Prenatal and early postnatal β-adrenergic receptor-mediated increase of cyclic AMP in slices of rat brain

Brain Research, 177 (1979) 515-522

515

c© Elsewer/North-Holland Biomedlcal Press

PRENATAL AND EARLY POSTNATAL fi-ADRENERGIC R E C E P T O R - M E D I A T E D I N C R E A S E OF C Y C L I C A M P IN SLICES OF RAT BRAIN

KENNETH G WALTON, EDITH MILLER and ROSS J. BALDESSARINI Department of P~ychtatry, Harvard Medical School and LaboratorlesJor Psychlatrw Research, Madman Research Center, McLean Dtvl~ton oJ Massachusetts General Hospltal, Behnont, Ma3s 02178 ( U.S A )

(Accepted March 8th, 1979)

SUMMARY The levels of cyclic A M P m shces of cerebral cortex and cerebellum from newborn rats were significantly, but transiently, increased by exposure to the fiadrenerglc agomst, lsoproterenol, lsobutylmethyxanthlne, an inhibitor of phosphodiesterase, enhanced this effect and permatted its detection in cerebral cortex obtained from the prenatal rat. These results are consistent with the possibilitaes that functional noradrenerglc synapses are formed early in the ontogeny of the CNS, and that norepmephrine may exert cyclic AMP-mediated influences on brain development.

INTRODUCTION The rich noradrenergm innervation of the neocortex is a key example of the monoamine-medlated, non-specific afferent systems arising in the brain stem reticular formation and reaching virtually all areas of the central nervous system (CNS) 2a. The normal functions of this and other wide-spread monoamine systems in the CNS are not clear, even in the adult 24. Recently, mounting evidence has Indicated that such diffuse pathways are among the earhest to appear m brain development, both in the rat6,v,9,',l,26,ao,38 and in man28, 29 Furthermore, several hnes of evidence indicate that the pathways are functional at an earlier age than was previously thought~,10, 50, giving rise to speculahon that they may play a disproportionately important role in the function or development of the fetal mammalian brain. In the rat on the 15th day of gestahon, when the catecholammes norepmephrme and dopamme first become clearly measurable in the cerebral cortex 9, many cells in this area are involved in the processes of mitosis, migration, differentmtion and synaptogenesas 5,2°. Furthermore, as many as 50°~ of the earhest synapses m the cerebral cortex appear to be noradrenerg~c, with input oragmatmg an the pontme

516 nucleus locus coeruleus10,5o. The question of what role this noradrenergtc mnervatlon might play in brain development remains open. However, from the viewpoint of function, in the developing visual cortex of the cat, norepinephrine is reported to be necessary for neurons to change their ocular dominance following monocular occlusionis,z4. Although the mechanism for such an effect is not known, catecholamine stimulation of adenylate cyclase, the enzyme catalyzing synthesis of cyclic AMP, is important in the actions of catecholamines15.17,19, and studies of the effects of cychc AMP have already suggested several possible mechanisms for catecholamine involvement m plasticity or development of the CNS. Specifically, cyclic AMP has been implicated in the regulation of neuronal specialization35,46, axogenests 37, the differential expression of neurotransmitter-synthesizing enzymes4°,41, and even mitotic activity of CNS cellsa6. Further reformation on cyclic AMP-related phenomena might help in elucidating the role of an early noradrenergic mnervation in the ontogeny of the CNS. Adenylate cyclase activity stimulated by dopamme or norepmephrme has been detected at birth in homogenates of rat brain s.42,45. On the other hand, studies of brain slices have failed, until several days after birth, to detect cyclase stimulation via the fJadrenergic receptors thought to mediate noradrenergic neurotransmission L4,33,39.This result seemed anomalous, especially in the cerebellum; norepinephrine inhibits electrophysiologic stimulation of Purkinje cells through a /5-adrenergic receptor mechanism and cyclic AMP in the adult 15, but identical inhibition Ls produced in newborn rats 49 even though a fl-adrenergic-mediated increase in cyclic AMP appears not to be present at this age 39. Due to the seemingly anomalous character of these results and to the potential importance of a cyclic AMP-mediated, noradrenergic regulation of CNS developmental processes, we have re-examined the ontogeny of fl-adrenergic receptoradenylate cyclase activity in slices of rat cerebral cortex and cerebellum using conditions different from those used previously14,33,39. Our results indicate that this receptor-adenylate cyclase system is detectable even prenatally, at least as early as the 18th day of gestation, and suggest that previous failures to detect activity at such early ages were due to the transient nature of the stimulation. (A preliminary report of this work has appearedaS.) METHODS Sprague-Dawley rat pups of both sexes were killed by decapitation and dissected on ice. Tissues were manually diced until pieces were 0.5 mm, or less, on all sides. The remainder of the procedure was similar to that previously reported for striatal slices47. After a 60 mm preincubation at 37 °C in Krebs-Ringer-bicarbonate, 150/zl aliquots of the tissue suspension were incubated with or without drugs, as described in the figure legends. The incubation was stopped by placing tubes in a 96 °C water bath for 5 min, and 20/~1 portions of the supernatants were assayed for cyclic AMP by the procedure of Brown et alP, with the following modification. Instead of separating bound from free cyclic AMP using activated charcoal, we used 25 mm diameter filters (Millipore,

