Enhanced dopamine cell survival in reaggregates containing telencephalic target cells

Dissociated, 14-day-old embryonic cells of the rostral mesencephalic tegmentum (RMT), including the dopamine neurons of this region, were allowed to reaggregate and develop in rotatory culture for 7 days in the presence of dissociated embryonic cells from the target areas of the dopaminergic neurons, corpus striatum (CS) or frontal cortex (FCx). Alternatively, RMT cells were allowed to reaggregate by themselves or in the presence of dissociated cells from a telencephalic area, occipital cortex (OCx), or mesencephalic area, tectum (T), which are not target areas for the dopamine neurons. Histofluorescence analysis revealed the number of dopamine neurons contained within reaggregates of any given type. Approximately 4 times as many dopamine neurons were found in RMT-CS coaggregates and 1.5 times as many in RMT-FCx coaggregates than in aggregates constituted from cells of the RMT either alone, or in coaggregates from RMT-OCx or RMT-T. Since axonal process formation and maintenance can only be observed in RMT-CS and RMT-FCx coaggregates, the enhanced dopamine neuron survival is probably due to an interaction of dopaminergic axonal processes with target cells within the reaggregates.     

Differential effects of phencyclidine and methamphetamine on dopamine metabolism in rat frontal cortex and striatum as revealed by in vivo dialysis

We have examined the effects of schizophrenomimetic drugs including phencyclidine (PCP) and methamphetamine (MAP) on cortical and striatal dopamine (DA) metabolism using an in vivo dialysis technique in the rat. An acute systemic injection of PCP (2.5–10 mg/kg, intraperitoneally (i.p.)) dramatically increased concentrations of DA, 3,4-dihydroxy-phenylacetic acid, and homovanillic acid in the dialysates from the medial frontal cortex in a dose-dependent fashion. However, PCP (2.5–10 mg/kg, i.p.) caused a much lower augmentation of extracellular DA release, with a significant decrease in dialysate DOPAC levels in the striatum. Moreover, continuous infusion of tetrodotoxin (TTX, 10⁻⁵ M) into the prefrontal or striatal region through the microdialysis tube completely blocked the ability of PCP (10 mg/kg, i.p.) to alter the extracellular release of DA and its metabolites in the respective areas. In contrast, MAP (4.8 mg/kg, i.p.) elicited a marked and tetrodotoxin-resistant increase in DA levels with a significant loss of DOPAC contents in the extracellular space of both the frontal cortex and the striatum. The present results clearly demonstrate the differential effects of PCP on cortical and striatal DA transmission, suggesting that PCP may facilitate DA release in the medial frontal cortex by increasing impulse flow in the DA neurons projecting to the cortical area, whereas PCP-induced elevation of extracellular DA in the striatum may be caused mainly by reuptake inhibition of DA liberated by basal activity of the striatal DA neurons. The regional variation in PCP-induced DA release would be due to the combination of NMDA (N-methyl-D-aspartate) receptor blocking and DA reuptake inhibition by the drug. The uniform and TTX-resistant nature of MAP-induced changes in brain DA metabolism may result from the direct actions of MAP at DA nerve terminals. © 1996 Wiley-Liss, Inc.     

Biochemical and morphological studies on GABA neurons in reaggregate culture

Dissociated cells from the 14-day fetal mouse corpus striatum (CS), rostral mesencephalic tegmentum (RMT) and tectum were reaggregated in rotation-mediated cultures in the following combinations: CS alone, RMT-CS, RMT-tectum, and tectum alone. Reaggregates were cultured for 1-4 weeks. An even distribution of gamma-aminobutyric acid (GABA)-positive cells was observed in all the reaggregate combinations that were cultured for 1 and 2 weeks. With increasing time in culture, cellular staining was decreased while positive fiber staining increased. CS and RMT-CS co-cultures increased their capacity to take up [3H]GABA with time in culture. All reaggregates reached a maximum uptake/accumulation capacity of 30-40 pmol/mg protein/30 min, by 4 weeks in vitro. There were no significant differences between the various co-aggregate combinations in the accumulation capacity. [3H]GABA accumulation in the reaggregates was largely blocked by the putative neuronal GABA uptake inhibitor, diaminobutyric acid, and was inhibited to a much lesser degree by the putative glial uptake inhibitor beta-alanine. All reaggregates released [3H]GABA to 70 mM potassium depolarization, in a calcium-dependent manner. One-three week CS reaggregates released more [3H]GABA in response to the potassium-induced depolarization than RMT-CS co-cultures. Since nigral dopamine neurons within the RMT proliferate processes and actively release dopamine only when co-cultured with CS target cells, it is suggested that these dopamine neurons might chronically inhibit striatal GABA neurons in the RMT-CS co-cultures, thereby depressing stimulated release of [3H]GABA.     

