Journal of Neurochemisrry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Synaptic Regulation of Immediate Early Gene Expression in Primary Cultures of Cortical Neurons *T. H. Murphy, *JfP.F. Worley, $IIY. Nakabeppu, $IIB. Christy, IJ. Gastel, and *§J. M. Baraban Departments of *Neuroscience, tNeurology, $Molecular Biology and Genetics, and $Psychiatry and Behavioral Sciences, and 11 Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, and YDepartment ofEnvironmenta1 Health Sciences, School of Hygiene and Public Health, Baltimore, Maryland, U.S.A.

Abstract: Neuronal stimulation can rapidly activate several immediate early genes that code for transcription factors. We have used primary cortical cultures to study the regulation of four of these genes, c-fos, c-jun, jun-B, and ~$268. Immunocytochemical studies with antibodies to Jun-B, c-Jun, and c-Fos demonstrate intense staining in the nuclei of a subset of cortical neurons in mature cultures (2 1-25 days in vitro) but not young cultures (3-7 days in vitro). To assess whether this immunoreactivity may be induced by spontaneous synaptic activity that develops with a similar profile, we examined the effects of agents that reduce this synaptic activity. Tetrodotoxin or N-methyl-D-aspartate receptor antagonists suppress basal immunoreactivity to Jun-B and cFos, but not c-Jun, indicating that the basal level of c-Jun expression is not dependent on electrical activity. Picrotoxin,

an agent that increases synaptic excitation indirectly by blocking inhibitory synaptic currents mediated by y-aminobutyric acidAreceptors, markedly increases the percentage of neurons displaying immunoreactivity to c-Fos, c-Jun, JunB, and Zif268. Northern analysis suggests that the increases in immunostaining induced by picrotoxin are secondary to a rapid increase in mRNA for these proteins. These findings provide evidence for rapid transcriptional regulation of immediate early genes in cortical neurons by synaptic activity. Key Words: N-Methyl-D-aspartate-Transcription factorCortex-c-Fos-c-Jun-Plasticity. Murphy T. H. et al. Synaptic regulation of immediate early gene expression in primary cultures of cortical neurons. J. Neurochem. 57, 18621872 (1 99 1).

Cell surface receptor stimulation by growth factors or neurotransmitters can trigger rapid activation of a set of genes referred to as immediate early genes (IEG) (Greenberg et al., 1985, 1986; Lau and Nathans, 1985; Morgan and Curran, 1986). Many of these genes code for transcriptional regulatory factors and are therefore thought to coordinate changes in gene expression underlying long-term alterations in cellular activity elicited by these signaling agents (Goelet et al., 1986; Morgan and Curran, 1989; Lau and Nathans, 1991; Sheng and Greenberg, 1990). The proposed involvement of transcription-factor immediate early genes in neuronal plasticity has focused attention on understanding the synaptic activation of these genes in brain neurons. To pursue this question, we have examined this process in vitro using primary cortical cultures. As these cultures mature, the neurons form functional synapses

and exhibit spontaneous activity (Dichter, 1978), making them well-suited for investigation of synaptic regulation of immediate early genes. In this study, we have used pharmacological agents to modulate synaptic activity in these cultures and assessed their effects on the expression of four transcription factors, c-Fos, cJun, Jun-B, and Zif268, which are induced rapidly by neuronal stimulation in vivo (Saffen et al., 1988; Morgan and Curran, 1989; Cole et al., 1990; for review, see Sheng and Greenberg, 1990; Wisden et al., 1990). c J u n (Maki et al., 1987)and Jun-B (Ryder et al., 1988) are homologous proteins that form heterodimers with c-Fos or other members of the Fos family (for review, see Curran and Franza, 1988). These complexes bind to DNA in a sequence-specificfashion to regulate target gene expression. Zif268 (Christy et al., 1988), also referred to as NGFIA (Milbrandt, 1987), Krox-24 (Le-

Received February 11, 199 1; revised manuscript received April 24, 199 1; accepted April 24, 199 1. Address correspondence and reprint requests to Dr. T. H. Murphy

6-cyano-7-nitroquinoxaline-2,3-dione; DIV, days in vitro; DMSO, dimethyl sulfoxide; GABAA,y-aminobutyric acid, ;IEG, immediate early genes; MEM, minimal essential medium; NMDA, N-methylD-aSpartate; PDA, phorbol diacetate; SDS, sodium dodecyl sulfate; SSC, saline-sodium citrate; TBS, 0.05 M Tris-C1, pH 7.4/15 g/L NaCI; TTX, tetrodotoxin.

at Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. Abbrevialions used: APV, 2-amino-4-phosphonovalerate; CNQX,

