An in vitro autoradiographic analysis of mu and delta opioid binding in the hippocampal formation of kindled rats

Brain Research, 412 (1987) 343-351

343

Elsevier BRE 12603

An in vitro autoradiographic analysis of mu and delta opioid binding in the hippocampal formation of kindled rats Barbara J. Crain 1'2'6, Kwen-Jen Chang 3'7 and James O. McNamara 4,5 Departments of 1pathology, 2Anatomy, 3Anesthesiology, 4Medicine (Neurology), and 5Pharmacology, Duke University Medical Center, Durham, NC27710 (U.S.A.), 6Epilepsy Research Unit, Veterans Administration Medical Center, Durham, NC27705 (U.S.A.), and 7Department of Molecular Biology, Wellcome Research Laboratories, Research Triangle Park, NC27705 (U.S.A.) (Accepted 21 October 1986)

Key words: Opioid binding; Hippocampus; Kindling; Autoradiography; Mu opioid peptide; Delta opioid peptide

Recent studies have shown that opioid peptide levels are altered in hippocampal formation of kindled animals. We therefore studied the distributions of mu and delta opioid binding sites in hippocampal formation of kindled and control rats using quantitative in vitro autoradiography. Animals received daily stimulations of the amygdala until they experienced 3 class 5 seizures. Paired control animals underwent implantation of electrodes but were not stimulated. Mu binding sites were labeled with 125I-FK-33824.Twenty-four hours after the last kindled seizure, mu binding was decreased by 32% in stratum pyramidale of CA 1and stratum radiatum of CA2 and by 17-27% throughout most of the rest of CA D CA2, and CA3. Few, if any, differences were seen between kindled and control animals at 7 or 28 days after the last kindled seizure. Delta binding sites were labeled with 125I-[D-AlaZ,D-LeuS]enkephalinin the presence of the morphiceptin analog PL-032. Twenty-four hours after the last kindled seizure, delta binding was decreased only in stratum moleculare of the dentate gyrus. Seven days after the last kindled seizure, delta binding was decreased by 11-17% throughout CA1, CA3, and the dentate gyrus. At 28 days after the last seizure, however, no differences were found between kindled and control animals. Since the decreases in mu and delta opioid binding are transient, they are unlikely to be the molecular basis of the permanent kindling phenomenon. Rather, these changes in opioid binding may represent responses to repeated seizures.

INTRODUCTION The kindling p h e n o m e n o n represents an animal model for epilepsy in which repeated administration of an initially subconvulsive electrical stimulus results in progressive intensification of seizure activity and culminates in a generalized (class 5) seizure 16. Once established, this effect is irreversible, and kindling has therefore become widely utilized for the study of partial seizures, epileptogenesis, and neuronal plasticity. Although the precise spatial extent of the underlying n e u r o n a l circuitry remains poorly understood, many nuclei of the limbic system and basal ganglia are probably involved33. Since endogenous opioid peptides are present in many of these same regions 47, n u m e r o u s studies have been performed to determine whether the development or the expression of kindled seizures can be modified by

opiate administration. As reviewed elsewhere 33, morphine, a mu agonist with little or no effect on the rate of kindling or on the expression of fully kindled seizures, exacerbates the spontaneous interictal spiking and the postictal behavioral depression seen after kindled seizures 1'14'2°' 31,41. The behavioral arrest, twitching, and wet-dog shakes seen after intracerebroventricular (i.c.v.) morphine or enkephalin are similar to postictal behavior after a kindled seizure 9AS'a9,21,39,4°'44. The fact that in some cases naloxone alone decreases the behavioral depression seen after a kindled seizure 14'41 suggests that endogenous opioids may mediate postictal events in kindled animals. Although the site or sites of this postulated enhanced opioid neuronal communication is u n d e t e r m i n e d , the hippocampal formation is a leading candidate since it is one of the first areas to show heavy labeling after an i.c.v, injec-

Correspondence: B.J. Crain, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B .V. (Biomedical Division)

344 tion of [3H]enkephalinl9. Previous biochemical studies of opioids in kindling have focused on the endogenous opioid peptides. Leu-enkephalin and MetS-enkephalin-Arg6-Gly7Leu 8 immunoreactivity nearly doubles in hippocampus 1 day after the last kindled seizure 25'37. Similarly, Met-enkephalin levels are doubled for at least 2 weeks in hippocampus, caudate, and septum after multiple seizures induced by intracerebral kainic acid, repeated electroconvulsive shock, and isoniazid 18'24. These changes appear to be due to increased enkephalin synthesis 34. Opioid receptors constitute another important biochemical index of opioid function. We therefore quantitated opioid receptor binding in the hippocampal formation with a radiohistochemical method. Specifically, we examined binding to mu and delta opioid receptors in kindled and control animals 24 h, 7 days, and 28 days after completion of kindling. Together, these two subpopulations account for approximately 80% of opioid binding in the hippocampal formation 7. MATERIALS AND METHODS

