The role of dopamine signaling in epileptogenesis

1Epilepsy is a chronic neurological disorder, characterized by spontaneous and recurrent bursts of neuronal hyperactivity (seizures) generally arising in restricted regions of the brain. Seizures may remain confined to their area of origin (“focal” or “partial” seizures) or spread to the whole cerebral hemispheres (“generalized” seizures). The behavioral outcome of seizures strictly depends on the brain regions that are affected by hyperactivity. Seizures have been traditionally characterized as an imbalance between excitatory (glutamatergic) and inhibitory (GABAergic) transmission. The role of glutamate and GABA in epileptogenesis (i.e., the process by which a normal brain develops epilepsy) has been extensively addressed elsewhere (see for example; ) and will not be further discussed here. Clinical and experimental studies investigated the role of the major neuromodulatory systems in epilepsy. Acetylcholine, serotonin, noradrenaline, and dopamine (DA;,; ) are all known to regulate seizure activity. In this review, we will focus on the role of DA in seizure onset and spread discussing evidence obtained in human and animal studies. We present a unifying hypothesis on the intracellular signaling cascades triggered by DA and involved in long-term epileptogenesis.

2Molecules that stimulate the dopaminergic (DAergic) system such as apomorphine, amphetamines, L-DOPA (L-3,4-dihydroxyphenylalanine), and anti-parkinsonian drugs (e.g., pergolide and bromocriptine) have anti-epileptic action and anti-convulsant effects. Seizures involving the limbic system appear to be the most critically affected by modulation of DA signaling. Brain areas receiving afferents from the mesolimbic DAergic pathway express different types of DA receptors. Interestingly, while pharmacological studies using animal models support the anti-convulsant effects of DA on limbic seizures, contrasting biochemical evidence has been obtained for the presence of DAergic dysfunctions either in the brain of epileptic patients or in animal models of seizure and epilepsy. This suggests that the involvement of DA in seizure and epilepsy is likely due to a dysfunctional control of DA levels or an alteration in the expression of specific receptors. Indeed, levels of DA and its metabolites markedly vary depending on the type of epilepsy and animal models considered. However, it is interesting to note that increased levels of DA as well as increased firing of DA neurons were detected in rodent models of temporal lobe epilepsy (TLE). These findings suggest also that variations of DA levels very likely alter the neuromodulatory action of DA on brain circuits of the limbic system.

3For instance, a glutamate–DA interaction has been proposed to explain individual susceptibility to epilepsy in limbic areas. According to this hypothesis, paroxysmal activity of the cerebral cortex in the epileptic brain would increase the tonic excitation of DA neurons by glutamate. This would then induce phasic release of DA, possibly leading to downregulation or desensitization of DA receptors and subsequently decreased phasic responses. Indeed, DA exerts a marked inhibitory effect on hippocampal excitability through activation of DA D2 receptors (D2Rs). Anti-psychotics (i.e., DAergic D2-like antagonists) lower seizure threshold in epileptic patients and promote seizures in patients with no previous history of the disease. Conversely, seizure inhibition occurs in patients administered anti-parkinsonian drugs such as pergolide and bromocriptine, which both act by stimulating D2Rs. Further observations supported the anti-convulsant effect of a low dose treatment with bromocriptine. According to the Starr’s hypothesis, a low dose of a D2R agonist would act through stimulation of presynaptic D2 autoreceptors leading to decreased DA release, while preventing the downregulation of postsynaptic D2R. Based on our results using mice lacking D2R (D2R-/- mice), we postulated that D2R activation might exert a neuroprotective action on hippocampal and DAergic neurons against excitotoxicity. Conversely, activation of DA D1 receptors (D1Rs) has a proconvulsant effect, lowering seizure threshold. The opposite action of D2R and D1R signaling might also be explained by the glutamate–DA interaction hypothesis for limbic epileptogenesis. Indeed, the activation of D1R in cortical tissue samples obtained from children undergoing epilepsy surgery has been shown to induce glutamate receptor-mediated neuronal hyperexcitability. More recent studies performed in animal models during seizures support these results showing a D1R-mediated activation of glutamatergic neurons (; see also below).

