Serotonin hyperinnervation abolishes seizure susceptibility in Otx2 conditional mutant mice

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 (En1^cre/+; Otx2^flox/flox), show a reduced number of dopaminergic neurons and an increased number of serotonergic neurons in the ventral midbrain. Despite these developmental anatomical alterations, En1^cre/+; Otx2 ^flox/flox adult mice display normal motor function. Here, we further investigated the neurological consequences of Otx2 inactivation in adult En1^cre/+; Otx2^flox/flox mice. Adult En1^cre/+; Otx2^flox/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, En1^cre/+; Otx2^flox/flox mice were resistant to generalized seizures induced by the glutamate agonist kainic acid (KA). Indeed, prolonged pretreatment of En1^cre/+; Otx2^flox/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 En1^Cre/+; Otx2^flox/flox mice, whereas a widespread c-fos mRNA labeling was observed throughout the brain of En1^Cre/+; Otx2^flox/flox mice pretreated with pCPA. These results clearly show that increased brain 5-HT levels are responsible for seizure resistance in En1^cre/+; Otx2 ^flox/flox mice and confirm the important role of 5-HT in the control of seizure spread.