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Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling
Neuroscience Letters, 81) (t 987) 298 31)2 Elsevier Scientific Publishers Ireland Ltd
298
NSL 04835
Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling B.J. Morris l, M.E. Moneta 2, G. ten Bruggencate 2 and V. H611t2 Department of Neuropharmacology, Max-Planck-lnstitut fiir Psychiatrie, Planegg-Martinsried ( F. R.G. ) and 2Physiologisches Institut, UniversiRit Miinchen, Miinchen ( F.R.G. ) (Received 23 April 1987; Revised version received 4 June 1987; Accepted 5 June 1987)
Key words." Hippocampus; Kindling; Opioid peptide; Dynorphin; In situ hybridization The effect of hippocampal kindling on the levels of prodynorphin mRNA in rat hippocampus was examined by in situ hybridization using a synthetic oligonucleotide probe. Cryostat tissue sections were hybridised with a 32p-labelled 100 mer DNA probe complementary to the coding region of rat prodynorphin mRNA, and exposed to X-ray film. In rats exhibiting stage 4 seizures, the levels of prodynorphin mRNA in the dentate gyrus were dramatically reduced compared to control animals. This suggests that the development of kindling is accompanied by a reduction in the rate of synthesis of peptides derived from prodynorphin.
Repeated, periodic, initially subconvulsive electrical stimulation of certain brain areas such as the amygdala or hippocampus results in the progressive development of generalized convulsive seizures. This process, termed kindling [5] provides an experimental model of temporal lobe epilepsy. There has been considerable interest in the role of the opioid peptides in the induction and maintenance of kindling. Opiates will affect the severity of the kindled seizures and the duration of the postictal depression [3, 6, 12, 19], while a number of reports indicate alterations in the hippocampal content of opioid peptides following seizure activity. Following amygdaloid kindling, the levels of Met-enkephalin and related peptides derived from proenkephalin (proenkephalin A) are increased, [8, 14, 21] and there is a concomitant increase in the content of proenkephalin mRNA in various brain regions [16], suggesting increased synthesis of proenkephalin peptides. Peptides derived from prodynorphin (proenkephalin B), are known to be synthesised in the granule cells of the hippocampal dentate gyrus [15] and transported and stored in the mossy fibre pathway which arises from the granule cells [13]. Amygdaloid-kindled seizures have been reported to decrease [8, 14] or decrease and later increase [10] the hippocampal content of dynorphin-related peptides.
Correspondence." B.J. Morris, Department of Neuropharmacology, Max-Planck-Institut f/Jr Psychiatrie, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
299 Since it is difficult to interpret changes in peptide levels in terms of the altered functional activity of neurones, we have studied the effect of kindled seizures on the content ofprodynorphin m R N A in the rat dentate gyrus. The use of in situ nucleic acid hybridization [4, 7] provides the anatomical resolution necessary to estimate changes in the prodynorphin m R N A content of this cell group following hippocampal kindling. Male Sprague Dawley rats (250-270 g) were implanted bilaterally with bipolar stainless-steel electrodes (0.2/tm diameter) in the hippocampus (dentate gyrus). Epidural spherical silver electrodes (1 mm diameter) served as EEG recording electrodes. One week after surgery rats were stimulated in the left hippocampus 3 times a day with interstimulus intervals not shorter than 3 h. The stimuli consisted of a 1-s train of 0.2-ms pulses delivered at 52 Hz; their intensity ranged from 300 to 600/zA and was adjusted individually to evoke a brief afterdischarge. One day after the development of stage 4 of the kindling procedure, rats were killed along with corresponding sham-operated controls. Brains were frozen on dry ice and 10-/~m cryostat sections thaw-mounted onto polylysine-coated slides. Some sections were stained with Cresyl