4These data clearly point to a prominent role of the DAergic circuits in limbic epileptogenesis. Classical pharmacological studies supporting this view have been extensively reviewed by, ), to which the reader is referred for a more detailed description. In the next paragraphs, we will summarize recent human studies in support of this hypothesis. Animal studies will then be discussed to highlight the role of specific DA receptor signaling pathways in seizure onset and spread. We will also describe the mechanisms by which DA receptor signaling may affect neuronal excitability and epileptogenesis in the long-term. The potential importance of DA receptor-based drugs for the treatment of epilepsy will be finally discussed.

5Recent studies performed on human epileptic patients (Table 1) confirm the role of DA-mediated neurotransmission in epilepsy. The role of DA in epilepsy is most likely mediated by the neuromodulatory effect of this molecule on structures belonging to the basal ganglia and elements of the limbic system. These structures are strongly interconnected and defective DA signaling either in the basal ganglia or in the limbic system might affect the electric properties of neurons located at distal sites through either direct interactions or through feedback mechanisms connecting the cortex to the striatum or other areas. In agreement, it has been postulated that the DAergic transmission in the basal ganglia may provide an inhibitory role. Indeed, the basal ganglia are not able to generate specific epileptic activity, as detected by electroencephalographic (EEG) recordings. However, seizures originating in the mesiotemporal lobe of TLE patients can induce EEG changes in the basal ganglia, that may act as filter to the further spread of ictal activity.

6Dopaminergic pathway arising from the ventral mesencephalon [substantia nigra and ventral tegmental area (VTA)] innervate the basal ganglia, the limbic system, and the cerebral cortex. Several imaging studies demonstrate that reduced DAergic activity is present in various forms of epilepsy. Reduced [18F]-fluoro-L-DOPA uptake (indicating a reduced binding to the DA transporter, DAT) was detected in the basal ganglia of patients suffering of ring20 epilepsy, resistant generalized “absence-like” epilepsy and drug-resistant TLE with hippocampal sclerosis. In TLE patients, [18F]-fluoro-L-DOPA uptake was reduced in the caudate, putamen, and substantia nigra. Reduced DAT has also been detected in patients with juvenile myoclonic epilepsy and epilepsy with tonic–clonic seizures only (GTCS; ).

7Alterations of both D1-like and D2-like receptors have been associated to different forms of epilepsy. For example, positron emission tomography (PET) with [11C]-SCH23390 revealed a reduced striatal D1R binding in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE; ), suggesting that neurotransmitter alterations in nigrostriatal DA circuits may contribute to nocturnal paroxysmal motor activity in ADNFLE. A reduced D2R/D3R density (as evaluated by PET using the high-affinity DA D2R/D3R ligand [18F]fallypride) was instead found in the temporal lobe of TLE with hippocampal sclerosis. Interestingly, the reduction of [18F]fallypride binding did not correlate with hippocampal atrophy, indicating that reduced D2R/D3R density is not just a consequence of the degenerative process, but might play a specific role in the pathophysiology of mesial TLE. The same authors also detected a reduction in D2R/D3R binding in the putamen of patients with juvenile myoclonic epilepsy. A recent study evaluated the expression and binding of both D1R and D2R cerebral cortex samples from surgically treated patients with TLE associated with mesial sclerosis (MTLE). As compared to control samples, higher D1R expression and binding and decreased D2R expression were detected in the neocortex of MTLE patients, whereas D2R binding was unaffected. MTLE samples also presented elevated DAT binding and low tissue content of DA. It is interesting to note that in this study, D1R binding negatively correlated with seizure onset age and frequency, and positively with epilepsy duration; conversely, D2R binding positively correlated with seizure onset age and negatively with epilepsy duration. These results are in agreement with several data from animal models of TLE (see below), respectively, showing a pro-epileptic and anti-epileptic role of D1R and D2R, and confirm that an altered function of the DAergic system might contribute to TLE.

