Diurnal variations of opioid peptides and synenkephalin in vitro release in the amygdala of kindled rats

Neuropeptides(1998) 32 (3), 293-299 © Harcourt Brace and Co. Ltd 1998

Diurnal variations of opioid peptides and synenkephalin in vitro release in the a m y g d a l a of kindled rats M. Asai, M. Zubieta, G. Matamoros-Trejo, G. Linares, P. Agustin Laboratorio de An~tlisis Quimicos, Instituto Mexicano de Psiquiatria, Antiguo Camino a Xochimilco, M6xico

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats (250-300 g) by daily intraperitoneal injections of 35 mg/kg of the convulsant agent. Immunoreactive (IR)-Met-enkephalin 0R-ME), IR-Leu-enkephalin (IRI.E), IR-heptapeptide (IR-HE), IR-octapeptide (IR-OC) and IR-synenkephalin (IR-Syn) in vitro release was measured from amygdala slices 24 h after the last stimulus, in groups of eight rats, every 4 h beginning at 08.:00 h. Opioid peptides in vitro release displayed diurnal variations. IR-ME and IR-Syn showed maximal levels before the onset of darkness (16:00 h). IR-LE and IR-OC release was enhanced 4 h later (20:00 h), no changes were detected for IR-HE. These results show that endogenous opioid system (EOS) release displays diurnal variations. The peak for the analysed peptides was reached before and during the dark phase. It is suggested that EOS release enhancement in PTZ-kindled rats, seems to be due to a compensatory mechanism against the excitation induced by the blockade of the GABAergic transmission. Summary

INTRODUCTION

Since an earlier report by Dr R. Frederickson et al, who demonstrated that diurnal rhythm was observed in the responsiveness of mice to nociceptive stimuli and in the hyperalgesic activity of naloxone, 1 it has been accepted that the endogenous opioid system (EOS), is subject to an internal physiological control. Diurnal variations for brain opiate receptors number, 2 [3-endorphin 3 and dynorphin 4 have been described, as well as Met-enkephalin and Leu-enkephalin in spinal cord, adrenal gland, 5 rat brain, C7 human plasma and monkey cerebrospinal fluid.8 In all the studies mentioned above, maximal concentration of opioid peptides and the number of receptors were reached during the dark phase. Moreover, EOS increased during hibernation 9 and showed selective modifications during seasonal variations, in molluscans 10as well as in mammals. 1~ On the other hand, it is well established that pentylenetetrazol (PTZ)-induced kindling selectively enhances opioid peptide tissue levels in rat brain, 12,~3 Received 8 January 1998 Accepted 23 January 1998 Correspondence to: Dr M. Asai, Laboratorio de Anfilisis Qufmicos, Instituto Mexicano de Psiquiatria, Antiguo Camino a Xochimilco, 101 Mexico DF 14370.

induces in vitro release of Met-enkephalin in striatum, 14 reduces g- and &opioid receptor binding 15 and produces a Met-enkephalin tissue content increase in basolateral amygdala during photoperiod. 7 Several authors have suggested a protective functional release of opioid peptides to prevent seizure activity in humans16 and animals.lZ18 It may be hypothesized that opioid peptides release displayed diurnal variations and could be enhanced after full pharmacological kindling. The present study assessed diurnal variations of in vitro release of immunoreactive (IR)-Met-enkephalin (IR-ME), IR-Leuenkephalin (IR-LE), IR-ME-Arg6-Phe 7 (IR-HE) and IR-MEArg6-GlyT-Leus (IR-OC) from basolateral amygdala after PTZ kindling in rats. IR-Synenkephalin (IR-Syn), the nonopioid portion of proenkephalin A, was also measured in the same paradigm. MATERIALS A N D METHODS

Male Wistar rats weighing 220-280 g, were housed with a 12 h light:12 h dark cycle in a temperature controlled room (21 + 1°C); the illumination period started at 06:00 h. Water and food pellets were available ad libitum. Experimental rats were injected intraperRoneally (i.p.) with 35mg/kg of PTZ every 2 4 h (10:30-11:30 a.m.) over a period of 30 days. Control animals received an 293

