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Home , Dynorphin, Dynorphin B, Leumorphin, Neoendorphin, Prodynorphin

Neuropeptides (1993) 25, 131-138 0 Longman Group UK Ltd 1993

Expression of -derived and mRNA in Guinea-pig Cortex

C. D. RAMSDELL and J. H. MEADOR-WOODRUFF

Mental Healrh Research Institute, Department of Psychiatry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720, USA (Reprint requests to JHMW)

Abstract-The distributions and extent of processing of four prodynorphin-derived peptides ( A (l-171, (l-81, , and a-) were determined in ten regions of the cortex as well as in the of the guinea-pig. There were significant differences between concentrations of these peptides in most cortical regions, with a- neoendorphin being several times more abundant than the other peptides, and dynorphin A (I-17) being present in the least amount. There were significant between-region differences in concentration for each , although most regions had concentrations similar to those seen in the striatum. Concentrations of each peptide tended to be higher in piriform, entorhinal, motor, and auditory cortex than in other cortical regions. The extent of processing of prodynorphin varied across cortical regions as well, primarily due to the extent of processing to a-neoendorphin. Prodynorphin mRNA levels were not significantly different between cortical regions or from the amount observed in the striatum. Although specific regional variation exists, it appears that in general prodynorphin is expressed and processed in a similar manner in the cortex as in the striatum.

Introduction brain regions.’ can be processed to the opkd active , and pro-opio- The endogenous peptides are produced by to a number of functional peptides post-translational processing of one of three propep- including P-endorphin. Prodyn contains three major tides, proenkephalin, pro-opiomelanocortin, or pro- functional domains which can produce a variety of dynorphin (prodyn). Each of these precursor active ligands (Fig. 1). The neoendorphin (NE) molecules can undergo tissue-specific post-transla- domain consists of a-NE, which can be further tional processing to produce distinct mixes of active processed to P-NE. The dynorphin (dyn) A domain, peptide products, which are often unique for given which is dyn A (l-1 7), can be further processed to Date received 3 March 1993 dyn A (l-8). The dyn B domain (leurnorphin) can Date accepted 3 March 1993 be processed to dyn B. All three of these domains Correspondence to: Dr. James H. Meador-Woodruff, Mental contain leucine , which is a theoretical Health Research Institute, Department of Psychiatry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48 109-0720, final processing product. USA The endogenous opioid ligands have varying

