Deuelopmental Brain Research, 70 (199I) 2;9L-2:97© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00

BRESD 60480

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Morphine regulates DNA synthesis in rat cerebellar neuroblasts in vitro

Kurt F. Hauser

Department of Anatomy and Neurobiology, University of Kentucky School of Medicine, Lexington, Ky 40536-0084 (USA)

(Accepted 25 August 1992)

Key words.. Endogenous opioid system; Bromodeoxyuridine; Protein kinase C; External granular layer; Cell proliferation; Organotypic culture;Rat cerebellum; Drug abuse

The effects of morphine on DNA synthesis by external granular layer (EGL) neuroblasts was examined in whole-mount organotypic culturesisolated from 10-day-old rat cerebella using bromodeoxyuridine (BrdU). After 24 h in vitro, explants were treated for 24 h with 10 nM, 1 or 100HLM morphine, morphine plus 30 nM, 3 or 300 HLM of the opiate antagonist naloxone, respectively, or those concentrations of naloxone alone.BrdU was added during the last 4 h of drug treatment. EGL neuroblasts were unambiguously identified by size and morphology, location and byprotein kipase C II immunocytochemistry. The proportion of EGL neuroblasts incorporating BrdU was significantly reduced in the presence of 1pM morphine, while 100 HM morphine had little additional effect. The concentration of morphine predicted to cause a half-maximal reductionin BrdU labeling index was 22.5 nM. Morphine,s ability to reduce BrdU incorporation by EGL neuroblasts was concentration dependent and wasprevented by concomitant treatment with naloxone, implicating the involvement of opioid receptors. The results suggest that morphine candirectly regulate the growth of the developing cerebellum by inhibiting neuroblast proliferation within the EGL.

Endogenous opioid systems are present duringdevelopment4,17,21.22.27.32,43,47.48,50,52 (for review, see ref.

29) and can modify nervous system matura-tion7,15.16,26,36.42.44.49-51 (for review, see ref. 12). Dur-

ing development, endogenous opioid neuropeptidestypically act by inhibiting the growth of the nervoussystem in a regionally dependent man-ner7,15,l6,26.36,44,49-5l. opr'ate drugs also affect neuraldevelopmentlo.l3.l8.23.30.41.45 presumably by disrupting

normal opioid-opioid receptor interactions.Opioids and opiate drugs affect neural cell numbers

in the developing nervous system. Most often this isreported to result from altered cell divi-sion7,23,26,36.41,42.44, 51. However, there is conflicting evi-

dence whether cell proliferation is directly or indirectlyaffectedl8,23,36. Treatment with the opioid antagonistnaltrexone causes long-term alterations in cell prolifer-ation that may be mediated in part through non-opioidmechanisms36. Glial numbers in the cerebellum can bealtered by opioids in vivo50 and astrocyte numbers canbe modified by opiates in cerebellar explant culturesl8.

Yet, there are temporal and regional differences in theability of [Met5]-enkephalin to inhibit the growth of

type 1 astrocytesl8. These differences may be depen-dent on local growth factors and/or cell-cell interac-tionsl8. Moreover, all opioid-responsive cells do notrespond similarly to opioids during development. Opi-ates affect neuronal numbers in the avian ciliary gan-glion by altering survival, not prolifcration30. Also,whether growth is accelerated or retarded is dependenton the duration of opioid exposure7.l6.50,5l. For exam-

ple, in mesencephalic raphe cultures from fetal rats,the number of serotonergic neurons is decreased afterchronic exposure, but increased following acute expo-sure to opioids7. Thus, there are considerable differ-ences in the cellular response to opioids during on-togeny. It is as yet unclear whether opioids affectdevelopment through a single underlying mechanism.

The rodent cerebellum is likely to provide importantclues about the developmental role of opioids. First,subpopulations of developing cerebellar neurons andglia express proenkephalin mRNA and/or peptide

Correspomdcflce fo.. K.F. Hauser, Department of Anatomy and Neurobiology, University of Kentucky School of Medicine, 800 Rose Street,Lexington, KY 40536-0084, USA. Fax: (1) (606) 258-5946.