Enhancement of [H]dopamine release and its [H]metabolites in rat striatum by nicotinic drugs

Recent evidence has identified directly muscarinic acetylcholine receptor (m-ACh R) and nicotinic acetylcholine receptor (n-ACh R) in the brain utilizing receptor binding assay. Several studies suggest that release of dopamine (DA) in the striatum is regulated by presynaptic receptors present on dopaminergic terminals. In the present study, the effects of cholinergic drugs on [³H]DA release were examined using micropunched tissue and synaptosomes obtained from rat striatum. ACh (5 × 10⁻⁴M) significantly increased spontaneous [³H]DA release, and the overflow was partially inhibited by d-tubocurarine (1 mM) but not atropine. Nicotine, lobeline, coniine and spartein, nicotinic agonists, significantly increased spontaneous and 25 mM K⁺ evoked [³H]DA release almost in a dose-dependent manner. In contrast, oxotremorine (2 × 10⁻⁴M), muscarinic agonist, did not any change in [³H]DA efflux. Furthermore, the metabolites of [³H]DA were separated by column chromatography. The main metabolite of [³H]DA in the spontaneous release from rat striatal synaptosomes was [³H]DOPAC (3,4-dihydroxyphenylacetic acid). Lobeline (5 × 10⁻⁵M) accelerated the outflow of [³H]DOPAC and [³H]OMDA metabolites (O-methylated and deaminated metabolites).These results could give rise to the suggestion that there was n-ACh R on the dopaminergic nerve terminals in the striatum and n-ACh R might have related to a directly excitatory effect on the DA release.     

Potassium Channels: A Potential Therapeutic Target for Parkinson’s Disease

The pathogenesis of the second major neurodegenerative disorder, Parkinson’s disease (PD), is closely associated with the dysfunction of potassium (K⁺) channels. Therefore, PD is also considered to be an ion channel disease or neuronal channelopathy. Mounting evidence has shown that K⁺ channels play crucial roles in the regulations of neurotransmitter release, neuronal excitability, and cell volume. Inhibition of K⁺ channels enhances the spontaneous firing frequency of nigral dopamine (DA) neurons, induces a transition from tonic firing to burst discharge, and promotes the release of DA in the striatum. Recently, three K⁺ channels have been identified to protect DA neurons and to improve the motor and non-motor symptoms in PD animal models: small conductance (SK) channels, A-type K⁺ channels, and K_V7/KCNQ channels. In this review, we summarize the physiological and pharmacological effects of the three K⁺ channels. We also describe in detail the laboratory investigations regarding K⁺ channels as a potential therapeutic target for PD.     

Dopamine release in the substantia nigra and striatum at presymptomatic and early symptomatic stages in parkinsonian mice

The degeneration of dopaminergic (DA-ergic) neurons of the nigrostriatal system results in the development of Parkinson’s disease (PD). It is assumed that the increase of DA release from survived DA-ergic neurons might be one of the compensatory processes serving to maintain the DA concentration in the extracellular space at a normal level and thereby providing a long-lasting period of asymptomatic development of the disease. The goal of this study was to estimate DA release in the substantia nigra (SN) containing somas of DA-ergic neurons and in the striatum containing their axonal terminals under the different extents of the degradation of the nigrostriatal system corresponding to either presymptomatic or symptomatic stage of parkinsonism in mice. In the ex vivo studies of the brain slices under perfusion, it has been shown that the values of both spontaneous and K⁺-stimulated release of DA in the SN at both stages of parkinsonism were the same as those in the controls suggesting the increase of DA release from the survived somas of DA-ergic neurons. In the striatum at presymptomatic and symptomatic stages, no compensatory modification of spontaneous DA release was observed whereas K⁺-stimulated release of DA from the survived axonal terminals increased significantly. However, the total amount of DA released at symptomatic stage was below the control level that was in contrast to that at presymptomatic stage. This observation is probably among the reasons of the appearance of the initial symptoms of parkinsonism.     

