MN9D Cells Research Articles

Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase and MEK.

Parkinson's disease is a neurodegenerative disorder associated with the selective death of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) can protect dopaminergic neurons in several parkinsonian models. We used the dopaminergic cell line MN9D to explore the mechanisms underlying GDNF-mediated protection against the neurotoxin 6-hydroxydopamine (6-OHDA). MN9D cell viability was decreased 24 hr after a 15-min exposure to 6-OHDA (50-1000 microM) as revealed by staining with Hoechst reagent and Trypan blue. The addition of GDNF (10 ng/ml) before, during, and after exposure to 6-OHDA significantly increased the number of viable cells as assessed by Hoechst staining. In contrast, 6-OHDA-induced cell membrane damage was unaffected as measured by Trypan blue exclusion. The PI3K specific inhibitor LY294002 (10-50 microM) blocked GDNF-mediated protection against nuclear condensation, as did the MAPK kinase (MEK) inhibitor U0126 (5- 20 microM). These studies suggest that GDNF can protect dopaminergic cells against some but not all aspects of 6-OHDA-induced toxicity by acting through both PI3K and MAPK signaling pathways.

Functional selectivity of dopamine receptor agonists. II. Actions of dihydrexidine in D2L receptor-transfected MN9D cells and pituitary lactotrophs.

D(2)-like dopamine receptors mediate functional changes via activation of inhibitory G proteins, including those that affect adenylate cyclase activity, and potassium and calcium channels. Although it is assumed that the binding of a drug to a single isoform of a D(2)-like receptor will cause similar changes in all receptor-mediated functions, it has been demonstrated in brain that the dopamine agonists dihydrexidine (DHX) and N-n-propyl-DHX are "functionally selective". The current study explores the underlying mechanism using transfected MN9D cells and D(2)-producing anterior pituitary lactotrophs. Both dopamine and DHX inhibited adenylate cyclase activity in a concentration-dependent manner in both systems, effects blocked by D(2), but not D(1), antagonists. In the MN9D cells, quinpirole and R-(-)-N-propylnorapomorphine (NPA) also inhibited the K(+)-stimulated release of [(3)H]dopamine in a concentration-responsive, antagonist-reversible manner. Conversely, neither DHX, nor its analogs, inhibited K(+)-stimulated [(3)H]dopamine release, although they antagonized the effects of quinpirole. S-(+)-NPA actually had the reverse functional selectivity profile from DHX (i.e., it was a full agonist at D(2L) receptors coupled to inhibition of dopamine release, but a weak partial agonist at D(2L) receptor-mediated inhibition of adenylate cyclase). In lactotrophs, DHX had little intrinsic activity at D(2) receptors coupled to G protein-coupled inwardly rectifying potassium channels, and actually antagonized the effects of dopamine at these D(2) receptors. Together, these findings provide compelling evidence for agonist-induced functional selectivity with the D(2L) receptor. Although the underlying molecular mechanism is controversial (e.g., "conformational induction" versus "drug-active state selection"), such data are irreconcilable with the widely held view that drugs have "intrinsic efficacy".

A role for alpha-synuclein in the regulation of dopamine biosynthesis.

The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

Dopaminergic neurons are protected from ionizing irradiation and 6-hydroxydopamine-induced apoptosis by overexpression of the Manganese Superoxide Dismutase (MnSOD) transgene

Dopaminergic neurons are protected from ionizing irradiation and 6-hydroxydopamine-induced apoptosis by overexpression of the Manganese Superoxide Dismutase (MnSOD) transgene

Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells.

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.

