Temporal and spatial preferences of c-fos mRNA expression in the rat brain following cortical lesion

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Brain Research, 60 (1993) 230-241) © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$(16.t)~)

BRES 18447

Temporal and spatial preferences of c-los mRNA expression in the rat brain following cortical lesion S a b e r a R u z d i j i c a, S a n j a P e k o v i c

a,

S e l m a K a n a z i r a, S a n j a Ivkovic and Ljubisav Rakic b

a,

M i r j a n a Stojiljkovic a

'~Department of Neurobiology and Immunology, Institute of Biological Research, University of Belgrade, Belgrade (Yugoslacia) and b School of Medicine, Clinical Center, Universityof Belgrade, Belgrade (Yugoslavia) (Accepted 18 August 1992)

Key words: c-fos mRNA expression; Ca 2+ uptake; Membrane potential; Cortical lesion; In situ hybridization

The expression of the proto-oncogene c-los is increased in neuronal cells by a number of stimuli and the usefulness of this gene as a marker of neuronal activity has been demonstrated. The temporal and spatial expression of c-los mRNA following the induction of a unilateral cortical lesion have been investigated in the rat brain by Northern blot analysis and in situ hybridization histochemistry. It was observed that the lesion evoked a rapid increase (20-fold) in the content of c-los mRNA in the ipsilateral cortex, whereas in the contralateral cortex c-los mRNA expression was more modest (7-fold). In the whole hippocampus a large and very rapid increase (17-fold) of c-los mRNA expression was detected. The effect of a cortical lesion on Ca 2÷ uptake and membrane potential was also investigated. Using synaptosomes as a model system, we have provided evidence that Ca 2+ entry via membrane depolarization increases in coordination with c-los gene expression in neuronal cells. The principal conclusions from this study are that cortical lesions induce transient expression of the c-los gene in specific neuronal cells of the rat brain.

INTRODUCTION Extracellular stimuli can elicit rapid transcriptional activation of a number of neuronal immediate-early genes. Several reports suggest that the products of these genes are crucial intermediates in a complex series of reactions that link membrane stimulation to long-term alterations in the cellular phenotype 42. The proto-oncogene c-los encodes a nuclear phosphoprotein Fos, belonging to the immediate-early gene families involved in stimulus-transcription coupling in the central nervous system (CNS) under diverse circumstances 43. Since ions, neurotransmitters and growth factors p r o m o t e c-los gene expression 1'22'24'29'30'39'41'51-56'57'63 it is not surprising that many other stimuli, including cutaneous stimulation, pharmacological seizures 5'4°'52'53'58, nerve transection 26, and cortical lesions also induce c-fos mRNA and Fos protein synthesis in brain 9,tl'13A4,15,5°. Previous studies have established that c-fos is expressed in adult neurons after seizures, cerebral is-

chemia and brain injury 14,15,40,45 Seizures and cerebral ischemia both induce c-los transcription and both of these pathological stimuli involve the release of excitatory amino acids and activation of N-methyl-D-aspartate (NMDA) receptors 17'49. The NMDA subset of glutamate receptors, which are coupled to cation channels and primarily regulate the transmembrane flux of calcium, have been postulated to play a role in neuronal excitability, memory and cell death 6°. The consequence of NMDA receptor stimulation is the increase of intracellular calcium, which is reported to induce c-los transcription 1'39'41"57'61. Induction of c-los mRNA and Fos protein by a number of stimuli, such as electrical or chemical depolarization and surgical trauma, can be blocked by the NMDA receptor antagonists, MK80116 or ketamine 15. Also, excitatory amino acids receptor agonists such kainic acid or NMDA increase the expression of c-fos mRNA31'4°, and Fos protein 48'59. Cell surface stimulation generates a second messenger signal that results in the transcriptional activation of c-los and the other immediate-early genes 54. Recent

Correspondence: S. Ruzdijic, Department of Neurobiology and Immunology, Institute of Biological Research, University of?Belgrade, 29. Novembra 142, 11,000 Belgrade, Yugoslavia. Fax: (38)(11)761-433.