8Despite the notion that several genetic factors are predisposing to epilepsy, little evidence is available in favor of a direct link between epilepsy and variation of genes coding for protein involved in DAergic neurotransmission. Two studies reported the association of DNA polymorphisms in the DAT gene and idiopathic absence epilepsy and alcohol-withdrawal seizures, indicating that genetic variation of the DAT gene may modulate neuronal network excitability and contribute to epileptogenesis. More recently, the genetic variation in DAergic function has been associated with the risk of adverse effect of anti-epileptic drug treatment. Specifically, chronic epileptic patients carrying genetic variants associated with decreased DAergic activity (DA-β-hydroxylase, DBH; catechol-O-methyltransferase, COMT and D2R) showed a higher susceptibility to adverse psychotropic effects of levetiracetam. This suggests that reduced DAergic transmission in epileptic patients might contribute to worsen the outcome of specific anti-epileptic medications.

9Pharmacological studies demonstrated that the activation of different DA receptor subtypes plays distinct roles in the onset and spread of limbic seizures. DA acts through two different types of G-protein-coupled receptors (GPCRs), named D1-like and D2-like. Activation of D1-like (D1 and D5) receptors results in reduction of seizure threshold and increased seizure severity. Conversely, the effect of D2-like (including D2, D3, and D4) receptors on seizure modulation is mainly inhibitory. Administration of D2-like receptor agonists lowers seizure activity, whereas blockade of these receptors has proconvulsant effects. More recently, studies performed on DA receptor knockout mice (Table 2) and the use of compounds acting on specific DA receptor subtypes contributed to dissect the intracellular pathways activated by different DA receptors in response to seizures (Figure 1).

10The D1R agonist SKF38393 has a proconvulsant action; D1-like receptors (D1R and D5R)-mediated signaling increases cAMP levels and protein kinase A (PKA) activity via the stimulation of adenylyl cyclase (AC) by stimulatory G-proteins. DA and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) is a critical downstream target of D1R- and D5R-mediated signaling. PKA-catalyzed phosphorylation activates DARPP-32, and converts it into an inhibitor of protein phosphatase-1 (PP-1). Phosphorylated DARPP-32, by inhibiting PP-1, activates a series of signaling cascades that are important in regulating neuronal excitability. In mice, D1-like receptor agonist administration induces seizures and DARPP-32 phosphorylation. Accordingly, seizure behavior is absent or greatly reduced in both D1R and DARPP-32 knockout mice, thus highlighting the crucial role of this signaling pathway in mediating DAergic control of seizures. In addition to its direct effect on DARPP-32, D1R-dependent activation of PKA signaling also leads to phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2). Accordingly, seizure-induced ERK activation in the granule cell layer of the dentate gyrus is absent in D1R knockout mice. Seizures resulting from D1R activation depend on the specific coupling of D1R to the PKA–DARPP-32–ERK pathway. D1-type receptor agonists stimulating the AC pathway increase the levels of Zif268 and Arc/Arg3.1 [two immediate early genes (IEGs) involved in transcriptional regulation and synaptic plasticity] in the dentate gyrus, with a time-course that parallels that of ERK phosphorylation. Conversely, D1 agonists that stimulate phospholipase C (PLC) but not AC do not induce seizure behaviors. These results clearly indicate that activation of D1R-dependent signaling has a proconvulsant activity. However, it must be pointed out that postnatal ablation of D1R-expressing striatal neurons results in spontaneous seizures in mice, suggesting seizure control may depend on the anatomical integrity of DAergic striatal pathways.

