Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures

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Molecular Brain Research 36 (1996) 114-126

Research report

Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures Krystyna R. Isaacs a.*, Gabriel de Erausquin b, Kenneth I. Strauss ~, David M. Jacobowitz ~, Ingeborg Hanbauer c a Laboratory of Clinical Science, NIMH, Building 10, Room 3D-48, 10 Center Drit.,e, Bethesda, MD 20892, USA h Psychiatry Department, Yale Uni~,ersity School of Medicine, West Hat.en, CT 06516, USA c Laboratory of Molecular Immunology, NHLBL Bethesda, MD 20892, USA

Accepted 20 September 1995

Abstract In mesencephalic primary cultures derived from El4 rat embryos, calretinin- and tyrosine hydroxylase-immunoreactive neurons comprised 2% and 5% of the total cell population, respectively, at 6-7 days in vitro. The number of calretinin-immunoreactive neurons was unchanged after a 12- or 24-h exposure to 500/zM kainic acid (KA), but a 50% cell loss was detected after a 48-h exposure to KA. Tyrosine hydroxylase-immunoreactive neurons demonstrated a 50% and 67% cell loss at 24- and 48-h exposures to 500 /zM KA. A 500 /zM N-methyI-D-aspartic acid (NMDA) incubation for 24 h had no effect on calretinin-immunoreactive cell number, but did significantly reduce tyrosine hydroxylase-immunoreactive cell numbers by 26%. In tyrosine hydroxylase-immunoreactive cells, exposure to KA appeared to stimulate the retraction of the neuritic tree and to cause somatic swelling. In contrast, calretinin-immunoreactive neurons developed larger and more complex neuritic trees after a 24-h exposure to 500 /zM KA but not NMDA. lmmunohistochemical coiocalization studies revealed that all tyrosine hydroxylase-immunoreactive and the majority of calretinin-immunoreactive neurons expressed the glutamate receptor subunits GluR2-R3. Very low levels of NMDARI receptor subunits were detected on cells in this culture and GluR4 receptor subunits were not detectable. Our experiments showed that glutamate receptors present in both calretinin- and tyrosine hydroxylase-immunoreactive cells were functional, since phosphorylated cAMP/Ca 2+ response element-binding protein levels were increased in both cell types after 10 or 30 min exposures to 500 /xM KA. The present results indicate that in the mesencephalic cultures tyrosine hydroxylase-immunoreactive cells are more vulnerable to KA excitotoxicity than calretinin-immunoreactive neurons. Keywordw Calcium-binding protein; Dopamine; Kainic acid; N-MethyI-D-aspartic acid; (+)-ot-Amino-3-hydroxy-5-mcthylisoxazole-4-propionic Cytotoxicity; Phosphorylated cAMP/Ca 2÷ response element-binding protein

1. Introduction It is well established that prolonged exposure to excitatory amino acids causes Ca 2÷ influx and delayed cell death in neuronal cultures [7,8]. Single cell imaging studies have demonstrated that the ionotropic glutamate receptor agonists kainic acid ( K A ) and ( + ) - a - a m i n o - 3 - h y d r o x y - 5 methylisoxazole-4-propionic acid ( A M P A ) increase the concentration of ionized intracellular Ca 2÷ ([Ca 2÷ ]i), in a dose-dependent manner, in cultured mesencephalic neurons from 14-day-old rat embryos [10]. In dopaminergic neurons, excessive AMPA/KA receptor stimulation irre-

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versibly increased [Ca 2+ ]i leading to cell death whereas, non-dopaminergic neurons of the same culture survived the increase in [ C a 2 ' ] i [10]. Blockade of voltage-gated L-type Ca 2 ~ channels, glutamate receptors, or inhibition of Ca 2~ release from intracellular stores attenuated AMPA and KA toxicity in dopaminergic neurons [10]. From these findings it was inferred that the regulation of [Ca 2÷]i homeostasis was a complex mechanism that involved Ca 2 ÷-buffering proteins and the action of specialized membrane processes including neurotransmitter receptors, specific ion channels, C a " ' transporters and pumps. In the brain, the calcium-binding protein calretinin (CR) is found in specific neuronal subpopulations [1,19,44] and shares a 5 0 - 6 0 % sequence homology with calbindin D-28k [40,49,45,55], a protein frequently postulated to have a calcium-buffering and/or neuroprotective function

