Krox20 may play a key role in the stabilization of long-term potentiation
J. Williams a M. Dragunow c,,, p. Lawlor c, S. Mason b, W.C. Abraham b j. Leah d, R. Bravo J. Demmer a, W. Tate a
a Department of Biochemistry, Dunedin, New Zealand b Department of Psychology, University ofOtago, Dunedin, New Zealand
c Department of Pharmacology, School of Medicine, University of Auckland, Auckland, New Zealand d School of Science, Griffith University, Brisbane, Australia
e Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research, Princeton, NJ, USA
e
Accepted 16 August 1994
Abstract
Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.
Keywords: Immediate-early gene; Learning; Memory
1. Introduct ion
Recent studies have shown that high-frequency stimulation of the perforant path, which produces long-term potentiation (LTP) of perforant path-de- ntate granule cell synapses, is associated with an induc- tion of immediate-early genes (IEGs) in granule cells [9,11,12,13,15,20,21,28]. The products of these IEGs are transcription factors that may regulate neuronal gene expression, leading to long-term adaptive re- sponses to synaptic or other forms of stimulation [10,12,24]. IEGs do not regulate the induction of LTP since the protein levels increase well after the high- frequency stimulation, and the extent of their expres- sion is not well correlated with the amount of LTP produced [15,22,23]. On the other hand, recent studies indicate that these molecules may be involved in the biochemical cascade leading to the stabilization of LTP, i.e. providing a durable form of LTP decaying with a time constant of several weeks (LTP3) [1,2,11,15,22].
Krox24 [12], also known as zif /268 [8], egr-1 [25], TIS 8 [26], and NGFI-A [5], is the immediate-early gene that is most readily induced after LTP [9,22,28,29]. Krox24, a member of the zinc-finger-containing tran- scription factor family, is activated by NMDA receptor stimulation and its expression is tightly correlated with the persistence of LTP [22]. Krox20 [6,7], also known as egr-2 [16], is another member of this family although it has homology only in the zinc-finger DNA binding domain, and it recognizes the same DNA binding sequence as Krox24 [18]. Krox20 is expressed in devel- oping hindbrain [27], basally in rat brain in neurons and non-nerve ceils [19], and in adult rat brain after seizure activity, dopamine receptor activation and opi- ate withdrawal [4], although others have found that seizures do not induce Krox20 [19]. The expression of Krox20 after LTP produced by perforant path tetaniza- tion was investigated to see if this member of the Krox family of transcription factors may play a role in the LTP stabilization processes. In the present study, LTP was induced in awake rats because we have found that sodium pentobarbital anaesthesia interferes with both lEG induction [11,13,15,22] and the stability of LTP [15].
88 Z Williams et al. / Molecular Brain Research 28 (1995) 87-93
2. Materials and methods
Methods for the induction and measurement of LTP in awake animals have been previously described [11,15,22]. The standard tetanization protocol to induce LTP involved 50 trains (400 Hz. 25 ms, 250 # s duration pulses) presented in bursts of 5 trains at 1 Hz with 1 min between bursts. This standard protocol was used for all of the time-course analysis. Only animals showing LTP 20 min post-in- duction were included (i.e. > 10% increase in population EPSP slope and > 2 mV increase in population spike amplitude [22]). In other conditions, stimulation trains were delivered as either 10, 20 or 30 trains on 1 day and 10 trains delivered on 2, 3 or 5 successive days. For some animals, the standard 50 train stimulation protocol was delivered in the presence of pentobarbital (60 mg/kg) ; CPP (10 mg/kg , i.p.) or MK-801 (1 mg /kg , i.p.). Low-frequency stimulation consisted of 50 trains of 10 pulses at 1 Hz, with a 20 s inter-train interval. Evoked responses to test stimuli (150 /xs, 1 /20 s) were recorded for 10 min before and for 20 min after tetanization, with population EPSP slope and population spike amplitude being mea- sured to assess the amount of LTP induced [15].