11D5R activation triggers both cAMP and PLC signaling. Similarly to D1R, D5R-mediated signaling through the cAMP pathway seems to be mainly involved in seizure control. D5R-/- mice treated with the proconvulsant D1R agonist SKF83822, showed an increased latency to first seizure and a reduced total time spent in EEG seizures when compared to wild-type (WT) mice. However, it must be pointed out that D5R seems to have less pronounced effects than D1R in regulating synaptic activity, as also confirmed by other studies.

12Several pharmacological lines of evidence indicate that D2R is the major DA receptor subtype involved in the anti-epileptogenic action of DA in limbic areas. In accordance with imaging studies performed in epileptic patients (Table 1), animal studies confirmed that reduced levels of D2R expression are detected in epileptogenic areas in seizing rodents. For example, D2-like binding sites were reduced in the caudate–putamen (CP) of pilocarpine-treated rats and genetically epileptic GAERS (genetic absence epilepsy rat from Strasbourg; ) and WAG/Rij (Wistar Albino Glaxo rats from Rijswijk; ) rats. Interestingly, WAG/Rij rats also showed a reduced D2-like binding in the CA3 region, confirming a prominent role of D2R signaling in limbic epileptogenesis. The crucial role of D2R signaling in the prevention of hippocampal epileptogenesis is highlighted by the observation that intra-hippocampal administration of remoxipride (a selective D2R antagonist) completely abolished the protective effects of DA against limbic seizures induced by pilocarpine in adult rats.

13D2-like receptor stimulation has an antagonistic effect to D1-like stimulation. D2-like receptors are coupled to Gi proteins that inhibit AC activity. Gi protein activation following DA binding to D2R leads to a decrease of cAMP production and subsequent modulation of PKA/ERK signaling. Accordingly, D2R activation is able to counterbalance DARPP-32 activity. In the hippocampal kindling model, an increased activation of Gi protein coupled to D2-like receptors was detected in the hippocampus and other brain areas, as evaluated by increased [35S]GTPγS in situ binding. Ligand stimulation of G-protein-coupled receptors results into the activation of the associated G-protein and binding of GTP to the catalytic α-subunit. Measuring the binding of [35S]GTPγS (a radiolabeled GTP analog) is therefore considered a reliable tool to quantify GPCR activation. Increased activation of Gi signaling downstream of D2R autoreceptors might explain the lower DA release detected in the hippocampus of fully kindled animals, which might facilitate seizures. Accordingly, hippocampal administration of the D2-like receptor antagonist sulpiride induces enhanced DA release and longer seizure duration in kindled animals. Genetic inactivation of the D2R gene and the consequent impairment of D2R-mediated signaling results in more severe limbic seizures. D2R-/- mice have an increased susceptibility to seizures induced by kainic acid (KA; ) and pilocarpine: D2R-/- mice experience generalized limbic motor seizures at doses that are not convulsant in WT mice. The canonical D2R-mediated signaling pathway negatively regulates ERK activity through reduction of cAMP levels and PKA activity, thereby modulating the expression of cAMP-responsive IEGs. Accordingly, KA administration in D2R-/- mice induces a massive c-fos expression (a typical cAMP-responsive gene; ), at a dose lower than in WT mice (Figure 2A). KA-induced c-fos mRNA upregulation mainly involves the DG-CA3 hippocampal circuit (, and Figure 2A), thus indicating that the D2R-mediated seizure control mainly involves this limbic circuit. A more rapid and longer-lasting ERK phosphorylation (consistent with the time-course of c-fos mRNA induction; Figure 2A and ) is detectable in the hippocampus of D2R-/- mice, as compared to WT controls (Figure 2B). In addition, KA-induced seizures result in a stronger and longer-lasting c-Fos protein upregulation in the D2R-/- hippocampus as compared to WT (Figure 2C; see also ). Taken together, these data confirm the critical role of the D2R cAMP-dependent signaling in mediating the first steps of DAergic control of hippocampal activity during seizures.

