An increase in intracellular levels of cyclic AMP produces trophic effects on striatal neurons developing in culture

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Neuroscience Vol. 82, No. 4, pp. 1009–1020, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00349-7

AN INCREASE IN INTRACELLULAR LEVELS OF CYCLIC AMP PRODUCES TROPHIC EFFECTS ON STRIATAL NEURONS DEVELOPING IN CULTURE N. NAKAO Department of Neurological Surgery, Wakayama Medical College, 7-27, Wakayama 640, Japan Abstract––Cyclic AMP-dependent kinases have been suggested to constitute signal transduction pathways involved in the regulation of neuronal development and survival. The present study examined whether elevated levels of cyclic AMP exhibit trophic activities on rat striatal neurons grown under serum-free culture conditions. Treatment with dibutyryl cyclic AMP, a permeable cyclic AMP, increased GABA uptake and immunocytochemically detectable levels of proteins such as c-Fos and calbindin-D28k. Neuronal survival was promoted by dibutyryl cyclic AMP only in lower density cultures. Chronic exposure of neurons to dibutyryl cyclic AMP enhanced the morphological development of calbindinD28k-positive neurons. Furthermore, pretreatment with dibutyryl cyclic AMP afforded neuroprotection against N-methyl--aspartate-induced excitotoxicity. The dibutyryl cyclic AMP-induced trophic effects above were blocked by adenosine 3*,5*-cyclic monophosphothioate, a specific inhibitor of cyclic AMPdependent kinases. We also examined whether cyclic AMP is involved in trophic effects provided by membrane depolarization induced by high K+ and growth factors such as basic fibroblast growth factor and insulin-like growth factor-1. Depolarization, but not the growth factors, increased intracellular levels of cyclic AMP. Adenosine 3*,5*-cyclic monophosphothioate diminished depolarization increases in GABA uptake, whereas it did not affect the trophic effect of the growth factors. Co-treatment with the growth factors and dibutyryl cyclic AMP produced additive effects on both increases in GABA uptake and neuroprotection against excitotoxicity. The present results indicate that cyclic AMP-dependent kinases play roles in mediating differentiation and survival of developing striatal neurons. Signalling pathways activated by either basic fibroblast growth factor or insulin-like growth factor-1 are independent of those involving cyclic AMP. In contrast, depolarization-induced trophic effects are mediated, at least in part, by cyclic AMP-dependent pathways. Protective actions of dibutyryl cyclic AMP against excitotoxic injury as well as the additive effects with the growth factors are of potential interest in the experimental therapy of acute or chronic neurodegenerative diseases. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: protein kinase A, cell death, neuronal development, growth factor, neurotrophic factor, signal transduction.

There is growing evidence to suggest that extracellular signals regulate the development and survival of neurons in the central and peripheral nervous systems. Those signals include growth factors,23 cytokines,51a neurotransmitters31,32,38,57,58 and 18,24,36,53,54 neural activity. It is believed that effects of these signals on neurons are mediated by several intracellular signal transduction pathways. These pathways involve protein kinase C, cyclic AMP (cAMP) and calcium, all of which have been suggested to play an important role in the expression of actions of the environmental signals.19,44 Abbreviations: ANOVA, analysis of variance; bFGF, basic fibroblast growth factor; cAMP, cyclic AMP; dbcAMP, dibutyryl cyclic AMP; dbcGMP, dibutyryl cyclic GMP; GAD, glutamate decarboxylase; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazineN*-2-ethanesulphonic acid; IGF-1, insulin-like growth factor-1; MEM, minimum essential medium with Earle’s salts; NMDA, N-methyl--aspartate; PBS, phosphatebuffered saline; PKA, protein kinase A; Rp-cAMPS, adenosine 3*,5*-cyclic monophosphothioate; VSCC, L-type voltage-sensitive calcium channel.

Previous in vitro studies have demonstrated that agents that increase intracellular cAMP levels promote the survival and morphological development of various types of neurons, including spinal cord neurons,7 hippocampal pyramidal neurons,39 retinal ganglion cells,24 mesencephalic dopamine neurons,22 serotonergic neurons14 and central noradrenergic neurons.62 Dibutyryl cAMP (dbcAMP), the permeant cAMP analogue, prevents apoptotic cell death induced by deprivation of trophic support in vitro.28,56 These findings suggest that in certain types of neurons the cAMP-dependent pathway, i.e. the protein kinase A (PKA) system, is implicated in the expression of various trophic effects of the extracellular signals. Simpson and Morris60 found that in striatal cultures the stimulation of D1 dopamine receptors induces the expression of the immediate-early genes, including c-fos and zif/268, through the cAMPdependent kinase pathway. Recent studies have also revealed that dbcAMP and forskolin themselves induce the expression of c-fos in striatal neurons

