Reduced striatal dopamine underlies the attention system dysfunction in neurofibromatosis-1 mutant mice

Abstract

Learning and behavioral abnormalities are among the most common clinical problems in children with the neurofibromatosis-1 (NF1) inherited cancer syndrome. Recent studies using Nf1 genetically engineered mice (GEM) have been instructive for partly elucidating the cellular and molecular defects underlying these cognitive deficits; however, no current model has shed light on the more frequently encountered attention system abnormalities seen in children with NF1. Using an Nf1 optic glioma (OPG) GEM model, we report novel defects in non-selective and selective attention without an accompanying hyperactivity phenotype. Specifically, Nf1 OPG mice exhibit reduced rearing in response to novel objects and environmental stimuli. Similar to children with NF1, the attention system dysfunction in these mice is reversed by treatment with methylphenidate (MPH), suggesting a defect in brain catecholamine homeostasis. We further demonstrate that this attention system abnormality is the consequence of reduced dopamine (DA) levels in the striatum, which is normalized following either MPH or l-dopa administration. The reduction in striatal DA levels in Nf1 OPG mice is associated with reduced striatal expression of tyrosine hydroxylase, the rate-limited enzyme in DA synthesis, without any associated dopaminergic cell loss in the substantia nigra. Moreover, we demonstrate a cell-autonomous defect in Nf1+/- dopaminergic neuron growth cone areas and neurite extension in vitro, which results in decreased dopaminergic cell projections to the striatum in Nf1 OPG mice in vivo. Collectively, these data establish abnormal DA homeostasis as the primary biochemical defect underlying the attention system dysfunction in Nf1 GEM relevant to children with NF1.

Figure 1.

Nf1 optic glioma (OPG) mice exhibit spatial learning deficits. (A) Control mice (CT; gray squares) and Nf1 OPG mice (black diamonds) show equivalent performance on cued trials for path length (course taken to platform), latency (time to reach platform) and swimming speed. (B) Although Nf1 OPG mice tended to exhibit inferior performance relative to the littermate control (CT) group during the place trials with regard to the distance traveled and time taken to navigate to the platform, these differences were not statistically significant. (C) However, when the platform was removed during probe trial 1, the Nf1 OPG mice spent significantly less time in the target quadrant (tg) compared with the littermate control (CT) group (*P = 0.002). Nf1 OPG mice showed little preference for the target quadrant (in terms of time spent in) compared with the right, left and opposite quadrants (rt, lt, opp), whereas the littermate control (CT) mice showed a significant spatial bias for the target quadrant as shown by them spending significantly more time in the target quadrant versus the time spent in the other quadrants (**P < 0.0002).The dotted line represents the time spent in each quadrant expected by chance alone. (D) In addition, the percentage of the distance swum in the target quadrant (out of the total distance traveled in the entire pool during the probe trial) was significantly lower in Nf1 OPG mice (*P = 0.01). (E) While not statistically significant, Nf1 OPG mice showed a strong trend toward fewer platform crossings compared with the littermate control (CT) group.

Figure 2.

Nf1 OPG mice exhibit reduced ambulatory activity and exploratory behaviors in the 1h locomotor activity test and show evidence of attention deficits. (A) Total ambulations, as a measure of general activity in a novel environment, were significantly lower in Nf1 OPG mice relative to littermate control (CT) mice (P = 0.008). (B) Rearing, as a measure of exploration and non-selective attention to environmental change, was also significantly reduced in Nf1 OPG mice compared with littermate control (CT) mice (P = 0.0039). Note that differences were greatest early on in the test session, suggesting an attenuated response to novelty on the part of the Nf1 OPG mice. (C) Nf1 OPG mice also showed evidence of altered emotionality by exhibiting fewer entries into the center than littermate control (CT) mice (P = 0.01). (D) Similarly, Nf1 OPG mice spent less time exploring the center compared with littermate control (CT) mice (P = 0.0001). (E) When normalized to the total distance traveled during the test session, there is still a significant reduction in distance traveled in the center for Nf1 OPG mice (P = 0.001).

Figure 3.

Nf1 OPG mice have no abnormalities in sensorimotor function or visual impairment. (A) Walking initiation is unaffected in Nf1 OPG mice. (B) Nf1 OPG mice were not impaired relative to littermate control (CT) mice on the small platform (balance) test. (C) There were no differences between littermate control (CT) and Nf1 OPG mice on the inverted screen test, suggesting that grip strength was intact in Nf1 OPG mice. Nf1 OPG mice performed equivalently to littermate control (CT) mice on rotarod tests of balance and dexterity involving (D) constant and (E) accelerating speeds. (F) No significant differences between littermate control (CT) and Nf1 OPG mice were observed using VOS to measure visual acuity (P = 0.96).

