excitotoxic death of a subset of embryonic rat motor neurons in vitro

Excitotoxic Death of a Subset of Embryonic Rat Motor NeuronsIn Vitro

*Hugh J. L. Fryer, ‡Ronald J. Knox, *†Stephen M. Strittmatter, and †‡Robert G. Kalb

*Section of Neurobiology and Departments of†Neurology and‡Pharmacology,Yale University School of Medicine, New Haven, Connecticut, U.S.A.

Abstract: We have used cultures of purified embryonic ratspinal cord motor neurons to study the neurotoxic effects ofprolonged ionotropic glutamate receptor activation. NMDAand non-NMDA glutamate receptor agonists kill a maximumof 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor sub-type-specific antagonists. Subunit-specific antibodies stainall of the motor neurons with approximately the same inten-sity and for the same repertoire of subunits, suggesting thatthe survival of the nonvulnerable population is unlikely to bedue to the lack of glutamate receptor expression. Extracel-lular Ca21 is required for excitotoxicity, and the route ofentry initiated by activation of non-NMDA, but not NMDA,receptors is L-type Ca21 channels. Ca21 imaging of motorneurons after application of specific glutamate receptoragonists reveals a sustained rise in intracellular Ca21 thatis present to a similar degree in most motor neurons, andcan be blocked by appropriate receptor/channel antag-onists. Although the lethal effects of glutamate receptoragonists are seen in only a subset of cultured motorneurons, the basis of this selectivity is unlikely to besimply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca21. KeyWords: Ionotropic glutamate receptors—Motor neu-rons—Excitotoxicity—Cell culture—L-type Ca21 chan-nels—Ca21-dependent cell death—Ca21 imaging.J. Neurochem. 72, 500–513 (1999).

A major advance in the understanding of neuronaldeath in the vertebrate CNS is the recognition of the roleof stimulation of excitatory amino acid (EAA) receptors,i.e., excitotoxicity. In both naturally occurring develop-mental neuron death and model systems of acute andchronic neuronal injury, antagonists of EAA receptors(Choi, 1988, 1990; Lees, 1993; Lipton and Rosenberg,1994; Shaw, 1994; Caldero´ et al., 1997; Solum et al.,1997) can attenuate neuron death. Investigation into thecellular and molecular events within stimulated neuronsthat lead to death will have broad impact on our under-standing of the regulation of neuron number under phys-iological and pathophysiological circumstances.

During normal development, an excessive number ofneurons is generated. Recent evidence suggests that ex-

citotoxicity may participate in regulating cell death dur-ing a circumscribed period of embryonic life. Severallines of evidence support this view: (a) Glutamate recep-tors (the major excitatory neurotransmitter system invertebrates) are expressed at high levels throughout theneuraxis at the time of synaptogenesis (Ziskind-Con-haim, 1990; Walton et al., 1993; Watanabe et al., 1994).Thus, the means exist to activate cells during the natu-rally occurring period of cell death. (b) In a variety of invitro systems, embryonic neurons can die an excitotoxicdeath (Stewart et al., 1991; Estevez et al., 1995). Thus,the immature state of embryonic neurons does not makethem resistant to excitotoxicity. (c) In vivo, applicationof glutamate receptor antagonists decreases the extent ofnaturally occurring cell death, whereas glutamate recep-tor agonists exacerbate cell death (Caldero´ et al., 1997;Solum et al., 1997). These results suggest that activationof glutamate receptors is likely to be a component of thephysiological mechanism by which reductions in cellnumber occur in embryonic life.

One of the fundamental problems with many experi-mental models of excitotoxicity is distinguishing directfrom indirect actions of EAAs. The most parsimoniousview is that excitotoxic injury is due to the direct acti-vation of EAA receptors expressed by the neuron underscrutiny. For example, in cultures of embryonic cerebralcortex, which contain several distinct types of neuronsand glia, it is thought that the direct activation of corticalneuron glutamate receptors is neurotoxic (Choi et al.,

Received June 23, 1998; revised manuscript received August 14,1998; accepted August 26, 1998.

Address correspondence and reprint requests to Dr. R. G. Kalb atDepartment of Neurology, Yale University School of Medicine, P.O.Box 208018, 333 Cedar St., New Haven, CT 06520-8018, U.S.A.

Abbreviations used: AMPA, a-amino-3-hydroxy-5-methylisox-azole-4-propionic acid; APV,D-2-amino-5-phosphonopentanoic acid;BDNF, brain-derived neurotrophic factor; [Ca21]i, intracellular cal-cium concentration; CMF-PBS, Ca21/Mg21-free phosphate-bufferedsaline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EAA, excitatoryamino acid; MK-801, 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate; NO, nitric oxide; NT, neurotro-phin.

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Journal of NeurochemistryLippincott Williams & Wilkins, Philadelphia© 1999 International Society for Neurochemistry

1987; Koh et al., 1990). There is a body of evidence,however, suggesting that the neurotoxic effects of EAAscan be indirect. Activation of glutamate receptors on asmall group of neurons in cultures of embryonic cortical(Dawson et al., 1991, 1993; Dawson and Dawson, 1996),striatal (Strijbos et al., 1996), and cerebellar (Brorson etal., 1994) cells leads to the production and release ofneurotoxic substances [such as nitric oxide (NO)] thatkill neighboring cells.

To begin to identify the determinants of neuronalexcitotoxic vulnerability, we have used cultures of motorneurons purified from the spinal cords of embryonic rats(Camu and Henderson, 1992, 1994). To avoid cell–celland synaptic contact-mediated effects, cells were cul-tured at low densities. This culture system has enabled usto examine the direct effects of glutamate receptor ago-nists on isolated motor neurons and begin to study theionic mechanisms underlying excitotoxic death.

MATERIALS AND METHODS

MaterialsTimed pregnant Sprague–Dawley rats were obtained from

Charles River (Kingstown, NY, U.S.A.). Leibowitz L15medium, glutamate, penicillin/streptomycin, Ca21/Mg21-free phosphate-buffered saline (CMF-PBS), mouse laminin,and sodium bicarbonate were purchased from GibcoBRL(Grand Island, NY, U.S.A.). All other culture reagents,kainate, and NMDA were purchased from Sigma (St. Louis,MO, U.S.A.). Glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801),D-2-amino-5-phosphonopentanoic acid (APV),a-amino-3-hy-droxy-5-methylisoxazole-4-propionic acid (AMPA), andnifedipine were purchased from Research BiochemicalsInternational (Natick, MA, U.S.A). Dr. Eugene Johnson(Washington University, St. Louis, MO, U.S.A.) providedthe hybridoma cell line 192. Recombinant human brain-derived neurotrophic factor (BDNF) was provided byCephalon (West Chester, PA, U.S.A) and neurotrophins(NT) 3 and 4/5 by Genentech (San Francisco, CA, U.S.A.).Anti-rat GluR6/7 and KA2 were obtained from UpstateBiotechnology Inc. (Lake Placid, NY, U.S.A.), and anti-ratGluR1, 2/3, and 4, NMDAR1, and NMDAR 2A/B were agift of Dr. Robert Wenthold (NINDCD, NIH, Bethesda, MD,U.S.A.). All glutamate receptor antibodies bind to intracel-lular epitopes.