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A(l-17) L A (l-8) 2% Fig. 1 Schematic of prodynorphin molecule. The major opioid-active domains of prodyn are shown. a-neoendorphin can be fbrther processed to P-neoendorphin, dynorphin A (1-17) to dynorphin A (l-8), and to dynorphin B. affinities for the different subtypes of opioid recep- past studies have shown that prodyn-derived prod- tors.2,3 Proenkephalin and pro-opiomelanocortin ucts as well as mRNA can be found in much of the both produce peptide products that have activity at cortex, a systematic study of the extent of distribu- the p and 6 receptors. Prodyn, however, is a partic- tion and processing of dyn has not been reported. ularly rich precursor molecule: in addition to pro- Does the cortex handle prodyn in a manner similar ducing ligands active at the p and 6 sites, this to the nigrostriatal system, or is either the synthesis propeptide can also produce peptides with relatively or post-translational processing different? Is prodyn high affinities for the K receptor. For example, dyn distributed and processed the same in all of cortex, A (l-17), dyn B, and a-NE are all active at the K or do certain regions express and/or process this receptor, having affinities for the K receptor about 5 propeptide uniquely? The purpose of the current times higher than for the p receptor, and about 20 study was to determine the distribution of prodyn times higher than for the 8 receptor. Compared with mRNA and peptides, as well as the extent of post- dyn A (l-l 7), however, dyn A (l-8) has a relatively translational processing, in multiple cortical regions higher af8nity for the 6 receptor. Thus, the extent of of the rodent, and to compare these data with that processing of the dyn A domain partially determines found in the striatum. We have selected the guinea- the relative 6 vs K functional activity of this domain pig for this study, as this animal has been reported ofprodyn. The combination of abundance and extent to have a higher density of K receptors in the cortex of processing of prodyn in any given brain region relative to other rodent species,Lo~19~20thus potentially thus contributes to the specific overall opioid tone reflecting relatively abundant prodyn expression as in that region. well. Prodyn as well as K receptors are fairly wide- spread in the mammalian brain.2,4J+13The striatum, , , amygdala, and Materials and methods are particularly enriched in these Tissue preparation molecules. The characterization of brain prodyn has Six adult male Hartley guinea-pigs weighing concentrated on deep brain structures; the nigro- between 300-350 g were sacrificed by rapid decap- striatal system has been particularly well-character- itation, and their brains were immediately removed ized.l”16 The striatum contains numerous prodyn- and placed on wet ice. The brains were sliced in the synthesizing cells, which project to the substantia coronal plane and punches were taken from ten cor- nigra. In this system in the rat, relatively high tical areas (Table 1) as well as from the caudate- levels of prodyn are found, and the dyn A domain is putamen (CPU). These punches were stored at -8O’C found primarily expressed as dyn A( l-8) until they were extracted. The cortex of most species has also been demon- strated to contain relatively high levels of both K receptor binding as well as prodyn-containing neu- Tissue extraction rons.17Js While the nigrostriatal system has been Each frozen tissue sample was weighed and fairly well-characterized, however, little is known extracted simultaneously for peptides and mRNA concerning the prodyn found in the cortex. Although using a guanidine isothiocyanate/LiCl extraction PRODYNORPHNDERIVED PEPTIDES AND mRNA IN GUINEA-PIG CORTEX 133