14In addition to their increased susceptibility to KA-induced seizures, D2R-/- mice also display increased susceptibility to KA-induced CA3 hippocampal cell death. This death occurs by apoptosis, as indicated by Bax and Caspase-3 upregulation in the hippocampus of KA-treated D2R-/- mice. We recently investigated the intracellular pathways involved in D2R-mediated control of seizure-induced CA3 hippocampal cell death. Several studies show that D2R may also trigger a cAMP-independent pathway. Activation of this pathway following DA binding to D2R results in the inhibition of Akt activity, by dephosphorylation of the threonine 308 (Thr308) residue, leading to activation of glycogen synthase kinase 3β (GSK-3β;, ). Accordingly, we observed GSK-3β activation in the D2R-/- hippocampus after KA, suggesting that upregulation of GSK-3β activity might contribute to increased susceptibility to seizure-induced cell death observed in these mice. However, GSK-3β upregulation in KA-treated D2R-/- mice was independent of Akt phosphorylation at Thr308, implicating that alternative pathways might contribute to modulate GSK-3β in the hippocampus during epileptic activity. The p38 mitogen-activated protein kinase (MAPK) and Wnt pathways, which have been implicated as potential alternative pathways in regulating GSK-3β activity, are not affected in KA-treated D2R-/- mice. We were able to show that following KA, phosphorylation of Akt occurs at the serine 473 residue (Ser473) in the CA3 region of WT but not of D2R-/- mice (; CA3 neuron loss following KA is detected in D2R-/- but not WT mice;; ). Conversely, a strong induction of Akt (Ser473) phosphorylation after KA was detected in the CA1 subregion (where no neuronal cell loss is detected after KA) of both WT and D2R-/- mice. We therefore proposed that loss of D2R signaling results in reduced Akt (Ser473) phosphorylation, rendering CA3 neurons more vulnerable to apoptosis. Further investigation is required to fully elucidate the Akt/GSK-3β targets involved in D2R-mediated response to excitotoxicity (see also below).

15Contrasting results were obtained about the role of D3R signaling in seizure modulation. D3R are mainly expressed in the limbic forebrain (; see also below). However, stimulation of D3R has a minimal inhibitory effect on limbic seizures: intra-accumbens pretreatment with D3 agonists delayed the onset of limbic seizures induced by pilocarpine, without any effect on their frequency and severity. In the same model, D2R agonists exerted an anti-convulsant action. Thus, the protective effect of DA on seizure propagation through the limbic forebrain is predominantly mediated by D2R rather than D3R. It has been proposed that D3R participates in D2R cAMP-independent pathway by enhancing D2R-mediated Akt (Thr308) phosphorylation. Signaling cascades downstream of D3R also involve Ca2+/calmodulin-dependent protein kinase II (CaMKII), that binds to the N-terminal region of the third intracellular loop of D3R, as well as ERK and CREB (cAMP response element-binding protein; ) phosphorylation, whose activity is negatively modulated by DR3.

16As the other members of the D2-like receptor family, D4R have a prominent inhibitory role on neuronal hyperexcitability. The frequency of spontaneous synaptic activity and the frequency and duration of epileptic discharges induced by 4-aminopyridine or bicuculline were increased in cortical slices from D4R-/- mice, as well as in brain slices from WT mice treated with a selective D4R antagonist. In vivo, D4R-/- mice showed a reduction of SKF83822-induced seizures, indicating that D4R interacts with AC-coupled D1R to positively regulate D1R-mediated seizures.

17The results reported in the previous sections clearly highlight the opposite neuromodulatory role of D1- and D2-like receptors on seizures arising in the limbic system. However, most of (if not all) these studies investigated the role of DA signaling on the modulation of acute seizures (such as those occurring during an experimentally induced status epilepticus). Few data are available suggesting a direct link between DA signaling and epileptogenesis, i.e., the establishment of a chronic epileptic condition following an initial precipitating injury. In the next paragraphs we will present current understanding of how altered DA signaling might contribute to a chronic epileptic condition. In the attempt to build up a common signaling network for DA in epileptogenesis, we will first consider (i) the (co)expression of DA receptors in epileptogenic brain areas and (ii) what we know about DAergic modulation of chronic seizures in appropriate animal models of epileptogenesis. Then we will try to explain how altered DA neurotransmission in epileptogenic brain areas might interfere with intracellular pathways involved in long-term hyperexcitability. Finally, we will propose a novel, testable hypothesis on the role of DA receptor signaling in epileptogenesis.

