(Received for publication, May 22, 1995)
From the
Exposure of primary cultures of embryonic rat striatal neurons to agents releasing nitric oxide (NO), including sin-1 molsidomine, S-nitroso-n-acetyl-penicillamine (SNAP), and S-nitrosoglutathione, resulted in an increase in the levels of expression of the immediate early genes c-fos and zif/268 in the cultured neurons. The membrane-permeable cGMP analogue, 8-bromo-cGMP, did not significantly affect c-fos and zif/268 mRNA levels, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the elevation in c-fos and zif/268 mRNA levels induced by SNAP. The induction of c-fos by the calcium ionophore A23187 was reduced by treatment with SNAP or 8-bromo-cGMP. Inhibitors of ADP-ribosyltransferases attenuated the stimulation of c-fos expression by SNAP. These results demonstrate for the first time that NO can induce immediate early gene expression in neurons, suggesting that NO may act as a mediator of neuronal plasticity via alterations in the expression of downstream genes. In addition, the results suggest that NO may exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.
A number of transcription factors have been identified that have
the capacity to alter the rate of transcription of specific target
genes in response to receptor-mediated signaling events at the cell
membrane(1) . Many of these putative transcription factors are
rapidly and transiently induced following receptor activation and
therefore can be classified as immediate early genes (IEGs). ()The pattern of IEG induction after a particular stimulus
can then be viewed as resulting in a corresponding shift in the level
of expression of the target (late response) genes some hours later.
The regulation of IEG expression has consequently attracted considerable interest, particularly in those cell types where functionally important changes in late response gene transcription are known to occur. Dramatic changes in the levels of peptide neurotransmitter mRNAs are observed in striatal neurons following manipulation of dopaminergic activity(2, 3, 4, 5) , and it seems that these changes contribute to disturbances in extrapyramidal motor control. A considerable research effort has therefore been directed at identifying the factors regulating IEG expression in striatal neurons.
The IEGs c-fos(6) and zif/268(7) , also known as egr1, Krox24, and NGFIA, are thought to be involved in functional plasticity in rat striatal neurons in that they are induced in vivo by treatment with dopaminergic drugs(8, 9, 10) . They may therefore be involved in coupling activity at dopamine receptors to subsequent alterations in the pattern of late response gene expression in striatal neurons. However, dopamine is unlikely to be the only important influence regulating striatal gene expression.
Nitric oxide (NO) is now known to act as an intercellular mediator in the brain(11) . It is released by cells containing the enzyme nitric oxide synthase following an increase in intracellular calcium levels and can then diffuse to affect neighboring cells. Many, but not all, of the actions of NO in the target cells involve the activation of guanylate cyclase, with subsequent increases in intracellular cGMP levels and stimulation of cGMP-dependent protein kinase(12, 13) . Recent work has shown that NO is a powerful stimulant of c-fos and zif/268 expression in PC12 cells(14, 15) , raising the possibility that NO may act to regulate gene expression in those brain areas containing nitric oxide synthase activity. Indeed, we have recently reported that NO is able dramatically to alter the pattern of gene expression in hippocampal granule cells(16, 17) .
Although the striatum contains a relatively small number of nitric oxide synthase-containing cells(18) , the entire striatal neurophil is a dense interwoven mesh of nitric oxide synthase-containing fibers(18, 19) . The anatomical framework is therefore present for NO to exert a powerful influence on the regulation of striatal gene expression.
In this study, I have used primary cultures of embryonic rat striatum to investigate the possibility that NO is able to induce IEG expression in striatal neurons. These cultures have been shown to contain all the cellular phenotypes present in the normal adult striatum (20, 21) and to show an induction of IEGs in response to dopamine receptor stimulation that matches the in vivo situation very closely (22) . I demonstrate here that NO is able to stimulate the expression of both c-fos and zif/268 in striatal neurons, and I provide evidence that the activation of guanylate cyclase and cGMP-dependent protein kinase does not play a major role in this effect.
The method for in situ hybridization was as described previously(22, 23, 24) . The culture slides were incubated with hybridization buffer (50% deionized formamide, 50 mM sodium phosphate buffer, 600 mM sodium chloride, 60 mM sodium citrate, 100 mg/ml polyadenylic acid, 40 mM dithiothreitol, 10% dextran sulfate) containing the labeled oligonucleotide at a concentration of 1 pg/ml for 16 h at 42 °C. The slides were then washed for 1 h in 150 mM sodium chloride, 15 mM sodium citrate, at 55 °C, dehydrated in ethanol, and either exposed to x-ray film for up to 1 week or dipped in Ilford K5 liquid photographic emulsion and exposed for 5 weeks. After development, the slides were counterstained with cresyl violet or neutral red.