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in vitro and in vivo.34,61 These findings suggest the involvement of cAMP-dependent kinases in the response of striatal neurons to environmental stimuli. The striatum is one of the major targets of nigral dopamine neurons, and whether the dopaminergic input might play an important role in the development of striatum has been a controversial issue. It has recently been shown that activation of dopamine D1 receptor that is positively linked to adenylate cyclase, the cAMP-synthesizing enzyme,26 alters several morphological aspects of striatal neurons in vitro.57 There is therefore considerable interest in the potential role of the cAMP-dependent pathway in the regulation of the differentiation and survival of striatal neurons. The general aim of the current study was to explore whether an increase in intracellular cAMP levels may yield neurotrophic effects on striatal neurons in vitro. Thus, experiments were designed to test whether agents that elevate intracellular cAMP levels may promote the survival and the morphological and biochemical differentiation of striatal neurons under serum-free culture conditions. The present study also investigated the possible involvement of cAMP in established neurotrophic actions provided by highK+-induced depolarization,27 or growth factors such as basic fibroblast growth factor (bFGF) and insulinlike growth factor-1 (IGF-1).49,64 Further experiments dealt with the issue of whether dbcAMP may protect striatal neurons against neuronal insult such as excitotoxic injury induced by a toxic dose of N-methyl--aspartate (NMDA).47,49

percentage of glial cells present in the cultures is around 0.5% at day 5 as revealed by glial fibrillary acidic protein (GFAP) immunocytochemistry. Measurement of GABA uptake GABA uptake was measured according to modifications of the procedure described previously.63 Briefly, cultures were preincubated for 20 min at 37)C with minimum essential medium with Earle’s salts (MEM; Gibco) containing 5 mM glucose and 0.1 mM amino-oxyacetic acid. [3H]GABA (50 Ci/mmol; New England Nuclear) was then added at a final concentration of 10 nM, and cells were incubated for 15 min at 37)C. Non-specific uptake was determined in sister cultures in the presence of 1 mM nipecotic acid. The reaction was terminated by removal of the incubation media, followed by the addition of 0.1 M NaOH. The cultures were lysed in PBS containing 1% Triton X-100 and 10% perchloric acid, and the [3H]GABA was measured by liquid scintillation counting. Specific uptake was calculated by subtracting non-specific from total uptake values. Measurement of intracellular cyclic AMP levels Striatal cultures were grown for 24 h in order to allow cells to attach well to culture plates, and thereafter they were incubated with test agents for 15 min. Control cultures were treated with PBS in an identical fashion. After the incubation, the culture media were removed and the cultures were then incubated with 0.5 M HClO4 for 1 h at 4)C. Cells were scraped and 5 M KHCO3 was added to each well. The cell suspension was centrifuged at 1000–1200 r.p.m. for 3 min. The supernatant was subjected to the determination of cAMP levels using a commercially available kit (New England Nuclear). N-Methyl--aspartate excitotoxic insult

EXPERIMENTAL PROCEDURES

Test agents dbcAMP (Sigma), dibutyryl cyclic GMP (dbcGMP; Sigma), adenosine 3*,5*-cyclic monophosphothioate (RpcAMPS; Calbiochem) and NMDA (Sigma) were dissolved in phosphate-buffered saline (PBS). Forskolin (Sigma), Ro-201724 (Calbiochem) and nifedipine (Calbiochem) were stored as solutions in dimethylsulphoxide, and these solutions were further diluted in PBS at the desired concentrations just before use. Human recombinant bFGF (Boehringer Mannheim) and IGF-1 (Boehringer Mannheim) were prepared in a solution of PBS containing 1% bovine serum albumin. Striatal cell cultures Primary striatal cell cultures were prepared from the lateral ganglionic eminences of 14-day-old rat embryos (Sprague–Dawley strain, Charles River) using established procedures,45 with minor modifications. Briefly, dissociated cells were plated on to four-well chamber slides (Nunc, U.S.A.), precoated with 10 µg/ml poly--lysine (Sigma). The plating density was 1.0#105 cells/cm2 (178,000 cells/well) unless stated otherwise. Cell cultures were grown in serumfree, chemically defined N2 medium6 at 37)C in a 95% air/5% CO2 humidified atmosphere. Test agents were added to the culture at the time of cell plating, and replenished every other day during the culturing period, unless specified otherwise. Control cultures were supplemented with PBS. Under the serum-free culture conditions employed here, the