violet, while others were fixed in 4% paraformaldehyde and processed for in situ hybridization as previously described [15]. Briefly, a 100 mer synthetic oligonucleotide corresponding to nucleotides 488-587 of the rat prodynorphin gene message sequence was hybridized to a synthetic 15 mer comprising nucleotides 573-587 of the anti-sense strand [2]. The complementary strand was extended using Klenow fragment and 32p-dATP (3000 Ci/mmol, Amersham) to a specific activity of 108 cpm//tg. Prehybridization treatments (0.2 N HCI, proteinase K, acetic anhydride and paraformaldehyde) were as described previously [15]. The sections were then hybridized with the prodynorphin c D N A probe (104 cpm//A) for 24 h at 50°C. After washing at 50°C as described, the sections were dehydrated in ethanol and apposed to Cronex M R F 32 film (Dupont). Densitometry was performed using a Leitz TAS microscope-linked densitometer. Inspection of Nissl-stained sections showed that there was no evidence of cell loss in the hippocampi from kindled animals. In sham-operated animals, a strong hybridization signal was obtained in the dentate gyrus, in the striatum, and in portions of the hypothalamus (Fig. la, c). Various controls indicated the specificity of the hybridization reaction. Pretreatment of section with RNase A (50 /zg/ml, 30 min) abolished the hybridization signal, while Northern analysis of extracted hippocampal RNA showed a single band with a size of 2600 nucleotides that expected for prodynorphin mRNA. In addition, the distribution of the hybridization signal in other regions of rat brain corresponds to the density ofdynorphin-immunoreactive perikarya [15], while hybridization with other DNA probes (proenkephalin and proopiomelanocortin) gives a completely different autoradiographic distribution. In animals exhibiting stage 4 seizures, there was a dramatic reduction in the hybridization signal from the dentate gyrus in both stimulated and unstimulated hemispheres (Fig. l b, d), indicating a marked decrease in the levels of prodynorphin mRNA. This occurred at both rostral and caudal levels. No decrease was seen in the striatum or dorsomedial hypothalamus. Densitometry confirmed the marked loss of prodynorphin m R N A in the dentate gyrus (Fig. 2).
300
Fig. t. In situ hybridization using the prodynorphin cDNA probe with sections from a sham-operated (a, c) and stage 4 kindled (b, d) animal, at rostral (a, b) and caudal (c, d) levels of the hippocampus. Exposure time 36 days, magnification × 3.5.
120
O.D. Units 80
40
i
D entat • Gyrus
DMH
Fig. 2. Optical density (O.D.) measurements from in situ hybridization autoradiographs. Open bars, shamoperated animals; closed bars, stage 4 kindled animals. Background optical density is subtracted from total density in each region. Three animals per group. DMH, dorsomedial hypothalamus. *P<0.05 compared to sham-operated group, significance assessed by Student's two-tailed t-test.
301 The present study confirms the utility of in situ hybridization as a high-resolution method for the detection of changes in m R N A levels in individual brain regions [18, 20]. The high sensitivity of the technique, along with the ability to study small, discrete areas of nervous tissue, gives considerable advantages over established 'in vitro' methods for the estimation of m R N A levels. The development of kindling in rats is known to be accompanied by increases in the hippocampal levels of peptides derived from proenkephalin [8, 14, 21], and concurrent increases in the levels of proenkephalin m R N A [16]. This suggests that there is a general increase in the activity of enkephalinergic neurones. Recent reports have shown that the hippocampal level of peptides derived from prodynorphin are decreased one day after stage 4 or 5 seizures [8, 14]. However, this decrease can be interpreted either as an increase or decrease in dynorphinergic activity. The results reported here show that the levels of prodynorphin m R N A are reduced in the granule cells of the dentate gyrus one day following stage 4 kindled seizures. While this