The homeobox-containing transcription factor Otx2 is crucially involved in fate determination of midbrain neurons. Mutant mice, in which Otx2 was conditionally inactivated by a Cre recombinase expressed under the transcriptional control of the Engrailed1 (En1) gene (En1cre/+; Otx2flox/flox), show a reduced number of dopaminergic neurons and an increased number of serotonergic neurons in the ventral midbrain. Despite these developmental anatomical alterations, En1cre/+; Otx2flox/flox adult mice display normal motor function. Here, we further investigated the neurological consequences of Otx2 inactivation in adult En1cre/+; Otx2flox/flox mice. Adult En1cre/+; Otx2flox/flox mice showed increased serotonin (5-HT) levels in the pons, ventral midbrain, hippocampus (CA3 subfield), and cerebral cortex, as indicated by HPLC and immunohistochemistry. Conversely, SERT (5-HT transporter) levels were decreased in conditional mutant brains. As a consequence of this increased 5-HT hyperinnervation, En1cre/+; Otx2flox/flox mice were resistant to generalized seizures induced by the glutamate agonist kainic acid (KA). Indeed, prolonged pretreatment of En1cre/+; Otx2flox/flox mice with the 5-HT synthesis inhibitor para-chlorophenylalanine (pCPA) restored brain 5-HT content to control levels, fully reestablishing KA seizure susceptibility. Accordingly, c-fos mRNA induction after KA was restricted to the hippocampus in En1cre/+; Otx2flox/flox mice, whereas a widespread c-fos mRNA labeling was observed throughout the brain of En1cre/+; Otx2flox/flox mice pretreated with pCPA. These results clearly show that increased brain 5-HT levels are responsible for seizure resistance in En1cre/+; Otx2flox/flox mice and confirm the important role of 5-HT in the control of seizure spread.
The homeobox-containing transcription factor Otx2 is required for regionalization, patterning, and neuronal differentiation in the midbrain (Simeone et al., 2002; Simeone, 2005). At the midbrain/hindbrain boundary, Otx2 specifies identity and number of dopaminergic versus serotonergic progenitors by antagonizing the Fgf8 (fibroblast growth factor 8) and Shh (sonic hedgehog) pathways and preventing ventral de-repression of the Nkx2.2 transcription factor (Puelles et al., 2003, 2004; Prakash et al., 2006). In dopaminergic progenitors, Otx2 is coexpressed with Engrailed 1 (En1) (Puelles et al., 2004; our unpublished observations). In conditional mutant mice, in which Otx2 was inactivated by a Cre recombinase expressed under the control of the En1 promoter (En1cre/+; Otx2flox/flox), midbrain dopaminergic neurons were greatly reduced in number and most of their precursors underwent neurotransmitter fate switch, generating serotonin (5-HT)-positive neurons (Puelles et al., 2004). This alteration is maintained throughout life, because En1cre/+; Otx2flox/flox adult mice still display reduced dopamine (DA) and increased 5-HT levels in the striatum and cerebral cortex (Borgkvist et al., 2006).
Psychiatric and neurological conditions, such as affective disorders and epilepsy, depend on dysfunctions of the serotonergic system. As regards the role of 5-HT in epilepsy, it is known that agents that elevate extracellular 5-HT levels (such as 5-HT reuptake blockers) inhibit seizures, whereas 5-HT depletion results in the lowering of seizure threshold (Mazarati et al., 2005; Bagdy et al., 2007). The ventral midbrain and limbic system are crucially involved in 5-HT-mediated control of seizures (Bagdy et al., 2007).
In this study, we characterized the effects of serotonergic hyperinnervation in En1cre/+; Otx2flox/flox mice, investigating whether elevated 5-HT might contribute to decrease seizure susceptibility in these animals. Seizures were evoked by systemic administration of the glutamate receptor agonist kainic acid (KA), a widely used model to study behavioral, anatomical, and cellular consequences of seizures in rodents (Lothman and Collins, 1981; Ben-Ari, 1985; Schauwecker and Steward, 1997). Here, we show that 5-HT hyperinnervation protects En1cre/+; Otx2flox/flox mice against KA-induced seizures.