Striatal cell cultures were performed as described earlier except that the seeding density was increased to 2.0#105/ cm2. A higher density of initially plated cells (>2.0#105/ cm2) yields more surviving and differentiated striatal neurons as compared to lower plating densities.49 Since a cell injury model requires a relatively high baseline number of well differentiated neurons, a plating density of 2.0#105/ cm2 was chosen in this part of the experiment. At 10 days in vitro, the striatal cultures were washed four times with sterile PBS and exposed to 100 µM NMDA (Sigma) for 24 h. Exposure media were 15 mM HEPES (Gibco)buffered MEM (Gibco) and 20 mM glucose. Parallel normal cultures were incubated with the same media without the addition of NMDA. Test agents were added to cultures 12 h prior to the exposure to NMDA, and were also included in the NMDA-containing MEM. After the 24 h exposure, cultures were subjected to cell viability assay as described below. Immunocytochemistry The procedures for immunocytochemistry were similar to those described previously.45 Briefly, the cell cultures were fixed for 20 min with 4% paraformaldehyde in 0.1 M phosphate buffer. Cells were permeabilized with 0.2% Triton X-100 in the presence of 5% blocking serum for 1 h. Incubation with the following primary antibodies was performed overnight at 4)C: monoclonal calbindin-D28k (1:500, Chemicon), polyclonal glutamate decarboxylase (GAD67; 1:500, Sigma), polyclonal GFAP (1:1000, DAKO) and polyclonal c-Fos (1:5000, Chembridge Research Biochemicals). Cells were then incubated with the

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corresponding biotinylated secondary antibodies (1:200) for 1 h and the bound antibodies were visualized using the Vectastain ABC kit (Vector Labs). Cell counting and morphological analyses Viability of cultured neurons was assessed using acridine orange/ethidium bromide as a vital stain. After washing cultures with 0.1 M PBS, 500 µl of a mixture of acridine orange (Sigma) and ethidium bromide (Sigma; 3.4 µg/ml of each component in 0.9% saline) was added to the cultures. acridine orange stains living cells green, whereas ethidium bromide enters dying cells through the leaky membranes, giving them an orange colour under epi-illumination at 390 nm. Only viable neurons were counted using a fluorescence microscope at #200 magnification. The numbers of c-Fos- and calbindin-D28k-positive cells were counted at #200 magnification in bright-field microscopy. In the excitotoxic experiments involving exposure to NMDA, the number of surviving neurons was expressed as a percentage of the cell count in normal control cultures. Cell counting in all the above experiments was performed on blind-coded slides in 24 previously determined fields of view in each culture well. The following morphological parameters were evaluated for individual calbindin-positive neurons at three days in vitro: total neurite length, number of branching points on neurites, number of primary neurites and cell body area. For estimation of neurite length, 100 neurons were randomly selected per well from two independent quadruplicate cultures. The total neurite length was measured at #200 magnification using Macintosh-based computerized image processing (Image software; version 1.52, NIH), as described previously.46,47,49 Numbers of branching points and primary neurites per neuron were counted on 60 neurons randomly selected from each group. Cell soma area was determined in 30 randomly selected neurons in each group using the aforementioned computerized image processing. Statistical analyses All values except for those regarding some of the morphological parameters were expressed as the mean&S.E.M. of four independent experiments, each comprising quadruplicate culture wells. Multiple comparisons between groups were made using one-way analysis of variance (ANOVA) with post-hoc Scheffe´ test. Group comparison of classes of morphological parameters including the number of primary neurite and branching points was performed using a ÷2 test. The total neurite length and cell soma area are expressed as the mean&S.E.M., and data were compared between control and dbcAMP-treated groups using unpaired two-tailed Student t-test. Other statistical analyses used are specified in the text. A probability value of less than 0.05 was considered significant. RESULTS

Effects of dibutyryl cyclic AMP on the development and survival of striatal neurons in serum-free cultures Striatal neurons degenerated gradually and progressively with time in culture. In control cultures, the number of surviving cells at day 2 was approximately 80% of that of initially plated cells (1.0#105 cells/ cm2), which decreased to 10% at day 9 under serumfree culture conditions (Fig. 1). Incubation with 1.0 mM of dbcAMP failed to counteract the progressive loss of striatal neurons under these conditions (Fig. 1A; P>0.05, two-way ANOVA with time#group interaction). Parallel to the survival

Fig. 1. Effects of dbcAMP (1 mM) on neuronal survival (A) and GABA uptake (B) in striatal cultures at a plating cell density of 1.0#105/cm2. Data are the mean&S.E.M. of four independent experiments with quadruplicate wells. For detailed statistical analyses see Results.

experiments, the uptake of GABA, which is a neurotransmitter for the majority of striatal neurons, was determined in sister cultures grown under identical conditions (Fig. 1B). GABA uptake per well in cultures without dbcAMP increased from days 1 to 5, indicating that biochemical differentiation of individual neurons occurred with time. The uptake then decreased, probably due to the degree of loss of neurons exceeding that of development of individual neurons after six days in vitro. In contrast to the absence of the survival-promoting effect of dbcAMP, it significantly enhanced the rate of development of GABA uptake (Fig. 1B; P<0.001, two-way ANOVA with time#group interaction). A significant difference in uptake between control and dbcAMP-treated groups was noted at day 3 (P<0.001, unpaired twotailed Student t-test), but not at six and nine days in culture. In the currently employed serum-free cultures, there were very few non-neuronal cells with large, flat and multipolar morphology. GFAPpositive glial cells constituted less than 1% of all cells throughout the culture period. Incubation with dbcAMP had no significant effect on the number of cells with non-neuronal morphology or GFAP-positive cells (data not shown). Neuronal survival in vitro is largely dependent on the density of initially plated cells, and survivalpromoting effects of increased cell density could therefore mask the potential survival effects of