Overexpression of Calbindin-D28K Induces Neurite Outgrowth in Dopaminergic Neuronal Cells via Activation of p38 MAPK

An MN9D dopaminergic neuronal cell line overexpressing calbindin-D28K (MN9D/Calbindin) was established in order to investigate directly the potential role of calcium-binding protein in neuronal differentiation. Overexpression of calbindin-D28K in MN9D cells resulted in significant increases in the number of neurites, the length of primary neurites, and the total extent of neurites. This robust neurite outgrowth occurred without cessation of cell division. Analysis of immunoblots revealed that this morphological differentiation was accompanied by increased expression of such markers of maturation as the synaptosomal protein SNAP-25. During calbindin-D28K-evoked neurite outgrowth in MN9D cells, phosphorylation of p38 mitogen-activated protein kinase (MAPK) dramatically increased while the levels and extent of phosphorylation of such other MAPKs as c-Jun N-terminal kinase (JNK) or extracellular response kinase (ERK) were not altered. Consequently, calbindin-D28K-induced neurite outgrowth was largely abolished by treatment with a p38 inhibitor, PD 169316, while the level of SNAP-25 in MN9D/Calbindin cells was not altered by this treatment. These data support an idea that calbindin-D28K and its associated p38 signaling pathway play a role in dopaminergic neuronal differentiation.

MPP+ Downregulates Mitochondrially Encoded Gene Transcripts and Their Activities in Dopaminergic Neuronal Cells: Protective Role of Bcl-2

The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 μM N-methyl-4-phenylpyridinium (MPP+) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP+ but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP+-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP+, overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP+-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP+-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP+-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP+-induced dopaminergic neuronal death.

Characterization of MPP+-Induced Cell Death in a Dopaminergic Neuronal Cell Line: Role of Macromolecule Synthesis, Cytosolic Calcium, Caspase, and Bcl-2-Related Proteins

To further characterize MPP+-induced cell death and to explore the role of Bcl-2-related proteins in this death paradigm, we utilized a mesencephalon-derived dopaminergic neuronal cell line (MN9D) stably transfected with human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Δ22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP+ underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP+-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50–400 μM) or Boc-aspartyl(OMe)-fluoromethylketone (50–200 μM) did not attenuate MPP+-induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP+-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP+ treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP+ treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP+-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP+ induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP+-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.

Sequential cleavage of poly(ADP-ribose)polymerase and appearance of a small Bax-immunoreactive protein are blocked by Bcl-X(L) and caspase inhibitors during staurosporine-induced dopaminergic neuronal apoptosis.

To assess the role of Bcl-X(L) and its splice derivative, Bcl-X(S), in staurosporine-induced cell death, we used a dopaminergic cell line, MN9D, transfected with bcl-xL (MN9D/Bcl-X(L)), bcl-xS (MN9D/Bcl-X(S)), or control vector (MN9D/Neo). Only 8.6% of MN9D/Neo cells survived after 24 h of 1 microM staurosporine treatment. Caspase activity was implicated because a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), attenuated staurosporine-induced cell death. Bcl-X(L) rescued MN9D cells from death (89.4% viable cells), whereas Bcl-X(S) had little or no effect. Bcl-X(L) prevented morphologically apoptotic changes as well as cleavage of poly(ADP-ribose)polymerase (PARP) induced by staurosporine. It is interesting that a small Bax-immunoreactive protein appeared 4-8 h after PARP cleavage in MN9D/Neo cells. The appearance of the small Bax-immunoreactive protein, however, may be cell type-specific as it was not observed in PC12 cells after staurosporine treatment. The sequential cleavage of PARP and the appearance of the small Bax-immunoreactive protein in MN9D cells were blocked either by Z-VAD-fmk or by Bcl-X(L). Thus, our present study suggests that Bcl-X(L) but not Bcl-X(S) prevents staurosporine-induced apoptosis by inhibiting the caspase activation that may be directly or indirectly responsible for the appearance of the small Bax-immunoreactive protein in some types of neurons.