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SYNAPTIC REGULATION OF IMMEDIATE EARLY GENE EXPRESSION maire et al., 1988), or egrl (Sukhatme et al., 1988), is a member of the zinc finger family of DNA binding proteins. Previous studies have demonstrated differential regulation of these genes in neuronal systems both in vivo and in vitro (Bartel et al., 1989; Worley et al., 199 1). For example, synaptic activation of dentate granule cells by perforant path stimulation induces zif268 but not c-fos (Cole et al., 1989; Wisden et al., 1990). Accordingly, in this study we have investigated regulation of several transcription factor genes. EXPERIMENTAL PROCEDURES Cell cultures and media Cell cultures were prepared from gestation day 17 SpragueDawley rat fetal cerebral cortex, using a papain (EC 3.4.22.2) dissociation method. The dissociated cells were resuspended at a density of 1.2 X lo6cells/ml in minimal essential medium (MEM) supplemented with 5.5 g/L glucose, 2 mM glutamine, 10%fetal calf serum, 5% heat-inactivated horse serum, 50 U/ml penicillin, and 0.05 mg/ml streptomycin, plated onto polylysine-coated (10 pg/ml) 35-mm culture dishes in 1.5-2 ml of medium or 12-well dishes (1 ml medium), and placed in a 37°C CO2-buffered incubator. The cultures were fed by the addition of MEM with 5.5 g/L glucose, 5% heat-inactivated horse serum, and 2 mM glutamine, after about 4-6, 12- 14, 16- 17, and 19-2 1 days in culture, with removal and replacement of approximately 60% of the medium. Cultures were not fed for at least 24 h before fixation for immunostaining to avoid any possible effects of refeeding on expression of transcription-factor proteins. During experimental treatments prior to immunostaining or RNA measurements, cultures were maintained in MEM with 5% heat-inactivated horse serum in a 37°C 5% COz incubator. Compounds of interest were added directly to the cultures in 50-200X stock solutions. All compounds used were dissolved in water, with the exception of actinomycin D and cycloheximide, which were dissolved in dimethyl sulfoxide (DMSO), diluted to 10% with water, and then added to cultures at 1OOX. Vehicle controls were included in each experiment. The addition of water (0.5-270 of final volume) or DMSO (0.1%)had no detectable effect on basal or stimulated IEG immunostaining. Handling of cultures during experiments (6 h prior to fixation) failed to induce c-Fos compared to cultures that were left unperturbed until immediately prior to fixation. Basal c-Fos immunoreactivity [21- to 25-days in vitro (DIV) cultures] was examined after switching cultures to serum-free MEM for up to 2 days and found to be identical to that observed in cultures fed with MEM containing 5% heat-inactivated horse serum. Cell viability was assessed following treatments used to modulate expression of transcription factor proteins in 22DIV cultures, using the membrane-impermeant fluorescent compound propidium iodide (4 pg/ml) as described by Krishan (1975). After treating cells with tetrodotoxin (TTX) ( 1 pM, 8 h), picrotoxin (10 pM, 8 h), phorbol diacetate (PDA) (10 pM, 4 h), or vehicle (water), neuronal viability was greater than 95% in all cases (compound applied in IOOX stock solutions).

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137 NaQ5.4 KC1,2.5 CaCl,, I .O MgS04, contained (in d) 0.44 KH2P04, 0.34 Na2HP04 (7H20), 10 Na+-HEPES, I NaHC03, 0.01 glycine, and 5 glucose (pH 7.4 and 340 mOsm). All recordings were made using the whole-cell variant of the patch-clamp technique at room temperature (Hamill et al., 198I), using 4- to 8-MQ glass micropipettes ( 1B/20 F4; World Precision Instruments) and an Axopatch IC patchclamp amplifier as described (Murphy and Baraban, 1990). The pipette solution used for current-clamp and voltageclamp experiments contained (in mM) 140 potassium methyl sulfate, 2 CaC12, 1 1 potassium ethyleneglycol-bis-N,N,N,N'tetraacetic acid, and 10 K+-HEPES. In some experiments, the following pipette solution was used to reverse spontaneous currents (in d): 120 CsC1, 20 tetraethylammonium chloride, 2 MgCI2, 1 CaC12,2.0 EGTA, 10 HEPES-NaOH. Seal resistances were greater than 0.5 GQ for all cells used. Unless noted all cells were voltage-clamped at -60 mV for all determinations and ages in culture. Recordings were made in a static bath (1-2 ml) in a 35-mm tissue culture dish. Antagonists were applied either by pressure ejection (1 5-20 psi for 0.5-5 s) from 2- to 6-pm-tip diameter glass pipettes (compounds diluted into bathing medium), positioned 250-500 pm from the cell of interest, or by addition directly into the bathing medium at a 1.00-fold concentration dissolved in bathing medium. Direct addition of bathing medium or water alone (20 p1 to a 2-ml bath) failed to affect ongoing electrical activity or cell stability.