Table 1 Cortical regions studied dyn A (l-8), dyn A (l-17), dyn B, and leucine- enkephalin, and has an IGO of 80 fmol/tube. Each Region Corresponding Function studied cortical areas* or common name sample was assayed in triplicate, and the means from three different assays were used for subsequent data Fr Frl, Fr2, Fr3 motor analysis. Em Ent entorhinal Te Tel, Te3 primary auditory Cg Cgl, Cg2 anterior cingulate (prefrontal) Northern analysis AI AI, Te2 insular cortexlauditoty association Par1 Parl, FL, HL primary somatosensory RNA samples were chromatographed overnight on Par2 Par2 secondary somatosensoty 1% agarose/5% formamide gels; 5 pg of total RNA Pir Pir pirifoml from each region was loaded on the gels. The next RSG RSA, RSG retrosplenial (posterior cingulate) oc Oc l,Oc2L, Oc2M visual day, RNA bands were transferred to Nytran mem- branes by blotting. The membranes were subse- *Corresponding areas are approximate cytoarchitecyural regions quently probed with a [32P]-labelled 733 base contained in each punch using the descriptive nomenclature of Zillesz3 riboprobe to rat prodyn. The membranes were then briefly shaken in 0.1 x SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.2), 0.1% NaDodS04, 1 mM procedure.21 The supernatant from the initial pre- EDTA, then washed in 1.5L of the same solution at cipitation (peptide phase) was acidified with acetic 70°C for 2 h. The membranes were apposed to acid, and applied to Sep Pak CU cartridges, washed Kodak X-OMAT film for several weeks at -80°C. with 0.1% trifluoroacetic acid (TFA), and eluted Following film development, the resulting bands with 60% acetonitile in 0.1% TFA. The eluant was were analyzed by quantitative densitometry. dried by rotary evaporation, and the pellet was resus- pended in methanol: O.lN HCl (50 : 50, v: v) for Data analysis radioimmunoassay. The mRNA fraction was puri- Mean values were obtained for individual peptide fied and ethanol-precipitated, and resuspended in concentrations in each brain region. A single value water for Northern analysis. for mRNA (expressed in units of optical density from densitometric analysis) was obtained for each Peptide assays region as well. To estimate the extent of interdomain processing of prodyn, the ratios of a-NEldyn A, a- Previously standardized radioimmunoassays’6~22 NE/dyn B, and dyn A/dyn B were calculated. Total were performed on the peptide extracts using anti- dyn A was calculated as the sum of dyn A (1-17) bodies specific for the opioid-active peptides dyn A and dyn A ( l-8). The ratio dyn A ( 1- 17)ldyn A ( l-8) (l-17), dyn A (l-8), dyn B, and a-NE, using 1251- was also calculated as an estimate of intradomain labeled tracers. The dyn A (1-17) antiserum is processing for the dyn A region; as these two pep- directed against the C-terminus of this peptide. The tides have different affinities for the recep- ICSOofthis assay is approximately 10 fmol/tube. This tors, differences in the processing of the ‘A’ domain antiserum has less than 0.00 1% cross-reactivity with may be physiologically relevant. All data were ana- dyn A (l-8), leucine-enkephalin, or or-NE. The dyn lyzed by one-way analysis of variance with post-hoc A (l-8) antiserum is directed against the C-termi- comparisons by Dunnett’s t-test, with p < 0.05 used nal region of this molecule, and the IGo of this assay to defme significant results. is approximately 6 fmol/tube. This antiserum has less than 0.00 1% cross-reactivity with dyn A (l-l 7), dyn B or leucine-enkephalin, and less than 0.01% Results cross-reactivity with a-NE. The dyn B antiserum is approximately 0.002% cross-reactive with dyn A Peptide content (l-8), less than 0.001% cross-reactive with dyn A Dyn A (1-17) levels were significantly different (1-17) or a-NE, and has an IGO of approximately across the studied regions (Fig. 2). Levels of this 15 fmol/tube. The a-NE antiserum is C-terminus peptide tended to be higher in piriform (Pir), entorhi- directed, has less than 0.001% cross-reactivity with nal (Ent), and primary auditory (Te) cortices than in 134 NEUROPEPTIDES