Materials and Methods
The generation and genotyping of En1cre/+; Otx2flox/flox mutant mice have been reported previously (Puelles et al., 2003, 2004). In particular, the En1Cre/+ and the Otx2flox/+ mouse strains were maintained through continued breeding of at least seven generations with C57BL/6 × DBA2 F1 mice. Then the two strains were mated to generate parental mice (En1cre/+; Otx2flox/+ and Otx2flox/flox). Otx2flox/flox mice were chosen as controls, because they do not show any anatomical or behavioral abnormality with respect to wild-type animals (Puelles et al., 2003; Borgkvist et al., 2006). Adult (6 months of age; weight, 20–35 g) mice of both sexes were used. Animals were housed in a 12 h light/dark cycle with food and water available ad libitum. Experiments were conducted in conformity with the European Communities Council Directive of November 24, 1986 (86/609/EEC).
Drug-free Otx2flox/flox and En1cre/+; Otx2flox/flox mice (n = 3 per genotype) were used for initial 5-HT and 5-HT transporter (SERT) immunohistochemistry. To deplete 5-HT, mice (n = 5 per genotype) received para-chlorophenylalanine (pCPA) (4-chloro-l-phenylalanine hydrochloride; Sigma-Aldrich; 10 mg/ml stock in saline) twice a day (at ≈10:00 A.M. and 6:00 P.M.) at a dose of 100 mg/kg intraperitoneally for 3 consecutive days (Rantamäki et al., 2007). Five Otx2flox/flox and five En1cre/+; Otx2flox/flox mice received saline with the same schedule and served as controls. Sixteen hours after the last pCPA/saline injection, brains were dissected. One hemisphere was used for immunohistochemistry, and the other for HPLC. For seizure studies, 10 Otx2flox/flox and 10 En1cre/+; Otx2flox/flox mice received a single intraperitoneal injection of KA (Ocean Produce International; dissolved in saline) at 20 mg/kg. For pCPA plus KA treatments, mice (n = 10 per genotype), received the same dose of KA 16 h after the last pCPA injection. All experiments were performed blind to genotype and treatment.
Brains were fixed by immersion in 4% paraformaldehyde. Coronal or sagittal sections (50 μm; cut on a freezing microtome) were incubated overnight with anti-5-HT (1:5000; Sigma-Aldrich; or 1:200; Millipore Bioscience Research Reagents) or anti-SERT (1:5000; Calbiochem) polyclonal antibodies, diluted in a PBS solution containing 1% serum and 0.1% Triton X-100. Sections were then reacted with a biotinylated secondary antibody (Vector Laboratories) followed by avidin–biotin–peroxidase complex (ABC kit; Vector Laboratories) and diaminobenzidine reaction. Quantitative analyses of immunohistochemistry experiments was performed as reported in the supplemental material (available at www.jneurosci.org).
5-HT was measured according to Atkinson et al. (2006). Brain areas (pons/ventral midbrain, hippocampus, and cerebral cortex) were dissected on ice, weighed to the milligram sensitivity, and extracted with a buffer containing 8.2% ascorbic acid, 1.64% Na2S2O5, and 0.83 m HClO4. Extraction buffer volume (in microliters) corresponded to three times the weight in milligrams of the specimen. Homogenates were centrifuged (30 min; 18,000 rpm; 4°C), and supernatants were used as samples for HPLC. Standard solutions were prepared dissolving 5-HT and tryptophan (Sigma-Aldrich) in extraction buffer. Twenty microliters of samples or standards were injected into a Synergy Hydro-RP separation column, fitted with a C18 cartridge column (Phenomenex). The column was eluted isocratically (0.8 ml/min; 29°C) with mobile phase (100 mm ammonium acetate, pH 4.5:methanol; 12.5:1 v/v) in a Waters Alliance HPLC apparatus. Detection was performed with a Waters 474 scanning fluorescence detector (excitation and emission wavelengths, 290 and 337 nm, respectively), and data analysis was performed with Waters Millenium software. Values (±SE) were reported as picomoles of 5-HT per milligram of wet tissue. Statistical analysis was performed by one-way ANOVA followed by post hoc Tukey's test.