K.R. Isaacs et al. / Molecular Brain Research 36 (1996) 114-126

[2,13,16,17]. Approximately 50% of tyrosine hydroxylase immunoreactive (TH-IR) neurons co-express CR in the substantia nigra of adult rats [18,46]. In cultures of cortical neurons CR-IR cells were more resistant than non-CR-IR cells to glutamate excitotoxicity [29], but damage to CR-IR neurons in hippocampal cultures was reported by M6ckel and Fischer [37] after exposure to the membrane-perforating calcium ionophore A23187. Although CR has been shown to bind Ca 2 ' [6,50], its capacity to buffer Ca 2+ in response to elevated [Ca 2 + ]i levels elicited by excitotoxins has yet to be addressed. In this study we examined the expression of CR and TH in primary cultures of mesencephalic neurons from El4 rat embryos and the co-expression of various subunits of ionotropic glutamate receptors. The present results document the expression of A M P A / K A receptor subtypes in both CR- and TH-containing neurons using antisera to glutamate receptors. In addition, we verified that these receptors were stimulated in both cell types by documenting the immunoreactivity of the phosphorylated form of the cAMP/Ca -'÷ response element-binding protein (CREB) [12,48]. CR-IR neurons were relatively resistant to excitatory amino acid toxicity until prolonged exposure to KA, whereas TH-IR neurons showed significant reductions in cell number as early as 24 h after exposure to KA or N-methyI-D-aspartic acid (NMDA).

2. Materials and methods

2.1. E l 4 mesencephalic cultures The mesencephalic region was dissected from El4 rat embryos in sterile phosphate-buffered saline (PBS) containing 6 m g / m l glucose and mechanically dissociated in culture medium consisting of equal volumes of MEM and HAM-F12 with 12-15% equine serum (Hyclone), 2 mM glutamine, 6 m g / m l glucose and antibiotics. Cells were plated in the above culture medium onto chamber slides (I.5 X 105/8-well or 3 x 105/2-well, Nunc slides) coated with poly-o-lysine (15 /zg/ml) and laminin (10 /xg/ml). After 5 days in vitro (DIV), 10 ~M Ara-C (cytosine a-D-arabinofuranoside) was added to retard glial growth. 2.2. lmmunohistochemist~. On 6 - 7 DIV, either KA, NMDA or AMPA (100 or 500 p,M) was added to the culture medium at 37°C in 4 of the 8 wells and the cells were exposed to the drugs over a time range from I to 48 h. When the exposure lasted 1 h, a 23-h incubation in excitatory amino acid (EAA)-free medium followed. After rinsing in PBS, the slides were fixed with 4% paraformaldehyde for 20 min at room temperature, rinsed again in PBS and then immersed in 0.3% Triton X-100 in PBS for 3 days at 4°C with mouse anti-TH (1:2000, lncstar) a n d / o r rabbit polyclonal anti-CR (1:5000

