Autoradiographic studies on the uptake of 3H-dopamine by neurons and astrocytes in explant and primary cultures of rat CNS: effects of uptake inhibitors

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Pergamon

Int[ J[ Devl Neuroscience\ Vol[ 04\ No[0\ pp[ 34Ð42\ 0886 ISDN Copyright Þ 0886 Published by Elsevier Science Ltd Printed in Great Britain 9625Ð4637:86 ,06[99¦9[99

PII] S9625Ð4637"85#99969Ð5

AUTORADIOGRAPHIC STUDIES ON THE UPTAKE OF H!DOPAMINE BY NEURONS AND ASTROCYTES IN EXPLANT AND PRIMARY CULTURES OF RAT CNS] EFFECTS OF UPTAKE INHIBITORS

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 SLI and L[ HO  SLI ELISABETH HO Department of Physiology\ University of Basel\ Vesalgasse 0\ CH!3940Basel\ Switzerland "Received 15 February 0885^ revised 8 April 0885^ accepted 12 May 0885# Abstract*The cellular localization of the uptake of 2H!dopamine was studied in explant and primary cultures from various regions of rat central nervous system by means of autoradiography[ In explant cultures of substantia nigra\ 2H!dopamine was taken up by cell bodies and processes of many neurons[ In cultures from striatum\ cerebellum and spinal cord\ neuronal cell bodies were not labelled\ whereas outgrowing nerve _bres revealed intense uptake of the monoamine[ Uptake of 2H!dopamine by neurons was Na¦! and temperature!dependent\ suggesting an active uptake mechanism[ In explant cultures\ astro! cytes did not accumulate 2H!dopamine\ whereas in primary cultures\ which were prepared from the same regions of rat central nervous system as the explant cultures\ astrocytes also revealed uptake of this monoamine[ The intensity of labelling was dependent on the incubation time[ Little uptake of 2H!dopamine was observed after an incubation time of 4 min and only after 09Ð04 min did the astrocytes show moderate labelling[ Uptake of 2H!dopamine by astrocytes was not Na¦! and temperature!dependent\ indicating that glial cells do not possess an active uptake mechanism for this monoamine[ This is consistent with bio! chemical investigations by other laboratories\ demonstrating that astrocytes accumulate 2H!dopamine by a facilitated di}usion system[ Addition of the uptake inhibitors nomifensine or GBR 01898 to explant cultures markedly reduced or inhibited uptake of 2H!dopamine by neurons at a concentration of 09−5 M[ In contrast\ accumulation of 2H!dopamine by astrocytes in primary cultures was only slightly a}ected by nomifensine at 09−5 M[ At the highest concentration used "09−4 M#\ nomifensine also blocked the uptake of 2H!dopamine by astrocytes[ Our _nding that GBR 01898 almost completely inhibited the uptake of 2H! dopamine by astrocytes already at 09−5 M suggests that this compound is a more potent inhibitor of the glial uptake of dopamine than nomifensine[ Key words] antidepressants\ astrocytes\ autoradiography\ 2H!dopamine\ uptake[

It is well known that the principal mode of inactivation of neurotransmitters such as amino acids and monoamines at central synapses is by an active uptake mechanism[5\09\03\10\16 There is also much evidence that astrocytes possess a high a.nity uptake mechanism for amino acid transmitters2\05\19 and to a lesser extent for monoamines[00\01\08\19\11\15 Recent investigations from our laboratory have shown a di}erence in the uptake pattern of noradrenaline and serotonin by astrocytes in explant cultures and in primary cultures[08 In explant cultures\ where both neurons and glial cells are present\ no uptake of the monoamines by astrocytes was detected\ whereas in primary cultures of rat central nervous system "CNS#\ astrocytes also revealed an active accumulation of 2H!noradrenaline and 2 H!serotonin[08 It was therefore of interest to compare the cellular localization of the uptake of another monoamine 2H!dopamine in explant cultures and in primary astrocyte cultures from various parts of rat CNS by means of autoradiography[ Many antidepressants exert their action by blocking the uptake of noradrenaline and serotonin\ whereas they are ine}ective against dopamine uptake[8\12\14\29 Furthermore\ the thymoleptic com! pound nomifensine "7!amino!1!methyl!3!phenyl!l\1\2\3!tetrahydroisoquinoline hydrogen maleate#\ which blocks the uptake of dopamine by rat brain synaptosomes\ is not speci_c\ since it also inhibits the uptake of noradrenaline to a similar extent[14 The inhibitor GBR 01898 "0!ð1!"diphenyl!methoxy# ethylŁ 3!"2!phenyl!1!propenyl#!piperazine# is a more potent and selective dopamine reuptake inhibi! tor[0\02\18 In the present investigations\ we studied the e}ect of both compounds "nomifensine and GBR 01898# on the uptake of 2H!dopamine by neurons and astrocytes in explant and in primary cultures[