Immunostaining Affinity-purified rabbit polyclonal antisera to c-Fos (Oncogene Science), Jun-B, c-Jun, and Zif268 were used for immunostaining. These antisera were prepared against residues 4-17 of human c-Fos (not contained in Fra proteins), a synthetic peptide correspondingto amino acids 45 to 6 1 of mouse Jun-B (Nathans et al., 1988), and c J u n and Zif268 expressed in Escherichia coli (Christy and Nathans, 1989; Nakabeppu et al., in preparation). Jun-B and c-Jun antisera were prepared by Y. Nakabeppu. Zif268 antiserum was prepared by B. Christy. To purify each antibody, the synthetic peptide or purified protein was linked to Sepharose 4B and used as an affinity column. Specificity of these antisera has been demonstrated using recombinantly expressed proteins. For immunostaining, cells were fixed for 1 h in 4% formalin, 0.1 M phosphate buffer (pH 7.4), washed with 0.05 M Tris-C1, pH 7.4/15 g/ L NaCl (TBS), permeabilized with 0.2% Triton X-100 (in TBS), and incubated with 3% normal goat serum for I h and then with primary antisera in 1% normal goat serum at 4°C overnight. Primary antisera were used at the following concentrations (in pg of affinity-purified protein/ml): 1 .O Zif268, 0.4 c-Jun, 0.4 Jun-B, and 0.1 c-Fos. After removal of primary antisera, cultures were processed using the avidin-biotinperoxidase method of antibody detection (Vector Labs), with the addition of 0.8 mg/ml NiCI, in the color development step. Omission of primary antisera or preadsorption of antisera with their respective antigens (approximately 1OX molar excess of antigen) totally abolished staining. Incubation of cFos antiserum (0.1 pg/ml) with the corresponding Fos-B peptide at 1 pg/ml did not result in a detectable reduction in c-Fos immunostaining.

Electrophysiology

Quantitation of immunostaining

For electrophysiological measurements, cells were switched to a Hank's balanced salt solution (by triple exchange) that

After immunostaining, cells were preserved in glycerol and examined under Hoffman modulation/bright-field optics. To

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estimate the percentage of cells exhibiting nuclear immunostaining, three to five fields at 50X magnification in duplicate wells were picked at random and the number of presumed neurons was counted. In these cultures, neurons tend to form distinct clumps of rounded, process-bearing cells, which are readily differentiated from the flat, underlying, confluent nonneuronal cells using Hoffman optics. These cells can be considered neurons since they display the following characteristic features: staining with neuron-specific enolase antiserum (Marangos et al., 1979; Murphy et al., 1990), spontaneous electrical activity, fast inward and outward voltage-sensitive currents (measured under voltage clamp), and sensitivity to N-methyl-D-aspartate (NMDA) toxicity [nonneuronal cells in cortical cultures are resistant (Choi et al., 1987)l. After estimating the number of neurons per 50X field within an experiment, the number of stained neurons was counted in at least three fields in each of two duplicate wells (=200-400 neurons). As bright-field optics are preferable for visualization of immunostaining, and Hoffman optics for identification of neuronal morphology, counting of stained neurons was performed by alternating between these optical modes.

Northern blot hybridization Extraction and preparation of RNA were performed as described (Cole et a]., 1990). Briefly, 20 pg of RNA (approximately 2 or 3 wells of a 12-well plate) was used for each lane. The amount of RNA was estimated by measuring absorbance at 260 nm. Ethidium bromide fluorescence of 18s and 28s ribosomal RNAs indicated that equivalent amounts of RNA were present in each lane of the blots used for cDNA hybridization. Blots were prehybridized at 42°C for 3 h and hybridized overnight at 42°C as described (Cole et al., 1990). The blots were washed twice for 30 min each in 2X saline-sodium citrate (SSC), 0.1%sodium dodecyl sulfate (SDS) at room temperature, followed by two 15-min washes in 0.2X SSC, 0.1% SDS at 55°C. cDNA probes were prepared by nick translation of inserts prepared from Bluescript plasmids containing coding regions of c-fos, ,jun-B, and zlf268.