Selective association of dopamine axons with their striatal target cells in vitro

The purpose of the present study was to examine the distribution of dopamine (DA) axons after simultaneously confronting DA cells with both target cells of the corpus striatum (CS) and with non-target cells of the tectum (T). Dissociated fetal cells of rostral mesencephalic tegmentum (RMT) containing DA neurons and dissociated non-target tectal cells were exposed to wheat germ agglutinin conjugated to the fluorescent dye, rhodamine, prior to reaggregation in rotatory culture in order to distinguish these cells from non-dyed CS cells in the resulting reaggregates. After 9 days in culture, RMT-T-CS reaggregates were exposed to 10(-5)M DA and processed for DA-fluorescence histochemistry. Single reaggregates were serially sectioned and color photomicrographs prepared from each section. It was found that non-target cells (red rhodamine fluorescence) segregated from the undyed target cells, forming discrete areas containing the two cell types. DA nerve cell bodies and their processes could be distinguished by their green fluorescence. Since it is not possible to distinguish axonal from dendritic processes in a single section, the extent of neuronal dendritic arborization was estimated from reaggregates prepared from cells of the RMT and non-target T cells, in which there is no extensive proliferation of DA axons and fluorescent DA processes (presumably dendrites) are confined to the area near the cell body. Following exclusion of DA dendritic fluorescence, it was found that 85.2% of the presumed axonal fibers were present in the non-dyed areas containing striatal target cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Striatal dopamine release stimulated by amphetamine or potassium: influence of ovarian hormones and the light-dark cycle

The release of endogenous dopamine (DA) from rat striatal tissue was studied in an in vitro superfusion system following hormonal manipulations in vivo. Progesterone treatment in estrogen-primed ovariectomized female rats potentiated DA release stimulated either by amphetamine or potassium (K⁺). In addition, the amount of striatal DA released in response to K⁺-stimulation was influenced by the light-dark cycle. We conclude that striatal DA release is modulated by ovarian hormones, and suggest that ovarian hormone modulation of presynaptic striatal DA activity may contribute to well-known estrous cycle dependent variations in some non-reproductive behaviors.     

In vivo release of dopamine in the nucleus accumbens of the rat: modulation by cholecystokinin

We observed that the release of endogenous dopamine (DA), induced by perfusion of a 55 mM K⁺-containing buffer in the nucleus accumbens, was Ca²⁺-dependent and confined to a local region. We also demonstrated that the sulphated form of cholecystokinin octapeptide, but not the unsulphated form, suppressed this stimulated release of dopamine in a concentration-dependent manner. This suggests that cholecystokinin may act as a functional antagonist to dopamine within this structure.     

Footshock treatment activates catecholamine synthesis in slices of mouse brain regions

Synthesis of catecholamines was measured in slices of frontal cortex, hypothalamus, striatum, hippocampus and brainstem by the accumulation of [³H]norepinephrine (NE) and [³H]dopamine (DA) following incubation with [³H]tyrosine.Following acute footshock (60 shocks, 0.3 mA 30 min), consistent increases in [³H]DA accumulation were seen in frontal cortex slices, but no significant effect was seen in striatal slices. The accumulation of [³H]NE was not altered consistently in frontal cortex, hypothalamus, hippocampus or brainstem. Brain slices from mice adrenalectomized 24–48 h before footshock showed similar results. However, in hypophysectomized mice, footshock did not increase the [³H]DA accumulation in slices of frontal cortex. Administration of dexamethasone before footshock prevented the footshock-induced increase in frontal cortex [³H]DA accumulation, but footshock then significantly increased [³H]NE accumulation in the hypothalamus and brainstem. Chronic footshock (5 days) had little effect on frontal cortex [³H]catecholamine accumulation but produced a consistent elevation of [³H]NE accumulation in slices from the hypothalamus.In confirmation that the slices data reflected in vivo metabolism, both dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in the frontal cortex but not the striatum of mice receiving acute footshock.Since previous studies have shown that ACTH administered intracerebroventricularly also accelerated [³H]DA accumulation in frontal cortex slices, these results are consistent with the involvement of ACTH in the effects of footshock on frontal cortex DA. The effects of chronic footshock are consistent with the activation of hypothalamic tyrosine hydroxylase by corticosterone.     