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Fig. 2. Effects of dbcAMP (1 mM) on cell viability at three days in vitro at different plating densities. Values represent the mean&S.E.M. of four independent cultures with quadruplicate wells. *P<0.001, significant difference from untreated control cultures (one-way ANOVA with post-hoc Scheffe´ test).

exogenous test agents.1,27,49,50 Effects of dbcAMP (1 mM) on the survival of striatal neurons were examined at three days in vitro in cultures with different, lower seeding densities, ranging from 0.1#105 to 0.5#105 cells/cm2. A significant survival effect of dbcAMP was manifest at plating densities of 0.1#105 (P<0.01, unpaired two-tailed Student t-test) and 0.25#105 (P<0.05; Fig. 2). Dose–response studies were performed with respect to the effects of either dbcAMP or forskolin on the uptake of [3H]GABA in striatal cultures (Fig. 3). When cultures were grown for three days in the presence of dbcAMP at various concentrations from 1 to 1000 µM, the factor enhanced the uptake in a dose-dependent manner. When compared to untreated control cultures, significant differences in uptake were noted in the dbcAMP-treated groups, starting at a concentration of 50 µM. Stimulation of adenylate cyclase with forskolin also produced a similar increase in uptake, reaching a maximal effect at 50 µM. Trophic effects of dibutyryl cyclic AMP via the protein kinase A system GABA uptake was determined in cultures that had been treated with test agents for three days. As shown in Table 1, the effects of dbcAMP (1 mM) and forskolin (50 µM) on GABA uptake were not mimicked by the permeable cyclic GMP analogue, dbcGMP (1 mM). The effect of forskolin was significantly potentiated by the addition of 10 µM of Ro-201724. Ro-201724 is an inhibitor of type IV phosphodiesterase that specifically degrades cAMP.3 These results indicate that the observed effects of dbcAMP and forskolin were specific for cAMP, but not generalized to cyclic nucleotides. The effect of dbcAMP (1 mM) on uptake was significantly attenuated by co-treatment with 1 mM of Rp-cAMPS (Table 1). Treatment with Rp-cAMPS alone did not

Fig. 3. Dose–response study of effects of dbcAMP or forskolin on GABA uptake of striatal neurons. Cultures were grown in the presence of the test agents at the concentrations indicated for three days. Data represent the mean&S.E.M. of four independent cultures, each comprising quadruplicate wells. *P<0.001, significant difference from untreated control cultures (one-way ANOVA with post-hoc Scheffe´ test). Table 1. Effects of dibutyryl cyclic AMP or forskolin on GABA uptake of cultured striatal neurons GABA uptake (% of control) Control dbcAMP (1 mM) dbcGMP (1 mM) Rp-cAMPS (1 mM) dbcAMP (1 mM)+Rp-cAMPS (1 mM) Forskolin (50 µM) Forskolin (50 µM)+Ro-201724 (10 µM)

100.0&3.8 165.1&7.6* 99.3&4.5 103.2&6.1 126.5&6.2 149.1&5.4* 196.2&8.3**

Striatal cultures were grown in the presence of test agents for three days and then subjected to GABA uptake measurement. Data represent the mean&S.E.M. of four independent cultures with quadruplicate wells. *P<0.001, significant difference from controls; **P<0.001, significant difference from forskolin-treated cultures (oneway ANOVA with post-hoc Scheffe´ test).

significantly affect uptake. Rp-cAMPS, which is membrane permeable, blocks the activation of cAMP-dependent protein kinases by competing with cAMP for binding the regulatory subunits of these