517

no. HAWP-02500, 0 45 #m pore size). After the assay tubes (containing samples or cychc AMP standards, tntlated cychc AMP (New England Nuclear, 33 Ci/mmol), binding protein, and buffer, at a total vol. of 50 ffl/tube) had incubated for 1 5-3 h at 0 ~C, 2.5 ml of ice-cold, 0.1 M Tris buffer (pH 7.4) was added to each. The mixture was then rapidly filtered, and the filters were rinsed 3 times with buffer before removing to wals for scintillation spectrometry. The lower hmlt of the assay was about 50 fmol of cychc AMP/tube. RESULTS

To achieve inhibition of cychc AMP breakdown, we used 3-isobutyl-l-methyl xanthme (IBMX), a phosphodlesterase inhibitor that is particularly effective in brain ttssue al. Fig. IA shows that 2.5 mM IBMX increased the cyclic AMP level in shces of A

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Fig I lsoproterenol-lnduced increase m cyclic A M P as a function of time m cortical slices with (A) and without (B) I B M X , a n d m cerebellar shces with I B M X (C), all prepared from rats In their first postnatal day The concentration of L-isoproterenol was 1 ffM (A a n d C) a n d 30/~M (B). The I B M X concentration was 2 5 raM, a n d the incubation was at 37 'C I B M X was a d d e d in 100 ttl of K r e b s R i n g e r - b i c a r b o n a t e buffer a n d lsoproterenol ~- alprenolol was a d d e d in 10 itl o f N2-gassed delomzed water The total v o l u m e m each sample was 260 td a n d each tube contained approximately 5 m g of tissue Each point represents the m e a n ± S E M of 3 separate experiments, each experiment, In turn, represented the assay o f 3 rephcate samples Error bars are omitted when S E M IS smaller than the radius of the symbol

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Fig. 2. Ontogeny of the /~-adrenergic receptor-adenylate cylase activity m rat cerebral cortex, as estimated from the stimulation of cyclic AMP levels in slices by isoproterenol Slices were incubated for 15 rain with 1 and 30 IbM L-isoproterenol in the presence and absence, respectively, of 2.5 mM IBMX. Separate experiments (3-5 replicate samples per condition) were performed at each age. Samples from the adult were included for comparison in most experiments The percent stimulation in theyoung was compared w~th the mean percent stimulatton in the adult.