Figure 4.

Nf1 OPG mice have reduced rearing frequency and duration during the sample object trial in the object recognition test. (A) Nf1 OPG (n = 20) and littermate control (CT) (n = 17) mice did not differ in the times devoted to investigating the two identical sample objects. However, the total rearing frequency (B) and rearing frequency in the field away from the objects (C) were both significantly reduced in Nf1 OPG mice (P = 0.0001) during the sample objects trial. (D) Rearing frequency during investigation of the sample objects was also significantly reduced in Nf1 OPG mice (P = 0.0025). The average rearing duration per episode was also significantly lower in Nf1 OPG mice in terms of (E) total rearings and (F) rearings in the field (P = 0.034 and 0.0017, respectively). (G) In contrast, average rearing duration was not different between groups during sample object investigation.

Figure 5.

Nf1 OPG mice exhibit object recognition retention impairments and attention abnormalities. (A) Littermate control (CT) mice spent significantly more time investigating the novel object versus the familiar object during the test trial (P = 0.0013), while the object investigation times (novel versus familiar) were not different for the Nf1 OPG mice. (B) Nf1 OPG mice spent significantly less time investigating the moved object compared with the littermate control (CT) group during the second test trial (P = 0.044). Nf1 OPG mice showed significantly reduced rearing frequency of (C) total rearings and (D) rearings in the field (P = 0.006 and 0.004, respectively) during the novel versus familiar test trial. (E) Nf1 OPG mice reared significantly less often than the littermate control (CT) group during investigation of the novel object (P = 0.009), but the groups did not differ in terms of rearing frequency shown during investigation of the familiar object. Both the littermate control (CT) and Nf1 OPG mice reared significantly more often when investigating the novel object compared with the familiar one (P = 0.002 and 0.019, respectively). (F) Nf1 OPG mice showed a non-significant trend toward decreased average rearing duration compared with the littermate control (CT) group during the novel versus familiar test trial.

Figure 6.

Treatment with methylphenidate (MPH) or l-dopa improves exploratory and attention behaviors in Nf1 OPG mice. (A) Total ambulations of Nf1 OPG and littermate control (CT) mice increased after a single IP injection of MPH (20 mg/kg) (P = 0.02). (B) Rearings in only Nf1 OPG mice increased after MPH administration (P = 0.05). (C) While not as strong an inducer of activity as MPH, l-dopa (50 mg/kg) also increased total ambulations (P = 0.04). (D) Rearings were increased in Nf1 OPG mice following l-dopa injection (P = 0.02).

Figure 7.

Nf1 OPG mice have normal numbers of dopaminergic neurons, but express reduced TH mRNA levels in the striatum. (A) TH-positive neuronal cell counts in the substantia nigra were similar in Nf1 OPG mice and littermate control (CT) mice (n = 6). (B) qPCR analysis of Th mRNA levels in the corpus striatum showed a significant decrease in Nf1 OPG mice compared with littermate controls (P = 0.02).

Figure 8.

Nf1+/− dopaminergic neurons have reduced neurite lengths and smaller growth cone areas. (A) TH-immunoreactive neurons from the ventral midbrain of E13 wild-type (WT) and Nf1+/− mice. (B) Nf1+/− TH-immunoreactive neurons have shorter neurite lengths compared with WT controls (P = 0.001; n = 30). (C) Nf1+/− TH-immunoreactive neurons have smaller growth cone areas relative to WT controls (P = 0.001; n = 30). Scale bars = 50 µm.

Figure 9.

Nf1 OPG mice have reduced dopaminergic projections in the striatum. (A) No change in the numbers of TH-immunoreactive neurons was observed in the substantia nigra of Nf1 OPG mice relative to littermate controls. (B) No differences in TH staining intensity were observed in the immediate neuronal projections from the substantia nigra in Nf1 OPG mice relative to littermate controls. (C) Synaptophysin immunolabeling of neurons in the striatum demonstrated a 9.5% reduction in mean intensity in Nf1 OPG mice relative to littermate controls (P = 0.0001; n = 8). (D) TH immunolabeling of neurons in the striatum demonstrated a 9% reduction in mean intensity in Nf1 OPG mice relative to littermate controls (P = 0.0001; n = 8). Scale bars = 100 µm.