Animals Male Sprague-Dawley rats initially weighing 300-425 g were used in all experiments. Stimulating electrodes were placed in the right amygdala and stainless steel screws for surface electroencephalographic recordings were inserted as previously described 11'32. After at least 7 days of recovery, kindling stimulations were administered daily until the animals exhibited 3 class 5 seizures. Following the completion of kindling, the experimental animals and their paired controls were killed by decapitation 24 h (n = 4), 7 days (n = 3) or 28 days (n = 3) after the last kindled seizure. In all cases, paired control animals were electrode-implanted but not stimulated. The brains were quickly removed, frozen in isopentane chilled in a methanol-dry ice bath, and stored at -60 °C. Serial frozen sections through the dorsal hippocampus were cut on a cryostat and again stored at -60 °C until the day of incubation. Sections adjacent to those used in the present study were labeled using [3H]flunitrazepam to label benzodiazepine binding sites or [3H]muscimol to label GABA-

ergic binding sites 38.

Autoradiography Mu opioid binding sites were labeled in 2-4 sections from the dorsal hippocampus in each case by 0.7 nM 125I-FK-33824 (initially 2 mCi/nmol), an enkephalin derivative highly selective for the mu subpopulation of opioid receptors 4,s,1°. Specific binding was defined as the difference between binding of a25I-FK33824 alone ('total binding') and binding of 125I-FK33824 in the presence of 1/~M diprenorphine ('nonspecific binding'). Under the incubation conditions used in this procedure, specific binding was 90-97% of total binding, 99% of specific binding was to mu opioid binding sites, and 47% of mu binding sites were labeled in hippocampal formation 1°,17. Labeled sections were exposed to LKB Ultrofilm 3H for 10-21 days. Delta opioid binding sites were labeled by 0.5 nM 125I-[o-Ala2,I>LeuS]enkephalin (initially 2 mCi/nmol) in the presence of 1 ¢zM PL-032 (Tyr-Pro-Phe-D-ProNH2), a morphiceptin analog which is highly selective for mu binding sites 8'1°. Non-specific binding was defined as the binding remaining in the presence of 1 tiM diprenorphine. Under the incubation conditions used in this procedure, specific binding was 65-82% of total binding, 99% of specific binding in hippocampal formation was to delta opioid binding sites, and 23% of delta binding sites were labeled 1°. Labeled sections from animals sacrificed 24 h or 7 days after the last kindled seizure were exposed to film for 3-8 weeks. Labeled sections from animals sacrificed 28 days after the last seizure were apposed to emulsioncoated coverslips5° (NTB-2, Kodak) for 9 weeks, developed, and counter-stained with methyl-green Pyronine-Y. In each experiment, the sections from an experimental animal and its paired control were incubated together and exposed to the same piece of film.

Quantification Enkephalin-labeled sections from kindled and control animals were exposed to film along with 3Hlabeled brain paste standards 43 which had been calibrated to 125I-labeled brain paste standards as previously described 1°. The optical density produced on the film by each standard section was determined using a Zeiss optical densitometer and a Rockwell

345 computer. Microscopic fields 100 /~m in d i a m e t e r were analyzed. ( D u e to the size of the silver grains, measurements of smaller fields were highly variable.) F o r each region examined, 5 - 9 separate readings were averaged, with standard errors of the mean less than 5% of the m e a n for each section. The optical densities of the 3H-standards were similarly measured. The amount of lzsI-ligand b o u n d (fmol/mg protein) was then calculated for each region in each animal. The left and right h i p p o c a m p a l formations were analyzed separately. The specific binding (total minus non-specific binding) was calculated for each region. D a t a from experimental and control animals was c o m p a r e d using a p a i r e d Student's t-test. F o r the 125I-DADL-labeled sections from animals sacrificed 28 days after the last kindled seizure, grain counts were p e r f o r m e d on the emulsion-coated cov-

erslip preparations. Microscopic fields 20 ktm × 20 ktm were analyzed. F o r each region, 5 - 6 separate readings were averaged, with standard errors of the mean less than 10% of the m e a n for each section. RESULTS

Laminar analysis The boundaries defining C A l, C A 2 and C A 3 in the present study are indicated in Figs. 1 and 3. C A l corresponds to that portion of the h i p p o c a m p a l formation in which the pyramidal cells are densely p a c k e d with a row of neurons readily visible in the apical portion of stratum pyramidale. The neurons in C A 2 are also densely p a c k e d but with no evidence of an apical row of cells within stratum pyramidale. In contrast, in C A 3 the p y r a m i d a l neurons are larger and m o r e widely spaced.

