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Correlations between immediate early gene induction and the persistence of long-term potentiation
NeuroscienceVol. 56, No. 3, pp. 711-727, 1993
0306-4522/93 $6.00 + 0.00
Pmgamon Press Ltd 0 1993 IBRO
Printed in Great Britain
CORRELATIONS BETWEEN IMMEDIATE EARLY GENE INDUCTION AND THE PERSISTENCE OF LONG-TERM POTENTIATION W. C. ARR.AHAM,*~ S. E. MASON,? J. -,I J. M. WILLIAM&$C. L. RICHARIXON,$ W. P. TAT&$ P. A. LAWJ.OR§and M. DRAGWNOW~ tDepartment of Psychology and the Neuroscience Research Center, and *Department of Biochemistry and the Center for Gene Research, University of Gtago, P.O. Box 56, Dunedin, New Zealand SDepartment of Pharmacology and Clinical Pharmacology, University of Auckland Medical School, Private Bag, Auckland, New Zealand Abxtraet-The duration of long-term potentiation in the dentate gyrus of awake rata was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate early genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of xif/268. Increasing the number of stimulus trains resulted in a greater p~~bility of eliciting long-term ~t~tiation with a time constant of several weeks (long-term potent&ion 3), as well as increasing the induction of xif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the xif/268 protein levels showed associated changes. These dam provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered gene expression, initiated by immediate early gene transcription factors such as -&f/268and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabili~tion or maintenan~ of long-term potentiation 3.
Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that may contribute to memory storage processes in the brain.’
the time constant
of LTP decay in the dentate
gyfus*s.6.~8>35
While the persistence of LTP over days or weeks is a feature fundamental to its putative role in memory mechanisms, little detailed information is available regarding the longevity of LTP. A careful analysis of the reported LTP decay rates in the dentate gyrus revealed a clustering of decay time instants of longer than one day into two distinct groups. These have been termed “LTP2”, with an average decay constant of about four days,3’2gand “LTP3”, with an average decay constant of about 23 days3 They stand in contrast to a much faster decaying phase of LTP with a time constant of about 2 h, termed “LTP1”.29 The factors that govern how long LTP will persist in any particular experiment are poorly understood, but it is known that the age of the animal, number of stimulus trains delivered, the number of days over which stimulation is given, and the presence of pentobarbital all influence *To whom correspondence should be addressed. EPSP, excitatory postsynaptic potential; IEG, immediate early gene; IEGP, immediate early gene product; LTP, long-term potent&ion; MOPS, 3-[N-morpholine]propanesulphonic acid; PBS, phosphate-bu&red saline; SDS, sodium dodecyl sulphate; SSPE, sodium ~o~&/s~i~ ph~p~te/e#yl~~aminetetra-acetic acid.
Abbreviations:
717
Thtre is good evidence that the synthesis of new macromolecules is important for the development of the longer-lasting forms of LTP, but not for LTP1.20,27,2* Although LTP2 and LTP3 both appear to require new protein synthesis, the extent to which they involve separate mechanisms remains an open question. We have proposed that altered gene expression is critical for LTP3, but not LTP2,*e3and this is supported by the fact that the mRNA inhibitor actinomycin D does not affect at least the early stages of LTP2 maintenance. nJs This proposal, however, still requires more direct experimental support, particularly since both LTP2 and LTP3 appear to be well described by single exponential functions, rather than the sum of two exponentials as might be expected if LTP3 incorporated a mechanism independent of that involved in LTP2. If altered gene expression is involved in LTP maintenance, it is likely that this will be initiated by the induction of immediate early genes (IEGS), coding for transcription factors. These genes have been proposed to serve as third messengers in the sequence of events leading from postsynaptic depolarization to adaptive gene responses.25 In the LTP model, a number of IEGs show rapid and transient increases in expression following s~mula~on, including c_Jun,” jun-B,ii,is*s8 Jun-D,*s raf-I ,*’ Fos-related genes*5J8
718
W. C’. AHRAHAUc’! (I/.
and zif/268:“,J”.‘x also known as NGH-A.” Krox 24.” and Egr-l.36 Of these. zif/268 has been most reliably induced in correspondence with the induction of LTP,“.‘X but both Fos-related genes and Jun family members are also consistently induced under certain conditions.‘? Little is known about the target genes regulated by these transcription factors. However, a high-affinity DNA binding site for zifi268 has been characterized.‘” and Fos and Jun family members form homodimers (Jun) and heterodimers (FosJun) that bind to the activator protein-l recognition site.‘h If IEG induction is a critical step in the sequence of events leading to the stabilization of LTP. the relation of IEG induction to LTP ‘induction’ is less important than its relation to the ‘persistence’ of LTP. In this study we examined the extent to which the induction of a number of IEGs, and their protein products (IEGPs), correlate with the persistence of LTP, as measured in separate groups of animals across a variety of stimulus conditions. Our aim was to look for a pattern of IEG responses that was consistently associated with the specific appearance of LTP3, and thus provide support for the hypothesis that LTP3 involves a mechanism qualitatively differTIS8”
ent from LTP2.
EXPERIMENTAL PROCEDURES Adult male Sprague -Dawley rats (3W500 g) were anaesthetized with sodium pentobarbital(60 mg/kg) and placed in a stereotaxic frame for initial electrode implantation. They were prepared bilaterally with monopolar recording electrodes in the dentate hilus, and monopolar stimulating electrodes in the perforant path, as described previously.” The wire electrodes, plus screw indifferent electrodes. were connected to a plastic headcap and the whole assembly fixed IO the skull with dental acrylic. At the end of the surgical procedure, penicillin was administered i.m. All operated animals were given a minimum two-week recovery period before the quality of the evoked field potentials was assessed while the animals were quietly awake. Recordings, sampled and recorded by microcomputer, were made in response to diphasic stimulus pulses (150 ps half-wave duration) that were generated by programmable constant current stimulators, set to a current to elicit a 3--S mV population spike.‘” Tetanization consisted of l&50 trains (400 Hz, 25 ms. 250 ps duration pulses) presented as bursts of five trains at 1 Hz (1 min between bursts). Trains were given either all on one day, or as IO trains on two, three or five consecutive days (referred to as 2 x IO, 3 x IO and 5 x 10 train groups). Measurements of excitatory postsynaptic potential (EPSP) initial slope and population spike amplitude (responses recorded at 0.05 Hz) were made during the 5 min before the first train and 20min after the last train to determine the degree of LTP induced.’ EPSP LTP was calculated as the per cent change from the average pretetanization value. Population spike LTP was calculated as the mV difference in amplitude from the prctetanization value. A few animals did not show LTP ( i 10% EPSP change and kss than 2 mV spike change) and were excluded from the study. Animals for immunohistochemical analysis received stimulation in only one hemisphere. while animals for Northern analysis generally received bilateral stimulation (since the immunohistochemistry demonstrated no contralateral effects of
stimulation and pooled tissue was requlrcd for the Northern blots). Animals used for LTP decay analysis received either unilateral or bilateral stimulation. depending on the accuracy of electrode placements.
The procedures for assessing LTP decay were identical 10 those described by J&ery er 01.” Briefly, baseline dentate gyrus evoked potentials were obtained m response to 4.5 test pulses to the perforant path delivered at 20-s intervals at the same time of day and in the same room for each ral. The animals were generally still but awake during this time. Once stable recordings were obtained for at least one week (three to four baseline recording sessions). tetamiration was deli\ered according to one of the protocols described abo~. After tetani;ration. recordings of the test pulse responses were made on post-tetanization days I. 2. 3.4. 7. IO. 14 and weekly thereafter until either the baselme was regamed or the recordings became unstable. Some hemispheres where recordings returned to a stable baseline and remained so for three to four weeks were retetanized using a new paradigm. The EPSP and soike values for the nest-tetanilstion dabs were expressed as the per Cent change, or mV difference. for the average of the lash four pretetanization baseline valun. The EPSP and sDike data for each hemisohere. from dav I post-tetanization onward, were plotted’ separately on a scattergram. A single negative exponential curve was fitted to the points. according to the equation J’ 7 Be. xi, where 8 is the y-intercept. k is the rate constant of dmy and I is the number of days post-tetanization. Tbe decay time constant (T) equals I!k and represents the time (in days) for LTP to decay by 63%. Log(k) was used to normalize the decay rates for statistical analysis. Hemispheres which showed sudden substantial drops in response amplitude. decayed more than l5”& below baseline or had data too variable for curvefitting were excluded from the analysi\
A specific oligonucleotide complementary to 28s rRNA (26mer; 5’-AACGATGAGAGTAGT0GTAmCACC~3’) was hybridized to all northern blots.’ The oligonucleotide was end-labelled with [y-“P]ATP (3000Cilmmol. Amersham) using T4 polynucleotide kinase. The zif/268” clone was a kind gift from Dr D. Nathans. The Jun-B (cat. no. 63205?-‘)and Jun-D (cat. no. 632tW) probes were obtained from American Type Culture Collection. The mouse c-Jun clone’? was a kind gift from Dr 1. M. Verma. Inserts from the zif/268 and Junclones were random prime labelled with [a-‘2PvCTP (3000 Ci/mmol; Amersham. PB. 10205) using an Amenham kit (RPN. 16002).