cerebral cortex from one-day-old rats by 3-4 times. A concentration of IBMX which would completely block cyclic AMP breakdown could not be achieved within the limit of solubility (about 7 mM) of the drug. After a 5 min preincubation with IBMX, the selective fl-adrenergic agonist L-isoproterenol (1 #M) was added to some of the tubes, and a second, sharper, rise of the cyclic AMP level occurred, stabilizing at 2-3 times the level in the presence of IBMX alone. To further verify that the receptor system involved in this stimulatton in oneday-old rats was indeed the fl-adrenergic receptor-cyclase we incubated slices for 15 min with 2.5 mM 1BMX and 1 # M isoproterenol in the presence of the selective fladrenergic-receptor antagonist, alprenoloP,2L In 3 experiments, stimulation by isoproterenol was inhibited by an average of 93 % by this agent at a concentration of 10 btM. In a second set of experiments IBMX was omitted from the incubation mixture. Fig. 1B shows that while some stimulation of the cyclic AMP level in slices of cerebral cortex from one-day-old rats seemed to occur, this stimulation was small, variable, and of short duration. The duration of exposure to agonists in previous investigations, where stimulation was not seen, varied from 6 min a9 to 30 min 14. Our experiments thus suggest that a transient effect might have gone unnoticed in these earlier studies. Fig. 1C shows that cyclic AMP levels in cerebellar slices from the newborn rat were also increased by 1 # M isoproterenol m the presence of IBMX. As inthe cerebral cortex, significant, but lesser, stimulation was observed in the absence of t B M X as well, provided that incubation times less than 15 min were used (not shown). Other results of earlier workers indicated a large increase in /3-adrenergicmediated cyclic AMP accumulation during the second postnatal week 14,zz,39. To test these results against our findings we examined the stimulation of cyclic AMP formation in the presence and absence of IBMX in cerebral cortical slices of several

519 ages. A 15 m m incubation with isoproterenol was chosen so that the experimental conditions in the absence of l BMX would closely resemble those of the earher studies. Our results (Fig. 2) agree with the earlier work in showing a marked increase of lsoproterenol-induced cyclic A M P accumulation during the second postnatal week, and this increase was seen whether or not IBMX was present. However, IBMX enhanced the apparent stimulation at all ages under 13 postnatal days and permitted us to observe stimulatmn in cerebral cortex on the 18th gestanonal day, the earliest age tested. DISCUSSION The apparently transient nature of the/#adrenerglc receptor-mediated increase of cychc A M P which we have observed m the brain of the newborn rat may represent an example of rapid 'refractoriness'16,2a, 44 of this receptor system. Refractoriness is a phenomenon, observed for several receptor-adenylate cyclase systems, in which the initial increase in cyclic A M P level in response to an agomst is greater than the increase caused by a subsequent addltmn or by continuous exposure to the agonist Recent reformation obtained on refractoriness m cultured cells indicates that in some cases multiple phases are involved, each with Its own mechanism16.32, 4~. One mechanism sometimes contributing to refractoriness is an activatmn of phosphodiesterase 16. Our observation that stimulation by isoproterenol appears to be both enhanced and prolonged by a potent phosphodiesterase inhibitor may suggest that at least part of what may be rapid refractoriness in newborn brain could be due to an activation of phosphod~esterase. It ~s noteworthy in this regard that none of the earlier studies of the ontogeny of rat brain adenylate cyclase stimulation via the /3adrenerg~c receptor used an inhibitor of phosphodiesterase, a factor which probably contributed to thmr failure to observe stimulation in newborns. A Ca ~ +-dependent mechanism for the raptd regulanon of phosphodlesterase in well known m brain ~. However, m addition to the possible contribution of phosphodlesterase actlvatmn to the apparently transient nature of the stimulation, other mechanisms for a rapid refractoriness exist16, 2a and might also be involved. A detectable, but low level of/3-adrenerg~c receptors m cerebral cortex of the newborn rat has been mdxcated recently by measurements of the binding of the radmhgand [tzsI]iodohydroxybenzylpindolol to this tissue 14. If the mechanism of the ~soproterenol-induced stimulation m developing brain is similar to that in intact ghoma cells in culture, which requires an agonist to occupy a very small number (less than I ° o) of ~-adrenergic receptor binding sites to produce half-maximal stimulation of cyclic A M P production 43, then it is likely that the number of receptors detected m the binding studies in the newborn rat could account for the increase in cyclic A M P which we observed Our present demonstration of a /]-adrenergic receptor-mediated increase of cychc AM P in cerebral cortex and cerebellum of fetal and newborn rats is compatible with the hypothesis that noreplnephrine influences neuronal development through )ts effects on cyclic A M P levels in postsynaptic neurons. However, since gha as well as

