268, and long-term potentiation in the dentate gyrus

Brain Research, 580 (1992) 147-154 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

147

BRES 17717

Correlation between the induction of an immediate early gene, zif/268, and long-term potentiation in the dentate gyrus C.L. Richardson 1, W.P. Tate 1, S.E. Mason 2, P.A. Lawlor 3, M. Dragunow 3 and W.C. Abraham 2 aDepartment of Biochemistry, Universityof Otago, Dunedin (New Zealand), bDepartmentof Psychology and the Neuroscience Research Centre, Universityof Otago, Dunedin (New Zealand) and CDepartmentof Pharmacology and Clinical Pharmacology, Universityof Auckland Medical School Auckland (New Zealand) (Accepted 24 December 1991)

Key words: Immediate early gene; Long-term potentiation; Dentate gyrus; Immunohistochemistry; Northern blot; Awake rat

Expression of the immediate early gene zif/268 (also termed NGFI-A, Krox 24, TIS8 and Egr-1) was investigated in awake rats following various long-term potentiation (LTP) induction protocols, zif/268 mRNA (Northern blots) and protein (immunohistochemistry) levels sharply increased following LTP, and followed a time course characteristic of other immediate early genes. When measured across 3 tetanization protocols known to produce differing degrees of LTP persistence, zif/268 induction was found to be more highly correlated with LTP duration than with the magnitude of initial LTP. These data support the hypothesis that the immediate early gene zif/268 plays a role as a third messenger in the cascade of cellular and nuclear events that govern the persistence of LTP. INTRODUCTION

Brief episodes of tetanic activation of the perforant path input to the dentate gyrus results in a persistent increase, or long-term potentiation (LTP), in the synaptic efficacy of this monosynaptic excitatory pathway 1°'2°. LTP exhibits a number of characteristics that suggests it may be a mechanism underlying information storage in the brain, but principal among these is the fact that LTP can last for weeks or months following its induction 8'9' 23. There is considerable variability in the reported rate of LTP decay, however, and factors which appear to affect LTP persistence include the number of stimulation episodes, the number of days over which they are given, and the age of the animals (for a review see ref. 3). Protein kinase C inhibitors 26'2s, protein synthesis inhibit o r s 24'27 and sodium pentobarbita123 have also been shown to influence LTP decay rate. Since the LTP decay rate can be considerably longer than the half-lives of brain m R N A ( < 4-20 h 5) or proteins (mostly between 2.5 and 9 days12), interest has begun to focus on whether LTP-inducing stimulation initiates transcription from a set of genes important for the subsequent persistence of LTP. Of particular interest are those genes showing rapid (within minutes), protein-synthesis independent, but transient increases in transcription following certain extracellular trigger stimuli, such

as growth factors or depolarization. Many such immediate early genes (IEGs) code for transcription factors and thus may serve as nuclear third messengers regulating the expression of other target genes and thereby the long-term adapative responses of neurons to external stimuli 1s,29. Several IEGs, including a c-fos-related gene 21, jun-B and zif[26817"35, have been shown to undergo a transient increase in transcription in the granule cells of the dentate gyrus following the induction of LTP by perforant path stimulation. Of these, zif/26814 (also known as NGFI-A 11, Krox 2425, TIS833 and Egr-132) is the gene whose m R N A is increased most consistently, although so far this has only been demonstrated in anesthetized animals. Target genes regulated by the zif/268 transcription factor have not yet been identified, although it is known that the consensus D N A sequence that binds zif/268 is a high-affinity site, G C G T G G G G C G 16. The increased zif/ 268 expression is related to LTP since it is blocked by various treatments that also block the induction of LTP, including N-methyl-D-aspartate receptor antagonism and stimulation of the inhibitory commissural input to the dentate gyrus during perforant path tetanization 17'35. We present here an investigation of zif/268 m R N A and protein induction following LTP in the dentate gyrus, and show that not only does this gene increase expression in awake animals, and with a time course char-

Correspondence: W.C. Abraham, Department of Psychology, University of Otago, Box 56, Dunedin, New Zealand. Fax: (64) (3) 479-8335.