Rats were deeply anaesthelized utth ether and then decapitated 20min post-tetanization. The brain was removed and the hippocampus and entorhinal cortex were dissected out. The dentate gyms was then dissected from the rest of the hippocampus. Total RNA was isolated from tissue and pooled across four to eight hemispheres at a time. using the method of Chomczynski and Sac&i.’ Total RNA (IO pg/lane) was separated by electrophonsis in 1% agarose, I x MOPS,0.6 M formaldehyde gels.” The RNA was then transferred directly on to nylon membrane (Hybond N + , Amersham) using a low pressure vacuum transfer system (Pharmacia-LKB) and fixed onto the membrane with 50mM sodium hydroxide. Blots were hybridized in turn, with the oligonucleotide and random primed probes at 65’C overnight in a buffer containing 4 x SSPE (I x SSPE: I50 mM sodium chloride. 9 mM sodium dihydrogen phosacid), 0.194 phate, 1mM ethylnediaminetetra-acttic sodium pyrophosphate, 0.S m&ml hcparin and 0.1% sodium dodecyl suiphate (SDS). Membranes wterewasbed to a final stringency of 0.2 x SSPE (or 2 x for the oli@tucleotide). 0. I % SDS at 65°C and exposed to prefiasbed Cronex
Immediate early genes and long-term potentiation
719
8lrn with intensifying screens at -80°C. Autoradiographs were qua&&d by laser densitometry (LKB 2222-020 UltraScan XL). Individual blots were stripped in boiing 0.1% SDS and returned to room temperature. The signals obtained for the IEG analyses were normalixed for the signal obtained from the rRNA oligonucleotide.’ Results of northern analysis were contirmed on two to three separate blots and the rRNA-normalized gene responses were averaged across blots.
rates for these two measures have been averaged to give a single decay value for each hemisphere tested. A typical response before tetanization, after tetanixation and after LTP decay is shown in Fig. IA. A monotonic increase in the persistence of LTP was observed as the number of stimulus trains given on one day and was increased from 10 to SO; example data from one animal given 10 stimulus trains and from one animal given 30 stimulus trains are shown Rats were deeply anaesthetized with ether 2 h postin Fig. 1B. Mean time constants increased from 1.5 tetanixation, and perfused transcardially with saline folto 21.3 days as illustrated in Fig. 2A, with some lowed bv 4% uaraformaldehvde in 0.1 M nhosnhate buffer. evidence for saturation occurring in the 40 and 50 pH 7.4. -Bra& were removed and then kctidns were cut train groups. Within most groups there was a concoronally (7Ocm) on a Vibratome; during sationing the siderable range of LTP decay rates. As the number of brain was immersed in 0.01 M phosphate-buffered saline (PBS). Sections were incubated in 1% hydrogen peroxide in trains was increased, however, there was a gradual 100% methanol for lOmitt, then washed with PBS and increase in the consistency with which LTP3 (decay incubated for 48 h with one of a series of antibodies to IEG time constants of three to four weeks) was observed. proteins: a rabbit polyclonal antibody to xif/268 (Krox 24, generously provided by R. Bravo) at a dilution of 1: 50,CQO This latter effect is apparent from the decrease in the in immunobutkr (0.01 M PBS, 1% normal goat serum and standard deviation across the 30-50 train groups methiolate, 0.04 mg/ml); a rabbit polyclonal antibody that (Fig. 2A, see also Table 2). detects both Fos and Fos-t&ted genes (no. PCOS), OncoAn increase in LTP persistence was also observed gene science (B. Moore, personal communication) diluted when 10 trains were repeatedly delivered across a 1:lOOO in immunobutfer; and Jun-speciIlc antibodies to number of days (mean time constants increasing from c-Jun (no. 607/S, 1:lOOO in immunobutfer), Jun-B (no. 725/3, 1:20,000 in immunobtier) and Jun-D (no. 783/3, 1.5 days for the 10 train group to 12.6 days for the 1: 20,000 in immunobuffer), which have been characterized 5 x 10 train group). The magnitude of this effect, previously using immunoblots2) The Jun family of antihowever, was less than when the 10 trains were bodies were generously provided by J. Leah, and were produced and characterized by Dr R. Bravo.” The nonspecific Fos-related gene antibody is different from that used in earlier reports,‘~~” but is the same as that used by Demmer et af.” The next sections were washed in PBS and incubated with biotinylated goat anti-rabbit serum (Sigma) in immunobutfer (1: 500dilution) 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 diaminobenxidine (Sigma) containing hydrogen peroxide. Some sections were incubated in immunobt&r without antiserum but containing normal rabbit sermn. These showed no staining, confirming the speciticity of our immunostaining. Immunoreactivity was visually assessed blind by M.D. or P.A.L. using a six-point rating scale ranging from 0 (extremely low immunoreactivity across the dentate gyms) to 5 (very high immunoreactivity). Sections from the dorsal dentate gyms (about 4mm posterior to bregma) were analysed; the judged degree of immunoreactivity in the dentate gyms 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 three and six animals.
A
_A_qpA
RESULTS
Long-term potent&ion of trains
decay following multiple sets
exponentials,
were
10
20 Days
Data for LTP decay in the dentate gyrus, and their fitted
0
obtained
from
39 hemi-
spheres, and combined with data from 20 hemispheres reported previously in Jeffery et al.” [the 20 hemispheres were distributed among the 10 (n = S), 50 (n = 7) and 5 x 10 (n = 8) train groups]. As previously observed in the dentate gyrus, LTP decayed to baseline for all rats. The decay of LTP was comparable for the EPSP slope and population spike amplitude (see also Jeffery et al’*), and so the decay
30
40
50
60
post-tetanization
Fig. 1. (A) Waveforms for a single rat from the 30 train group at successive stages of decay, from left to right: just prior to LTP (i.e. one of several base line responses used to calculate the mean baseline value); 20 min aRer induction of LTP, day 59 post-tetanization. Each waveform is the average of 10 responses. (B) Scattergratn of the tiy data for two animals given 10 (open circles) and 30 (closed circles) stimulus trains. A single negative exponential curve was fitted to the points giving decay time constants of r = 1.54 days for the IO train animal and T = 26.1 days for the 30 train animal (calculated as described in Experimental Procedures).
W. C. ABRAHAM et al.
720
delivered repeatedly on one day (Fig. 2A). LTP3 occurred frequently in the 5 x 10 group, but the average LTP decay time constant for this group was most like that of the group receiving 30, rather than 50, trains on one day.
-m
-1 50
-
131 6 d )
-1 25
-
(178dl
100-
(100dI
A
It should be noted that the various paradigms all "induced" substantial LTP initially, measured 20 min post-tetanization for both the EPSP (Fig. 2B) and population spike (Fig. 2C). There were no clear correlations across the paradigms between the degree of LTP initially induced and the subsequent rate of decay. Distribution of decay rates
m
2.
...
u
$ a
5o, 0
-075-
0 50
-
-
156dl
(3 2 d )
1
same day
0 - - - - aover days
I I
I
I
I
I
1
2
3
4
5
Sets of 10 trams
Number of stimulus trains
Number of stimulus trains
Fig. 2. (A) The decay rate of LTP for each stimulation paradigm. The mean log decay rate (corresponding decay time constants are given in parentheses) were calculated for each paradigm: sets of 10 trains of stimulation given on one day (solid line) or as one set given each day over several days (dotted line). (B, C) Comparisons of the amounts of EPSP and spike LTP induction 20 min post-tetanization produced by all animals in each of the eight stimulation paradigms. Group designations are explained in Experimental Procedures. Although the 30 and 40 stimulation train groups showed somewhat less EPSP LTP than the others, this was not mirrored in the decay rates for these groups. Overall there was little correlation between mean induction of LTP and mean decay rate of LTP across groups. The values are means and bars represent S.E.M. Ten train group, n = 7; 20 trains, n = 7; 30 trains, n = 6; 40 trains, n = 7; 50 trains, n = 11; 2 x 10 trains, n = 6; 3 x 10 trains, n = 7; 5 x 10 trains, n = 8.