Reduced levels of catalase activity potentiate MPP +-induced toxicity: comparison between MN9D cells and CHO cells

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to be toxic by inducing oxygen free radicals in the mammalian nervous system, especially in the nigrostriatal dopaminergic system. The present study was designed to compare the toxic effects of MPP +, the active metabolite of MPTP, in MN9D neuronal cells that exhibit relatively low levels of catalase activity, as compared to CHO cells, which exhibit high levels of catalase activity. The survival of the MN9D cells in the presence of 250 μM MPP + was less than 10%, whereas CHO cells exhibited 70% survival at the same concentration of MPP +. The ED 50 values of MPP + in MN9D and CHO cell lines were 60–600 μM, respectively. MN9D cells contain less catalase, an enzyme believed to be involved in the detoxification of free radicals compared to CHO cells. The catalase activity was 2 Units/mg protein in MN9D cells and 30 U/mg protein in CHO cells. The catalase activity in CHO cells increased with increasing MPP + concentrations from 100–500 μM, however, it decreased at 1 mM MPP +. In contrast, catalase activity in MN9D remained the same at all MPP + concentrations. When the CHO cells were pre-treated with 10–25 mM 3-aminotriazole (3-AT), which inhibits catalase activity, and exposed to MPP + at various concentrations, they became susceptible to MPP +. It is evident from these data that the differential susceptibility to MPP + in these two cell lines are due to differences in catalase activity. In addition, the inhibition of constituentive catalase activity in CHO cells by 3-AT treatment enhances their susceptibility. In conclusion, the study demonstrates that catalase activity represents an important defence mechanism in MPTP-induced toxicity.

Overexpression of HA-Bax but not Bcl-2 or Bcl-XL attenuates 6-hydroxydopamine-induced neuronal apoptosis.

Bax, a member of the Bcl-2 gene family, is known to promote apoptosis in many cases but to block cell death under certain conditions. To investigate the potential role of Bax in 6-hydroxydopamine (6-OHDA)-induced cell death, we first established and characterized a dopaminergic neuronal cell line (MN9D) stably overexpressing hemagglutinin epitope-tagged Bax (MN9D/HA-Bax) as well as control clones (MN9D/Neo). Treatment of MN9D/Neo cells with 6-OHDA induced typical apoptotic cell death accompanied by shrinkage of the cell, nuclear condensation, and DNA fragmentation as demonstrated by light microscopy and agarose gel analysis. Overexpression of HA-Bax in MN9D cells was shown to attenuate 6-OHDA-induced cell death as determined by the MTT reduction assay and agarose gel analysis for DNA fragmentation. Western blot analysis revealed that cleavage of poly(ADP-ribose)polymerase induced by 6-OHDA was attenuated in MN9D/HA-Bax cells. In contrast, overexpression of a well-known cell death-inhibiting protein such as Bcl-2 or Bcl-XL did not attenuate 6-OHDA-induced cell death. Interestingly, cell death induced by hydrogen peroxide (0.25-2.0 mM) was significantly accelerated, whereas the rate of cell death induced by menadione (10-50 microM) was not affected in MN9D/HA-Bax cells. Thus, our present data suggest that the functionally diverse roles of Bax may be determined by the type of stress applied to the cell.

Mechanisms of peroxynitrite-induced [Ca2+]i increase in single neuronal cell

The mechanism of peroxynitrite (ONOO−)-induced [ca2+]i increase in single MN9D cell (Dopaminergic neuroblastoma cell line) was studied by using Fura-2 microfluorometric technique. The results show that ONOO− caused a rapid increase of [Ca2+]i when ONOO− was puffed to the cell. Removing Ca2+ from the bath or using calcium channel antagonist (CdCl2, Nifedipine) greatly inhibited the [Ca2+]i increase induced by ONOO−1, suggesting that the opening of L-Ca2+ channel makes a great contribution to the [Ca2+]i increase. The effect of sulfhydryl reductive agent (DTT) on ONOO−-induced [Ca2+]i increase suggests that ONOO−-activating L-Ca2+ channel is partly related to its oxidative speciality.