18All DA receptor subtypes are expressed in epileptogenic brain areas. D1Rs are expressed at high levels in the CP, nucleus accumbens (NAc), substantia nigra pars reticulata (SNr), amygdala, and cerebral cortex, and to a lower level in the hippocampus. D5Rs are expressed in the entorhinal cortex, SNr, and hippocampus mainly in the dentate gyrus). Lower levels of expression are found in the NAc and CP neurons. D2Rs are mainly expressed in the CP, NAc, SN pars compacta (SNc), in the ventral tegmental area, cerebral cortex, amygdala, and hippocampus. D3R expression is mainly restricted to areas of the limbic system (NAc, islands of Calleja), but is also present in the SNc, VTA, hippocampus, and cerebral cortex. Finally, D4R expression in epileptogenic areas is limited to the frontal cortex, amygdala, hippocampus, and SN.

19This brief summary clearly shows that most DA receptor subtypes are present in epileptogenic areas within the limbic system. In these areas, DA receptors are generally expressed in different subsets of neurons, but co-expression of different subtypes has also been detected in restricted neuronal populations. For example, D1R and D2R are generally expressed in distinct subpopulations of striatal medium spiny neurons, but a small percentage (5–15%) of these neurons has been shown to co-express both receptors; similarly, 20–25% of pyramidal neurons of the prefrontal cortex do co-express D1R and D2R. In the hippocampus, D1R mRNA is predominantly expressed in the granule cell layer of dentate gyrus, whereas the protein is localized in the molecular layer. D2R mRNA is also expressed in granule cells of the dentate gyrus, but its expression is also detectable in CA1–CA3 pyramidal layers; D2R protein is instead localized in the hilus and stratum lacunosum moleculare. According to the expression profile of DA receptor subtypes, it is therefore likely that the signaling cascades depicted in Figure 1 may cooperate at least in some, restricted neuronal subpopulations within the limbic system, such as dentate granule cells in the hippocampal formation. Indeed, DA has been shown to markedly regulate neuronal excitability in the dentate gyrus, as well as other limbic regions via D1-like and D2-like signaling pathways.

20The vast majority of studies demonstrating a DAergic modulation of seizure onset and spread were performed on animal models of acute but not chronic seizures. Thus, a direct demonstration of a neuromodulatory effect of DA in epileptogenesis is substantially lacking. However, some important indications may be obtained from the limbic kindling model. Limbic kindling consists in the repeated, subthreshold electrical stimulation of the amygdala or hippocampus, that ultimately leads to the expression of chronic seizures. Kindling has been extensively used for the preclinical evaluation of anti-epileptic drugs; many studies demonstrated that drugs showing anti-epileptic effects against limbic kindling also have an anti-epileptic efficacy in clinical TLE. The effect of DAergic drugs on kindled seizures is well-documented. Non-selective DA agonists (such as amphetamines) have an anti-epileptic action. Interestingly, while the prototypical D1R agonist SKF38393 has no effect in this model, D2R-selective compounds do modify seizure threshold. D2R agonists (lisuride) are protective, whereas D2R blockers (haloperidol) exacerbate kindled seizures.