To whom correspondence should be addressed[ 34

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EXPERIMENTAL PROCEDURES Explant cultures were prepared from spinal cord of foetal rats "06Ð07 days in utero# and from substantia nigra\ striatum and cerebellum of newborn rats[ The cultures were placed on collagen! coated ACLAR coverslips and grown in Roller tubes at 24>C for 01Ð39 days[06 Primary astrocyte cultures were prepared from the same CNS areas as those for explant cultures and grown in serum! free media essentially according to the method described by Fischer[7 For the uptake studies\ both explant and primary astrocyte cultures of the same age in vitro were rinsed several times in Gey|s solution "26>C#[ They were then incubated in Gey|s solution containing 2H!dopamine "speci_c activity 35[6 and 37 Ci:mmol# at a concentration of 09−6 M "26>C#[ To prevent degradation of dopamine\ the monoamine oxidase inhibitor pargyline "9[0 mM# was added to the incubation medium[ The incubation time ranged from 4 to 04 min[ To investigate whether the radioligand is taken up by an active transport mechanism\ studies were carried out at 9Ð1>C and in Na¦!free incubation medium "Na¦ being replaced by choline and Tris#[ The uptake inhibitors nomifensine and GBR 01898 were added to the incubation medium at concentrations of 09−5 and 09−4 M[ After incubation with 2H!dopamine\ the cultures were rinsed in 9[0) phosphate bu}er\ _xed for 29 min at 3>C in 1) paraformaldehyde\ 9[0) glutaraldehyde and 9[0) tannic acid in phosphate bu}er\ and washed in ice!cold phosphate bu}er and distilled water[ Afterwards\ the cultures were dehydrated to 099) ethanol\ mounted on object slides and air!dried[ Then they were covered with Ilford L3 emulsion by the loop technique and stored for 1Ð2 weeks at 3>C[06 To identify astrocytes and neurons\ many explant and primary cultures were stained either with a monoclonal antibody against glial _brillary acidic protein "GFAP#1 or with cresyl violet "Nissl# before application of the Ilford L3 emulsion[07\08 The autoradiographs were developed with a Kodak D 08 developer[ Materials The radioligand 2H!dopamine was purchased from New England Nuclear Co[ and the monoclonal antibody against GFAP from Amersham[ The antidepressants nomifensin and GBR 01898 were kindly provided by Hoechst\ Frankfurt am Main\ Germany and Novo Nordisk\ Bagsvaerd\ Denmark\ respectively[ RESULTS Uptake of 2H!dopamine in explant cultures The characteristic morphological appearance of neurons and astrocytes in explant cultures of rat CNS by phase contrast microscopy has been described in previous publications[07\08\19 The various cell types were identi_ed by speci_c staining properties\ e[g[ Nissl staining for neurons and GFAP for astrocytes[1\07\08 Dopamine!containing neurons in the mesencephalon and in the substantia nigra are the origin of most of the known central ascending and descending dopaminergic pathways[3\4\6\17 After incubation of explant cultures from the substantia ni`ra with 2H!dopamine "4 and 09 min#\ many neurons with long processes were intensely labelled ðFig[ 0"A# and "B#Ł[ In cultures from striatum\ cerebellum and spinal cord\ no uptake of 2H!dopamine was observed by neuronal cell bodies\ whereas in the outgrowth zone\ many nerve _bres with varicosity!like structures revealed intense labelling[ Figure