520 n e u r o n s exhibit fl-adrenergic a d e n y l a t e cyclase activity 12,13, we c a n n o t discount the possibility t h a t at least s o m e o f the effect we o b s e r v e d was o f glial origin. Even so, c a t e c h o l a m i n e s induce glial-specific e n z y m a t i c activity in cultured g l i o m a cells 27, and m a y even c o n t r o l p r o l i f e r a t i o n or survival o f c e r t a i n types o f glial cells25, 31,36. Glia, in turn, p r o d u c e factors which s u p p o r t the survival a n d g r o w t h o f n e u r o n s 2. Thus. regardless o f the type o f cell in which the fl-adrenergic r e c e p t o r - a d e n y l a t e cyclase system m a y reside, its presence at a very early age a n d its discrete time course o f d e v e l o p m e n t a p p e a r to lend s u p p o r t to the possibility o f cyclic A M P - m e d i a t e d influences o f n o r e p i n e p h r m e in b r a i n development. ACKNOWLEDGEMENTS S u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t s MH-3051 ! a n d M H 31154; T h e Scottish Rite Benevolent F o u n d a t i o n , N o r t h e r n M a s o n i c J u r i s d i c t i o n o f the U . S . A . ; N a t i o n a l Institutes o f H e a l t h C a r e e r R e s e a r c h Scientist A w a r d M H - 4 7 3 7 0 (R.J.B.) a n d a R e s e a r c h F e l l o w s h i p f r o m the M e d i c a l F o u n d a t i o n , B o s t o n ( K . G . W . ) .

REFERENCES 1 Alexander, R. W., Davis, J. N. and Lefkowitz, R. J., Direct identification and characterization o f fl-adrenerglc receptors in rat brain, Nature (Lond.), 258 (1975) 437-440. 2 Barde, Y. A., Lindsay, R. M., Monard, D. and Thoenen, H., New factor released by cultured glioma cells supporting survival and growth of sensory neurons, Nature (Lond.), 274 (1978) 818. 3 Brown, B. L., Albano, J D. i . , Ekins, R. P., Sgherzi, A. M. and Tampion, W., A simple and sensitive saturation assay method for the measurement of adenosine 3',5'-cyclic monophosphate Blochem J., 121 (1971) 561-562. 4 Cheung, W. Y., Lynch, T. J. and Wallace, R. W., An endogenous Ca2+-dependent activator protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. In W J. George and L. J Ignarro (Eds.), Advances in Cyclic Nucleotide Research, Vol. 9, Raven Press, New York, 1978, pp. 233-251. 5 Coyle, J. T., Biochemical aspects ofneurotransmlssion in the developing brain, Int. Rev. Neurobtol., 20 (1977) 65-103. 6 Coyle, .1".T. and Axelrod, J., Dopamme-fl-hydroxylase m the rat brain: developmental characteristics, J. Neurochem., 19 (1972) 449-459. 7 Coyle, J. T. and Axelrod, J , Tyrosine hydroxylase in rat brain: developmental characteristics J Neurochem, 19 (1972) 1117-1123. 8 Coyle, J. T. and Campochiaro, P., Ontogenesis of dopaminergic-cholinergm interactions m the rat striatum: a neurochemical study, J. Neurochem., 27 (1976) 673-678. 9 Coyle, J. T. and Henry, D., Catecholamines in fetal and newborn rat brmn, J. Neurochem, 21 (1973) 61-67. 10 Coyle, J. T. and Molliver, M. E., Major innervatlon of newborn rat cortex by monoammergic neurons, Scwnce, 196 (1977) 444-447. 11 Forn, l., Krueger, B. K. and Greengard, P., Adenosine 3',5'-monophosphate content in rat caudate nucleus: increase associated with synaptic transmission, Science, 186 0974) 942-944 12 Gilman, A. G., Regulation of cyclic AMP metabolism in cultured cells of the nervous system. In P. Greengard and G. A. Robison (Eds.) Advanc. Cyclic Nucleotide Res, Vol l, Raven Press, New York, 1972 pp. 389-410 13 Gilman, A. G. and Nirenberg, M., Effect of catecholamines on the adenosine 3',5"-cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. Sci (Wash), 68 (1971) 2165-2168. 14 Harden, T. K., Wolfe, B. B., Sporn, .I.R., Perkins, J'. P. and Molinoff, P. B , Ontogeny of/~-adrenergic receptors in rat cerebral cortex, Brain Research, 125 (1977) 99-108.