Trophic effects of cAMP on striatal neurons

kinases.5 The enhancing effect of dbcAMP on GABA uptake was therefore mediated by the PKA system. Effects of dibutyryl cyclic AMP on protein expression in striatal neurons An extracellular signal-induced activation of signal transduction pathways usually results in the rapid and transient activation of immediate-early genes such as c-fos.59 Striatal cultures were grown for three days in the absence of any test agents and then incubated with dbcAMP (1 mM) for 30, 60 and 120 min. The number of c-Fos-immunoreactive neurons was increased with time after addition of dbcAMP (Fig. 4A, B). Approximately 40% of the total cells were found to exhibit c-Fos immunoreactivity at 120 min after the treatment. It was determined whether chronic exposure to dbcAMP may promote the expression of GAD67. GAD67, an isoform of the GABA-synthesizing enzyme,25 is localized in all of the striatal GABAergic neurons,41 and the level of its mRNA increases with time from embryonic stages to adulthood.21 Compared to controls, there seemed to be more GAD67immunopositive cells in the cultures treated with dbcAMP for six days. In both the control and dbcAMP-treated cultures, there was a gradient of immunostaining intensity among cells, although comparison with control experiments involving incubation with non-specific immunoglobulins indicated that stained cells, to varying degrees, were all GAD67-positive. Since it was difficult to discriminate very faintly stained from non-stained cells, no attempt was made to count the number of immunopositive cells in this part of the study. Since a subset of striatal spiny projection neurons express calbindin-D28k, a calcium-binding protein,17 the effect of dbcAMP on the expression of calbindinD28k in striatal neurons in vitro was also examined. The time-course of changes in the number of cells expressing immunocytochemically detectable levels of calbindin-D28k is illustrated in Fig. 5A. In control cultures, the number of calbindin-D28k-positive neurons increased progressively with time in culture, and at day 6 approximately 19% of the total cell population were calbindin-D28k-positive (Fig. 5A). Continuous exposure of cultures to dbcAMP (1 mM) induced a significant increase in the number of cells exhibiting detectable levels of this protein compared to control cultures at each time-point (P<0.001 at day 3, P<0.001 at day 6, unpaired two-tailed Student t-test; Fig. 5A–C). Since calbindin immunocytochemistry employed here clearly delineated both the soma and neurites of positive cells (Fig. 5B, C), morphogenetic effects of dbcAMP on calbindin-D28k-positive neurons were also investigated at three days in vitro. In the dbcAMP-treated culture, calbindin-D28k-positive neurons had longer neurites and larger cell bodies than in non-treated control cultures (Table 2). Treat-

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ment with dbcAMP also significantly increased the number of branching points (P<0.001, ÷2 test; Fig. 6A). There was no significant difference in the number of primary neurites per neuron between the two groups (P>0.05, ÷2 test; Fig. 6B). Relationship between neurotrophic factors and the protein kinase A system It has been shown that bFGF and IGF-1 yield trophic effects on cultured striatal neurons by promoting neuronal survival and development.49,64 Treatment with either bFGF (10 ng/ml) or IGF-1 (50 ng/ml) for three days enhanced GABA uptake by around 50% compared to controls (Table 3). Combination of either of the two growth factors and dbcAMP yielded a significant, additive effect on the uptake (Table 3). The GABA uptake-promoting effects of these growth factors were not significantly blocked by Rp-cAMPS (1 mM) (Table 3). These findings strongly suggest that trophic actions of the growth factors on striatal neurons are mediated by mechanisms other than those involving cAMPdependent kinases. Indeed, incubation with either bFGF (10 ng/ml) or IGF-1 (50 ng/ml) for 15 min did not significantly elevate intracellular levels of cAMP (Table 5). Neuronal depolarization induced by high K+ produces trophic effects on several types of neurons, including striatal neurons.27 Incubation with 50 mM KCl for three days significantly enhanced GABA uptake, which was blocked by 1 mM of Rp-cAMPS (Table 4). No additive effects were produced by simultaneous application of high K+ and dbcAMP (Table 4). Despite the increased GABA uptake, depolarization by high K+ did not promote neuronal survival (data not shown). Notably, 1 µM of nifedipine, the L-type voltage-sensitive calcium channel (VSCC) blocker, significantly attenuated the depolarization-induced trophic effect, whereas it did not affect the uptake enhanced by dbcAMP (Table 4). Neither GABA uptake nor cell survival were influenced by treatment with nifedipine (1 µM) alone. Addition of K+ (50 mM) significantly increased intracellular cAMP levels, and co-treatment with nifedipine (1 µM) prevented this elevation (Table 5). The results indicated that high K+-induced depolarization promotes the development of striatal neurons, at least in part, via the protein kinase A system, and that the influx of calcium through the VSCC is involved in the depolarization-induced elevation of cAMP. Protective effects of dibutyryl cyclic AMP against excitotoxic injury It was explored whether treatment with dbcAMP may increase the resistance of striatal neurons to NMDA-induced neurotoxicity. Exposure of 10-dayold cultures to NMDA (100 µM) for 24 h led to a

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Fig. 4. Time-course of the effects of dbcAMP (1 mM) on the expression of c-Fos in striatal cultures. Striatal cultures were grown for three days without any test drugs and then treated with 1 mM of dbcAMP for 30–120 min. Immunocytochemically detectable levels of c-Fos were increased with time after addition of dbcAMP (A). Representative pictures of c-Fos-immunoreactive neurons in cultures exposed to dbcAMP for 30 min (B), 60 min (C) or 120 min (D). Photomicrographs were taken in bright field with increased contrast in order to visualize non-immunostained cells. Scale bar=50 µm.