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equivalent volume of saline solution under a similar schedule. After injection, each animal was returned to its cage and observed for I h. The behavioural changes were evaluated following the criteria of Ito et al:~9 stage 1, no convulsions; stage 2, head twitching; stage 3, clonic convulsions; stage 4, kangaroo posture (violent convulsions); stage 5, kangaroo posture with falling back. Those animals that reached stage 5 at least three times were used for in vitro release experiments. The time course of chemical kindling development has been reported previously. 13 In vitro release Eight rats were sacrificed by decapitation, brains were removed and put in saline solution at 4°C. The amygdala dissection was made following the procedure of Engel et al?o Tissue was sliced in two directions at 90 ° at 300 ~tm with a tissue chopper (McIlwain). Slices from groups of eight rats were incubated for 15 rain in 25 ml of Krebsbicarbonate medium at 37°C and pH Z4 saturated with a mixture of 95% 02-5% CO 2. The composition of the Krebs-bicarbonate medium was as follows (mM): NaC1 118, KC1 4.8, CaC12 0.5, KH2PO4 1.2, MgSO 4 1.2, NaHCO 3 25, glucose 10. Amygdala slices from two rats were put in plastic superfusion chambers and superfused with oxygenated Krebs-bicarbonate at a flow rate of 1 ml/min. After a preliminary wash period of 15 rain, slices were superfused for another 20 min before switching to a medium with high potassium concentration. All experiments were conducted simultaneously with four superfusing chambers (two control versus two experimental) every 6 h during a 24 h cycle and were repeated three times. In order to avoid opioid peptide degradation, PheMa was added to superfusion media. Opioid peptides and Synenkephalin release from amygdala were analysed in the presence of Phe-Ala 1 mM and 55 mM K+, which served as depolarizing stimulus. Controls with and without calcium were performed as described previously. ~s Perfusates of 10 ml were collected at 10 min intervals in siliconized glass tubes containing 1 ml of 0.1 N HC1. Remaining slices were homogenized and centrifuged at 50 000 x g at 4°C for 1 h. Supernates were concentrated, resuspended in water and stored at -20°C for later IRopioid peptide evaluation. Opioid peptides and synenkephalin assays Opioid peptides and synenkephalin were assayed in triplicate by radioimmunoassay as described previously. 18,2~ Iodinated opioid peptides and [Tyr43](Syn 63-70) were used as tracers. IR-ME, IR-LE, IR-OC and IR-HE antisera cross-reactivities are shown in Table 1. Synenkephalin antiserum to [Tyr63](Syn 63-70) was donated by Dr O. Neuropeptides (1998) 32(3), 293-299

Table 1 IR-opioid peptides antisera cross-reactivities % Cross-reactivity Peptide

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Met-(o)-enkephalin 100.00 Met-enkephalin 2.90 Leu-enkephalin 0.01 Met-enkephalin-Arg 0.76 Leu-enkephalin-Arg <0.01 Met-enkephalin-Arg-Phe <0.01 Met-enkephalin-Arg-Gly-Leu <0.01 Dynorphin 1-8 <0.01 (z-endorphin <0.01 J3-endorphin <0.01 7-endorphin <0.01

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<0.01 3.83 100.00 0.53 4.33 0.37 <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 <0.01 <0.01 <0.01 <0.01 100.00 <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 100.00 <0.01 <0.01 -

Vindrola and showed cross-reactivity of 1% with native 8.6 kDa peptide (proenkephalin 1-77). No cross-reactivity was observed with Met-enkephalin, Met-enkephalinArg ~, Met-enkephalin-Arg~-Phe 7, Met-enkephalin-Arg 6GlyT-Leu8, Leu-enkephalin peptide E and F, dynorphin B, amidorphin and metorphamide? 2 Protein content was determined by the method of Lowry et al23 with bovine serum albumin as a reference standard. Peptide contents in perfusates and tissue content were expressed as pmol of IR-peptide/mg of protein. Statistics Statistical differences between groups were calculated by two-way ANOVA and Student's t-test. A P < 0.05 level or less was accepted as significant. RESULTS IR-ME release in the control group showed diurnal variations, where maximal concentration was reached between 16:00 and 20:00 h. IR-ME release in the kindling group showed a significant increase over the control group during photoperiod (two-way ANOVA F(1,5) = 3.39, P < 0.009 for the hour a day, and F(1,5) = 4.87, P < 0.031 for treatment). Student's t-test showed a significant increase ( P < 0.05) at 12:00 and 24:00 h compared with the control group. IR-ME content in the remaining slices showed a significant enhancement in PTZ-kindled rats during photoperiod, except at 04:00 h (Fig. 1). IR-LE release was parallel between the control and experimental group; maximal concentration was reached at 24:00 h. IR-LE levels did not differ with treatment. A significant change was observed for the hour F(1,5) = 9.50, P < 0.000. IR-LE levels in tissue slices showed a significant increase at 16:00 and 20:00 h, P < 0.047, P < 0.01, respectively (Fig. 2). IR-OC showed a defined circadian rhythm, its release was similar between groups in all the © Harcourt Brace and Co. Ltd 1998