Seizures were scored according to Racine (1972): stage 0, normal behavior; stage 1, immobility; stage 2, forelimb and/or tail extension, rigid posture; stage 3, repetitive movements, head bobbing; stage 4, forelimb clonus with rearing and falling (limbic motor seizure); stage 5, continuous rearing and falling; stage 6, severe whole-body convulsions (tonic–clonic seizures); stage 7, death. For each animal, seizure severity was scored every 20 min for 2 h after KA administration. The maximum rating scale values reached by each animal over each 20 min interval were used to calculate the rating scale value (±SE) for each treatment group. Statistical analysis was performed by two-way repeated-measures ANOVA followed by post hoc Holm–Sidak test.
In situ hybridization.
Mice (n = 3 per genotype and treatment group, chosen among those used for behavioral analysis) were killed at 2 h after KA injection. Nonradioactive in situ hybridization was performed on brain coronal sections using a digoxigenin-labeled c-fos riboprobe, as described previously (Costantin et al., 2005).
We first analyzed the distribution of 5-HT and SERT in the ventral midbrain and hippocampus of drug-free En1cre/+; Otx2flox/flox mutant and Otx2flox/flox control adult mice. Immunohistochemistry experiments confirmed the presence of 5-HT-positive neurons in the ventral midbrain of En1cre/+; Otx2flox/flox but not Otx2flox/flox mice (Fig. 1A) (Puelles et al., 2004). In the hippocampus, 5-HT staining was increased in the CA3 area of Otx2 conditional mutant mice, compared with control mice (Fig. 1A). These findings were confirmed by quantitative analysis (see supplemental material, available at www.jneurosci.org). Conversely, SERT levels in serotonergic fibers were markedly reduced in these areas in En1cre/+; Otx2flox/flox mice, when compared with control animals (Fig. 1B).
We next determined the 5-HT levels in different brain areas of En1cre/+; Otx2flox/flox and Otx2flox/flox mice (n = 5 per genotype). HPLC analysis in Otx2 conditional mutant mice revealed a significant increase of 5-HT content in the pons/ventral midbrain, compared with control mice (Fig. 2A, saline-treated groups) (one-way ANOVA, p < 0.05; post hoc Tukey's test, control vs mutant, p < 0.05). A slight but not significant increase of 5-HT content was detected in the whole hippocampus of En1cre/+; Otx2flox/flox compared with Otx2flox/flox mice (one-way ANOVA, p > 0.05). According to our previous study (Borgkvist et al., 2006), 5-HT levels were also increased in the cerebral cortex of mutant mice (picomoles of 5-HT/milligram of tissue: saline-treated Otx2flox/flox, 1.71 ± 0.19; saline-treated En1cre/+; Otx2flox/flox, 4.8 ± 0.77; one-way ANOVA, p < 0.05; post hoc Tukey's test, p < 0.05). In both control and Otx2 conditional mutant mice (n = 5 per genotype), prolonged treatment with the 5-HT synthesis inhibitor pCPA significantly reduced 5-HT levels in the pons, ventral midbrain, and hippocampus (Fig. 2A). pCPA also decreased 5-HT content in the cerebral cortex in both genotypes (picomoles of 5-HT/milligram of tissue: pCPA-treated Otx2flox/flox, 0.98 ± 0.16; pCPA-treated En1cre/+; Otx2flox/flox, 1.7 ± 0.5; one-way ANOVA, p < 0.05; post hoc Tukey's test, pCPA vs saline of same genotype, p < 0.05). 5-HT levels in pCPA-treated En1cre/+; Otx2flox/flox mice did not significantly differ from those detected in saline-treated Otx2flox/flox animals (Fig. 2A). 5-HT immunohistochemistry performed on brain sagittal sections from saline- and pCPA-treated Otx2flox/flox and En1cre/+; Otx2flox/flox mice confirmed these findings. According to our previous results (Fig. 1A,B) (Borgkvist et al., 2006), increased 5-HT staining was detected in several areas including ventral midbrain, basal forebrain, cerebral cortex (Fig. 2B), and pons (raphe nuclei) (Fig. 2B,C) of saline-treated En1cre/+; Otx2flox/flox mutants, compared with saline-treated Otx2flox/flox controls. Treatment with pCPA markedly reduced 5-HT staining in all these areas in both genotypes (Fig. 2B,C). Quantitative analysis confirmed these findings (see supplemental material, available at www.jneurosci.org).