For the groups receiving stimulus trains all on one day, there are two striking observations (Fig. 2A). Firstly, a distinct increase in average LTP persistence occurs between the 10 and 20 train groups. This is due to some hemispheres in the 10 train group showing very little remaining LTP by the first day posttetanization, but none showing this effect in the 20 train group. Secondly, it is clear that the persistence of LTP steadily increases as the number of trains is increased. This latter observation could reflect a consistent increase in LTP persistence for all hemispheres as the number of trains increases, or may reflect a changing mixture of hemispheres showing short (LTP2) and long (LTP3) decay constants. To examine this issue qualitatively, the decay rates for all hemispheres receiving trains on one day were plotted on a frequency histogram, irrespective of the number of stimulus trains, to see whether a unimodal or bimodal distribution of decay rates was evident (four hemispheres from the 10 train group showing decay constants less than 1.1 days were excluded from this analysis). Figure 3A shows that there was an apparent bimodal distribution of the decay rates. This bimodality is not due to an over-representation of hemispheres receiving relatively mild (10 trains) or intense (50 trains) high-frequency stimulation, since restriction of the analysis to the 20, 30 and 40 train groups resulted in a similar frequency distribution (data not shown). A double Gaussian function was fitted to the data in Fig. 2A, and two peaks were found at 5.8 and 30.7 days. These values are similar to the previously calculated mean LTP2 and LTP3 time constants of four and 23 days, respectively.' The low point between the two peaks was 12 days, and this was taken as the dividing point between LTP2 and LTP3 for subsequent analyses. A different frequency distribution was apparent for the histogram constructed for the trains administered across days, i.e. the 10, 2 x 10, 3 x 10 and 5 x 10 groups. As illustrated in Fig. 3B, there was no evidence for bimodality in this distribution. Instead, a weighting of the distribution toward rapid decay times is apparent, perhaps reflecting the relative inability of these stimulus protocols to generate LTP3. Immediate early gene responses to long-term poten tiation-inducing stimulation
IEG responses to the different stimulation protocols were monitored using northern blot analysis of mRNA levels (Fig. 4A). zif/268 showed a very strong
Immediate early genes and long-term potentiation increase in expression in response to LTP-inducing stimulation and Jun-B also showed a consistent increase. The c-Jun gene gave a modest increase in expression, due in part perhaps to the fact that its level before tetanization was much higher than for the other two 1EG.s. As the number of trains delivered was increased from 10 to 50, all the inducible IEGs showed mrresponding increases in transcription. For example, the zif{Z68 response varied from about a two-fold increase after 10 trains to a lo-fold increase after 50 trains. On the other hand, a cJun mRNA response was not detectable after 10 trains but there was about a two-fold increase after 50 trains. In Fig. 4B, the RNA loads in each lane have bee.n normalized to the rRNA oligonucleotide signals. A sizable response atso occurs for Jun-D, but at a time later than the 20-min time point used For the northern blot analysis in the present experiment.‘? The above pattern of mRNA changes was mirrored by the IEGP changes, and a similar response pattern was observed for Fos-related genes as welt. An example is shown with the antibody aa;ainst the zif/268 protein (Fig. J), and a summary of the data
7
6
I
Trains on om day
Trains across days
6
I
1.6
4.0
10.0
25. t
for all of the IEGPs is given in Table 1. A very different pattern of gene responses occurred when tuition consisted of 10 trains delivered on up to five cmsewtive hp. The zif/268 protein was the only IEGP showing a positive response on the second, third and fifth days of tetanization, in addition to the response on the first day. The increased protein levels on days 3 and 5 were. not simply a residual response carried over from the previous days, since in two animals given stimulation for two days and killed 24 h later, there was no observable zif/268 response. Neither c-Jun, Jun-B, Jun-D nor Fos-related genes showed a detectable increase after the first day of 10 trains, and no response was evident for any of these IEGPs on subsequent days of stimulation. Six animals that were tested for LTP persis&nce following 40 or 50 trains of stimulation were subsequently retetanized and used for immunohistochemical analysis. Because several of the antibodies were not available at this time, we have complete data for only Fos-retated genes and a partial set of data for zif/268, These six rats all displayed LTP3 (mean decay constant = 25.7 days). Two animals subsequently received 50 trains and scored 1 and 2 for Fos-related gene imm~nore~ti~ty. Two animals received 30 trains and scored 0 and 1 for Fos-related gene immuno~ctivity. Two animals received 10 trains and showed no change in Fos-retated gene immunor~~vity. A similar pattern of results was seen for zif/268. These data are in accord with the mean results shown in Table 1, and illustrate a within-animal correlation between LTP3-inducing stimulation and IEGP responsiveness. ~rnrned~at~ early gene resportses and persistence of lcmg-term potentiation
Log LTP decay rate 7
721
63.1
LTP decay time constant(days)
Fig. 3. Frequency histogram of LTP log decay rates for each of the types of stimulation paradigms. The number of animals showing particular decay rates for LTP when the &nut&on tins are @en to&r on one day are shown in the upper panel, and those for an&& g&en IOtrains on swxxssive days are shown in the lower panel. Note the bimodaI distribution of decay rates when trains are given on one day. Dots represent values obtained by fitting a double Gaussian function to the data. Data are taken from the same animals as shown in Fig. 2, minus four hemispheres from the 10 train group which had decay constants < 1.1 days. Trains in one day, n = 34; trains across days, n = 21.
To determine whether any of the gene responses were particularly related to the persistence of LTP, correlations were performed across tetanization conditions between the mean LTP decay rates (log-transformed) on the one hand, and the mean mRNA and protein responses on the other. Such an analysis for the groups receiving IO-SO trains on one day showed no clear preferential correlation for a particular gene. All correlations were high, ranging between -0.82 and -0.96 for the various IEGPs, with &f/268 showing the highest correlation (Fig. 6A). Similar correlations using data from groups with multiple days of tetanization could only be done for zif/268, since the other genes showed virtualty no responses under these conditions. For zif/268, the correlations between the gene response on the last day of stimulation and LTP decay rate (mRNA, t = 0.50; protein, F -- 0.31) were low compared to the same correlations for groups receiving the stimulation all on one day. When stimulation is given across days, however, the cumulative (i.e. summed) amount of zifJ268 transcription factor elicited should give a better prediction of LTP perkten~ than the response on just the last day of tetanization. When
722
W. C. ABRAHAM et al.
rRNA
r
w.
7
Uw
Fig. 4. Induction of IEG mRNA in awake rats. (A) Northern blot analysis showing induction of zif1268, cJun and Jun-B mRNA in the dentate gyrus, 20 min after receiving the stimulation. Blots were stripped of the particular probes and rehybridized with an oligonucleotide specific for 28s rRNA (indicated here as the rRNA). Note that Jun-B was from a separate blot and has a separate rRNA internal control. (B) Bar graphs of the relative amount of IEG mRNA in the dentate gyrus when normalized against the quantity of rRNA in each lane. Each lane contained approximately 10 pg of total RNA, prepared from six to eight hemispheres.
the cumulative zif/268 was calculated for each day of stimulation (using linear interpolation between the mean 3 x 10 and 5 x 10 values to estimate the day 4 zif/268 response), the correlation between zif/268 and LTP decay rate was extremely high (r = 0.998, P < 0.005; Fig. 6B). The cumulative zif/268 mRNA levels were not so well correlated with LTP decay rate (r = -0.68, n.s.). Frequency distribution of immediate early gene product responses One important question is whether any genes show responses that correlate specifically with the presence of LTP2 or LTP3. To address this question we tabulated the frequency of hemispheres showing IEGP responses irrespective of response amplitude, and compared this with the frequency of hemispheres showing LTP2 or LTP3. (By simplifying the immunohistochemical analysis to one of whether an observable change in staining had occurred or not we avoid possible problems associated with our normal ratingscale estimation of response magnitude.) As can be seen in Table 2, LTP3 was not produced in any hemispheres receiving 10 trains, but its frequency of occurrence gradually increased with numbers of
trains, levelling off for the 40 and 50 train groups (71-73% of hemispheres). The corresponding data for the Fos-related gene and Jun responses showed a similar pattern, i.e. no hemispheres showed detectable responses after 10 trains, and a gradual increment in the percentage of responding hemispheres as the number of stimulus trains increased. The zif/268 response, however, had a lower threshold than for the appearance of either LTP3 or the Fos-related gene and Jun responses. Fifty per cent of hemispheres showed a zif/268 response after 10 trains, even though none would have been expected to develop LTP3. After 30 trains, 100% of the hemispheres showed a zif/268 response, when only 50% would have been expected to show LTP3. Instead, the zif/268 response had a threshold corresponding to that for LTP2, since about half of the 10 train hemispheres developed LTP2 (three of seven) and a similar fraction showed a zif/268 response (two of four). A similar correlation was observed when trains were given over a number of days (Table 2). This correlation between the thresholds for zif/268 expression and LTP2 is in contrast to the Fos and Jun responses, which correlate with the appearance of LTP3, and only when stimulation is given on one day.
Immediate early genes and lons_term poteotiation
723
Fig. 5. ~0~~~0~~~ showing xif/%8 imrn~o~t~~st~ following different LW induction protocols. (a) The kvel of zif/268 protein present after two days of it3 stimulus trains/day. (b) After three days of 10 trains/day. (c) After five days of IO trains/day. (d) The level of protein after 10 trains. (e) After 20 tmins on one day. (f) After 30 trains on one day. (g) After Zo trains on one day. LTP-induciug &m&ion was applied to one hemisphere chosen at mndom. In each case the animal was killed 2 h after the last set of stimuhrs trains. The stimulated hemisphere is shown on the kfk side of each part of the figure. Note that zif/268 shows a response on each day of stimuhttion (a-c), and that the level of response graduahy incresses as the munber of trriius on one day increases. Sesie bar in c=gOO$Un.
Table 1. protein expression in response to long-term ~~~~-j~~
lotion
Protein (mean f S.D. rating) LTP protocol
Fos-related gtne
c-Jon
Jun-B 0 0.2 i
10 20
O.l:O.2
0.2O*0.3
:
0.3 1.42f 0.5 l.1
0.2 0.9 f* 0.2 0.3
2 X 10 3 x 10 5x 10
0 0 0
0 -0.3 f 0.6 -0.1 f 0.2
Jun”D
a.5
1.1 1.2i0.9 fO.6 -0.2 f 0.3 0 0
xifj26g 0.8 It LO
0.i0&O*2 Es* 1.0 0.5 0.8 f 5.6 1.0
2.1 2.8 kO.8 f 1.2
-0.7 f 0.6 -0.2 f 0.3 0
2.3 f 0.6 1.2 f 1.3 I.7 f 1.2
The extent of expression of IEGFs 2 h after the different LTP-inducing stimutstion prvadigmsisshown~~ratingonascaIeof0_5(meafiof~to$xaaimafsper condition), and after cormcting for the expression in the control hemisphere.
124
W. C. ABRAHAM
A -LTP ZiW266 P--Qc.Jun D----OJtJfl-8 0 . . . . . . . . . 0 J,,,_D
p m” P D 5
2* 5 ti g 1 = B r)
0 10
20
30
40
50
Number
of stimulus trains on one day
(17.8d)
LTP decay o----oprOt‘%n
B
0 1
2 3 Days of 10 trains
tv (I/
Instead, a bimodal distribution of decay time constants was observed, with peaks at 5.8 and 30.7 days. both well within the range of constants previously calculated for LTP2 and LTP3. respectively.2 A blmodal distribution is not a necessary outcome fol LTP3 to involve a different mechanism from LTP2. as there could be a gradual recruitment of the additional mechanism as more trains are delivered, bur its presence does give solid support for the hypothesis of an additional mechanism (LTP3), and further suggests that there is a threshold governing ils recruitment. Spacing the sets of IO trains by one day instead of‘ I min also resulted in a gradual prolongation of LTP. although the cumulative effect of stimulation on consecutive days was less than when all trains were delivered on one day. This is consistent with the observation that LTP decay is relatively rapid after only 10 stimulus trains. Thus, by the lime stimulation was delivered on the second day, relatively little LTP would have been present to interact with the newly induced LTP. These results are consistent with previous studies in which LTP maintenance was prolonged by giving small numbers of trains on consecutive days,S.‘Wbut not consistent with DeJongc and Racine,” who found no additional increase when 70 trains were delivered daily for five days. In thi> latter study, it is possible that the mechanisms relating to LTP persistence had already been saturated after one day of stimulation.