21The advantages of the limbic kindling model of epileptogenesis are multiple: a precise, focal activation of specific brain areas; a reliable development of chronic epileptogenesis; and a rapid and consistent pattern of seizure propagation and generalization. However, the kindling procedure is labor intensive, and spontaneous seizures develop only after a very large number of stimulations. For these reasons, DAergic modulation of seizure onset and spread has been more extensively studied in pharmacological models of limbic epileptogenesis, namely seizures induced by the muscarinic agonist pilocarpine and glutamatergic agonist KA. These two drugs induce very similar epileptic activity despite their distinct mechanism of action. Pilocarpine and KA initially provoke signs of focal epilepsy (stereotyped pre-convulsive behaviors), due to the activation of limbic areas (dentate gyrus, hippocampal formation, amygdala, entorhinal cortex). From these areas, epileptic activity rapidly propagates to the whole cerebral cortex, culminating in acute motor seizures and status epilepticus. Most importantly, pilocarpine- and KA-induced seizures result in extensive neurodegeneration in specific regions of the brain and may lead to the occurrence of spontaneous chronic seizures in the long-term. Using these models, a clear effect of D1R and D2R signaling on the genesis of limbic seizures has been observed, as described in previous paragraphs. However, several questions remain open. Specifically, do DA drugs (namely, D1R antagonists and D2R agonists) have a disease-modifying effect? Do they reduce or stop the occurrence of chronic seizures? The first issue could be addressed by administering DA compounds after pilocarpine- or KA-induced status epilepticus, and recording the occurrence of spontaneous chronic seizures. To test the anti-convulsant effect of DA drugs onto chronic seizures, D1R- and D2R-selective compounds should instead be administered during the occurrence of spontaneous seizures in appropriate models of chronic epilepsy (such as that resulting from intrahippocampal administration of KA; ).

22Evidence discussed above supports a neuromodulatory role of DA signaling in limbic epileptogenesis. However, the mechanisms by which DA signaling affects neuronal excitability and epileptogenesis in the long-term remain largely unknown. Here we propose that activation of neuronal cell death pathways (a well-known causal factor of limbic epileptogenesis;; ) following altered DA signaling might contribute to chronic epilepsy. As summarized in Figures 1 and 3, stimulation of D1R and blockade of D2R signaling can lead to the activation of neuronal cell death pathways. This phenomenon essentially involves two intracellular cascades: the PKA/ERK/Fos/Jun pathway and the mammalian target of rapamycin (mTOR) pathway.

23Canonical, cAMP-dependent signaling through D1R and D2R activates the expression of the IEGs c-fos and c-jun. Treatment with D1R agonists results in a robust Fos-like immunoreactivity in basal ganglia and limbic structures of rats undergoing pilocarpine-induced generalized seizures. Recent studies indicate that D1R signaling through the G protein αolfβ2γ7 might contribute to seizure-induced neuropathology. Similarly, D2R receptor blockade by haloperidol induces Fos and Jun B expression during status epilepticus in the hippocampus and striatum; accordingly, KA seizures in D2R-/- mice markedly induce c-fos and c-jun expression (; Figure 2). The protein products of c-fos/c-jun form the AP-1 transcription factor, whose activation regulates the expression of a wide number of cell death genes. The prolonged activation of c-fos after acute seizures was proposed as one of the crucial steps that trigger long-term neuronal death. Jun phosphorylation (mediated by the c-Jun N-terminal kinase, JNK) activates Jun transcriptional activity and triggers apoptotic neuronal cell death after seizures.

24D2R signaling also occurs through a cAMP-independent, Akt/GSK-3β-dependent pathway (; see also Figure 1 and references above). Loss of D2R signaling in D2R-/- mice results in reduced Akt (Ser473) phosphorylation and subsequent overactivity of GSK-3β, thus rendering CA3 neurons more susceptible to apoptosis. GSK-3β hyperactivity is known to induce hippocampal neurodegeneration, through mechanisms involving the blockade of the pro-survival β-catenin pathway as well as the activation of the mTOR pathway. KA seizures do not alter the β-catenin pathway in the D2R-/- hippocampus. Thus, it is likely that GSK-3β hyperactivity in KA-treated D2R-/- mice results in the activation of the mTOR pathway. Several evidences support the crucial role of this pathway in epileptogenesis. For example, the components of the mTOR pathway are upregulated after seizures and, most importantly, inhibition of mTOR by rapamycin may ameliorate the development of epilepsy-related pathology and reduce the expression of spontaneous seizures in TLE models. In addition, there is strong evidence that rapamycin may prevent epilepsy and ameliorate its progression in mice lacking the tuberous sclerosis complex genes 1 and 2 (TSC1/2), which act as negative regulators of mTOR. The mechanisms through which mTOR overactivity promotes epileptogenesis and neurodegeneration remain to be understood; according to the multiple action of the mTOR targets, these might involve altered cell growth and morphology, dysregulation of glutamatergic neurotransmission, inflammation, axonal sprouting, and remodeling of epileptogenic circuits (; see also Figure 3).