The 45-base oligonucleotide probes used to detect
c-fos mRNA and zif/268 mRNA were as described (10, 22, 23, 25) and were labeled to
a specific activity of around 10 dpm/µg using
S-dATP (Dupont NEN) and terminal deoxynucleotidyl
transferase (Pharmacia Biotech Inc.). In some experiments, after the
fixation step, the culture slides were processed for
immunohistochemistry using antisera against glial fibrillary acidic
protein or neuron-specific enolase (Affiniti), according to standard
procedures.
The intensity of the film autoradiographic signal was
measured using the MCID image analysis system, and results were
expressed as relative optical density units 10
. In
every culture slide used in this investigation, at least one well was
treated with vehicle to allow a direct comparison with the drug-treated
wells. Between two and four wells received each treatment tested in the
experiment, and the mean hybridization signal for the particular
treatment in that culture was obtained by pooling the mean
hybridization signals for each well that had received the treatment.
The treatment was then repeated on a number of different cultures. The
results are presented as mean ± S.E. from the number of separate
determinations shown. Statistical analysis was performed using ANOVA
with posthoc Fisher's test for pairwise
comparisons.
The probes used in this study have been extensively characterized under the conditions used(10, 22, 23, 25) . Nevertheless, the specificity of the in situ hybridization signal for each probe was confirmed in various preliminary experiments. Northern analysis of extracted RNA, performed at the same stringency as the in situ hybridization, showed a single band of the expected size in each case. Hybridization of cultures with sense probes, labeled to the same specific activity, gave no signal, and the addition of a 25-fold excess of unlabeled antisense oligonucleotide to the hybridization buffer completely displaced the hybridization signal (not shown).
The primary neuronal cultures of rat striatum have also been extensively characterized(21) . Non-neuronal cells represent a small percentage of the total number of cells following cytosine arabinoside treatment and can be readily distinguished from neurons in the counterstained preparations used for analysis(21, 22, 23) .
Preliminary experiments established that 45 min after drug application represented the time of maximal induction of both c-fos and zif/268 mRNAs (not shown). This time point was consequently used for all subsequent experiments.
In vehicle-treated cultures, consistent with striatal tissue in vivo, the hybridization signal representing c-fos mRNA was generally low, with the zif/268 signal being slightly higher (Fig. 1, a and d). Addition of SNAP (200 µM) to the cultures resulted in an increase in the density of silver grains representing c-fos mRNA and zif/268 mRNA (Fig. 1, b and e, Table 1). Similar increases in the hybridization signal were observed following treatment with sin-1 (Fig. 1c, Table 1), and S-nitrosoglutathione (Fig. 1f, Table 1). However, over a range of doses, 8-Br-cGMP proved unable to increase the c-fos hybridization signal (not shown) or the zif/268 hybridization signal (Fig. 2).
Figure 1: Effect of NO-releasing agents on c-fos and zif/268 mRNA levels in cultured striatal neurons. Photomicrographs show the hybridization signal (blacksilvergrains) representing c-fos mRNA (a-c) and zif/268 mRNA (d-f) overlying counterstained neuronal cell bodies, following treatment with vehicle (a and d), 200 µM SNAP (b and e), 500 µMsin-1 (c), or 200 µMS-nitrosoglutathione (f). Scalebar represents 25 µm. Note that an increased density of silver grains is observed over some neurons (examples indicated by arrows) in panels b, c, e, and f but not over other neurons (examples indicated by arrowheads).
Figure 2:
Effect of SNAP () and 8-Br-cGMP
(
) on the zif/268 hybridization signal in cultured
striatal neurons. Results are expressed as the relative optical density (R.O.D.) of the film hybridization signal and are the mean
± S.E. of between three and eight
determinations.
In every case, the increase in silver grain density was found to occur over the smaller, more intensely counterstained cells in the cultures. These were identified in preliminary immunocytochemical experiments as neurons. While a large percentage of neurons were labeled following application of NO-releasing agents, there were still many neurons that did not show a significant accumulation of silver grains (i.e.Fig. 1, b, c, e, and f). No increase in silver grain density was detected over the larger, more weakly counterstained cells, representing non-neuronal cells.