In situ hybridization was performed as described using a 35S-labeledantisense RNA probe to the full-length rat zif268 clone (Cole et al., 1990). For in situ hybridization, cells were plated on 13-mm round-glass coverslips (Gold Seal; polylysine coated, 50 pg/ml, 1 h; Clay Adams), which were contained in 24-well plates and covered with 0.5 ml of medium. After experimental treatment of these cultures (within the wells), cells were fixed in 4% paraformaldehyde, 0.1 M sodium phosphate, pH 7.0, for 10 min, rinsed with 2X SSC, acetylated in triethanolamine/acetic anhydride for 10 min, rinsed with H 2 0 for 30 s, and dehydrated with the following alcohols: 70, 95, 100, and 95% ethanol (30 s in each). The coverslips were then removed and glued to microscope slides with silicone. Riboprobe ( lo6 cpm) in 50 jd of hybridization solution (Cole et al., 1990) was then added and slides were coverslipped and incubated at 56°C overnight in a humidified chamber. The coverslips were removed under 2X SSC and the slides were washed twice in 2X SSC at room temperature ( I5 min each), then treated with 10 pg/ml RNase A for 30 min at 30"C, washed for another 30 min in 2X SSC, and dehydrated with ethanol (see above). The slides were then coated with Kodak NTB-2 emulsion (diluted by 5%) and developed using Dektol after 5- 15 days of exposure.

J Neitrorlicw., I'ol 57. No. 6 , 1991

RESULTS Basal levels of immunostaining In initial experiments, we examined the basal level of expression of c-Fos, c-Jun, and Jun-B proteins as these cultures were allowed to mature for up to 25 DIV (Fig. 1A-D). After 1-2 weeks in culture, immunocytochemical staining for each of these proteins was observed over the nuclei of a subset of presumed neurons (< I O%), with little or no staining detected in glial elements. This immunostaining was distinct from background levels but less intense than that observed in basal or stimulated mature cultures in parallel experiments. In cultures maintained for 21-25 days, approximately 20% of neurons display c-Fos, c-Jun, and Jun-B staining under basal conditions (Fig. 1E). Basal Jun-B and c-Fos immunostaining was largely absent from glial cells in 21- to 25-day cultures, while c J u n showed a variable, low level of glial expression. As observed for c-Fos and Jun-B, basal staining for Zif268 was restricted to nuclei of presumed neuronal cells. In contrast to these other proteins, the percentage of neurons expressing Zif268 immunoreactivity under basal conditions in mature (21- to 25-DIV) cultures varied to a much greater extent, over a range of 1-10%. Spontaneous electrical activity In parallel experiments, we examined the spontaneous electrical activity displayed by neurons in these cultures. In whole-cell recordings, spontaneous synaptic activity was present in greater than 85% of neurons cultured for 2 1-25 days (-60-mV holding potential, in the presence of physiological Ca2+and Mg2+),which was primarily in the form of fast inward currents (m200-ms duration) that reversed near 0 mV (Fig. 2A and B). Current-clamp records indicate that the synaptic currents were of sufficient magnitude to elicit regenerative responses (Fig. 4A). Spontaneous activity was not reliably observed until approximately 7 DIV (Fig. 2). The frequency and amplitude of the fast inward currents increased significantly between 14 and 2 1-25 DIV (Fig. 2B). Electrical activity correlates with c-Fos and Jun-B expression To assess directly whether basal levels of immunoreactivity reflect activation of these genes by spontaneous electrical activity, we examined whether TTX would lower basal expression of these transcription factors. The addition of TTX (1-10 p M ) led to a reduction in the percentage of Jun-B- and c-Fos-positive presumed neurons, without affecting c-Jun immunoreactivity (Fig. 3). As expected, following the addition of TTX spontaneous electrical activity was barely detectable. Previous studies have suggested that N M D A receptor activation may play a key role in eliciting immediate early gene activation (Szekely et al., 1988; Cole et al.,

SYNAPTIC REGULATION OF IMMEDIATE EARLY GENE EXPRESSION

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FIG. 1. c-Fos, Jun-8, c-Jun, and neuron-specific enolase immunoreactivity in untreated 21- to 25-DIV

C-JUN C-FOS cortical Neuron-specific cultures.enolase. A: c-Fos. E: 8: Development Jun-B. C: c-Jun. of Jun-B, D:

d u n , and c-Fos immunoreactivity in primary cortical neurons. Data shown are the mean percentages of stained neuronal nuclei from at least three separate experiments performed in duplicate from different cell platings. Analyses of variance indicateda significant effect of age in culture ( p < 0.05). Calibration bar = 30 pm.