0000000000000000000000000000000000000000000000000000 4 Fig[ 0[ Uptake of 2H!dopamine in explant cultures of rat CNS[ "A# and "B# Autoradiographic localization of the uptake of 2H!dopamine "09−6 M\ 09 min# in an explant culture from substantia nigra which was stained simultaneously by a monoclonal antibody against glial _brillary acidic protein ðGFAP^ "A# bright! _eld^ "B# dark!_eld illumination micrographsŁ[ The neurons are labelled intensely over the cell bodies and processes by 2H!dopamine\ whereas the underlying astrocytes remain unlabelled "arrows^ culture 11 days in vitro#[ "C# and "D# Outgrowth zone of an explant culture of spinal cord ð"C# bright!_eld^ "D# dark!_eld illumination micrographsŁ[ The outgrowing nerve _bres reveal intense uptake of 2H!dopamine "09−6 M\ 09 min#\ whereas the GFAP!positive astrocytes are unlabelled "arrows^ culture 13 days in vitro#[ "E# Bright! _eld^ "F# dark!_eld illumination micrographs of an explant culture of striatum[ Many outgrowing nerve _bres are intensely labelled by 2H!dopamine "09−6 M\ 09 min#\ whereas glial cells did not reveal any uptake of the monoamine "culture 03 days in vitro#[ Bars\ 29 mm[

Uptake of dopamine in CNS cultures

COLOUR * FOR POSITION ONLY

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0"C#Ð"F# illustrate outgrowing nerve _bres from a spinal cord ð"C# and "D#Ł and a striatal culture ð"E# and "F#Ł which are labelled by 2H!dopamine[ In contrast to neuronal cell bodies and processes\ astrocytes in explant cultures did not accumulate 2 H!dopamine[ As shown in Fig[ 0"A#Ð"D#\ GFAP!positive astrocytes surrounding the labelled neurons and nerve _bres did not reveal any uptake of the radioligand[ This is consistent with previous observations on the cellular localization of the uptake of 2H!noradrenaline and 2H! serotonin\ demonstrating that glial cells in explant cultures remained unlabelled[04\08\19 The uptake of 2H!dopamine by neuronal cell bodies and _bres was Na¦! and temperature! dependent\ being signi_cantly reduced or blocked in Na¦!free incubation medium ðFig[ 2"A#Ł or at low temperature "9Ð1>C\ not illustrated#[

Fig[ 1[ Uptake of 2H!dopamine by astrocytes in primary cultures[ "A# Bright!_eld and "B# dark!_eld illumination micrograph of GFAP!positive astrocytes in a primary culture of cerebellum which are weakly labelled by 2H!dopamine "09−6 M\ 09 min#[ Note that only some astrocytes are labelled "arrows# whereas other GFAP!positive cells do not show any uptake of the monoamine "culture 04 days in vitro#[ "C# and "D# Uptake of 2H!dopamine "09−6 M\ 04 min# by astrocytes in a primary culture of spinal cord ðdark!_eld illumination micrograph^ "D#Ł which is simultaneously stained for GFAP "C#[ The arrow shows a GFAP! positive astrocyte which is almost unlabelled\ whereas the neighbouring glial cell reveals uptake of 2H! dopamine "culture 13 days in vitro#[ Bars\ 29 mm[

Uptake of dopamine in CNS cultures

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Fig[ 2[ Uptake of 2H!dopamine by neurons and astrocytes in Na¦!free incubation medium[ "A# Di}erential interference micrograph of an explant culture of substantia nigra[ Incubation in Na¦!free solution inhibited the uptake of 2H!dopamine "09−6 M\ 09 min# by neurons and outgrowing nerve _bres "explant culture 19 days in vitro#[ "B# Na¦!free incubation medium did not signi_cantly reduce the uptake of 2H!dopamine "09−6 M\ 09 min# by astrocytes in primary cultures "cerebellar culture\ 04 days in vitro#[ Action of the uptake inhibitors nomifensine and GBR 01898 on the accumulation of 2H!dopamine by neurons in explant cultures[ Addition of the uptake inhibitors nomifensine "C# and GBR 01898 "D# at concentrations of 09−5 M completely blocked the uptake of 2H!dopamine "09−6 M\ 09 min# by neuronal cell bodies and _bres in explant cultures of substantia nigra "cultures 11 days in vitro#[ Bars\ 29 mM[