148 acteristic of o t h e r I E G s , but that this e x p r e s s i o n is well c o r r e l a t e d with the persistence of LTP.

MATERIALS AND METHODS

Electrophysiology Adult male Sprague-Dawley rats (300-500 g) were anesthetized with sodium pentobarbital (60 mg/kg) and placed in a stereotaxic frame (tooth bar set at -3.0 mm). A midline scalp incision was made and the skull exposed. Monopolar recording electrodes (75 #m stainless steel wires were implanted bilaterally through burr holes in the skull into the dentate hilus 3.8 mm posterior and 2.5 mm lateral to bregma. Monopolar stimulating electrodes (125/~m stainless steel wires) were implanted into the perforant path fibers running in the angular bundle 4.5 mm lateral to lambda. The wire electrodes, plus screw indifferent electrodes, were connected to a plastic headcap and the whole assembly fixed to the skull with dental acrylic. At the end of the surgical procedure, penicillin was administered i.m. All operated animals were given a minimum 2 week recovery period before the quality of the evoked field potentials was assessed while the animals were quietly awake. Animals showing large positive synaptic waves prior to population spike initiation, plus population spike thresholds < 300/,tA were used for the stimulation groups. Animals without acceptable recordings were used as implanted, unstimulated controls (but handled and otherwise treated identically to stimulated animals). The evoked potentials were recorded through field effect transistors, amplified and filtered (0.1 Hz to 3 kHz) prior to sampling at 10 kHz by a PDP micro-ll computer. Diphasic stimulus pulses (150 ~s half-wave duration) were generated by programmable constant current stimulators, and set to a current eliciting a 3-5 mV population spike. Animals for immunohistochemical analysis received stimulation in only one hemisphere, while animals for Northern analysis received bilateral stimulation (since the immunohistochemistry demonstrated no contralateral effects of stimulation and pooled tissue was required for the Northern blots). Evoked responses to test stimuli (every 20 s) were recorded for 10 min prior and for 20 min following tetanization. Tetanization consisted of 50 trains (400 Hz, 25 ms, 250/~s duration pulses) presented either in bursts of 5 trains at 1 Hz (1 min between bursts, group 5013) or equally spaced 20 s apart (group 50S). Low-frequency stimulation consisted of 50 trains of 10 pulses at 1 Hz, with a 20 s inter-train interval. For anesthetized groups, the previously operated animals were anesthetized with sodium pentobarbital (60 mg/kg) on the experiment day 4 h prior to tetanization, and remained anesthetized until sacrifice (group A50B). All intervals between tetanization and sacrifice are presented as time after the last stimulus train. Measurements of excitatory postsynaptic potential (EPSP) initial slope and population spike amplitude were made just before and 20 min after tetanization to determine the degree of LTP induced 1.

then decapitated at the appropriate time post-tetanization. The brain was removed and the hippocampus and entorhinal cortex were dissected out. The dorsal hippocampus was further dissected into the dentate gyrus and the rest of the hippocampus, giving 3 brain regions in total for analysis. Total RNA was isolated from tissue pooled from 6-8 hemispheres at a time, using the method of Chomczynski and Sacchi ~3. 5/~g of total RNA was denatured with glyoxal and electrophoresed through 1.2% agarose gels. The RNA was then transferred directly onto nylon membrane (Hybond N+ Amersham) using a low pressure vacuum transfer system (Pharmacia-LKB). The RNA was fixed to the membrane on filter paper soaked in 50 mM sodium hydroxide. Blots were hybridized with oligonucleotide probes or random primed probes at 65°C overnight in a buffer containing 4 x SSPE (1 × SSPE: 150 mM sodium chloride, 9 mM sodium dihydrogen phosphate, 1 mM ethylene diamine tetra acetic acid), 0.1% sodium pyrophosphate, 0.5 mg/ml heparin, 0.1% SDS. Membranes were washed to a final stringency of 0.7 × SSPE, 0.1% SDS at 65°C. Membranes were exposed to preflashed Cronex film with intensifying screens at -80°C. Autoradiographs were quantitated by densitometry (LKB 2222-020 UltraScan XL). The signals obtained for the zif/268 analyses were normalized for the signal obtained from the rRNA oligonucleotide7.

Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital at the appropriate time post-tetanization, and perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were removed and then sections were cut coronally (70 #m) on a vibratome; during sectioning the brain was immersed in 0.01 M phosphate-buffered saline (PBS). Sections were incubated in 1% hydrogen peroxide in 100% methanol for 10 min, washed with PBS and then incubated for 48 h with a rabbit polyclonal antibody to Krox 24 (generously provided by R. Bravo) at a dilution of 1:50,000 in immunobuffer (0.01 M PBS, 1% normal goat serum and methiolate, 0.04 mg/ml). Next sections were washed in PBS and incubated with biotinylated goat anti-rabbit serum (Sigma) in immunobuffer (1:500 dilution) for 24 h. After washing in PBS, sections were incubated with extravidin (Sigma, 1:500 dilution in immunobuffer) for 2 h, washed in PBS and placed in the chromogen diaminobenzidine (Sigma) containing hydrogen peroxide. Some sections were incubated in immunobuffer without Krox 24 antiserum and these showed no staining, confirming the specificity of our Krox 24 immunostaining. Immunoreactivity was visually assessed blind by M.D. and P.A.L. using a 6 point rating scale ranging from 0 (extremely low immunoreactivity across the dentate gyrus) to 5 (maximal immunoreactivity). Sections from the dorsal dentate gyrus (about 4 mm posterior to bregma) were analyzed; the judged degree of immunoreactivity for the control, unstimulated hemisphere was subtracted from that for the experimental hemisphere to obtain a final corrected value for each animal. Each treatment group for immunohistochemistry contained between 3 and 6 animals.

Oligonucleotide probes A specific oligonucleotide complementary to 28S rRNA was synthesized on an Applied Biosystems 330B DNA synthesizer using phosphoramidite chemistry. This probe sequence is as follows: (26mer) 5'-AACGATGAGAGTAGTGGTATI"I'CACC-3'.The oligonucleotide was end-labelled with [y-32p]dATP (3000 Ci/mmol, Amersham) using T4 polynucleotide kinase.

Random primed probes A 1.6 kb BglII fragment from plasmid 268BS65 containing DNA encoding zif/268TM was purified from an agarose gel by electroelution and labelled with [ct-32p]dATP (3000 Ci/mmol, Amersham) using random oligonucleotide primers and the Klenow fragment of DNA polymerase (MultiprimeT M Amersham).

Northern blots Rats were deeply anesthetized with sodium pentobarbital and

RESULTS

Induction of zif/268 mRNA and protein in awake animals O u r initial e x p e r i m e n t s a i m e d to establish w h e t h e r zif/ 268 (zif) e x p r e s s i o n increases following LTP in awake animals. Fig. 1A shows the effect of 50 h i g h - f r e q u e n c y trains (400 H z ) p r e s e n t e d in a 'burst' t y p e p a r a d i g m (see M a t e r i a l s and M e t h o d s ) , on zif p r o t e i n levels 2 h later, as d e t e c t e d i m m u n o h i s t o c h e m i c a l l y w i t h an a n t i b o d y dir e c t e d against t h e e n t i r e zif p r o t e i n . T h e r e was a clear increase in i m m u n o r e a c t i v i t y t h r o u g h o u t the d e n t a t e g r a n u l e cell layer of t h e s t i m u l a t e d h e m i s p h e r e r e l a t i v e

149 to the implanted but unstimulated contralateral hemisphere. Scattered cells both in the dentate molecular layer and the dentate hilus also show increased immunoreactivity ipsilaterally. There were no apparent differences elsewhere in the hippocampus or cortex between

600 -

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Fig. 2. Time course of zif induction in awake animals. There was a sequential, transient induction of zif mRNA and protein following 50B stimulation. The mRNA data points represent the results from tissue pooled from at least 8 hemispheres, and have been normalized against the the amount of rRNA in each sample. Each protein data point is the mean result from 3-6 animals, with standard error bars presented when they are greater than 0.