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with expression of these proteins, since the addition of a phorbol ester, 12,13-PDA(2 h), to cultures pretreated with MK-801 for 4 h resulted in a robust increase in Jun-B and c-Fos immunoreactivity (>50%of neurons stained) (data not shown). In contrast to the effects of these glutamate receptor antagonists, antagonists of other transmitter receptors including mianserin (serotonin), atropine (muscarinic), haloperidol (dopamine), and timolol (adrenergic) at 1 pM failed to affect basal c-Fos expression in 21- to 25-day cultures, while 1 pM MK-80 1 or TTX reduced basal immunoreactivity by more than 80% in the same experiment (data not shown). To determine whether blockade of nonNMDA glutamate receptors may also reduce basal immunostaining, we examined the effect of 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) (Honore et al., 1988). At 10 pM this drug was without effect, and at

24 DIV Corilcal Neurons

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FIG. 2. Spontaneous electricalactivity in primary cortical neurons. A Representativevoltageclamp records from cortical neurons after 3, 8 , 15, and 21 days in culture. Cells were voltage-clamped at

-60 mV as described in Experimental Procedures. Calibration bars = 100 pA and 10 s. B: Aggregate data on spontaneous electrical activity from voltage-clamped neurons (-60 mV) of the indicated age. At 3 days in culture, 0 of 7 cells exhibited spontaneous activity; at 8 days, 9 of 11 cells; at 15 days, 9 of 13 cells; and at 21-25 days, 31 of 36 cells. 0

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1989). Accordingly, we examined the effects of NMDA receptor antagonists on both immunostaining and spontaneous electrical activity in 2 1- to 25-day cultures. In contrast to TTX, the NMDA receptor antagonist 2amino-4-phosphonovalerate (APV) ( 100-300 p M ) added to the bathing medium did not totally suppress, but markedly reduced, the peak amplitude of spontaneously occurring electrical activity (72 f 15% reduction in amplitude; n = 6 cells; p < 0.05). APV (300 p M ) applied to cultures for 6 h reduced the percentage of neurons displayingc-Fos and Jun-B immunostaining by more than 70% without significantly affecting c J u n (Fig. 3B). Half-maximal effects of APV on c-Fos staining were observed at concentrations less than 30 pM. The noncompetitive NMDA receptor antagonist, MK801 (10 p M ) (Wong et al., 1986), added to cultures also produced a time-dependent reduction in Jun-B and c-Fos immunoreactivity without affecting c J u n immunoreactivity (Fig. 3). Half-maximal effects of MK-801 on c-Fos staining were observed at concentrations less than 1 pM. The ability of NMDA receptor antagonists to decrease basal levels of Jun-B and c-Fos immunoreactivity does not appear to reflect nonspecific interference J.Neurochem.. Vol. 57, No. 6. 1991

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FIG. 3. Reduction in basal Jun-B and c-Fos expression by TTX or NMDA antagonists. A: The effect of TTX (10 &I MK-801 ), (10 f l ) and , cycloheximide(10 pg/ml) on spontaneous expressionof c-Jun and Jun-B imrnunoreactivityin 21- to 22-DIV cultures (the means of duplicate experiments from two separate cell platings). Cyclohex., cyclohexirnide. B Effect of 6 h of MK-801 (10 APV (300 pM), or TTX (1-10 treatment on basal c-Fos, Jun-B, or c-Jun immunostaining. Values shown are the means SEM of at least three separate duplicate experiments.' p < 0.05, pairedt test comparing the percentageimmunoreactive neurons in the control to that with the indicated treatments.

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SYNAPTIC REGULATION OF IMMEDIATE EARLY GENE EXPRESSION

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FIG. 4. Properties of spontaneous electrical activity in 21- to 25DIV neurons. A: Spontaneous activity measured under current clamp (resting potential = -53 rnV; calibration = 50 ms/l5 mV). 8: Effect of picrotoxin (10 p M ) and TTX (1 pM) under voltage clamp (holdingpotential = -60 mV; calibration = 10 s / l O O PA).C: NMDA antagonists (100 f l APV) reduce spontaneous activity (10 pM

picrotoxin present). APV was applied by pressure ejection as described in Experimental Procedures (holdingpotential = -60 mV; calibration = 10 s/200 PA). D: An example of a picrotoxin-treated cell (10 p M ) that has a partial response to pressure-ejectedAPV (300pM) (holding potential = -60 rnV; calibration = 2 s / l O O PA). The break in the record following APV application indicates 1 min of recovery.