Fig. 1. The laminar distribution of mu opioid binding sites in a control animal. The boundaries between CA 1 and C A 2 and between CA2 and CA 3 are indicated by stars. The laminae are labeled as follows: o, stratum oriens; p, stratum pyramidale; l-m, stratum lacunosum-moleculare; l, stratum lucidum; m-s, stratum moleculare, suprapyramidal blade; m-i, stratum moleculare, infrapyramidal blade. The dentate gyrus (DG) is also indicated. Scale bar = 1 ram. (Modified from Fig. 2 in ref. 10.)

346 The boundaries of the laminae in CA1, C A 2 and CA3 and the dentate gyrus are also indicated in Figs. 1 and 3. These have been illustrated and described in more detail in a previous study m.

Binding to mu opioid binding sites The normal distribution of 125I-FK-33824 (mu) binding sites has been described previously 1°,17,42 and is illustrated in Fig. 1. Binding is greatest in stratum pyramidale and stratum lacunosum-moleculare of CA2 and CA 3 and is also relatively high in stratum oriens and stratum radiatum of CA 2, stratum oriens of CA 3, and stratum pyramidale of C A 1. Twenty-four hours after the last kindled seizure, mu opioid binding was decreased by 17-32% throughout CA D C A 2 , and CA 3 (Fig. 2). The largest changes were in stratum radiatum of CA 2 (32% _+ 4%, P < 0.001) and stratum pyramidale of CA I (32% MU OPIOID BINDING AFTER KINDLED SEIZURES (Mean % change from control± SE) 50 24, Hrs. ,o

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+ 6%, P < 0.01). No significant changes were seen in stratum moleculare of the dentate gyrus. By 7 days and 28 days after the last kindled seizure, the early decrease in mu opioid binding had disappeared. In contrast, there was a slight tendency for increased binding in the experimental animals, although this was only statistically significant in stratum oriens of CA2 at 7 days (15% -+ 4%, P < 0.05) and stratum pyramidale of CA 3 at 28 days (19% _+ 5%, P < 0.05).

Binding to delta opioid binding sites The normal distribution of delta binding sites labeled by 125I-DADL in the presence of PL-032 is illustrated in Fig. 3 and has been described previously1°. Delta binding sites are numerous in stratum pyramidale and stratum lacunosum-moleculare. Compared to mu binding, delta binding is relatively enriched in stratum moleculare of the dentate gyrus. Like mu opioid binding, delta opioid binding decreases in the hippocampal formation after kindled seizures. However, the time course and the distribution of the changes differ somewhat. Twenty-four hours after the last kindled seizure, many layers of the hippocampal formation showed small decreases in delta opioid binding. These changes reached statistical significance only in stratum moleculare of the dentate gyrus (12% + 4%, P < 0.05, in the suprapyramidal blade and 13% + 4%, P < 0.05, in the infrapyramidal blade, see Fig. 4). By 7 days after the last kindled seizure, decreased binding in the order of 11-17% was present throughout CA D CA 3, and the dentate gyrus, but n o t C A 2. Twenty-eight days after the last seizure, there were no statistically significant differences between the experimental and the control groups. An apparent tendency for increased delta opioid binding was seen in only one of the 3 pairs of animals. DISCUSSION

Opioid peptide levels in kindled animals Fig. 2. M u opioid binding after kindled seizures. The m e a n percent differences in m u opioid binding between kindled animals and their paired controls are illustrated 24 h, 7 days, and 28 days after the last kindled seizure. Statistical significance on a two-tailed paired Student's t-test is indicated: * P < 0.05; ** P < 0.01; *** P < 0.001. T h e abbreviations are the same as in Fig. 1.