We next investigated whether increased 5-HT levels might alter seizure susceptibility in Otx2 conditional mutant mice. Adult En1cre/+; Otx2flox/flox and Otx2flox/flox mice (n = 10 per genotype) received a single systemic injection of KA (20 mg/kg) and were observed for 2 h. KA treatment had a strong convulsant effect in Otx2flox/flox mice. All mice showed initial immobility, rapidly followed by repeated generalized (stage 4–6) seizures (Fig. 3). The mean latency to the first generalized seizure in KA-treated Otx2flox/flox mice was 18.9 ± 8.7 min (Table 1). Progression of clinical signs was dramatically different in En1cre/+; Otx2flox/flox animals (Fig. 3). Indeed, the trajectory in behavior score of En1cre/+; Otx2flox/flox mice differed from that of control mice starting from 20 min after KA administration (two-way repeated-measures ANOVA, p < 0.001; post hoc Holm–Sidak test, En1cre/+; Otx2flox/flox vs Otx2flox/flox mice, p < 0.001). The majority of Otx2 conditional mutant mice displayed only preconvulsive behaviors, never showing any sign of generalized seizure activity (Fig. 3). Only 1 of 10 En1cre/+; Otx2flox/flox mice showed forelimb clonus with rearing and falling, followed by a single, brief tonic–clonic seizure. In this animal, latency to the first generalized seizure was 52 min (Table 1). En1cre/+; Otx2flox/flox never showed any sign of generalized seizure activity also at later times (>2 h) after KA administration (data not shown). Depletion of endogenous 5-HT by pretreatment with pCPA in Otx2 conditional mutant mice (n = 10) resulted in the occurrence of strong KA-induced generalized seizures, as observed in Otx2flox/flox mice (Fig. 3). Indeed, seizure severity in En1cre/+; Otx2flox/flox pretreated with pCPA was significantly different from that observed in the same mice without pCPA (two-way repeated-measures ANOVA, p < 0.001; post hoc Holm–Sidak test, En1cre/+; Otx2flox/flox plus pCPA vs En1cre/+; Otx2flox/flox, p < 0.001; En1cre/+; Otx2flox/flox plus pCPA vs Otx2flox/flox, p > 0.05). Latency to first generalized seizure in Otx2 conditional mutant mice pretreated with pCPA was comparable with that observed in Otx2flox/flox mice (Table 1). Pretreatment with pCPA in Otx2flox/flox mice (n = 10) resulted in the same severity of KA-induced behavioral seizures as observed in Otx2flox/flox mice without pCPA and En1cre/+; Otx2flox/flox mice pretreated with pCPA (two-way repeated-measures ANOVA, p > 0.05; post hoc Holm–Sidak test, Otx2flox/flox plus pCPA vs Otx2flox/flox or En1cre/+; Otx2flox/flox plus pCPA, p > 0.05). Saline-treated animals of all genotypes never showed any sign of seizure activity (data not shown).
We next used c-fos mRNA in situ hybridization to study the pattern of brain activation at 2 h after KA. A strong c-fos mRNA labeling was observed in the septum, caudate–putamen, cerebral cortex, amygdala, hypothalamus, and hippocampus of Otx2flox/flox mice, whereas c-fos mRNA induction was restricted to the hippocampus in En1cre/+; Otx2flox/flox mice (Fig. 4). Conversely, En1cre/+; Otx2flox/flox mice pretreated with pCPA showed the same widespread c-fos mRNA labeling as observed in Otx2flox/flox mice. Pretreatment with pCPA did not alter the pattern of KA-induced c-fos mRNA expression in Otx2flox/flox mice (Fig. 4). Saline-treated animals of both genotypes did not show any c-fos mRNA labeling throughout the brain (data not shown).