521 15 Hoffer, B. J., Slggms, G. R., Ohver, A. P and Bloom, F. E., Cyclic adenosine monophosphate mediated adrenerglc synapses to cerebellar Purkinje cells. In P. Greengard and G. A. Roblson (Eds.), Advanc. Cychc Nucleottde Res., Vol. 1, Raven Press, New York, 1972, pp. 4 1 1 4 2 3 16 Johnson, G. L., Wolfe, B. B , Harden, T K., Mohnoff, P. B. and Perkins, J. P., Role of fl-adrenerglc receptors m catecholamme-lnduced desensitization of adenylate cyclase m human astrocytoma cells, J. btol Chem., 253 (1978) 1472-1480. 17 Kakmchi, S and Rall, T. W., The mfluence of chemical agents on the accumulation of adenosine 3',5'-monophosphate m shces of rabbit cerebellum, Molec. PharmacoL, 4 (1968) 367-378. 18 Kasamatsu, T. and Pett~grew, J. D , Depletion of brain catecholammes: failure of ocular dominance shift after monocular occlusion m kittens, Scgence, 194 (1976) 206-209. 19 Kebablan, J. W., Petzold, G. L. and Greengard, P , Dopamme-sensitive adenylate cyclase m the caudate nucleus of rat brain and its similarity to the 'dopamme receptor', Proc. nat. Acad S~1. (Wash.), 69 (1972) 2145-2149. 20 Komg, N , Roch, G. and Marty, R., The onset of synaptogenesls m rat temporal cortex, Anal EmbryoL, 148 (1975) 73-87. 21 Lauder, J M and Bloom, F. E., Ontogeny of monoamme neurons m the locus coeruleus, raphe nuclei and substantm mgra of the rat. 1. Cell differentiation, J. comp. Neurol, 155 (1974) 469-482. 22 Lefkowitz, R J", Mukherjee, C , Coverstone, M and Caron, M G., Stereoslzeclfic [ZH](--)-alprenolol binding sites,/~-adrenerglc receptors and adenylate cyclase, Btochem btophy~. Re~. Con,nmn, 60 (1974) 703-709. 23 Lefkowltz, R J. and Williams, L. T , Molecular mechanisms of activation and desensitization of adenylate cyclase coupled I:eta-adrenerglc receptors. In W. J. George and L. J lgnarro (Eds), Advanc Cychc Nucleonde Res, Vol 9, Raven Press, New York, 1978 pp 1-17. 24 Levitt, P. and Moore, R. Y., Noradrenahne neuron innervatlon of the neocortex m the rat, Brain Re.warch, 139 (1978) 219-231 25 LewJs, P. D , Patel, A J., B6ndek, G. and 13al~izs, R., Effect of reserpine on cell proliferation in the develolzmg rat brain: a quantltatwe histological study, Brim Bese~rch, 129 (1977) 299-308. 26 Maeda, T and Dresse, A , Recherches sur le d6velolzement du locus coeruleus. 1 Etude des catecholammes au microscope de fluorescence, Acta neurol. Belg, 69 (1909) 5-10. 27 McMoms, F. A , Norepmephrme induces ghal-sl0ecific enzyme activity m cultured ghoma cells, Proc nat. Acad. Sci (Wash), 74 (1977) 4501-4504 28 Nobln, A and BJorklund, A , Topography of the rnonoamme neuron systems m the human brain as revealed m fetuses, Acta phy~lol stand., Suppl, 388 (1973) 1-40 29 Olson, L , Bor6us, L O and Selger, A., HJstochem~cal demonstration and mapping of 5-hydroxytryptamme- and catecholamme-contammg neuron systems m the human fetal brain, Z Anat EntwukI-Ge.sch., 139 (1973) 259-282 30 Olson, L and Seiger, A., Early prenatal ontogeny of central monoamme neurons m the rat fluorescence hlstochemJcal observations, Z Anat EntwtcM.-Gesch, 137 (1972) 301-316. 31 Patel, A J , B6ndek, G , Bal~izs, R. and Lewis, P D , Effect of reserpine on cell ptohferat,on in the developing rat brain: a biochemical study, Brain Re6earch, 129 (1977) 283-297. 32 Perkins, J P , Johnson, G. L and Harden, T K , Drug-induced modification of the responsiveness ofadenylate cyclase to hormones. In W J George and L J. Ignarro (Eds), Advanc. Cychc Nucleotide Res' Vol 9, Raven Press, New York, 1978, pp 19-32 33 Perkins, J P and Moore, M. M , Regulation of the adenosine cychc 3',5'-monophosphate content of rat cerebral cortex: ontogenet~c development of the responsweness to catecholamines and adenosine, Molee. PharmacoL, 9 (1973) 774-782. 34 Pettlgrew, J D. and Kasamatsu, T , Local norepmephrine perfuslon and effects of monocular depravation m 6-OHDA-treated kittens, Neuroaci Abstr, 3 (1977) 572. 35 Prasad, K N., Kumar, S , Gilmer, K and Vernadakls, A., Cyclic AMP-reduced differentiated neuroblastoma cells, changes m total nucleic acid and protein contents, Btochem blophy~. Re.~. Comnum , 50 (1973) 973-977. 36 Raft, M C , Hornby-Smttb, A and Brockes, J P , Cychc A M P as a mltogemc signal for cultured rat Schwann cells, Nature (Lond), 273 (1978) 672-673. 37 Rolsen, F J , Murphy, R A. and Braden, W G , Dlbutyryl cyclic adenosine monophosphate stlrnulatlon of colcemid-mhlblted axonal elongation, Science, 177 (1972) 809-811. 38 Schlumpf, M , Shoemaker, W. J and Bloom, F E., The development of catecholamme fibers m the prenatal cerebral cortex of the rat, Neuroscl. Abstr., 3 (1977) 118. 39 Schmidt, M J , Palmer, E. C., Dettbarn, W -D. and Robmon, G. A., Cychc A M P and adenyl cyclase m the developing rat brain, Develop. PsyehobloL, 3 (1970) 53-67.