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Fig. 1. Induction of zif in awake animals. A: section through the dorsal hippocampus showing increased zif immunoreactivity in the dentate gyrus granule cell layer ipsilateral to 50B stimulation of the perforant path (left side), 2 h after stimulation. Bar = 1 mm. B: Northern blot analysis showing zif induction only in the dentate gyrus, 20 min after receiving 50B stimulation. The blot was stripped and reprobed for ribosomal RNA to normalize the zif data (see histogram), thus accounting for variations in the loadings of the various lanes. Small differences observable in B for the hippocampus and entorhinal cortex samples after 50B stimulation, and in the dentate gyrus after low-frequency stimulation, fall within the normal variations seen with this analysis and are not believed to be significant. Each lane represents RNA pooled from at least 8 hemispheres. DG, dentate gyrus; HIP, rest of the hippocampus; EC, entorhinal cortex; No, implanted but unstimulated; Lo, 20 rain after low-frequency stimulated (1 Hz); Hi, 20 min after 50B stimulation.

the two hemispheres. It is inferred that the observed increase in zif protein occurs as a result of de novo translation of newly synthesized mRNA, since zifmRNA also increased specifically in the dentate gyrus 20 min after LTP induction, with no obvious changes in the entorhinal cortex or the rest of the hippocampus (Northern blot analysis, data normalized to rRNA in each sample, Fig. 1B). Equal numbers of stimulus pulses presented at lowfrequency (1 Hz) did not produce LTP and had no effect on zif. The time course of the increased zif expression was assessed by sacrificing animals at various intervals (0, 20 rain or 1, 2, 4, 8, or 24 h) after the end of the 50 stimulus trains, as shown in Fig. 2. A very slight increase in mRNA and protein was apparent immediately after tetanization. The mRNA levels rose to a peak around 20 rain post-tetanization, but had returned to baseline within 2 h. As expected, the increase in zif immunoreactivity (assessed visually on a 6 point rating scale) was delayed relative to the mRNA, peaking around 2 h and returning to baseline levels by 8 h post-tetanization. This delayed time course of the protein response is consistent with the hypothesis that it results from the increased mRNA levels which peaked earlier in time. The short half-lives of the mRNA and protein are consistent with zif functioning as an IEG in this system, with potential to play a role in regulating the genomic response to LTPinducing stimulation. An increase in zif expression was observed immunohistochemically for all 10 animals killed at the 1 and 2 h time points, indicating the reliability of eliciting a zif response with these stimulation parameters in awake rats. Northern analysis is not able to provide similar information regarding response reliability across individual rats.

150 However, across several Northern blots, a 6-9-fold increase in zif m R N A has been consistently observed for the 20 min post-tetanization interval. This increase in zif m R N A is measured relative to a very low basal level of zif m R N A , and the variation in the quantitation of this low initial value accounts for the variation in the calculated fold increase of zif m R N A .