Motor neuron purification, drug treatments, andquantification of cell survival

Motor neurons from embryonic rat embryos were purified aspreviously described (Camu and Henderson, 1992, 1994). Inbrief, in CMF-PBS, ventral spinal cords were dissected fromthe embryos of 15-day pregnant rats. After digestion with0.05% (wt/vol) trypsin in CMF-PBS, minced spinal cord pieceswere gently dissociated in L15 containing 2% (vol/vol) horseserum (Hyclone) and 0.1 mg/ml DNase using a 1-ml plasticmicropipette tip. Purified motor neurons were then obtainedusing a two-step procedure. In the first step, the low-buoyant-density motor neurons were aspirated from the interface be-tween L15 medium and 6.5% (wt/vol) metrizamide (Boehr-inger Ingelheim, Niederlassung, Germany). In embryonic day

14 through postnatal day 5 rat ventral spinal cord, only motorneurons express p75, the low-affinity nerve growth factor re-ceptor (Yan and Johnson, 1988). Thus, in the second step of thepurification, metrizamide-isolated cells were purified by immu-nopanning on 100-mm plates (Greiner Labortechnik, Fricken-hausen, Germany) that had been coated previously with goatanti-mouse polyclonal antiserum (Cappel-Organon Teknika,Durham, NC, U.S.A.), followed by the spent medium condi-tioned by the hybridoma cell line 192, which produces themonoclonal antibody to p75. The purified motor neurons elutedfrom the immunopanning plates with monoclonal antibody 192were diluted with L15 medium supplemented with 0.63 mg/mlsodium bicarbonate, 100 IU/ml penicillin, 100mg/ml strepto-mycin, 2% (vol/vol) horse serum, 20 mM glucose, 5mg/mlinsulin, 0.1 mM putrescine, 20 nM progesterone, 0.1 mg/mlconalbumin, 30 nM sodium selenite, and 0.5, 1, and 1 ng/ml ofrecombinant BDNF, NT4/5, and NT3, respectively. The cellswere seeded at low density (4,500 cells or 4.5 cells/mm2) ontoeither 35-mm plates (Nunc, Boston, MA, U.S.A.) or 14-mmround glass coverslips (12 cells/mm2; Assistent, Germany),both of which had been coated with 0.1 mg/ml poly-D-orni-thine followed by 2mg/ml mouse laminin.

Cell death assays were performed after motor neurons werecultured overnight at 37°C in a humid 5% CO2 incubator. Stocksolutions of AMPA, APV, glutamate, and glycine were pre-pared in Locke’s buffer (see below); NMDA, kainate, andCNQX in 0.1 M NaOH; and MK-801 and nifedipine in di-methyl sulfoxide. For toxicity assays, the culture medium wasreplaced with Locke’s buffer (mM: 134 NaCl, 25 KCl, 2.3Ca21, 5 dextrose, 4 NaHCO3, and 5 HEPES, pH 7.2) contain-ing the various drugs (see Results), and the cultures wereincubated at 37°C in 5% CO2. After 1 h, the drug-containingLocke’s buffer was removed, the plates were washed threetimes with fresh Locke’s buffer, and the original medium wasreplaced. In some experiments, the duration of agonist expo-sure was varied. The cells were cultured for an additional day(20–24 h) prior to quantification (see below). See figure leg-ends for controls for each experiment.

Cell survival was quantified for 20–24 h following drugincubations, using a Nikon TMS inverted phase-contrast mi-croscope at a magnification of 1003 (see Results). Survivingcells from at least three nonadjacent 33 3-mm grids wereaveraged from each plate. Each data point (6 SEM) representsthe average of at least three plates. Significance was determinedusing ANOVA with Scheffe´’s post hoc analysis.

ImmunocytochemistryCells grown on coverglass were fixed with 4% (wt/vol)

paraformaldehyde and 0.1% (vol/vol) glutaraldehyde in 0.1Mphosphate buffer (pH 7.4) for 20 min at room temperature.Following several washes in phosphate buffer, cells wereblocked and permeabilized in antibody dilution buffer [Dulbec-co’s modified Eagle’s medium containing 5% (vol/vol) fetalcalf serum and 0.2% (wt/vol) sodium azide] containing 0.2%(vol/vol) Triton X-100 for 10–20 min at room temperature.Coverglass were incubated at 4°C for 1–3 days in antiserumdiluted in dilution buffer. Bound antibody was visualized usinga species-specific fluorescein isothiocyanate-conjugated sec-ondary antibody (Jackson ImmunoResearch Laboratories).Stained cells were mounted on glass slides in Vectashield(Vector Laboratories) and photographed on a Zeiss Axioscope.

J. Neurochem., Vol. 72, No. 2, 1999

501EXCITOTOXIC EMBRYONIC MOTOR NEURON DEATH

Single-cell intracellular Ca21 concentration([Ca21]i) imaging

Because it is leakage resistant and less susceptible to intraor-ganelle compartmentalization compared with fura-2, fura-PE3was used for measuring [Ca21]i (Vorndran et al., 1995). TheKD for the Ca21/fura-PE3 complex is 250 nM, which makesfura-PE3 suitable for measuring Ca21 concentrations in therange of 25 nM to 2.5 mM. Purified motor neurons werecultured on 12-mm glass coverglass that had been glued to35-mm plates with holes drilled out to accommodate the cov-erglass. Preparation of the growth substratum was as describedabove. After 24–48 h of culture (see above), the cells wereloaded for 1 h at 37°C with 20mM fura-PE3/AM (TefLabs,Austin, TX, U.S.A.), which was prepared by combining 1volume of fura-PE3/AM with an equal volume of 20% (wt/vol)pluronic acid solution and then diluted with culture medium.The excess extracellular dye was then washed away (23 3-mlrinse in Locke’s buffer). Neurons were left to equilibrate for 30min in Locke’s buffer prior to Ca21 imaging.