5
6. Correlation of LTP decay rate with IEGP expression. (A) The decay time constants of LTP for the different stimulation paradigms (IO- 50 trains given on one day) are compared with the expression of each of the proteins for zif/268, c-Jun. Jun-B, Jun-D and FRA for the same paradigms. (B) The decay time constants for LTP when 10 trains of stimulation are given each day for successive days (closed circles) are compared with the expression of ziQ268 protein (open circles). The cumulative sum of zifl268 over the successive days is also shown (triangles) Fig.
Table 2. Frequency of hemispheres showmg long-term potentiation 2,/3 or immediate early gene product responses for each stimulalion paradigm Number
LTP3
mechanisms
jar
long-term
potmriuticm
The LTP2jLTP3 distinction was originally derived from an analysis of decay rates in single experiments reported by a number of laboratories, all using somewhat different stimulation protocols.’ The experiments reported here have identified how the LTP decay rate changes following systematic manipulation of the tetanization parameters. As expected from preliminary experiments,‘* the average LTP decay time constant showed a gradual increase as the number of stimulus trains on one day was increased from 10 lo 50. However. a corresponding gradual increase was not apparent from the frequency distribution of decay rates, plotted across individual hemispheres, regardless of the tetanization paradigm.
0%
28%
(017)
(2:7)
0% Kb4)
141% (I ‘7)
C-.lUll
I% 10.4)
(I 31
Jun-B
0% W4)
25% (I 4)
Jun-D
O’h (O/4)
2 5 %I (1.‘4)
LTP2 01
43% (3.7)
I oo”io
LTP3 LiTi
50%
86% (6i7)
2
und 31)
20
Fos-related gene
DIS<‘tiSSION
Multiple
of stimulus trains on one day
IO
(24)
zif/268
or
.s(i
; 3 ? I,
(7 ‘7)
(8:X)
(-5’5)
0% (O/7) 43% (3,7)
83% (Si6)
86”,, (h;7\
I( u “4
50% (2!4)
100%
6090 (39,
IOO%J (1’3)
I
LTP2 LTP3
40
of days of IO train stimulation .__ ._.-.. i 2 4 -62% 0% 145.0 (I ?I I5 8) (o:h)
Number
LTP3
;o
(3i3)
(X.8)
Immediate early genes and long-term potentiation The frequency distribution of LTP decay rates after multiple days of stimulation did not show obvious bimodality. This could be due to the fact that relatively few hemispheres actually displayed LTP time constants in the LTP3 range. It is also possible that, for individual hemispheres, L’IP induction at a number of discrete points in time produced LTP that decayed at different rates for different synapses. This could have had the effect of broadening the range of time constants calculated using single exponential functions, thereby smoothing the frequency distribution Given the relatively small numbers, and skewedness, of observations in this portion of our data, the interpretation of this frequency distribution remains ambiguous. Relation between immediate early gene responses and tong-term potentiation persistence As the number of trains delivered on one day was increased from 10 to 50, all IEGs studied showed associated increases in expression. ~~~n~ngly, large positive correlations between LTP persistence and IEG induction were found when the data were grouped according to the number of stimulus trains delivered, with zif/268 showing the greatest correlation as well as the largest overall response. The relationship between IEG responses and LTP persistence is rather different when tetanization is delivered as 10 trains on consecutive days. In this case the only transcription factor that consistently shows increased expression on each day of stimulation is zifi268. In contrast, none of the other proteins showed a detectable change on any day of stimulation. Thus, it would appear that if IEGs are at all involved in stabilizing LIP, ziff268 is the best candidate primary effector. One of our working hypotheses is that altered gene expression is necessary for the development of LTP3, but not LTPZ. Since the distinction between LTP2 and LTP3 received support from the bimodal frequency distribution of decay rates, it was of interest to see whether the pattern of transcription factor responses also supported the hypothesis. One clear outcome of this analysis is that, contrary to expectation, a sizeable gene response occurred for zif/268 under conditions when LTP2, but not LTP3, would have been expected to be induced (i.e. after 10 stimulus trains). Furthermore, the frequency of positive zif1268 responses in this condition was similar to the frequency of appearance of LTP2 (cf. Table 2). This zif/268 response is similar to that seen after 50 trains in ~ntobarbital-ana~theti~ animals, also a condition in which only LTP2 is produced. In contrast, the Fos-related gene and Jun proteins did not appear to increase in the IO train condition, nor in the presence of pentobarbital.‘3~‘4*‘8 However, a small response for these IEGPs under such conditions, below the detection level of our i~uno~st~he~cal techniques, cannot be ruled out. In line with our working hypothesis, a much larger IEG response occurred after stimulation expected to
725
evoke LTP3. In this conditions, the zif/268 response was considerably greater than that following LTPZinducing stimulation, and the fos-related gene and jun mRNA and proteins were also prominently induced. zif/268 induction showed the best correlation with LTP persistence, particularly given the failure of the other IEGs to respond when stimulation was given on consecutive days. However, it remains possible that the greater LTP persistence observed when all trains are given on one day occurs when a coordinated response of several IEGs is generated. While it is clear that zif/268 induction is generated by LTP%inducing stimulation, it is not yet clear whether this response is sufficient to elicit an effective target gene response. Thus, it remains uncertain whether altered gene expression plays a role in LTP2 induction. On the other hand, it seems very likely that gene expression changes are important for LTP3, given the extensive pattern of IEG response correlated with LIP3 induction, and the dramatic downregulation of IEG responses and LTP persistence caused by pentobarbital. Given that zif/268, at least, shows increased expression in association with LTP2, a threshold IEGP response may be necessary in order to elicit target gene responses suflicient to play a role in LTP persistence. interpretation of correlational data There are major difBculties in establishing that IEGs participate in a biochemical cascade critical for stabilizing or maintaining LTP. Of most importance is the fact that the IEGP responses are transient, occurring over a much briefer time course than the LTP itself. Thus, it is not possible to measure IEGP levels and LTP persistence in the same animals, following a given tetanization episode. Nor is it yet possible to specifically manipulate IEGP responses to tetanic stimulation while at the same time measuring LTP persistence. These difficulties are not so great when IEG responses are compared to the initial degree of LTP induction, and indeed, widely varying degrees of correlation have been reported between these two variables.“~*“*38 However, poor or negative correlations between these variables are not decisive. By participating in a cascade of events involving target effector genes, IEGs are more likely to contribute to events later than the initial induction of LTI’, such as stabilization of the physiological or morphological alterations already established locally at the synapses.” While LTP may be generated in the absence of IEG induction in certain experimental paradigms, any LTP so induced might be relatively short-lasting (as shown in the present experiments). Conversely, IEG induction in the absence. of observable LTP may merely indicate that some other triggering event has occurred, such as a seizure or tissue damage,16 and thus does not preclude an important role for these same genes in the regulation of LTP persistence. We have taken the approach, themfore, that the correlation of most importance is that between LTP
W <‘. AIMAHAM ci
726
“persistence” and the degree of IEG mRNA and protein induction. By necessity this involves the use of separate groups of animals to assess these two variables. Furthermore, the critical correlations are ones made across stimulus conditions that all cause LTP initially. but which then last over distinctly different periods of time. By taking this approach in the present study we have shown that the zif!268 reswnse correlates well with the induction of LTP2 and LTP3, and fos-related gene and jun induction correlates to a lesser extent with LTP3. While these data are correlational in nature, the strength of the relationships reported here provide support for the
ul
working hypothesis that gene expression is critical for at least LTP3. and that further work to determine the nature of effector gene responses is warranted. Acknowledguments-We thank N. Lucas l’or help in collecting and analysing some of the LTP persistence data, and Dr P. Smith for help with the statistical analysis. We thank Dr R. Bravo and Dr J. Leah for kindly donating antibodies. and Dr D. Nathans for the 214268 clone. This research was supported by grants from the Health Research Council 01 N.-g.
and
the
Human
Frontier
Science
Programme
to
W.C.A. and W.P.T.. grants from the Health Research Council of N.Z. and the neurological Foundation to M.D.. and a postgraduate fellowship from the Neurological Foundation to J.W.