Correlation of zif induction with LTP persistence A critical question regarding l E G induction is whether it correlates in any way, qualitatively or quantitatively, with the induction or the persistence of LTP as induced by a variety of stimulus conditions. One way to approach this issue is to correlate, in separate groups of animals, I E G induction shortly after tetanization with LTP persistence, as observed following various tetanization protocols. For the present experiment, zif m R N A (20 min post-tetanization) and protein (2 h post-tetanization) were measured in animals receiving one of 3 types of high-frequency stimulation (as described in Materials and Methods): 50 trains in a burst paradigm (50B), 50 trains in a 'spaced' paradigm (50S), or 50B trains delivered while the animal was anesthetized with sodium pentobarbital (60 mg/kg, A50B). Initial LTP induction was measured 20 min post-tetanization in all these animals. Animals that did not show LTP of either the EPSP ( > 10% increase) or population spike ( > 2 mV increase) were not used for Northern or immunohistochemical analyses. Values for the decay rate of LTP following such stimulation have been taken from our findings in separate groups of animals, as previously published 23. LTP induction was slightly, but not significantly, increased in the 50S condition compared to the 50B condition for both the EPSP and the population spike (combining across animals used for immunohistochemistry and Northern analysis, Table I). LTP induction under sodium pentobarbital was somewhat reduced compared to the other two groups, but again, this effect was not statistically significant. On the other hand, zif m R N A

and protein induction were consistently lower for the 50S group relative to the 50B group, and were significantly reduced for the A50B group to about 15% of the 50B group response (immunohistochemistry, P < 0.05, Fig. 3). The within-animal correlation between zif immunoreactivity and LTP induction across the 3 groups was not high either for the EPSP (r = 0.474, P > 0.1, n = 13) or the population spike (r = 0.390, P > 0.1, n = 13). The correlations between the group mean LTP induction and the mean protein and m R N A induction were substantially better (protein correlation with EPSP, r = 0.958 and spike, r = 0.829; m R N A correlation with EPSP, r = 0.949 and spike, r = 0.813), but they were not sufficiently high to be statistically significant for the small number of groups (3) being compared. In contrast, when mean zif induction and mean LTP decay rate were compared across the 3 stimulus conditions, striking relationships were observed (Fig. 3C). The correlation between protein induction and log rate of LTP decay was -0.999 (P < 0.05, LTP decay rates averaged for the EPSP and spike data) as was the correlation between m R N A induction and log rate of LTP decay (P < 0.05). Not surprisingly, the levels of m R N A and protein induction were also highly correlated (r = 0.999, P < 0.05). These data, showing that the correlation between zif induction and LTP decay rate between animals is higher than the correlation between zif induction and initial LTP induction within animals is strong evidence for the hypothesis that zif plays a role in establishing the persistence of, or stabilizing, LTP. A c o m m o n feature of zif immunoreactivity in hemispheres where expression was only slightly increased by a stimulus condition (e.g. for the A50B animals) was that it was greater in the lower blade than in the upper blade of the dentate gyrus, and greater in the lateral portions of the two blades (Fig. 3A). This finding is consistent with our prior studies of Fos-like immunoreactivity, which showed similar disparities within the dentate gyrus following tetanization 23. While anatomical varia-

TABLE I

LTP Induction, zif/268 induction and LTP decay following various tetanization protocols

EPSP = mean + S.E.M. % change from baseline; Spike --- mean + S.E.M. difference from baseline in mV; mRNA = mean % change from non-stimulated controls (values averaged across two separate Northern blots); Protein = mean + S.E.M. judged difference from control hemisphere; LTP decay rate = log decay averaged across EPSP and population spike (equivalent time constant in days), taken from Jeffery et al. 23.

Group

50B 50S A50B

LTP Induction

zif/268

LTP decay rate

EPSP

Spike

mRNA

Protein

35 + 4% 35 + 6% 30 + 6%

6.8 + 0.7 7.9 + 1.0 4.5 + 1.0

670% 500% 100%

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-1.31 -1.13 -0.51

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151

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Fig. 3. zif induction following various tetanization protocols. A: zif immunoreactivity in one animal 2 h after receiving A50B stimulation (left side), i.e. 50 trains delivered in burst-type fashion in the presence of pentobarbital. Bar = i mm. B: Northern blot showing the zif mRNA response 20 min after the various tetanization protocols in the dentate gyrus and hippocampus. The histogram shows the zif response normalized for levels of rRNA. C: histogram comparing the mean LTP log rate of decay, zif mRNA levels (using data from the Northern blot shown in B) and zif protein levels across tetanization conditions, mRNA ns are > 8 hemispheres; protein ns are 50B = 5, 50S = 3, A50B = 5; LTP decay rates are from Jeffery et al. 21, as given in Table I. Standard errors, where appropriate for these data, are given in Table I.

