The number of cells expressing dopamine D2 receptor mRNA in rat brain caudate putamen is higher in oestrus

Abstract

Dopamine D2 receptors (D2-Rs) in the central nervous system are involved in the control of locomotion, cognition, emotion and neuroendocrine secretion. The intensity of cellular responses to specific stimuli is dependent on the concentration of dopamine or its agonist, and the availability, as well as the concentration, of all the other components of the signalling pathway in the cell, including the receptors. Many factors can influence the level of mRNA encoding the receptors. In order to study the changes in the level of expression of the D2-R mRNA in the brain of female rats at different stages of the oestrous cycle, we used a quantitative in situ hybridization technique. Four groups of animals were analysed: rats in prooestrus (POE), oestrus (OE), dioestrus 1 (DOE1) and dioestrus 2 (DOE2). A 35S-labelled riboprobe was transcribed in vitro from the 1.5-kb D2-R cDNA. The caudate putamen of the rats, which shows the highest level of expression of D2-R mRNA in the brain, was examined. The number of silver grains per cell, representing hybridization of riboprobe, and the number of cells expressing the D2-R mRNA, were counted with the computer-assisted image analysis system Lucia-M. Our results show that the expression of the D2-R mRNA in the lateral striatum varies during the oestrous cycle, with the highest expression measured during DOE2. The number of cells expressing the D2-R mRNA also changes during the different phases, with the highest number being detected in OE. This indicates that during OE more cells transcribe the D2-R mRNA. The results suggest that the variations in the concentration of the D2-R mRNA in the caudate putamen of the rat brain at different stages of the reproductive cycle are caused by the combination of variable expression of the mRNA per cell and different number of the cells that express the mRNA.

Fig. 1

Scheme of the transverse section of the rat brain (upper), 0.8 mm caudal to bregma, Plate 21 (Paxinos & Watson 1997) in comparison to photo composed of photomicrographs of the rat brain transverse section hybridized with a ³⁵S-labelled riboprobe complementary to the rat D₂-R mRNA, under dark-field microscopy (lower). The silver regions on the upper part of the figure represent the area in the caudate putamen that was measured in our study. The bright dots on the lower part represent the specific hybridization in the striatum region. 3V, 3rd ventricle; AA, anterior amygdaloid area; cc, corpus callosum; cg, cingulum; CpU, caudate putamen; ec, external capsule; f, fornix; GP, globus pallidus; ic, internal capsule; lo, lateral olfactory tract; LV, lateral ventricle; ox, optic chiasm; Pe, periventricular hypoth nucleus; Pir, piriform cortex; RF, rhinal fissure; Sfi, septofimbrial nucleus; SI, substantia innominata; SO, supraoptic nucleus; st, stria terminalis; TS, triangular septal nucleus; Tu, olfactory tubercle; VP, ventral pallidum.

Fig. 2

Photomicrographs of the caudate putamen in rats hybridized with a ³⁵S-labelled riboprobe complementary to the rat D₂-R mRNA (objective magnification 40×, HE), as shown under light-field microscopy (2a upper – OE, 2b upper – DOE2) and the same sections under dark-field microscopy (2a lower – OE, 2b lower – DOE2). The stronger hybridization signal/cell (more than five grains over the nucleus) is seen in 2b. Figure 2(c) shows background hybridization with the ³⁵S-labelled sense riboprobe.

Fig. 3

Number of silver grains which represent the specific hybridization per cell at different phases of the oestrous cycle in the striatum (DOE1, dioestrus 1; DOE2, dioestrus 2; POE, prooestrus; OE, oestrus). Columns show mean values ± standard errors (SEM). Asterisk denotes groups significantly different from group of animals in OE at the 0.05 level.

Fig. 4

Number of cells expressing D₂-R mRNA (more than five silver grains per cell) at different phases of the oestrous cycle in the striatum (DOE1, dioestrus 1; DOE2, dioestrus 2; POE, prooestrus; OE, oestrus). Columns present mean values ± standard errors (SEM). Asterisk denotes groups significantly different from group of animals in OE at the 0.05 level.