could be due either to increased degradation of prodynorphin m R N A or to decreased gene transcription, the data suggest that the rate of synthesis of dynorphin-related peptides is decreased. It is clear that, in this situation, there is a dissociation between the effects on proenkephalin synthesis and prodynorphin synthesis. Differences in the metabolic regulation of the two opioid peptide families in the hippocampus have been noted in the seizure-sensitive Mongolian gerbil [11]. It therefore seems likely that dynorphin and enkephalin-related peptides fulfil contrasting functions in a situation of recurrent seizure activity. It is conceivable that the effects on dynorphin synthesis are uncoupled from events at the axon terminal, and that the dramatic fall in dynorphin levels is due to unchanged or increased release combined with reduced synthesis. However, the evidence from other tissue systems (e.g. refs. 1, 9 and 17) suggests that it is more likely that changes in the levels of m R N A directly reflect changes in the rate of peptidergic neurone activity. This would imply that, with the development of kindling, there is a decrease in the activity of the dynorphinergic component of the mossy fibre pathway. Whether this effect is a cause or a result of the kindling phenomenon remains to be determined. Interestingly, the decrease in prodynorphin m R N A levels is seen in both hemispheres, stimulated and unstimulated. This implies that the decrease is not due to the local effect of electrical stimulation, but to the state of higher seizure susceptibility that will involve the dentate gyrus of both hemispheres. In summary, this report represents the first evidence that induction of kindled seizures leads to a dramatic decrease in the levels of prodynorphin m R N A in the dentate gyrus.
We thank Professor A. Herz for his support throughout this study. We are also grateful to Irmgard Dohle for secretarial assistance. This project was supported by D F G (SPP 'Neuropeptides', and SFB 220).
302 1 Chen, C.L., Dione, F.T. and Roberts, J.L., Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds, Proc. Natl. Acad. Sci. USA, 80 (1983) 2211--2215. 2 Civelli, O., Douglass, J., Goldstein, A. and Herbert, E., Sequence and expression of the rat prodynorphin gene, Proc. Natl. Acad. Sci. USA, 82 (1985) 42914295. 3 Frenk, H., Enget, J., Ackermann, R.F., Shavit, Y. and Liebeskind, J.C., Endogenous opioids may mediate postictal behavioral depression in amygdaloid-kindled rats, Brain Res., t 67 (1974) 435-440. 4 Gall, J.G. and Pardue, M.L., Nucleic acid hybridization in cytological preparations, Meth. Enzymol., 38 (1971) 470-480. 5 Goddard, G., Development of epileptic seizure through brain stimulation at low intensity, Nature (London), 214 (1967) 1020-1020. 6 Hardy, C., Punksepp, J., Rossi, J. and Zolovick, A.J., Naloxone facilitates amygdaloid-kindling in rats, Brain Res., 194 (1980) 293 297. 7 Hudson, P., Penschow, J., Shine, J., Ryan, C., Niall, H. and Coghlan, J., Hybridization histochemistry: use of recombinant DNA as a homing probe for tissue localization of specific mRNA populations, Endocrinology, 108 (1981) 353 356. 8 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 kindling, Brain Res., 365 (1986) 185-191. 9 Kley, N., Loeffler, J.Ph., Pittius, C.W. and H611t, V., Proenkephalin A gene expression in bovine adrenal chromaffin cells is regulated by changes in electrical activity, EMBO J., 5 (1986) 967-970. 10 Lason, W., Przewlocka, B., Stala, L. and Przewlocki, R., Changes in hippocampal immunoreactive dynorphin and neoendorphin content following intraamygdalar kainic acid-induced seizures, Neuropeptides, 3 (1983) 399-404. 11 Lee, R.J., Hong, J.-S., McGinty, J.F. and Lomax, P., Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure-sensitive Mongolian gerbils, Brain Res., 401 (1987) 353-358. 12 LeGal LaSalle, G., Calvino, B. and Ben-Ari, Y., Morphine enhances amygdaloid seizures and increases inter-ictal spike frequency in kindled rats, Neurosci. Lett., 6 (1977) 255-260. 