For Ca21 imaging, the plates were placed on the stage of aNikon Diaphot inverted microscope equipped with a 403 ob-jective (Nikon Plan Fluor numerical aperture5 1.3). Theillumination system consisted of a 75-W xenon arc lamp and acomputer-controlled monochromator-based excitation (PhotonTechnology, South Brunswick, NJ, U.S.A.) that was coupled tothe microscope via a fiberoptic cable. Fluorescent images wereacquired with a Hamamatsu C2400 iCCD camera. The culturedensities were such that 20–40 motor neurons could be mon-itored in a single field. Single-field ratio images were acquiredsequentially for 1–4 s at 345- and 380-nm excitation wave-lengths. [Ca21]i sampling was performed at 30- to 60-s inter-vals. The camera gain voltage was adjusted according to theinitial fluorescence intensity of the cells at the beginning ofeach experiment and maintained constant thereafter. Betweenacquisition episodes, the excitation illumination was blockedby automatic shutter control. Pixel/pixel intensity ratio imageswere converted to free [Ca21]i from the relationship [Ca21]i5 Q * KD * [( R 2 Rmin)/(Rmax 2 R)] (Grynkiewicz et al.,1985).Rmin andRmax were determined in intact motor neuronsby applying 1–10mM digitonin in Ca21-free buffer containing0.5 mM EGTA, followed by perfusion with medium containing2.3 mM Ca21. KD is the dissociation constant of the fura-PE3/Ca21 complex (250 nM). The constantQ was determined fromthe ratio of 380-nm evoked fura-PE3 fluorescence in 0 and 2.3mM Ca21-containing buffer. Corrections for background fluo-rescence and camera dark current were carried out as describedpreviously (Knox et al., 1996) and incorporated into the on-lineacquisition program.

In a typical experiment, baseline [Ca21]i values were estab-lished. Motor neurons were then depolarized with KCl or gluta-mate agonists, which were added from a 10-fold concentratedstock solution to the incubation buffer. [Ca21]i was sampled at 30-or 60-s intervals and plotted as line drawings using Image Mastersoftware (PTI, South Brunswick, NJ, U.S.A.).

RESULTS

In this study, we used cultures of purified embryonicday 15 rat spinal cord motor neurons to study excitotoxicneuronal death due to the activation of the motor neu-ron’s ionotropic glutamate receptor. Twenty-four hoursafter plating,;95% of the cells stained for islet 1/2 (Fig.1A) and the low-affinity NT receptor p75 (not shown),

proteins specifically expressed by embryonic motor neu-rons (Yan and Johnson, 1988; Ericson et al., 1992). Thus,we confirm that the purity of the cells in our cultures isthe same as that already described (Henderson et al.,1993). To avoid cell contract-mediated effects, we cul-tured the cells at low densities (;4.5 cells/mm2).

Killing of a subset of cultured, purified motorneurons by activation of ionotropic glutamatereceptors

We first evaluated the toxicity of glutamate to culturedmotor neurons. Purified embryonic rat motor neurons,which had been cultured for 18–24 h following plating(1-day cultures), were exposed for 1 h to various dilu-tions of glutamate. To ensure that NMDA, as well asnon-NMDA, receptors were maximally activated, neu-rons were exposed to glutamate in Locke’s buffer con-taining depolarizing concentrations of K1 but lackingMg21. Identical results were obtained upon stimulatingcells using Locke’s buffer with 5 mM KCl. Control cellswere incubated in Locke’s buffer without glutamate re-ceptor agonists. Cell survival was assessed the day fol-lowing exposure. All neurons with smooth, noncrenatedcell bodies and intact neuritic processes were counted asliving neurons (Fig. 1C). Morphologic criteria for cellsurvival were confirmed using trypan blue exclusion (notshown). Glutamate led to a dose-dependent death ofmotor neurons (Fig. 2A), with a half-maximal (EC50)response of;20 mM. A maximum of 40% of purifiedmotor neurons was sensitive to glutamate-induced celldeath. The same percentage of the cells was vulnerable toglutamate receptor-mediated toxicity in culture grownfor 4 days in vitro (62.16 3.8% survival) as in 1-daycultures. These results indicate that direct activation ofmotor neuron glutamate receptors is toxic.

We investigated the effects of preferentially activatingsubtypes of ionotropic glutamate receptors using thesynthetic glutamate receptor agonists AMPA, kainate,and NMDA (Fig. 2B, C, and D, respectively). Saturatingdoses of either AMPA (.100 mM) or kainate (.500mM), like glutamate, caused the death of;40% of thepurified motor neurons, and AMPA (EC50 5 8 mM) was10-fold more potent that kainate (EC50 5 80 mM). Con-sidering these EC50 values, it is likely that AMPA wasacting exclusively at AMPA-preferring receptors. Withan EC50 5 80 mM, kainate is likely to be acting atAMPA-preferring receptors but potentially has addi-tional actions at kainate-preferring receptors.

To confirm the involvement of non-NMDA glutamatereceptors in inducing motor neuron cell death, CNQX(20 mM), a competitive antagonist of both subtypes ofnon-NMDA glutamate receptors (Watkins et al., 1990),was added to cultures (Fig. 3A). CNQX blocked kainate-and AMPA-induced cell death. As would be expected ofa competitive inhibitor, CNQX was less effective inblocking higher doses of AMPA. To test the possibilitythat AMPA and kainate may be acting indirectly byevoking the release of glutamate, which can then activate


502 H. J. L. FRYER ET AL.

the NMDA receptor, the noncompetitive inhibitor of theNMDA receptor, MK-801, was included in the Locke’sbuffer during the incubation with AMPA or kainate.MK-801 did not affect AMPA- or kainate-induced tox-icity (not shown). These results indicate that excitotoxicdeath of motor neurons can be mediated by the activationof the non-NMDA subtypes of glutamate receptor.

In addition to non-NMDA glutamate receptor-medi-ated motor neuron death, NMDA is also neurotoxic. Thetoxicity of NMDA is dose dependent (Fig. 2D), with anEC50 of ;10 mM. Saturation of the response occurs atconcentrations of NMDA above 300mM, resulting in thedeath of;40% of the purified motor neurons.

The ability of NMDA receptor antagonists to abrogatethe effect of NMDA confirms the participation of theNMDA receptor in excitotoxic motor neuron cell death(Fig. 3B). Activation of the NMDA receptor requires

glycine, an obligatory co-agonist that binds to an allo-steric site on the receptor (Johnson and Ascher, 1987). Inthe absence of glycine, NMDA does not induce motorneuron death. The toxicity of 300mM NMDA can beabrogated with the noncompetitive inhibitor MK-801 (at20 or 100mM) or with the competitive inhibitor APV(300 mM). Thus, activation of the NMDA receptor isequally as effective in causing cell death as activation ofthe other glutamate receptor subfamilies.