The effect of methamphetamine on the release of glutamate from striatal slices

Summary One postulated role of dopamine in the striatum is to reduce neuronal activity in cortico-striatal glutamergic terminals. We investigated the effects of methamphetamine, which displaces dopamine, on glutamate release from rat striatal slices. Methamphetamine significantly reduced K⁺-stimulated (45 mM) glutamate release. In slices prepared from rats treated 8 days previously with methamphetamine there was enhanced (approximately 200%) release of glutamate. This study demonstrates that dopamine has a modulatory effect on glutamate release in the striatum.     

Intrastriatal iron perfusion releases dopamine: an in-vivo microdialysis study

Summary. We perfused iron as FeCl₃ directly into the striatum of normal rats and used the in vivo microdialysis technique to monitor striatal levels of dopamine (DA). KCl was perfused to assess the functional integrity of the DA receptors at the end of each dialysis experiment. Cu⁺² (as CuSO₄) and Cl⁻ (as NaCl) were perfused to compare the effects of Fe⁺³ to that of other heavy metal and donors of Cl⁻ anion. Perfusion of FeCl₃ (1 mM for 15 min) produced a 250% increase in striatal levels of DA. Perfusion of CuSO₄ (1 mM for 15 min) or NaCl (10 mM for 15 min) did not affect striatal DA levels. There was a significant increase in DA levels with KCl stimulation (56 mM for 15 min) after perfusion with FeCl₃. We conclude that iron releases DA from striatal nerve endings without the immediate destruction of the DA terminals. The implications of chronic release of dopamine as a cause of dopaminergic cell death are discussed. Received November 13, 2001; accepted January 18, 2002     

Release of endogenous catecholamines from slices of hypothalamus of rats

The release of endogenous catecholamines from superfused slices of rat hypothalamus was studied under basal conditions and during release evoked by 40 mM K⁺. Catecholamines in superfusates, and in extracts of the tissue after stimulation, were isolated by column chromatography and quantitated by liquid chromatography with electrochemical detection. Norepinephrine (NE) was not consistently demonstrable in superfusate collected under basal conditions, but 40 mM K⁺ caused the release of from 2 to 4 ng/g of tissue per min. The addition of cocaine to the superfusate caused increases in basal and evoked release of NE. Epinephrine (E) could be measured in superfusates of slices from male but not female rats and then only when cocaine was added to the superfusate. Accordingly, the concentration of E in hypothalamus was greater in male rats than in female rats. Dopamine (DA) was not consistently measurable in the spontaneous overflow from slices either in the presence or absence of cocaine. K⁺-evoked release of DA could be demonstrated in slices from female rats. The addition of cocaine increased the evoked release of DA from slices from both sexes. Corticosterone, added to cocaine, had no effects on the efflux of any of the catecholamines. The experiments suggest that neuronal reuptake of all catecholamines is very efficient in the hypothalamus both under basal conditions and during evoked release.     

Age-related changes in rat brain monoamines release: Peculiarity of dopamine release

Our aim has been to investigate the ability of the rat brain to retain its level of neurotransmitter release over life. We have investigated the neurotransmitter release from the rat brain synaptosomes prelabeled with ³H-DA, ³H-NA, or ³H-5HT, and perfused with Krebs-Ringer medium alone (basal release) or containing a high K⁺, calcium ionophore, tyramine or amphetamine (evoked release). Brain areas have been dissected of animals 45 days and 4, 6, and 11 months old. The results have shown a gradual reduction of the ³H-NA release evoked by a high K⁺ from 45 days to 6 months, which is stabilized until 11 months of age. The reduction rate has been relatively different from the brain areas investigated (36% for the frontal cortex and 26% for the hippocampus and cerebellar cortex). A similar reduction has been seen with ³H-5HT released from synaptosomes of the frontal cortex, hippocampus, and striatum. Surprisingly, the ³H-DA release that was evoked by high K⁺ was greater in rats 11 months old than in younger rats; this effect has been seen in synaptosomes from the caudate and the frontal cortex. The calcium ionophore A23187 has shown a releasing picture similar to a high K⁺. When we analyzed a nonexocitotic, but probably carrier mediated, release (evoked by tyramine or amphetamine), there was reduced release of all of the above neurotransmitters from 45 days to 11 months of age. We presume that there have been adaptative changes in neurotransmitter evoked release due to changes in Ca⁺⁺ utilization, as inferred from the results from calcium ionophore experiments and carrier performance. Aging seems to have preserved the extracellular neurotransmitter concentrations of Na and 5HT, compensating for the decreased carrier performance with a reduced depolarization rate of nerve terminals. DA homeostasis seems to have been altered with normal aging and may produce an elevation in extracellular DA and in its metabolite, yielding free radicals. This mechanism may play a role in degenerative changes associated with normal aging. © 1993 Wiley-Liss, Inc.     

