Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Phencyclidine (PCP), a drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist with potent psychotomimetic properties (Johnson and Jones, 1990). PCP has been shown to exacerbate psychotic symptoms in schizophrenia and has been proposed to model both the positive and negative symptoms of schizophrenia (Javitt and Zukin, 1991).

It has been demonstrated that NMDA receptors are involved in a variety of physiological and pathological processes, including memory and learning (Collingridge et al., 1983; Ripley and Little, 1995), neuronal development (D'Souza et al., 1993), epileptiform seizures, synaptic plasticity (Meldrum and Garthwaite, 1990), and acute neuropathologies such as stroke and trauma (Beal, 1992). There is also evidence for its involvement in chronic neuropathologies such as Alzheimer's (Cotman et al., 1989), Parkinson's, and Huntington's diseases (Meldrum and Garthwaite, 1990; Greenamyre, 1993), and mental illnesses such as schizophrenia and anxiety disorder (Meldrum and Garthwaite, 1990).

In recent years, it has been demonstrated that NMDA receptor antagonists such as PCP and MK-801 cause neurodegeneration in rat brain (Olney et al., 1991; Sharp et al., 1994). Previous in vivo studies from this laboratory have demonstrated that chronic administration of PCP results in a sensitized locomotor response in rats to PCP challenge (Johnson et al., 1998). This sensitization is associated with apoptotic cell death and an increase in NMDA receptor NR1 subunit mRNA and immunoreactivity, as well as an altered functionality of the NMDA receptor in rat forebrain (Hanania et al., 1999; Wang et al., 1999).

A growing family of genes that share homology with the Bcl-2 proto-oncogene is involved in the regulation of cell death. Various homodimers and heterodimers formed by proteins of this family can either promote or inhibit apoptosis. A physiological role for Bcl-2 and Bcl-X_L in neuron survival has been shown (Merry et al., 1994). Bcl-X_L is the major form of Bcl-2 family expressed in the murine nervous system in embryonic and adult brain (Gonzalez-Garcia et al., 1995). Bax (Bcl-2-associated protein X) was the first Bcl-2-related protein to be isolated that showed homology with Bcl-2 throughout two highly conserved regions. Bax homodimers are known to be proapoptotic, but Bax dimers with Bcl-2 or Bcl-X_L are inactive in this regard. Thus, the ratio of Bcl-2/Bax or Bcl-X_L/Bax is an index that can determine whether an apoptotic stimulus results in the life or death of a cell.

The goal of this study was to determine whether chronic PCP administration in vitro increased the neurotoxic potential of NMDA and if so, to determine whether the neurotoxicity was associated with an increase in markers of apoptosis such as DNA fragmentation and Bcl-2 family proteins. We also sought to determine whether chronic PCP in vitro caused an increase in the amount of NR1 subunit mRNA as previously observed in vivo. It was observed that treatment of forebrain cultures with PCP increased the level of NR1 mRNA. This was associated with a decrease in the apoptotic threshold for NMDA as measured by an increase in DNA associated with histone, fragmented DNA as assessed by nick-end labeling, and the Bcl-X_L/Bax ratio. It is suggested that this system is a suitable model of PCP-induced apoptosis in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Drugs and Other Materials. PCP was obtained from the National Institute on Drug Abuse (Rockville, MD). PCP was dissolved in Dulbecco's modified Eagle's medium (DMEM). NMDA was purchased from Tocris Neuramin (Bristol, UK). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Sigma (St. Louis, MO). The medium and fetal bovine serum were purchased from Life Technologies (Grand Island, NY). All other kits and enzymes were obtained from Boehringer Mannheim (Indianapolis, IN). Other chemicals were obtained from Sigma.

Primary Cell Culture. Primary forebrain cultures were prepared from newborn rats (Sprague-Dawley) as described by Kiss et al. (1994). Briefly, forebrains were dissected from brains and dissociated in cold Hanks solution without Mg²⁺ and Ca²⁺. Cultures were grown on polylysine-coated coverslips in DMEM supplemented with 10% (v/v) fetal bovine serum. Glial proliferation was stopped with a mitotic inhibitor, cytosine -D-arabinofuranoside (beginning on the third day of culture). Cells were treated with 3 µM PCP or normal medium at 37°C for 48 h (days 4-6). After the treatment, cultures were rinsed with serum-free defined medium. Cultures were then exposed to NMDA (30 or 100 µM, or control) on day 6 in serum-free defined medium (prepared by adding a supplement mixture consisting of 15 µg/ml insulin, 20 µg/ml transferrin, 20 nM progesterone, 100 µM putrescine, and 30 nM sodium selenite to DMEM). Neurotoxicity was evaluated in four different assays 20 h after the addition of NMDA in Mg²⁺-containing medium.