tion in I E G responsiveness may simply reflect the manner in which the stimulation electrodes have been placed in the angular bundle, optimizing excitation near the recording electrode site, it could be more significantly reflecting uncharted variations in the degree of local circuit inhibition, second messenger activity or some other factor affecting the induction of IEGs.

Constitutive expression of zif During the course of the above experiments we observed that there was substantial zif immunoreactivity present throughout the cerebral cortex (layers 2-6) and in area CA1 of the hippocampus of both hemispheres, regardless of Stimulus condition (e.g. Fig. 1A). This is suggestive of constitutive production of the protein in these areas, and contrasts with the virtually complete absence of immunoreactivity in the dentate gyrus and area CA3 of control hemispheres. O n the other hand,

control unstimulated animals showed about the same level of zif m R N A in the dentate gyrus as in the rest of the hippocampus (cf. Fig. 1B). To assess whether the apparently constitutive protein was in fact elicited by the surgical or handling procedures used in the above experiments, we compared cortical and CA1 zif immunoreactivity in separate animals with and without surgical intervention, with and without handling prior to sacrifice, or with pentobarbital administration prior to sacrifice (2 animals per group). The resuits consistently showed a similar high degree of basal immunoreactivity in the cortex and area CA1 for all animals, except those that had been previously anesthetized for 4 h with sodium pentobarbital. Thus there is substantial constitutive expression of zif in cortex and CA1 that is not secondary to the surgical or handling procedures used in our normal experiments. The fact that pentobarbital reduced this constitutive expression is

152 consistent with our finding that the same treatment reduced LTP-associated increases in zif expression. DISCUSSION The results of the present study clearly demonstrate that LTP-inducing stimulation can increase zif m R N A and protein levels in the dentate gyrus granule cell layer of awake rats, thus confirming and extending the previous findings by two groups using anesthetized animals 17' 35. The results are similar to those previous studies in that the zif induction was confined to the granule cell layer in the stimulated hemisphere, and was not observed elsewhere in the hippocampus nor in the contralateral hemisphere. We presume from the pattern of immunohistochemical staining in the cell layer that the zif induction was occurring at least in the granule cells; local interneurons probably also exhibited the effect, as scattered cells in the dentate molecular layer also showed zif induction. The time course of zif m R N A and protein induction was rapid in onset and persisted for only a few hours, a characteristic feature of I E G responses to extracellular stimuli ~6. This probably reflects a transient enhancement of zif transcription, but we cannot rule out the possibility that the m R N A or protein turnover rate has been affected. It was also notable that the frequency and pattem of stimulation was important for inducing both zif and LTP: short trains of 400 Hz pulses was an efficient stimulus while an equal number of pulses at 1 Hz was not.

Relation of zif/268 to LTP processes The question of major concern to this study is whether zif plays a role in any aspect of the LTP process. The time course data exclude certain possibilities; for example, the delay in the onset of increased translation is probably too long to be important for the initial induction of LTP, which peaks within a minute or two following tetanization 22. Thus it is not surprising that LTP induction and zif expression show varying degrees of correlation across different experimental paradigms 17'3°. Furthermore, the early return to baseline levels indicates that zif is not directly involved in the synaptic expression of LTP. It appears more likely, as others have hypothesized, that zif (and possibly other IEGs) serves as a third messenger in the cascade of cellular and nuclear events that govern the stability of LTP. The fact that zif protein is a likely transcription factor 3~ and is localized in the LTP experiments to the granule cell somata supports this hypothesis. The present experiments provide the first direct evidence, however, that zif induction is related to LTP maintenance processes. Indeed there was a striking cor-