decrease in the number of viable neurons to approximately 25% of normal control cultures. Cultures were pretreated with dbcAMP (1 mM) for 12 h, and thereafter exposed to NMDA (100 µM) for 24 h, with simultaneous addition of dbcAMP. Treatment with dbcAMP significantly attenuated the neuronal loss induced by NMDA (Table 6). A similar treatment with either bFGF (10 ng/ml) or IGF-1 (50 ng/ml) also ameliorated NMDA-induced cell death. The extent of cell survival was comparable among cultures treated with those factors alone: approximately 50% of normal control cultures. Incubation with 1 mM of Rp-cAMPS significantly attenuated the protective effects of dbcAMP, but not of the growth factors (data not shown). Combination of dbcAMP and either of the two growth factors afforded a significantly greater neuroprotection than each factor alone (Table 6). Neuronal survival in cultures treated

simultaneously with dbcAMP and either bFGF or IGF-1 was similar, and increased to around 80% of normal controls. DISCUSSION

Cyclic AMP-dependent trophic effects In general, neurotrophic activities comprise: (i) promotion of survival and/or development; (ii) regulation of the synthesis of proteins for differential functions of neurons; and (iii) maintenance of structural integrity of neurons under unfavourable conditions such as exposure to neuronal insults.23 In the present study, an increase in intracellular cAMP levels by either forskolin or dbcAMP produced trophic effects on striatal neurons in vitro, as indicated by enhancement of neuronal survival, GABA uptake, the expression of proteins such as c-Fos,

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Fig. 5. Time-course of the effects of dbcAMP (1 mM) on the expression of calbindin in striatal neurons developing in culture (A). Treatment with dbcAMP (1 mM) was started at the time of cell plating and cultures were processed for calbindin immunocytochemistry at three and six days in vitro. Data are the mean&S.E.M. of four independent experiments with quadruplicate wells. Representative examples of calbindin-immunostained cells in control (B) and dbcAMP-treated cultures (C) at three days in vitro. Both the soma and neurites are intensely immunostained with the calbindin antibody. Photomicrographs were taken in bright field with increased contrast from the condenser in order to visualize non-immunostained cells. Scale bar=50 µm.

calbindin-D28k and possibly GAD67, the morphological development of calbindin-D28k-positive neurons, and the resistance of neurons against NMDA-induced excitotoxic injury. The present striatal cultures contain a negligible number of glial cells (<1% of total cells). Treatment with dbcAMP did not affect the number of cells with non-neuronal morphology or GFAP-positive cells. Therefore, the neurotrophic effects above are most likely due to direct actions of the test agents. Cell survival, in contrast to other parameters, was promoted by dbcAMP only in lower cell density cultures. Previous cell culture experiments have also

Table 2. Effects of dibutyryl cyclic AMP (1 mM) on the total neurite length and the cell body area of calbindin-positive neurons developing in culture for three days

Control dbcAMP

Total neurite length (µm)

Cell body area (µm2)

158.2&47.3 358.8&51.4*

102.5&5.3 141.8&9.6*

Data represent the mean&S.E.M. *P<0.001, significant difference from controls (unpaired two-tailed Student t-test).

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N. Nakao Table 4. Effects of depolarization on GABA uptake in striatal cultures GABA uptake (% of control) Control KCl (50 mM) dbcAMP (1 mM) KCl (50 mM)+dbcAMP (1 mM) KCl (50 mM)+Rp-cAMPS (1 mM) KCl (50 mM)+nifedipine (1 µM) dbcAMP (1 mM)+nifedipine (1 µM)

100.0&2.1 140.3&5.4* 155.2&5.1* 146.5&3.7* 121.1&5.3 113.2&4.5 149.2&6.2*

Test agents were added to cultures at the time of plating, and three days thereafter GABA uptake was measured. Data represent the mean&S.E.M. of four independent cultures with quadruplicate wells. *P<0.001, significant difference from controls (one-way ANOVA with post-hoc Scheffe´ test). Table 5. Effects of neurotrophic factors on intracellular levels of cyclic AMP Intracellular levels of cAMP (% of control)

Fig. 6. Effects of dbcAMP (1 mM) on the number of branching points (A) and the number of primary neurites (B) of calbindin-positive neurons grown for three days in culture. Histograms show distribution of each morphological parameter, as shown by the percentage of the neurons examined. Table 3. Effects of growth factors on GABA uptake of cultured striatal neurons GABA uptake (% of control) Control bFGF (10 ng/ml) IGF-1 (50 ng/ml) dbcAMP (1 mM) bFGF (10 ng/ml)+dbcAMP (1 mM) IGF-1 (50 ng/ml)+dbcAMP (1 mM) bFGF (10 ng/ml)+Rp-cAMPS (1 mM) IGF-1 (50 ng/ml)+Rp-cAMPS (1 mM) dbcAMP (1 mM)+Rp-cAMPS (1 mM)

100.0&3.2 160.2&11.0* 154.4&13.1* 178.1&9.7* 231.5&13.2** 222.6&19.5** 178.2&15.6* 159.3&12.1* 112.8&8.3

