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.