Dymrphin A (I-17) Dynmphin A (l-8) 5, I 30 , 1

a 25

iI 43 20

j 2 15

i 1 10

5

0 0 c!Fu R Ent Te cg AxPsrlPlK2I4rRsGoC CPU Fr Eat Te Cg AIPsrlPfr2PirR!3OOC

0 CPU Fr Ent Te Cg CPo R Em Te Cg AIPmlFW2PirRSGOC

Fig. 2 Concentrations of prodynorphin-derived peptides in cortical regions and in the striatum (CPU). For each of the four peptides, there were significant regional differences in concentration. For dynorphin A (l-17), there were significant differences for region (F = 4.88, p < 0.0001). Significant post-hoc comparisons were: Pir > all other cortical regions, but equal to Ent; Ent > all cortical regions except Fr, Te, and Pir; Te > Par1 and Cg. Only Pir and Ent were significantly different from CPU. For dynorphin A (l-8) there were also significant regional differences (F = 6.13, p < 0.0001). Significant post-hoc comparisons were: Pir > all other cortical regions except Ent; Ent > all cortical regions except Pir, Fr, Te; Te > all cortical regions except Ent, Pir, Par1 , Cg, RSG; and Fr > Par 1, Cg, RSG, OC. As with dynorphin A (l-17), only Pir and Ent were significantly different from CPU. Dynotphin B also exhibited a main effect for region (F = 4.83, p < 0.0001). Post-hoc comparisons revealed Pir > all other cortical regions; and Fr > all cortical regions except Ent and Te. Only Pir significantly differed from CPU. Finally, a-neoendorphin concentrations differed across regions (F = 2.13, p < 0.05). Post-hoc comparisons indicated Pir > all other cortical regions except Ent and Te; and Ent > Par2. Only Pir > CPU; all other cortical regions were not significantly different from CPU. For abbreviations used. see Table 1. other cortical regions. Both piriform and entorhinal icantly between regions (Fig. 2). The highest levels cortices had significantly higher levels of dyn (A of dyn B were found in the piriform and motor cor- (1-17) than the striatum (CPU), but other cortical tices, and the highest levels of a-NE in piriform and regions had levels of this peptide similar to those entorhinal cortices. Of all cortical regions, only the measured in the striatum. piriform cortex had significantly higher dyn B and Dyn A (1-8) had a very similar pattern of distri- a-NE levels than the striatum. bution to dyn A (l-l 7), and also showed similar sig- Examination of the relative contribution of each of nificant differences between cortical regions (Fig. the four prodyn-derived peptides to the total amount 2). Piriform, entorhinal, and primary auditory cor- of peptide measured revealed that the greatest con- tices tended to be higher than other cortical areas, tribution in every region studied came from a-NE and the motor area (Fr) had a higher concentration (Fig. 3). The next most abundant domain in all regions than several other regions. As was found for dyn A was dyn A, which was predominantly expressed as (l-l 7), the levels of dyn A (l-8) in the piriform and dyn A (l-8). Those regions which consistently had entorhinal cortices were significantly elevated over the highest individual peptide levels (piriform, the level found in the striatum. entorhinal, primary auditory and motor areas), had Dyn B and o-NE levels also both differed signif- the smallest relative contribution from a-NE. PRODYNORPHIN-DERIVED PEPTIDES AND mRNA IN GUINEA-PIG CORTEX 135

1.00 Messenger RNA levels l m To examine the possible effects of transcription on 0.1s : E,,, the regional variation observed in peptide con- 1 0 AU-13 centrations and processing, mRNA levels were 1 0.50 determined in the same regions. As shown in f 0.2s

15

10 Fig. 3 Relative contribution of each of the four prodynorphin- i derived peptides to total prodynorpbin expression. Note that cz- 5 neoendorphin is the most abundant fotm in each region of cortex as well as in the striatum (CPU). The dynorphin A domain is the next most abundant, comprised in large part of dynorphin A (l-8). CPU R cg AI Pul ml Pk In general, the relative contributions of each peptide in the cor- tical regions is quite similar to what is observed in the shiatum.

Peptide processing To estimate the extent of the processing of prodyn within different cortical regions, ratios of peptide concentrations were calculated for each region. Both between-domain processing (Fig. 4) and within- domain processing (of the ‘A’ domain, Fig. 5) com- parisons were made. The ratio a-NEldyn B was uniformly greater than one, and ranged from 5-18 (Fig. 4). There were significant regional differences in this ratio. In general, this ratio was significantly lower in pri- mary auditory, piriform and motor cortices than in other cortical regions. This ratio was significantly lower in piriform and motor cortex than in the stria- turn, but did not differ from the striatum in other cortical regions. The ratio ol-NE/dyn A was sig- nificantly different across regions as well, with 0 Bm Te Cg Al Pb Rso oc higher values in primary somatosensory (Par l), visual (OC), and retrosplenial (RSG) cortices than Fig. 4 Between-domain processing of prodynorphin. The ratios in other cortical regions. All cortical regions had a of each domain to each of the other two are demonstrated as an estimate of the extent of between-domain processing of pro- similar value of this ratio compared to the striatum, dynorphin. Regional differences were found for the ratio a-neoen- except for primary somatosensory and retrosple- dorphmdynorphin B (F = 3.30, p < 0.005). Significant post-hoc nial cortices, in which this ratio was higher than in comparisons included: Te, Pir, and Fr < Par 1,RSG, OC, and AI; and Fr < Par2. The CPU was only significantly different from Fr the striatum. The ratio dyn A/dyn B was not sig- and Pir, and similar to all other cortical regions. Similarly, the ratio nificantly different between the cortical regions or of c+neoendorphin/dynorphin A was significantly different across the striatum. regions (F = 4.97, p < 0.0001). Significant post-hoc comparisons included: Parl, RSG, OC > Fr, Ent, Te, Pir; Par1 > Par2 and Cg; Within-domain processing was estimated for the and RSG > AI > Fr. Only Par1 and RSG were significantly dif- ‘A’ domain of prodyn (Fig. 5), by determining the ferent from CPU. Although there were significant regional differ- ratio of dyn A (I-17)/dyn A(l-8). No significant ences in the ratios containing a-neoendorphin, the ratio of dynorphinA/dynorphinB wasnotsignificantlydifferent(F=0.71) differences were found either between cortical across regions. Note that these ratios in cortex are very similar to regions or between any region and the striatum. those in the striatum (CPU) in each case. 136 NEUROPEPTIDES

