Synthesis, protein levels, activity, and phosphorylation state of tyrosine hydroxylase in mesoaccumbens and nigrostriatal dopamine pathways of chronically food-restricted rats

Abstract

Chronic food restriction (FR) enhances the rewarding and motor-activating effects of abused drugs, and is accompanied by changes in dopamine (DA) dynamics and increased D-1 DA receptor-mediated cell signaling and transcriptional responses in nucleus accumbens (NAc). However, little is known about effects of FR on DA synthetic activity in the mesoaccumbens and nigrostriatal pathways. In Experiment 1 of the present study, tyrosine hydroxylase (TH) gene expression was measured in ventral tegmental area and substantia nigra, using real-time RT-PCR and in situ hybridization; no differences were observed between FR and ad libitum fed (AL) rats. In Experiment 2, TH protein levels, determined by Western blot, were found to be elevated in NAc and caudate-putamen (CPu) of FR relative to AL rats. In the absence of increased transcription, this may reflect a slowing of TH degradation. In Experiments 3 and 4, DA synthetic activity was assessed by Western blot measurement of TH phosphorylation at Ser40, and HPLC measurement of in vivo tyrosine hydroxylation rate, as reflected by DOPA accumulation following administration of a decarboxylase inhibitor (NSD-1015; 100 mg/kg, i.p.). Basal phospho-(Ser40)-TH levels did not differ between groups but DOPA accumulation was decreased by FR. Decreased DOPA synthesis, despite increased levels of TH protein, may reflect the inhibitory effect of increased DA binding to TH protein or decreased concentrations of cofactor tetrahydrobiopterin. Finally, in response to D-amphetamine (0.5 and 5.0 mg/kg, i.p.), phospho-(Ser40)-TH was selectively decreased in NAc of FR rats. This suggests increased feedback inhibition of DA synthesis-a possible consequence of postsynaptic receptor hypersensitivity, or increased extracellular DA concentration. These results indicate that FR increases TH protein levels, but may decrease the capacity for DA synthesis by decreasing TH activity. According to this scheme, the previously observed upregulation of striatal cell signaling and transcriptional responses to DA receptor agonist administration may include compensatory neuroadaptations.

Figure 1

Effects of chronic food restriction (FR) on tyrosine hydroxylase mRNA levels in substantia nigra (SN) and ventral tegmental area (VTA) determined by real-time RT-PCR analysis. All data have been normalized for levels of β-Actin expression within the same sample and represent group means ± SEM (n= 7 per group) of fold-induction over the control ad libitum (AL) fed group.

Figure 2

Effects of chronic food restriction (FR) on tyrosine hydroxylase mRNA levels in substantia nigra (SN) and ventral tegmental area (VTA) determed by in situ hybridization. Left: mean ± SEM (n=5 per group) optical density (OD) expressed in comparison to the normalized control ad libitum (AL) fed group. Right: Tyrosine hydroxylase mRNA expression in SN and VTA of representative AL and FR rats.

Figure 3

Mean ± SEM (n_AL=6; n_FR=5) ratio of total tyrosine hydroxylase protein/tubulin in substantia nigra (SN) and ventral tegmental area (VTA) of food-restricted (FR) rats determined by Western blot and expressed in comparison to the normalized control ad libitum (AL) fed group. Graphed results are displayed with representative immunoblots.

Figure 4

Mean ± SEM (n = 11 per group) ratio of total tyrosine hydroxylase protein/tubulin in caudate-putamen (CPu) and nucleus accumbens (NAc) of food-restricted (FR) rats determined by Western blot and expressed in comparison to the normalized control ad libitum (AL) fed group. Graphed results are displayed with representative immunoblots. ^*p=.05; ^**p=.001

Figure 5

Mean ± SEM (n_AL=13; n_FR = 11) ratio of phospho-(Ser40)-tyrosine hydroxylase/tubulin in caudate-putamen (CPu) and nucleus accumbens (NAc) of food-restricted (FR) rats determined by Western blot and expressed in comparison to the normalized control ad libitum (AL) fed group.

Figure 6

Mean ± SEM (n= 5-6 per group) ratio of phospho-(Ser40)-tyrosine hydroxylase/tubulin in caudate-putamen (CPu) and nucleus accumbens (NAc) of food-restricted (FR) rats injected 20 min prior to sacrifice with 0.0, 0.5 or 5.0 mg/kg d-amphetamine (i.p.) and expressed in comparison to the normalized control ad libitum (AL) fed group injected with saline vehicle. Graphed results are displayed with representative immunoblots. ^*p<.05; ^**p<.01

Figure 7

Mean ± SEM (n =13-14 per group) DOPA concentration (ng/mg tissue), determined by HPLC, in caudate-putamen (CPu) and nucleus accumbens (NAc) of ad libitum fed (AL) and food-restricted (FR) rats treated with the DOPA decarboxylase inhibitor, NSD-1015 (100 mg/kg, i.p). ^*p<.05.