It is striking that activation of either non-NMDA orNMDA glutamate receptor causes the death of;40% ofcultured purified motor neurons (Figs. 2 and 3). Al-though it is possible that the NMDA-sensitive motorneurons were distinct from the non-NMDA-sensitivepopulation, we found that addition of all three agonistswas toxic to only 40% of the neurons (not shown). Thiswas further supported by the finding that glutamate, the

FIG. 1. Immunocytochemistry and appearance ofpurified motor neurons. Cultured, purified rat motorneurons, which were fixed after 48 h in culture, wereincubated with antibodies generated against islet1/2 (A) or nothing (B) and then visualized with sec-ondary antibodies conjugated to fluorescein isothio-cyanate. More than 95% of the cultured cellsstained for islet 1/2. C: The appearance of living anddead motor neurons under phase-contrast optics.Viable cells (filled arrows) have smooth perikaryaand intact neuritic processes, whereas dead or dy-ing cells (open arrows) have beaded neurites andcrenellated cell bodies. Bars 5 50 mm (B) and 32 mm(C).



endogenous ligand that activates both NMDA and non-NMDA receptors, kills only 40% of the cells (Fig. 2A).To inhibit the toxicity of glutamate, antagonists of bothNMDA (MK-801) and non-NMDA (CNQX) glutamatereceptors had to be used concurrently (Fig. 4); neitheralone was sufficient. Thus, glutamate can kill a singlepopulation of motor neurons by activating either NMDAor non-NMDA receptors.

Glutamine toxicityUsing the identical motor neuron purification method and

similar culture conditions, Metzger et al. (1998) have re-cently shown that glutamate is not toxic to motor neurons.In their study, however, glutamine was included as a sup-plement to their culture medium. Because glutamine can betoxic to neurons in other culture systems (Rosenberg,1991), we determined its effect on motor neurons.

We found that glutamine caused a dose-dependentdeath of motor neurons (Fig. 5) with an EC50 5 10 mM(Fig. 5A). Because glutamine is metabolized to gluta-mate by neuronal mitochondrial glutaminase (Curthoysand Watford, 1993; Roberg et al., 1995), it seemedconceivable that motor neurons were converting the glu-tamine to glutamate that, when released, activated gluta-mate receptors. To test this possibility, we included MK-801 and CNQX in the Locke’s buffer during the glu-tamine incubation and found a complete blockade ofglutamine toxicity (Fig. 5B). Either alone, however, didnot block glutamine toxicity. The toxic effects of glu-

tamine were apparent soon after plating cells. Threehours following plating, cultures in which glutamine hadbeen included in the incubation medium had 30% fewercells than cells plated without glutamine (Fig. 5B). Inlight of our data, it seems plausible that the vulnerablemotor neurons in the study of Metzger et al. (1998) havebeen eliminated at the time of plating.

Expression of glutamate receptor subunits bycultured purified motor neurons

Because 40% of the cultured motor neurons die as aresult of glutamate receptor activation, it is possible thatthe glutamate-resistant population does not express iono-tropic glutamate receptors or expresses glutamate recep-tors with distinctive subunit composition. To address thisissue, motor neurons were stained with previously char-acterized antisera generated against intracellular epitopesof the various ionotropic glutamate receptor subunits(Petralia and Wenthold, 1992; Petralia et al., 1994a,b;Blahos and Wenthold, 1996) (Fig. 6).

The relative staining density of motor neurons withanti-glutamate subunit-specific antibodies ranged fromlight (score of1 in Table 1) to heavy (score of11). InFig. 6, staining for GluR4, GluR6/7, and NR1 is shown.For any particular anti-subunit-specific antiserum, therelative staining density was the same for all neurons inthe culture, thus indicating no major quantitative differ-ences in the expression of particular subunits amongneurons in our culture.

FIG. 2. Glutamate receptor agonists cause a dose-dependent death of purified motor neurons. Purified rat motor neurons cultured for1 day were exposed for 1 h to various concentrations of glutamate (EC50 5 20 mM) (A), AMPA (EC50 5 8 mM) (B), kainate (EC50 5 80mM) (C), or NMDA (EC50 5 9 mM) (D) in Locke’s buffer (see Materials and Methods). Control cultures were treated identically, but withoutagonists. One day following exposure to glutamate receptor agonists or not, the number of surviving cells was assessed. Data arereported as percentage of control. At saturating concentrations of glutamate receptor agonists, ;40% of the motor neurons die. Thedata points (6 SEM) have been fit to the Michaelis–Menten equation.



High levels of immunoreactivity (Fig. 6; Table 1) wereseen with antibodies to GluR5/6/7, GluR2/3/4c, GluR4,KA-2, and NR2A/B. Lower levels of immunoreactivitywere seen with antibodies to NR1. Anti-GluR1 stainedcultured motor neurons barely above the backgroundlevel. Examples of immunocytological findings are dis-played in Fig. 6 and summarized in Table 1.

In summary, our cultured motor neurons express sub-units for each of the three subtypes of ionotropic gluta-mate receptors. Thus, motor neurons that die from pro-longed exposure to glutamate receptor agonists expressthe cognate ionotropic glutamate receptors. In addition,because all of the subunits are, essentially, uniformlyexpressed on all of the motor neurons, we cannot distin-guish the vulnerable (40%) from the resistant (60%)population of motor neurons based upon apparent differ-ences in expression of ionotropic glutamate receptorsubunits. Of note, the repertoire of glutamate subunitsexpressed by our cultured motor neurons is similar tothat expressed by motor neurons of adult rats in vivo(Jakowec et al., 1995).

Same durations of exposure to glutamate receptoragonists are required to cause significant amountsof cell death

In cultures of cortical neurons containing mixed celltypes, a 5-min exposure to agonists of the NMDA recep-tor is sufficient to cause 90% of the cells to die, whereasa much longer exposure (.3 h) is required for agonists of

non-NMDA glutamate receptors (Koh et al., 1990). Thusfor cortical neurons, excitotoxicity induced by NMDAreceptor activation can be distinguished from activationof non-NMDA receptors on the basis of the duration ofexposure to receptor agonists.

To determine if activation of NMDA or non-NMDAglutamate receptors on motor neurons can be similarlydistinguished, motor neurons were exposed to AMPA,kainate, or NMDA for various lengths of time in Locke’sbuffer, and cell survival was assessed 24 h later (Fig. 7).Though for each agonist increasing the time of exposureresulted in increasing neuronal death, only for exposuresof $1 h was the difference from control significant.Longer exposures did not result in greater amounts ofcell death: the number of cells surviving an overnightexposure to kainate (53.256 5.25%), which was in-cluded in the culture medium, was not significantly dif-ferent from the number of surviving cells exposed for 1 h(Fig. 7). Thus, in contrast to mixed cortical neuron cul-tures, differences in the excitotoxic mechanisms causedby the activation of various glutamate receptor subtypescannot be distinguished by the time required for expo-sure to agonists.