Dyncqhin A (l-l7)/DynorpMnA (I-8) tently higher peptide levels, and several regions 0.25 having relatively less contribution to total peptide T I levels from a-NE, than other cortical regions and 0.2 the striatum. As has been previously demonstrated in the rodent 0.15 nigrostriatal system, I6 levels of a-NE were consid- i erably higher than levels of any of the products 0.1 derived from the A & B domains of prodyn in all regions studied. The structure of prodyn suggests 0.05 that the three domains might be expected to be pre- sent in equal concentrations, yet a-NE was consis- 0 tently higher. Trujillo et alI6 suggested that this CPU Fr Ent Te Cg AI Par1 F&2 ptr I& dc phenomenon might be due to the primary Fig. 5 Ratio of dynorphin A (1-17)idynoqhin A (l-8). This sequence of the prodyn molecule. Each functional ratio is presented as an estimate of within-domain processing for domain contains leucine enkephalin as a theoretical the ‘A’ domain. This ratio was not significantly different (F = 0.67) between cortical regions nor from that seen in the striatum. product. In both the dyn A & B domains, the cleav- age site for the liberation of enkephalin is the diba- Table 2, there were no statistically significant dif- sic amino acid pair, arginine-arginine. In the ferences between cortical regions or between any neoendorphin domain, however, this cleavage site region and the striatnm (F = 1.33, p = n.s.>. is arginine-lysine. The discrepancy in content that has been previously reported and observed in this study may reflect the differential preference of endogenous processing enzymes for these dibasic Discussion cleavage sites. This would suggest that arginine- This study was designed to examine prodyn expres- arginine is more favored for enzyme action than sion and processing in the cortex of the guinea-pig, arginine-lysine; accordingly, the prodyn A & B and to compare these findings with those seen in the domains may be further processed to leucine more well-understood striatum. For the most part, enkephalin, while the neoendorphin domain may not the cortex and the striatum appear to handle prodyn be favored for continued processing to enkephalin. in a similar manner, at the levels of mRNA expres- This scenario would result in an apparent accumu- sion, peptide content, and apparent extent of post- lation of neoendorphin products relative to longer translational processing. Some regional variation in peptide fragments derivied from the A & B domains. peptide levels and extent of processing were found, Whatever the mechanism, however, it appears that however, with a few cortical regions having consis- the cortex processes the dynorphin precursor in a manner similar to the striatnm. Table 2 Prodynorphin mRNA content in ten cortical After a-NE, the most abundant domain was dyn regions and in the striatum A. Dyn A exists as dyn A (l-17), or can be further processed to dyn A (1-g). Dyn A (1-S) was found Region t?lRNA in higher concentrations than dyn A (l-l 7), which CPU 265 f 10 suggests a relatively greater 6 versus K opioid tone Fr 183 f 30 associated with the A domain in the cortex. Further, Ent 276 f 28 Te 204 f 13 the cortex appears to process the A domain much cg 169 f 42 like the striatum. AI 262 f 32 Despite the similarity of prodyn expression Pir 170 f 84 RSG 262 f 37 within the cortex and between cortical regions and oc 96 k 55 the striatum, there were several significant differ- ences. Four regions of cortex (piriform, entorhinal, Values are expressed as mean f SEM in units of optical density. There were no significant differences between the individual primary auditory and motor areas), tended to have regions. higher peptide levels than other regions. Of these PRODYNORPHIN-DERIVED PEPTIDES AND mRNA IN GUINEA-PIG CORTEX 137 four areas, the piriform and entorhinal were the Acknowledgements highest, and were the only areas to consistently have The authors would like to acknowledge the generous gift of anti- levels significantly higher than the stiatum. Both sera used in this study which was provided by Dr , as the piriform and entorhinal regions are associated well as to thank her for her guidance, training, and encourage- ment. Dr Meador-Woodruff was supported by a Research with limbic integration functions, and this consis- Scientist Development Award from the National Institute of tent elevation of peptide levels may be associated Mental Health (KOl MHOO818). Mr Ramsdell was the recipient with this functional role of these regions. It is not of a Student Biomedical Research Fellowship from the University of Michigan Medical School (T35 HLO7690). clear, however, why primary auditory and motor areas might also have increased peptide levels; References these differences will need to be the subject of future investigation. 