Animals (3-6 animals per group) were sacrificed by ether over- dose 0 min, 20 min, 1, 2, 4, 8, 24 or 48 h post-tetanization. For immunohistochemistry, the animals were perfused through the heart with 60 ml of saline followed by 120 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed, left to stand in the above fixative for 2 -4 days and then cut coronally (70 p.m thick sections) on a vibroslice machine. Immunohistochemistry was performed as previously described [22]. For quantification pur- poses, the degree of immunoreactivity was visually assessed blind using a 6 point rating scale ranging from 0 (extremely low immunore- activity across the dentate gyrus) to 5 (high immunoreactivity). Sec- tions from the dorsal dentate gyrus (about 4 mm posterior to bregma) were analyzed; the judged degree of immunoreactivity for the con- trol, unstimulated hemisphere was subtracted from that for the experimental, st imulated hemisphere to obtain a final corrected value for each animal [22].
The two antisera that were used for these studies (316/5, 715/5) recognize Krox20 but do not cross-react with Krox24 (antibodies 802/1 and 804/1) as described previously in immunoblot studies [14].
Northern blot analysis was performed as previously described [11]. Membranes were hybridized at 60°C overnight with the mouse Krox20 probe in heparin hybridization buffer. Northern blots were washed to a final stringency of 0.7 × SSPE, 0.1% SDS. Membranes were autoradiographed with preflashed Cronex 4 X-ray film for 48 h at - 7 0 ° C in X-ray cassettes containing two intensifying screens. Autoradiographs were scanned with a LKB UltroScan laser densito- meter (2222-020). The membranes were then stripped of the Krox20 probe and hybridized with a 28S rR NA oligonucleotide probe [3]. The Krox20 probe was a kind gift from Dr. Patrick Charnay. Inserts from the Krox20 clones were random prime labeled with [o~ 32 P]dCTP (3000 Ci /mmol ; Amersham, PB.10205) using an Amersham kit (RPN.16002).
3. Results
3.1. High-frequency stimulation induces Krox20
LTP of the perforant path synapses can be reliably induced, without seizures, in the dentate gyrus of awake, freely moving rats following the application of 50 stimulus trains to the perforant path. In association with LTP induction, there was a dramatic increase
both in the level of Krox20 mRNA expression (Fig. 1A,B) and the degree of Krox20-1ike immunostaining (Fig. 2A,B). The immunohistochemical analysis showed nuclear staining only in the dentate gyrus granule cells, demonstrating that the increase in expression was spe- cific to the post synaptic neurons. The increased im- munostaining occurred only ipsilateral to the high- frequency stimulation, with no apparent increase above the low basal levels in the contralateral hemisphere, which served as a non-stimulated but implanted con- trol. Staining was higher in the lower blade of the dentate, as previously reported for other transcription
A Krox 20
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Fig. 1. Timecourse of Krox20 m R N A induction in awake animals. A: Northern blot analysis showing a rapid and transient increase in the dentate gyrus of Krox20 m R N A 20 rain and 1 h following LTP-induc- ing stimulation of the perforant path. Autoradiographs show: Krox20 m R N A signal and 28S rRNA signal. B: histogram showing increase in Krox20 expression normalized to the loadings of total RNA. Lane 1, unstimulated; lane 2, L T P + 2 0 min; lane 3, L T P + 1 hour; lane 4, L T P + 2 h; lane 5, L T P + 4 h; lane 6, L T P + 8 h; lane 7, L T P + 2 4 h; lane 8, LTP + 48 h.
J. Williams et al. / Molecular Brain Research 28 (1995) 87-93 89
factors [11]. The highest basal levels of Krox 20 were found in layers 2-3 of the neocortex.
3.2. Time course of Krox20 induction
Northern blot analysis demonstrated an increase in Krox20 mRNA expression at 20 and 60 rain post- tetanization, but not at later timepoints (Fig. 1A,B). This time course of induction was similar to that previ- ously reported for the related molecule, Krox24 [9,22,28], although the basal levels of Krox20 expres- sion appeared to be lower than Krox24 and the abso- lute level of induction of Krox20 did not appear to be as great. The time course of Krox20 protein induction, while showing the expected early onset of response, indicated a surprising persistence after tetanization. Increased immunostaining was apparent by 20 min post-tetanization, remained at peak levels from 1-8 h and was still apparent at low levels at 24 h before declining to control levels by 48 h (Fig. 2B). For com- parison, we show here that the Krox24 response in the same animals lasted between 4 and 8 h, as previously reported (Fig. 2B, and [22]). In particular, at 8 h Krox
24 is back to baseline, whereas Krox 20 levels are still strongly elevated. This direct comparison in the same animals indicated that the antibodies used to detect Krox24 and Krox20 were recognizing distinctly differ- ent proteins.