231 studies in the pheochromocytoma PC12 cell line, a model system for studying neuronal signal transduction, established that membrane depolarization elicits induction of the c-los gene by voltage-gated calcium influx 1'6'39'51'56'57. Depolarization of PC12 cells is correlated with rapid phosphorylation of the CRE-binding p r o t e i n ( C R E B ) 55'64'65. The ability of CREB to activate c-los gene transcription suggests that CREB may mediate transcriptional induction by Ca 2÷ as well as by c A M p 5 5 , 56.

The evidence suggesting that membrane depolarization and intracellular Ca 2÷ modulate immediate-early gene expression led us to examine c-fos levels in vivo following cortical lesions. One of the difficulties in studying c-fos induction in vivo is that membrane potential and Ca 2÷ influx are difficult to measure in the intact brain. In an attempt to establish a useful model for measuring Ca 2÷ influx in nerve cells we have studied the effect of cortical lesions on Ca 2+ uptake in isolated nerve terminals: synaptosomes. Synaptosomes are functionally 'intact' in that they retain many of the properties of in situ presynaptic nerve terminals 2'3'37. Consequently, these preparations are very useful for analyzing the Ca 2÷ transport systems in nerve terminals of the CNS. In this paper we have demonstrated that membrane depolarization resulting from cortical lesions produces a transient induction of the c-los gene in specific regions of the brain. A relationship between c-fos gene expression and Ca 2÷ influx in synaptosomes has also been observed. These data provide a biochemical framework for the stimulus-transcription coupling cascade that may represent a component of the signal transduction pathways in neurons. MATERIALS AND METHODS

Animals and surgery Adult male Mill Hill Hooded rats (250-300 g) were anesthetized for a very short period (less than 5 min) with ether and a 1 mm wide hole was unilaterally drilled with a dental drill through the skull on the left side. The lesions were performed through the sensorimotor cortex area (1.5 mm lateral from the midline, 2.5 mm posterior to bregma). The drill was inserted approximately 1.5 mm below the underlying cortical region 11's°. The skull wound was packed with gel foam, and the skin overlying the cranium covered with gel foam. At various times (0, 15, 30, 60, 120 and 240 min) after cortical injury rats were sacrificed by decapitation. The left and right cortex and whole hippocampus were quickly dissected and immediately used throughout the experiments. Sham-lesioned animals were similarly anesthetized and skin overlying the cranium was cut. Unlesioned and unanesthetized (control) animals were also examined.

In situ hybridization In situ hybridization was performed according to a modified method as described previously 25. Rats were sacrificed by decapitation and the brains frozen on dry ice. Coronal sections (16/zm) were cut at the region of brain lesions and the thawed section onto

poly-L-lysin-coated slides. Following fixation for 15 min with 4% formaldehyde in phosphate-buffered saline, pH 7.4 (PBS) and treatment with proteinase K (1 tzg/ml) for 1 h at 37°C, sections were treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCI, pH 8.0 for 10 min, to reduce non-specific adsorption of the probe, and delipidized in increasing concentrations of ethanol followed by chloroform. Final mapping studies were done using the asS-labeled c-los riboprobe at a concentration of 1-1.5 x 106cpm per section. For the preparation of antisense RNA probes, EcoRI/ HinlII fragments (about 1.2 kb) were excised from the cloned c-los rat DNA62 and subcloned into EcoRI/HinlII cleaved pGEM-4Z (Promega) vectors respectively. Antisense RNA probes were than prepared from these clones by linearizing the DNA with EcoRl and transcribing with T7 RNA polymerase in the presence of 3ss-labeled UTP. All hybridization reactions were carried out in 50% formamide, 10% dextran sulphate, 0.6 M NaCI, 10 mM Tris-HCI, pH 7.2, 1 ×Denhardt's (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 1 mM EDTA, 100 ~ g / m l salmon DNA, 50 /~g/ml yeast tRNA, 0.1% SDS and 0.1 M DTT at 55°C fo~ approximately 3 h. This was followed by incubation at 37°C with RNAase A (20 p.g/ml) for 30 min. Finally the sections were washed overnight with 2× SSC, followed by several hours of washing in 0.2× SSC at room temperature. The sections were rinsed in 70%, 80% and 90% ethanol with 0.3 M ammonium acetate and in 100% ethanol before air drying for autoradiography. Hybridization was evaluated by using both Amersham beta-Max film and Kodak NTB2 emulsion autoradiography with exposure times of 3-6 days and 3-6 weeks, respectively. Autoradiograms were developed according to the Manufacturer's recommendations.