Numerous brain regions which are likely to be important in the development and maintenance of kindling contain one or more endogenous opioid peptides. These include the nucleus accumbens, caudate, putamen, substantia nigra, amygdala, hippocampal formation, pyriform cortex, and entorhinal

347

Fig. 3. The laminar distribution of delta opioid binding sites in a control animal. The boundaries between CA 1 and CA2 and between CA2 and C A 3 a r e indicated by stars. The laminae are Labeled as follows: o, stratum oriens; p, stratum pyramidale; r, stratum radiatum; l-m, stratum lacunosum-moleculare; m-s, stratum moleculare, suprapyramidal blade; m-i, stratum moleculare, infrapyramidal blade. The dentate gyrus (DG) is also indicated. Stratum oriens and stratum lucidum could not always be distinguished from the adjacent layers and were therefore not analyzed. Scale bar = 1 mm. (Modified from Fig. 5 in ref. 10.)

cortex 47. Radioimmunoassays of opioid peptide levels have therefore been performed on samples from some of these areas after amygdala kindling or after seizures induced by other methods. Levels of both Met- and Leu-enkephalin were elevated 40% in both cerebral hemispheres 28 h after the last kindled seizure 4s. The rise in Leu-enkephalin levels during the kindling process began first in the stimulated hemisphere and then spread bilaterally, while Met-enkephalin levels were unaltered until the animals had experienced at least 5 class 5 kindled seizures 46. Using specimens from amygdala-kindled rabbits, Przewlocki et al. found 230% elevations in hippocampal formation of immunoreactive dynorphin A and 70-100% increases in a-neoendorphin and Leuenkephalin, but not fl-endorphin, 24 h after the last

fully kindled seizure 37. Dynorphin levels remained elevated by 280% 1 month later. The increase was specific for hippocampus, with no changes in striatum, hypothalamus, pituitary, thalamus, medulla/ pons, cortex, or cerebellum. Whether the elevations in Leu-enkephalin and ct-neoendorphin levels showed similar persistence and spatial selectivity was not determined. In a similar experiment using amygdala-kindled rats with 20 class 4 or class 5 seizures, Iadarola et al. showed an 82% increase in hippocampal MetS-en kephalin-Arg6-Gly7-Leu s 24 h after the last seizure 25. In contrast to the large permanent increase in immunoreactive dynorphin A in rabbits, dynorphin A levels in rats decreased by 55% 24 h after the last kindled seizure. This apparent discrepancy may reflect biphasic changes similar to those seen after oth-

348 D E L T A OPIOID BINDING A F T E R KINDLED SEIZURES (Mean % change

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Fig. 4. Delta opioid binding after kindled seizures. T h e m e a n percent differences in delta opioid binding between kindled animals and their paired control animals are illustrated 24 h, 7 days, and 28 days after the last kindled seizure. Statistical significance on a two-tailed paired Student's t-test is indicated: * P < 0.05; ** P < 0.01. T h e abbreviations are the same as in Fig. 3.

er types of seizures 28'36. The kindled-seizure related increases in many, but not all, opioid peptides could be associated with the kindling phenomenon itself, but they are more likely to be responses to repeated seizures. For example, hippocampal and pituitary levels of dynorphin are elevated after seizures produced by 7-hydroxybutyrate 27, and decreased 24 h after repeated electroconvulsive shocks 26. Met-enkephalin levels are elevated by 50-100% for 24 h to at least 2 weeks in hippocampus, caudate, and septum, but not in cortex or pons/medulla, after multiple seizures induced by intracerebral kainic acid injections, repeated electroconvulsive shocks, and i s o n i a z i d 1s'24'26,36.

Opioid binding sites in kindled animals Since opioid receptors, like opioid peptides, are found throughout the nervous system 17'22 and may be

altered in different regions in different ways, the effects of kindling on opioid binding must be considered separately in each area of the brain. In addition, since most brain regions contain more than one subpopulation of opioid receptors 7, each subpopulation of binding sites must be analyzed separately. As a first step towards understanding the effects of kindling on opiate receptors, and also as a step towards understanding the significance of the reported changes in opioid peptide levels, binding to mu and delta opioid receptors was examined autoradiographically in the dorsal hippocampal formation of the rat 24 h, 7 days and 28 days after the last amygdata-kindled seizure. In contrast to the large and possibly permanent increases in hippocampal opioid peptide levels25'37, decreases in binding to mu and delta opioid receptors were transient and relatively small. Thus, binding to mu opioid binding sites was decreased by 17-32% throughout CA1, CA 2, and CA3, but not the dentate gyrus, 24 h after the last kindled seizure. This change disappears by seven days. Compared to the decrease in mu opioid binding, the decrease in binding to delta opioid sites appeared slightly later, at seven days after the last seizure, and was even smaller, with binding decreased by 11-17% in CA1, CA3, and the dentate gyrus. Opiate receptor binding in kindled rats has also been described using in vivo autoradiography in a preliminary report by Weiss et al. 48. Immediately after the last kindled seizure, [3H]diprenorphine was injected intravenously. This ligand labels all subtypes of opioid receptors 7. The rats were sacrificed 20 min later. With this paradigm, [3H]diprenorphine binding was decreased by 35-40% in cingulate cortex, nucleus accumbens, and dorsal hippocampus, by 48% in the basolateral amygdala, and by 20-30% in ventral hippocampus, cortical amygdaloid nucleus, hypothalamus, and sensory cortex. No changes were seen in caudate or motor cortex4q Since this experiment was done in vivo, endogenous opioid peptides were not washed out. Thus, the decreased occupancy of binding sites by [3H]diprenorphine immediately after the seizure may in part reflect increased occupation of binding sites by endogenous opioids released during the seizure, although alterations in the receptors themselves and alterations in 3H-ligand access to tissue are also possible. In the present in vitro autoradiographic experi-