115

[55]) with 1% goat serum. After rinsing in PBS, the slides were subsequently incubated for 30 rain in 0.3% Triton X-100 containing goat anti-mouse Texas Red (1:100, Cappel) a n d / o r goat anti-rabbit fluorescein isothiocyanate (FITC, 1:300, Cappel) secondary antibodies. The sections were washed in PBS with 0.2% Triton X-100, mounted with glycerol containing p-phenylenediamine dihydrochloride and viewed with a fluorescent microscope. Additional tissue culture cells were grown on slides and incubated for 2 days at 4°C with mouse anti-NMDARI (1:1000, Pharmingen), rabbit NMDAR1 (1:50(I, Chemicon), rabbit anti-GluR2-R3 (1:500, Chemicon), or rabbit anti-GiuR4 (1:500, Chemicon) antibodies in conjunction with either mouse anti-TH (1:2000, lncstar), rabbit anti-TH (1:21100, PelFreeze) or goat anti-calretinin (1:3000 [52]) antibodies and 1% donkey serum. Following a series of rinses and a 30 min incubation with donkey anti-goat Texas Red (1:100, Jackson Immunoresearch) and donkey anti-rabbit or anti-mouse FITC (1:300, Accurate Labs), the immunoreactive cells were examined with a fluorescent microscope. Control slides were treated with incubation solutions without primary antibodies or with goat or rabbit cairetinin antibodies pre-adsorbed with recombinant CR. To verify that KA exposure had a functional effect on both CR-IR and TH-IR cells, the nuclei of these cells were examined for the presence of the phosphorylated-CREB (PCREB). This PCREB antibody has been reported to recognize both the phosphorylated form of CREB, thc phosphorylated activating transcription factor 1 (ATFIL and the Ca2+/cAMP response element-binding modulator (CREM) isoforms [14]. Further references to this antibody will be to PCREB-like immunoreactivity (PCREB-IR). Mesencephalic primary cultures (6 DIV) were fixed after 10 or 30 min exposures to 50() /xM KA and immunoreacted with rabbit anti-PCREB (1:25(}0, Upstate Biotechnologies Inc.) and goat anti-CR (1:2000) or with rabbit anti-PCREB and mouse anti-TH (1:20(10) antibodies. PCREB-IR was visualized with secondary antibodies conjugated with FITC and CR or TH was visualized with secondary antibodies conjugated with Texas Red. 2.3. Detection of NMDAR1 and GIuR2-R3 receptor subtypes with immunoblots Mesencephalic primary cultures grown for 6 days in 6-well cluster trays (750000 cells/well; Costar) were rinsed two times in PBS, then scraped into vials, sonicated, and mixed with 2 X sample buffer [26] and stored at -80°C. For comparison purposes, micropunched tissue from frozen adult rat cerebellum was also collected in sample buffer, sonicated and frozen at -80°C. Tissue culture samples were lyophilized and reconstituted in 2 X sample buffer and SDS-PAGE was performed on 7.5% precast minigels (Bio-Rad) with Tris/glycine buffer, pH 8.3. Immunobiotting was performed using either 1:5000 GluR2-R3 (Chemicon) or 1:1000 NMDARI (Pharmingen)

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and visualized with a chemiluminescence kit (Kirkegaard and Perry Laboratories). 2.4. Neurite process morphometrics

Three representative photographs were made at 32 x from each well of control cells and cells exposed for 24 h to 500 /zM NMDA, AMPA or KA (Leitz fluorescent microscope and Leitz Orthomat E camera). Negatives were coded numerically and cell process number, length and branch frequency were analyzed blindly. Negatives were digitized using a homogeneous light source and analyzed using NIH Image 1.4. lmmunoreactive cells were identified and processes were traced by hand using a mouse and branching points as well as the number of processes were counted manually. All measurements except length were averaged to give mean measurements per cell/high power photographic field. Length measurements were recorded as length/cell. Statistical comparisons were performed by two-way ANOVA for unbalanced designs (when comparing measures individually) followed by Scheff6 post-hoc comparisons where appropriate. Student's t-test was used for simple comparisons. To compare global effects of pharmacological treatments on cell morphology, discriminant analysis was performed on a matrix consisting of mean length x branching points x number of dendrites using treatment as the classification criterion. The autocorrelation (within group) tables demonstrated significant correlation between neuritic number and branching, and the negative correlation between neurite length and branching (not shown). The first significant discriminant function was used to compare between treatments, because it always accounted for the majority of the variance of the whole dataset and was taken to represent the complexity of the network. STATGRAPH (Statistical Graphics Corp., Bethesda, MD) or STATVIEW (Abacus Concepts, Inc., Berkeley, CA) were used for statistical calculations. 2.5. Quantification o f neurotoxicity

Cultures were exposed to 500 /xM KA for 12 h (n = 7 wells), 24 h (n = 8 wells) or 48 h (n = 6 wells) or to 500 /zM NMDA for 24 h (n = 4 wells) or to equal volumes of water (controls: n = 14 wells for KA experiments, n = 4 wells for NMDA experiments). Cells were subsequently identified by CR and TH immunofluorescence and counted in 10 microscopic fields per well directly from the microscope with a 25 X objective. The data represents 2 - 3 independent experiments for each time point. A non-radioactive lactate dehydrogenase activity (LDH) assay (Promega, Cytotox96) was used to compare levels of cytotoxicity in cultures after treatment with 500 p-M KA for 12, 24 or 48 h to control cultures (n = 8 wells/condition; two replications). Aliquots of the media from cultures were removed after exposure to KA. Thus, it was possible to generate an accurate estimate of total cell death from the

same cultures being assayed for specific CR-IR or TH-IR neuron number and appearance. An estimate of cell death derived by counting directly from the slides was also used to provide an alternate measure of cytotoxicity after exposure of the cells in culture to KA or NMDA. Control and experimental cultures were examined for the uptake of propidium iodide (Molecular Probes), a dye which penetrates only non-viable cells and intercalates with single- and double-stranded DNA. Propidium iodide (500 / z g / m l H20) was added to the media after a 23-h exposure to 500 p,M KA (n = 8), 500 /xM NMDA (n = 8) or to control cultures ( n - - 7 ) . After a 1-h incubation the cultures were rinsed and fixed in 4% paraformaldehyde, and coverslipped with glycerol containing p-phenylenediamine dihydrochloride. Cells that had incorporated the fluorescent dye were counted directly from the microscope with a 25 X objective.