Effects of the uptake inhibitors nomifensine and GBR 01898 on the uptake of 2H!dopamine in neurons and nerve _bres of explant cultures Addition of nomifensine or GBR 01898 to the incubation medium at a concentration of 09−5 M completely inhibited the uptake of 2H!dopamine "09−6 M# by neurons in explant cultures[ Figure 2"C# and "D# illustrate that\ in cultures of substantia nigra\ the uptake of 2H!dopamine by neuronal cell bodies and _bres was blocked by nomifensine "C# and GBR 01898 "D# both at concentrations of 09−5 M[ Uptake of 2H!dopamine by astrocytes in primary cultures The cell bodies of astrocytes in primary cultures varied considerably in size and shape and revealed multiple processes[ Staining of primary astrocyte cultures with a monoclonal antibody against

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GFAP has revealed that more than 84) of the cells were GFAP!positive and are therefore identi_ed as astrocytes[08 After incubation with 2H!dopamine "09−6 M# for 4 min\ no or little uptake by astrocytes was observed\ whereas after an incubation time of 09Ð04 min\ the glial cells were weakly to moderately labelled by the monoamine ðFig[ 1"A# and "C#^ Fig[ 3"A# and "B#Ł[ Furthermore\ a signi_cant uptake of 2H!dopamine only occurred in approximately one!third of the astrocytes[ Figure 1"A# and "C# illustrate GFAP!positive astrocytes in primary cultures of cerebellum and spinal cord\ respectively[ The dark!_eld illumination micrographs ðFig[ 1 "B# and "D#Ł show that only some astrocytes "marked by arrows# have taken up 2H!dopamine\ whereas other cells remain unlabelled[ This is in contrast to previous autoradiographic studies from our laboratory demonstrating that 2H!noradrenaline and 2 H!serotonin were taken up by almost all astrocytes in primary cultures and that the intensity of labelling was much stronger than with 2H!dopamine[07 There was no obvious di}erence in the number of labelled astrocytes or in the intensity of labelling between cultures originating from the di}erent CNS areas studied[ Furthermore\ uptake of 2H!dopamine by the astrocytes was not signi_cantly reduced in Na¦!free incubation medium ðFig[ 2"B#Ł or at low temperature "9Ð1>C\ not illustrated#[ Effects of the uptake inhibitors nomifensine and GBR 01898 on the uptake of 2H!dopamine by astrocytes in primary cultures Nomifensine appears to have a weak e}ect on astroglial dopamine uptake\ since addition of this compound at 09−5 M to the incubation medium failed to signi_cantly reduce the accumulation of 2 H!dopamine by astrocytes in primary cultures ðFig[ 3"C#Ł[ Only at a higher concentration "09−4 M# did the antidepressant completely block the uptake of 2H!dopamine by these cells ðFig[ 3"E#Ł[ After addition of the more speci_c and potent dopamine uptake inhibitor GBR 01898\ uptake of 2H! dopamine by astrocytes was signi_cantly already reduced or inhibited at a concentration of 09−5 M ðFig[ 3"D#Ł and completely blocked at 09−4 M ðFig[ 3"F#Ł[ DISCUSSION A great number of investigations indicate that\ in addition to neurons\ astrocytes play a role in the inactivation of amino acid transmitters\2\05\19 whereas there are controversial data for the uptake of monoamines by these cells[00\01\04\19\11\15 Recent autoradiographic studies from our laboratory have demonstrated that there is a di}erence between astrocytes in explant cultures and in primary cultures with respect to the uptake of noradrenaline and serotonin[08 Almost all astrocytes in primary cultures showed an active uptake of both monoamines\ whereas glial cells in explant cultures failed to accumulate these substances[04\08\19 In the present study\ similar observations were made on the uptake of 2H!dopamine[ In explant cultures\ 2H!dopamine was actively accumulated by neuronal cell bodies and _bres but not by glial cells\ whereas in primary cultures\ astrocytes showed uptake of the monoamine[ The intensity of labelling of the astrocytes by 2H!dopamine was\ however\ much weaker than by 2H!noradrenaline and 2H!serotonin[ Furthermore\ 2H!dopamine was only taken up in approximately one!third of the astrocytes\ suggesting that only a certain type or subpopulation of glial cells is involved in the accumulation of this monoamine[ These _ndings are in agreement with autoradiographic studies by Semeno} and Kimelberg\15 describing that uptake of 2H!dopamine was not uniform throughout the culture\ and intense labelling was only observed in approximately 19) of the total cell area[ In contrast to neurons which possess an active uptake mechanism for 2 H!dopamine\03 the present study\ as well as biochemical investigations by other laboratories\00\01