REFERENCES
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25 Morgan J. 1. and Curran T. (1986) Role of ion flux in the control of c-fos expression. NaJurf 322, 552-555. 26 Nakabenou Y.. Rvder K. and Nathans D. ( 1988) DNA binding activities of three murine jun proteins: stiinulation by fos. celi 54, 907-415
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27. Otani S. and Abraham W. C. (1989) Inhibition of protein synthesis in the dentate gyms, but not the entorhinal cortex, blocks maintenance of long-term potentiation in rats. Neurosci. Z.ert. 106, 175-180. 28. Otani S., Marshall C. J., Tate W. P., Goddard G. V. and Abraham W. C. (1989) Maintenance of long-term potentiation in rat dentate gyms requires protein synthesis but not messenger RNA synthesis immediately post-tetanixation. Neuroscience 28. 519526. 29. Racine R. J., Milgram N. W. and Hafner S. (1983) Long-term potentiation phenomena in the rat hmbic forebrain. Brain Res. 260, 217-232. 30. Richardson C. L., Tate W. P., Mason S. E., Lawlor P. A., Dragunow M. and Abraham W. C. (1992) Correlation between the induction of an immediate early gene, xif/268, and long-term potentiation in the dentate gyrus. Brain Res. 580, 147-154. 31. Ryder K., Lanahan A., Perez-Albueme E. and Nathans D. (1989) Jun-D: a third member of the jun gene family. Proc. natn. Acad. Sci. U.S.A. 86, 1500-1503. 32. Ryder K., Lau L. F. and Nathans D. (1988) A gene activated by growth factors is related to the oncogene v-jun. Proc. natn. Acad. Sci. U.S.A. 85, 1487-1491. 33. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring
Harbor Laboratory Press, New York. 34. Schreiber S. S., Maren S., Tocco G., Shors T. J. and Thompson R. F. (1991) A negative correlation between the induction of long-term potentiation and activation of immediate early genes. Molec. Brain Res. 11, 89-91. 35. Staubli U. and Lynch G. (1987) Stable long-term potentiation elicited by “theta” pattern stimulation. Brain Res. 435, 221-234. 36. Sukhatme V. P., Cao Z., Chang L. C., Tsai-Morris C. H., Stamenkovich D., Ferreira P. C. P., Cohen D. R., Edwards
S. A., Shows T. B., Curran T., Le Beau M. M. and Adamson E. D. (1988) A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 53, 3743. 37. Tippetts M. T., Vamum B. C., Lim R. W. and Herschman H. R. (1988) Tumor promoter-inducible genes are differentially expressed in the 16 developing mouse. Molec. Cell Biol. 8, 45704572. 38. Wisden W., Errington M. L., Williams S., Dunnett S. B., Waters C., Hitchcock D., Evan G., Bliss T. V. P. and Hunt S. P. (1990) Differential expression of immediate early genes in the hippocampus and spinal cord. Neuron 4, 603614. (Accepted 8 June 1993)
0306-4522/93 $6.00 + 0.00
Pmgamon Press Ltd 0 1993 IBRO
Printed in Great Britain
CORRELATIONS BETWEEN IMMEDIATE EARLY GENE INDUCTION AND THE PERSISTENCE OF LONG-TERM POTENTIATION W. C. ARR.AHAM,*~ S. E. MASON,? J. -,I J. M. WILLIAM&$C. L. RICHARIXON,$ W. P. TAT&$ P. A. LAWJ.OR§and M. DRAGWNOW~ tDepartment of Psychology and the Neuroscience Research Center, and *Department of Biochemistry and the Center for Gene Research, University of Gtago, P.O. Box 56, Dunedin, New Zealand SDepartment of Pharmacology and Clinical Pharmacology, University of Auckland Medical School, Private Bag, Auckland, New Zealand Abxtraet-The duration of long-term potentiation in the dentate gyrus of awake rata was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate early genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of xif/268. Increasing the number of stimulus trains resulted in a greater p~~bility of eliciting long-term ~t~tiation with a time constant of several weeks (long-term potent&ion 3), as well as increasing the induction of xif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the xif/268 protein levels showed associated changes. These dam provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered gene expression, initiated by immediate early gene transcription factors such as -&f/268and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabili~tion or maintenan~ of long-term potentiation 3.
Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that may contribute to memory storage processes in the brain.’
the time constant
of LTP decay in the dentate
gyfus*s.6.~8>35
While the persistence of LTP over days or weeks is a feature fundamental to its putative role in memory mechanisms, little detailed information is available regarding the longevity of LTP. A careful analysis of the reported LTP decay rates in the dentate gyrus revealed a clustering of decay time instants of longer than one day into two distinct groups. These have been termed “LTP2”, with an average decay constant of about four days,3’2gand “LTP3”, with an average decay constant of about 23 days3 They stand in contrast to a much faster decaying phase of LTP with a time constant of about 2 h, termed “LTP1”.29 The factors that govern how long LTP will persist in any particular experiment are poorly understood, but it is known that the age of the animal, number of stimulus trains delivered, the number of days over which stimulation is given, and the presence of pentobarbital all influence *To whom correspondence should be addressed. EPSP, excitatory postsynaptic potential; IEG, immediate early gene; IEGP, immediate early gene product; LTP, long-term potent&ion; MOPS, 3-[N-morpholine]propanesulphonic acid; PBS, phosphate-bu&red saline; SDS, sodium dodecyl sulphate; SSPE, sodium ~o~&/s~i~ ph~p~te/e#yl~~aminetetra-acetic acid.
Abbreviations:
717
Thtre is good evidence that the synthesis of new macromolecules is important for the development of the longer-lasting forms of LTP, but not for LTP1.20,27,2* Although LTP2 and LTP3 both appear to require new protein synthesis, the extent to which they involve separate mechanisms remains an open question. We have proposed that altered gene expression is critical for LTP3, but not LTP2,*e3and this is supported by the fact that the mRNA inhibitor actinomycin D does not affect at least the early stages of LTP2 maintenance. nJs This proposal, however, still requires more direct experimental support, particularly since both LTP2 and LTP3 appear to be well described by single exponential functions, rather than the sum of two exponentials as might be expected if LTP3 incorporated a mechanism independent of that involved in LTP2. If altered gene expression is involved in LTP maintenance, it is likely that this will be initiated by the induction of immediate early genes (IEGS), coding for transcription factors. These genes have been proposed to serve as third messengers in the sequence of events leading from postsynaptic depolarization to adaptive gene responses.25 In the LTP model, a number of IEGs show rapid and transient increases in expression following s~mula~on, including c_Jun,” jun-B,ii,is*s8 Jun-D,*s raf-I ,*’ Fos-related genes*5J8
718
W. C’. AHRAHAUc’! (I/.
and zif/268:“,J”.‘x also known as NGH-A.” Krox 24.” and Egr-l.36 Of these. zif/268 has been most reliably induced in correspondence with the induction of LTP,“.‘X but both Fos-related genes and Jun family members are also consistently induced under certain conditions.‘? Little is known about the target genes regulated by these transcription factors. However, a high-affinity DNA binding site for zifi268 has been characterized.‘” and Fos and Jun family members form homodimers (Jun) and heterodimers (FosJun) that bind to the activator protein-l recognition site.‘h If IEG induction is a critical step in the sequence of events leading to the stabilization of LTP. the relation of IEG induction to LTP ‘induction’ is less important than its relation to the ‘persistence’ of LTP. In this study we examined the extent to which the induction of a number of IEGs, and their protein products (IEGPs), correlate with the persistence of LTP, as measured in separate groups of animals across a variety of stimulus conditions. Our aim was to look for a pattern of IEG responses that was consistently associated with the specific appearance of LTP3, and thus provide support for the hypothesis that LTP3 involves a mechanism qualitatively differTIS8”
ent from LTP2.
EXPERIMENTAL PROCEDURES Adult male Sprague -Dawley rats (3W500 g) were anaesthetized with sodium pentobarbital(60 mg/kg) and placed in a stereotaxic frame for initial electrode implantation. They were prepared bilaterally with monopolar recording electrodes in the dentate hilus, and monopolar stimulating electrodes in the perforant path, as described previously.” The wire electrodes, plus screw indifferent electrodes. were connected to a plastic headcap and the whole assembly fixed IO the skull with dental acrylic. At the end of the surgical procedure, penicillin was administered i.m. All operated animals were given a minimum two-week recovery period before the quality of the evoked field potentials was assessed while the animals were quietly awake. Recordings, sampled and recorded by microcomputer, were made in response to diphasic stimulus pulses (150 ps half-wave duration) that were generated by programmable constant current stimulators, set to a current to elicit a 3--S mV population spike.‘” Tetanization consisted of l&50 trains (400 Hz, 25 ms. 250 ps duration pulses) presented as bursts of five trains at 1 Hz (1 min between bursts). Trains were given either all on one day, or as IO trains on two, three or five consecutive days (referred to as 2 x IO, 3 x IO and 5 x 10 train groups). Measurements of excitatory postsynaptic potential (EPSP) initial slope and population spike amplitude (responses recorded at 0.05 Hz) were made during the 5 min before the first train and 20min after the last train to determine the degree of LTP induced.’ EPSP LTP was calculated as the per cent change from the average pretetanization value. Population spike LTP was calculated as the mV difference in amplitude from the prctetanization value. A few animals did not show LTP ( i 10% EPSP change and kss than 2 mV spike change) and were excluded from the study. Animals for immunohistochemical analysis received stimulation in only one hemisphere. while animals for Northern analysis generally received bilateral stimulation (since the immunohistochemistry demonstrated no contralateral effects of
stimulation and pooled tissue was requlrcd for the Northern blots). Animals used for LTP decay analysis received either unilateral or bilateral stimulation. depending on the accuracy of electrode placements.
The procedures for assessing LTP decay were identical 10 those described by J&ery er 01.” Briefly, baseline dentate gyrus evoked potentials were obtained m response to 4.5 test pulses to the perforant path delivered at 20-s intervals at the same time of day and in the same room for each ral. The animals were generally still but awake during this time. Once stable recordings were obtained for at least one week (three to four baseline recording sessions). tetamiration was deli\ered according to one of the protocols described abo~. After tetani;ration. recordings of the test pulse responses were made on post-tetanization days I. 2. 3.4. 7. IO. 14 and weekly thereafter until either the baselme was regamed or the recordings became unstable. Some hemispheres where recordings returned to a stable baseline and remained so for three to four weeks were retetanized using a new paradigm. The EPSP and soike values for the nest-tetanilstion dabs were expressed as the per Cent change, or mV difference. for the average of the lash four pretetanization baseline valun. The EPSP and sDike data for each hemisohere. from dav I post-tetanization onward, were plotted’ separately on a scattergram. A single negative exponential curve was fitted to the points. according to the equation J’ 7 Be. xi, where 8 is the y-intercept. k is the rate constant of dmy and I is the number of days post-tetanization. Tbe decay time constant (T) equals I!k and represents the time (in days) for LTP to decay by 63%. Log(k) was used to normalize the decay rates for statistical analysis. Hemispheres which showed sudden substantial drops in response amplitude. decayed more than l5”& below baseline or had data too variable for curvefitting were excluded from the analysi\
A specific oligonucleotide complementary to 28s rRNA (26mer; 5’-AACGATGAGAGTAGT0GTAmCACC~3’) was hybridized to all northern blots.’ The oligonucleotide was end-labelled with [y-“P]ATP (3000Cilmmol. Amersham) using T4 polynucleotide kinase. The zif/268” clone was a kind gift from Dr D. Nathans. The Jun-B (cat. no. 63205?-‘)and Jun-D (cat. no. 632tW) probes were obtained from American Type Culture Collection. The mouse c-Jun clone’? was a kind gift from Dr 1. M. Verma. Inserts from the zif/268 and Junclones were random prime labelled with [a-‘2PvCTP (3000 Ci/mmol; Amersham. PB. 10205) using an Amenham kit (RPN. 16002).
