The Cholinergic Stimulating Effects of Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Are Mediated by Protein Kinase C

Abstract: The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC-depleted sympathetic neurons. The inhibition of CNTF-stimulated ChAT by phorbol ester occurred in a dose-dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate. These observations suggest that a common PKC-dependent pathway, which is independent of phosphatidylinositol 4,5-bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell-cell contact in cultured sympathetic neurons.     

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple cholinergic differentiation factors are present in footpad extracts: comparison with known cholinergic factors.

Sweat glands in rat footpads contain a neuronal differentiation activity that switches the phenotype of sympathetic neurons from noradrenergic to cholinergic during normal development in vivo. Extracts of developing and adult sweat glands induce changes in neurotransmitter properties in cultured sympathetic neurons that mimic those observed in vivo. We have characterized further the factors present in the extract and compared their properties to those of known cholinergic factors. When assayed on cultured rat sympathetic neurons, the major activities in footpad extracts from postnatal day 21 rat pups that induce choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP) and reduce catecholamines and neuropeptide Y (NPY) are associated with a soluble protein of 22-26 x 10(3) M(r) and a pI of 5.0. These properties are similar to those of ciliary neurotrophic factor (CNTF). Moreover, the purified fraction from footpads has ciliary neurotrophic activity. Antibodies to CNTF that immunoprecipitate all differentiation activity from sciatic nerve extracts, a rich source of CNTF, immunoprecipitate 80% of the cholinergic activity in the footpad extracts, 50% of the VIP and 20% of the NPY activities. Neither CNTF protein nor CNTF mRNA, however, can be detected in immunoblot and northern analysis of footpads even though both CNTF protein and mRNA are evident in sciatic nerve. CNTF-immunoreactivity is associated with a sparse plexus of sensory fibers in the footpad but not with sweat glands or the Schwann cells associated with them. In addition, in situ hybridization studies with oligonucleotide probes failed to reveal CNTF mRNA in sweat glands. Comparison of the sweat gland differentiation activity with the cholinergic differentiation factor from heart cells (CDF; also known as leukemia inhibitory factor or LIF) suggests that most of the cholinergic activity in foot pads is biochemically distinct from CDF/LIF. Further, antibodies that block the activity of CDF/LIF purified from heart-cell-conditioned medium do not block the ChAT-inducing activity present in footpad extracts of postnatal day 8 animals. A differentiation factor isolated from skeletal muscle did not induce cholinergic properties in sympathetic neuron cultures and therefore is unlikely to be the cholinergic differentiation factor produced by sweat glands. Taken together, our data suggest that there are at least two differentiation molecules present in the extracts and that the major cholinergic activity obtained from footpads is related to, but distinct from, CNTF. The second factor remains to be characterized. In addition, CNTF associated with sensory fibers may make a minor contribution to the cholinergic inducing activity present in the extract.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Cholinergic neuronal differentiation factors: evidence for the presence of both CNTF-like and non-CNTF-like factors in developing rat footpad.

Catecholaminergic sympathetic neurons are able to change their transmitter phenotype during development and to acquire cholinergic properties. Cholinergic sympathetic differentiation is only observed in fibers innervating specific targets like the sweat glands in the rat footpad. A function for ciliary neurotrophic factor (CNTF) in this process has been implied as it is able to induce cholinergic properties (ChAT, VIP) in cultured chick and rat neurons. We show here that a CNTF-like, VIP-inducing activity is present in rat footpads and that its increases 6-fold during the period of cholinergic sympathetic differentiation. Immunohistochemical analysis of P21 rat footpads demonstrated CNTF-like immunoreactivity in Schwann cells but not in sweat glands, the target tissue of cholinergic sympathetic neurons. The expression of this factor in footpads seems to be dependent on the presence of intact nerve axons, as nerve transection results in a loss of CNTF-like cholinergic activity and immunoreactivity. Immunoprecipitation experiments with rat footpad extracts provided evidence for the presence of ChAT-inducing factors other than CNTF, which may independently or together with CNTF be involved in the determination of sympathetic neuron phenotype.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Pineal cells enhance choline acetyltransferase activity in sympathetic neurons

Cells derived from the neonatal rat pineal gland were cocultured with cells derived from neonatal rat superior cervical ganglia (SCG) in an attempt to determine whether a sympathetic target organ with only adrenergic properties could enhance the development of adrenergic transmitter properties in sympathetic neurons in tissue culture. Choline acetyltransferase was measured as an index of cholinergic differentiation, and tyrosine hydroxylase was measured as an index of adrenergic differentiation. As indices of total cell number and cellular volume, DNA and protein, respectively, were also measured. We found that the pineal-SCG cocultures contained ten times greater choline acetyltransferase activity than sister neuronal cultures cultured without pineal cells, thus indicating that the pineal cells enhanced cholinergic properties in the sympathetic neurons. This cholinergic enhancement was dependent upon the presence of nerve growth factor and could not be obtained with pineal-conditioned medium. Tyrosine hydroxylase activity, measured on cultures sister to those mentioned above, was low in all cultures and decreased somewhat in SCGs cultured alone. TH activity in the pineal-SCG cocultures, however, increased slightly. Some tyrosine hydroxylating activity developed in pineals cultured alone, however, and may have been responsible for the small increase in tyrosine hydroxylase activity noted in the pineal-SCG cocultures. The implications of these results for a determination of the role that target organ plays in the development of the transmitter properties of sympathetic neurons are discussed.     