0000000000000000000000000000000000000000000000000000 4 Fig[ 3[ Action of the uptake inhibitors nomifensine ð"A#\ "C# and "E#Ł and GBR 01898 ð"B#\ "D# and "F#Ł on the accumulation of 2H!dopamine by astrocytes in primary cultures[ "A# Uptake of 2H!dopamine "09−6 M\ 04 min# by astrocytes in a primary culture of substantia nigra "11 days in vitro#[ "C# and "E# After addition of nomifensin at a concentration of 09−5 M\ uptake of 2H!dopamine "09−6 M\ 04 min# by astrocytes was only reduced "C# whereas it was completely blocked at 09−4 M "E#[ "B# Uptake of 2H!dopamine "09−6 M\ 04 min# by astrocytes in a primary culture of substantia nigra "10 days in vitro#[ "D# and "F# The more speci_c dopamine uptake inhibitor GBR 01898 blocked the accumulation of 2H!dopamine "09−6 M\ 04 min# by astrocytes at both concentrations 09−5 M "D# and 09−4 M "F#[ Bars\ 29 mM[

Uptake of dopamine in CNS cultures

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demonstrate that accumulation of the monoamine by astrocytes in primary cultures was not Na¦! and temperature!dependent\ suggesting that the uptake of 2H!dopamine is due to a facilitated di}usion mechanism rather than to an active transport[00\01 Most antidepressant agents are selective inhibitors of either noradrenaline or serotonin uptake and have only a minimal e}ect on dopamine uptake[12\13\29 The thymoleptic drug nomifensine was one of the _rst antidepressants known to block the uptake of dopamine in the CNS[14\29 This substance is\ however\ also a powerful inhibitor of noradrenaline uptake[0\8\14\29 A considerable e}ort therefore has been made to develop drugs which are selective dopamine!uptake blockers[ The inhibitor GBR 01898\ which is a member of a series of diphenyl!substituted piperazine derivatives\ selectively inhibits the uptake of dopamine with a 099!fold lower a.nity for noradrenaline[0\8\02\29 It is also a more potent inhibitor of 2H!dopamine uptake "099 nM# by homogenates of rat brain "IC49 0 nM# than nomifensine "IC49 023 nM#[0\8 Our studies have shown that in explant cultures\ uptake of 2 H!dopamine by neuronal cell bodies and _bres was blocked by both nomifensine and GBR 01898 at a concentration of 09−5 M[ In contrast\ nomifensine was less e}ective in inhibiting dopamine uptake by glial cells[ Accumulation of 2H!dopamine by astrocytes in primary cultures was only slightly reduced by nomifensine at 09−5 M[ At a concentration of 09−4 M\ nomifensine also blocked the uptake of 2H!dopamine by these cells[ GBR 01898\ however\ inhibited the uptake of 2H! dopamine by astrocytes already at a concentration of 09−5 M\ suggesting that this compound is a more potent inhibitor of the uptake of 2H!dopamine by astrocytes than nomifensine[ In a previous publication\ the di}erence between astrocytes in explant cultures and in primary cultures with respect to the uptake of 2H!noradrenaline and 2H!serotonin was discussed[08 The present investigations demonstrate that a similar di}erence exists between these two culture systems concerning the uptake of 2H!dopamine[ It is\ however\ suggested that uptake of 2H!dopamine by astrocytes is due to a facilitated di}usion mechanism and not to an active uptake process\ as this is the case for the glial uptake of 2H!noradrenaline and 2H!serotonin[ Acknowled`ements*We should like to thank Prof[ L[ Ma(¼tre\ Basel:Switzerland and Prof[ U[ Otten\ Dept of Physiology\ University of Basel\ Basel:Switzerland for their valuable comments and suggestions on the manuscript[ We are grateful to Mrs C[ Fehlmann for skilful technical assistance and to Mr M[ Wymann for photographic work[ We should also like to thank Hoechst\ Frankfurt am Main\ Germany and Novo Nordisk\ Bagsvaerd\ Denmark\ for providing the uptake inhibitors nomifensine and GBR!01898\ respectively[