Cell death by activation of each glutamate receptorsubtype: Dependence on Ca21 influx

Ca21 influx has been shown to play a critical role inthe excitotoxic death of cultured Purkinje cells (Brorsonet al., 1995), cerebral cortical (Choi, 1987; Hartley et al.,

FIG. 3. Antagonists of NMDA and non-NMDA glu-tamate receptors abrogate the toxicity of NMDA,AMPA, and kainate. Motor neurons were treatedand quantified as in Fig. 2. A: Motor neuron survivalwas assessed in cultures exposed for 1 h to twoconcentrations of either kainate (KA) or AMPA, inthe presence (lightly shaded columns) or absence(darkly shaded columns) of 20 mM CNQX. Culturesof cells exposed to nothing or CNQX alone servedas controls. CNQX blocks the toxicity of AMPA andkainate but is not toxic. B: Prevention of NMDAreceptor activation abrogates NMDA-induced tox-icity. During the 1-h incubation period with 300 mMNMDA, either 20 mM glycine was added or not or15 mM MK-801 or 100 mM APV was included in theLocke’s buffer. Cells treated similarly but withoutNMDA served as controls. (1) indicates whether adrug was included in the incubation buffer. Treat-ments that prevent NMDA receptor activation(NMDA exposure in the absence of glycine or in thepresence of APV or MK-801) prevent NMDA-in-duced toxicity. The numbers over the columns rep-resent the significance (p , 0.001) as comparedusing ANOVA with Scheffe’s post hoc analysis.



1993), spinal cord (Tymianski et al., 1993), and hip-pocampal (Abele et al., 1990) neurons. In spinal cordculture systems, exposure to EAAs in high extracellularCa21 exacerbates cell death, and conversely, in the ab-sence of extracellular Ca21, cell death is prevented(Tymianski et al., 1993). We wanted to determine ifextracellular Ca21 plays a direct role in motor neuronexcitotoxicity and if the dependence on extracellularCa21 can distinguish the neurotoxic effects of activating

NMDA versus non-NMDA glutamate receptors. To ad-dress these issues, motor neurons were exposed to glu-tamate receptor agonists in incubation buffer containingnominally free, normal (2.3 mM), or high (10 mM) Ca21

(Fig. 8A). In the absence of extracellular Ca21, neitherkainate, AMPA, nor NMDA was toxic. In contrast, inhigh Ca21, the toxicity of these drugs was increasedrelative to that of normal extracellular Ca21. Thus, thecell death induced by excessive activation of each of thesubfamilies of ionotropic glutamate receptor is equallydependent on extracellular Ca21 concentrations.

We next examined the route of Ca21 entry into motorneurons following activation of glutamate receptors. Be-cause purified motor neurons in culture do not possessCa21-permeable AMPA/kainate glutamate receptors(Neve et al., 1997), we investigated the possibility thatCa21 entry upon AMPA/kainate receptor stimulation isvia voltage-sensitive Ca21 channels. To block L-typeCa21 channels, the dihydropyridine nifedipine was in-cluded during the incubation of motor neurons withAMPA, kainate, or NMDA. Nifedipine (20mM) abol-ished the cell death induced by either AMPA or kainate,whereas NMDA receptor-mediated cell death was unaf-fected (Fig. 8B). MK-801, the open channel antagonist ofthe NMDA receptor, did antagonize NMDA-mediatedmotor neuron toxicity (Fig. 3) but had no effect onAMPA or kainate neurotoxicity. These results indicatethat the entry of Ca21 into motor neurons, which isnecessary for non-NMDA receptor-mediated excitotoxicinjury, is via L-type Ca21 channels. Ca21 entry neces-sary for motor neuron death following activation ofNMDA receptors presumably occurs via the Ca21-per-meable NMDA receptor.

Although Ca21 entry into motor neurons is neces-sary for cell death, we wanted to determine if it alone

FIG. 4. Protection against glutamate-induced toxicity requiresthe concurrent antagonism of both NMDA and non-NMDA glu-tamate receptor subtypes. Cultured cells were treated with 1mM glutamate, 20 mM glycine, 15 mM MK-801, or 20 mM CNQXin various combinations as indicated in the figure. (1) indicateswhether a drug was included in the Locke’s buffer. Blockade ofeither NMDA or non-NMDA receptor alone does not prevent celldeath. Concurrent antagonism of all subtypes of ionotropic glu-tamate receptors is required. Asterisks indicate significancefrom controls, which were similarly treated cells but in the ab-sence of any added drug in the Locke’s buffer (*p , 0.05; **p, 0.005; N.S., not significant). Comparisons were made usingANOVA with Scheffe’s post hoc analysis.

FIG. 5. Glutamine is toxic to cultured embryonic motor neurons. A: Cultured motor neurons were treated with various doses of glutamine with20 mM glycine added to ensure activation of NMDA receptors. Glutamine shows a concentration-dependent toxicity with an EC50 of 10 mMand causes the death of ;40% of the cultured motor neurons at doses of $100 mM. B: Blockade of both NMDA (100 mM MK-801) andnon-NMDA (50 mM CNQX) receptors prevents cell death induced by 100 mM glutamine 1 20 mM glycine, suggesting that glutamine ismetabolized to glutamate by motor neurons, which is released and causes glutamate-induced toxicity. Administration of either antagonistalone does not inhibit glutamine toxicity. Additionally, within 3 h following the plating of motor neurons, glutamine incubated with purifiedmotor neurons causes the death of ;30% of the neurons compared with cells cultured in medium lacking glutamine.



is sufficient. To test this hypothesis, we exposed mo-tor neurons to glutamate receptor agonists in normaland depolarizing concentrations of K1. It has beenshown previously that 25 mM extracellular K1

depolarizes purified motor neurons, causing a sus-tained rise in [Ca21] i through the activation of volt-age-sensitive Ca21 channels (Hivert et al., 1995). Inthe absence of glutamate receptor agonists, cell sur-vival in 25 mM K1 is indistinguishable from that innormal (5 mM) K1 (Fig. 9). In addition, the extent ofcell death caused by glutamate receptor agonists issimilar in either depolarizing or normal concentra-tions of K1.