Diumal variations of opioid peptides and synenkephalin re~ease 295

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Fig. 1 IR-Met-enkephalin release and slices content from basolateral amygdala. (A) Release of IR-ME evoked by a 10 min ,exposure to 55 mM K+; control group (ll) and kindling group ([E). Each point represents the mean _+SEM of 3 experiments. (B) IR-ME content in tissue slices in the same conditions as described in panel A. Shaded area represents the dark period. Statistical analysis: two-way ANOVA for panel (A) hour: F(1,5) = 3.398, P < 0.009; treatment: F(1,5) = 4.87, P < 0.031. Student's t-test for panels A and B. * P < 0.05, **P< 0.01, ***P< 0.001 compared with control group.

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Fig. 3 IR-heptapeptide release and slices content from basolateral amygdala. (A) Release of IR-HE evoked by a 10 min exposure to 55 mM K+; control group (1) and kindling group (R). Each point represents the mean _+SEM of 3 experiments. (B) IR-HE content in tissue slices in the same conditions as described in panel A. Shaded area represents the dark period. Statistical analysis: two-way ANOVA panel (A) hour: F(1,5) = 14.700, P < 0.000; treatment F(1,5) = 0.001, P < 0.975. Student's t-test for panels A and B: * P < 0.05, **P< 0.03 compared with control group.

analysed hours, except at 20:00 h. At this hour IR-OC levels increased in both groups, but the kindling group displayed significant increase when compared with the control group (P< 0.05). IR-OC tissue content in the kindling group was higher than the control group during photoperiod (Fig. 4). IR-HE release did not show circadian rhythm, its values in both control and experimental groups showed similar concentrations. Two-way ANOVA analysis showed statistical differences for the hour F(1,5) = 14.7, P < 0.000, but no changes were found as a result of the treatment. IR-HE content in slices was higher in the experimental than in the control group (Fig. 3). Finally, IR-Syn release showed defined variations during photoperiod. The peak for their release was reached at 16:00 h. There were no significant changes between the control and the PTZ-kindied group by Student's t-test. However, ANOVA analysis showed significant differences as a result of treatment F(1,5) = 5.49, P < 0.022, as well as for the hour F(1,5) = 16.12, P < 0.000. IR-Syn tissue content was only detected at 16:00 and 20:00 h (Fig. 5). DISCUSSION The results presented here show that opioid peptides and synenkephalin release display diurnal variations in both control and experimental groups, where maximal concentration was reached before and during dark phase. Neuropeptides (1998) 32(3), 293-299

The present study found that IR-ME and IR-Syn from control groups displayed maximal opioid peptide release 2 h before the onset of darkness (16:00 h), while IR-LE, IR-OC and IR-HE release was enhanced during the dark phase. Diurnal rhythm of opioid peptide release in the control group resembles that observed in conditions wher~ EOS is involved. Rats display variations during photoperiod for opiate receptors in the rat forebrain, 2 and for peptide levels in several brain structures, e.g. hypothalamus, e basolateral amygdalaz and in the spinal cord? On the other hand it .has been shown that the kindling phenomenon in rats, induced by amygdala stimulation, is facilitated when animals are stimulated during the dark phase and it has been suggested that opioid peptides may be related to the convulsant activity in naive animals.24 This study showed an increased release of IRME, IR-LE and IR-OC in amygdala of PTZ-kindled groups during dark phase, as weU as in whole amygdala tissue content as described, z This apparent discrepancy could be explained if some differences between both experimental models of epilepsy are considered. Rats with electrical kindled seizures have enhanced benzodiazepine receptor binding in limbic structures 24 h after the last stimulation and binding is reduced in cingulate cortex at this time. 25-2zIn contrast, a single or chronic non-convulsive administration of PTZ reduced benzodiazepine receptor binding in specific amygdaloid nuclei) ~ It has © Harcourt Brace and Co. Ltd 1998

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Fig. 5 IR-Synenkephalin release and slices content from basolateral amygdala. (A) Release of IR-Syn evoked by a 10 min exposure to 55 mM K+; control group (m) and kindling group (C]). Each point represents the mean + SEM of 3 experiments. (B) IR-Syn content in tissue slices in the same conditions as described in panel A. Shaded area represents the dark period. Statistical analysis: two-way ANOVA panel (A) hour: F(1,5) = 16.12, P < 0.000, treatment F(1,5) = 5.498, P < 0.022.