REFERENCES 0[ Andersen P[ H[ "0878# The dopamine uptake inhibitor GBR 01898] selectivity and molecular mechanism of action[ Eur[ J[ Pharmacol[ 055\ 382Ð493[ 1[ Bologa L[\ Bisconte J[ C[\ Joubert R[\ Marangos P[ J[\ Derbin C[\ Rioux F[ and Herschkowitz N[ "0871# Accelerated di}erentiation of oligodendrocytes in neuronal!rich embryonic mouse brain cell cultures[ Brain Res[ 141\ 018Ð025[ 2[ Chaudhry F[ A[\ Lehre K[ P[\ v[ Lookeren Campagne M[\ Ottersen O[ P[\ Danbolt N[ C[ and Storm!Mathisen J[ "0884# Glutamate transporters in glial plasma membranes] highly di}erentiated localizations revealed by quantitative ultrastructural immunocytochemistry[ Neuron 04\ 600Ð619[ 3[ Commissiong J[ W[ and Sedgwick E[ M[ "0863# On the presence of dopamine in the mammalian spinal cord[ Br[ J[ Pharmacol[ 40\ 007PÐ008P[ 4[ Dahlstrom A[ and Fuxe K[ "0854# Evidence for the existence of monoamine!containing neurons in the central nervous system[ I[ Demonstration of monoamines in the cell bodies of brain stem neurons[ Acta Physiol[ Scand[ 51\ 0Ð44[ 5[ De Feudis F[ V[ "0864# Amino acids as central neurotransmitters[ Ann[ Rev[ Pharmacol[ 04\ 094Ð029[ 6[ Dupelj M[ and Geber J[ "0870# Dopamine as a possible neurotransmitter in the spinal cord[ Neuropharmacolo`y 19\ 034Ð037[ 7[ Fischer G[ "0873# Growth requirements of immature astrocytes in serum!free hormonally de_ned media[ J[ Neurosci[ Res[ 01\ 432Ð441[ 8[ Gianutsos G[\ Morrow G[\ Light St[ and Sweeney M[ J[ "0871# Dopaminergic properties of nomifensine[ Pharmacol[ Biochem[ Behav[ 06\ 840Ð843[ 09[ Graefe K[!H[ and Bonisch H[ "0877# The transport of amines across the axonal membranes of noradrenergic and dopaminergic neurons[ In Handbook of Experimental Pharmacolo`y Vol[ 89:I\ pp[ 082Ð134[ Springer\ Berlin[ 00[ Hansson E[ "0874# Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures[ Neurochem[ Res[ 09\ 556Ð564[ 01[ Hansson E[\ Eriksson P[ and Nilsson M[ "0874# Amino acid and monoamine transport in primary astroglial cultures from de_ned brain regions[ Neurochem[ Res[ 09\ 0224Ð0230[ 02[ Heikkila R[ E[ and Manzino L[ "0873# Behavioral properties of GBR 01898\ GBR 02958 and GBR 02987] speci_c inhibitors of dopamine uptake[ Eur[ J[ Pharmacol[ 092\ 130Ð137[ 03[ Horn A[ S[ "0889# Dopamine uptake] a review of progress in the last decade[ Pro`r[ Neurobiol[ 23\ 276Ð399[ 04[ Hosli E[\ Bucher U[ M[ and Hosli L[ "0864# Uptake of 2H!noradrenaline and 2H!4!hydroxytryptamine in cultured rat brainstem[ Experientia 20\ 243Ð245[