Greater sustained [Ca21]i elevation with glutamatereceptor activation than with K 1-induceddepolarization

Although all neurons express the proteins required forthe assembly of AMPA, kainate, and NMDA receptors, itis possible that not all neurons express functional cellsurface receptors. To examine this issue, we studied thedynamics of [Ca21]i transients in motor neurons stimu-lated with glutamate receptor agonists. To determine ifmotor neurons of this age have functional voltage-acti-vated Ca21 channels, we also determined the [Ca21]i

dynamics of neurons depolarized with 25 mM K1. Com-paring the [Ca21]i transients induced by activation of

FIG. 6. Expression of ionotropic gluta-mate receptor subunits by cultured mo-tor neurons. Fixed motor neurons wereincubated with antiserum to either GluR4(A), GluR6/7 (B), NR1 (C), or with no pri-mary antibody (D) and stained with fluo-rescein isothiocyanate-labeled second-ary antibodies. Antisera to GluR4 andGluR6/7 stained all cells with approxi-mately the same intensity. In Table 1,cells stained with this intensity werescored as 11. Cells stained with anti-serum to NR1 stained very lightly (scoreof 1 in Table 1). All of the cells in aculture stained with a given ionotropicglutamate receptor antiserum have thesame relative staining density. Bar 5 75mm.



non-NMDA glutamate receptors with those induced bydepolarization with 25 mM K1 was of particular interestbecause glutamate receptor activation is toxic whereasK1 depolarization is not, yet both activate L-type Ca21

channels.Motor neurons, cultured at a density that permitted

imaging of 20–40 cells simultaneously, were loadedwith fura-PE3, and agonist-evoked changes in [Ca21]itransients were examined. At this density, the toxicity ofglutamate receptor agonists was the same as found withlower-density cultures (not shown). The free [Ca21]i ofunstimulated motor neurons is;100 nM. Figure 10shows [Ca21]i data from individual neurons. The filledsymbols plotted on each of the graphs represent the meanresponses averaged from all responsive neurons in afield.

Thirty to 60 s following the addition of K1 to theincubation buffer (25 mM final concentration), the mean

[Ca21]i rose sharply to;960 nM. Variations in theresponses of individuals cells were noted. Within 2–3min, [Ca21]i dropped significantly to a sustained level of;270 nM. By 25–35 min following the elevation ofextracellular K1, the variation in the individual re-sponses from the average was reduced and [Ca21]ireached a new baseline of;220 nM. Upon return of theextracellular K1 to normal concentrations, [Ca21]i re-turned to the prestimulus baseline (;80 nM). Most motorneurons responded to elevation of extracellular K1 withthe same pattern of [Ca21]i elevation with the exceptionof a small subpopulation (;15%) of nonrespondingcells. These results were obtained in four separate prep-arations (96 cells).

Stimulation of motor neurons with glutamate recep-tor agonists induced a mean peak rise in [Ca21] i to;430, 540, 460, and 550 nM for glutamate, AMPA,kainate, and NMDA, respectively (Fig. 10). As wasfound for high K1, glutamate, AMPA, kainate, andNMDA all caused a large rise in [Ca21] i, followed by

FIG. 7. The extent of glutamate receptor agonist toxicity de-pends on the duration to which motor neurons are exposed.Cultures of motor neurons were exposed to 100 mM AMPA(darkly shaded columns), 1 mM kainate (lightly shaded columns),or 300 mM NMDA (open columns) for either 0, 10, 30, 60, or 180min in Locke’s buffer and quantified as in Fig. 2. Twenty micro-molar glycine was added to the Locke’s buffer during the incu-bation of cells with NMDA. Cells incubated in Locke’s bufferwithout agonists served as controls. Long periods of exposure(.1 h) were required for agonist-induced cell death.

TABLE 1. Density of staining of various ionotropicglutamate receptor subunits

Glutamate receptor subtype Relative staining densitya

AMPA subunitsGluR1 0GluR2/3 11GluR4 11

Kainate subunitsGluR5/6/7 11KA2 11

NMDA subunitsNR1 1NR2A/2B 11

a Arbitrary units: 0, no staining;1, light staining;11, medium toheavy staining. See Fig. 6 for examples of staining.

FIG. 8. The toxicity of glutamate receptor agonists depends onCa21 influx. A: Cultured cells were exposed to 100 mM AMPA(darkly shaded columns), 1 mM kainate (lightly shaded columns),or 300 mM NMDA (open columns) in Locke’s buffer containing noadded Ca21 or with 2.5 or 10 mM added Ca21. Cells incubatedin Locke’s buffer with varying concentrations of Ca21 but lackingagonists served as controls. In the absence of extracellularCa21, neither AMPA, kainate, nor NMDA was neurotoxic. B:Cultured motor neurons were exposed to 100 mM AMPA, 1 mMkainate, or 300 mM NMDA with (hatched columns) or without(filled columns) 20 mM nifedipine. The L-type Ca21 channelantagonist nifedipine was able to prevent the cell death in-duced by AMPA and kainate but did not prevent NMDA-induceddeath.



a lower sustained elevation. In contrast to the twofoldsustained increase over baseline [Ca21] i levels in-duced by 25 mM K1, all of the glutamate receptoragonists induced on average a threefold increase overbaseline. As was also found for high K1, there was alow percentage of cells that did not respond to gluta-mate receptor stimulation (;15%). In general, all re-sponding cells displayed similar dynamics of [Ca21] itransients. Although the distribution of glutamate-evoked sustained [Ca21] i transients within motor neu-rons was broad, we found no evidence for a distincttype of response that might correspond to an excito-toxicity-sensitive group of cells.

As can be seen in Fig. 10, the pattern of [Ca21]itransient displayed by motor neurons varied as a functionof the specific glutamate receptor agonists applied. Al-though the trial-to-trial responses to the individual recep-tor agonists were very similar, the peak [Ca21]i of theinitial transient was more variable. The responses to bothAMPA and kainate could be inhibited by CNQX but notby MK-801, whereas the response to NMDA wasblocked by MK-801 (not shown).

DISCUSSION

Vulnerability of a subset of embryonic motorneurons to glutamate-induced excitotoxicity

Using specific agonists and antagonists, we havefound that prolonged activation of NMDA or non-NMDA ionotropic glutamate receptors causes excito-toxic death of a population of purified embryonic motorneurons in vitro. Kainate, AMPA, or NMDA, individu-ally or in combination, induces the death of;40% of thecells (Fig. 2), identical to the fraction induced to die byglutamate itself. Therefore, within the vulnerable popu-lation of embryonic motor neurons, it is unlikely thatthere are subpopulations that selectively respond to ac-tivation of only one of the subtypes of glutamate recep-tors.