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productsl7,32.40.52. second, opioid receptors are presentin the developing cerebellum2l.43.47,48. Lastly, the growth

of cerebellar neurons and/or glia can be modified bymanipulating endogenous opioid systemsl5,l6.l8.26.45,50.5l

The rate of thymidine incorporation by neuroblasts(i.e., neuronal precursors capable of undergoing mito-sis) in the external granular layer (EGL) can be af-fected by opioid agonists and antagonists in vivo5l. Inculture, opiates can intrinsically affect the outgrowth ofcells from cerebellar explant culturesl8.45 which consistof neurons and glial8. The growth and survival ofPurkinje cells is also affected by opiates in explantcultures (Hauser, in preparation). Yet, despite findingsthat the developing cerebellum is sensitive to opioids,the direct effects of opiates on DNA synthesis incerebellar neuroblasts in culture has not been previ-ously studied. The preliminary results herein supportthe hypothesis that opiates per se can inhibit the rateof proliferation of neuronal progenitors within theEGL. These findings have implications regardingchronic licit or illicit opiate drug use during pregnancyand early childhood.

Cerebella obtained from 24, 10-day-old male,Sprague-Dawley rats (Harlan Sprague-Dawley, Indi-anapolis, IN) were dissected into small [(0.3-0.5) x 1mm2] explants and maintained for 48 h in vitro beforeharvesting. There are considerable differences in the

growth of different regions within the rat cerebellumand in the expression of endogenous opioids by cere-bellar cells in developing rats (see ref. 32). Thus, ex-

plants were only taken from the lateral portions of crusI and II (i.e., between the posterior superior fissureand the ansoparamedian fissure24). To measure thelabeling index within the EGL, explants were trimmedparasagittally using a micro-surgery scalpel (4 mmblade) aided with 40 x dissecting microscope and posi-tioned so the EGL was at their peripheral border aspreviously describedl7.l8. Explants were maintained inorganotypic culture in 12-well (22 mm diameter) plastictissue culture chambers as previously describedl7.l8.Cultures were treated continuously for the last 24 hwith media alone (untreated), media containing mor-phine sulfate (NIDA) (10 nM, 1 or 100 4tM) alone orwith (-)-naloxone (DuPont, Wilmington, DE) (30 nM,3 or 300 4tM, respectively)) or media containing theabove concentrations of naloxone alone. During thelast 4 h of opiate treatment, 5-bromodeoryuridine(BrdU; Sigma, St. Irouis, MO) was added to the mediato a final concentration of 50 mg/ml.

After 48 h in vitro, explants were fixed z'# sztL/ On thecoverslips on which they were grown. It was importantto optimize the BrdU signal-to-noise ratio while pre-serving cellular morphology (EGL cells are identified

based on their unique size and morphology). There-fore, several previously described5.9.l1,3l.38 BrdU im-

munocytochemical procedures were compared in pre-liminary studies. In the present study) explants werefixed at 4oC in 2% paraformaldehyde38 for 30 min or in7097o ethanol overnight31. Explants were later perme-abilized by treatment in 5097o ethanol in 0.1 M PBS(pH 7.4) (30 min), 7097o ethanol in 0.1 M PBS (30 min)and 50% ethanol in 0.1 M PBS (30 min) as modifiedfrom Jaeger et al.20. permeabilization was followed bybrief DNA denaturing (0.07 M NaOH or 2 N HCl for0.5 or 1 h)5,38. In some cases, DNA was denaturedbefore alcohol-PBS permeabilization. Primary mono-clonal antibodies against BrdU (Chemicon, Temecula,CA) diluted 1:50 or 1:100 (w/v) in PBS (phosphatebuffered saline), pH 7.4, containing 0.197o Triton-X 100and 1% crystalline grade bovine serum albumin (BSA;Calbiochem, CA). Tissues were incubated with antiserafor 48 h at 4oC on an orbital shaker at 40-60 r.p.m.Anti-BrdU antibodies were detected using a biotin-avidin-peroxidase detection system as directed (Vecta-stain ABC kit, Vector Laboratories, Burlingame, CA).The reaction was visualized using a nickel-intensifiedDAB chromagen consisting of freshly made 2.5%Nickel ammonium sulfate, 0.35% diaminobenzidine and0.012% H2O2 in 0.1 M sodium acetate, pH 6.0 (ref.14). Approximately 2,000 randomly selected EGL cellswere sampled from explants from each rat. The label-ing index [labeled cells/(labeled + unlabeled cells)] wasdetermined using a 100 x oil immersion objective withHoffman modulation contrast optics as modified fromGao et al.9 who studied BrdU-labeled EGL cells inreaggregate culture. Labeling index was only deter-mined when all cells (labeled and unlabeled) werevisible within a defined EGL. Multiple explants fromindividual rats were distributed across experimental

groups. Each value represents explants sampled from# - 4 to fl - 6 rats. Data were analyzed using ANOVAand reported as the mean ± S.E. Post hoe comparisonswere made using Newman-Keuls test. To estimate theconcentration of morphine that would half-maximallysuppress the labeling index in EGL neuroblasts, non-linear, least-squares regression analysis was performed(GraphPad; GraphPad Software).