522 40 Schner, B. K. and Shapiro, D. L., Effects of N6-monobutyryl-cychc AMP on glutamate decarboxylase activity in fetal rat brain cells and glial tumor cells in culture, Exp. Cell Res., 80 (1973) 459--462 41 Siman-Tov, R. and Sachs, L., Enzyme regulation in neuroblastoma cells, selection of clones with low acetylcholinesterase activity and the independent control of acetylcholinesterase and cholineO-acetyltransferase, Europ. J. Biochem., 30 (1972) 123-129. 42 Spano, P. F., Kumakura, K., Govoni, S. and Trabucehi, M , Ontogenetic development of neostriatal dopamine receptors in the rat, J Neurochem., 27 (1976) 621-624. 43 Terasaki, W. L. and Brooker, G., [12~I]Iodohydroxybenzylpindolol binding sites on intact rat glioma ceils, J. biol. Chem., 253 (1978) 5418-5425 44 Terasaki, W. L , Brooker, G. de Vellis, J., Inglish, D., Hsu, C -Y. and Moylan, R. D., Involvement of cyclic AMP and protein synthesis in catecholamine refractoriness. In W. J. George and L. J lgnarro (Eds.), Advanc. Cyclic Nucleotide Res, Vol. 9, Raven Press, New York 1978, pp. 33-52 45 Von Hungen, K., Roberts, S. and Hill, D. F., Developmental and regional variations in neurotransmitter-sensitive adenylate cyelase systems in cell-free preparations from rat brain, J Neurochem., 22 (1974) 8l 1-819 46 Walicke, P. A. and Patterson, P. H., Effects of neuronal activity and cyclic nucleotlde derivatives on the development of transmitter function in cultured sympathetic neurons, Neurosci Abstr, 4 (1978) 129 47 Walton, K. G. and Baldessarini, R. J., Effects of Mn 2+ and other &valent cations on adenytate cyclase actwity m rat brain, J Neurochem., 27 (1976) 557-564. 48 Walton, K. G , Miller, E. and Baldessarini, R. J., fl-Adrenergic receptor: presence m cortex and cerebellum of newborn rat, Neurosci.. Abstr, 3 (1977) 123. 49 Woodward, D. J., Hoffer, B. J., Siggins, G. R. and Bloom, F. E., The ontogenetlc development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar Purkinje cells, Brain Research, 34 (1971) 73-97 50 Zecevic, N. R. and Molliver, M. E., The origin of the monoaminerg~c innervation of immature rat neocortex: an ultrastructural analysis following lesions, Brain Research, 150 (1978) 387-397.