25Taken together, these data lead us to propose that loss of D2R signaling (induced by pharmacological blockade or genetic inactivation of D2R) might contribute to epileptogenesis via the activation of the mTOR pathway. This hypothesis might be tested by checking whether targets of the mTOR complex are upregulated in D2R-/- mice following KA- or pilocarpine-induced seizures. It would be then possible to investigate whether the mTOR inhibitor rapamycin is able to prevent seizures in KA or pilocarpine-treated D2R-/- mice. It is interesting to point out that seizure induction by activation of D1R might also converge onto the mTOR pathway; indeed, this pathway is also activated via PKA/ERK signaling (Figure 3), and the D1R agonist SKF81297 was shown to increase the phosphorylation of the mTOR target ribosomal protein S6 in the dentate gyrus, in an ERK-dependent manner. However, SKF81297 administration did not activate (but rather suppressed) the mTORC1/S6 kinase pathway, suggesting that S6 phosphorylation occurs independently of mTOR. Further investigation is therefore needed to understand whether stimulation of D1R signaling may promote epileptogenesis via activation of the mTOR pathway.

26Modulation of limbic seizures by DAergic drugs, as detected in the kindling, pilocarpine and KA models (see above) might predict a similar effect of these drugs on clinical epileptogenesis. However, no known DAergic drug is currently used to treat epilepsy, despite anti-epileptic effects of DA agonists have been reported in epileptic patients. The lack of a systematic investigation of the anti-epileptic efficacy of DA agonists is certainly due to their severe neurological and neuropsychiatric side effects. However, some studies investigated the potential use of the D2R-selective agonist bromocriptine in some forms of epilepsy. Bromocriptine was originally reported to have an anti-epileptic effect in a case of self-induced, drug-resistant epilepsy. Other studies subsequently confirmed that bromocriptine was highly effective in reducing seizure frequency in TLE patients affected by pituitary prolactinomas. It is important to observe that these studies did not report severe side effects of prolonged bromocriptine treatment (see also ). Interestingly, in D2R-/- mice, increased seizure susceptibility is accompanied by the progressive development of pituitary prolactinomas, suggesting that altered D2R signaling might be a common cause of these two conditions. These observations definitely prompt for a better investigation of the anti-epileptogenic efficacy of D2R-selective agonists. Indeed, different D2R agonists (including bromocriptine) have neuroprotective efficacy against KA-induced brain damage, and recent studies promisingly show that lisuride may reduce seizures occurring after traumatic brain injury. Further investigation in both animal models and clinical settings is needed to establish the anti-epileptogenic efficacy of D2R agonists.

27In this review, we described recent evidence from both human and animal studies supporting the opposite role of D1-like and D2-like receptor signaling in limbic epilepsy. These studies indicate that increased D1R and decreased D2R function might be involved in limbic epilepsy. We propose that altered D1R and D2R signaling might contribute to epileptogenesis via the activation of the neuronal cell death cascades, activated by the PKA/ERK and mTOR pathways. The possible therapeutic application of these findings has been long disregarded, mainly due the severe side effects of DAergic drugs. However, the beneficial effects of selective D2R agonists observed in both animal and human epilepsy would deserve more attention.

28The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.