been proposed that PTZ binds to the picrotoxin site of the GABA receptor complex, 28 impairing GABA/benzodiazepine-coupled chloride channel activity29 and blocking GABA-mediated inhibition. We suggest that GABA recep© Harcourt Brace and Co. Ltd 1998

tor blockade by PTZ activates the EOS through disinhibition of the excitatory amino acid transmission. Moreover, [3-endorphin administration counteracts the decreases in rat brain [3H]-flunitrazepam binding to GABA receptor Neuropeptides (1998) 32(3), 293-299

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after p i n e a l e c t o m y , 3° a surgical p r o c e d u r e t h a t causes p r o c o n v u l s a n t activity, d i s r u p t s t h e c i r c a d i a n r h y t h m of GABA3~ a n d r e d u c e s t h e e n k e p h a l i n tissue c o n t e n t in rat h y p o t h a l a m u s ? These results are c o m p a t i b l e w i t h t h e view t h a t EOS a n d GABAergic t r a n s m i s s i o n are closely related neurotransmitter systems sharing a common p r o t e c t i v e effect a g a i n s t epileptic activity. To t h e b e s t of o u r k n o w l e d g e , no d a t a h a v e b e e n r e p o r t e d of a p h a r m a c o l o g i c a l k i n d l i n g effect over s y n e n k e p h a l i n release during photoperiod. Synenkephalin, the non-opioid portion of P r o e n k e p h a l i n , 32 is p r o d u c e d a n d r e l e a s e d m a i n l y as a n i n t a c t m o l e c u l e in t h e a d u l t rat b r a i n a n d is n o t destroyed, in c o n t r a s t to o p i o i d peptides, w h i c h are r e a d ily m e t a b o l i z e d w h e n released. 3~ It h a s b e e n s u g g e s t e d t h a t IR-Syn assays are a b e t t e r i n d e x of t h e activity of e n k e p h a l i n n e u r o n s t h a n t h e a s s a y of e n k e p h a l i n s themselves. 34 The p r e s e n t s t u d y f o u n d t h a t IR-Syn release d i s p l a y e d d i u r n a l variations. In b o t h c o n t r o l a n d k i n d l e d rats, release was specifically e n h a n c e d before t h e d a r k p e r i o d (16:00 h). In addition, t h e s t u d y was n o t able to d e t e c t IR-Syn in tissue slices, e x c e p t at 16:00 a n d 20:00 h. If IR-Syn tissue c o n t e n t is e n h a n c e d at 16:00 h, it is p o s s i b l e t h a t at this h o u r t h e EOS c o u l d b e activated, w i t h a c o n c o m i t a n t o p i o i d p e p t i d e release a n d tissue c o n t e n t e n h a n c e m e n t . It is i n t e r e s t i n g to n o t e t h a t once s y n e n k e p h a l i n was d e t e c t e d (16:00 h), o n l y IR-ME release was increased, t h e release of o t h e r p e p t i d e s was a u g m e n t e d 4 h later (20:00 h). A p r e v i o u s r e p o r t f o u n d t h a t in t h e b a s o l a t e r a l a m y g d a l a of t h e rat brain, t h e IRME tissue c o n t e n t significantly d e c r e a s e d 15 rain after t h e ictal phase, while IR-LE a n d IR-OC d e c r e a s e d after 30 min, s u g g e s t i n g a differential release d u r i n g t h e postkictal d e p r e s s i o n t i m e course. 13 The p h y s i o l o g i c a l relev a n c e of IR-Syn in epfleptogenesis r e m a i n s u n k n o w n . However, it h a s b e e n s h o w n t h a t s y n e n h e p h a l i n release c o u l d be i n v o l v e d in n e r v e cell proliferation, 35 a n d in DNA s y n t h e s i s of h u m a n peripheral m o n o n u c l e a r cellsY We c o n c l u d e t h a t EOS in vitro release d i s p l a y e d d i u r n a l variations, a n d s u g g e s t t h a t EOS c o u l d b e a c t i v a t e d before t h e d a r k p h a s e (16:00). In P T Z - k i n d l e d rats t h e i n c r e a s e d release e n h a n c e m e n t d u r i n g d a r k n e s s c o u l d serve as a c o m p e n s a t o r y m e c h a n i s m a g a i n s t t h e e x c i t a t i o n i n d u c e d b y t h e GABAergic t r a n s m i s s i o n failure.

ACKNOWLEDGEMENTS The authors thank Dr Osvaldo Vindrola for s y n e n k e p h a l i n a n t i s e r u m a n d Mrs C a r m e n N a v a r r o for t e c h n i c a l assistance. This w o r k was partially s u p p o r t e d b y g r a n t 3 2 3 6 M - P 9 6 9 7 from CONACyT M4xico to M. Asai.

Neuropeptides (1998) 32(3), 293-299

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