Dopamine D4 receptor knockout mice exhibit neurochemical changes consistent with decreased dopamine release.

Dopamine D4 receptor (D4R) knockout mice (D4R-/-) provided for unique neurochemical studies designed to understand D4R contributions to dopamine (DA) regulation. In this study, post-mortem brain tissue content of DA did not differ between D4R+/+ and D4R-/- mice in the striatum (Str) or nucleus accumbens core (NAc). However, there was a significant decrease (82%) in the content of 3,4-dihydoxyphenylacetic acid (DOPAC), a major metabolite of DA, in the NAc of D4R-/- mice. Microdialysis studies performed in a region of brain spanning of the dorsal Str and NAc showed lower baseline levels of DA and a significant reduction in KCl-evoked overflow of DA in the D4R-/- mice. Baseline extracellular levels of DOPAC and homovanillic acid were also significantly lower in the D4R-/- mice. In vivo chronoamperometric recordings of KCl-evoked release of DA also showed decreased release of DA in the Str and NAc of the D4R-/- mice. These studies demonstrate a role of D4Rs in presynaptic DA regulation and support the hypothesis that alterations in D4Rs may lead to diminished DA function.

Cerebellar neurotransmission in attention-deficit/hyperactivity disorder: does dopamine neurotransmission occur in the cerebellar vermis?

Children and adolescents with attention-deficit/hyperactivity disorder (ADHD) have smaller cerebellar volumes, particularly in the posterior-inferior cerebellar vermis (lobules VIII-X). Functional activation of the human cerebellar vermis following stimulant administration has also been repeatedly demonstrated. There is no well-characterized dopaminergic pathway that projects to the posterior-inferior cerebellar vermis, although the dopamine transporter (DAT) and tyrosine hydroxylase (TH) have been localized in the posterior-inferior vermis in the non-human primate by immunohistochemistry. We hypothesized that DA neurotransmission may occur in localized "hot spots" in the cerebellar vermis, and if so, that differences in such neurotransmission might be relevant to the pathophysiology of ADHD. To investigate this hypothesis, cerebellar tissue was obtained from rats and non-human primates. Catecholamines were extracted and analyzed using HPLC with coulometric detection. A regional gradient of norepinephrine (NE) and DA was found throughout the cerebellum with NE levels always roughly 10-40-fold higher than DA in both rats and monkeys. In addition, in vivo microdialysis studies were performed in the rat posterior-inferior cerebellar vermis in anesthetized animals. Significant NE overflow was observed over baseline following reverse microdialysis induced release by potassium or d-amphetamine. DA overflow was not observed over baseline for potassium stimulation, but was significant for d-amphetamine stimulation. These studies refute the hypothesis that DA neurotransmission normally occurs in the rat cerebellar vermis, but highlight that vermal DA is released by d-amphetamine. The presence of DAT may therefore allow for enhanced regulation of NE and not regulation of released DA.

Neurochemical investigations of dopamine neuronal systems in iron-regulatory protein 2 (IRP-2) knockout mice.

Abnormal iron accumulations are frequently observed in the brains of patients with Parkinson's disease and in normal aging. Iron metabolism is regulated in the CNS by iron regulatory proteins (IRP-1 and IRP-2). Mice engineered to lack IRP-2 develop abnormal motoric behaviors including tremors at rest, abnormal gait, and bradykinesia at middle to late age (18 to 24 months). To further characterize the dopamine (DA) systems of IRP-2 -/- mice, we harvested CNS tissue from age-matched wild type and IRP-2 -/- (16-19 months) and analyzed the protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter (VMAT2), and DA levels in dorsal striatum, ventral striatum (including the core and shell of nucleus accumbens), and midbrain. We further analyzed the phosphorylation of TH in striatum at serine 40, serine 31, and serine 19. In both dorsal and ventral striatum of IRP-2 knockout mice, there was a 20-25% loss of TH protein and accompanied by a approximately 50% increase in serine 40 phosphorylation above wild-type levels. No change in serine 31 phosphorylation was observed. In the ventral striatum, there was also a significant loss (approximately 40%) of DAT and VMAT2. Levels of DA were decreased (approximately 20%) in dorsal striatum, but turnover of DA was also elevated ( approximately 30%) in dorsal striatum of IRP-2 -/- mice. We conclude that iron misregulation associated with the loss of IRP-2 protein affects DA regulation in the striatum. However, the modest loss of DA and DA-regulating proteins does not reflect the pathology of PD or animal models of PD. Instead, these observations support that the IRP-2 -/- genotype may enable neurobiological events associated with aging.

Aged Fischer 344 rats exhibit altered orolingual motor function: relationships with nigrostriatal neurochemical measures.