Rats were deeply anaesthelized utth ether and then decapitated 20min post-tetanization. The brain was removed and the hippocampus and entorhinal cortex were dissected out. The dentate gyms was then dissected from the rest of the hippocampus. Total RNA was isolated from tissue and pooled across four to eight hemispheres at a time. using the method of Chomczynski and Sac&i.’ Total RNA (IO pg/lane) was separated by electrophonsis in 1% agarose, I x MOPS,0.6 M formaldehyde gels.” The RNA was then transferred directly on to nylon membrane (Hybond N + , Amersham) using a low pressure vacuum transfer system (Pharmacia-LKB) and fixed onto the membrane with 50mM sodium hydroxide. Blots were hybridized in turn, with the oligonucleotide and random primed probes at 65’C overnight in a buffer containing 4 x SSPE (I x SSPE: I50 mM sodium chloride. 9 mM sodium dihydrogen phosacid), 0.194 phate, 1mM ethylnediaminetetra-acttic sodium pyrophosphate, 0.S m&ml hcparin and 0.1% sodium dodecyl suiphate (SDS). Membranes wterewasbed to a final stringency of 0.2 x SSPE (or 2 x for the oli@tucleotide). 0. I % SDS at 65°C and exposed to prefiasbed Cronex
Immediate early genes and long-term potentiation
719
8lrn with intensifying screens at -80°C. Autoradiographs were qua&&d by laser densitometry (LKB 2222-020 UltraScan XL). Individual blots were stripped in boiing 0.1% SDS and returned to room temperature. The signals obtained for the IEG analyses were normalixed for the signal obtained from the rRNA oligonucleotide.’ Results of northern analysis were contirmed on two to three separate blots and the rRNA-normalized gene responses were averaged across blots.
rates for these two measures have been averaged to give a single decay value for each hemisphere tested. A typical response before tetanization, after tetanixation and after LTP decay is shown in Fig. IA. A monotonic increase in the persistence of LTP was observed as the number of stimulus trains given on one day and was increased from 10 to SO; example data from one animal given 10 stimulus trains and from one animal given 30 stimulus trains are shown Rats were deeply anaesthetized with ether 2 h postin Fig. 1B. Mean time constants increased from 1.5 tetanixation, and perfused transcardially with saline folto 21.3 days as illustrated in Fig. 2A, with some lowed bv 4% uaraformaldehvde in 0.1 M nhosnhate buffer. evidence for saturation occurring in the 40 and 50 pH 7.4. -Bra& were removed and then kctidns were cut train groups. Within most groups there was a concoronally (7Ocm) on a Vibratome; during sationing the siderable range of LTP decay rates. As the number of brain was immersed in 0.01 M phosphate-buffered saline (PBS). Sections were incubated in 1% hydrogen peroxide in trains was increased, however, there was a gradual 100% methanol for lOmitt, then washed with PBS and increase in the consistency with which LTP3 (decay incubated for 48 h with one of a series of antibodies to IEG time constants of three to four weeks) was observed. proteins: a rabbit polyclonal antibody to xif/268 (Krox 24, generously provided by R. Bravo) at a dilution of 1: 50,CQO This latter effect is apparent from the decrease in the in immunobutkr (0.01 M PBS, 1% normal goat serum and standard deviation across the 30-50 train groups methiolate, 0.04 mg/ml); a rabbit polyclonal antibody that (Fig. 2A, see also Table 2). detects both Fos and Fos-t&ted genes (no. PCOS), OncoAn increase in LTP persistence was also observed gene science (B. Moore, personal communication) diluted when 10 trains were repeatedly delivered across a 1:lOOO in immunobutfer; and Jun-speciIlc antibodies to number of days (mean time constants increasing from c-Jun (no. 607/S, 1:lOOO in immunobutfer), Jun-B (no. 725/3, 1:20,000 in immunobtier) and Jun-D (no. 783/3, 1.5 days for the 10 train group to 12.6 days for the 1: 20,000 in immunobuffer), which have been characterized 5 x 10 train group). The magnitude of this effect, previously using immunoblots2) The Jun family of antihowever, was less than when the 10 trains were bodies were generously provided by J. Leah, and were produced and characterized by Dr R. Bravo.” The nonspecific Fos-related gene antibody is different from that used in earlier reports,‘~~” but is the same as that used by Demmer et af.” The next sections were washed in PBS and incubated with biotinylated goat anti-rabbit serum (Sigma) in immunobutfer (1: 500dilution) 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 diaminobenxidine (Sigma) containing hydrogen peroxide. Some sections were incubated in immunobt&r without antiserum but containing normal rabbit sermn. These showed no staining, confirming the speciticity of our immunostaining. Immunoreactivity was visually assessed blind by M.D. or P.A.L. using a six-point rating scale ranging from 0 (extremely low immunoreactivity across the dentate gyms) to 5 (very high immunoreactivity). Sections from the dorsal dentate gyms (about 4mm posterior to bregma) were analysed; the judged degree of immunoreactivity in the dentate gyms 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 three and six animals.
A
_A_qpA
RESULTS
Long-term potent&ion of trains
decay following multiple sets
exponentials,
were
10
20 Days
Data for LTP decay in the dentate gyrus, and their fitted
0
obtained
from
39 hemi-
spheres, and combined with data from 20 hemispheres reported previously in Jeffery et al.” [the 20 hemispheres were distributed among the 10 (n = S), 50 (n = 7) and 5 x 10 (n = 8) train groups]. As previously observed in the dentate gyrus, LTP decayed to baseline for all rats. The decay of LTP was comparable for the EPSP slope and population spike amplitude (see also Jeffery et al’*), and so the decay
30
40
50
60
post-tetanization
Fig. 1. (A) Waveforms for a single rat from the 30 train group at successive stages of decay, from left to right: just prior to LTP (i.e. one of several base line responses used to calculate the mean baseline value); 20 min aRer induction of LTP, day 59 post-tetanization. Each waveform is the average of 10 responses. (B) Scattergratn of the tiy data for two animals given 10 (open circles) and 30 (closed circles) stimulus trains. A single negative exponential curve was fitted to the points giving decay time constants of r = 1.54 days for the IO train animal and T = 26.1 days for the 30 train animal (calculated as described in Experimental Procedures).
W. C. ABRAHAM et al.
720
delivered repeatedly on one day (Fig. 2A). LTP3 occurred frequently in the 5 x 10 group, but the average LTP decay time constant for this group was most like that of the group receiving 30, rather than 50, trains on one day.
-m
-1 50
-
131 6 d )
-1 25
-
(178dl
100-
(100dI
A
It should be noted that the various paradigms all "induced" substantial LTP initially, measured 20 min post-tetanization for both the EPSP (Fig. 2B) and population spike (Fig. 2C). There were no clear correlations across the paradigms between the degree of LTP initially induced and the subsequent rate of decay. Distribution of decay rates
m
2.
...
u
$ a
5o, 0
-075-
0 50
-
-
156dl
(3 2 d )
1
same day
0 - - - - aover days
I I
I
I
I
I
1
2
3
4
5
Sets of 10 trams
Number of stimulus trains
Number of stimulus trains
Fig. 2. (A) The decay rate of LTP for each stimulation paradigm. The mean log decay rate (corresponding decay time constants are given in parentheses) were calculated for each paradigm: sets of 10 trains of stimulation given on one day (solid line) or as one set given each day over several days (dotted line). (B, C) Comparisons of the amounts of EPSP and spike LTP induction 20 min post-tetanization produced by all animals in each of the eight stimulation paradigms. Group designations are explained in Experimental Procedures. Although the 30 and 40 stimulation train groups showed somewhat less EPSP LTP than the others, this was not mirrored in the decay rates for these groups. Overall there was little correlation between mean induction of LTP and mean decay rate of LTP across groups. The values are means and bars represent S.E.M. Ten train group, n = 7; 20 trains, n = 7; 30 trains, n = 6; 40 trains, n = 7; 50 trains, n = 11; 2 x 10 trains, n = 6; 3 x 10 trains, n = 7; 5 x 10 trains, n = 8.