3. Results 3. I. Characterization of neuronal phenotypes

Approximately 2% of the total neurons in the mesencephalic culture were CR-IR and approx. 5% were TH-IR. Dual staining at 3 - 1 2 DIV revealed many TH-IR and CR-IR cells, but little colocalization was apparent. In mesencephalic cultures at 7 DIV, multipolar and bipolar CR-1R cells with diffuse cytoplasmic and nuclear immunoreactivity and numerous long, branching varicose processes were frequently observed (Fig. 1). Morphological heterogeneity was also evident in TH-IR neurons. TH-IR cells were either bipolar with oval-shaped cell bodies or multipolar with pyramidal-shaped somas (Fig. 2). TH-immunoreactivity was present in the cytoplasm but the nucleus was unstained. Many TH-containing cells were individually dispersed throughout the well, but 'clusters' of TH-IR cells were also noted. lmmunoblots from cultured mesencephalic neurons revealed an abundance of GIuR2-R3 receptors but no NMDAR1 receptors (Fig. 3). lmmunohistochemical studies of 6 - 7 DIV neurons revealed that the anti-GluR2-R3 antibody labeled cell bodies and proximal fibers but not fine varicosities or nuclei in many cultured mesencephalic neurons including CR-IR and TH-IR cells (Fig. 4). The intensity of the GIuR2-R3 label varied significantly within the cell culture. While all TH-IR cells and the majority of the CR-IR cells were labeled with anti-GluR2-R3 antibodies, a small population of CR-IR neurons failed to label with GluR2-R3 antibodies at detectable levels (Fig. 4). GluR4 receptors were not detected in any cells of these cultures with immunohistochemistry. We used two different antibodies in an attempt to detect NMDARI receptors. With immunohistochemistry, very low levels of NMDARI receptors were detected with the Pharmingen antibody, but no cells were labeled with the antibody from Chemicon. Under the same incubation conditions, cortical neurons in

K.R. lsaacs et al. / Molecular Brain Re.search 36 (1906) 1 1 4 - 1 2 6

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The receptors were detected with monoclonal antibodies to NMDARI (l:ll)00, Pharmingen) and polyclonal antibodies to GIuR2-R3 (1:5000, Chemicon) using a chemiluminescence kit (Lumiglo, KPL).

Cultured C R - I R and T H - I R neurons s h o w e d no morphological signs o f delayed neurotoxicity f o l l o w i n g a continuous 12-h exposure to 500 g,M KA. A l-h exposure to either 100 tzM KA, A M P A , or N M D A f o l l o w e d by 23 h in E A A - f r e e media caused a slight s w e l l i n g in cell bodies and processes in T H - I R cells and a m o d e r a t e extension of neuritic processes in C R - I R cells. Since these m o r p h o l o g i cal changes were marginal and inconsistent, they will not be described in detail. 3.2.1. K a i n i c acid A 24-h exposure to 100 /zM K A failed to decrease the n u m b e r o f C R - I R cells, but did increase the n u m b e r of C R - I R fibers (Fig. 5A). Large swellings in the processes

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Fig. 4. All TH-IR cells examined were immunoreactive for the glutamate receptor subtype GIuR2-R3. Arrows point to representative TH-IR cells which also contain GluR2-R3 receptors. The majority of CR-IR cells contained the GIuR2-R3 receptor (filled arrows), but a very small proportion of the CR-IR cells showed no immunoreactivity for the GluR2-R3 receptor (open arrow). Measurement bar equals 57 ,ttm.