Treatment with test agents was started at the time of cell seeding, and cultures were processed for GABA uptake measurement at three days in vitro. Data represent the mean&S.E.M. of four independent cultures with quadruplicate wells. *P<0.001, significant difference from controls; **P<0.001, significant difference from cultures treated with each growth factor alone (one-way ANOVA with post-hoc Scheffe´ test).

demonstrated that a significant survival effect of test agents becomes manifest with decreasing plating density.1,20,50 This could be explained by the fact that there is a greater dependency on exogenous factors to

Control bFGF (10 ng/ml) IGF-1 (50 ng/ml) KCl (50 mM) KCl (50 mM)+nifedipine (1 µM)

100.0&5.3 106.2&7.4 103.1&6.3 148.5&5.2* 112.1&4.1

Cultures grown for 24 h after plating were treated with test agents for 15 min, and thereafter intracellular levels of cAMP were determined. Data are expressed as the mean&S.E.M. of four independent experiments with each comprising quadruplicate wells. *P<0.001, significant difference from controls (one-way ANOVA with post-hoc Scheffe´ test).

prevent cell death in lower density cultures, since in such cultures survival is less supported by reciprocal trophic signals among neighbouring neurons via soluble factors and/or direct cell to cell contacts. Permeant cAMP analogues have been shown to prevent apoptosis induced by trophic deprivation in cultures of sympathetic neurons and septal cholinergic neurons.28,56 It remains to be clarified whether the type of cell death that occurred under the present low-density culture conditions is apoptosis. Trophic activities and cyclic AMP-dependent signalling pathway It has been shown in non-neuronal cell cultures that bFGF regulates cAMP levels51 and requires cAMP for its mitogenic activity.9,35 A recent study revealed that, in retinal ganglion cell cultures, peptide growth factors including neurotrophins, ciliary neurotrophic factor, bFGF and IGF-1 exhibit no significant trophic activities unless cAMP levels are concurrently elevated.42 These findings all indicate that there is cross-talk between the cAMP-dependent

Trophic effects of cAMP on striatal neurons Table 6. Effects of dibutyryl cyclic AMP, basic fibroblast growth factor, insulin-like growth factor-1 or a combination of dibutyryl cyclic AMP and either of the growth factors on the survival of striatal neurons in cultures exposed to N-methyl--aspartate-induced excitotoxic insult Cell survival (% of normal) Normal NMDA NMDA+dbcAMP (1 mM) NMDA+bFGF (10 ng/ml) NMDA+IGF-1 (50 ng/ml) NMDA+dbcAMP (1 mM)+bFGF (10 ng/ml) NMDA+dbcAMP (1 mM)+IGF-1 (50 ng/ml)

100.0&2.7 24.6&2.1 50.5&3.8* 60.1&2.2* 56.4&2.6* 82.3&3.1** 80.5&2.2**

Cultures were pretreated with the test agents for 12 h, and thereafter exposed to NMDA (100 mM) for 24 h in the presence of the agents. Cell viability is normalized by setting the number of surviving neurons in normal cultures as 100%. Data represent the mean&S.E.M. of four independent cultures with quadruplicate wells. *P<0.001, significant difference from untreated control cultures subjected to NMDA excitotoxicity (one-way ANOVA with post-hoc Scheffe´ test). **P<0.001, significant difference from NMDA-exposed culture treated with each factor alone (one-way ANOVA with post-hoc Scheffe´ test).

pathway and the signal transduction pathway activated by growth factors. In the present study, trophic actions of bFGF and IGF-1 were not blocked by a specific PKA inhibitor, nor did these growth factors alter cAMP levels. This suggests that bFGF and IGF-1 act on striatal neurons independently of cAMP-dependent pathways, which is supported by the findings that dbcAMP and either of the growth factors gave rise to additive trophic effects. It is possible that there are differences in the mode of intracellular signal transduction among cell types. Nonetheless, the observed additive effects could be produced by a synergy between the pathways activated by dbcAMP and the growth factors. Thus, cAMP could potentiate the growth factor-related signal transduction pathways. In support of this view, similar results have been obtained previously in cell culture experiments with other neuron types. It has been shown in PC12 cells that cAMP analogues potentiate the activation of mitogen-activated protein kinase induced by nerve growth factor,16 and that dbcAMP promotes nerve growth factor-induced survival and differentiation.43 Synergistic trophic effects of dbcAMP and growth factors have also been demonstrated in hypothalamic and mesencephalic dopamine neurons.22,52 Membrane depolarization induced by high K+ mimicked the trophic action produced by dbcAMP. Thus, application of high K+ enhanced GABA uptake, which was blocked by a specific PKA inhibitor. The findings lead to the view that membrane depolarization yields neurotrophic effects, at least in part,