50 pM it markedly reduced basal c-Fos staining (52 -+ 12% reduction; p < 0.05, paired t test). These experiments were performed in the presence of 1 mM glycine, making it unlikely that CNQX was antagonizing NMDA receptors (Pellegrini-Giampietro et al., 1989). The selective insensitivity of c J u n immunoreactivity to treatment with TTX or NMDA receptor antagonists may reflect merely differences in protein stability. However, this explanation seems unlikely, as treatment with cycloheximide, a protein synthesis inhibitor, rapidly decreased levels of c J u n and Jun-B proteins in parallel (Fig. 3A). Picrotoxin increases expression To determine whether raising the level of excitatory synaptic activity would increase the fraction of presumed neurons expressing these transcription factors, we examined the effect of blocking y-aminobutyric acidA(GABAA) receptor-mediated inhibition with picrotoxin. We found that, at relatively low concentrations, picrotoxin (10 p M ) substantially increased the frequency or amplitude of inward currents in all cells examined (n = 11; Fig. 4B), and the addition of 1 p M TTX blocked these large currents (five of five cells;

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-60-mV holding potential). At more positive holding potentials (-20 mV), outward spontaneous currents were apparent in control cells. Picrotoxin (10 p M ) reduced these currents, consistent with its ability to block inhibitory synaptic currents mediated by GABAA receptors. Treatment of 24-DIV cultures with 10 pMpicrotoxin for 6 h increased the expression of c-Fos, Jun-B, and c-Jun. The fraction of Jun-B- and c-Fos-positive presumed neurons more than doubled, while c-Jun-positive neurons were increased to a lesser extent (Figs. 5 and 6). These increases in both Jun-B and c-Fos immunoreactivity following picrotoxin treatment were detectable within 2 h, reached maximal levels after 46 h, and were still detectable after 24 h. The percentage of presumed neurons displaying Zif268 immunostaining increased from an average basal level of 2% to a level of 51% (n = 3 separate experiments) by 2 h of picrotoxin treatment (Fig. 7). With more prolonged treatment (6 h), smaller increases in Zif268 immunoreactivity were observed. Treatment of cells with TTX just prior to picrotoxin completely blocked the increase in staining for all four proteins and, as noted before, lowered Jun-B and c-Fos staining below basal levels. In a similar fashion, pretreatment with MK-80 1 and APV ( N 10 min) also blocked the increase in immunostaining induced by picrotoxin (Jun-B, c-Jun, and c-Fos; 6 h, 10 p M ) and lowered Jun-B and c-Fos staining below basal levels (Figs. 5 and 6). As observed in examining the effect of bath-applied APV on spontaneous activity, local application of APV by pressure ejection (0.5-5 s; 100-2,000 p M pipette solution, but actual concentration at cells is lower) in cultures exposed to picrotoxin resulted in a reversible decrease in the peak amplitude of spontaneous electrical activity (>30%) (Fig. 4C and D) in five of nine cells examined.

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FIG. 5. Picrotoxin (Picro)increases c-Fos, c-Jun, and Jun-B protein immunoreactivity.Values shown are the means 5 SEM of at least three separate duplicate experiments with each antibody performed on 21- to 25-DIV cultures. ' p i0.05, paired t test comparing immunostaining in picrotoxin-treated cultures to that under all other conditions for each serum indicated. Cells were treated with 1-1 0 f l TTX, 10 f l MK-801,or 300 pM APV 10 min prior to picrotoxin (6 h; 10 f l )as indicated.

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FIG. 6. Activity-dependent regulation of c-Fos, Jun-B, and c-Jun immunoreactivity in 21- to 25-DIV primary cortical cultures. Calibration bar = 30 pm. A: Basal c-Fos immunoreactivity.B: c-Fos immunoreactivity after picrotoxin (10 pM; 6 h). C c-Fos irnmunoreactivity after picrotoxin (10 pM) and TTX (1 pM) for 6 h. D: Basal Jun-B immunoreactivity.E: Jun-B imrnunoreactivity after picrotoxin (10 fiM; 6 h). F: Jun-B imrnunoreactivityafter picrotoxin (10 pM) and TTX (1 pM) for 6 h. G: Basal c-Jun irnmunoreactivity.H: c-Jun imrnunoreactivityafter picrotoxin (10 pM; 6 h). I: c-Jun immunoreactivityafter 10 pM picrotoxin and 10 pM MK-801 (6 h).

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FIG. 7. Zif268 expression. A Zif268 in situ hybridization following 1 h of 10 pM picrotoxin (left, Hoffman modulation optics; right, bright field). B: Zif268 in situ hybridization in untreated cells. Calibration bar = 30 pm. C: Zif268 immunoreactivityin control cultures. D: Zif268 immunoreactivity after 10 pA4 picrotoxin (2 h). E: Zif268 immunoreactivityafter 2 h of picrotoxin (10 p M ) and TTX (1 pM) added 5 min before picrotoxin. Calibration bar = 30 pm.