RNA isolation and Northern blot analysis RNA was isolated by the guanidine isothiocyanate (GTC)/cesium chloride centrifugation method 34'36. After dissection on ice, the left and right cerebral cortex and hippocampus (from 7-8 animals) were homogenized in 7 ml of 5 M GTC, 4% sarcosil, 50 mM sodium acetate, pH 5.0 and 1% 2-mercaptoethanol. To the homogenate dry CsCI 0.5 g/ml was added and then layered on 3 ml of CsCI, density 1.7 g / c m 3, for ultracentrifugation (20 h at 40,000 rpm in a Ti-50 rotor). The resulting pellets were suspended in 0.3 M sodium acetate, pH 5.0, 0.1% SDS, 10 mM EDTA and the RNA was then precipitated with 2.5 vols. of ethanol at - 20°C. After microcentrifugation (15 min, 10,000 rpm, 4°C) the pellets were resuspended in 100 ~1 of sterile water. The levels of total RNA were quantified by optical density at 260 nm (absorbance of 1.0 = 40/zg/ml), Levels of c-los mRNA were determined by Northern blot analysis using the oligonucleotide priming method 1s,34. In brief, 10 ~g of RNA were electrophoresed on 1% agarose gels containing 0.2 M MOPS, pH 7.0, 50 mM sodium acetate, 10 mM EDTA and 1 M formaldehyde. Ethidium bromide (0.5 ~g/ml) was included in the gels to allow visualization of stained RNA samples with UV light and to ensure that all lines contained equivalent amounts of intact RNA. The RNA was transferred to GeneScreen membranes (NEN Research Products, Boston) and cross-linked to the nylon by UV irradiation. The membranes were then incubated with 32P-labeled fragments for 2 h at 65°C in Rapid Hybridization Buffer (Amersham). Prehybridization, hybridization and washing conditions were as recommended by the manufacturer. The membranes were air-dried and autoradiographed for 16-75 h. The amount of c-los mRNA in the bands was determined by quantitative densitometry. After autoradiography, membranes were stripped of radioactivity by boiling in 10 mM Tris, pH 7.5, 1 mM EDTA and 0.1% SDS before reprobing with labeled DNA containing actin sequences.

Preparation of synaptosomes Synaptosomes from rat cerebral cortices (left and right; LCx, RCx), and hippocampus (Hippo) were prepared as described previously 47. The tissue was homogenized in ice-cold 0.32 M sucrose (buffered with 5 mM Tris-HCl, pH 7.4) to give a 10% (w/v) preparation. The homogenates were centrifuged at 1000 g for 10 min at 4°C and the resulting supernatants were centrifuged at 12,000 g for 30 rain. The pellets consisting of synaptosomes2° were gently resus-

232 pended in preincubation solution (PS) containing (in mM): 142 NaCI, 3 NaHCO3, 0.4 KI-I2PO 4, 1.3 MgCI 2, 10 glucose (D), 3.6 KCI, 20 Tris-HCl, pH 7.4, and were left on ice until use (no longer than 1 h). The protein concentration of each sample was determined using bovine serum albumin as standard 33'35.

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Ca: + uptake of synaptosomes Just prior to their use in the experiments synaptosomes were further diluted with PS to adjust the protein concentration to 1 m g / m l . After preincubation at 32°C for 10 min, synaptosomes were chilled on ice and immediately used to determine the 45Ca2+ uptake 3s'47. The non-depolarized (resting) accumulation of 45Ca2+ was performed in the presence of 0.8 m M CaCI2, 0.4/zCi 45CAC12 (0.85 /xCi/tzmol = 31.45 kBq/t~mol, A m e r s h a m ) and other components remained as in PS, in a final volume of 200 #1. The reaction was initiated by addition of 50/xl of synaptosomal suspension. After 40 s at 32°C the incubation was terminated by dilution with 2 ml chilled PS containing 2 m M CaCI2. Each sample was rapidly filtered through 'a Millipore filter (0.45/zm pore size). The filters were rinsed 2 times with 5 ml of the same buffer, dried and then transferred to vials containing scintillation fluid. The radioactivity was measured in a LKB 1219 RackBeta liquid scintillation counter. The results were expressed as nmol of accumulated C a 2 + / m g protein/rain. Nonspecific absorption on the filters was subtracted.

