Immediate-early gene responses in the avian song control system: cloning and expression analysis of the canary c-jun cDNA

iiii

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 27 (1994) 299-309

Research report

Immediate-early gene responses in the avian song control system: cloning and expression analysis of the canary c-jun cDNA Kent L. Nastiuk a, Claudio V. Mello b, Julia M. George c, David F. Clayton c,. a Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NYIO021, USA b Laboratory of Animal Behavior, The Rockefeller University, New York, N Y 10021, USA c Department of Cell and Structural Biology, and The Beckman Institute, University of Illinois, Urbana, IL 61801, USA Accepted 2 August 1994

Abstract

Previous studies have shown that song presentation results in a rapid rise in mRNA levels for the ZENK gene (the avian homologue of zif-268, Egr-1, NGFI-A, and Krox-24) in specific parts of the songbird forebrain. Metrazole-induced seizures also cause an increase in ZENK mRNA, even more widely throughout the telencephalon. Surprisingly, however, little or no ZENK induction by either stimulus was observed in several forebrain areas involved in auditory processing and song production. To learn whether this pattern of regulation is specific to ZENK, we examined the response of another 'immediate-early' gene, c-jun. Here we first describe the identification, cloning and sequence analysis of a canary cDNA encoding c-jun. Then, by in situ hybridization we show that c-jun is also induced by song or seizure, and in a pattern mostly similar to ZENK. As with ZENK, no induction of c-jun is observed in the androgen receptor-containing song nuclei or within the primary thalamo-recipient auditory area of the forebrain. Thus common immediate early gene responses appear to be selectively uncoupled from physiological activation in these specific forebrain regions, which are also characterized by tight developmental, hormonal and seasonal regulation.

Keywords: Immediate early gene; Zebra finch; Canary; c-jun; zif-268; NGFI-A; Metrazole; Songbird

I. Introduction

The songbird is one of the most intensively studied models of the relationship between brain anatomy and behavior [6,13,38,58]. A specific set of interconnected b r a i n nuclei evolved in the telencephalon of oscine songbirds [27] and is necessary for song learning and production [14,59,66]. In the zebra finch, canary and many other songbirds, the major telencephalic song control nuclei differ in size, as defined by Nissl-staining boundaries, between males (who sing) and normal females (who sing less frequently or not at all) [26,57]. This sexual dimorphism of neural structure arises at least in part through the expression of androgen and e s t r o g e n r e c e p t o r s in s o m e of t h e s e nuclei [5,7,8,10,27,55]. The function of the song system must

* Corresponding author. 506 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801, USA. Fax: (1) (217) 244-1648; e-mail: davidclayton@qmsl .life.uiuc.edu 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 1 7 6 - 6

also be sensitive to the behavioral experience of the individual, as exposure to specific songs during critical periods in juvenile development is essential for the normal development of the bird's own song [23,35,43, 70]. Functional plasticity in the song control system appears to be highly regulated, as the song of some species (such as the zebra finch) is virtually unchanging once learned, whereas other species (such as the canary) display distinctive cycles of song learning, production and 'forgetting' [58]. Despite considerable progress in identifying the neural circuitry involved in song production, our understanding of the molecular and cellular mechanisms underlying song learning, song processing or song-induced behavioral responses remains undeveloped. A significant advance towards a molecular-level analysis of plasticity in the song system came recently with the discovery that specific portions of the brain of canaries and zebra finches respond to the sound of birdsong with a rapid increase in transcription of a specific gene,

