Attention-deficit/hyperactivity phenotype in mice lacking the cyclin-dependent kinase 5 cofactor p35

Abstract

Background: Attention-deficit/hyperactivity disorder (ADHD) may result from delayed establishment of corticolimbic circuitry or perturbed dopamine (DA) neurotransmission. Despite the widespread use of stimulants to treat ADHD, little is known regarding their long-term effects on neurotransmitter levels and metabolism. Cyclin-dependent kinase 5 (Cdk5) regulates DA signaling through control of synthesis, postsynaptic responses, and vesicle release. Mice lacking the Cdk5-activating cofactor p35 are deficient in cortical lamination, suggesting altered motor/reward circuitry.

Methods: We employed mice lacking p35 to study the effect of altered circuitry in vivo. Positron emission tomography measured glucose metabolism in the cerebral cortex using 2-deoxy-2-[¹⁸F] fluoro-d-glucose as the radiotracer. Retrograde dye tracing and tyrosine hydroxylase immunostains assessed the effect of p35 knockout on the medial prefrontal cortex (PFC), especially in relation to mesolimbic circuit formation. We defined the influence of Cdk5/p35 activity on catecholaminergic neurotransmission and motor activity via examination of locomotor responses to psychostimulants, monoamine neurotransmitter levels, and DA signal transduction.

Results: Here, we report that mice deficient in p35 display increased glucose uptake in the cerebral cortex, basal hyperactivity, and paradoxical decreased locomotion in response to chronic injection of cocaine or methylphenidate. Knockout mice also exhibited an increased susceptibility to changes in PFC neurotransmitter content after chronic methylphenidate exposure and altered basal DAergic activity in acute striatal and PFC slices.

Conclusions: Our findings suggest that dysregulation of Cdk5/p35 activity during development may contribute to ADHD pathology, as indicated by the behavioral phenotype, improperly established mesolimbic circuitry, and aberrations in striatal and PFC catecholaminergic signaling in p35 knockout mice.

Figure 1

P35 knockout results in increased rate of cortical glucose uptake. (A)Representative coronal and sagittal positron emission tomography images of global brain glucose metabolism in control and p35^−/− mice. Images represent the average values of a 60 min dynamic scan conducted after administration of 2-deoxy-2-[¹⁸F]fluoro-D-glucose (FDG). The color scale for each image is proportional to the percent Injected dose per gram (%ID/g) within the animal: white represents the highest, and red, yellow, green, and blue represent sequentially lower concentrations. (B) Time course of FDG uptake in the cerebral cortex. FDG uptake values (%ID/g) in the cerebral cortex are normalized to FDG uptake in muscle. Graph depicts mean values ± standard deviation of the cortex:muscle ratio in 5 min increments. (C) Rate of glucose uptake over the time-span of the 60 min scan. Values represent the mean slopes ± standard error of the best-fit lines derived from B (***p < 0.001, analysis of covariance, n = 5-7).

Figure 2

Upregulation of mesolimbic connectivity after p35 knockout. (A) Representative fluorescent Nissl stain of the prefrontal cortex in p35^−/− and control mice at low magnification. Boxes indicate anatomical region of higher magnification shown in B and C. (B) Representative fluorescent stain of the medial prefrontal cortex (mPFC) for Nissl (green) and the retrograde neuronal tracer, Fluorogold (FG) (red), after injection of FG into the nucleus accumbens shell of control and p35^−/− mice at high magnification. (C) Representative fluorescent stain of the mPFC for tyrosine hydroxylase (TH) at high magnification. Scale bars: 500 μm, A; 100 μm, B and C.

Figure 3

p35 knockout induces a locomotor profile reminiscent of ADHD. (A) Time course of the locomotor response to saline injection in control and p35^−/− mice. Mean locomotor counts ± standard error for the 60 min following saline injection are shown (left). Each time point represents the average sum of locomotor counts from the previous five minutes, with the first point depicting 5 minutes of post-injection activity. Total locomotor counts ± standard error over the 60 min are also depicted (right) (***p < 0.001, Student’s unpaired t test, n = 5-7). (B) Time course of the locomotor response to chronic administration of methylphenidate (MPH, 10 mg/kg). Graph on the left shows mean locomotor counts ± standard error for 60 min intervals over a period of 5 days. Histogram at the right depicts total activity for each day ± standard error for the first 30 min of each session (*p < 0.05, Student’s paired t test, n = 5). (C) Time course of the locomotor response to chronic cocaine (20 mg/kg). Graph on the left shows mean locomotor counts ± standard error for 60 min intervals over a period of 5 days. Histogram at the right depicts total activity for each day ± standard error (*p < 0.05, Student’s unpaired t test, n = 5-7).

Figure 4

Effect of p35 knockout and chronic methylphenidate on catecholamine levels and metabolism in the prefrontal cortex. (A) Dopamine levels and metabolism in control and p35^−/− mice before and after 5 consecutive injections of methylphenidate (MPH, 10 mg/kg). Graphs depict picomoles of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and DA turnover (ratio of homovanillic acid over DA) normalized to tissue weight ± standard error (*p < 0.05, **p < 0.01, ***p < 0.001, Student’s unpaired t test, n = 5-7 per group). (B) Serotonin levels and metabolism in control and p35^−/− mice before and after chronic MPH. Graphs show picomoles of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT turnover (ratio of 5-HIAA over 5-HT) normalized to tissue weight ± standard error (*p < 0.05, **p < 0.01, Student’s unpaired t test, n = 5-7 per group).

Figure 5

Effect of p35 knockout on dopaminergic neurotransmission in acute striatal and prefrontal cortical slices. (A) Immunoblot analysis of phospho-Ser845 GluR1 in striatal slices from control and p35^−/− mice treated with SKF-81297 (1 μM, 5min). Blots were probed for phospho-Ser845, and total GluR1. Quantitation of the phosphoprotein, total protein, and the phospho/total protein ratio ± standard error normalized to wild-type controls are shown (*p < 0.05, **p < 0.01, Student’s unpaired t test, n = 4-6 per group). (B) Immunoblot analysis of phospho-Thr34 DARPP-32 in striatal slices. Blots were probed for phospho-phospho-Thr34 of DARPP-32, and total DARPP-32 (**p < 0.01, Student’s unpaired t test, n = 4-6 per group). (C) Immunoblot analysis of phospho-Ser845 GluR1 in acute prefrontal cortical slices (*p < 0.05, Student’s unpaired t test, n = 5-9 per group).