For the groups receiving stimulus trains all on one day, there are two striking observations (Fig. 2A). Firstly, a distinct increase in average LTP persistence occurs between the 10 and 20 train groups. This is due to some hemispheres in the 10 train group showing very little remaining LTP by the first day posttetanization, but none showing this effect in the 20 train group. Secondly, it is clear that the persistence of LTP steadily increases as the number of trains is increased. This latter observation could reflect a consistent increase in LTP persistence for all hemispheres as the number of trains increases, or may reflect a changing mixture of hemispheres showing short (LTP2) and long (LTP3) decay constants. To examine this issue qualitatively, the decay rates for all hemispheres receiving trains on one day were plotted on a frequency histogram, irrespective of the number of stimulus trains, to see whether a unimodal or bimodal distribution of decay rates was evident (four hemispheres from the 10 train group showing decay constants less than 1.1 days were excluded from this analysis). Figure 3A shows that there was an apparent bimodal distribution of the decay rates. This bimodality is not due to an over-representation of hemispheres receiving relatively mild (10 trains) or intense (50 trains) high-frequency stimulation, since restriction of the analysis to the 20, 30 and 40 train groups resulted in a similar frequency distribution (data not shown). A double Gaussian function was fitted to the data in Fig. 2A, and two peaks were found at 5.8 and 30.7 days. These values are similar to the previously calculated mean LTP2 and LTP3 time constants of four and 23 days, respectively.' The low point between the two peaks was 12 days, and this was taken as the dividing point between LTP2 and LTP3 for subsequent analyses. A different frequency distribution was apparent for the histogram constructed for the trains administered across days, i.e. the 10, 2 x 10, 3 x 10 and 5 x 10 groups. As illustrated in Fig. 3B, there was no evidence for bimodality in this distribution. Instead, a weighting of the distribution toward rapid decay times is apparent, perhaps reflecting the relative inability of these stimulus protocols to generate LTP3. Immediate early gene responses to long-term poten tiation-inducing stimulation
IEG responses to the different stimulation protocols were monitored using northern blot analysis of mRNA levels (Fig. 4A). zif/268 showed a very strong
Immediate early genes and long-term potentiation increase in expression in response to LTP-inducing stimulation and Jun-B also showed a consistent increase. The c-Jun gene gave a modest increase in expression, due in part perhaps to the fact that its level before tetanization was much higher than for the other two 1EG.s. As the number of trains delivered was increased from 10 to 50, all the inducible IEGs showed mrresponding increases in transcription. For example, the zif{Z68 response varied from about a two-fold increase after 10 trains to a lo-fold increase after 50 trains. On the other hand, a cJun mRNA response was not detectable after 10 trains but there was about a two-fold increase after 50 trains. In Fig. 4B, the RNA loads in each lane have bee.n normalized to the rRNA oligonucleotide signals. A sizable response atso occurs for Jun-D, but at a time later than the 20-min time point used For the northern blot analysis in the present experiment.‘? The above pattern of mRNA changes was mirrored by the IEGP changes, and a similar response pattern was observed for Fos-related genes as welt. An example is shown with the antibody aa;ainst the zif/268 protein (Fig. J), and a summary of the data
7
6
I
Trains on om day
Trains across days
6
I
1.6
4.0
10.0
25. t
for all of the IEGPs is given in Table 1. A very different pattern of gene responses occurred when tuition consisted of 10 trains delivered on up to five cmsewtive hp. The zif/268 protein was the only IEGP showing a positive response on the second, third and fifth days of tetanization, in addition to the response on the first day. The increased protein levels on days 3 and 5 were. not simply a residual response carried over from the previous days, since in two animals given stimulation for two days and killed 24 h later, there was no observable zif/268 response. Neither c-Jun, Jun-B, Jun-D nor Fos-related genes showed a detectable increase after the first day of 10 trains, and no response was evident for any of these IEGPs on subsequent days of stimulation. Six animals that were tested for LTP persis&nce following 40 or 50 trains of stimulation were subsequently retetanized and used for immunohistochemical analysis. Because several of the antibodies were not available at this time, we have complete data for only Fos-retated genes and a partial set of data for zif/268, These six rats all displayed LTP3 (mean decay constant = 25.7 days). Two animals subsequently received 50 trains and scored 1 and 2 for Fos-related gene imm~nore~ti~ty. Two animals received 30 trains and scored 0 and 1 for Fos-related gene immuno~ctivity. Two animals received 10 trains and showed no change in Fos-retated gene immunor~~vity. A similar pattern of results was seen for zif/268. These data are in accord with the mean results shown in Table 1, and illustrate a within-animal correlation between LTP3-inducing stimulation and IEGP responsiveness. ~rnrned~at~ early gene resportses and persistence of lcmg-term potentiation
Log LTP decay rate 7
721
63.1
LTP decay time constant(days)
Fig. 3. Frequency histogram of LTP log decay rates for each of the types of stimulation paradigms. The number of animals showing particular decay rates for LTP when the &nut&on tins are @en to&r on one day are shown in the upper panel, and those for an&& g&en IOtrains on swxxssive days are shown in the lower panel. Note the bimodaI distribution of decay rates when trains are given on one day. Dots represent values obtained by fitting a double Gaussian function to the data. Data are taken from the same animals as shown in Fig. 2, minus four hemispheres from the 10 train group which had decay constants < 1.1 days. Trains in one day, n = 34; trains across days, n = 21.
To determine whether any of the gene responses were particularly related to the persistence of LTP, correlations were performed across tetanization conditions between the mean LTP decay rates (log-transformed) on the one hand, and the mean mRNA and protein responses on the other. Such an analysis for the groups receiving IO-SO trains on one day showed no clear preferential correlation for a particular gene. All correlations were high, ranging between -0.82 and -0.96 for the various IEGPs, with &f/268 showing the highest correlation (Fig. 6A). Similar correlations using data from groups with multiple days of tetanization could only be done for zif/268, since the other genes showed virtualty no responses under these conditions. For zif/268, the correlations between the gene response on the last day of stimulation and LTP decay rate (mRNA, t = 0.50; protein, F -- 0.31) were low compared to the same correlations for groups receiving the stimulation all on one day. When stimulation is given across days, however, the cumulative (i.e. summed) amount of zifJ268 transcription factor elicited should give a better prediction of LTP perkten~ than the response on just the last day of tetanization. When
722
W. C. ABRAHAM et al.
rRNA
r
w.
7
Uw
Fig. 4. Induction of IEG mRNA in awake rats. (A) Northern blot analysis showing induction of zif1268, cJun and Jun-B mRNA in the dentate gyrus, 20 min after receiving the stimulation. Blots were stripped of the particular probes and rehybridized with an oligonucleotide specific for 28s rRNA (indicated here as the rRNA). Note that Jun-B was from a separate blot and has a separate rRNA internal control. (B) Bar graphs of the relative amount of IEG mRNA in the dentate gyrus when normalized against the quantity of rRNA in each lane. Each lane contained approximately 10 pg of total RNA, prepared from six to eight hemispheres.
the cumulative zif/268 was calculated for each day of stimulation (using linear interpolation between the mean 3 x 10 and 5 x 10 values to estimate the day 4 zif/268 response), the correlation between zif/268 and LTP decay rate was extremely high (r = 0.998, P < 0.005; Fig. 6B). The cumulative zif/268 mRNA levels were not so well correlated with LTP decay rate (r = -0.68, n.s.). Frequency distribution of immediate early gene product responses One important question is whether any genes show responses that correlate specifically with the presence of LTP2 or LTP3. To address this question we tabulated the frequency of hemispheres showing IEGP responses irrespective of response amplitude, and compared this with the frequency of hemispheres showing LTP2 or LTP3. (By simplifying the immunohistochemical analysis to one of whether an observable change in staining had occurred or not we avoid possible problems associated with our normal ratingscale estimation of response magnitude.) As can be seen in Table 2, LTP3 was not produced in any hemispheres receiving 10 trains, but its frequency of occurrence gradually increased with numbers of
trains, levelling off for the 40 and 50 train groups (71-73% of hemispheres). The corresponding data for the Fos-related gene and Jun responses showed a similar pattern, i.e. no hemispheres showed detectable responses after 10 trains, and a gradual increment in the percentage of responding hemispheres as the number of stimulus trains increased. The zif/268 response, however, had a lower threshold than for the appearance of either LTP3 or the Fos-related gene and Jun responses. Fifty per cent of hemispheres showed a zif/268 response after 10 trains, even though none would have been expected to develop LTP3. After 30 trains, 100% of the hemispheres showed a zif/268 response, when only 50% would have been expected to show LTP3. Instead, the zif/268 response had a threshold corresponding to that for LTP2, since about half of the 10 train hemispheres developed LTP2 (three of seven) and a similar fraction showed a zif/268 response (two of four). A similar correlation was observed when trains were given over a number of days (Table 2). This correlation between the thresholds for zif/268 expression and LTP2 is in contrast to the Fos and Jun responses, which correlate with the appearance of LTP3, and only when stimulation is given on one day.
Immediate early genes and lons_term poteotiation
723
Fig. 5. ~0~~~0~~~ showing xif/%8 imrn~o~t~~st~ following different LW induction protocols. (a) The kvel of zif/268 protein present after two days of it3 stimulus trains/day. (b) After three days of 10 trains/day. (c) After five days of IO trains/day. (d) The level of protein after 10 trains. (e) After 20 tmins on one day. (f) After 30 trains on one day. (g) After Zo trains on one day. LTP-induciug &m&ion was applied to one hemisphere chosen at mndom. In each case the animal was killed 2 h after the last set of stimuhrs trains. The stimulated hemisphere is shown on the kfk side of each part of the figure. Note that zif/268 shows a response on each day of stimuhttion (a-c), and that the level of response graduahy incresses as the munber of trriius on one day increases. Sesie bar in c=gOO$Un.
Table 1. protein expression in response to long-term ~~~~-j~~
lotion
Protein (mean f S.D. rating) LTP protocol
Fos-related gtne
c-Jon
Jun-B 0 0.2 i
10 20
O.l:O.2
0.2O*0.3
:
0.3 1.42f 0.5 l.1
0.2 0.9 f* 0.2 0.3
2 X 10 3 x 10 5x 10
0 0 0
0 -0.3 f 0.6 -0.1 f 0.2
Jun”D
a.5
1.1 1.2i0.9 fO.6 -0.2 f 0.3 0 0
xifj26g 0.8 It LO
0.i0&O*2 Es* 1.0 0.5 0.8 f 5.6 1.0
2.1 2.8 kO.8 f 1.2
-0.7 f 0.6 -0.2 f 0.3 0
2.3 f 0.6 1.2 f 1.3 I.7 f 1.2
The extent of expression of IEGFs 2 h after the different LTP-inducing stimutstion prvadigmsisshown~~ratingonascaIeof0_5(meafiof~to$xaaimafsper condition), and after cormcting for the expression in the control hemisphere.