Release of endogenous catecholamines from the striatum and bed nucleus of stria terminalis evoked by potassium and N-methyl-D-aspartate: In vitro microdialysis studies

Induced release of endogenous dopamine and noradrenaline from coronal slices containing the striatum and the bed nucleus of the stria terminalis, respectively, was studied by means of in vitro microdialysis. A Ca⁺²-dependent and reserpine-sensitive K⁺-induced release of catecholamines was detected in both nuclei. We confirmed that N-methyl D-aspartate (2.5 and 5.0 mM in the dialysis perfusion solution) induces the release of dopamine from the striatum, and this effect was blocked by prior dialysis perfusion with 500 μM MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist. Infusion of N-methyl-D-aspartate (1–10 mM) or glutamate through the dialysis probe did not produce any detectable modification in the extracellular levels of noradrenaline in the bed nucleus of the stria terminalis. In addition, perfusion with D-serine (100 μM)alone or in the presence of desipramine (10 μM) resulted in a slight increase in extracellular noradrenaline in the bed nucleus of the stria terminalis. However, N-methyl-D-aspartate in the presence of D-serine and desipramine produced a marked increase in extracellular noradrenaline from the bed nucleus of the stria terminalis. These results indicate that N-methyl-D-aspartate receptors might regulate the release of noradrenaline from the bed nucleus of the stria terminalis as is the case of dopamine release in the striatum. The in vitro microdialysis seems to be a suitable complement to the in vivo microdialysis for the study of catecholamine release in discrete regions of the central nervous system and its local regulation by excitatory amino acid receptors. © 1995 Wiley-Liss, Inc.     

Dopamine inhibits the release of immunoreactive β-endorphin from rat hypothalamus in vitro

Mediobasal hypothalamus tissue (MBH) from adult male rats was incubated in Krebs-Ringer bicarbonate medium (KRB). KRB was changed at 15 min intervals and the concentration of immunoreactive β-endorphin (β-ENDi) in the medium was measured by radioimmunoassay. Incubation of MBH tissue in normal KRB resulted in a constant release rate of β-ENDi of approximately 1% of the tissue content per h. KRB containing 45 mM K⁺ causes a two fold increase in the release rate of β-ENDi which was Ca²⁺ dependent. Dopamine (0.01–1.0 μM) inhibits both the spontaneous and the K⁺-stimulated release of β-ENDi in a dose related manner. The dopamine receptor blocking agent haloperidol prevents this inhibitory effect of dopamine. The selective D-1 receptor agonist SKF 38393 does not affect the release rate of β-ENDi; whereas the selective D-2 receptor agonist LY 141865 inhibits both the spontaneous and K⁺-stimulated release of β-ENDi. The effects of LY 141865 can be blocked by (−)-sulpiride, a selective D-2 receptor antagonist. Norepinephrine only weakly inhibits the K⁺-stimulated release of β-ENDi, and effect that can be blocked by haloperidol but not by the α-adrenoceptor blocker phentolamine. At concentrations tested (0.01–1.0 μM), isoproterenol, 5-hydroxytryptamine, carbachol and 8-Br-cAMP (1.0 μM) do not affect β-ENDi release. It is concluded that dopamine can inhibit the release of β-ENDi from hypothalamic neurons via a D-2 receptor mechanism.     

Coordinated Postnatal Maturation of Striatal Cholinergic Interneurons and Dopamine Release Dynamics in Mice