Cytotoxicity Detection Assay. The release of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as a generic index of cell death. Twenty hours after exposure to NMDA or control medium, the medium was collected and assayed for LDH activity with a cytotoxicity detection kit from Boehringer Mannheim. Briefly, LDH catalyzes the conversion of lactate to pyruvate on reduction of NAD⁺ to NADH/H⁺; the added tetrazolium salt (yellow) is then reduced to formazan (red). The amount of formazan formed correlates to LDH activity. The formazan product is measured with a microtiter plate reader at an absorption wavelength of 490 nm.

MTT Reduction Cell Viability Assay. The dye MTT is taken up and metabolized to a colored product by viable mitochondria. Thus, the measurement of this metabolic reduction reaction was used as a marker of mitochondrial viability. Briefly, 100 µl of MTT (5 mg/10 ml of medium) was added to each well, and the plate was incubated for 4 h at 37°C. The MTT solution was removed, 100 µl of dimethyl sulfoxide was added to each well, and the color intensity was assessed with an enzyme-linked immunosorbent assay (ELISA) plate reader at a wavelength of 590 nm.

Fragmented DNA Detection by ELISA. Although the LDH release assay and the MTT reduction assay are reliable indices of cell death, neither is specific for apoptotic cells. Such cells are better characterized by DNA fragmentation that is the result of internucleosomal cleavage of DNA by apoptosis-specific activation of endonucleases. The presence of fragmented DNA associated with nucleosomal histone in chronic PCP-treated and control cells was assessed by a specific two-site ELISA using an antihistone primary antibody and a secondary anti-DNA antibody according to the manufacturer's instructions (Boehringer Mannheim). Briefly, cells were grown in 10-cm tissue culture dishes. After the treatment regimen, cells were spun and resuspended in 3 ml of lysis buffer and incubated for 30 min at room temperature. After centrifugation, the supernatants (cytosol containing low-molecular mass, fragmented DNA) were diluted 1:2 (v/v) with lysis buffer. Then, 20 µl from each sample was transferred to a plate reader well precoated with antihistone antibody, and 80 µl of immunoreagent mix, including the secondary antibody, was added. After incubation and washes, the wells were treated with the chromogen substrate, and the intensity of the color that developed was assayed at 405/490 nm wavelength.

Terminal dUTP Nick-End Labeling (TUNEL) Assay. This assay is widely used to assess apoptosis in situ. It relies on the detection of fragmented DNA strands, but because fragmentation can occur via nonapoptotic mechanisms, it is not absolutely specific for apoptosis. After the treatment regimen, the cells were rinsed with PBS; fixed by ice-cold (4°C) 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2; and processed for evaluation of nuclei containing fragmented DNA in situ. Terminal deoxynucleotidyl transferase, a template-independent polymerase, was used to incorporate biotinylated nucleotides at sites of DNA breaks as previously described (Johnson et al., 1998). The cells were photographed with the use of an Olympus light microscope. The percentage of TUNEL-positive cells was estimated in five 0.24-mm² fields in each of three dishes in each treatment condition. Each condition was assessed at least in triplicate and experiments were repeated three times independently. Data are presented as the mean ± S.E. A probability of P < .05 was considered significant (one-way ANOVA).

Light-Microscopic Immunocytochemistry and Nuclear Staining. This experiment evaluated the nuclear morphology of PCP-treated and control cells, with Hoechst 33258 to visualize the nucleus and polysialic acid-neuronal cell adhesion molecule (PSA-NCAM) immunocytochemistry to mark neurons. A mouse monoclonal antibody (Meningococcus group B) to PSA-NCAM was used (1:500 dilution). After the treatment regimen, the cells were rinsed with PBS; fixed by ice-cold (4°C) 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2; and the indirect immunofluorescence technique was used to visualize immunoreactivities. The cells were incubated with the primary antibody (diluted in PBS/0.5% BSA solution) at 4°C overnight. Bound antibodies were revealed with fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Boehringer Mannheim) secondary antibody (diluted 1:80 in PBS/0.5% BSA solution). The cells were examined with an Olympus light microscope equipped with epifluorescence.