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K.R. Isaacs et al. / Molecular Brain Research 36 (1996) 114-126

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Fig. 5. A: CR-IR neurons and fibers after 100 /zM KA for 24 h. Note the extensive fiber ramifications. B: TH-IR neurons after 100 t~M AMPA for 1 h. Note the extensive process ramifications and swollen ,soma in one TH-IR neuron and the normal appearing one beside it. C, D: TH-IR neurons after 10~l txM KA for 24 h. Numerous TH-IR cells in this condition have spoke-like processes (C) emerging directly from the cell body. D: In this panel, the very long processes ~ e n after treatment of low doses of KA are apparent (arrowheads). Compare with normal cells in Fig. 1 (CR) and Fig. 2 (Ttt). Measurement bar is 83 /zm in (A,BoD) and 21 /.Lm in (C).

(also called torpedoes) were infrequent. TH-IR cells in the same cultures exhibited a rumpled and swollen appearance with numerous very short processes emanating directly from the cell body in a 'spoke-like' manner (Fig. 5C,D). This dose also elicited an increase in the branching of TH-IR varicose fibers (arrowheads, Fig. 5D). The number of CR-IR cells was unchanged after a 12or 24-h exposure to 500 /xM KA but was significantly reduced (down 50% from control values) after a 48-h exposure to 500 /xM KA (F3.31 = 4.86, P < 0.01, Fig. 6). After a 24-h exposure to KA, prominent long CR-IR varicose processes with intensely fluorescent varicosities were also visible (Fig. 1). Quantification of neurite branch and length in CR-IR cells revealed a two-fold increase in average process length and increases in branch point numbers (Table 1). There was a marked reduction in the number of TH-IR cells after a 500 /zM KA treatment for

24 h (down 50% from control values) and 48 h (down 66.5% from control values) (F3.31 = 15.07, P < 0.0001, Fig. 6). Many of the remaining TH-IR cells displayed a spoke-like appearance created by a reduction in number and a shortening of the original distal processes and the production of very short proximal ramifications originating from the soma similar to the effects of the 100 /xM KA shown in Fig. 5C. Occasional long processes with torpedoes were observed but could rarely be traced back to a fluorescent cell body. When quantified, the overall effect was a significant shrinkage of the TH-IR neuritic tree (Table 1). Granular material (identified as autofluorescent lipofuscin) was present in the perikarya of a population of KA-treated cells (Fig. 2, filled arrows). All CR-IR and TH-IR nuclei were immunoreactive for the PCREB family of transcription factors after l(I or 30 min exposures to 500 # M KA (Fig. 7).

K.R. Isaacs et al. / Molecular Brain Research 36 (19961 114-126

Effects of Kainic Acid on Mesencephalic Neurons

emanating from the cell bodies. This increase in TH-IR fibers was most prominent after exposure to 100 /zM KA for 1 h (Fig. 5B). Although the overall discriminant analysis indicated the CR-IR cell morphometrics were modified by AMPA exposure, post-hoe analyses revealed no significant effects on CR-IR neuron number, branch points or neurite length after a 24-h exposure to 500 /xM AMPA (Table 1). No torpedoes or spoke-like processes were visible on CR-IR cells (Fig. 1). In contrast, TH-IR cells appeared more compromised because greater swelling and more spokes were detected. Long varicose TH-IR fibers with only a few torpedoes were also found as a result of 24-h 500 # M AMPA treatment. A subpopulation of normal appearing TH-IR cells also existed in these cultures (arrow, Fig. 2).

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Fig. 6. The n u m b e r of C R - I R cells did not c h a n g c significantly after 12 or 24 h exposure to 5 0 0 ~.M KA. A significant decreasc from control levels of 4 9 . 5 % w a s detected after 48 h, however. In contrast, approx. 5 0 % (24 h) and 6 6 % (48 h) o f T H - I R cells were lost after treatment with 5 0 0 p_M KA. L D H activity levels increased steadily within the culture ( ' " P < 0.01. Scheff~, mean ~z SEM).

3.2.2. ( + )-cr-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid After a 24-h exposure to 100 /xM AMPA a small increase in CR-IR fiber ramifications with occasional large torpedoes on varicose fibers was detected. No CR-IR cells exhibited a spoke-like appearance that would indicate neuronal damage. In contrast, at this dose, a subpopulation of TH-IR cells displayed increased numbers of varicose fibers filled with torpedoes and sporadic spoke-like processes

When mesencephalic cultures were exposed for 24 h to 100 /xM NMDA, no changes in CR-IR and TH-IR cell number or TH-IR neurite number were noted. In contrast, CR-IR neurite number appeared moderately increased (data not shown). A 24-h exposure to 500 /xM NMDA had no effect on CR-IR cell number (t-test, P > 0.1 ), process length, branch points or dendrite number (Table 1). At the same dose, TH-IR process length and branch points were unchanged (Table 1) but a 26% decrease in TH-IR cell number was detected (t-test, P < 0.01).