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' Z E N K ' [45,47]. This induction of Z E N K is a clear example of the 'immediate-early gene' ( I E G ) response, that occurs in neurons and other cell types following various forms of stimulation, including growth factor treatment, neurotransmitter receptor activation and depolarization [4,11,20,33,34,48,51,63,76]. In mammals, the I E G response typically involves the concomitant induction of many genes, and estimates have placed the total n u m b e r of different I E G s at 100 or m o r e [69]. Many of these IEGs, including Z E N K , encode D N A binding proteins with transcriptional regulatory properties, and their expression following cellular stimulation may represent the first stage in a biochemical cascade leading to longer-term changes in gene expression and cellular properties [52]. In the nervous system, such a mechanism has been widely hypothesized to play a role in neuronal modifications underlying long-term m e m ory formation [2,31,69]. Regardless of its function, many investigators have found this response to be useful as a tool for m a p p i n g brain areas that are activated in various behavioral or pharmacological paradigms (e.g. [32,49,50,61,64,73]. Against this background, our studies of Z E N K induction in the songbird brain [44-47] raised a curious paradox. W e found a significant divergence between the anatomical pattern of Z E N K induction, and that predicted on the basis of existing physiological and anatomical information. O n e set of experiments involved administration of a G A B A antagonist, metrazole, which resulted in widespread Z E N K induction throughout most of the telencephalon [44,46]. However, induction did not occur (or occurred at much lower levels) in certain specific telencephalic regions, including the primary auditory thalamo-recipient zone (Field L2a) and the a n d r o g e n receptor-containing song control nuclei. In another set of experiments we used taped birdsong as the stimulus [45,47]. Song presentation caused a specific activation of the Z E N K gene in the caudo-medial portions of the neostriatum and hyperstriatum ventrale, brain areas that had not been specifically implicated in song processing, although they were known to show responses to complex auditory stimuli. Surprisingly, Z E N K induction was still not observed in Field L2a or in the song nuclei, despite the fact that these brain areas are known to be physiologically activated by song stimulation, as shown previously with both electrophysiology and metabolic labeling techniques. Thus we failed to see Z E N K induction following song stimulation in several areas where Z E N K induction would have b e e n most expected [45]. T h e observation of a lack of a Z E N K response in some specific brain areas that are undergoing physiological activation immediately raised the question of whether other l E G s might be undergoing induction in these areas instead. With this in mind, we have cloned the canary h o m o l o g of another well-studied l E G , c-jun,

and used this to probe the response of the c-jun gene in songbird brain following stimulation either by song or by metrazole. T h e protein e n c o d e d by the c-jun gene is also a transcriptional regulator. Unlike the Z E N K protein however (which directly binds D N A by virtue of specific zinc finger motifs), the c-jun protein acts by forming dimers with other 'leucine zipper' transcriptional proteins, most notably c-fos, to form the AP-1 D N A - b i n d i n g complex [39,56]. c-jun's D N A binding specificity is thus different from that of Z E N K , and c-jun could be involved in functional aspects of the l E G response distinct from Z E N K . Here, we first describe the isolation and characterization of the canary h o m o l o g of c-jun. Next we investigate w h e t h e r c-jun is also induced in the songbird brain by song or metrazole treatment, and if so, w h e t h e r the induction pattern differs significantly from the Z E N K induction patterns. In particular, does c-jun induction occur in areas that lack Z E N K induction in response to physiological activation?

2. Materials and methods 2.1. cDNA cloning

The cDNA library used to isolate the canary homolog of c-jun was constructed in lambda gtl0, and contains approximately 3 x 106 independent clones (described in [28]). The library was screened by plating at approximately 20,000 pfu per 150 mm plate and the plaques were allowed to develop to about 0.5 mm diameter, and lifted in duplicate onto nylon filters (Colony/Plaque Screen, NEN). The filters were hybridized essentially according to Westneat [74]. Briefly, they were rinsed in 2 x SSC and pre-hybridized in a solution containing 7% SDS, 1 mM disodium EDTA, 263 mM sodium phosphate, and 1% BSA. Chicken c-jun cDNA (a kind gift of D. Foster) was random primed [24,25], denatured, added directly to the pre-hybridization solution, and incubated at 55°C overnight in a shaking water bath. The filters were then rinsed in 2xSSC, 0.1% SDS at room temperature and finally washed in 1×SSC, 0.1% SDS at the hybridization temperature. The filters were then exposed to XAR-5 film at - 70°C with intensifying screens. Positive clones were isolated through two additional rounds of screening, and their inserts subcloned into pBluescript (Stratagene) by standard methods [65]. 2.2. Sequence analysis"

The cDNA clone was sequenced on both strands using Sequenase (USB) and synthetic oligonucleotide primers. Each strand of the plasmid was sequenced from both the T7 and T3 primers, and subsequently from internal synthetic oligonucleotide primers (17 or 18 nt) determined by the previous sequencing results. The sequences were assembled on DNASIS (Hitachi) and all comparisons were carried out using the FASTA suite of programs for the PC [65]. 2.3. Southern blot analysis-