The present study utilized a novel behavioral preparation to measure differences in orolingual motor function between young (6 months) and aged (24 months) Fischer 344 (F344) rats. Rats were trained to lick an isometric force-sensing operandum for water reinforcement so that the number of licks per session, licking rhythm and lick force could be compared between the two groups. The aged rats exhibited a greater number of licks per session, but a slowed licking rhythm, compared to the young rats. Lick force did not differ significantly between the groups. The dopamine (DA) uptake inhibitor nomifensine decreased all three measures in both groups. Analyses of whole brain tissue content of DA, 3,4 dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the substantia nigra and dorsal striatum revealed no significant differences between the two age groups. Differences were observed between the two groups with respect to relationships between behavioral and neurochemical tissue measures. Striatal DA content and the number of licks per session were positively correlated for the young rats but not for the aged rats. In the aged rats, but not the young rats, positive correlations were also observed between licking rhythm and the DOPAC+HVA/DA ratio in the substantia nigra. These findings suggest that age-related alterations in orolingual motor function may relate in part to functional changes in DA neuronal circuits.

Effects of chronic intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) in aged Rhesus monkeys.

In this study, 17-23 year old Rhesus monkeys were used as an early model of Parkinson's disease (PD). Four animals received chronic infusions of GDNF and four received vehicle infusions into the right putamen via programmable pumps for 8 weeks. Weekly videotaping was performed to record general motor performance and a monkey movement analysis panel (mMAP) was used to quantify fine and coarse upper limb motor performance. The GDNF-treated animals showed significant improvements in their overall motor performance in the last 3 weeks of the study compared to controls. Fine motor time of the upper limbs improved significantly in both the GDNF-treated and control animals. After 8 weeks of drug administration, the animals were euthanized and tissue punches were taken from the basal ganglia for measures of dopamine (DA) and DA metabolite levels. In the right putamen, GDNF infusion produced a 217% increase in homovanillic acid (HVA) levels. In addition, DA levels increased by 50% in the right caudate nucleus and there were 122 and 76% increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the right and left caudate nucleus, respectively. HVA levels were also seen to be increased by 212% in the right caudate nucleus. Finally, changes were seen in the right globus pallidus, with 390 and 171% increases in DA and HVA levels, respectively. These data support the hypothesis that GDNF may be beneficial for the treatment of damaged or degenerating DA neurons in aged monkeys and possibly in aged humans.

Aged Fischer 344 rats exhibit altered locomotion in the absence of decreased locomotor activity: exacerbation by nomifensine.

A novel force plate actometer was used to measure locomotor activity and gait in young (6 months) versus aged (24 months) Fischer 344 rats. The actometer revealed altered gait in the aged rats in the absence of decreased locomotor activity. The catecholamine uptake inhibitor, nomifensine increased locomotor activity in both groups and exacerbated the gait alteration in the aged group. Analyses of whole brain tissue levels of dopamine (DA), 3,-4 dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the substantia nigra and dorsal striatum revealed no significant differences between the two age groups. In the young (but not aged) rats, distance traveled was negatively correlated with striatal DOPAC + HVA/DA tissue ratios (a measure of DA turnover). In the aged (but not the young) rats, positive correlations were observed between distance traveled and DOPAC + HVA/DA ratios in the substantia nigra. Neither striatal nor nigral DA content was significantly correlated with distance traveled in either age group. These findings demonstrate that aged rats may exhibit functional changes in locomotor activity in the absence of quantitative changes in nigrostriatal DA content.

Enhanced dopamine transporter activity in middle-aged Gdnf heterozygous mice.

Glial cell line-derived neurotrophic factor (GDNF) supports the viability of midbrain dopamine (DA) neurons that degenerate in Parkinson's disease. Middle-aged, 12 month old, Gdnf heterozygous (Gdnf(+/-)) mice have diminished spontaneous locomotor activity and enhanced synaptosomal DA uptake compared with wild type mice. In this study, dopamine transporter (DAT) function in middle-aged, 12 month old Gdnf(+/-) mice was more thoroughly investigated using in vivo electrochemistry. Gdnf(+/-) mice injected with the DAT inhibitor, nomifensine, exhibited significantly more locomotor activity than wild type mice. In vivo electrochemistry with carbon fiber microelectrodes demonstrated enhanced clearance of DA in the striatum of Gdnf(+/-) mice, suggesting greater surface expression of DAT than in wild type littermates. Additionally, 12 month old Gdnf(+/-) mice expressed greater D(2) receptor mRNA and protein in the striatum than wild type mice. Neurochemical analyses of striatal tissue samples indicated significant reductions in DA and a faster DA metabolic rate in Gdnf(+/-) mice than in wild type mice. Altogether, these data support an important role for GDNF in the regulation of uptake, synthesis, and metabolism of DA during aging.