349 ments, the slide-mounted sections were preincubated in buffer containing GDP and sodium chloride. A similar wash step effectively removes endogenous opioid peptides in membrane studies 5, and thus it is likely that the decreased opioid binding in hippocampal formation 24 h (mu) or 7 days (delta) after kindled seizures is due to a change in receptor number rather than occupancy. In the present study, binding isotherms conducted with quantitative radiohistochemical methods could not distinguish changes in affinity from changes in numbers of binding sites because the differences between experimental and control were not large enough (data not presented). However, since the 7-20% decreases in opioid binding to hippocampal membranes from animals receiving multiple electroconvulsive shocks is due to a change in receptor number 34, the decreased binding seen after kindled seizures is most likely due to a decrease in receptor number as well. As described in the preceding paragraphs, the present in vitro autoradiographic study in hippocampal formation of kindled animals, the in vivo autoradiographic study of multiple brain regions of kindled animals a8 and the study in hippocampal membranes of animals receiving multiple electroconvulsive s h o c k s 34 all reported decreased opioid binding of approximately the same magnitude after seizures. In contrast, there has been one report of 30% increases in the number of [3H]DADL binding sites in synaptosomal preparations from whole rat brain (minus cerebellum) after repeated electroconvulsive shocks 23. Identifying the source of this discrepancy will require further study. One distinct possibility is that a decrease in binding to hippocampal membranes was masked by large increases in other sites. The general observation that hippocampal opioid binding is modified in numerous seizure models suggests that the changes seen in kindled animals are a response to seizures rather than a kindling-specific change.

Altered opioid neuronal communication after kindled seizures The changes in opioid binding suggest that opioid neuronal communication is altered after kindled seizures. Three particularly interesting aspects of this alteration are its cellular localization, its mechanism, and its effect on opioid synaptic efficacy.

Careful laminar analysis of the distribution of opioid binding in the hippocampal formation reveals that it parallels the distribution of both interneuron cell bodies and glutamic acid decarboxylase-positive terminals 2'1°. This qualitative morphologic observation that opioid receptors may be associated with interneurons is supported by electrophysiologic studies3°35'51. Thus, the decreased opioid binding after kindling may reflect changes in multiple populations of hippocampal interneurons. If only certain subpopulations of interneurons in each layer are associated with the decreased opioid binding, the functional significance of the decreased binding might be greater than its overall small magnitude might suggest. If the markedly increased opioid peptide levels found in hippocampal formation after kindled seizures 25'37 reflect increased enkephalin synthesis and release, then the transiently decreased mu and delta opioid binding may reflect down-regulation of the receptors in response to increased efflux of opioid peptides during or after kindled seizures. Such a mechanism has been proposed to explain the decreased opioid binding seen after seizures produced by electroconvulsive shock, a seizure model in which increased preproenkephalin synthesis suggests increased opioid peptide synthesis and release 49. Application of opioid peptides can induce downregulation of opioid binding both in hippocampal slices in vitro 13 and in cultured neuroblastoma cells629. Decreased binding could also be due to other mechanisms. For example, kindling induced decreases in muscarinic cholinergic binding are not induced by the ligand 11. In any case, whether opioid synaptic efficacy shows a net increase depends both on presynaptic factors like increased peptide release, which may produce increased excitation of pyramidal cells by inhibiting interneurons 12'3°'35'51, and on postsynaptic factors like decreases in peptide binding sites, which may reflect decreased pyramidal cell excitability. Direct electrophysiological measurements of opioid synaptic efficacy are required to make this determination.

ACKNOWLEDGEMENTS This work was supported by the United States Veterans Administration.

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