In this study, we show that conditional inactivation of the Otx2 gene in ventral midbrain results in increased 5-HT levels in several brain areas of En1cre/+; Otx2flox/flox adult mice. Behavioral and gene expression analyses showed that mutant mice were markedly resistant to KA-induced seizures. Seizure resistance was attributable to increased 5-HT levels, because 5-HT depletion by pCPA fully reestablished KA seizure susceptibility in En1cre/+; Otx2flox/flox mice.
A link between 5-HT and seizure inhibition was originally suggested by the observation that anticonvulsant drugs elevated brain 5-HT levels (Bonnycastle et al., 1957). Several studies then confirmed a crucial role of 5-HT in the control of seizures. It is now generally accepted that drugs elevating extracellular 5-HT levels [such as selective serotonin reuptake inhibitors (SSRIs)] exert a powerful antiepileptic action. For example, the anticonvulsant effect of the SSRI fluoxetine has been clearly demonstrated in a wide variety of experimentally induced seizure models, as well as in genetically epileptic animals. Conversely, depletion of brain 5-HT content is known to decrease the threshold to evoked seizures in several experimental paradigms (Bagdy et al., 2007). Our study confirms and expands this notion. Indeed, we showed that genetically induced increase of 5-HT levels results in marked protection against KA-induced seizures.
Different 5-HT receptors have been shown to modulate seizure susceptibility and development of epilepsy. More specifically, the 5-HT1A, 5-HT2C, 5-HT3, and 5-HT7 subtypes, which are all expressed in epileptogenic brain areas, are the most relevant in epilepsy (Bagdy et al., 2007). With respect to KA-induced seizures, the role of 5-HT1A receptors has been clearly elucidated. Administration of 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] (a specific 5-HT1A agonist) reduces KA-evoked seizures in rats (Gariboldi et al., 1996), whereas increased lethality after KA seizures is observed in mice with targeted inactivation of the 5-HT1A gene (Sarnyai et al., 2000). Mice lacking 5-HT2C receptors also develop epilepsy (Tecott et al., 1995; Brennan et al., 1997). In KA seizure-resistant En1cre/+; Otx2flox/flox mice, the role of 5-HT receptor signaling pathways remains to be elucidated.
Among the seizure models in which 5-HT plays a prominent role, DBA/2 mice have been well characterized. These mice are extremely prone to audiogenic or KA-induced seizures (Collins, 1972; Ferraro et al., 1995). Serotonin has been proposed to control seizure outcome in DBA/2 mice, because fluoxetine reduces respiratory arrest after audiogenic seizures (Tupal and Faingold, 2006). We used control and Otx2 conditional mutant mice of a mixed DBA/2 × C57BL/6 background. Otx2flox/flox control mice never showed spontaneous seizures, but displayed high susceptibility to seizures induced by 20 mg/kg KA, similarly to what observed in DBA/2 mice (Ferraro et al., 1995; McLin and Steward, 2006) and mice with a mixed DBA/2 × C57BL/6 background (Dell'agnello et al., 2007). Conversely, En1cre/+; Otx2flox/flox mice showed a marked resistance to KA seizures, indicating a protective effect of 5-HT in the mixed DBA/2 × C57BL/6 background.
Seizure resistance in En1cre/+; Otx2flox/flox mice was also confirmed by gene expression studies. A precise correlation exists between the pattern of c-fos induction and the appearance of generalized seizures after KA. Preconvulsive behaviors (stages 1–3 of Racine's scale) induce c-fos mainly in the hippocampus, whereas generalized seizures (stages 4–6) result in a widespread expression in several brain areas (Willoughby et al., 1997; Bozzi et al., 2000). Accordingly, En1cre/+; Otx2flox/flox mice never experienced generalized seizures after KA, showing c-fos mRNA induction restricted to the hippocampus. Conversely, En1cre/+; Otx2flox/flox mice treated with pCPA, as well as Otx2flox/flox mice, showed KA-induced generalized seizures and widespread induction of c-fos mRNA.