Genomic DNA was analyzed by standard techniques [65]. Samples of DNA were digested with 10 units restriction endonucleases (New England Biolabs) in 10 ~l l x appropriate restriction endonuclease buffer (per/xg DNA) for three hours, and EDTA added to 15

E L . Nastiuk et al. / Molecular Brain Research 27 (1994) 299-309 mM to stop the reaction. Protein was then precipitated by adding ammonium acetate to 2.5 M and centrifugation at 15,000 x g for 15 min at room temperature. DNA in the supernatant was precipitated by adding 2.5 volumes ethanol and centrifugation at 15,000 × g for 15 min at 4°C. The pellet was washed with 70% ethanol and dried under vacuum. The samples were resuspended at 0.1 mg/ml, heated to 65°C for 10 min, cooled on ice, and loaded onto a 0.8% LE agarose gel and electrophoresed in TAE buffer [65]. The DNA was transferred to a nylon membrane (Zeta-Probe, Biorad) according to

301

Westneat [74]. Hybridizations using the random primed canary c-jun cDNA were carried out as for cDNA library screens (above) but the hybridization temperature was increased to 60°C.

2.4. RNA preparation Total cellular RNA was prepared from all canary tissues using either by the guanidinium-CsC1 method [16,19,30,36] or by acid guanidinium thiocyanate phenol chloroform extraction [17]. RNA

Canary c-jun eDNA

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26

384

G I y ~alr A s n A s n Ala Lys Val Leu Lys G l n A a n M e t T h r L e u | A a n Leu Set|Asp Pro Set Set Asn heu Lys Pro H i s Leu A r g A s n byB A S h A l a

58

G G A T A C A A T A A C G C C A A G G T G C T G A A G C A G A A C A T G A C G C T G A A C C'DG T C C G A C C C C T C C AGC A A C C T G A A G C C G C A C C T G A G G A A C A A G A A T G C C

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T h r Pro ~ n r Pro Thr G l n Phe Leu Cys Pro hys A S n V a l T h r A S p G l u

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A C G C C G A C C C O G ACG C A G TTC C T G TGC C C C A A G A A T G T C A C C G A C G A G C A G G A G G G G T r C GCC G A G G G C T ~ C G T G A G A G C T T ~ G G C T G A G ~

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122 672

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Val Pro G l u Met

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GGA TAC GCG CCC CAG CAT CAC ATA AAC CCC CAG ATG CCC GTG CAG CAT OCC CGG C/T CAG GCT ~

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TCC CCC A T

GAC ATG GAG ~CA CAG GAG AGA ATC AAA GCC GAG AGA AAG CGC ATG AGG AAC AGA ATC GCG GCG ~2C

Lys C y s A r g Lys A r g Lys Leu G l u A r g Ile A l a A r g Leu G l u G I u Lya val Lys Thr Leu Lys Ala G l n A S h S e t G I u Leu A l a Set T h r Ala ASh A A A T ~ C O G G A A A A G G A A G "FfG G A A A G G A~'~ G C C C G G T T G G A A G A A A A A G T G A A A A C T ~

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GGA AGT GCT GTT CCT GCT CGG ATG AGA TGT CAG AT

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TAA CAT TGA CCA AGA CCT GCA TGG ACC TAA CAT TCG ATG ATC ATT CAG TAT TAA

T A T G T A G T A T T C C ' ~ A A G A A A A A A A A A A G T G G G A G G G A G G'FF T G T G G G A G G ~

A A A A A A A A A C.AA C T ~ T T C T G C C'DG C C T T C A A G T A A A ~

TGT ATG TAC ATA TCT TIT ~

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T A C ~'~C A T G A C T T D G T A A G"IT Aq'~ T I T A T G ~"I~GT T T A T T T G G G C A CI,G C C C A G ~ A T T G T Y ~

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AGG CTG AGT CCT GCC ~GT GCT CCG CAA AGC GCA TGT GTG GAA AGA CTG GCA AAG CCT TCA ~

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Glycosylation

poly A

degradation

Fig. 1. Canary c-jun cDNA. Sequence of the canary c-jun clone (Genbank accession # L35273), with the encoded amino acids and surmised glycosylation sites, the potential rapid degradation site, and the poly(A) addition signal sites indicated.

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K.L. Nastiuk et aL / Molecular Brain Research 27 (1994) 299-309

(measured by absorbance at 260 nm) was recovered at approximately 1 mg per gram tissue. Poly(A) + RNA was isolated by oligo(dT) cellulose chromatography [65].