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05[ Hosli E[\ Hosli L[ and Schousboe A[ "0875# Amino Acid Uptake\ pp[ 022Ð042[ Academic Press\ New York[ 06[ Hosli E[ and Hosli L[ "0878# Autoradiographic localization of binding sites for neurotransmitters in explant cultures of rat central nervous system[ In A Dissection and Tissue Culture Manual of the Nervous System\ pp[ 232Ð234[ Alan R[ Liss\ Inc[ 07[ Hosli E[ and Hosli L[ "0882# Autoradiographic localization of binding sites for neuropeptide Y and bradykinin on astrocytes[ Neuroreport 3\ 048Ð051[ 08[ Hosli E[ and Hosli L[ "0884# Autoradiographic studies on the uptake of 2H!noradrenaline and 2H!serotonin by neurons and astrocytes in explant and primary cultures of rat CNS] e}ects of antidepressants[ Intl J[ Dev[ Neurosci[ 02\ 786Ð897[ 19[ Hosli L[ and Hosli E[ "0867# Action and uptake of neurotransmitters in CNS tissue culture[ Rev[ Physiol[ Biochem[ Pharmacol[ 70\ 024Ð077[ 10[ Iversen L[ L[ "0856# The Uptake and Stora`e of Noradrenaline in Sympathetic Nerves\ p[ 142[ Cambridge University Press\ Cambridge[ 11[ Kimelberg H[ K[ and Katz D[ M[ "0875# Regional di}erences in 4!hydroxytryptamine and catecholamine uptake in primary astrocyte cultures[ J[ Neurochem[ 36\ 0536Ð0541[ 12[ Nielsen E[ B[ and Scheel!Kruger J[ "0877# Central nervous system stimulants] neuropharmacological mechanisms[ In Transduction Mechanisms of Dru` Stimuli "eds Balster B[ and Colpaert F[ C[#\ pp[ 46Ð61[ Springer!Verlag\ Berlin[ 13[ Riederer P[\ Laux G[ and Poldinger W[ "0882# Neuro!Psychopharmaka\ Band 2] Antidepressiva und Phasenprophylaktika\ p[ 500[ Springer!Verlag\ Wien[ 14[ Schacht U[ and Heptner W[ "0863# E}ect of nomifensine "HOE 873#\ a new antidepressant\ on uptake of noradrenaline and serotonin and on release of noradrenaline in rat brain synaptosomes[ Biochem[ Pharmacol[ 12\ 2302Ð2311[ 15[ Semeno} D[ and Kimelberg H[ K[ "0874# Autoradiography of high a.nity uptake of catecholamines by primary astrocyte cultures[ Brain Res[ 237\ 014Ð025[ 16[ Snyder S[ H[\ Kuhar M[ J[\ Green A[ I[\ Coyle J[ T[ and Shaskan E[ G[ "0869# Uptake and subcellular localization of neurotransmitters in the brain[ Intl Rev[ Neurobiol[ 02\ 016Ð047[ 17[ Ungerstedt U[ "0860# Stereotaxic mapping of the monoamine pathways in the rat brain[ Acta Physiol[ Scand[ Suppl[ 256\ 0Ð37[ 18[ Van der Zee P[\ Koger H[ S[\ Gootjes J[ and Hespe W[ "0879# Aryl 0\3!dialk"en#yl!piperazines as selective and very potent inhibitors of dopamine uptake[ Eur[ J[ Med[ Chem[ ! Chim[ Therapeut[ 04\ 252Ð269[ 29[ Willner P[ "0872# Dopamine and depression] a review of recent evidence[ III[ The e}ects of antidepressant treatments[ Brain Res[ Rev[ 5\ 126Ð135[