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In some cells, local application of APV did not affect the amplitude of spontaneous currents but reduced their duration, suggesting involvement of both NMDA and non-NMDA receptors in mediating these currents (Fig. 4D). In the presence of picrotoxin, continuous application of 300 p M APV (by addition to the static bathing medium) decreased the peak amplitude of spontaneous currents by 72 k 16% (n = 4 cells; p < 0.05). The increase in immunostaining induced by picrotoxin appears to reflect increased levels of mRNA for these transcription factors, as stimulation of 2 1-DIV cultures with 10 pM piarotoxin for 1.25 h increased mRNA for c-fos, jun-B, and zif268 (Figs. 7 and 8). As found with immunostaining, 10-min pretreatment with TTX completely blocked the increase in mRNA levels induced by picrotoxin. To confirm that the rise in mRNA is confined to neurons, as predicted by the immunostaining studies, we performed in situ hybridization with a [35S]zij268antisense riboprobe. These studies demonstrate that picrotoxin treatment induced a marked increase in zif268 mRNA that is localized to presumed neurons (Fig. 7). Using standard blot analysis, we were unable to detect reliably basal levels of mRNA for these IEGs and, therefore, could not directly assess whether TTX reduced mRNA levels. However, the addition of 4 pg/ ml of the transcription inhibitor, actinomycin D, significantly reduced basal immunoreactivity for c-Jun, Jun-B, and c-Fos within 6 h, indicating that basal levels of immunostaining reflect ongoing transcriptional activation of these genes (Table 1). As expected, actinomycin D treatment also totally blocked the increase in c-Fos, Jun-B, and c J u n immunostaining produced by either PDA or picrotoxin (Table 1).

FIG. 8. Picrotoxin (Picro) and PDA increase zif268, c-fos, and junB mRNA. Lane 1 : control (Con). Lane 2: PDA (10 pV), 1.25 h. Lane 3: T T X (1 pM), 4.0 h. Lane 4: picrotoxin (10 f l ) 1.25 , h. Lane 5: picrotoxin TTX (1.25 h, 1 0 pM; and 4.0 h, 1 p M ) . Arrowheads indicate migration of 18s and 2 8 s rRNAs.

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TABLE 1. Percentage of immunoreactive neurons 21-25 DIV cJun Control Picrotoxin, 10 pM PDA, 8 p M Act D, 4 pg/ml Act D and picrotoxin Act D and PDA

2 0 t 4(5) 31 f 7 (3) 59 k 4 (3) 6 & 2 (5)" 5 2 (3)" 8 f 3 (3)"

*

Jun-B 19 f 2 40 f 8

c-Fos

(6)

(3) 56 k 4 (4) 5 f 1.6 (6)" 5 2 1 (3)" 7.8 f 1 (4)"

14

3

(6)

40 +- 9 (4) 59 t 8 (3) 7 t 1.6 (6)" 7 f 2 (4)" 11 k 4.5 (3)"

The means t SEM of (n) separate duplicate experiments in which 21- to 25-DIV cortical cultures were treated as indicated in the presence or absence of actinomycin D (Act D), 4 pg/ml. Actinomycin D was added 6 h prior to fixation, picrotoxin added for 5.75 h, and PDA added for 2 h. p < 0.05 (paired t test comparing the percentage immunoreactive neurons for each condition in the presence and absence of actinomycin D).

DISCUSSION We have used primary cortical cultures to investigate regulation of immediate early genes in brain neurons. Although previous studies focused on transient expression of these genes (for review, see Sheng and Greenberg, 1990), our results demonstrate that c-Jun, JunB, and c-Fos are spontaneously expressed in a substantial fraction of neurons as these primary cultures mature. These findings are consistent with the presence of basal expression of these genes in adult cortex, whereas at birth, basal levels are low (Worley et al., 1990). In cortical cultures, the basal expression of JunB and c-Fos appears to be driven by synaptic transmission, as it correlates with development of electrical activity and is markedly reduced by incubation with TTX or NMDA receptor antagonists. In contrast, basal c J u n expression is not reduced by these agents, suggesting that its expression is dependent on other factors. Basal expression of Zif268 varied greatly among experiments, making it difficult to analyze whether it is dependent on electrical activity. Although more than 85% of neurons in 21- to 25-DIV cultures display spontaneous electrical activity, only approximately 20% exhibit Jun-B or c-Fos expression. Studies with picrotoxin indicate that this disparity may reflect, in part, insufficient synaptic stimulation to trigger expression of these genes. Low concentrations of this agent, which selectively reduce inhibitory GABA-ergic synaptic potentials in cortical neurons (Andrews and Johnston, 1979; Barnes and Dichter, 1984), indirectly increase the frequency and magnitude of inward excitatory currents and, also, significantly increase the expression of all four transcription factor proteins studied, Zif268, cJun, Jun-B, and c-Fos. Although cJun basal expression is not reduced by TTX, the rise induced by picrotoxin is blocked by TTX, indicating that it can respond to increased synaptic activity. Our results add to growing evidence for differential