Measurement of membrane potential The membrane potential was measured indirectly by following the distribution of the lipophilic cation (3H)tetraphenylphosphonium (TPP ÷ ) across the membrane. The uptake of 3HTPP ÷ (as bromide salt) was conducted under the same conditions as the 45Ca 2+ uptake except that labeled CaCI 2 was omitted 3s. The reaction solution contained 0.2 /zCi 3HTPP + (23 m C i / ~ z m o l = 8 5 1 M B q / p . m o l , Amersham). After incubation at 32°C for 10 rain the reaction was stopped by dilution with 2 ml chilled PS containing 2 m M CaCI 2, and then a rapid filtration through prewashed Sartorius glass fiber filters was performed. The filters are treated and radioactivity was counted as described above. The results obtained were presented as pmol T P P + / m g p r o t e i n / m i n . Non-specific absorption on the filters was subtracted.

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Northern blot analysis of c-fos mRNA expression Numerous studies have demonstrated that c-los gene expression is induced during stimulation of the intact n e r v o u s s y s t e m 5'tt'26'27'28'5°'52'53. Furthermore, various forms of seizure, lesions to the CNS, as well as nerve transection increase the c-fos m R N A and protein levels in a temporal manner and to a degree varying with brain location. The current studies were designed to determine specifically how cortical lesions influence the timing of c-los gene expression in various parts of the brain by measuring the appearance of c-los mRNA. Tissues from left and right cortex (LCx, RCx) and whole hippocampus were surveyed for levels of c-los mRNA, the gene product that is known to be activated in the brain after cortical lesions. Using Northern blot analysis we observed a dramatic increase in cortical c-fos m R N A 15 min following the lesion (Fig. IA, lanes 3 and 4). A large elevation (20-fold on the left side and 7-fold on the right side) of c-fos m R N A levels occurred in the cerebral cortex in comparison to levels in con-

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Fig. 1. Time course of c-fos m R N A expression in the left and right cortex following a cortical lesion. A: total R N A was isolated as pooled samples from left and right cortical tissues. Ten ~zg of total R N A were hybridized with oligo primed 32p-labeled of cofos probe. Blots were exposed to X-ray film for 75 h. A single band for c-fos m R N A hybridization was observed. Numbers above the lanes (1, 3, 5, 7, 9, and 11 are the left side, 2, 4, 6, 8, 10, and 12 are the right side) are codes left and right side of the cortex at the indicated time. Control and different time points are included below the autoradiogram. B: photographs of the ethidium bromide stained R N A gels are included to demonstrate that equal amounts of intact R N A were applied to each line of the gel. Position of 28 S and 18 S ribosomal R N A are indicated. C: relative c-los m R N A concentrations were determined by densitometric scanning of the autoradiogram in A. The values represent the ratio of induction c-fosmRNA in left (LCx) and right (RCx) cortex to the basal expression of c-fos m R N A in control animals. Autoradiograms and densitometry of blots stripped and rehybridized with actin probe served to normalize R N A content between lines are not included.

trois (Fig. 1C, LCx, RCx). Analysis of the time course of c-los m R N A formation demonstrated a rapid response of nerve cells in the ipsilateral cortex, reaching

233 maximal levels within 15 min (Fig. 1A, lanes 3 and 4), and barely detectable at 240 min (Fig. 1A, lanes 11 and 12) after lesion. Comparison of c-fos mRNA production in the left (ipsilateral) and right (contralateral) cortex by densitometric scanning of the autoradiographs (Fig. 1C) revealed that the induction of c-los gene in the contralateral cortex was clearly reduced (Fig. 1C, RCx) in contrast to ipsilateral cortex (Fig. 1C, LCx). The effect of cortical lesions on the expression of the c-fos gene in whole hippocampus also was examined by Northern blot analysis. It was found that the formation of c-los mRNA, measured 15 min after lesion was elevated 18-fold (Fig. 2, lane 2 and C) compared to control (Fig. 2, lane 1 and C). Following maximum induction at 15 min, little or no c-fos mRNA could be detected 240 min after cortical lesion (Fig. 2, line 6). These results demonstrated that formation of a unilateral cortical lesion induced temporal and spatial preferences for c-los mRNA expression in the rat brain.