13 McGinty, J.F., Herinksen, S.J.. Goldstein, A., Terenius, L. and Bloom, F.E., Dynorphin is contained within hippocampal mossy fibres: immunochemical alterations after kainic acid administration and colchicine-induced neurotoxicity, Proc. Natl. Acad. Sci. USA, 80 (1983) 589 593. 14 McGinty, J.F., Kanamatsu, T., Obie, J., Dyer, R.S., Mitchell, C.L. and Hong, J,S., Amygdaloid kindling increases enkephalin-like immunoreactivity but decreases dynorphin-like immunoreactivity in rat hippocampus, Neurosci. Lett., 71 (1986) 31-36. 15 Morris, B.J., Haarmann, I., Kempter, B., H611t, 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. 16 Naranjo, J.R., Iadarola, M.J. and Costa, E., Changes in the dynomic state of brain proenkephalinderived peptides during amygdaloid kindling, J. Neurosci. Res., 16 (1986) 75 -87. 17 Quach, T.T., Tang, F., Kageyama, H., Mocchetti, I., Guidotti, A., Meek, J.L., Costa, E. and Schwartz, J.P., Enkephalin biosynthesis in adrenal medulla, Mol. Pharmacol., 26 (1984) 255-260. 18 Sherman, T.G., McKelvy, J.F. and Watson, S.J., Vasopressin mRNA regulation in individual hypothalamic nuclei: a Northern and in-situ hybridization analysis, J. Neurosci., 6 (1986) 1685-1644. 19 Stone. W.S., Eggteton, C.E. and Berman, R.F., Opiate modification of amygdaloid-kindted seizures in rats, Pharmacol. Biochem. Behav., 16 (1982) 751-756. 20 Uht, G.R., Zingg, H.H. and Habener, J.F., Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus, Proc. Natl. Acad. Sci. USA, 82 (1985) 5555-5559. 21 Vindrola. O., Briones, R., Asai, M. and Fernandez-Guardiola, A., Amygdaloid kindling enhances the enkephalin content in the rat brain, Neurosci. Lett., 21 (1981) 39--43.
298
NSL 04835
Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling B.J. Morris l, M.E. Moneta 2, G. ten Bruggencate 2 and V. H611t2 Department of Neuropharmacology, Max-Planck-lnstitut fiir Psychiatrie, Planegg-Martinsried ( F. R.G. ) and 2Physiologisches Institut, UniversiRit Miinchen, Miinchen ( F.R.G. ) (Received 23 April 1987; Revised version received 4 June 1987; Accepted 5 June 1987)
Key words." Hippocampus; Kindling; Opioid peptide; Dynorphin; In situ hybridization The effect of hippocampal kindling on the levels of prodynorphin mRNA in rat hippocampus was examined by in situ hybridization using a synthetic oligonucleotide probe. Cryostat tissue sections were hybridised with a 32p-labelled 100 mer DNA probe complementary to the coding region of rat prodynorphin mRNA, and exposed to X-ray film. In rats exhibiting stage 4 seizures, the levels of prodynorphin mRNA in the dentate gyrus were dramatically reduced compared to control animals. This suggests that the development of kindling is accompanied by a reduction in the rate of synthesis of peptides derived from prodynorphin.
Repeated, periodic, initially subconvulsive electrical stimulation of certain brain areas such as the amygdala or hippocampus results in the progressive development of generalized convulsive seizures. This process, termed kindling [5] provides an experimental model of temporal lobe epilepsy. There has been considerable interest in the role of the opioid peptides in the induction and maintenance of kindling. Opiates will affect the severity of the kindled seizures and the duration of the postictal depression [3, 6, 12, 19], while a number of reports indicate alterations in the hippocampal content of opioid peptides following seizure activity. Following amygdaloid kindling, the levels of Met-enkephalin and related peptides derived from proenkephalin (proenkephalin A) are increased, [8, 14, 21] and there is a concomitant increase in the content of proenkephalin mRNA in various brain regions [16], suggesting increased synthesis of proenkephalin peptides. Peptides derived from prodynorphin (proenkephalin B), are known to be synthesised in the granule cells of the hippocampal dentate gyrus [15] and transported and stored in the mossy fibre pathway which arises from the granule cells [13]. Amygdaloid-kindled seizures have been reported to decrease [8, 14] or decrease and later increase [10] the hippocampal content of dynorphin-related peptides.