2.5. Northern blot analysis Total or poly(A) + RNA was electrophoresed in 1.2% LE agarose gels containing 2% formaldehyde and 67 n g / m l ethidium bromide. The RNA was transferred in 10x SSC to a nylon membrane (Nytran, S&S) essentially as in [19], 32p-labeled riboprobes were synthesized for c-jun using [a-32p]UTP (800 Ci/mmol, NEN). After purification through Sephadex G-50, the probe was added directly to a hybridization solution, based on the solution used by [18], as modified by Amasino [3], containing 250 mM sodium chloride, 250 mM sodium phosphate (pH 7.2), 7% SDS, 10% polyethylene glycol (MW 8000), 1 mM EDTA, 50% formamide, 20 /xg/ml polyadenylic acid, and l0 s' dpm/ml probe. After hybridization overnight ( > 16 h) at 65°C, the filters were sequentially washed at 65°C in 0.1xSSC/0.1% SDS, 0.1 x SSC/0.1% SDS, and exposed to XAR-5 at -700C with intensifying screens. In order to assure the quality of the RNA samples, the integrity of the 28S rRNA band was examined by ethidium bromide staining.

taining 0,1% /3-mercaptoethanol (BME). Sections were then washed for 1 hour at room temperature in fresh 2 x S S P E / 0 . 1 % BME, followed by a 1 h wash at 65°C in 50% formamide/2xSSPE/0.1% BME. The slides were then equilibrated with 2XSSC for 30 min (three 10 rain washes) and incubated for 30 rain at 37°C in a solution containing 20 g g / m l of RNAse A, followed by three rinses in 2 x SSC at room temperature. Slides were then dehydrated, air dried and exposed to X-ray films for 1 to 4 weeks. Alternate sections were routinely hybridized with either sense or antisense strand c-jun riboprobes; following this protocol, no signal is detected on X-ray film autoradiograms for the sense strand riboprobes.

2.10. Quantitation of autoradiograms X-ray film autoradiograms were quantified using NIH's Image software and a Macintosh based image analysis system. Measurements were taken over several brain areas including NCM and other neostriatal areas, the paleostriatum, the hyperstriatum and the archistriatum. After subtracting the background, the values obtained in brains stimulated with song or metrazole were divided by values obtained in the same areas in unstimulated controls and numbers are expressed as fold-induction above control levels (for further details see [45,46]).

2.6. Metrazole administration Metrazole (40-50 mg/kg for canaries and 60-70 mg/kg for zebra finches: dose adjusted to induce moderate seizure activity without death within the first 30 rain) was injected intramuscularly (pectoral muscle); injections were made from a freshly prepared stock at the concentration of 20 mg/ml. Controls were uninjected, as no significant ZENK induction was previously observed in controls injected with saline [46].

2. Z Song stimulation Song playbacks were performed as described in detail elsewhere [47]. Briefly, after 24 h of acoustic isolation, birds were stimulated with tape-recorded conspecific song for 30 min (each minute of the stimulus contained 15 s of song followed by 45 s of silence); the birds' own songs were not included in the stimulus.

2.8. Tissue preparation Animals were sacrificed 30 min after the metrazole injection or after 30 min of playback by decapitation. The brains were quickly dissected from the skulls, frozen in Tissue-tek and stored at -70°C before sectioning. 10 ~m brain sections were cut in a cryostat at -18°C, thaw-mounted onto slides pre-coated with 3-aminopropyl triethoxysilane (TESPA), fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 5 min, rinsed in PBS, dehydrated and stored desiccated at -70°C.

2.9. In situ hybridization Brain sections were hybridized with 35S-labeled riboprobes according to Clayton et al. [19], with some modifications, as described below. Briefly, slides were acetylated, rinsed in 2xSSPE (buffered salt solution as defined in [65]), dehydrated and air dried. 3-sS-labeled riboprobes made for c-jun were diluted in hybridization solution containing 50% formamide, 2xSSPE, 2 /~g/~l of tRNA, 2 /xg//xl of bovine serum albumin (BSA), 400 to 1000 ng/pA of poly(A) and 100 mM of dithiothreitol (DTT); 16 tzl of this solution (0.25 to 0.5 × 106 dpm) was added to each section which was then coverslipped. Slides were incubated under oil for 3 h at 65°C and then rinsed with chloroform; coverslips were removed in 2×SSPE con-