3.3. Quantification of cytotoxici~ LDH activity in the supernatant steadily increased with prolonged exposure to 500 I~M KA and was significantly

Table 1 Effects of E A A s on C R - I R and T H - I R neuron m o r p h o l o g y C

KA

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9.8 + 1.9 1.0 ± 0.6 124.11 + 31.5

10.5 + 1.0 3.8±0.7 " 269.9 ± 80.0 " "

14.11 +__ 1.4 3 . 3 ± 1.0 157.4 ± 41.9

10.2 + 1.6 2.5+11.8 158.2 ± 62.2

9.3 + 2.4 2 . 2 ± 1.0 159.7 i 55.5

6.8 + 1.6 2.1 .+ 1.1 143.2 _+ 39.2

CR-IR neurons Neurite n u m b e r / H P F Branch p o i n t s / H P F Neurite l e n g t h / c e l l

F 4, = 4.55 P < I).111

b).14 = 4.24 t:' < 11.112

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TH-IR neurons Neuritc n u m b e r / H P F Branch p o i n t s / H P | : Neurite l e n g t h / c e l l

17.2 + 2.2 " " 5.11 _+ 1.1 " " 124.3 + 17.2 " "

36.8 ± 8.6 19.11 "1-4.7 221.4 ± 35.8

l"~lz = 5.46 P < 11.01

24.7 + 3.9 6.5 ± 1.6 171.2 ± 24.1

26.0 + 3.2 12.2 _+ 2.7 8.3 _+ 1.7 2.11 ± 1).6 156.8 ± 19.9 112.3 + 25.8 k].lz = 11.43 NS

15.8 .+ 1.1 2.3 +_ 0.4 88.0 i 8.8 t'].t ~ = 3.14 NS

Results of each experiment arc expressed as mean + SEM. All E A A s were applied tk)r 24 h at concentrations of 500 # M . Discriminant analyses were performed on each experiment separately. F values (with degrees of freedom) and c o r r e s p o n d i n g 1' values are provided below each comparison. 1" values in the discriminant analyses of 0.111 or less allowed correct classification of more than 9 0 % of the neurons, w h e r e a s P values of (I.05 or less allowed correct classification of about 8 0 % o f the neurons. N u m b e r of neurites and b r a n c h i n g points are expressed per c e l l / h i g h I:x)wer field (HPF). A v e r a g e neurite length is expressed per cell. " " P < O.{Jl, " 1' < 0.{)5, Scheff~.

K.R. Isaacs et al. / Molecular Brain Research 36 (1996) 114-126

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different from control levels at 48 h (Fig. 6; F3.22 = 12.81, P < 0.01). A significant increase in propidium iodide uptake by mesencephalic neurons was detected after a 24-h

exposure to 500 /xM KA as compared to neurons exposed for 24 h to vehicle (Fig. 8; F2,2o = 13.99, P < 0.01). A 24-h exposure to 500 /xM N M D A had no effect on

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Fig. 7. PCREB-Iike immunoreactivity is detected in the nuclei of all CR-IR and TH-IR cells in primary mesencephalic cultures after a 10 min exposure to 500 ~ M KA. Filled arrows point to examples of neurons with CR-IR and PCREB-IR or TH-IR and PCREB-IR. Note some cells in the control condition (C) are also immunoreactive for PCREB (open arrows). Measurement bar equals 80 /,tin.

K.R. Isaacs et al. / Molecular Brain Research 36 (1996) 114-126 8"

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Fig. 8. Propidium iodide incorporation in 6 DIV mesencephalic primary neuronal cultures after 24 h exposure to 500 # M KA, but not 5(~) /aM NMDA, revealed significantly more dead cells in the KA-treatcd group. • P < 0.01, Scheff~, mean _+SEM.

propidium iodide uptake by mesencephalic neurons (Fig. 8).

4. Discussion

Recent findings demonstrated that A M P A / K A receptor stimulation in primary cultures of rat embryonic mesencephalic neurons elicited an irreversible increase in [Ca :~ ]i in dopaminergic neurons that resulted in a delayed cell death, while other neurons within the same culture remained viable [10]. Here we addressed the question whether a higher degree of vulnerability was due to the expression of different glutamate receptor subtypes or the presence of other [Ca :+ ], regulatory mechanisms within different neuronal subpopulations.