For protein kinase C (PKC) II immunocytochem-istry, explants were fixed with 4% paraformaldehyde inSorenson,s phosphate buffer, pH 7.4. Explants werepermeabilized and incubated in goat anti-PKC II antis-era (a gift from Dr. F.L. Huang, NIH) diluted 1 : 1,500or 1 :2,000 as modified slightly from Huang et al.l9 foruse with an ABC kit (Vector Labs). In some cases,second antibodies conjugated to alkaline phosphatasewere visualized with a red reaction product (Vectastain

293

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Fig. 1. A-E: bromodeoxyuridine (BrdU) immunocytochemistry of organotypic explant cultures of 10-day-old rat cerebellum at 48 h in vitro thatwere incubated with BrdU for 4 h and then harvested. The cerebellar external granular layer (EGL) is a transient proliferative zone consisting ofneuroblasts and postmitotic neurons which can be readily identified in organotypic explants. The explant culture in A was sectioned tangentially(horizontally) through several folia at the lateral edge of the cerebellum and shows how the histiotypic organization of the EGL is maintained invitro. In the present study, explants were much thinner and sagittally sectioned so that the EGL was at the peripheral border of the explantwhere cells are easily counted (B). EGL cell bodies are typically 5-8 pm in diameter and have only a thin rim of cytoplasm (B). The nuclei ofreplicating EGL neuroblasts incorporate BrdU and dividing cells often form clusters (B,C). D,E: the outgrowth from cerebellar explants containssome BrdU-immunoreactive EGL progeny (arrow) that are positioned outside the EGL. Granule cells (EGL progeny) migrate from the EGLboth inwardly into the explant and outwardly into the outgrowthl. Larger non-neuronal cells (arrowheads) resemble astroblasts. Phase-contrast

(D) and brightfield (E) micrographs of the same field. Scale bars: A, 100 pm; B, 10 prm; C, 25 pm; D,E, 25 prm.

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Ill Kit). Immunoreactivity was not seen when eitherBrdU or PKC II primary antibodies were excludedfrom the reaction.

BrdU labeling indexes appeared comparable irre-spective of whether the tissue was fixed in ethanol or

paraformaldehyde. Ethanol fixation gave a better BrdUsignal-to-noise ratio, but with slight loss in cellularmorphology. However, because our past studies haveidentified EGL cells based on their morphology in

paraformaldehyde fixed explant cultures, BrdU incor-poration was also assessed in paraformaldehyde fixedtissue. Although more prolonged (1 h) DNA denatur-ing) as well as the use of a more concentrated (i.e.,1 : 50 dilution) of the BrdU primary antibody, improvedthe BrdU signal, paraformaldehyde fixation still re-sulted in increased non-specific staining compared toethanol fixation. Therefore, the reported measure-ments were made on ethanol fixed tissue. To validatethe BrdU procedure, [3H]thymidine autoradiographywas performed in six untreated explants in parallelwith the present BrdU procedure and yielded similarresults (Hauser, unpublished results).

EGL neuroblasts and their progeny were readilyidentified in organotypic cultures based on criteria as

previously noted by myselfl7 and othersl.9.20.37. Thesecells have a distinct size (approximately 5-8 +m cellbody diameter) and morphology l`9W.20.37 and are some-times distributed in clusters at the edge of the explant(Fig. 1A-E). EGL progeny migrate both into and awayfrom the explantl. EGL cells are the only neuronal

precursors to incorporate BrdU, since all other cere-bellar neurons are postmitotic in the postnatal rat2.3Antisera against an isozyme of PKC II also labeledEGL cellsl9 and their progeny6.l9 further confirmedtheir identity. Some immature glia, however, also ex-

pressed PKC II. Because PKC II was not an exclusivemarker for granule cell progenitors in the presentculture system, these results are not reported. Non-neuronal cells were distinguishable from EGL cells(Fig. 1D,E). Replicating astrocyte progenitors werelarger (i.e., ll.8± 1.4 HLm diameter Cell body) thanEGL cellsl7. These immature astrocytes typically hadmultiple nucleoli within a cell with discernible cyto-

plasm, were GFAP immunoreactive and distributed atthe periphery of the outgrowthl7.

Morphine caused a concentration-dependent reduc-tion in BrdU incorporation by EGL cells (ANOVA;P<0.01) (Fig. 2). Treatment with 1 or 100 pM mor-

phine caused a significant reduction in the BrdU label-ing index of EGL neuroblasts compared to untreatedcultures (Fig. 2). It was predicted that 22.5 nM mor-

phine would produce a half-maximal reduction in BrdUincorporation by EGL neuroblasts using regression

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Untroatod 10nM 1 uM 100uM

Morphine Cone.