relation between the degree of zif m R N A and protein induction and the decay of LTP over days and weeks. It was significant that such correlations were found even though the molecular and physiological measurements were made in separate groups of animals. These data contrast with our previous study in which the induction of Fos-like immunoreactivity was poorly correlated with LTP decay rate. In particular, Fos-like immunoreactivity was significantly higher with spaced trains than with burst trains, despite rather similar LTP decay rates 23. The fact that zif was induced postsynaptically in the dentate granule cells suggests that not only initial induction mechanisms 34, but also putative stabilization mechanisms are being engaged postsynapticaUy with LTP in the dentate gyrus. This finding does not provide any further information, however, as to the pre- or postsynaptic locus of the LTP expression mechanism(s). In previous studies, we found that blockade of protein synthesis in the dentate gyrus by anisomycin during a critical window of about 15 min just after LTP induction prevented the maintenance of LTP for longer than 3 h 27. Since zif protein synthesis is induced rapidly in the first 20 min post-tetanization, it may be one of the critical proteins affected by anisomycin, However, those previous studies also suggested this critical new translation was transcription independent, and since it appears in the present experiment that the zif protein response is transcription dependent, it is more likely that tetanic synaptic activity triggers synthesis of some other critical protein(s) which governs LTP maintenance over the first 3-6 h post-tetanization. Despite the high correlation between zif induction and LTP decay rates, it is apparent that zif is only one step in the sequence of events initiated by high-frequency synaptic activity. There remain several major outstanding questions. For example, which transcription factors or second messenger pathways must be activated, or repressed, for LTP-related zif induction? The serum responsive element has been found upstream of the zif coding region and may provide an indication of such factors 15. It is also not clear which are the key genes whose transcription is being regulated by zif, although zif binding domains exist upstream of additional IEGs whose expression is induced by extraceUular stimuli in other systems. Such IEGs may also have functions important for LTP, perhaps in concert with zif. Finally, we can not yet be certain that zif activity is causally related to LTP persistence, instead of being spuriously correlated via linkage with some other critical LTP mechanism. There are a number of IEGs which have been shown in anesthetized animals not to be induced in the dentate gyrus following LTP-inducing stimulation of the perforant

153 path. These include c-los, c-jun, ]un-D, N G F I - B , PC4 and S R E W h e t h e r these genes are induced in a w a k e animals needs to be investigated, since it is clear that the induction of Fos-like immunoreactivity is completely blocked by urethane and pentobarbital anesthesia 19'21'23, and even the robust z i f response is significantly attenuated by pentobarbital (present results).

Constitutive expression o f zif/268 C o m p a r e d with the dentate gyrus and area CA3 of the hippocampus, area CA1 and the neocortex show relatively high basal levels of z i f immunoreactivity. These regional differences in z i f expression were not obvious for the m R N A in our N o r t h e r n blot analysis, but were apparent in previous studies using in situ hybridization35' 36. Such a pattern of constitutive expression may reflect

regional differences in spontaneous Nomethyl-D-aspartate receptor-dependent burst discharges 2'5'36, activity in second messenger pathways, or z i f turnover rates, among other possibilities. However, the fact that constitutive expression is reduced by pentobarbital, which enhances synaptic inhibition, suggests that it is activity d e p e n d e n t and may reflect a mode of neural activity that varies between brain regions during resting wakefulness.

Acknowledgements. We are grateful to R. Bravo for the Krox 24 antibody, and to D. Nathans for the zif/268 cDNA probe. We thank A. Jellie and J. Demmer for their help with the Northern analysis and J. Williams for her contributions to the manuscript. This research was supported by grants from the NZ Medical Research Council and the Human Frontiers in Science Program to W.C.A. and W.P.T., and from the NZ Medical Research Council and the NZ Lottery Board to M.D.

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