The cGMP-phosphodiesterase inhibitor MY5445 (26) did not potentiate the effects of SNAP (Table 1). The selective cGMP-dependent protein kinase inhibitor KT5823 (26, 27) applied alone to striatal cultures did not affect basal levels of c-fos or zif/268 mRNAs (not shown) or the increases observed following exposure to SNAP (Table 1). However, hemoglobin, which acts as a NO scavenger, blocked the ability of SNAP to elevate c-fos and zif/268 gene expression (Table 1). Ruthenium red, which has been proposed as an inhibitor of cADP-ribose-mediated mobilization of intracellular calcium, when applied to striatal cultures caused a massive induction of both c-fos and zif/268 mRNA levels (i.e. c-fos hybridization signal following vehicle treatment, -21.0 ± 1.3 relative optical density units, n = 14; following ruthenium red (30 µM) -126.4 ± 3.9 relative optical density units, n = 7, p < 0.001). The calcium ionophore A23187 also produced a large increase in c-fos and zif/268 mRNA levels (Table 2), and these increases were attenuated by cotreatment with low concentrations of SNAP or 8-Br-cGMP. Two inhibitors of ADP-ribosyltransferases, nicotinamide and phylloquinone(28) , were tested against the SNAP-induced increases in IEG expression. Phylloquinone appeared to reduce basal levels of expression of c-fos mRNA but not zif/268 mRNA (Table 3). Both nicotinamide and phylloquinone attenuated the effect of SNAP on c-fos mRNA levels, whereas only nicotinamide reduced the stimulation of zif/268 expression by SNAP (Table 3).
We report here that exposure of rat striatal primary cultures to agents releasing nitric oxide resulted in dramatic increases in the levels of c-fos and zif/268 mRNAs. The use of in situ hybridization to monitor these changes has the advantage that the cell types where the changes in gene expression occur can be identified with some confidence. It is worth noting that, despite the small proportion of non-neuronal cells in such cultures, some pharmacological agents can stimulate dramatic increases in IEG expression in the glial cells(23) . Nitric oxide-releasing agents cause increases in c-fos mRNA levels and zif/268 mRNA levels in the same population of cells that exhibit immunoreactivity for neuron-specific enolase. The induction of IEG expression is therefore occurring in the striatal neurons. We have previously shown that a high proportion of striatal neurons in primary culture express either the proenkephalin gene or the protachykinin gene (21) and therefore are likely to represent the medium spiny projection neurons characterized in the mature striatum(29) . Interneurons are thought to constitute only a small percentage of the number of neurons in the normal striatum(29) , and the corresponding neurochemical phenotypes represent a similarly small proportion of neurons in striatal primary culture(21) . The high proportion of neurons containing c-fos mRNA or zif/268 mRNA in these experiments after treatment with NO-releasing agents suggests that IEG induction is taking place in the culture counterpart of the medium spiny neuron. Nevertheless, there are clearly many neurons that do not show increased c-fos or zif/268 mRNA levels after exposure to NO (Fig. 1). This suggests that the ability of NO to enhance IEG expression is dependent on some intracellular factors that, by their presence only in a subpopulation of neurons, serve to target the transcriptional effects of NO to these cells.
It has been reported that certain drugs that liberate NO in solution can also give rise to nonspecific effects unrelated to NO release. A number of NO-releasing agents, from different structural classes, were therefore tested in these experiments. The ability of all of them to increase IEG expression, when NO release is their only common feature, strongly implicates NO in these effects, as does the observation that hemoglobin, which scavenges NO, reduces the effects of SNAP.
It is clear that, in a number of different cell types, increased transcription of both the c-fos and zif/268 genes can be observed following activation of either cAMP-dependent protein kinase or protein kinase C(1) . This is in line with the presence of cAMP-responsive enhancers and phorbol ester-responsive enhancers in the promoter regions of these genes. Indeed, our own results show that activation of either cAMP-dependent protein kinase or protein kinase C can produce a dramatic elevation in c-fos mRNA and zif/268 mRNA levels in primary striatal cultures(22) . Recent evidence has demonstrated that, in PC12 cells, NO can also increase c-fos mRNA and zif/268 mRNA levels(14, 15) . In one case, NO-releasing agents were powerful stimulants of IEG expression, and this action appeared to involve activation of guanylate cyclase and cGMP-dependent protein kinase(14) , while in the other case NO release had no effect alone but acted to potentiate the stimulation of IEG expression following calcium influx due to A23187(15) . The results presented here confirm the hypothesis, based on the results with PC12 cells, that NO can induce c-fos and zif/268 expression in neurons.