124
W. C. ABRAHAM
A -LTP ZiW266 P--Qc.Jun D----OJtJfl-8 0 . . . . . . . . . 0 J,,,_D
p m” P D 5
2* 5 ti g 1 = B r)
0 10
20
30
40
50
Number
of stimulus trains on one day
(17.8d)
LTP decay o----oprOt‘%n
B
0 1
2 3 Days of 10 trains
tv (I/
Instead, a bimodal distribution of decay time constants was observed, with peaks at 5.8 and 30.7 days. both well within the range of constants previously calculated for LTP2 and LTP3. respectively.2 A blmodal distribution is not a necessary outcome fol LTP3 to involve a different mechanism from LTP2. as there could be a gradual recruitment of the additional mechanism as more trains are delivered, bur its presence does give solid support for the hypothesis of an additional mechanism (LTP3), and further suggests that there is a threshold governing ils recruitment. Spacing the sets of IO trains by one day instead of‘ I min also resulted in a gradual prolongation of LTP. although the cumulative effect of stimulation on consecutive days was less than when all trains were delivered on one day. This is consistent with the observation that LTP decay is relatively rapid after only 10 stimulus trains. Thus, by the lime stimulation was delivered on the second day, relatively little LTP would have been present to interact with the newly induced LTP. These results are consistent with previous studies in which LTP maintenance was prolonged by giving small numbers of trains on consecutive days,S.‘Wbut not consistent with DeJongc and Racine,” who found no additional increase when 70 trains were delivered daily for five days. In thi> latter study, it is possible that the mechanisms relating to LTP persistence had already been saturated after one day of stimulation.
5
6. Correlation of LTP decay rate with IEGP expression. (A) The decay time constants of LTP for the different stimulation paradigms (IO- 50 trains given on one day) are compared with the expression of each of the proteins for zif/268, c-Jun. Jun-B, Jun-D and FRA for the same paradigms. (B) The decay time constants for LTP when 10 trains of stimulation are given each day for successive days (closed circles) are compared with the expression of ziQ268 protein (open circles). The cumulative sum of zifl268 over the successive days is also shown (triangles) Fig.
Table 2. Frequency of hemispheres showmg long-term potentiation 2,/3 or immediate early gene product responses for each stimulalion paradigm Number
LTP3
mechanisms
jar
long-term
potmriuticm
The LTP2jLTP3 distinction was originally derived from an analysis of decay rates in single experiments reported by a number of laboratories, all using somewhat different stimulation protocols.’ The experiments reported here have identified how the LTP decay rate changes following systematic manipulation of the tetanization parameters. As expected from preliminary experiments,‘* the average LTP decay time constant showed a gradual increase as the number of stimulus trains on one day was increased from 10 lo 50. However. a corresponding gradual increase was not apparent from the frequency distribution of decay rates, plotted across individual hemispheres, regardless of the tetanization paradigm.
0%
28%
(017)
(2:7)
0% Kb4)
141% (I ‘7)
C-.lUll
I% 10.4)
(I 31
Jun-B
0% W4)
25% (I 4)
Jun-D
O’h (O/4)
2 5 %I (1.‘4)
LTP2 01
43% (3.7)
I oo”io
LTP3 LiTi
50%
86% (6i7)
2
und 31)
20
Fos-related gene
DIS<‘tiSSION
Multiple
of stimulus trains on one day
IO
(24)
zif/268
or
.s(i
; 3 ? I,
(7 ‘7)
(8:X)
(-5’5)
0% (O/7) 43% (3,7)
83% (Si6)
86”,, (h;7\
I( u “4
50% (2!4)
100%
6090 (39,
IOO%J (1’3)
I
LTP2 LTP3
40
of days of IO train stimulation .__ ._.-.. i 2 4 -62% 0% 145.0 (I ?I I5 8) (o:h)
Number
LTP3
;o
(3i3)
(X.8)
Immediate early genes and long-term potentiation The frequency distribution of LTP decay rates after multiple days of stimulation did not show obvious bimodality. This could be due to the fact that relatively few hemispheres actually displayed LTP time constants in the LTP3 range. It is also possible that, for individual hemispheres, L’IP induction at a number of discrete points in time produced LTP that decayed at different rates for different synapses. This could have had the effect of broadening the range of time constants calculated using single exponential functions, thereby smoothing the frequency distribution Given the relatively small numbers, and skewedness, of observations in this portion of our data, the interpretation of this frequency distribution remains ambiguous. Relation between immediate early gene responses and tong-term potentiation persistence As the number of trains delivered on one day was increased from 10 to 50, all IEGs studied showed associated increases in expression. ~~~n~ngly, large positive correlations between LTP persistence and IEG induction were found when the data were grouped according to the number of stimulus trains delivered, with zif/268 showing the greatest correlation as well as the largest overall response. The relationship between IEG responses and LTP persistence is rather different when tetanization is delivered as 10 trains on consecutive days. In this case the only transcription factor that consistently shows increased expression on each day of stimulation is zifi268. In contrast, none of the other proteins showed a detectable change on any day of stimulation. Thus, it would appear that if IEGs are at all involved in stabilizing LIP, ziff268 is the best candidate primary effector. One of our working hypotheses is that altered gene expression is necessary for the development of LTP3, but not LTPZ. Since the distinction between LTP2 and LTP3 received support from the bimodal frequency distribution of decay rates, it was of interest to see whether the pattern of transcription factor responses also supported the hypothesis. One clear outcome of this analysis is that, contrary to expectation, a sizeable gene response occurred for zif/268 under conditions when LTP2, but not LTP3, would have been expected to be induced (i.e. after 10 stimulus trains). Furthermore, the frequency of positive zif1268 responses in this condition was similar to the frequency of appearance of LTP2 (cf. Table 2). This zif/268 response is similar to that seen after 50 trains in ~ntobarbital-ana~theti~ animals, also a condition in which only LTP2 is produced. In contrast, the Fos-related gene and Jun proteins did not appear to increase in the IO train condition, nor in the presence of pentobarbital.‘3~‘4*‘8 However, a small response for these IEGPs under such conditions, below the detection level of our i~uno~st~he~cal techniques, cannot be ruled out. In line with our working hypothesis, a much larger IEG response occurred after stimulation expected to
725
evoke LTP3. In this conditions, the zif/268 response was considerably greater than that following LTPZinducing stimulation, and the fos-related gene and jun mRNA and proteins were also prominently induced. zif/268 induction showed the best correlation with LTP persistence, particularly given the failure of the other IEGs to respond when stimulation was given on consecutive days. However, it remains possible that the greater LTP persistence observed when all trains are given on one day occurs when a coordinated response of several IEGs is generated. While it is clear that zif/268 induction is generated by LTP%inducing stimulation, it is not yet clear whether this response is sufficient to elicit an effective target gene response. Thus, it remains uncertain whether altered gene expression plays a role in LTP2 induction. On the other hand, it seems very likely that gene expression changes are important for LTP3, given the extensive pattern of IEG response correlated with LIP3 induction, and the dramatic downregulation of IEG responses and LTP persistence caused by pentobarbital. Given that zif/268, at least, shows increased expression in association with LTP2, a threshold IEGP response may be necessary in order to elicit target gene responses suflicient to play a role in LTP persistence. interpretation of correlational data There are major difBculties in establishing that IEGs participate in a biochemical cascade critical for stabilizing or maintaining LTP. Of most importance is the fact that the IEGP responses are transient, occurring over a much briefer time course than the LTP itself. Thus, it is not possible to measure IEGP levels and LTP persistence in the same animals, following a given tetanization episode. Nor is it yet possible to specifically manipulate IEGP responses to tetanic stimulation while at the same time measuring LTP persistence. These difficulties are not so great when IEG responses are compared to the initial degree of LTP induction, and indeed, widely varying degrees of correlation have been reported between these two variables.“~*“*38 However, poor or negative correlations between these variables are not decisive. By participating in a cascade of events involving target effector genes, IEGs are more likely to contribute to events later than the initial induction of LTI’, such as stabilization of the physiological or morphological alterations already established locally at the synapses.” While LTP may be generated in the absence of IEG induction in certain experimental paradigms, any LTP so induced might be relatively short-lasting (as shown in the present experiments). Conversely, IEG induction in the absence. of observable LTP may merely indicate that some other triggering event has occurred, such as a seizure or tissue damage,16 and thus does not preclude an important role for these same genes in the regulation of LTP persistence. We have taken the approach, themfore, that the correlation of most importance is that between LTP
W <‘. AIMAHAM ci
726
“persistence” and the degree of IEG mRNA and protein induction. By necessity this involves the use of separate groups of animals to assess these two variables. Furthermore, the critical correlations are ones made across stimulus conditions that all cause LTP initially. but which then last over distinctly different periods of time. By taking this approach in the present study we have shown that the zif!268 reswnse correlates well with the induction of LTP2 and LTP3, and fos-related gene and jun induction correlates to a lesser extent with LTP3. While these data are correlational in nature, the strength of the relationships reported here provide support for the
ul
working hypothesis that gene expression is critical for at least LTP3. and that further work to determine the nature of effector gene responses is warranted. Acknowledguments-We thank N. Lucas l’or help in collecting and analysing some of the LTP persistence data, and Dr P. Smith for help with the statistical analysis. We thank Dr R. Bravo and Dr J. Leah for kindly donating antibodies. and Dr D. Nathans for the 214268 clone. This research was supported by grants from the Health Research Council 01 N.-g.
and
the
Human
Frontier
Science
Programme
to
W.C.A. and W.P.T.. grants from the Health Research Council of N.Z. and the neurological Foundation to M.D.. and a postgraduate fellowship from the Neurological Foundation to J.W.
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