Regions outside of the Bcl-2 homology domains, BH1 and BH2 protect a dopaminergic neuronal cell line from staurosporine-induced cell death

Recent evidence demonstrates that the proto-oncogene product, Bcl-2 can protect cells from a variety of cell death-inducing stimuli. Because previous studies have demonstrated that protein kinase (PK) pathways may be involved in the regulation of cell death, we tested various PK inhibitors for their effects on cell death in a dopaminergic neuronal cell line, MN9D, as well as the potential of Bcl-2 family members and structural mutants to block this process. Cells expressing either human Bcl-2 (MN9D/Bcl-2), or neomycin (MN9D/Neo; control cells) were treated with either staurosporine (0.25–2 μM) or trifluoperazine (10–100 μM). In control MN9D/Neo cells, both reagents led to a dose-dependent cell death with morphological features of apoptosis. Overexpression of Bcl-2 rescued cells from staurosporine-induced but not trifluoperazine-induced apoptotic cell death. Cell death induced by the specific PKC inhibitor, calphostin C was also significantly attenuated in MN9D/Bcl-2 cells indicating that a PKC pathway represents one mechanism by which Bcl-2 prevents staurosporine-induced cell death. Similarly, the Bcl-2 family member, Bcl-X L also blocked staurosporine-induced cell death in MN9D cells whereas overexpression of Bcl-X S or Bax did not. Finally, staurosporine-induced cell death was still blocked by the expression of clones encoding mutations in the Bcl-2 homology domains, BH1 and BH2, as well as C-terminally truncated Bcl-2. These data suggest that in the staurosporine-mediated cell death model Bcl-2 is not heterodimerizing to related proteins through these highly conserved structural domains nor does it need to be membrane-anchored. Thus, in this paradigm, either Bcl-2 functions as a homodimer or essential sequences lie outside of the BH1 or BH2 domains.

The Rat D4 Dopamine Receptor Couples to Cone Transducin (Gαt2) to Inhibit Forskolin-stimulated cAMP Accumulation

Based on its expression pattern and pharmacology, the D4 dopamine receptor may play a role in schizophrenia. Thus it is of interest to know what signaling pathways are utilized by this receptor. Previously, we showed that activation of D4 receptors in a mouse mesencephalic neuronal cell line (MN9D) inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive (Ptx-sensitive) fashion. Of the known Ptx-sensitive G-protein alpha subunits, MN9D-expressed Galphai2, GalphaoA, and GalphaoB; however, none of these coupled to the D4 receptor. Using a low stringency polymerase chain reaction cloning method, we found an additional Ptx-sensitive G-protein cone transducin (Galphat2) expressed in the MN9D cells. We also found that Galphat2 mRNA is highly expressed in rat mesencephalic tissue. To test the hypothesis that the D4 receptor couples to Galphat2, we cotransfected MN9D cells with the D4 receptor and a mutagenized Ptx-resistant Galphat2 subunit (mGalphat2). Application of the dopaminergic agonist quinpirole to cotransfected cells inhibited forskolin-stimulated cAMP accumulation in the presence or absence of Ptx. To our knowledge, this is the first report demonstrating that the D4 dopamine receptor functionally couples to a specific G-protein and that a non-opsin-like receptor can couple with a transducin subunit.

Bax accelerates staurosporine-induced but suppresses nigericin-induced neuronal cell death.

Bax, a member of the Bcl-2 multigene family, is known to promote apoptosis. To investigate the role of Bax in an experimentally induced cell death of the murine dopaminergic neuronal cell line (MN9D), we established MN9D cells stably over-expressing murine Bax (MN9D/ Bax) or vector alone (MN9D/Neo). In MN9D/Neo cells treated with either 1 microM staurosporine or 0.1 microM nigericin, a ladder pattern of DNA fragmentation was induced. As expected, over-expression of Bax in MN9D cells accelerated staurosporine-induced cell death as measured by the MTT reduction assay (62.3% survival in MN9D/Neo vs 27.0% survival in MN9D/Bax). Surprizingly, both nigericin-induced cell death and its accompanying DNA fragmentation were largely attenuated in MN9D/Bax cells (22.0% survival in MN9D/Neo vs 86.7% survival in MN9D/Bax). Similar patterns were observed in two other MN9D/Bax cell lines. Cleavage of poly(ADP-ribose)polymerase caused by nigericin was greatly attenuated in MN9D/Bax cells suggesting that, like Bcl-2, Bax suppresses nigericin-induced cell death by inhibiting the activation of cysteine proteases. Thus, our data imply that Bax acts as a negative or positive regulator of cell death depending on the type of death stimulus applied to the cell.