However, the mechanisms involved do not correspond with those observed in the cell line. An effect mediated by cGMP and cGMP-dependent protein kinase would be mimicked by 8-Br-cGMP, potentiated by the cGMP phosphodiesterase inhibitor MY5445, and blocked by KT 5823. The IEG induction in striatal neurons by NO does not show these characteristics (Table 1). Similarly, no potentiation of the ability of the calcium ionophore A23187 to increase c-fos and zif/268 mRNA levels was observed following treatment with low concentrations of NO releasing agents (Table 2). On the contrary, SNAP decreased the degree of IEG induction by A23187. Since this inhibitory effect was reproduced by 8-Br-cGMP, increased cGMP levels in striatal neurons may act to suppress IEG induction due to calcium influx.
It is possible that this inhibitory effect of NO and cGMP on
c-fos induction involves protein kinase C. The increased
c-fos expression in neurons following calcium influx through
voltage-dependent calcium channels or NMDA receptors appears to be
partially mediated via stimulation of protein kinase C and the
c-fos promoter element responding to protein kinase
C(30, 31) . It is clear that A23187-induced calcium
influx activates neuronal phospholipase C (32) and that NO,
cGMP, and cGMP-dependent protein kinase are all able to interfere with
the stimulation of protein kinase C by phospholipase C activity (33, 34) . The reduction of the effect of A23187 on
c-fos expression in our experiments by 8-Br-cGMP could then be
explained by a functional antagonism at the level of phopholipase C.
Alternatively, it is possible that stimulation of cGMP-dependent
protein kinase activates a membrane Ca-pumping
ATPase(35, 36) , which attenuates the elevations in
intracellular Ca
due to A23187.
Hence, release of NO in the striatum appears to be exerting two opposing effects on IEG expression, a suppression (via guanylate cyclase activation) of the potentially dramatic induction following calcium influx and a direct, more modest elevation that occurs independent of guanylate cyclase. This inhibitory action would then be acting in opposition to the predominant effect of NO to enhance the expression of these IEGs.
Since the mechanisms through which NO raises c-fos and zif/268 mRNA levels in striatal neurons involve neither guanylate cyclase nor synergism with calcium influx, they differ from the mechanisms elevating c-fos and zif/268 expression in the PC12 cell line (14, 15) and from those regulating late response genes in hippocampal neurons(16, 17) . It has become clear that many of the cellular actions of NO are not mediated by cGMP and cGMP-dependent protein kinase(37, 38, 39, 40, 41) . For example, NO can stimulate ADP-ribosylation of intracellular target proteins(28, 38, 42, 43, 44, 45) , and such a mechanism could contribute to effects on gene expression. In fact, in the striatal neurons, the induction of c-fos by SNAP was markedly reduced by two inhibitors of cellular ADP-ribosylation, nicotinamide and phylloquinone. However, the induction of zif/268 was suppressed by nicotinamide but not by phylloquinone. This could be interpreted as evidence that different mechanisms regulate c-fos and zif/268 induction in response to NO. For example, the lower potency of phylloquinone in suppressing zif/268 induction could reflect its relatively weak affinity for poly(ADP-ribosyltransferases)(28, 46) . However, the specificity of these compounds as inhibitors of cellular ADP-ribosyltransferases is far from complete, and great caution should probably be exercised in the interpretation of these results. Nevertheless, the results may provide some of the first indications that ADP-ribosylation is involved in the regulation of neuronal gene expression.
There is also evidence that NO can liberate intracellular calcium stores via cADP-ribose(47) , a pathway blocked by ruthenium red. Attempts to determine whether the NO-induced increase in IEG mRNA levels was sensitive to ruthenium red were thwarted by the fact that ruthenium red itself caused a massive induction of c-fos and zif/268 expression. The reasons for this are not entirely clear at present but may suggest that caution should be observed when interpreting the effects of ruthenium red. However, the liberation of intracellular calcium stores by NO is thought to involve guanylate cyclase and cGMP-dependent protein kinase (47) and so is unlikely to contribute to the induction of IEGs reported here.
This leaves open the question of exactly how the increased expression of c-fos and zif/268 is achieved following NO release. An attractive possibility is that the action involves either direct nitrosation or ADP-ribosylation of transcription factors regulating IEG expression(37) , although the cellular specificity that we observe would require these transcription factors to be present only in a sub-population of neurons. It is clearly of some importance for our understanding of physiological and pathological plasticity in the striatum to identify the cellular mechanisms involved in these effects.