3. Results 3.1. The canary homolog o f c-jun I n o r d e r t o i s o l a t e t h e c a n a r y h o m o l o g o f c-jun, w e exploited the relatively close evolutionary relationship between chickens and canaries to identify cross hybridizing clones. A chicken cDNA clone containing the e n t i r e c o d i n g s e q u e n c e o f t h e c-jun g e n e w a s f i r s t shown to react with specific bands on a canary genomic Southern blot (see Materials and methods). Using the c h i c k e n c D N A as a p r o b e , w e t h e n s c r e e n e d 4 0 0 , 0 0 0 independent clones from a cDNA library derived from the HVC-associated telencephalon of male canaries [28]. O n e i n d e p e n d e n t c l o n e w a s i s o l a t e d w h i c h c o n t a i n e d a n i n s e r t o f a p p r o x i m a t e l y 2.0 kb. T h i s i n s e r t was subcloned into pBluescript and sequenced to conf i r m its i d e n t i t y . T h i s c a n a r y c D N A c l o n e c o n t a i n s 1958 b p (Fig. 1), w i t h 306 b p o f 5' u n t r a n s l a t e d s e q u e n c e , 9 4 2 b p o f p r e s u m p t i v e c o d i n g s e q u e n c e , a n d a 683 b p 3' u n t r a n s l a t e d r e g i o n (3' U T R ) . T h e 3' U T R c o n t a i n s a t l e a s t t h r e e p o t e n t i a l p o l y A a d d i t i o n sites, a n d t h e s e q u e n c e A T T T A , w h i c h is f o u n d in m a n y I E G s a n d o t h e r m R N A s w h i c h a r e r a p i d l y d e g r a d e d [68]. T r a n s l a t i o n of the 942 bp open reading frame yields a protein of 314 a m i n o a c i d s ( a a ) , w i t h a p r e d i c t e d m o l e c u l a r m a s s of approximately 34,500 daltons, exactly matching the c h i c k e n a n d c l o s e to t h e 37 k D a m a s s o f t h e 337 a m i n o a c i d r o u t i n e c-jun [39]. T h e p r e d i c t e d a a s e q u e n c e o f t h e c a n a r y c-jun c l o n e is 96.2 p e r c e n t i d e n t i c a l t o t h e c h i c k e n c-jun s e q u e n c e [40,56], a n d 1 0 0 % i d e n t i c a l in t h e D N A - b i n d i n g l e u c i n e z i p p e r r e g i o n (Fig. 2). T h e 1 0 0 % i d e n t i t y o f t h e s e d o m a i n s is m a i n t a i n e d w h e n t h e

K.L. Nastiuk et al. / Molecular Brain Research 27 (1994) 299-309

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Fig. 2. Homology of the canary c-jun with other c-jun genes. Top: graph of percent identity of amino acids (10 AA window) between canary and other receptors reported. Note the two regions of high homology. Insertions in the human and murine sequences are indicated by triangles where size is proportional to insert length; filled circles, canary vs. chicken; open circles, canary vs. mouse; diamonds, canary vs. human.

canary is compared to the murine and human c-jun homologs, but the N-terminal 213 aa of the molecule is only 69.5% identical (see Fig. 2). We examined this apparent c-jun cDNA for similarity with other canary genomic sequences, in order to assure that the signal measured using it as a hybridization probe was not contaminated by cross-reactions.

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Fig. 3. Canary c-jun genomic Southern blot. Canary genomic DNA was digested with restriction endonucleases, electrophoresed, blotted and probed with the radiolabeled canary c-jun cDNA (final wash: 60°C, 0.1XSSC, 0.1%SDS). Lane a, PstI; lane b, HindlII; lane c, EcoRI; size of molecular weight markers indicated on the left (in kb).

Fig. 4. Northern Blot analysis of c-jun RNA in canary and zebra finch brain. Three /xg cytoplasmic poly(A) ÷ RNA or 30 /zg total RNA was separated on a 1.2% formaldehyde/agarose gel and transferred to a nylon membrane. The membrane was probed with a riboprobe encoding canary c-jun and washed to 65°C, 0.1 x SSC, 0.1% SDS. Lane a, canary total cellular RNA; lane b, canary polyadenylated RNA; lane c, zebra finch polyadenylated RNA.