The effect of cortical lesions on time course of and 3HTPP + uptake

45Ca2 +

Since several groups have reported that Ca 2÷ influx induces c-fos t r a n s c r i p t i o n 1'6'39'56'57 w e postulated that

cortical lesions would change C a 2+ entry in parallel with c-los gene expression. To verify that cortical lesions affect transport of calcium ions, even in the absence of depolarizing agents, we study the effect of cortical lesions on resting (non-depolarized) accumulation of 45Ca2+ b y rat cortical synaptosomes. The time course of changes in resting 45Ca2+ uptake after left cortical lesions compared to the control values is shown in Fig. 3. The data revealed that the effect of unilateral cortical lesion was more than 3-fold higher on the injured compared to the non-injured side. Increase of 45Ca2+ uptake by cortical synaptosomes was the most extensive (166-170%) within the first 0-15 min time intervals following the lesion, with a sharp decrease at 30 min post injury. A further decrease in the rate of 45Ca2+ uptake to almost control levels was observed from 60 to 240 rain after lesion. These results suggest that increased ability of the cortical synaptosomes to a c c u m u l a t e C a 2+ immediately after cortical injury may be due to changes in membrane potential. To determine the relationship between membrane potential and C a 2+ transport in synaptosomes the uptake of 3HTPP+ was utilized. This lipophilic cation is known 4'19'32'38 to enter membranes and distribute according to electrical potential, so that its accumulation within cells indicates an increase in the interior negative transmembrane potential. In contrast, depolariza-

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Fig. 2. Time course of c-los mRNA expression in whole hippocampus following cortical lesion. A: total RNA samples were prepared from whole hippocampus and analyzed for c-los mRNA by Northern blot hybridization. Numbers (1-6) above the lanes indicate control and time points following lesion. B: autoradiogram of blot stripped and rehybridized with actin probe served to normalize RNA content between lanes. Photographs of the ethidium bromide stained RNA gel is not included. C: the concentrations of c-los mRNA were determined by densitometric scanning of A.

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Fig. 3. Time course of changes in resting 45Ca2+ uptake following cortical lesion. Synaptosomes (1 mg/ml) isolated from left and right cortex (LCx, RCx) at 0, 15, 30, 60 and 240 min post injury were preincubated for 10 min at 32° inPS (see Materials and Methods). Resting 45Ca2+ uptake was performed in the presence of 4 mM KCI, 0.8 mM CaCI 2, 0.4/zCi 45CAC12 (0.85~Ci/p, mol = 31.45 kBq//~mol) and other components remained as in PS. The uptake lasted for 40 s and was terminated by dilution with 2 ml of ice-cold PS containing 2 mM CaCI 2, followed by rapid filtration through a Millipore filter (0.45 /zm pore size). The results are expressed as % of control values.

tion of the membrane will be followed with reduced 3HTPP ÷ uptake by synaptosomes. The time course of change in 3HTPP + accumulation is shown in Fig. 4. An analysis of the data showed that during the first 30 min after lesion the concentration of 3HTPP + inside the cortical synaptosomes was significantly decreased 3°-4°~' when compared to the control values. This decrease is an indicator of depolarization of synaptic plasma membrane. From 60 to 240 min after lesion 3HTPP+ uptake reached control levels indicating the completion of membrane repolarization. The changes in 3HTPP + uptake were expressed markedly at the lesioned side. It seems reasonable to suppose therefore that the increase in 45Ca2+ uptake shortly after the lesion (0-30 min) is related to depolarization of the membrane of cortical synaptosomes. The time course of changes in 45Ca2+ and 3HTPP ÷ uptake by hippocampal synaptosomes exhibit the same trend as in the cortex (data not presented).