Western Blot Analysis. After the treatment regimen, the medium was first removed and the attached cells were washed with PBS. Protein extraction was accomplished by cell lysis with SDS. Protein samples were measured for protein concentration with BCA Protein Reagent (Pierce, Rockford, IL). Equal amounts of total protein (10 µg) were loaded on each lane and run on SDS-polyacrylamide gels with a Tris/glycine running buffer system and then transferred to a polyvinylidene difluoride membrane (0.2 µm) in a Mini Electrotransfer Unit (Bio-Rad, Richmond, CA). The blots were probed with an anti-Bcl-X_L (1:1000, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) antibody, anti-Bax (1:1000, polyclonal; Santa Cruz Biotechnology) antibody, and anti-actin (1:3000, monoclonal, house-keeping protein; Amersham). Immunoblot analysis was performed with horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG with the enhanced chemiluminescence Western blotting detection reagents (Amersham). The Bcl-X_L/Bax ratio was analyzed by the Lynx 5000 Imagine Analysis System.

In Situ Hybridization of NR1 mRNA. An oligonucleotide probe complementary to the mRNA encoding the NMDA glutamate receptor NR1 subunit was selected on the basis of cloned cDNA sequences. The sequence of the probe used for in situ hybridization was as follows: 5'-TTCCTCCTCCTCCTCACTGTTCACCTTGAATC-GGCCAAAGGGACT (this corresponds to a region that is constant across all NR1 splice variants, amino acids 566-580). It was 3'-end labeled by incubation with ³⁵S-deoxy-ATP (New England Nuclear, Boston, MA) and terminal deoxynucleotidyl transferase (Boehringer Mannheim) to attain specific activities of ~5-8 × 10⁸ cpm/µg. The specificity of the probe has been previously described (Monyer et al., 1992).

Quantitation of In Situ Autoradiographs. Images were acquired with a digital microscopy apparatus (Image Tools Software, University of Texas Health Science Center at San Antonio), saved as 640 × 480 × 8-bit grayscale Tif files and sent to a University core image laboratory for analysis. Briefly, the images were smoothed with a 3 × 3 square filter to remove noise, and regions of interest were selected using a threshold technique that segments the image into labeled cells and background. The threshold was determined interactively by the consensus of two trained observers (one was blind to the treatment) and then held constant for the cells in both the PCP group and the control group. The density of silver grains that exceeded the threshold was estimated in five 0.24-mm² fields in each of three dishes treated with either PCP or normal serum-free defined medium. Each condition was assessed at least in triplicate and experiments were repeated three times independently. A monotonic relationship was assumed to exist between measured labeling and the amount of mRNA labeled with the radioactive probe. This technique is similar to that used by several laboratories except that a fixed size rather than a variable size region of interest is used (Rudolf et al., 1996). Data are presented as the mean ± S.E. Statistical differences were determined by Student's t test. A probability of P < .05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Morphological Characterization of Primary Cell Cultures and NMDA-Induced Neurotoxicity. Two randomly selected regions of 1.8 mm² were selected from three culture dishes from independent experiments to assess the phenotype of the forebrain cells used in this study. Approximately 130 cells were counted in each region. The use of immunofluorescent staining of PSA-NCAM, a neuron-specific marker, and glial fibrillary acidic protein (a glial marker) in control rat forebrain culture revealed that 57 ± 3% of the cells in culture were neurons. Figure 1A illustrates the neuron-specific staining of cultured cells with PSA-NCAM. Figure 1B shows that all the nuclei of PSA-NCAM-positive neurons were stained with Hoechst 33258 as visualized with a fluorescence microscope. Glial fibrillary acidic protein-positive glia also were positively stained by Hoechst 33258 (data not shown). Neurons remained viable up to 2 weeks in culture, but were used in these experiments 7 days after plating. At day 6, in vitro, cells were exposed to NMDA for 20 h. Figure 1C shows NMDA-induced nuclear condensation and fragmentation, which are hallmarks of apoptosis. We also found that most of the apoptotic cells were neurons because those cells with condensed and fragmented nuclei were costained by PSA-NCAM (Fig. 1C).

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Effect of NMDA on nuclear morphology of forebrain neurons in primary culture. A, neuron-specific staining of cultured cells with PSA-NCAM as revealed by immunofluorescence of anti-mouse IgG conjugated to fluorescein isothiocyanate (scale bar, 110 µm). B and C, higher magnification of nuclei stained by Hoechst 33285 (with an excitation wavelength of 365 nm) after treatment with control medium (B) or 100 µM NMDA (in the presence of Mg2+) for 20 h (C). Closed arrows show fragmented nuclei and the open arrow points to a condensed nucleus, both of which are hallmarks of apoptosis. The faint outline of neurons in B and C is due to the partial overlap in PSA-NCAM fluorescence at 365 nm. Scale bars in B and C, 13.75 µm.

