Bm intervals from the frontal pole through the brainstem. Two out of three adjacent sections in a series were incubated with an antiserum to dynorphin A > 17(provided by L. Terenius, Uppsala, Sweden) or [LeuS]-enkephalin (LE) (provided by R.J. Miller, U. Chicago) followed by avidin-biotin-peroxidase immunoreagents as described TM, The third section in a series was stained with Richardson's methylene blue-azure II Nissl stain 22. Sections from each group were coded and analyzed in a blind manner. In some experiments, antiserum against dynorphin B or dynorphin AI_ s (provided by E. Weber, Stanford U.), was used instead of the dynorphin A l _ : antiserum. Similarly, the [Met5]-enkephalin antiserum used in R t A was employed instead of [LeuS]-enkephalin antiserum. However, the dynorphin Aj._ s and [MetS]-enkephalin antisera were not as effective for ICC as they were for RIA. Therefore, photomicrographs were prepared from tissue stained with dynorphin A~_ ~, and [LeuSl-enkephalin antisera only. [Met5]-enkephalin immunoreactivity in the hippocampus was significantly increased in the SS gerbils (postseizure) compared to SR gerbils (Table 1). The "FABLE I1 Dynorphin A: ,sirnmunoreactivity in the gerbil brain

Number of animal (n) = 6. Values are in ng/g wet wt. Brain region

Seizure resistant

Seizure-sensitive . . . . . . . . . . . . Preseizure Postseizure

Hippocampus Hypothalamus Brainstem Midbrain

6.68 -+ 0.37 28.0+ 1.2 2.96 + 0.15 3.31 _+0.4

10.1 + 0.98* 27.6 ±: 1.4 2.89 :~ (1.49 4.17 :.e 0.6

1(I.1 + 0.66* 29. l +- 3.6 2.97_+0.39 4.57:2 0.5

*P <01055 (Students t:tesii compared to seizure resistant_

355 m e a n level of M E - L I in SS (preseizure) gerbils also

M E - L I in the h i p p o c a m p u s of SS gerbils (postsei-

t e n d e d to be greater t h a n in S R gerbils b u t the differ-

zure) a p p e a r e d to be a specific change since o t h e r brain regions did n o t show any changes in M E - L I .

ence did not reach a significant level. This increase in

!i~¸;i!!iii!!i!ii d; ~ilJi:

]7 ¸

A

Fig. 1. LE-LI in the hippocampal formation of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. A: LE-LI in the perforant/ temporoammonic (PTA) pathway innervating the ventral dentate gyrus and hippocampal fields in a SR gerbil (×5(l); B: high magnification of A ( x 40); C: LE-LI in the PTA pathway of a SS gerbil 5 min after a spontaneous seizure. Note the increase in immunostaining compared to A (x83): D: high magnification of C (x66).

356

8 Fig. 2. Dynorphin Al_ 1- immunoreactivitv in mossy fibers of SS and SR gerbils. A: normal DN-LI in the m o s s ) fibers of a SR gerbil ( x 20); B: high magnification of the hilar area in A (× 22); C: DN-LI in the mossy fibers of a SS gerbil 5 min after a s p o n t a n e o u s seizure ( × 50). Note the increase in immunostaining compared to A: D: high magnification of the hilar area in C ( x 66).

357 Dynorphin Al_ s levels were significantly greater in the hippocampal region of both SS (preseizure) and SS (postseizure) gerbils compared to SR (Table II), whereas other brain regions did not show any significant change in DN-LI. Immunocytochemistry on perfused brain sections supported the results found with R I A . Enkephalin (LE-LI or ME-LI) immunoreactivity was increased in the perforant pathway originating in the entorhinal cortex of SS gerbils as compared to SR gerbils (Fig. 1). ME-LI or LE-LI was particularly intense in the perforant path of gerbils killed within 5 min of a seizure. No change in ME-LI or LE-LI was observed in the mossy fibers of SS gerbils. Dynorphin A immunoreactivity within the hippocampal mossy fibers was increased in SS gerbils, equally in pre- and postseizure states as compared to SR animals (Fig. 2). The present study demonstrates changes in brain enkephalinergic and dynorphinergic systems within the hippocampal region of SS gerbils compared to the SR strain. Consistent with previous reports which demonstrated increased enkephalin levels in the hippocampus following either electrically or chemically induced c o n v u l s i o n s 7"m'tl"j2"21, levels of both [MetS] or [LeuS]-enkephalin were increased in the hippocampus of SS gerbils as demonstrated by two independent techniques, i.e. R I A and ICC. Furthermore, levels of dynorphin A~ s or dynorphin Al_ w were increased in the mossy fibers of SS gerbils in contrast to depletion of dynorphin A immediately following electrically or chemically induced seizures. The increases in ME-LI and DN-LI observed in SS gerbils did not follow the same pattern. As seen in Table 1, only the SS (postseizure) gerbil had significantly increased ME-LI in the hippocampus as com-

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