4.1. Relative resistance of CR-IR cells and vulnerability of TH-IR cells to excitatory amino acids" Exposure of primary cultures of mesencephalic neurons to AMPA/KA-selective glutamate receptor agonists led to the selective cell death of dopaminergic neurons after 24 h. We detected that one of the neuronal subtypes that remained viable during this period of exposure was the CR-containing neurons. The TH-IR cell number was reduced to 50% of control values after a 24-h exposure to 500 /xM KA, whereas the CR-IR cell number was unaffected at this dose. The CR-IR cell number was reduced to 50% of control values after 48-h exposure to KA but TH-IR neurons were much more severely affected. Preliminary studies using a lysate ribonuclease protection assay

123

on primary mesencephalic cells indicated a reduction in TH mRNA after KA exposure (unpublished observations). However, the uptake of propidium iodide and LDH activity were increased after KA treatment suggesting that the reduced number of TH-IR cells was likely to be a reflection of cell death rather than solely a reduction in expression of TH. Protection of CR-IR cells from delayed excitotoxicity was also reported by Lukas and Jones [29]. In their studies, cortical CR-IR cells were resistant to excitatory amino acid or A23187 exposure when compared to cells which did not express CR. Mattson et al. [32] found calbindin immunoreactive (CB-IR) cells in hippocampal cultures were resistant to glutamate- or A23187-induced cell death, while M6ckel and Fischer [37] showed that in hippocampal cultures CR-IR, but not CB-IR cells, survived excitotoxic insult. In the latter report it was proposed that a low density of glutamate receptors may account for the apparent resistance of CR-IR and CB-IR cells to cxcitotoxins [37]. Our results, however, showed a similar distribution of glutamate receptor subtypes on both CR-IR and TH-IR neurons, the most abundant being the GluR2-R3 receptor subunit. The number of NMDAR1 receptors was very low and GIuR4 subunits were undetectable with immunohistochemistry, lmmunohistochemical studies in the substantia nigra compacta of adult rats indicated that both NMDA and A M P A / K A receptors exist in this nucleus [31),41,42]. In the substantia nigra of adult rats, GIuR2-R3 receptors were found colocalized in the majority of the TH-IR and CR-IR cells (lsaacs and Jacobowitz, unpublished observations). The abundant expression of GIuR2-R3 receptors and the absence or extremely low levels of NMDARI receptors in cultured mesencephalic neurons was further confirmed with immunoblotting. These data arc in line with a report by de Erausquin et al. [10], that showed a significant elevation of [Ca 2" ], in mesencephalic cultures after I min exposures to AMPA and KA but no response to NMDA exposure. Additional electrophysiological and pharmacological studies have provided functional evidence for the predominant role of A M P A / K A receptors in the glutamate action on dopaminergic neurons of the substantia nigra compacta [34,35]. Recently, it was shown that phosphorylation of Serine133 in CREB, CREM and AFTI is a consequence of elevated intracellular cAMP or Ca-" levels following transsynaptic stimulation and that PCREB regulates the transcription of early inducible genes [14,48]. The present results show that brief KA exposures increased the phosphorylation of CREB in TH-IR and CR-IR neurons indicating that the GluR2-R3 receptors were functional in both neuronal subtypes. After 24 h, KA-treated TH-IR cells exhibited neuronal swelling, as well as shortened and simplified neuritic arborizations which created a 'spoke-like' image (Fig. 54"). Michel et al. [36] found a similar morphological pattern of neurite retraction associated with cell loss in PCI2 cells

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exposed to various doses of A23187. Several reports in the literature indicated that changes of intracellular free ion homeostasis may account for neuronal swelling. In cultured cerebellar granule cells glutamate exposure abolishes the Na ~ gradient across neuronal membranes [21], thereby producing neuronal swelling [23,47]. The Na+Ca 2~ exchanger regulates [Ca 2~ ]i by facilitating C a 2 + efflux, but when intracellular Na + is elevated to high levels, the function of the Na+Ca 2' exchanger is overwhelmed and Ca 2+ is accumulated in mitochondria [22]. Such a mitochondrial Ca 2" increase may, in turn, lead to C a 2+ phosphate formation [11] which can decrease ATP formation and thereby reduce the function of ATP-dependent enzymes [43]. A role for inhibition of mitochondrial ATP synthesis has been suggested as a mechanism operating in cell death [4]. The destabilization of C a 2+ homeostasis disrupts the cells' energy metabolism regulated by Ca :+ and increases the formation of oxygen radicals that can lead to either necrosis or can initiate a cascade of events that causes apoptosis. While it is conceivable that the TH-IR cell death observed in this study involves apoptosis, studies with PCI2 cells exposed to A23187 excluded cell death via this pathway [36] and preliminary data (Scortegagna and Hanbauer, unpublished data) obtained with mesencephalic cultures revealed no characteristic DNA fragmentation elicited by AMPA or KA exposure. 4.2. After I£AA exposure, neurite growth was frequently detected in CR-IR neurons but only rarely in TH-IR neurons

