L-dopa reverses behavioral deficits in the Pitx3 mouse fetus

Abstract

Studies of fetal rodents have provided evidence that early emerging behaviors, such as the suckling response, are dependent on the developing dopaminergic system. Although connections have been made between manipulations of dopamine and altered behavioral responses, the specific neural pathways involved have yet to be discovered. In this study, we examined the neurobehavioral output of the nigrostriatal pathway, using the Pitx3ak/2J mouse model (Pitx3). Used extensively in the study of Parkinson's disease, the Pitx3 mouse has very specific prenatal loss of dopaminergic neurons solely in the nigrostriatal pathway. Because of this specificity, we hypothesized that behavioral deficits specific to the nigrostriatal pathway would be reversed with administration of the dopamine precursor 3,4-dihydroxyphenylalanine (L-dopa). To test this hypothesis, homozygous mutant and heterozygous control fetal subjects were administered 1 of 4 doses (0, 25, 50, or 75 mg/kg) of L-dopa on the day before birth. Quantification of fetal behavior was scored from video recordings of behavioral observations. The behavioral measures used were (a) spontaneous movement activity; (b) state organization, from quantifications of high- and low-amplitude movements; (c) interlimb movement synchrony, a measure of limb coordination; and (d) oral grasp, similar to a newborn infant suckling response. Specific behavioral deficits observed in the Pitx3 mutants were reversed by L-dopa administration in a dose-dependent manner. However, different deficits required dissimilar doses for reversal, suggesting that some early emerging behaviors may be more sensitive to the administration of L-dopa. Taken together, this study provides valuable information about prenatal behaviors dependent on the nigrostriatal pathway.

Figure 1

Schedule of L-Dopa delivery and behavioral observations. Bars depict the experimental timeline for the four fetal subjects observed from each pregnancy. The timing of L-Dopa administrations and behavioral observations were staggered, in order to facilitate separate video recordings for each subject. The gray segments denote the 1-min baseline (left) and 4 min spontaneous movement observation periods (right). The bar to the right of the 1-min baseline indicates the time point of L-Dopa administration. A period of 16-min was allowed for L-Dopa activation, immediately following the administration (white bar). The dark gray bar (far right) indicates the 1-min presentation of a non-nutritive artificial nipple, and the corresponding behavioral observation of the Oral Grasp Response. Inset: A mutant Pitx3 subject is shown gently restrained in a curved holding apparatus during observations of the Oral Grasp. Restraint is achieved by securing a soft strap across the thorax of the fetus at the beginning of the Oral Grasp observation. Subject above is exhibiting a typical grasp of the non-nutritive artificial nipple. Note the micropthalmic eye, visible through the semi-transparent skin. One uterine horn is also visible to the left of the fetal subject. The scale bar is 5 mm.

Figure 2

Forelimb activity during spontaneous movement. Bars represent the mean frequency/min of forelimb movement during the 4-min spontaneous movement observation period. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). Broken line denotes the mean level of forelimb activity from het subjects receiving the 0 mg/kg dose (saline controls). Error bars depict the SEM.

Figure 3

State cyclicity and proportion of low amplitude behavior. (A) Sections of the circle depict the mean durations of high amplitude (black), and low amplitude (gray) state for heterozygous controls. High amplitude activity includes awake-like behaviors such as stretching and peddling movements. Low amplitude activity is defined as twitch-like movement occurring during periods of upper body atonia. Periods of quiescence (white) occurred in between high and low amplitude states, with very brief transitions(< 1 s) between low amplitude and high amplitude behavior. Arrows represent the cyclic nature of state organization, with behavior alternating between high and low amplitudes, and transitional periods of quiescence. Subjects typically exhibited 3-4 cycles during the 4 min observation period. (B) Bars represent the mean proportion of low amplitude state activity during the spontaneous movement observation. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). The dotted line represents the mean level of low amplitude state for the het subjects at the 0 mg/kg dose (saline controls). Error bars depict the SEM.

Figure 4

Interlimb Movement Synchrony during low amplitude behavioral state. Profiles represent the mean relative frequency of Interlimb Movement Synchrony in the forelimbs at each of the 0.1 s intervals during low amplitude behavior. Low amplitude behaviors were defined as those possessing a twitch-like property during upper body muscle atonia. Quantification of synchronous forelimb behavior represents the lag between right and left forelimb movements. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). The dashed lines reflect the het subject profile at the 0 mg/kg dose (saline control). Error bars depict the SEM.

Figure 5

Latency to 1st grasp response. Bars depict the mean latency in seconds to the first grasp response after presentation of a non-nutritive artificial nipple. A grasp is defined as any firm closure of the jaw around the nipple, including those of brief duration. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). The broken line reflects the mean latency to 1st grasp for the het subjects at the 0 mg/kg dose (saline controls). Error bars depict the SEM.

Figure 6

Proportion of observation period attached to nipple. Bars represent the mean proportion of the 1-min observation period during which fetal subjects were attached to the non-nutritive artificial nipple in a grasping response. Grasps were defined as any firm closure of the jaw around the nipple, including those of short duration. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). The broken line reflects the mean proportion of grasp time for the het subjects at the 0 mg/kg dose (saline controls). Error bars depict the SEM.

Figure 7

Forelimb and hindlimb activity during the Oral Grasp Response. Bars depict the mean rates/min of movement frequency for forelimbs (top) and hind limbs (bottom) while grasping the non-nutritive artificial nipple. Fetal subjects from either mutant (mut) or heterozygous (het) groups were administered one of four doses of the dopamine precursor L-Dopa (0, 25, 50, or 75 mg/kg). The dotted lines reflect the level of limb activity for the het subjects at the 0 mg/kg dose (saline controls). Error bars depict the SEM.