Localization c-los mRNA in the rat brain after cortical lesions The localization of c-los mRNA-containing cells in the rat brain was detected by in situ hybridization histochemistry. The expression of c-los m R N A after cortical lesion is illustrated by representative coronal sections through the hippocampus in Fig. 5. Brain regions were determined from the stereotaxic atlas of Paxinos and Watson 46. Hybridization of the 35S-cRNA

probes to c-los mRNA-labeled cells in control (shamoperated) brain sections was very low (Fig. 5A). The amount of c-fos mRNA expression is significant, especially in the cortices, pyramidal cells of hippocampus and granule cells of the dentate gyrus. These data indicate that the induction of c-los mRNA may be elicited by a very subtle stimulation, such as the acute stress associated with the handling of the animals. In immediately sacrificed animals following cortical lesions, the distribution and the levels of c-fos mRNA were very similar to those obtained in sham-treated animals (Fig. 5B). In contrast, in animals sacrificed 15 min (Fig. 5C) and 30 min (Fig. 5D) following the lesion a dramatic increase in c-fos mRNA levels could be observed in some principal structures, including frontoparietal cortex, primary olfactory cortex, piriform cortex, granule cells of dentate gyrus (were the most intense labeling was detected) and hippocampal pyramidal cells (CA1, CA2, CA3, CA4). At this early time point, hybridization was elevated in the lateral amygdala and ventromedial hypothalamus. In animals sacrificed 1 h following the lesion, hybridization to c-los m R N A was significantly further increased within the principal cell layers of hippocampus as well as in dentate gyrus and cortices (Fig. 5E). However, as can be seen in Fig. 5F in animals sacrificed 4 h following the lesion the expression of c-fos mRNA was very low in all examined structures. These observations suggest that the specific and marked inductions of c-los mRNA observed following the lesion is a consequence of the cortical lesions themselves.

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235 A comparison of labeling was made between the ipsilateral (damaged side) and contralateral side of the cortex. As can be seen (Fig. 5C,D,E), the hybridization of c-fos probes to c-los mRNA-containing cells is clearly elevated in the ipsilateral cortex in contrast to the hybridization in the contralateral side of cortex.

To localize c-los mRNA-containing cells more precisely the tissue sections were exposed to photographic emulsion for 3 weeks and examined by light-field microscopy. As shown in Fig. 6, within the ipsilateral side of the cortex (Fig. 6B) there were dense concentrations of silver grains over the neuron-reach layers of the

Fig. 5. Localization of c-los mRNA-containing cells in rat brain analyzed by in situ hybridization histochemistry. Photomicrographs of coronal sections through the hippocampus of a sham-operated rat (A) and rats sacrificed immediately (B), 15 rain (C), 30 min (D), 1 h (E) and 4 h (F), following cortical lesions. Autoradiographs present 16/~m coronal sections after in situ hybridization with 35S-labeled antisense c-los probe. In sham-treated rats (Fig. 5A) low or moderate levels of hybridization can be seen. There is no difference in signals between sham-treated rats (A) and rats sacrificed immediately after lesion (B). Autoradiographic localization of c-los mRNA-containing cells in cortical regions: Fr, frontal; In, insular; Pir, piriform; Te, temporal; Am, amygdala; CA1, CA2, CA3, CA4, pyramidal cells of hippocampus; DG, dentate gyrus; Th, ventromedial hypothalamus; PO, primary olfactory cortex; Ipsi, ipsilateral side of brain; Contra, contralateral side of brain. Arrow indicates the location of the cortical lesion.

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Fig. 6. Cellular distribution of c-los mRNA hybridization in the cerebral cortex (A, B, C), CAI pyramidal cells of hippocampus (D, E, F), dentate gyrus (G, H, I), and hypothalamus (J, K, L) in animals sacrificed 30 min following cortical lesions. Photomicrographs from emulsion dipped slides of brain sections. Sections were stained with Cresyl violet following emulsion development. The sections A, D, G, and J are from control rats, ipsilateral side of the brain. A comparison was made between the ipsilateral (B, E, H, K) and the contralateral side (C, F, I, L) of the brain. Objective magnification, 100 ×.

