Serotonin hyperinnervation abolishes seizure susceptibility in Otx2 conditional mutant mice

Abstract

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.

Figure 1.

5-HT is increased and SERT is decreased in the ventral midbrain and hippocampus of Otx2 conditional mutant mice. A, B, Images show coronal sections through the ventral tegmental area of the midbrain (top) and CA3 region of the hippocampus (bottom) from Otx2^flox/flox and En1^cre/+; Otx2^flox/flox mice, stained with 5-HT (A) and SERT (B) antibodies. Scale bar, 150 μm.

Figure 2.

5-HT depletion in control and Otx2 conditional mutant mice. A, HPLC determination of 5-HT content in the pons/ventral midbrain (top) and hippocampus (bottom) from Otx2^flox/flox and En1^cre/+; Otx2^flox/flox mice, after a 3 d treatment with saline or pCPA. Data are reported as mean ± SE (n = 5 animals per group). *p < 0.05, post hoc Tukey's test. B, Representative low-magnification images showing 5-HT immunostaining on sagittal brain sections from control and Otx2 conditional mutant mice, treated with saline or pCPA. Genotypes and treatments are as indicated. Abbreviations: bf, Basal forebrain; ctx, cerebral cortex; dr, dorsal raphe nucleus; p, pons; vmb, ventral midbrain. Scale bar, 3.4 mm. C, Representative high-magnification images showing 5-HT immunostaining in the dorsal raphe nucleus from control and Otx2 conditional mutant mice, treated with saline or pCPA. Genotypes and treatments are as indicated. Scale bar, 150 μm.

Figure 3.

Resistance to KA-induced seizures in Otx2 conditional mutant mice is abolished by 5-HT depletion. Graph shows the progression of behavioral changes over a 2 h observation period after KA in control and Otx2 conditional mutant mice, with or without pCPA pretreatment. Genotypes and treatments are as indicated. Data are reported as mean seizure scores ± SE (n = 10 animals per group). **p < 0.001, post hoc Holm–Sidak test, En1^cre/+; Otx2^flox/flox versus the other three treatment groups.

Figure 4.

Effect of 5-HT depletion on c-fos mRNA expression in the brain of KA-treated control and Otx2 conditional mutant mice. The panels show c-fos mRNA in situ hybridizations on coronal sections at the level of the caudate–putamen (top) and dorsal hippocampus (bottom) from representative control and Otx2 conditional mutant mice (with or without pCPA pretreatment), 2 h after KA. Genotypes and treatments are as indicated. Abbreviations: amy, Amygdala; CPu, caudate–putamen; ctx, cerebral cortex; hip, hippocampus; ht, hypothalamus; sept, septum. Scale bar, 2 mm.