Ontogeny and dopaminergic regulation in brain of Ras homolog enriched in striatum (Rhes)

Abstract

Rhes is one of several signaling molecules preferentially expressed in the striatum. This GTP-binding protein affects dopamine-mediated signaling and behavior. Denervating the striatum of its dopaminergic inputs in adulthood reduces rhes mRNA expression. Here we show that dopamine depletion in adult rats by 6-hydroxydopamine caused a significant decrease in striatal Rhes protein levels as measured by Western blotting. The role of dopamine input on rhes mRNA induction during ontogeny was also examined. Rhes mRNA was measured on postnatal days 4, 6, 8, 10, 15, and 24 with in situ hybridization to determine its normal ontogeny. Signal in striatum was detectable, but very low, on postnatal day 4 and increased gradually to peak levels at days 15 and 24. Outside of the striatum, rhes mRNA was expressed at high levels in hippocampus and cerebellum during the postnatal period. Hippocampal signal was initially highest in CA3 and dentate gyrus, but shifted to higher expression in CA1 by the late postnatal period. Several other nuclei showed low levels of rhes mRNA during ontogeny. Depletion of dopamine by 6-hydroxydopamine injection on postnatal day 4 did not affect the ontogenetic development of rhes mRNA, such that expression did not differ statistically in lesioned versus vehicle-treated animals tested in adulthood. These findings suggest that although dopamine input is not necessary for the ontogenetic development of rhes mRNA expression, changes in both rhes mRNA and Rhes protein are integral components of the response of the adult striatum to dopamine depletion.

Figure 1

An anti-peptide antibody recognizes Rhes protein. (a) Western blot of increasing amounts of a recombinant GST-tagged Rhes protein (MW = 55.5 kD). (b) CHO-K1 cells were transfected with either empty vector (pcDNA3.1; lane 1, emp) or pcDNA3.1 with an insert encoding the 30 kD form of Rhes protein, and harvested either 24h or 48h later (lanes 2 and 3). Western blot of 20 μg of total protein from cell lysates shows that the Ab recognizes a ∼30 kD band specifically in the Rhes-transfected cells.

Figure 2

Rhes protein is significantly decreased in hemispheres of adult rats receiving 6-OHDA. (a) Western blot of Rhes protein from left and right striata of two representative rats. Lanes 1 and 2 are left and right striata, respectively, of a single rat, and lanes 3 and 4 are left and right striata, respectively, of a separate rat. (b) Western blot of TH in striata of the same rats as in (a). (c) Quantification of Western blots for Rhes protein indicating a significant decrease in Rhes protein in 6-OHDA-treated striata versus untreated striata. Data were analyzed by paired t-test; n = 8. * p<0.05.

Figure 3

Rhes mRNA expression in the neonatal period determined by in situ hybridization, sagittal view. Rat pups were sacrificed for analysis on postnatal days 6, 10, 15 and 24, and as adults. A, Medial and lateral sections from rats of the stated ages showing the medial-to-lateral gradient of rhes mRNA in CPu. Hippocampal and cerebellar signal is higher in neonates than in adults. B, Cresyl violet stained sections (top panel) delineate layers of rhes mRNA expression in the same sections (bottom panel). C, higher magnification of P15 from panel B. C1-C3 are cresyl violet-stained sections; C4-C6 show rhes mRNA in the same sections. scale bar = 1 mm (A, B) or 500 μM (C). GL = glomerular layer; DG = dentate gyrus; EGL = external germinal layer; ML = mitral cell layer; Sub = subiculum; GrL = granular cell layer.

Figure 4

Rhes mRNA expression in the neonatal period as determined by in situ hybridization, coronal view. Rat pups were sacrificed for analysis on postnatal days 4, 8, 10, 15, and 24. Rhes mRNA expression increased gradually during this time in striatum (B, C, D, and E), CA1 of hippocampal formation (F), and cerebellum (H). No signal was detected with a ³⁵S-labeled sense riboprobe in anterior CPu (I), posterior CPu (J), hippocampus (K), or cerebellum (L). OB, olfactory bulbs; NAc SH, shell of nucleus accumbens; NAc core, core of nucleus accumbens; ANT, anterior nucleus of the thalamus; DG, dentate gyrus; Sub, subiculum; MGN, medial geniculate nucleus; PN, pontine nuclei; IC inferior colliculus.

Figure 5

Quantification of rhes mRNA during neonatal development. Rhes mRNA increased gradually until day 15, at which point it stabilized at adult levels. Autoradiographs of in situ hybridization were quantified according to ¹⁴C standards exposed to the same film. Whole striata were manually outlined, and a value of mean density was obtained. For each animal, 2-4 samples were taken from adjacent striatal sections. n = 2-3.

Figure 6

Dopamine depletion of neonatal rat pups does not affect rhes mRNA expression. Rat pups were administered ICV 6-OHDA or vehicle on postnatal day 4 and sacrificed at 3 months in order to determine TH immunoreactivity and rhes mRNA expression. Rats that received neonatal 6-OHDA showed severe DA depletion, as measured by TH immunoreactivity, but rhes mRNA was still expressed, as measured by in situ hybridization of adjacent sections with a ³⁵S-labeled rhes riboprobe.

Figure 7

Quantification of rhes mRNA levels in rats treated with 6-OHDA or vehicle on postnatal day 4. Autoradiographs from in situ hybridization were quantified according to ¹⁴C standards exposed to the same film and expressed as percent of vehicle-treated animals from the same film. DA-depleted rats showed no statistical differences in rhes mRNA expression relative to vehicle-treated controls at any level of CPu or in NAc or OT. n = 6 for vehicle-treated, n = 11 for 6-OHDA.