237 cortex in comparison with the contralateral side (Fig. 6C) and control (Fig. 6A). As can be seen (Fig. 6B) c-los mRNA was increased dramatically in the ipsilateral cortex 30 min following cortical lesion, and showed a spatial distribution similar to that obtained with Northern blot analysis. Hybridization c-los mRNAcontaining cells was strongly increased bilaterally in granule cell layers of dentate gyrus (Fig. 6H, I) as well as in pyramidal cell layers of region CA1 (Fig. 6E, F) by 30 min following the lesion. At this time point hybridization was also slightly elevated in the ventromedial hypothalamus (Fig. 6K, L). Silver grains were hardly detectable on the control side in all examined structures (Fig. 6A, D, G, E). Analysis of these results reveals a high degree of variability in the density of silver grains overlying individual neurons of the brain regions, which gives us important insights into the functional significance of increased c-fos production following cortical lesions. DISCUSSION Induction of the c-fos proto-oncogene is likely to play a key role in the transduction of short-lived environmental signals into long-lasting changes in nerve cells by various forms of stimulation. A brief period of enhanced neuronal activity can result in the sequential production of c-fos mRNA and the Fos protein that participates directly as a transcriptional factor 43. In the present study we have described the induction of the c-fos proto-oncogene in different neuronal cells in response to a cortical lesion. Within the cortex and hippocampus c-fos mRNA levels increased rapidly (for only 15 min) and to varying degrees followed by a dramatic decline to control levels. In agreement with previous studies in situ hybridization of c-fos mRNA confirmed these results. A variety of pharmacological agents produce quite different patterns of c-fos induction. For example, pentylenetetrazol, NMDA and kainic acid-induced seizures increase c-fos expression in dentate gyrus of mice and rats 3~'4°'44'48, whereas amphetamine and cocaine induce c-los mRNA only in the striatum2L Furthermore, electroconvulsive shock increased c-fos mRNA in many brain structures (including discrete areas within the limbic system, hypothalamus and cerebellum) of mice 8. Additionally, Fos protein measured by immunohistochemistry was reported to have increased in the hippocampus and cortex, following electrically kindled seizure ~°,~2. Typically, traumatic cortical injury induces the formation of c-los gene products in entire ipsilateral cortex ~,~4.~5,26,5°.

The experimental results presented here demonstrate that the cortical lesions stimulate a large increase in the expression of c-los mRNA, by many different populations of adult forbrain neurons in a time-dependent manner. One brief cortical stimulation is sufficient to induce a large increase in c-los mRNA in many populations of cortical neurons (frontoparietal, temporal, olfactory and piriform cortex), in the granule cells of dentate gyrus and pyramidal cells of the hippocampus. The more interesting difference in the response of c-los mRNA levels to cortical lesions is the large increase of c-fos mRNA levels in the ipsilateral side of cortex. The 'cortical' pattern of c-fos mRNA expression include several cerebral areas with most marked expression which was observed in the frontoparietal, olfactory and piriform cortex. The cortical distribution was always unilateral on the damaged hemisphere, which is in agreement with the other reports 1~'15'26. The anatomical pattern observed by in situ hybridization was remarkably similar (Fig. 5) to that obtained with Northern blot analysis (Fig. 1), suggesting that c-fos mRNA may be coordinately expressed in the same set of neurons, after cortical lesion. A comparison of changes in levels of c-fos mRNA, in the cortex, hippocampus and thalamus reveals many differences in distribution of c-los mRNA expression. Hybridization to c-fos mRNA-containing cells is predominantly localized within stratum pyramidale in the hippocampus and granular ceils of dentate gyrus, as well as in the different layers of cortex (Fig. 6). The presence of relatively hybridized neurons in the ventromedial thalamus was observed. The cortical distribution was always unilateral, whereas hippocampal was usually bilateral. However, hippocampal distribution of c-fos mRNA could be also unilateral in the earliest phases of cortical lesion, which suggest that unilateral processes rapidly become bilateral later. Previous studies have established that traumatic brain injury leads to the release of excitatory amino acids in the brain 17. These results suggest that NMDA receptors which are coupled to ligand-gated ion channels primarily regulate the transmembrane flux of calcium. Using radiolabeled ligands, NMDA receptors have been visualized by autoradiography 6°. Highest densities of labeled NMDA receptors are observed in the CA1 region of the hippocampus. Relatively high densities of binding are in the cerebral cortex, whereas levels of binding in the thalamus, striatum and cerebellum are moderate by comparison 49,6°. The result of the NMDA receptor stimulation is increased intracellular calcium, which is reported to increase c-fos transcription39,41,49,56,57.