SYNAPTIC REGULATION OF IMMEDIATE EARLY GENE EXPRESSION regulation of c-Jun and Jun-B. Although both are induced by growth factors in PC12 cells, Jun-B is preferentially activated by depolarization (Bartel et al., 1989). In cortical cultures, there is a marked difference in the regulation of basal expression of these homologous members of the Jun family. Although Jun-B expression is blocked by agents that reduce synaptic activity, c J u n expression persists. We doubt that this difference reflects merely increased stability of c J u n relative to Jun-B, since treatment with cycloheximide reduces immunostaining for both proteins in parallel. Although in these cultures Jun-B expression appears to be dependent entirely on synaptic activity, the factors responsible for driving basal expression of c J u n remain to be identified. Conceivably, growth factors present in these cultures may be involved. In recent studies, we have observed a similar differential regulation of c-jun and ~$268in cortical neurons in vivo (Worley et al., 1991). Both ofthese transcription factors are expressed in cortical neurons in vivo in untreated rats. To assess whether these basal levels are dependent on afferent synaptic activity, TTX was administered in one eye to remove retinal input, and the effect on expression of c-jun and ~ $ 2 6 8monitored in the visual cortex. This treatment markedly reduced both ~ $ 2 6 8mRNA and protein in the contralateral visual cortex, whereas c-jun expression was unaffected. Previous studies have demonstrated that activation of these genes by growth factors or neurotransmitters occurs via increased transcription (for review, see Lau and Nathans, 1990; Sheng and Greenberg, 1990). The rapid increases in mRNA for ~ $ 2 6 8 ,c-fos, and jun-B following picrotoxin treatment are consistent with activation of these genes at the transcriptional level in this paradigm. Furthermore, as cycloheximide and actinomycin D both rapidly lower immunostaining, it appears that basal protein expression represents ongoing transcription and protein synthesis. Glutamate has been proposed to be the major excitatory neurotransmitter in the mammalian brain (Fonnum, 1984). In cortical brain slice preparations, both NMDA and non-NMDA glutamate receptors mediate prominent components of excitatory postsynaptic potentials (Jones and Baughman, 1988). The addition of NMDA receptor antagonists, MK-801 and APV, blocks both basal and picrotoxin-stimulated JunB and c-Fos immunoreactivity, suggesting coupling of this glutamate receptor subtype to activation of these genes. Furthermore, studies in cerebellar granule cells point to the involvement of NMDA receptors in activation of c-fos (Szekely et al., 1988).However, in preliminary studies on cortical cultures, application of NMDA receptor agonists has produced only slight increases in expression of c-Fos in these cultures. Due to prominent NMDA-induced toxicity in these cortical cultures, it may be difficult to dissociate NMDA-induced toxicity from possible effects of NMDA receptor activation on IEG expression. In contrast to NMDA,

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application of the non-NMDA glutamate receptor agonist kainate reliably increases c-fos expression in mature cortical cultures. As cortical neurons in culture exhibit extensive synaptic connections, conceivably NMDA receptor antagonists suppress gene activation via an indirect, presynaptic action that reduces excitatory synaptic currents mediated by kainate receptors. The results of this study demonstrate that cortical neurons in culture display rapid synaptic regulation of transcription-factor immediate early genes. We have also found that phorbol esters markedly increase expression of these genes as described in other cell types (for review, see Lau and Nathans, 1991). Recent studies have demonstrated that phorbol esters and depolarization act via distinct promoter sequences to induce activation of c-fos (Sheng et al., 1988, 1990).Accordingly, synaptic activation of these genes observed under basal conditions or following picrotoxin treatment may be mediated via multiple pathways. Further studies using this in vitro system may prove helpful in deciphering the intracellular pathways involved in linking synaptic activity impinging on the cell surface to gene transcription in the nucleus. Acknowledgment: We thank Darla Lawrence for excellent secretarial assistance and Andrew J. Cole for assistance with in situ and blot hybridization. This work was supported in part by NIMH Training Grant 5T32 MHl5330-11 (T.H.M.), an NRSA Fellowship (T.H.M.), USPHS Grants DA-00266 (J.M.B.) and 1ROl EY08900 (P.F.W.),a grant from the Lucille P. Markey Charitable Trust (J.M.B.), the Joseph P. Kennedy, Jr., Foundation (J.M.B.), and the Howard Hughes Medical Institute. J.M.B. is a Lucille P. Markey Scholar.

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