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via cAMP-dependent kinases. This is supported by the observation that high-K+-induced depolarization elevated cAMP levels in striatal neurons. Previous studies have demonstrated a positive correlation between electrical activity of neurons and cAMP levels. In several neuronal cell lines, depolarization has been shown to increase cAMP levels with a consequent enhancement of the responsiveness to trophic factors.4,37,55 Tetrodotoxin-induced electrical blockade decreased cAMP levels in retinal ganglion cells.24 There is a body of evidence indicating that depolarization exhibits neurotrophic activities, and that calcium influx through the VSCC plays a pivotal role in the initiation of a cascade eventually leading to the trophic actions.15,29,30 In the present study, the VSCC blocker nifedipine inhibited both depolarization-induced cAMP elevations and increases in GABA uptake. This further supports that cAMP could be a mediator of depolarizationdependent trophic actions on developing striatal neurons. Morphogenetic effects of dibutyryl cyclic AMP Treatment of cultured striatal neurons with dbcAMP increased the number of branching points, neurite length and cell soma area. Morphogenetic effects of agents that increase or mimic cAMP have been investigated in vitro on several types of neurons. It has been shown previously that cAMP analogues promote neurite outgrowth in cultures of sympathetic and dorsal root ganglion neurons.56 Agents that elevate cAMP levels enhance outgrowth rate of axons and dendrites of hippocampal pyramidal neurons in culture,39 whereas they inhibit neurite elongation in Helisoma neurons.40 Prolonged exposure of serotonergic neurons to dbcAMP has been demonstrated to make some aspects of their morphology resemble those of mature raphe neurons, in that most of the treated neurons possess only one process with extensive arborization.14 These findings, together with the present ones, strongly suggest that cAMP is involved in intracellular pathways mediating morphological development of vertebrate neurons. The observed morphogenetic effects of dbcAMP lend further support to the hypothesis that dopamine innervation may play an important role in the differentiation of striatal neurons in vivo, since cAMP is a second messenger for dopamine D1-like receptors.26 Protective effects of dibutyryl cyclic AMP against excitotoxicity Treatment with dbcAMP increased the resistance of striatal neurons to NMDA-induced excitotoxic injury, and the protective effects were diminished by a specific PKA inhibitor. This is in agreement with previous reports that agents that elevate cAMP levels protected hippocampal or cortical neurons against glutamate-induced toxicity in vitro.8,39 Dopamine D1

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receptor agonists that can activate adenylate cyclase also prevent neuronal death induced by exposure to glutamate.2 In the light of these findings, one could raise the possibility that cAMP-related signals operate somehow against a toxic cascade of events that follows an overstimulation of glutamate receptors. It remains to be fully elucidated how the activation of cAMP-dependent kinases enhances the resistance of neurons to excitotoxicity. A primary step of intracellular excitotoxic processes is believed to be an excessive influx of calcium through the glutamategated calcium channel.13,33 A possible mechanism of actions of factors capable of mitigating excitotoxic injury is that they can stabilize intracellular calcium homeostasis. It has been revealed that growth factors suppress a rise in intracellular calcium levels following exposure to excitatory amino acids by inducing the expression of calcium-binding proteins (e.g., calbindin) in neurons, and that they protect against excitotoxicity.10,11,12,48 In the current study, chronic exposure to dbcAMP enhanced immunocytochemically detectable levels of calbindin-D28k in striatal neurons, although it is not clear at present if only 12 h of dbcAMP treatment induces the up-regulation of calbindin levels sufficiently to provide neuroprotection. In a study by Amano et al.,2 on the other hand, dopamine D1 agonists prevented excitotoxic injury without affecting glutamate-induced influx of calcium. This suggests that cAMP may act on excitotoxicity-related events downstream from calcium influx. Neuroprotection conferred by bFGF or IGF-1 was not affected by Rp-cAMPS. As is the case

of the effects of the growth factors on GABA uptake, therefore, they are likely to afford neuroprotection through mechanisms that are independent of cAMPrelated pathways. Still, dbcAMP could potentiate the protective effects of bFGF or IGF-1.

CONCLUSIONS

The present study indicates that elevated levels of cAMP and subsequent activation of PKA yield several trophic effects on developing striatal neurons in vitro. This sheds light on the potential roles of cAMP-dependent kinases in the regulation of survival and development of striatal neurons in vivo. Of particular note is the fact that co-treatment with cAMP and growth factors, such as bFGF and IGF-1, produce additive trophic actions. If there is cross-talk between the pathways related to cAMP and the growth factors, further studies are necessary to illuminate which levels of the growth factor-related signalling pathways cAMP may positively regulate. The results on dbcAMP-mediated neuroprotection against excitotoxic insult have implications for the development of neuroprotective strategies in the treatment of excitotoxicity-related brain injuries, including cerebral ischaemia, trauma and Huntington’s disease.13 Acknowledgements—I am grateful to Professor Toru Itakura for invaluable support and scientific advice throughout this work.

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