Quantitation of NMDA-Induced Cell Death in Control and PCP-Treated Cultures. In these experiments, we evaluated two concentrations of NMDA (30 and 100 µM) in control cultures and in cultures pretreated for 48 h with PCP. In control cultures, after washout of PCP, 30 µM NMDA had no significant effect on the percentage of TUNEL-positive cells, but 100 µM NMDA caused a significant 3-fold increase in this measure (Figs. 2 and 3). In contrast, in PCP-treated cells, both 30 and 100 µM NMDA had a large and significant effect on the percentage of TUNEL-positive cells (Figs. 2 and 3A). These data suggest that pretreatment with PCP sensitizes these cells to the toxic effects of NMDA. This suggestion was validated by similar results from an experiment that measured the effects of PCP pretreatment on NMDA-induced increases in LDH release. Herein, the effect of both concentrations of NMDA were potentiated significantly by PCP pretreatment (Fig. 3B).

View larger version (125K): [in this window] [in a new window]   Fig. 2.   Effect of treatment with 3 µM PCP for 2 days on NMDA-induced apoptosis measured with TUNEL staining in primary cultures of forebrain. A and B, in normal medium; C and D, with 30 µM NMDA; and E and F, with 100 µM NMDA for 20 h immediately after PCP washout. Chronic PCP treatment (B, D, F) appeared to increase the number of TUNEL-positive neurons after NMDA treatment (quantitated in Fig. 3). Scale bar, 22.5 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Effect of 48-h incubation of forebrain cultures with 3 µM PCP. After incubation, PCP was washed out and the cultures were treated with either control medium or medium containing either 30 or 100 µM NMDA in the presence of physiological magnesium for 20 h. A, effect of PCP pretreatment on NMDA-evoked apoptosis expressed as the percentage of TUNEL-positive cells relative to the total number of cells measured in five randomly selected areas (0.055 mm2) from each culture. B, effect of PCP pretreatment on NMDA-induced LDH release expressed as percentage release of LDH relative to basal values in untreated control cells (defined as 100%). C, effect of chronic PCP treatment on the ability of 30 µM NMDA to reduce MTT uptake. D illustrates the effect of chronic PCP on the ability of NMDA to elicit an increase in fragmented DNA as measured by ELISA. Data are mean ± S.E. of three independent experiments, each performed in triplicate (*P < .05). In all four assays of neurotoxicity, 30 µM NMDA was nontoxic in normal control cultures but was markedly toxic after chronic PCP.

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Effect of chronic PCP treatment on the in situ hybridization signal of NMDAR1 subunit mRNA level in rat forebrain cultures. Cultures were grown on 2.2 × 2.2-cm coverslips in individual Petri dishes for 4 days before adding either control medium or medium containing 3 µM PCP for 2 days. NR1 mRNA was estimated in five 0.24-mm2 fields in each of three dishes under each treatment condition. A, quantitation of the NR1 mRNA signal from control and PCP-treated cultures. Data are mean ± S.E. of three independent experiments, each performed in triplicate. Representative autoradiographs from control and PCP-treated cultures are shown in B and C, respectively. *P < .05 (Student's t test).