238 P e r h a p s the most likely m e c h a n i s m by which a single cortical lesion might trigger c-los m R N A expression is through d e p o l a r i z a t i o n of n e u r o n a l m e m b r a n e s a n d the c o n s e q u e n t increase in intracellular calcium. T h e fact that calcium can stimulate c-los gene transcription i m m e d i a t e l y a n d w i t h o u t n e w p r o t e i n synthesis, suggests that calcium acts directly o n this gene 23. In

Biological Science, Canada) for their encouragement, thoughtful discussions and comments. We are grateful to Srdjan Spasojevic for help with photomicrographs and to Borka Ilic for excellent technical assistance. This work was supported by grants from the Scientific Foundation of Serbia (No. 0309), and the Vivian L. Smith Foundation for Restorative Neurology, Houston, Texas.

REFERENCES

this study we have d e m o n s t r a t e d that the time course of rapid a c c u m u l a t i o n of c-los m R N A (15 rain) following the lesion of the cortical n e u r o n s , is basically the same as that p r o d u c e d in PC12 cells by growth factors, or m e m b r a n e d e p o l a r i z a t i o n by potassium 1'7'51'56'57. F u r t h e r m o r e , m e m b r a n e d e p o l a r i z a t i o n in PC12 cell i n d u c e s a n elevation in i n t r a c e l l u l a r calcium, which is sufficient to evoke a n increase of c-los m R N A levels. Thus, our c u r r e n t observations are c o m p a t i b l e with the possibility that calcium a c c u m u l a t e s intracellularly in vivo following the lesion. However, conclusive proof of the i n v o l v e m e n t of Ca 2÷ influx via m e m b r a n e depolarization in the r e g u l a t i o n of a target gene in vivo is difficult. T h e r e f o r e , in f u r t h e r e x a m i n a t i o n s the effect of cortical lesions o n Ca 2 + influx a n d m e m b r a n e potential was followed using synaptosomes, as a m o d e l system, at various time intervals after lesion. M a x i m u m a c c u m u l a t i o n of calcium in synaptosomes was observed d u r i n g the first 15 m i n after the lesion, followed by a sharp decrease after 30 m i n (Fig. 3). Since Ca 2+ uptake was m e a s u r e d in the absence of depolarizing agents, we have c o n c l u d e d that the cortical lesion causes d e p o l a r i z a t i o n of n e u r o n a l m e m b r a n e . Such a conclusion was s u p p o r t e d by a strong decrease in m e m b r a n e p o t e n t i a l d u r i n g the same time course as monitored by the d i s t r i b u t i o n of 3 H T P P +. T h e r e f o r e , those alterations of m e m b r a n e p o t e n t i a l could be the cause of increased Ca 2+ uptake. T h e results p r e s e n t e d here, provide consistent evid e n c e that a cortical lesion induces a rapid a n d transient expression of c-los m R N A in c o o r d i n a t i o n with Ca 2* influx following m e m b r a n e depolarization. T h e o t h e r principal f i n d i n g in our study is the observation that a brief cortical lesion leads to a t e m p o r a l a n d localized expression of the c-los gene. T h e putative i n v o l v e m e n t of the c-fos gene in transd u c t i o n of brief s t i m u l a t i o n to l o n g - t e r m changes in the cell p h e n o t y p e t h r o u g h r e g u l a t i o n of g e n e expression suggests that i n d u c t i o n of c-fos gene might be a useful m a r k e r with which the effects of pharmacological, electrical a n d physiological stimuli may be traced in the CNS.

Acknowledgements. The authors wish to express their gratitude to Professors Dusan Kanazir (Serbian Academy of Sciences and Art, Belgrade) and Bruce H. Sells (University of Guelph, College of

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