Fig. 2. Treatment with morphine for 24 h caused a concentration-de-pendent reduction in BrdU labeling index in cerebellar EGL neurob-lasts in cerebellar explant cultures. Individual labeling indices weredetermined from 2 or 3 explants per rat. The mean labeling index±S.E. was determined from « -4 to « -6 rats in each group(see text)(* P < 0.05 compared to the value for untreated cultures; Newman-

Keuls test).

analysis. The effects of 100 prM morphine were pre-vented by co-administration of 300 pM naloxone(ANOVA; P<0.01). Naloxone treatment alone, atconcentrations up to 300 44M, had no effect on EGLcell labeling index (Fig. 3).

The results indicate that morphine,s action on cellu-lar proliferation was concentration dependent and pre-vented by the opiate antagonist naloxone and thereforemediated through opioid receptors. Morphine affectedDNA synthesis in neuroblasts within isolated explants,suggesting that opiates can intrinsically inhibit neurob-last proliferation in the cerebellum.

It was not surprising that proliferating EGL cellswould respond to opiates in vitro. First, [3H]thymidine

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Treatment

I control

I Morphine

I Morph/Nal

I Naloxone

Fig. 3. Treatment with loo peM morphine for 24 h significantlyreduced the percentage of external granular layer (EGL) neuroblastsincorporating bromodeoxyuridine (BrdU) compared to untreatedcerebellar explant cultures. The reduction in BrdU labeling indexcaused by 100 pM morphine was prevented by concurrent treatmentwith 300 pM naloxone (Morph/Nal), while naloxone (300 HM)alone had no effect on BrdU labeling index. Individual labelingindices were determined from 2 or 3 explants per rat. The meanlabeling index±S.I. was detemined from # -6 rats in each group

(see text)(* P < 0.05; NewmanxKeuls test).

incorporation by EGL cells is decreased by [Met5]-en-kephalin and increased by opioid antagonists in dosagesthat continuously block opioid receptors in vivo51.Purkinje cell development, in part dependent ontrophic input from the axons of granule cells (EGLprogeny)2.3.34,35,37.39, is affected by opioids and/or opi-ate drugs in vivol5,l6 and in vitro (Hauser, unpublishedresults). Second, there is increasing evidence that thecerebellum expresses opioid receptors during develop-ment4,21,43,47,48. This suggests that opiates may affectthe proliferation of EGL cells via a direct action at thecerebellum. Opioid receptors reported to be present inthe developing cerebellum include the p-, K- and f-and perhaps 6-subtypes4,47,48. K- and 6-42 and {-42.47.48opioid receptors have been implicated in growth.

However, P-funaltrexamine49 or DAGO42 sensitive pr-opioid receptors may not be involved in growth. Al-though morphine is a preferential p-opioid receptoragonist, this narcotic also displays limited cross-reactiv-ity with 6-opioid receptors and with the class of J-opioid receptor characterized in rat cerebellum (seerefs. 41,42). Thus, morphine might be acting throughone or more non-pr-sites. This notion is supported bystudies of opiate-dependent growth in astrocytes41,42.Recently, [3H]naloxone binding sites have been local-ized within the EGL in humans21, suggesting that EGLcells themselves may respond directly to opioids.

In many brain regions, opioids per se are proposedto dz.recr/y affect neural growth by inhibiting cell pro-liferation41,5l. However, in some regions of the nervoussystem opioids may only indirectly affect cellnumbers23.36. Kornblum et al.23 found that morphinedid not affect [3H]th)midine incorporation in isolatednervous tissue in vitro. DiCiccio-Bloom et al.8 foundthat [Met5]enkephalin, at o.1-10 peM conccntratjons,did not affect the proliferative rate of superior cervicalganglion neuroblasts from embryonic rats in vitro. Sim-ilarly) the growth of neurons in the hippocampus is

profoundly affected by opioids in vivo15,l6, yet theoutgrowth of cultured hippocampal explants is unaf-fected by 10 4tM morphinel8. In vitro, there are re-gional and temporal differences in the ability of opiatesto regulate the growth of type 1 astrocytes that are notevident in vivol8. Lastly, morphine increases neuronalnumbers in the ciliary ganglion, not by affecting repli-cation, but by increasing their rate of survival30. Thesefindings show that opiates can affect cell numbers, butsuggest that they can act both by direct and indirectmechanisms depending on the specific targetl8. proba-bly no single trophic factor acts alone to regulate anyevent during neural development28. Adrenal corticos-teroids46 and thyroid hormone25 affect the rate of