The glutamate receptor-induced death of spinal motorneurons has been studied using a number of differentsystems. Motor neurons of intact ex vivo spinal cords dierapidly when exposed for 90 min to NMDA, quisqualate,kainate, and other excitotoxins (Stewart et al., 1991). Inadult spinal cords, motor neurons are vulnerable to asingle intrathecal injection of AMPA, quisqualate, or

FIG. 9. Depolarization alone is insufficient to cause motor neu-ron toxicity. Cultured cells were exposed to 100 mM AMPA, 1mM kainate, or 300 mM NMDA in Locke’s buffer containing 5mM (filled columns) or 25 mM (open columns) K1. Culturesincubated with Locke’s buffer in 5 mM K1 served as controls.Depolarizing concentrations of K1 did not cause cell death andhad little effect on the extent of cell death induced by AMPA,kainate, or NMDA.

FIG. 10. K1 at 25 mM or glutamate receptoragonists evoke elevated [Ca21]i transients inpurified motor neurons. [Ca21]i values weredetermined from ratiometric fluorescencemeasurements made every 30 or 60 s (seeMaterials and Methods). After establishingbaseline [Ca21]i, 10-fold stocks of K1 orglutamate agonists were added to the incu-bation buffer. A: Incubation of motor neu-rons in high extracellular K1 (25 mM) evokeda sustained elevation of [Ca21]i that returnedto baseline concentrations when extracellu-lar K1 was reduced to normal concentra-tions (5 mM). B and C: Incubation of motorneurons in either (B) 200 mM glutamate 1 20mM glycine, or (C) 10 mM AMPA (E), 100 mMkainate (F), or 100 mM NMDA 1 20 mMglycine (‚) also evoked rises of [Ca21]i thatwere greater than that induced by high ex-tracellular K1. In A and B the dark line rep-resents the average response.



kainate (Hugon et al., 1989; Urca and Urca, 1990; Kwakand Nakamura, 1995) but require continuous intrathecaladministration of NMDA to induce cell death (Nag andRiopelle, 1990). Studies of cultured spinal cord neuronsalso show neuronal vulnerability to glutamate receptoractivation. With use of lactate dehydrogenase release asa measure of cell death, dissociated spinal cord neuronshave been shown to be vulnerable to AMPA-, kainate-,and NMDA-induced toxicity (Wells et al., 1994; Tsuji etal., 1995; Yin et al., 1995; Regan, 1996). Interestingly,the EC50 values for AMPA and kainate presented here(Fig. 2) are almost identical to those presented by Tsujiet al. (1995). Our results complement these studies byshowing that the direct activation of ionotropic glutamatereceptors on the cell surface of motor neurons by ago-nists can account for at least some of the neuron deathpreviously studied.

However, the extent of motor neuron death in oursystem is lower than that found by some investigatorsusing other culture systems. In particular, Weiss andcolleagues have shown that a.40-min exposure to kai-nate causes the death of 85% of motor neurons in mixedspinal cord cultures (Carriedo et al., 1995, 1996). Inaddition, a higher percentage of the immunocytochemi-cally identified motor neurons die when exposed to kai-nate than when exposed to NMDA. In comparison, pu-rified motor neurons are equally vulnerable to NMDA,kainate, or AMPA regardless of how long these cellsare exposed to these reagents (Fig. 7). What accounts forthe differences between our results and those of Carriedoet al.?

One possibility is the presence of several differentcell types in other culture systems. In mixed spinalcord cell cultures, glutamate receptor agonists maycause cell death both directly, by activating receptorson the susceptible neuron, and indirectly, by activatingreceptors on other cells in the culture that subse-quently release substances toxic to motor neurons. NOis one possible toxic substance (Bonfoco et al., 1995;Strijbos et al., 1996) because the spinal cord in younganimals is enriched in NO synthase-expressing cells(Kalb and Agostini, 1993; Wu et al., 1994; Wetts etal., 1995; Bruning and Mayer, 1996). When corticalneurons are cultured on a glial substrate, excitotoxiccell death occurs as a function of glutamate-dependentrelease of neurotoxic levels of NO (Samdani et al.,1997). Thus, in contrast to our results, glutamate-dependent release of NO or other toxic substances byneighboring cells in mixed spinal cord cultures maykill a larger number of motor neurons. Under ourculture conditions, we find that less than half of thecultured motor neurons succumb to the direct toxiceffects of excessive glutamate receptor activation.

Another possible difference between our results andthose of other investigators may be related to the state ofmaturation of the motor neurons. Whereas many inves-tigators employ cultures of embryonic neurons that havebeen maintained in vitro for 2–3 weeks, we have studied

motor neurons that have been cultured for only 1 daybefore exposure to glutamate receptor agonists. Sensitiv-ity to glutamate receptor-induced toxicity has beenshown to increase as a function of time in culture (Choiet al., 1987; Peterson et al., 1989; Kato et al., 1991;Regan and Choi, 1991). For example,.80% of embry-onic cortical or spinal cord neurons cultured for.18days die when exposed to glutamate, whereas only 25%are susceptible in 4-day cultures (Regan and Choi, 1991).Increasing sensitivity to glutamate toxicity may be re-lated to increasing physiological responsiveness to glu-tamate stimulation, as has been shown for cortical neuroncultures (Murphy and Baraban, 1990). Similarly, motorneurons of older embryonic spinal cord are also moreresponsive to glutamate receptor activation than motorneurons of younger embryos (Ziskind-Conhaim, 1990).To be able to determine if the susceptibility of purifiedmotor neurons to glutamate toxicity might vary as afunction of culture age greater than 1 week requireslonger-term cultures. Our current culture system cannotbe used to address this issue as 95% of the motor neuronsdie by 7 days in vitro.

Why are only a subset of embryonic motor neuronsvulnerable to ionotropic glutamate receptor-mediatedtoxicity? In contrast to hippocampal neurons, in whichvulnerability to NMDA increases in parallel with NMDAreceptor expression (Peterson et al., 1989), in our cul-tures it seems unlikely that the presence or absence ofparticular subunits, or a difference in the relativeamounts of each type of subunit, determines whether aneuron is vulnerable to ionotropic glutamate receptor-mediated toxicity (Fig. 6; Table 1). This extends thefindings of Kato et al. (1991), who have shown that theextent of excitotoxicity in cerebellar granule cell culturesdoes not correlate with the expression of kainate recep-tors.

Although all motor neurons express the same comple-ment of glutamate receptor subunits, perhaps only apopulation expresses functional cell surface receptors.As a measure of glutamate responsiveness, we studiedagonist-evoked rises in [Ca21]i. We found that the re-sponse of the majority of motor neurons to application ofagonist was quite similar. In the absence of a clear subsetof motor neurons with particularly high or low agonist-evoked [Ca21]i transients, it seems most likely that wher-ever the determinant of vulnerability lies, it is unlikely tobe tied to the differential expression of glutamate recep-tors or evoked rises in [Ca21]i.