Correspondence." B.J. Morris, Department of Neuropharmacology, Max-Planck-Institut f/Jr Psychiatrie, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
299 Since it is difficult to interpret changes in peptide levels in terms of the altered functional activity of neurones, we have studied the effect of kindled seizures on the content ofprodynorphin m R N A in the rat dentate gyrus. The use of in situ nucleic acid hybridization [4, 7] provides the anatomical resolution necessary to estimate changes in the prodynorphin m R N A content of this cell group following hippocampal kindling. Male Sprague Dawley rats (250-270 g) were implanted bilaterally with bipolar stainless-steel electrodes (0.2/tm diameter) in the hippocampus (dentate gyrus). Epidural spherical silver electrodes (1 mm diameter) served as EEG recording electrodes. One week after surgery rats were stimulated in the left hippocampus 3 times a day with interstimulus intervals not shorter than 3 h. The stimuli consisted of a 1-s train of 0.2-ms pulses delivered at 52 Hz; their intensity ranged from 300 to 600/zA and was adjusted individually to evoke a brief afterdischarge. One day after the development of stage 4 of the kindling procedure, rats were killed along with corresponding sham-operated controls. Brains were frozen on dry ice and 10-/~m cryostat sections thaw-mounted onto polylysine-coated slides. Some sections were stained with Cresyl violet, while others were fixed in 4% paraformaldehyde and processed for in situ hybridization as previously described [15]. Briefly, a 100 mer synthetic oligonucleotide corresponding to nucleotides 488-587 of the rat prodynorphin gene message sequence was hybridized to a synthetic 15 mer comprising nucleotides 573-587 of the anti-sense strand [2]. The complementary strand was extended using Klenow fragment and 32p-dATP (3000 Ci/mmol, Amersham) to a specific activity of 108 cpm//tg. Prehybridization treatments (0.2 N HCI, proteinase K, acetic anhydride and paraformaldehyde) were as described previously [15]. The sections were then hybridized with the prodynorphin c D N A probe (104 cpm//A) for 24 h at 50°C. After washing at 50°C as described, the sections were dehydrated in ethanol and apposed to Cronex M R F 32 film (Dupont). Densitometry was performed using a Leitz TAS microscope-linked densitometer. Inspection of Nissl-stained sections showed that there was no evidence of cell loss in the hippocampi from kindled animals. In sham-operated animals, a strong hybridization signal was obtained in the dentate gyrus, in the striatum, and in portions of the hypothalamus (Fig. la, c). Various controls indicated the specificity of the hybridization reaction. Pretreatment of section with RNase A (50 /zg/ml, 30 min) abolished the hybridization signal, while Northern analysis of extracted hippocampal RNA showed a single band with a size of 2600 nucleotides that expected for prodynorphin mRNA. In addition, the distribution of the hybridization signal in other regions of rat brain corresponds to the density ofdynorphin-immunoreactive perikarya [15], while hybridization with other DNA probes (proenkephalin and proopiomelanocortin) gives a completely different autoradiographic distribution. In animals exhibiting stage 4 seizures, there was a dramatic reduction in the hybridization signal from the dentate gyrus in both stimulated and unstimulated hemispheres (Fig. l b, d), indicating a marked decrease in the levels of prodynorphin mRNA. This occurred at both rostral and caudal levels. No decrease was seen in the striatum or dorsomedial hypothalamus. Densitometry confirmed the marked loss of prodynorphin m R N A in the dentate gyrus (Fig. 2).