We predicted that the entire c-jun coding sequence should be contained on a single exon, as this is the case in other species [56]. Therefore, we digested canary genomic DNA with restriction endonucleases which have no recognition sites in the cloned canary c-jun cDNA. After electrophoretic size-fractionation of the digested DNA, the D N A was 'Southern blotted' and hybridized under standard conditions to the radiolabeled canary c-jun cDNA (Fig. 3). A single prominent band was detected in each digest, as expected if the c-jun cDNA probe is hybridizing to a single exon in a single-copy gene. When similar Southern blots were hybridized and washed under less stringent conditions, additional bands appeared (as evidenced by the faint lower band in lane c, Fig. 3, and data not shown), probably due to similarities in the conserved leucine zipper region between c-jun and other members of the 'jun' family of genes [62]. To detect the m R N A complementary to the c-jun cDNA, RNA (northern) blotting experiments were conducted, using RNAs from both canaries and zebra finches and a radiolabeled riboprobe synthesized from the c-jun cDNA clone. In the RNA of both species, only one band is detected (Fig. 4). Thus, under our standard hybridization conditions, the c-jun probe does not show significant cross-reaction with other RNAs, and is suitable as a probe for either canaries or zebra finches.

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3.2. Induction o f c-jun by M e t r a z o l e A d m i n i s t r a t i o n T o analyze c-jun induction c o m p a r e d to Z E N K ind u c t i o n following a g e n e r a l d e p o l a r i z i n g stimulus in songbirds, we used in situ h y b r i d i z a t i o n m e t h o d s as d e s c r i b e d p r e v i o u s l y for Z E N K analysis [46,47]. P a r a s a g i t t a l b r a i n sections from m e t r a z o l e - t r e a t e d can a r i e s a n d z e b r a finches w e r e h y b r i d i z e d with 35Sl a b e l e d r i b o p r o b e s for each m R N A , followed by aut o r a d i o g r a p h y . T h e resulting p a t t e r n s w e r e very similar in b o t h c a n a r i e s (not shown) a n d z e b r a finches (Fig. 5). T h e d i a g r a m on the b o t t o m left o f Fig. 5 indicates the structures that can b e seen in the a u t o r a d i o g r a m s in

A

p a n e l s A - D . c-jun m R N A basal levels (Fig. 5C, uninj e c t e d control) are low t h r o u g h o u t the brain, t h o u g h they t e n d to be g e n e r a l l y h i g h e r t h a n Z E N K m R N A basal levels (Fig. 5A). A f t e r m e t r a z o l e t r e a t m e n t , a m a r k e d c-jun i n d u c t i o n occurs in the b r a i n ( c o m p a r e p a n e l s 5C a n d 5D). This i n d u c t i o n is r e s t r i c t e d to the t e l e n c e p h a l o n a n d has a d i s t r i b u t i o n which is similar to the p a t t e r n of Z E N K i n d u c t i o n ( c o m p a r e p a n e l s 5B a n d 5D). c-jun expression after m e t r a z o l e is highest in the L o b u s p a r a o l f a c t o r i u s ( L P O ) a n d is d e t e c t a b l e t h r o u g h o u t each of t h e m a i n t e l e n c e p h a l i c subdivisions including n e o s t r i a t u m a n d all h y p e r s t r i a t a l fields, alt h o u g h the overall level of i n d u c t i o n is s o m e w h a t lower

C

Fig. 5. c-jun induction after metrazole. Adjacent parasagittal brain sections of adult male zebra finches at the level of song control nuclei HVC and RA were hybridized with radiolabeled riboprobes for canary ZENK (A and B) or c-jun (C and D). X-ray film autoradiographs of sections from uninjected controls (A and C) and metrazole-injected birds (B and D) are shown. The schematic diagram on the bottom left represents the structures that can be seen in A to D. A short exposure of a more medial section (through the center of area X) is shown in panel E. Abbreviations: A, archistriatum; Cb, cerebellum; HA, hyperstriatum accessorium; HD, hyperstriatum dorsale; HV, hyperstriatum ventrale; HVC, high vocal center; L2, subfield L2 of Field L; IMAN, lateral magnocellular nucleus of the anterior neostriatum; LPO, lobus paraolfactorius; N, neostriatum; NB, nucleus basalis; NC, caudal neostriatum; PP, paleostriatum primitivum; RA, nucleus robustus archistriatalis; TeO, optic tectum; X, Area X.