Role of Bcl-2 Family Proteins and Reactive Oxygen Species (ROS) in NMDA Receptor-Mediated Apoptosis. To determine whether the expression pattern of key members of Bcl-2 family genes are correlated with the morphological and biochemical results, cell lysates were immunoblotted with rabbit antisera to Bcl-X_L and Bax. Figure 5 shows that the polyclonal anti-Bcl-X_L antibody recognized a single protein band at ~29 kDa, and the anti-Bax antibody recognized a single protein band at ~21 kDa. Visual inspection of lanes 1, 2, and 3 reveals that NMDA produced a concentration-dependent increase in Bax and concomitant decrease in Bcl-X_L. A similar effect was observed in PCP-treated cultures (lanes 4, 5, and 6). However, quantitative densitometry revealed that the effect of 30 µM NMDA on both Bax and Bcl- X_L was more marked in PCP-treated cultures than in control (data not shown). These densitometry measurements were used to calculate a ratio of Bcl-X_L to Bax in each lane in three independent experiments and the mean ± S.E. of these ratios is shown in Fig. 6. This ratio was markedly decreased by 100 µM NMDA in both control and PCP-treated cultures; however, the effect of 30 µM NMDA was equivalent to 100 µM NMDA after PCP treatment, whereas it had no significant effect in control cultures. This pattern is very similar to that observed when the neurotoxic effect of NMDA was assessed by either LDH release or MTT uptake (Table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of Bcl-XL and Bax expression in primary forebrain culture. Control cells were exposed to 100 (lane 1), 30 (lane 2), and 0 µM NMDA (lane 3). Chronic PCP-treated cells were exposed to 100 (lane 4), 30 NMDA (lane 5 and 7), and 0 µM NMDA (lane 6). Total cellular protein was extracted and analyzed by polyacrylamide gel electrophoresis (10 µg of protein per lane), electrotransferred, and then probed with anti-Bcl-XL (top) and anti-Bax (bottom). By comparison with molecular mass standards (Santa Cruz Biotechology), a band of ~29 kDa was identified as Bcl-XL and another band of ~21 kDa was identified as Bax according to their labeling by anti-Bcl-XL and anti-Bax antibodies, respectively. Results shown are representative of three independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Densitometric measures of the ratio of Bcl-XL to Bax. Values represent mean ± S.E. of three independent experiments. Statistical comparisons consisted of a one-way ANOVA (*P < .05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several laboratories have reported that a chronic regimen of PCP results in a sensitized locomotor response to PCP challenge, but the mechanism is still largely unknown. This laboratory has demonstrated that such sensitization is associated with apoptotic cell death in the cortex, an increase in NMDA receptor NR1 subunit protein and mRNA, and an increase in NMDA receptor function (Johnson et al., 1998; Hanania et al., 1999; Wang et al., 1999). This study sought to extend these findings to an in vitro model and to begin to define the possible underlying mechanisms at the cellular and molecular level. The goals of this study were to determine: 1) whether PCP treatment alone was neurotoxic; 2) whether PCP treatment increased NMDA receptor mRNA and/or function (i.e., NMDA-induced neurotoxicity); 3) whether NMDA treatment in the presence of physiological Mg²⁺ resulted in apoptotic neuronal death, and, if so; 4) its association with alterations in steady-state levels of Bcl-2 family proteins; and 5) whether the apoptotic mechanism involved the production of superoxide anion or other ROS.

PCP Neurotoxicity. Compared with buffer-treated cultures, we never observed a significant effect of treatment with 3 µM PCP alone. Thus, in this sense, the forebrain culture model does not mimic what we observe in vivo. Our interpretation of this finding is that even though the NMDA receptor is up-regulated (see below) as we observed in vivo, there is insufficient "NMDAergic" tone in the culture to produce toxicity. This conclusion is in keeping with the relative sparseness of synaptic connections in culture compared with the in vivo situation.

Effects of Chronic PCP on NMDA Receptor. In this study, an in situ hybridization technique was used to investigate whether NMDA NR1 subunit mRNA levels were changed after PCP treatment. The results of this study indicate that there is an increased density of the NR1 subunit mRNA after 3 µM PCP treatment for 48 h. NR1 mRNA density previously has been shown to be increased in cultured cortical neurons after exposure to several antagonists, including D-AP5, CGS 19755, MK-801, and ethanol, but not after exposure to the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (Grant et al., 1990; Williams et al., 1992; Hu et al., 1996). Although the NR1 protein was not measured herein, these data are consistent with the increase in NR1 mRNA and protein that was observed in vivo after chronic PCP treatment (Hanania et al., 1999; Wang et al., 1999). The mechanism by which PCP and other NMDA antagonists are able to increase NR1 mRNA and protein is currently unknown, but is certainly deserving of further research because this mechanism could be key to understanding neurotoxicity in this and other models.

Mechanisms of NMDA-Induced Cell Death after Chronic PCP. In addition to increases in intracellular sodium, chloride, and water observed after high concentrations of glutamate or NMDA, milder stimuli have been shown to be associated with a calcium-dependent increase in the formation of nitric oxide and other ROS or free radical generators such as superoxide anion and hydrogen peroxide (Gunasekar et al., 1995; Nicholls and Budd, 1998). NMDA receptor-mediated formation of ROS is blocked by inhibitors of mitochondrial electron transport such as rotenone (Dugan et al., 1995) and by a proton ionophore that blocks the uptake of Ca²⁺ into the mitochondria by dissipating the pH gradient (Reynolds and Hastings, 1995). This suggests that Ca²⁺ uptake by the mitochondria triggers the formation of ROS. How this occurs is not clear, but it has been suggested that superoxide anion is derived from complex I of the mitochondrial respiratory chain, at least in brain (Nicholls and Budd, 1998). Our observation that SOD, particularly in the presence of catalase, completely prevented NMDA-induced neurotoxicity, as well as the changes in Bcl-2 family proteins, suggest that superoxide anion may play a role in the regulation of Bax and/or Bcl-X_L. How exogenously administrated SOD/catalase retards this effect of intracellular ROS is not known, but this observation is not without precedence (Gunaseker et al., 1995).