D1R-Mediated Activation of ERK in the Hippocampus Figure 9. Zif268 and Arc/Arg3.1 expression induced by SKF 81297 is dependent on ERK activation. (A) Single confocal sections showing immunofluorescence detection of Zif268 and Arc/Arg3.1 protein expression at various times after SKF 81297 (5.0 mg/kg, i.p.) injection. Scale bar: 40 mm. (B and C) Quantification of Zif268 and Arc/Arg3.1 immunoreactive neurons in mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.) at various time points. Data (means 6 SEM; n = 3–6 mice per group) were analyzed using one-way ANOVA: Zif268 (F(5,12) = 36.50; p,0.001), Arc/Arg3.1 (F(5,21) = 23.50; p,0.001). (D) Immunohistochemical detection of Zif268 and Arc/Arg3.1 and protein expression in the granule cell layer of mice treated with SKF 81297 (5.0 mg/kg) alone or in combination with SL327 (50 mg/kg, i.p.). Scale bar: 40 mm. (E and F) Quantification of Zif268 and Arc/Arg3.1 immunoreactive neurons in mice treated with SKF 81297 (5.0 mg/kg) alone or in combination with SL327 (50 mg/kg, i.p.) at two time points. Data (means 6 SEM; n = 3–4 mice per group) were analyzed using two-way ANOVA: Zif268, effect of time (F(1,10) = 105.87, p,0.001), SL327 (F(1,10) = 239.57, p,0.001), interaction (F(1,10) = 42.45, p,0.001); Arc/Arg3.1, effect of time (F(1,12) = 123.88, p,0.001), SL327 (F(1,12) = 143.47, p,0.001), interaction (F(1,12) = 70.12, p,0.001). Bonferroni-Dunn test: *** p,0.001 vehicle vs. SL327. doi:10.1371/journal.pone.0019415.g009 produced by SKF 81297 was only partially reduced by SL327, a drug, which prevents the phosphorylation of ERK, indicating the involvement of other signaling cascades in D1R-mediated control of this particular downstream target. In contrast, SKF 81297- induced phosphorylation of histone H3 at Ser10 was abolished by pretreatment with SL327. Surprisingly, ERK activation was not accompanied by increased phosphorylation of MSK1 or MSK2 (data not shown), two major substrates of ERK critically involved in histone H3 phosphorylation [24,25,26]. Therefore, the molecular link between ERK activation and histone H3 phosphorylation remains to be identified. Induction of gene expression has been extensively studied in various experimental models of seizures. For instance, increased expression of genes coding for activity-regulated transcription factors, such as c-fos and zif268, has been demonstrated in the hippocampus after kindling [27,28], or following electrical or chemical induction of seizures [29,30,31,32,33,34]. Moreover a strong induction of c-Fos has been observed in the granule neurons of the DG following a spontaneous seizure [23]. In the present study, we found a large increase in levels of Zif268 in the nuclei of granule cells, which followed temporally the increase in ERK and histone H3 phosphorylation produced by SKF 81297. This effect was prevented by treatment with SL327, indicating that phosphorylation of ERK and, possibly, of histone H3 is implicated in the expression of this gene. However, previous studies showed that inhibition of MSK1, which prevents cocaine- induced phosphorylation of histone H3 at Ser10, did not prevent the ability of this drug to increase the expression of zif268 [24]. Thus, further studies will be necessary in order to demonstrate the involvement of histone H3 phosphorylation in the regulation of zif268 expression, in the dentate gyrus. In contrast to Zif268, we found only a small increase in c-Fos expression following D1R activation in the dentate gyrus. The pattern of immediate early gene induction observed with SKF 81297 is similar to that described following high frequency stimulation of the perforant pathway, which induces LTP in the DG. Thus, tetanising stimuli increase Zif268, without consistently affecting c-Fos expression [35,36,37,38]. Moreover, the increase in Zif268 expression associated with LTP is prevented by inhibition of ERK with SL327 [39]. Administration of SKF 81297 increases also the levels of Arc/ Arg3.1, another gene involved in multiple forms of neuronal plasticity, including LTP and LTD. Previous work proposed that the involvement of Zif-268 in the late phase of LTP is related to its ability to promote Arc/Arg3.1 transcription [40]. Levels of Arc/ Arg3.1 are also regulated at the translational level, following rapid transport of mRNA to dendrites [41]. Thus, the increased expression of Arc/Arg3.1 induced by activation of D1Rs may depend on concomitant ERK-dependent phosphorylation of histone H3, which may be implicated in the nucleosomal response, and rpS6, which may instead regulate local synaptic translation of mRNA encoded by Arc/Arg3.1. In support of this possibility, it has recently been shown that, in the hippocampus, activation of ERK promotes local Arc/Arg3.1 expression independently of other signaling pathways involved in translational control, such as the mammalian target of the rapamycin cascade [42]. Changes in the expression of zif268 and Arc/Arg3.1 reported here have been studied with respect to their involvement in hippocampal plasticity. Dopamine transmission has been impli- cated in various forms of learning and memory, in which the hippocampal formation is though to play an essential role. In many cases, the action of dopamine has been proposed to occur via activation of D1- or D2-type receptors located in various anatomical regions within the hippocampal formation. It will be interesting to define whether the effects described here are produced by a direct action of SKF 81297 on the granule cells of the DG, or by an indirect mechanism involving a polysynaptic circuit. In conclusion, the present study indicates that the epileptiform activity produced by systemic administration of a D1R agonist is associated with activation of ERK in the granule cells of the DG. This effect is accompanied, in the nucleus, by increased phosphorylation of histone H3 and expression of zif268. Moreover, administration of SKF 81297 enhances phosphoryla- tion of rpS6 in the cytoplasm and promotes the expression of Arc/ Arg3.1. These changes are distinct from those produced by chemoconvulsants, such as pilocarpine and KA, which cause a much more pervasive and prolonged activation of the DG and other hippocampal regions and which trigger the expression of additional immediate early genes. The more restricted action of SKF 81297 is consistent with the inability of this drug to induce SE, spontaneous seizures and neuronal cell death, and further supports the notion that the DG acts as a gate and sensor able to detect a wide range of stimuli impinging on the hippocampus. Future studies will be required to elucidate the significance of D1R-mediated molecular changes in hippocampal plasticity and function. Supporting Information Figure S1 Effect of SKF 81297 on c-Fos expression in the dentate gyrus. (A) Single confocal sections showing immuno- fluorescence detection of c-Fos protein expression at various times after SKF 81297 (5.0 mg/kg, i.p.) injection. Scale bar: 40 mm. (B) Quantification of c-Fos immunoreactive neurons in mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.) at various time points. Data (means 6 SEM; n = 3–6 mice per group) were analyzed using one-way ANOVA: c-Fos (F(5,12) = 6.14; p,0.05). Bonferroni- Dunn test: NS. (TIF) Figure S2 SKF 81297, SKF 83822 and SKF 83959 induce ERK phosphorylation in the dentate gyrus. (A) Single confocal section showing P-ERK in the DG following systemic administration of SKF 81297 (5.0 mg/kg, i.p.), SKF 83822 (2.0 mg/kg, i.p.) and SKF 83959 (2.0 mg/kg, i.p.). Scale bar: 40 mm. (B) Quantification of P-ERK immunoreactive neurons PLoS ONE | www.plosone.org 13 May 2011 | Volume 6 | Issue 5 | e19415