KL. Nastiuk et al. / Molecular Brain Research 27 (1994) 299-309

tl

Fig. 6. c-jun induction in the rostro-dorsal paleostriatum after metrazole. Adjacent parasagittal brain sections of adult male zebra finches injected with metrazole were hybridized with radiolabeled riboprobes for canary c-jun (A) or ZENK (B). Close-up views of X-ray film autoradiographs of sections at the level of Area X and surrounding tissue are shown.

305

than ZENK, especially in the hyperstriatum. (By densitometric analysis of X-ray film autoradiographs, the average level of c-jun induction in the telencephalon is 4-6 fold compared to 10-15 fold for ZENK). In contrast to these areas of c-jun induction, some specific areas have negligible c-jun signal (indistinguishable from control levels), including Field L2a; paleostriatum primitivatum (PP); and the androgen receptor-containing song control nuclei, 'High Vocal Center' (HVC), the 'robust' nucleus of the archistriaturn (RA) and lateral magnocellular nucleus of the anterior neostriatum (IMAN) (Fig. 5D). These areas also show low levels of ZENK induction [46] (Fig. 5B). Aside from the quantitative difference in overall induction levels described above, the c-jun and ZENK expression patterns after metrazole treatment differ in only one other obvious aspect: c-jun levels in song nucleus Area X after metrazole treatment are approximately 2-fold lower (by densitometry) than in the surrounding paleostriatum (Fig. 5E; Area X corresponds to the large area of relatively low c-jun signal in the rostro-dorsal portion of LPO). In contrast, ZENK is typically induced in Area X to levels equivalent to the adjacent paleostriatum, except for a thin rim of low induction surrounding Area X [45]. A side-by-side

Fig. 7. c-jun induction after song presentation. Adjacent parasagittal brain sections of adult male canaries at the level of the caudo-medial neostriatum (NCM) and just medial to Field L2 were hybridized with radiolabeled canary ZENK (A and B) or c-jun (C and D) riboprobes (see methods for details). X-ray film autoradiographs of sections from unstimulated controls (A and C) and song-stimulated birds (B and D) are shown.

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comparison of Z E N K and c-jun induction in the rostrodorsai portion of the paleostriatum, including Area X, is presented in Fig. 6. In general, the induction patterns were similar in both canaries and zebra finches.

3.3. c-jun Induction following song exposure To determine whether song presentation induces the c-jun gene, we again used in situ hybridization to examine brain sections of canaries and zebra finches that had been exposed to playbacks of tape-recorded song or silence [45,47]. To allow direct comparison of the c-jun and Z E N K induction patterns, adjacent sections from birds in each group were alternatively hybridized to c-jun probes or to Z E N K probes. Again, the resulting patterns were similar in canaries (Fig. 7) and zebra finches (not shown). In medial parasagittal sections of canary brains (about 150 mm from the midline), basal levels of Z E N K and c-jun are comparably low (Fig. 7A,C) and the pattern of c-jun induction (Fig. 7D) is very similar to the Z E N K pattern (Fig. 7B). This level includes the caudo-medial neostriatum (NCM), where we had previously identified a robust Z E N K induction response [45,47], and induction of both genes is again highest within NCM. Similar to the case with metrazole induction (Fig. 5), the average level of c-jun induction by song (2-3 fold) is lower than for Z E N K (6-9 fold). No consistent c-jun induction response is observed at more lateral brain levels, and no c-jun induction is detected within any of the song control nuclei or in Field L2a (not shown).

4. Discussion

The induction of Z E N K by birdsong [45,47] provided dramatic evidence that natural perceptual stimuli could cause rapid genomic responses in the brain. This earlier finding raised at least two pressing questions that are largely answered by the results reported here. First: is Z E N K the only gene that is induced by these stimuli, or are other genes also induced? Second: why did we fail to observe Z E N K induction in several brain areas known to be physiologically activated by song could that be a peculiarity specific to the Z E N K gene, or does it indicate a more general lack of l E G responses in these areas? To address these questions, we developed a songbird probe for another immediate-early gene which has been especially well characterized in mammalian systems. Our choice, c-jun, was also based in part on the fact that c-jun encodes a transcription factor protein of an entirely different structural (and presumably functional) class compared to ZENK. The c-jun gene was originally identified as a proto-oncogene, the cellular counterpart of a gene in an avian sarcoma virus [40,56].