Dynamic changes in motor abilities and motivated behaviors occur during the juvenile and adolescent periods. The striatum is a subcortical nucleus critical to action selection, motor learning, and reward processing. Its tonically active cholinergic interneuron (ChI) is an integral regulator of the synaptic activity of other striatal neurons, as well as afferent axonal projections of midbrain dopamine (DA) neurons; however, little is known about its development. Here, we report that ChI spontaneous activity increases during postnatal development of male and female mice, concomitant with a decreased afterhyperpolarization (AHP). We characterized the postnatal development of four currents that contribute to the spontaneous firing rate of ChIs, including I_SK, I_A, I_h, and I_NaP. We demonstrated that the developmental increase in I_NaP drives increased ChI firing rates during the postnatal period and can be reversed by the I_NaP inhibitor, ranolazine. We next addressed whether immature cholinergic signaling may lead to functional differences in DA release during the juvenile period. In the adult striatum, nicotinic acetylcholine receptors (nAChRs) prevent linear summation of DA release in response to trains of high-frequency stimuli. We show that, in contrast, during the second postnatal week, DA release linearly sums with trains of high-frequency stimuli. Consistently, nAChR antagonists exert little effect on dopamine release at postnatal day (P)10, but enhance the summation of evoked DA release in mice older than postnatal day P28. Together, these results reveal that postnatal maturation of ChI activity is due primarily to enhanced I_NaP and identify an interaction between developing cholinergic signaling and DA neurotransmission in the juvenile striatum.SIGNIFICANCE STATEMENT Motor skills and motivated behavior develop rapidly in juvenile rodents. Recent work has highlighted processes that contribute to the postnatal maturation of striatal principal neurons during development. The functional development of the striatal cholinergic interneuron (ChI), however, has been unexplored. In this study, we tracked the ontogeny of ChI activity and cellular morphology, as well as the developmental trajectory of specific conductances that contribute to the activity of these cells. We further report a link between cholinergic signaling and dopamine (DA) release, revealing a change in the frequency-dependence of DA release during the early postnatal period that is mediated by cholinergic signaling. This study provides evidence that striatal microcircuits are dynamic during the postnatal period and that they undergo coordinated maturation.     

Effects of metabolic alterations on dopamine release in an in vitro model of neostriatal ischaemia

Release of neurotransmitters, including dopamine (DA), plays a central role in neuronal death during cerebral ischaemia. We investigated the effects of changes in energy demand and supply on DA release in cerebral ischaemia in vitro. Rat striatal slices were superfused (400 ml/h) with an artificial cerebrospinal fluid at 34°C, unless otherwise stated. Ischaemia were mimicked by removal of O₂ and reduction in glucose concentration from 4 to 2 mM. DA release was monitored by voltammetry. The profile of ischaemia-induced DA release was temperature-dependent. Hypothermia (to 24°C) delayed, slowed, and reduced ischaemia-induced DA release relative to 34°C. Pretreatment of the slices for 3 h with creatine (25 mM) delayed and slowed ischaemia-induced DA release. Conversely, blockade of Na⁺/K⁺ ATPase with ouabain induced an anoxic depolarisation and rapid DA release similar to ischaemia. In summary, the onset of DA release in this model is controlled by the balance between energy supply and utilisation. Strategies that increase availability of energy substrates for the membrane sodium pump (i.e., pre-incubation with creatine) or decrease their utilisation (hypothermia) slow and delay DA release. Hypothermia may owe part of its neuroprotective effect to a delay and slowing of ischaemia-induced release of DA and/or other neurotransmitters.     

Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons.

To study development of the nigrostriatal pathway in an in vitro model system, organotypic slices obtained from rat pups (P4) and containing the striatum and the cortex were grown together with apposed embryonic (E13.5) mesencephalic blocks according to the static slice culture method of Stoppini et al. (1991; J. Neurosci. Methods 37:173-182). Under these conditions, mesencephalic dopaminergic (DA) fibers rapidly grow through the slice, preferentially its striatal portion. This innervation provides a true synaptic innervation to the striatum, as shown by the presence of DA terminals on striatal neurons. DA fibers are able to exert a functional influence, as seen by their ability to modulate c-Fos expression in striatal neurons in the same way as in vivo. Thus, blockade, under basal conditions, of the effect of spontaneously released dopamine by the D2 receptor antagonist haloperidol leads to the activation of c-Fos expression in the striatum. Furthermore, stimulation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striatal innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seems to be specific for DA neurons; no selectivity could be observed when noradrenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reveal any oriented outgrowth of DA fibers from the mesencephalon, suggesting that the selective innervation observed in the organotypic slices results from some contact-dependent, presumably adhesive interactions rather than from the presence of some diffusible substance orienting the growth of DA fibers towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the selective striatal innervation are not the same as those determining the adhesion of the DA neurons. These results show that cocultures of cortex-striatum and mesencephalic slices result in a system that displays a number of the morphological and functional traits of the normal nigrostriatal system and that can be relied on as a good in vitro model of in vivo development.