3.3. Relation of the Krox20 response to L TP induction
Expression of IEGs may respond to synaptic activity or membrane potential changes that accompany LTP without being specifically related to the LTP induction mechanisms. To determine how Krox20 induction cor- relates with LTP induction, the Krox20 response was investigated following five stimulus conditions, 20 min following LTP induction: (1) the normal 50 trains of high-frequency stimulation, sufficient to induce LTP; (2) low-frequency stimulation (1 Hz), insufficient to induce LTP; (3) 50 trains of high-frequency stimulation in the presence of sodium pentobarbital and (4) 50 trains of high-frequency stimulation in the presence of the NMDA antagonists CPP (10 mg/kg, i.p.) and MK- 801 (1 mg/kg, i.p.), which prevent LTP induction.
Both Northern (Fig. 3A) and immunohistochemical
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Fig. 2. Timecourse of Krox20 protein induction in awake animals. Top: immunohistochemical analysis of sections through dorsal hippocampus showing increased Krox20 immunoreactivity in the dentate gyrus granule cell layer 2 h following LTP-inducing stimulation of the perforant path. The increased immunostaining occurred only ipsilateral to the high-frequency stimulation (a), with no apparent increase above the low basal levels in the contralateral hemisphere (b), which served as a non-stimulated but implanted control. Bottom: graph showing the timecourse of Krox20 and Krox24 protein expression following LTP-inducing stimulation.
90 J. Williams et al. / Molecular Brain Research 28 (1995) 87-93
analyses (Fig. 3B) demonstrated significant Krox20 in- duction in the normal high-frequency group. Northern analysis demonstrated no Krox20 response when high-
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frequency stimulation was given in the presence of pentobarbital despite the induction of LTP in this group which was, however, less than that observed in awake animals (Fig. 3C). Krox20 was not induced in those animals in which no LTP resulted, i.e. the low- frequency and N M D A receptor antagonist groups. These data indicate that while Krox20 can be induced in conjunction with LTP, there are conditions (e.g, in the presence of pentobarbital) for which there is little or no Krox20 response following LTP induction. This same pattern of gene response in relation to LTP induction had been observed for Krox24 [22], jun B, jun D, c-jun and los-related genes [11].
3.4. Relationship o f the Krox20 response to L TP persis-
tence
Previously we have documented that LTP persis- tence depends, in part, on the number of stimulus trains delivered. LTP decays with a time constant of a few days after only 10 trains (LTP2), but its decay constant lengthens to several weeks (LTP3) if 50 trains are given [2,15]. LTP persistence reverts to the LTP2- type when the 50 trains are given in the presence of pentobarbital. Accordingly, we examined whether Krox20 induction, both m R N A and protein, was re- lated to stimulus conditions that induced the long-last- ing form of LTP, i.e. LTP3 or in the shorter form LTP2 (Fig. 4A,B). An increase in Krox20 protein expression was not detected after 10 stimulus trains (even though LTP was induced and there was a small increase in m R N A expression), but an increase which became progressively greater occurred as more stimulus trains were delivered, Small increases in Krox20 expression were observed when 10 trains were given on 2, 3 but not 5 consecutive days, however, the cumulative Krox20 response is parallel to the gradual increase in LTP persistence observed in other animals (and reproduced in Fig. 4C) given the same progressive increase of stimulus trains. Pentobarbital, which we have demon- strated to block Krox20 induction following 50 stimulus trains, also prevents the normally occurring LTP3, Ieav-
Fig. 3. Krox20 mRNA and protein expression and mean induction of LTP, following various stimulation paradigms. A: histogram derived from Northern blot analysis showing Krox20 mRNA expression nor- malized to the loadings of total RNA. B: histogram derived from Krox20 immunohistochemistry. C: histogram showing mean LTP (EPSP) induced following either: (1) low-frequency stimulation (LF); (2) high-frequency stimulation+sodium pentobarbital (pentobarb); (3) high-frequency stimulation+NMDA receptor antagonists, MK- 801 or CPP (CPP/MK-801); (4) high-frequency stimulation (HF).