1. Akil, H., Bronstein, D. and Mansour, A. (1988). Overview of the endogenous opioid systems: anatomical, biochemical In comparing peptide ratios to explore possible and functional issues. In: Rodgers, R. J. and Cooper, S. J. differences in apparent peptide processing, there (eds) , and Behavioural Processes. John Wiley and Sons, p l-23. was again similarity seen between the cortex and 2. Garzon, .I., Sanchez-Blazquez, P., Hollt, V., Lee, N. M. and striatum. While the a-NEldyn A and a-NEldyn B Loh, H. H. (1983). Endogenous opioid peptides: ratios showed some variability, the dyn A/dyn B, Comparative evaluation of their receptor affmities in the mouse brain. Life Sci. 33: 291-294. and dyn A (l-l 7)/dyn A( l-8) were remarkably sim- 3. Leslie, F. M. (1987). Methods used for the study of opioid ilar throughout cortex and striatum. Processing dif- receptors. Pharmacol. Rev. 39: 197-249. ferences appeared to be related to the extent of 4. Akil, H., Hewlett, W., Barchas, J. D. and Li, C. H. (1980). Binding of [3H]P-endorphin to rat brain membranes: expression of the neoendorphin domain: in those Characterization and opiate properties. Eur. J. Phatmacol. regions with the highest total peptide levels, the rel- 64: l-8. ative contribution from a-NE was the lowest, hence 5, Fallon, J. H. and Leslie, F. M. (1986). Distribution of dynor- phin and enkephalin peptides in the rat brain. J. Comp. the ratios containing a-NE were lower in those spe- Neurol. 249: 293-336. cific regions. One possible explanation forthis is that 6, Geary, W. A. and Wooten, G. F. (1983). Quantitative film autoradiography of opiate and antagonist binding in processing enzymes that convert dyn A and dyn B rat brain. J. Pbarmacol. Exp. Ther. 225: 234-240. into leucine enkephalin may be substrate saturated 7. Khachaturian, H., Lewis, M. E., Haber, S. N., Houghten, R. or otherwise less active in those regions of cortex A., Akil, H. and Watson, S. J. (1985). Prodynorphinpeptide immunocytochemistry in rhesus monkey brain. Peptides 6 with higher total dyn content. (Suppl. 2): 155-166. To determine at what level of prodyn gene regu- 8. Lewis, M. E., Khachaturian, H. and Watson, S. J. (1983). lation the observed content and processing differ- Comparative distribution of opiate receptors and three opi- oid peptide neuronal systems in rhesus monkey central ner- ences might occur, prodyn mRNA levels were also vous system. Life Sci. 33: 239-242. determined in the same regions. There were no sig- 9. Lewis, M. E., Pert, A., Pert, C. B. and Herkenham, M. (1983). nificant differences in prodyn mRNA between cor- Opiate receptor localization in rat cerebral cortex. J. Comp. Neurol. 216: 339-358. tical regions or between the cortex and the striatum. 10. McLean, S., Rothman, R. B., Jacobson, A. E., Rice, K. C. These data suggest that the variability found in pep- and Herkenham, M. (1987). Distribution of opiate receptor subtypes and enkephalin and dynorphin immunoreactivity in tide expression is not transcriptionally mediated, but the hippocampus of squirrel, guinea-pig, rat, and hamster. J. is rather determined at the level of translation or Comp. Neurol. 255: 497-510. post-translational processing. 11. Slater, P. and Cross, A. J. (1980). Autoradiographic distrib- ution of dynorphin l-9 binding sites in primate brain. These results indicate that with the exception of Neuropeptides 8: 71-76. a few cortical areas, prodyn is handled quite simi- 12. Suda, T., Tozawa, F., Tachibana, S., Demura, H. and larly in the cortex and the striatum of the guinea-pig. Shizume, K. (1982). Multiple forms of immunoreactive dynorphin in rat pituitary and brain. Life Sci. 3 1: 5 l-57. There were a few regional differences found, with 13. Tempei, A. and Zukin, R. S. (1987). Neuroanatomical pat- increased peptide levels being present in limbic, terns of the p, 6 and K opioid receptors of rat brain as deter- mined by quantitative in vitro autoradiography. Proc. Natl. auditory, and motor cortical regions, but the signif- Acad. Sci. USA 84: 430843 12. icance of this remains unclear. In general, however, 14. Fallon, J. H., Leslie, F. M. and Cone, R. I. (1985). Dynorphin- it appears that dynorphin may serve a general rather containing pathways in the substantia nigra and ventral tegmentum: a double labeling study using combined than a region-specific function throughout much of immunofluorescence and retrograde tracing. Neuropeptides the cortex. 5: 457460. 138 NEUROPEPTIDES