The c-jun gene has been cloned in a number of species, to which we can now add the canary. The principle source of uncertainty in identifying the c-jun homolog was the presumed presence of related members of the jun 'family' in the genome, as observed in organisms studied to date [62]. However, the combined weight of our DNA sequence, southern and northern blot analyses constitutes compelling evidence that we have identified, specifically, the canary c-jun gene homolog. Our data clearly demonstrate that c-jun is induced in the brain of songbirds in response to both metrazole and song stimulation, in patterns that are mostly similar to ZENK. Thus the first question above can be answered: No, Z E N K is not unique in its response to song, and conceivably many other lEGs are also part of the genomic program activated by song [2,11,12,31,32, 37,50,51,63]. The response patterns of c-jun and Z E N K are not precisely identical, however, and the metrazole experiment reveals one particularly interesting difference: c-jun shows a decreased level of induction in song control nucleus 'Area X' as compared to ZENK. This indicates that different combinations of lEGs may occasionally be induced in different brain nuclei, perhaps associated with the different functional properties of these nuclei. Mechanistically, this could result from differences in specific signal transduction components (necessary for l E G induction) in Area X compared to other brain regions, coupled with different sensitivities of the Z E N K and c-jun genes to these various signal transduction components. As the name 'Area X' suggests, the functional role of Area X in the song circuitry remains somewhat mysterious, though strong evidence exists for a role in the learning of song patterns in the juvenile [14,66]. Area X is also unusual in that, among the major sexually dimorphic song nuclei, it alone fails to express the androgen receptor gene and shows no androgen binding activity [6,10,55]. Despite this, the overwhelming impression conveyed by our data is that the activation patterns of Z E N K and c-jun are more similar than they are different. Of special note, c-jun shares with Z E N K a conspicuous lack of induction in the 3 androgen receptor-containing song nuclei, and in the primary auditory thalamo-recipient area of the telencephalon (Field L2a). These areas show little or no induction of either Z E N K or c-jun, whether the stimulus is a seizure (which induces both genes throughout most of the rest of the telencephalon) or a song (which is known to induce both electrophysiological and metabolic responses in these brain regions [22,41,42,53,54,67,71,72,75]). The trivial possibility that this apparent lack of induction reflects a non-specific decrease in hybridization signals in these areas (for example, due to decreased cell density) can be dismissed, given our previous evidence for other RNAs which either do not distinguish these areas from

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the rest of the brain [19,21,29] or which are enriched in some of these areas [55]. Thus the second question above can now be answered: the lack of Z E N K induction in these parts of the auditory and song control systems is not a peculiarity unique to the Z E N K gene itself. Instead, the androgen receptor-containing song nuclei and Field L2a must differ somehow from most of the rest of the telencephalon, such that synaptic activation (sufficient to induce detectable physiological responses) is not coupled to induction of at least 2 common immediate early genes. Neither the mechanistic basis nor the functional significance for this observation is known. However, this observation adds to the growing list of molecular attributes that differentiate some or all of these regions from the rest of the telencephalon. These attributes include apparently lower levels of both N M D A receptors [1,9] and GAP-43 gene expression (Clayton et al., ms. in prep.), as well as increased levels of several proteins that can buffer calcium [15,77]. Since both N M D A receptors and GAP-43 have been shown to participate in plastic change in the nervous system, mediated in part by intracellular signalling pathways involving calcium, each of these differences would appear at first glance to promote the stabilization of circuitry in these brain areas, perhaps making them more resistant to change. Clearly it remains a possibility that these brain regions express other novel lEGs, yet to be discovered, following song a n d / o r metrazole. It is also possible that these regions may be capable of activating presently known lEGs, but only during restricted windows of plasticity [29]. In any event, these particular auditory and vocal processing centers of the songbird appear to stand apart from most of the rest of the brain in the way lEGs are regulated. The song system may thus offer a unique perspective for gauging the functional role of immediate early gene induction in the brain.

Acknowledgements This work was supported by grants from the Whitehall Foundation and the NIH (NS-25742). We gratefully acknowledge the contributions of Fernando Nottebohm and the many members of his laboratory who have interacted with us.

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