Ciliary neurotrophic factor stimulates tyrosine hydroxylase activity

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.     

The cholinergic neuronal differentiation factor from heart cell conditioned medium is different from the cholinergic factors in sciatic nerve and spinal cord

Environmental cues play an important role in determining the transmitter phenotype of developing sympathetic neurons. Several factors have been described which can induce cholinergic function in cultured sympathetic neurons. We have compared certain biological and immunological properties of three of them, cholinergic differentiation factor (CDF), membrane-associated neurotransmitter-stimulating factor (MANS), and ciliary neurotrophic factor (CNTF), to determine whether they are different. As previously reported, all three increased acetylcholine synthesis in cultured sympathetic neurons. In addition, MANS as well as CNTF and CDF decreased catecholamine synthesis. CNTF and MANS, but not CDF, promoted the survival of embryonic chick ciliary neurons. Affinity-purified antibodies raised against a synthetic peptide corresponding to the N-terminal sequence of CDF immunoprecipitated CDF, but not MANS or CNTF. These results indicate that although CDF, MANS, and CNTF have similar effects on transmitter synthesis by cultured sympathetic neurons, CDF lacks the ciliary neurotrophic activity of MANS and CNTF. Further, CDF possesses an N-terminal epitope which is absent from both MANS and CNTF. Thus, CDF is distinct from MANS and CNTF, and at least two factors exist which can alter the transmitter phenotype of sympathetic neurons in vitro.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

Intracellular Storage and Evoked Release of Acetylcholine from Postnatal Rat Basal Forebrain Cholinergic Neurons in Culture with Nerve Growth Factor

Cholinergic neurons from the septum area, the vertical limb of the diagonal band of Broca, and the nucleus basalis of Meynert of postnatal 13-day-old rats were cultured with or without nerve growth factor (NGF) conditions. Total choline acetyltransferase (ChAT) activities, acetylcholine (ACh) contents, and survival numbers of cholinergic neurons in culture from each of three distinct regions were increased by NGF treatment, but little difference was found in cellular ChAT activities and ACh contents obtained in cultures with or without NGF. The result shows that NGF promotes the survival of cholinergic neurons from 13-day-old rats. Furthermore, the release of ACh from cultured neurons was investigated. The cells cultured with NGF showed a larger increase of the high K+-evoked ACh release than those cultured without NGF. However, NGF had no effect on spontaneous release. This suggests that NGF could regenerate and sustain the stimulation-evoked release mechanisms of ACh in cultured cholinergic neurons from postnatal rats.     

K-252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 n M K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.     

Induction of Cholinergic Expression in Developing Spinal Cord Cultures

The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (1 mM) produced a 2-3-fold stimulation of ChAT activity in developing cultures whereas 8-bromo cGMP had no effect. A phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, also increased (2-fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2-16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP-mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1-day exposure to dbcAMP. 125I-Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7-9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-Related Changes in Tyrosine Hydroxylase and Choline Acetyltransferase in Sympathetic Ganglia of a Rat Strain with Reduced Sympathetic Neuron Numbers

Abstract: In Wistar rats, a subpopulation of sympathetic ganglionic neurons dies during ageing, but in the GH strain, these same neurons die during the period of perinatal maturation. We have compared tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in superior cervical ganglia of GH and control rats at different ages. Ganglionic TH rose to near adult levels between postnatal weeks 1 and 2. No significant differences in TH values were seen between GH and control ganglia at any age, indicating that reduced neuron numbers are compensated for by increased cellular activity, Ganglionic ChAT rose initially in parallel with TH and then more slowly over postnatal weeks 3–4, reaching adult levels that were about 20° lower in GH than in normal ganglia. During ageing, TH remained constant but ChAT continued to rise slowly in GH ganglia, whereas ChAT in normal ganglia fell by about 10°. Both the strain difference in ChAT during development and the fall in ChAT during ageing in normal animals parallel the differences in ganglion cell numbers seen under these circumstances.     

Cholinergic differentiation of rat sympathetic neurons in culture: effects of factors applied to distal neurites.

Cholinergic properties are induced in sympathetic neurons by several factors applied to entire neurons in culture. Evidence from work with the rat sweat gland model indicates that factors located in target tissues can induce cholinergic differentiation in vivo. We now report that when leukemia inhibitory factor (LIF), heart cell-conditioned medium (HCCM), or dermal fibroblast-conditioned medium (DFCM) is applied to only distal neurites in compartmented cultures of rat sympathetic neurons, the neurons exhibit an increase in specific choline acetyltransferase activity and a concomitant decrease in levels of tyrosine hydroxylase. LIF, HCCM, and DFCM also induce neurite fasciculation, thus suggesting an additional role of cholinergic switching factors in regulating axon-axon and/or axon-substrate adhesion. These results demonstrate that rat sympathetic neurons have the cellular machinery to respond to cholinergic differentiation cues located in peripheral targets, analogous to the response to nerve growth factor.     

Toxicity of 1-Methyl-4-Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.