Glial-derived neurotrophic factor (GDNF) induced morphological differentiation of an immortalized monoclonal hybrid dopaminergic cell line of mesencephalic neuronal origin

Monoclonal hybrid cell lines derived from either mesencephalic or striatal neuronal parents were screened for the effects of glial-derived neurotrophic factor (GDNF) on morphological differentiation. The mesencephalic dopaminergic MN9D cell line responded to GDNF with a 3- to 4-fold increase in the number of differentiated solitary cells and provides an unlimited source of monoclonal cells for studies on the mechanism of action of GDNF and the nature of its receptor.

PTX-sensitive regulation of neurite outgrowth by the dopamine D3 receptor.

Neurotransmitter receptors are known to have direct roles in the modulation of neuronal morphogenesis. Previous work showed that clonal mesencephalic MN9D cells individually transfected with D2-like dopamine receptors show increased neurite outgrowth following long-term exposure to the D2-like receptor agonist quinpirole. In the current study, brief stimulation of D3 receptor-expressing cells also elicited increased neurite outgrowth, which could be mimicked by the Gi/G(o) protein activator mastoparan. Pretreatment with the Gi/G(o) protein inhibitor pertussis-toxin blocked the quinpirole- and mastoparan-mediated increases in outgrowth. These results suggest that dopamine D3 receptor stimulation has an immediate, G-protein-mediated role in neuronal morphogenesis.

Overexpression of Bcl-2 attenuates MPP+, but not 6-ODHA, induced cell death in a dopaminergic neuronal cell line

In order to investigate the role of Bcl-2 in dopaminergic cells, we established a dopaminergic neuronal cell line (MN9D) stably expressing human Bcl-2 (MN9D/Bcl-2) or neomycin (MN9D/Neo). Overexpression of Bcl-2 in MN9D cells attenuated cell death due to treatment of mitochondrial electron transport inhibitors includingN-methyl-4-phenylpyridinium, whereas it did not prevent cell death induced by reagents generating reactive oxygen species including 6-hydroxy-dopamine. Moreover, the rate of glucose uptake in MN9D/Bcl-2 was significantly lower than that in MN9D/Neo after MPP+treatment. Thus, Bcl-2 may counter aberrations in mitochondrial electron transfer processes by altering energy metabolism within the MN9D cells.

Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions.

MN9D is an immortalized dopamine-containing neuronal hybrid cell line. When MN9D cells were coaggregated with primary embryonic cells of optic tectum, a brain region that does not receive a dopaminergic innervation, there was a marked reduction in their dopamine content, tyrosine hydroxylase immunoreactivity, and tyrosine hydroxylase mRNA. Similar reductions in dopamine content were produced by coaggregation with cells from embryonic thalamus, another brain region devoid of dopaminergic innervation. Coaggregation of MN9D cells with dopaminoceptive cells from the corpus striatum or the cortex did not have a demonstrable stimulatory effect on the dopamine content of MN9D cells. The decrease in MN9D dopamine content produced by optic tectum cells was not reversed by addition of corpus striatum cells. Thus, the MN9D hybrid cells are able to respond to an inhibitory factor(s) from cells derived from brain areas that are not targets for dopaminergic neurons. Catecholamine-producing PC12 cells did not respond in a similar manner, suggesting that the response of MN9D cells is a function of their mesencephalic origin. Given the selective response of MN9D cells to different brain cell populations, this hybrid cell line should facilitate investigations of cell-cell interactions in the central nervous system that may be involved in the expression of neurotransmitter phenotype and establishment of specific neuronal connections.