proliferation of EGL neuroblasts. Cell-cell interactions

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also affect EGL cell proliferation9. opiates are likelyto interact with other factors to modulate key matura-tional events23,28. yet, because opiates per se can affectthe growth in isolated explants; this suggests that, inthe cerebellum, opioids might normally be an initiatingsignal that triggers later events. Opiates affect manyaspects of cerebellar development in vitro. For exam-ple, Purkinje cell dendritic length and complexity(Hauser, in preparation), glial cell numbersl8, and therate of EGL cell replication are intrinsically modifiedby opiates in culture. In conclusion, cerebellar histoge-nesis appears to be directly affected by opiates withEGL neuroblasts being a principle cellular target ofaction. Additional studies are needed to assess whetheropiates per se affect dividing neuroblasts in other brainregions.

It was interesting that naloxone treatment by itselfhad no effect on the labeling index of EGL neurob-lasts. Continuous blockade of opioid receptors by opi-oid antagonists speeds the rate of [3H]th)midine incor-poration by EGL5l and purkinje cell dendritic growthin ratsl5.l6. This and other findings support the hypoth-esis that opioids are normally present during cerebellardevelopment17,32.40,43,52 and function by tonically in-hibiting growthl5-l7,50-52. cerebellar astrocytesl7,40,Purkinje32 and EGL cells52 express proenkephalinmRNA and peptide products. Importantly) EGL neu-roblasts in 10-day-old rats52 and in explant culturesderived from newborn rats33, respectively) containenkephalin and proenkephalin immunoreactivity. Yet,if locally produced opioids can inhibit cerebellar growthand opioids are expressed in vitro, then why doesnaloxone alone have no effect in the present study?One explanation is that there are significant differ-ences jn aIllOunt Of endugenOuS OPiOidS that are avail-able to developing cells in different regions of thecerebellum at different times. osborne et al.32 foundusing in situ hybridization that proenkephalin mRNAis not expressed uniformly throughout the developingcerebellum in rats. Expression is greater in caudal32and lateral regions (Osborne and Hauser, unpublishedresults). There may be more opioids in lobule VIII in6-day-old rats (region examined in ref. 51) than inexplants from the lateral hemispheres of 10-day-oldrats. However, this explanation is inconsistent withsome preliminary findings. Fewer cells in 6-day-old ratsexpress proenkephalin products than in 10-day-oldrats32 and fewer cells within the lobule VIII express

proenkephalin mRNA than in the dorsal paraflocculus(Osborne and Hauser, in preparation) which is consid-ered to be a lateral extension of lobule vIII24. Thelocal expression of other opioid -genes, i.e., prodynor-

phin and proopiomelanocortin (POMC) might be in-

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volved in cerebellar development. POMC gene prod-ucts are likely to have developmental effects in thecerebellum26 as well as in other brain regions26.27.Alternative explanations might be that circulating opi-oids mediate the effect, or that opioids produced invitro might be diluted and/or degraded differentlyfrom those in vivo. Lastly) the EGL is a dynamicstructure2,3 and the response of dividing cells to opi-oids may differ with age. v6rtes et al.44 found that 200

HJg/100 g body weight of naloxone had no effect on[3H]thymidine incorporation at 1, 3, 6, 9 or 12 h aftertreatment in biochemical studies of ll-day-old rat cere-bellum. In the same study) they found that [3H]thymi-dine incorporation was significantly increased at 1and/or 3 h in the forebrain and hypothalamus44. Lor-ber et al.26 similarly found that i.c. administration of 3

4Lg/g brain Weight Of nalOXOne had nO affect On DNAsynthesis after 1 h in the cerebellum of 10-day-old rats.Further studies are needed to assess the role of locally

produced endogenous opioids in the developing cere-bellum.

In summary, the results provide evidence that opi-ates per se can regulate the proliferation of EGLneuroblasts through a direct action on the developingcerebellum. The finding that opiates can intrinsicallyinhibit neurogenesis has implications regarding chroniclicit or illicit opiate drug use during pregnancy andearly childhood.

I thank Ms. Carol Turbek for expert technical help and Ms. NancyMayfield and Ms. Elizabeth J. Bunzendahl for counting cells. Iespecially thank Dr. Freesia L. Huang for anti-PKC II antisera, Dr.Marilyn Duncan and Dr. John Osborne for helpful comments. Sup-ported by the National Institute on Drug Abuse (DA 06204).

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