In mesencephalic primary cultures, CR-IR cells were not only resistant to the excitotoxic effects of KA at 24 h as documented by the lack of cell death, but KA exposure caused a striking extension of neuronal processes. Only at doses of 100 /xM KA or less was a moderate extension of processes visible in TH-IR neurons. Low doses of glutamate, KA, AMPA, A23187, or elevations in K + have been shown to elicit neurite extension in cultured hippocampal pyramidal neurons, suggesting that small increases in [Ca2~ ]i concentrations promote neurite extension [31]. Growth factors combined with excitatory amino acids were also shown to promote Purkinje cell neurite branching in cerebellar cultures [9]. By facilitating Ca 2 ' action with the c-fos promoter calcium response element, PCREB may increase the late-response gene expression responsible for axon outgrowth or neurotransmitter synthesis [12]. The increase in immunoreactive fibers could be due to either increased CR or TH axoplasmic flow or to the sprouting of new fibers. Alternatively, since conformational changes in CR are elicited by Ca2+-binding [24], it is possible that altered antibody recognition by such conformational changes [53] would increase the visibility of thc smallest caliber immunostained fibers by fluorescence microscopy. Development of highly sensitive radioimmunoassays to determine CR or TH content in the pres-

ence of equivalent calcium concentrations will be necessary to address this question. 4.3. Functions of calcium-binding proteins in neurons

The degree of participation of CR and CB as regulators of calcium homeostasis and/or calcium-mediated cellular responses remains unknown. A buffer function for CB has been postulated by several investigators [3,20] and received cursory support from studies demonstrating changes in calcium-buffering capacity in anterior pituitary cells transfected with CB cDNA [28] and in sensory neurons injected with CB [5]. Results of other studies have suggested that CB and CR potentially interact with known (Ca 2+-, MgE+-ATPase, [38]) or unknown [54] cellular targets. Recent reports demonstrated that CR, in a manner similar to calmodulin, significantly changed its conformation upon binding Ca 2+ [24,25]. The results of these studies are not mutually exclusive and suggest that CR and CB can function as both calcium buffers and signal regulators. The selective loss of TH-IR cells following exposure to KA may be due to the lack of CR expression in TH-IR cells. The lack of co-expression of CR and TH in cultured cells was surprising considering the high levels found in the adult rat brain substantia nigra compacta [18]. We are currently investigating whether growth factors and/or other serum factors can affect the amount of colocalization. The destabilization of [Ca 2-]i homeostasis, which we previously have shown to occur preferentially in dopaminergic neurons in culture [10], could provide a mechanism for the increased vulnerability of mesencephalic dopaminergic neurons lacking CR in patients with Parkinson's disease. In a similar manner, the resistance of CR-IR neurons in culture to delayed neurotoxicity may be due to a contribution by CR to the modulation of [Ca-" ~]i homeostasis. With the redefinition of human substantia nigra compacta anatomical boundaries using substance P immunoreactivity [33], we estimate the number of CR-IR neurons in this region is higher than that of CB-IR neurons. Substantia nigra compacta neurons containing calcium binding proteins appear to be spared in Parkinson's disease [15,39,56] and in monkeys [27] exposed to 1-methyl-4phenyi-l,2,3,6-tetrahydropryridine (MPTP), a drug which elevates [Ca 2~ ]i in dopaminergic neurons [51]. It is possible that complete protection from neurotoxicity requires the presence of more than one calcium-binding protein to regulate signal transduction and/or calcium buffering.

Acknowledgements We thank Dr. Lois Winsky for her thought-provoking suggestions and review of this manuscript, as well as Quy Ha and Matthew Wolpoe for their technical assistance.

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