15. McLean, S., Bannon, M. J., Zamir, N. and Pert, C. B. tex. Brain Res. 541: 4149. (1985). Comparison of the - and dynorphin- 19. Foote, R. W. and Maurer, R. (1986). Distribution of opioid containing projections to the substantia nigra: a radioim- binding sites in the guinea-pig hippocampus as compared to munocytochemical and biochemical study. Brain Res. 361: the rat: A quantitative analysis. Neuroscience 19: 847-856. 185-192. 20. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H. and 16. Trujillo, K. A., Day, R. and Akil, H. (1990). Regulation of Watson, S. J. (1988). Anatomy of CNS opioid receptors. striatonigral prodynorphin peptides by dopaminergic agents. Trends Neurosci. 11: 308-3 14. Brain Res. 5 18: 244-256. 21. Cathala, G., Savouret, J. T., Mendez, B., West, B. L., Karin, 17. Taquet, H., Javoy-Agid, F., Mauborgne, A. et al. (1988). M., Martial, J. A. and Bexter, J. D. (1983). A method for the Biochemical mapping of -, substance P-, isolation of intact, translationally active ribonucleic acid. [met]enkephalin, [leulenkephalin- and dynorphin A (1-8)- DNA 2: 329-335. like immunoreactivities in the human cerebral cortex. 22. Day, R. and Akil, H. (1989). The post-translational process- Neuroscience 27: 871-883. ing of prodynotphin in the rat . 18. Sato, M., Morita, Y., Saika, T., Fuji& M., Ohhata, K. and Endocrinology 124: 2392-2405. Tohyama, M. (1991). Localization and ontogeny of cells 23. Zilles, K. (1985). The cortex of the rat: A stereotaxic atlas. expressing preprodynorphin mRNA in the rat cerebral cor- Springer-Verlag, Berlin.