300
Fig. t. In situ hybridization using the prodynorphin cDNA probe with sections from a sham-operated (a, c) and stage 4 kindled (b, d) animal, at rostral (a, b) and caudal (c, d) levels of the hippocampus. Exposure time 36 days, magnification × 3.5.
120
O.D. Units 80
40
i
D entat • Gyrus
DMH
Fig. 2. Optical density (O.D.) measurements from in situ hybridization autoradiographs. Open bars, shamoperated animals; closed bars, stage 4 kindled animals. Background optical density is subtracted from total density in each region. Three animals per group. DMH, dorsomedial hypothalamus. *P<0.05 compared to sham-operated group, significance assessed by Student's two-tailed t-test.
301 The present study confirms the utility of in situ hybridization as a high-resolution method for the detection of changes in m R N A levels in individual brain regions [18, 20]. The high sensitivity of the technique, along with the ability to study small, discrete areas of nervous tissue, gives considerable advantages over established 'in vitro' methods for the estimation of m R N A levels. The development of kindling in rats is known to be accompanied by increases in the hippocampal levels of peptides derived from proenkephalin [8, 14, 21], and concurrent increases in the levels of proenkephalin m R N A [16]. This suggests that there is a general increase in the activity of enkephalinergic neurones. Recent reports have shown that the hippocampal level of peptides derived from prodynorphin are decreased one day after stage 4 or 5 seizures [8, 14]. However, this decrease can be interpreted either as an increase or decrease in dynorphinergic activity. The results reported here show that the levels of prodynorphin m R N A are reduced in the granule cells of the dentate gyrus one day following stage 4 kindled seizures. While this could be due either to increased degradation of prodynorphin m R N A or to decreased gene transcription, the data suggest that the rate of synthesis of dynorphin-related peptides is decreased. It is clear that, in this situation, there is a dissociation between the effects on proenkephalin synthesis and prodynorphin synthesis. Differences in the metabolic regulation of the two opioid peptide families in the hippocampus have been noted in the seizure-sensitive Mongolian gerbil [11]. It therefore seems likely that dynorphin and enkephalin-related peptides fulfil contrasting functions in a situation of recurrent seizure activity. It is conceivable that the effects on dynorphin synthesis are uncoupled from events at the axon terminal, and that the dramatic fall in dynorphin levels is due to unchanged or increased release combined with reduced synthesis. However, the evidence from other tissue systems (e.g. refs. 1, 9 and 17) suggests that it is more likely that changes in the levels of m R N A directly reflect changes in the rate of peptidergic neurone activity. This would imply that, with the development of kindling, there is a decrease in the activity of the dynorphinergic component of the mossy fibre pathway. Whether this effect is a cause or a result of the kindling phenomenon remains to be determined. Interestingly, the decrease in prodynorphin m R N A levels is seen in both hemispheres, stimulated and unstimulated. This implies that the decrease is not due to the local effect of electrical stimulation, but to the state of higher seizure susceptibility that will involve the dentate gyrus of both hemispheres. In summary, this report represents the first evidence that induction of kindled seizures leads to a dramatic decrease in the levels of prodynorphin m R N A in the dentate gyrus.
We thank Professor A. Herz for his support throughout this study. We are also grateful to Irmgard Dohle for secretarial assistance. This project was supported by D F G (SPP 'Neuropeptides', and SFB 220).