Role of extracellular Ca21 in glutamate receptor-mediated toxicity

Other investigators have demonstrated the critical roleplayed by Ca21 in mediating excitotoxicity. We find thatmotor neuron death induced by agonists of ionotropicglutamate receptors is dependent upon extracellularCa21 (Fig. 8A). This is consistent with previous dataestablishing that extracellular Ca21 is required for exci-totoxicity (Choi, 1987, 1988, 1995; Abele et al., 1990;



Hartley et al., 1993; Brorson et al., 1995), including theexcitotoxic death of immunocytochemically identifiedmotor neurons in mixed spinal cord cultures (Carriedo etal., 1995, 1996). Lowering extracellular Ca21 preventsthe induced rise of [Ca21]i in motor neurons in ourcultures (not shown).

Although the elevation of [Ca21]i is clearly necessary,our data suggest it is not sufficient for the excitotoxicdeath of the vulnerable population of embryonic motorneurons. Activation of the L-type Ca21 channel is re-quired for AMPA/kainate toxicity (Fig. 8B), yet activa-tion of these channels alone using depolarizing concen-trations of K1 is insufficient for the induction of celldeath (Fig. 9). As was found by Tymianski et al. (Tymi-anski et al., 1993; Tymianski and Tator, 1996), ourresults show that glutamate receptor agonists induce arise in [Ca21]i within an order of magnitude of thatinduced by depolarizing levels of extracellular K1 (Fig.10). Although the changes of cytoplasmic Ca21 inducedby these two stimuli are not dramatically different, theglutamate receptor agonist-evoked rise in [Ca21]i mayexceed a threshold for triggering cell death in the vul-nerable population that is not reached when high con-centrations of extracellular K1 are used to depolarizeneurons. Alternatively, glutamate receptor agonists, butnot high K1, may evoke a rise in [Ca21]i within a distinctsubcellular microdomain that can trigger cell death. Thiswould be consistent with the idea that, for non-NMDAionotropic glutamate receptors, co-activation of the L-type Ca21 channel glutamate receptors is required forexcitotoxic death of motor neurons (Tymianski et al.,1993).

In contrast to non-NMDA ionotropic glutamate re-ceptors, the intrinsic Ca21 permeability of the NMDAreceptor is the most likely route of Ca21 entry uponactivation of these receptors (Fig. 8B). This indicatesthat either of these two different routes of Ca21 entrycan lead to excitotoxic death of motor neurons. Thisresult is of note because it has been shown that Ca21

entry via L-type Ca21 channels activates differentsignaling pathways from those activated by Ca21 entrythrough the NMDA receptor (Bading et al., 1993;Ghosh et al., 1994). In motor neurons, whether Ca21

entry via the NMDA receptors or L-type Ca21 chan-nels converge onto a single death pathway or distinctpathways that ultimately lead to death remains to bedetermined.

These observations bear on the issue of selective vul-nerability. The majority of motor neurons have similaragonist-evoked Ca21 dynamics. In addition, glutamatereceptor subtype-specific agonists evoke distinct patternsof rises in intracellular Ca21 (Fig. 10). Yet despite thesimilarities, only a subset of motor neurons is vulnerableto excitotoxic death. These results strongly suggest thatthe determinant of vulnerability is downstream of the risein intracellular agonist-evoked Ca21.

Role of excitotoxicity in developmentalneuronal death

The death of neurons during development is a normalprocess during the formation of the nervous system, andin vivo studies suggest the participation of glutamateexcitotoxicity in developmental death. For example, Op-penheim and colleagues showed that in neuromuscularlyblocked chicken embryos, increasing spinal cord synap-tic activity, using electrical stimulation, increases motorneuron cell death (LeRay et al., 1993). More directevidence comes from studies in which it has been shownthat NMDA receptor antagonists applied to chicken em-bryos during development reduce death of spinal motorneurons (and of other CNS neurons such as brainstemauditory neurons), whereas glutamate receptor agonistsexacerbate motor neuron death (Caldero´ et al., 1997;Solum et al., 1997). Excitotoxic mechanisms of naturallyoccurring death can be expanded to other excitatoryreceptor systems. Hory-Lee and Frank (1995) haveshown that nicotinic acetylcholine receptor antagonists atdoses that do not inhibit muscle activation but are capa-ble of inhibiting the neuronal nicotinic acetylcholinereceptor reduce the naturally occurring death of motorneurons. A mechanism common to excitatory recep-tor systems is the sustained, possibly toxic, levels of[Ca21]i. Indeed, it has been reported that raising [Ca21]iin vivo with Ca21 ionophores transiently increases therate of spinal motor neuron death during development(Ciutat et al., 1995). Thus, investigation into the mech-anisms by which glutamate kills immature neurons mayprovide insight into the life-and-death decisions regulat-ing neuron number throughout the developing nervoussystem.

At embryonic day 15, motor neuron cell death in therodent spinal cord is well underway, with more than halfof the cells that are destined to die having done so by thispoint (Flanagan, 1974; Oppenheim, 1986; Oppenheim etal., 1986). Our results suggest that determinants of sus-ceptibility to the toxic effects of glutamate downstreamof agonist-evoked rises in [Ca21]i are likely to be presentin motor neurons during the developmental death period.Identification of these molecular determinants in embry-onic neurons that confer susceptibility may also provideinsight into the mechanisms of pathologic cell death thatfollows trauma or degenerative nervous system disor-ders.

Acknowledgment:We thank Dr. Eugene Johnson for mono-clonal antibody 192 and Pat Lampe for help in growing thesecells; Dr. Robert Wenthold for polyclonal antisera to GluR1,GluR2/3, GluR4, NMDAR1, and NMDAR2A/B; Dr. ThomasJessell for monoclonal antibodies to islet 1/2; Cephalon forBDNF; and Genentech for NT4/5 and NT3. We especiallythank Drs. Chris Henderson and Vilma Arce for teaching us therat motor neuron purification technique. Thanks go also to Drs.James Howe and Thomas Hughes for their analysis and helpfuldiscussions of our work, Dr. Fiona Inglis for help with statis-tics, and Drs. Susan Stegenga and Joshua Brumberg for theirreview and comments on the manuscript. The work presented



was supported by PHS NS 29837 and NS 33467. S.M.S. is aJohn Merck Scholar in the Biology of Developmental Disabil-ities in Children.

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excitotoxic death of a subset of embryonic rat motor neurons in vitro

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