The ventral midbrain and limbic system are crucially involved in the serotonergic control of seizures. For example, endogenous 5-HT transmission in the substantia nigra is able to inhibit the spread of seizure activity generated in the limbic system (Pasini et al., 1996). In En1cre/+; Otx2flox/flox mice, we detected increased levels of 5-HT in several brain areas, including the ventral midbrain, basal forebrain, cerebral cortex, and hippocampal CA3 subfield. Prolonged pretreatment of En1cre/+; Otx2flox/flox mice with the 5-HT synthesis inhibitor pCPA restored brain 5-HT content to control levels and abolished seizure resistance in mutant mice. This indicates that increased availability of synaptic 5-HT is indeed protective against KA seizures in En1cre/+; Otx2flox/flox mice. Increased synaptic availability of 5-HT was also indicated by decreased SERT levels in the hippocampus and ventral midbrain of En1cre/+; Otx2flox/flox mice. SERT decrease has been demonstrated to occur after prolonged elevation of 5-HT levels in mice (Mirza et al., 2007). Thus, in Otx2 conditional mutant mice, decreased SERT levels might be a consequence of increased 5-HT innervation.
En1cre/+; Otx2flox/flox mice show a strong reduction of DA cell number in the ventral midbrain (Puelles et al., 2004; Borgkvist et al., 2006). Therefore, it might be questioned that reduction of DA cells could also contribute to modify seizure susceptibility in these animals. DA, as 5-HT, is clearly involved in the control of epileptic seizures (Starr, 1996). A large series of studies strongly support the idea of an antiepileptic action of DA. Indeed, DA agonists inhibit convulsive seizures, both in experimental animals and humans. For example, the prototypical mixed D1/D2 receptor stimulant apomorphine has long been known to exert an antiepileptic action in humans (Starr, 1996). Mesolimbic DAergic pathways have been proposed to exert this inhibitory control (La Grutta and Sabatino, 1990; Starr, 1996). According to this view, DA reduction in the mesolimbic system of En1cre/+; Otx2flox/flox mice would be expected to exacerbate seizure severity. In contrast, spontaneous or KA-induced seizures were never observed in En1cre/+; Otx2flox/flox mice, which instead displayed a marked resistance to KA. This suggests that, in this animal model, the effect of 5-HT hyperinnervation onto seizure control is more prominent than that of DA reduction.
Several studies suggest that there may be a link between epilepsy and depression, in which 5-HT would exert a crucial role. Epilepsy is often associated with depression in humans, and increased seizure susceptibility is accompanied by behavioral symptoms of depression in animal models (Mazarati et al., 2007). Moreover, serotonergic antidepressant drugs (such as fluoxetine) have anticonvulsant properties, and some antiepileptic drugs are effective in treating bipolar affective disorders (Jobe et al. 1999). We propose that En1cre/+; Otx2flox/flox mutant mice, in which 5-HT hyperinnervation results in marked resistance to experimentally induced seizures, might serve as a novel genetic model to investigate the mechanisms underlying mood disorders.
This work was supported by grants from the Italian Association for Cancer Research, the European Community FP6 (EuTRACC Integrated Project LSHG-CT-2006-037445), and Fondazione Cassa di Risparmio di Roma (A.S.). Y.B. is a recipient of research grants from Parents Against Childhood Epilepsy (New York, NY) and the National Research Council [Consiglio Nazionale delle Ricerche (CNR)–Ricerche Spontanee a Tema Libero program]. The financial support from Fondo per gli Investimenti della Ricerca di Base (CHEM-PROFARMA-NET Project) of the Italian Government to Istituto di Neuroscienze del CNR is also acknowledged. We thank Giulio Cesare Cappagli, Adriano Tacchi, and the technical/administrative staff of the Istituto di Neuroscienze for excellent assistance, and Matteo Caleo, Massimo Pasqualetti, and Manuela Scali for helpful discussions.
- Correspondence should be addressed to either of the following: Antonio Simeone, CEINGE-Biotecnologie Avanzate, via Comunale Margherita 482, 80145 Naples, Italy, ; or Yuri Bozzi, Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, via G. Moruzzi 1, 56100 Pisa, Italy,