302 1 Chen, C.L., Dione, F.T. and Roberts, J.L., Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds, Proc. Natl. Acad. Sci. USA, 80 (1983) 2211--2215. 2 Civelli, O., Douglass, J., Goldstein, A. and Herbert, E., Sequence and expression of the rat prodynorphin gene, Proc. Natl. Acad. Sci. USA, 82 (1985) 42914295. 3 Frenk, H., Enget, J., Ackermann, R.F., Shavit, Y. and Liebeskind, J.C., Endogenous opioids may mediate postictal behavioral depression in amygdaloid-kindled rats, Brain Res., t 67 (1974) 435-440. 4 Gall, J.G. and Pardue, M.L., Nucleic acid hybridization in cytological preparations, Meth. Enzymol., 38 (1971) 470-480. 5 Goddard, G., Development of epileptic seizure through brain stimulation at low intensity, Nature (London), 214 (1967) 1020-1020. 6 Hardy, C., Punksepp, J., Rossi, J. and Zolovick, A.J., Naloxone facilitates amygdaloid-kindling in rats, Brain Res., 194 (1980) 293 297. 7 Hudson, P., Penschow, J., Shine, J., Ryan, C., Niall, H. and Coghlan, J., Hybridization histochemistry: use of recombinant DNA as a homing probe for tissue localization of specific mRNA populations, Endocrinology, 108 (1981) 353 356. 8 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 kindling, Brain Res., 365 (1986) 185-191. 9 Kley, N., Loeffler, J.Ph., Pittius, C.W. and H611t, V., Proenkephalin A gene expression in bovine adrenal chromaffin cells is regulated by changes in electrical activity, EMBO J., 5 (1986) 967-970. 10 Lason, W., Przewlocka, B., Stala, L. and Przewlocki, R., Changes in hippocampal immunoreactive dynorphin and neoendorphin content following intraamygdalar kainic acid-induced seizures, Neuropeptides, 3 (1983) 399-404. 11 Lee, R.J., Hong, J.-S., McGinty, J.F. and Lomax, P., Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure-sensitive Mongolian gerbils, Brain Res., 401 (1987) 353-358. 12 LeGal LaSalle, G., Calvino, B. and Ben-Ari, Y., Morphine enhances amygdaloid seizures and increases inter-ictal spike frequency in kindled rats, Neurosci. Lett., 6 (1977) 255-260. 13 McGinty, J.F., Herinksen, S.J.. Goldstein, A., Terenius, L. and Bloom, F.E., Dynorphin is contained within hippocampal mossy fibres: immunochemical alterations after kainic acid administration and colchicine-induced neurotoxicity, Proc. Natl. Acad. Sci. USA, 80 (1983) 589 593. 14 McGinty, J.F., Kanamatsu, T., Obie, J., Dyer, R.S., Mitchell, C.L. and Hong, J,S., Amygdaloid kindling increases enkephalin-like immunoreactivity but decreases dynorphin-like immunoreactivity in rat hippocampus, Neurosci. Lett., 71 (1986) 31-36. 15 Morris, B.J., Haarmann, I., Kempter, B., H611t, 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. 16 Naranjo, J.R., Iadarola, M.J. and Costa, E., Changes in the dynomic state of brain proenkephalinderived peptides during amygdaloid kindling, J. Neurosci. Res., 16 (1986) 75 -87. 17 Quach, T.T., Tang, F., Kageyama, H., Mocchetti, I., Guidotti, A., Meek, J.L., Costa, E. and Schwartz, J.P., Enkephalin biosynthesis in adrenal medulla, Mol. Pharmacol., 26 (1984) 255-260. 18 Sherman, T.G., McKelvy, J.F. and Watson, S.J., Vasopressin mRNA regulation in individual hypothalamic nuclei: a Northern and in-situ hybridization analysis, J. Neurosci., 6 (1986) 1685-1644. 19 Stone. W.S., Eggteton, C.E. and Berman, R.F., Opiate modification of amygdaloid-kindted seizures in rats, Pharmacol. Biochem. Behav., 16 (1982) 751-756. 20 Uht, G.R., Zingg, H.H. and Habener, J.F., Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus, Proc. Natl. Acad. Sci. USA, 82 (1985) 5555-5559. 21 Vindrola. O., Briones, R., Asai, M. and Fernandez-Guardiola, A., Amygdaloid kindling enhances the enkephalin content in the rat brain, Neurosci. Lett., 21 (1981) 39--43.