J. Williams et a l . / Molecular Brain Research 28 (1995) 87-93 91
Fig. 4. Krox20 mRNA and protein expression and mean induction of LTP, following a titration of LTP-inducing stimulation. A: histogram derived from Northern blot analysis showing Krox20 mRNA expres- sion normalized to the loadings of total RNA. B: histogram derived from Krox20 immunohistochemistry. Hatching represents cumulative protein. C: histogram showing LTP (EPSP) decay time-constant following high-frequency stimulation consisting of either: (1) 10 trains; (2) 20 trains; (3) 30 trains; (4) 50 trains; (5) 10 trains on 2 consecutive days (2 x 10); (6) 10 trains on 3 consecutive days (3 x 10); (7) 10 trains on 5 consecutive days (5 × 10).
ing an LTP that persists with a time constant of two days (LTP2 [15]).
4. Discussion
The present experiments demonstrate that Krox20 shows a rapid induction in dentate granule cells follow- ing LTP-inducing stimulation of the perforant path. The unusual aspect of this response was that although the mRNA induction for Krox20 was transient and had a similar timecourse to Krox24 (cf. [24]), with levels back to baseline after 2 h, the increase in protein levels was greatly prolonged. Krox24 was back to baseline by 8 h yet Krox20 was still strongly expressed at 8 h and was still expressed at 24 h. Krox20 protein may be more stable than Krox24 and therefore is a good candi- date to regulate the cascade of secondary gene expres- sion that may be involved in stabilizing LTP for an extended time. Worley et al [29]. also recently found that krox20 mRNA is induced by a 50 train LTP stimulus, although these researchers did not investigate the time-course of the induction or protein levels.
Basal expression of both Krox20 and Krox24 genes are low in the dentate gyrus, in contrast to other hippocampal regions which exhibit low Krox20 mRNA and protein levels, but relatively high levels of Krox24 [22]. This suggests that Krox20 and Krox24 may sub- serve somewhat different physiological functions in hippocampal pyramidal neurons. We did not detect any Krox20 immunostaining in non-nerve cells in con- trast to the results of Mack et al. [19]. The reason for this difference is presently unclear.
The induction of Krox20, like that of the other immediate-early genes [9,11,13,22,28], was prevented by competitive and non-competitive NMDA receptor antagonists, which also prevent LTP induction. Thus, LTP induces Krox20 in dentate granule cells via activa- tion of the NMDA-subtype of glutamate receptors. Protein expression after LTP was prevented by sodium pentobarbital, which does not completely block the induction of LTP, but does dramatically decrease the persistence of LTP [15]. We suggest that Krox20, like Krox24, is involved in processes relating to the stabi- lization of LTP (see Abraham et al. [1] for a review of these ideas). This hypothesis is further supported by the observation that 10 trains of stimulation did not induce a detectable Krox20 response, whereas 50 trains produced a strong induction. We have previously shown that although both 10 and 50 trains induce substantial LTP, the LTP after the 10 train protocol decays with a time constant of a few days (LTP2), whereas the LTP following 50 trains lasts considerably longer, decaying with a time constant of about 3 weeks (LTP3 [15], although see also [29]).
The dependence of LTP3 on gene expression has
92 J. Williams et al. / Molecular Brain Research 28 (19951 87-93
been demonstrated in Abraham et al. [2], and it is envisaged that there would be a group of genes in- duced whose products are essential for the persistence of LTP. Krox 20 would be a strong candidate for a transcription factor mediating this effect. Both Krox20 and Krox24 recognize the cis-acting e lement G C G G G G G C G [8,18], suggesting that this element may be involved in the stabilization of LTP. The pro- longed induction of the Krox20 transcription factor in dentate granule cell nuclei after LTP which is NMDA receptor-mediated, and correlated v~ith the stability of LTP is indicative that this lEG product could be a key element in the pathway to establish persistent LTP. The target gene(s) in turn regulated by this Krox20 expression in these neurons await to be determined.
Acknowledgements
This work was supported by grants from the NZ Health Research Council, Lottery Board, Human Frontier Science Program, and New Zealand Neuro- logical Foundation. J. Williams was supported by a W.B. Miller Fellowship from the New Zealand Neuro- logical Foundation.
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