Critical role of the embryonic mid-hindbrain organizer in the behavioral response to amphetamine and methylphenidate

Abstract

The embryonic mid-hindbrain organizer, which is composed of a transient cell population in the brainstem, controls the development of dopaminergic and serotonergic neurons. Different genes determining the position and activity of this embryonic structure have been implicated in dopamine- and serotonin-associated disorders. Mouse mutants with a caudally shifted mid-hindbrain organizer, are hyperactive, show increased numbers of dopaminergic neurons and a reduction in serotonergic cells. In the present study we used these mutants to gain insights into the genetic and developmental mechanisms underlying motor activity and the response to psychostimulants. To this end, we studied the motor activity of these animals after exposure to methylphenidate and amphetamine and characterized their dopaminergic and serotonergic innervation. Saline-treated mutants showed increased locomotion, more stereotypic behavior and a decrease in rearing compared to wild-type mice. This baseline level of activity was similar to behaviors observed in wild-type animals treated with high doses of psychostimulants. In mutants methylphenidate (5 or 30 mg/kg) or amphetamine (2 or 4 mg/kg) did not further increase activity or even caused a decrease of locomotor activity, in contrast to wild-type mice. Fluoxetine (5 or 10 mg/kg) reduced hyperactivity of mutants to levels observed in wild-types. Transmitter measurements, dopamine and serotonin transporter binding assays and autoradiography, indicated a subtle increase in striatal dopaminergic innervation and a marked general decrease of serotonergic innervation in mutants. Taken together, our data suggest that mice with an aberrantly positioned mid-hindbrain organizer show altered sensitivity to psychostimulants and that an increase of serotonergic neurotransmission reverses their hyperactivity. We conclude that the mid-hindbrain organizer, by orchestrating the formation of dopaminergic and serotonergic neurons, is an essential developmental parameter of locomotor activity and psychostimulant response.

Fig. 1. Locomotor activity, rearing and stereotypies for animals treated with saline, 5 or 30 mg/kg MP

A. Effect of MP on locomotor activity measured in distance traveled per 5 min time intervals (Mean ± SEM). WT mice showed increased activity following 5 mg/kg and 30 mg/kg MP. The hyperactivity elicited by the high dose declined rapidly, as stereotypy increased. Locomotor activity of En1^+/Otx2 mutants after MP exposure was not significantly different from saline treated mutants. B. Time (sec) spent engaged in rearing behaviour (Mean ± SEM) following MP treatment. The first minute of each 10 minute interval was sampled. Mutant mice have less rearing behaviour compared to WT after saline and 5 mg/kg MP, but following 30 mg/kg MP there was no difference between mutant and WT mice. In the mutant mice, 30 mg/kg MP significantly reduced rearing, and 5 mg/kg had no effect. In WT mice, 5 mg/kg MP increased rearing and 30 mg/kg decreased rearing. C. Time (sec) spent engaged in stereotypic behaviour (Mean ± SEM) following MP treatment. The first minute of each 10 minute interval was sampled. WT animals treated with 5 or 30mg/kg MP show a significant increase in stereotypies. Mutant mice showed more stereotypy than WT mice following saline or the low dose of MP and less stereotypy than WT following the high dose of MP. Both saline groups n=14, all other groups, n=8

Fig. 2. Locomotor activity, rearing and stereotypies for animals treated with saline, 2 or 4mg/kg AMPH

A. Effect of AMPH on locomotor activity measured in distance traveled per 5 min time interval (Mean ± SEM). In WT mice 2 as well as 4 mg/kg AMPH led to an increase in locomotor activity. En1^+/Otx2 showed reduced activity after AMPH exposure and were more active than WT mice after saline treatment. B. Time (sec) spent engaged in rearing behaviour (Mean ± SEM) following AMPH treatment. The first minute of each 10 minute interval was sampled. In the mutant mice, both doses of AMPH led to a decrease in rearing, whereas in WT mice decreased rearing was observed after the higher dose. C. Time (sec) spent engaged in stereotypic behaviour (Mean ± SEM) following AMPH treatment. The first minute of each 10 minute interval was sampled. Stereotypies were significantly increased for wild-type as well as for mutants with the higher dose. Both saline groups, n=11, WT 2 mg/kg and 4 mg/kg AMPH and En1^+/Otx2 2 mg/kg, n=7, and En1^+/Otx2 4 mg/kg, n=6.

Fig. 3. Locomotor activity, rearing and stereotypies for animals treated with saline, 5 or 10 mg/kg FLU

A. Effect of FLU on locomotor activity measured in distance traveled per 5 min time interval (Mean ± SEM). Saline treated En1^+/Otx2 were more active than saline-treated WT mice. FLU treated mutants showed a dose dependant decrease in locomotor activity. B. Time (sec) spent engaged in rearing behaviour (Mean ± SEM) following FLU treatment. The first minute of each 10 minute interval was sampled. FLU did not affect rearing in WT but the higher dose of FLU reduced rearing in mutants. C. Time (sec) spent engaged in stereotypic behaviour (Mean ± SEM) following FLU treatment. The first minute of each 10 minute interval was sampled. There were 6 mice in each group.

Fig. 4. Representative autoradiograms of [¹²⁵I]RTI-55 binding to coronal sections of WT and mutant brains

A, B visualization of DAT. C, D visualization of SERT. SERT binding was significantly reduced in the dorsal as well as in the median raphe. Caudate Putamen (CPu), dorsal raphe nucleus (DR), median raphe nucleus (MR).

Fig. 5. Maximal binding capacity (Bmax) and affinity (Kd) of [³H]GBR12935 and [³H]Citalopram in WT and En1^+/Otx2 mutants

DAT measured by [³H]GBR12935 binding was significantly increased in the caudate putamen of mutants. SERT measured by [³H]Citalopram binding was significantly reduced in the caudate putamen and in the frontal cortex of mutants. Values are expressed as mean ± SD. Asterisks indicate significant difference in t testing (p<0.05)