Expression and function of dopamine receptors in the developing medial frontal cortex and striatum of the rat

Abstract

The timeline of dopamine (DA) system maturation and the signaling properties of DA receptors (DRs) during rat brain development are not fully characterized. We used in situ hybridization and quantitative PCR to map DR mRNA transcripts in the medial frontal cortex (mFC) and striatum (STR) of the rat from embryonic day (E) 15 to E21. The developmental trajectory of DR mRNAs revealed distinct patterns of DA receptors 1 and 2 (DRD1, DRD2) in these brain regions. Whereas the mFC had a steeper increase in DRD1 mRNA, the STR had a steeper increase in DRD2 mRNA. Both DR mRNAs were expressed at a higher level in the STR compared with the mFC. To identify the functional properties of DRs during embryonic development, the phosphorylation states of cyclic AMP response element binding protein, extracellular signal-regulated kinase 1/2, and glycogen synthase kinase 3 beta were examined after DR stimulation in primary neuronal cultures obtained from E15 and E18 embryos and cultured for 3 days to ensure a stable baseline level. DR-mediated signaling cascades were functional in E15 cultures in both brain regions. Because DA fibers do not reach the mFC by E15, and DA was not present in cultures, these data indicate that DRs can become functional in the absence of DA innervation. Because activation of DR signal transduction pathways can affect network organization of the developing brain, maternal exposure to drugs that affect DR activity may be liable to interfere with fetal brain development.

Figure 1. Generation of DR probes to measure mRNA transcripts in rat brain

Three non-overlapping RNA probes were generated for both DRD1 and DRD2 and combined for in situ hybridization studies. Each probe (labeled ‘1’, ‘2’, ‘3’) was examined individually in Northern blots using RNA from whole rat brain (A). Arrows indicate the 28S and 18S rRNA bands. Expected size of DRD1 mRNA is approximately 4 kB and DRD2 is approximately 2.7kB (Bunzow et al., 1988; Zhou et al., 1990). The approximate size of the 28S band in rats is 4.8 kB and 18S is 1.9 kB. (B) Schematic of an embryonic rodent brain. For in situ analyses, probes were hybridized to coronal sections taken from the mFC (1) or STR (2). D=dorsal, C=caudal. (C-E) Dissection strategy for primary neuronal cultures, QPCR and Western blots. (C) Dorsal view with the first three cuts (1-3) that removed septum and midbrain. (D) Hemispheres were rotated to a sagittal view from the lateral ventricle onto the inside of the cortex. The part of the hippocampus that was not removed by cuts 2 or 3 was lifted up and FC was cut out as shown (cuts 4 and 5). (E) Coronal view to show how striatum was removed from RH and NC (cut 6). (F) Coronal brain slices from E15 to E21. The lighter colored regions indicate the approximate area of mFC and STR dissected. mFC was dissected rostral to this area, and STR caudal to this area. Abbreviations: FC: frontal cortex; HP, hippocampus; LV, lateral ventricle; NC, neocortex; OB, olfactory bulb; RH, rhinencephalon; SP, septum; ST, striatum. Scale bar 500μm.

Figure 2. In situ hybridization of DRD1 development in rat mFC and STR

Representative photomicrographs of in situ hybridization of coronal sections of embryonic rat brains at E15, E17, E19, and 21. A combination of three DRD1 probes was used to visualize the expression of receptors in mFC (left panel) and STR (right panel). For each time point, a representative antisense slice was placed next to a sense slice to show background levels of hybridization. Boxes indicate the regions in which in situ densitometry was quantitated. Scale bar is 500μm.

Figure 3. In situ hybridization of DRD2 development in rat mFC and STR

Representative photomicrographs of in situ hybridization of DRD2 receptors in coronal sections of embryonic rat brains from E15 to E21. A combination of three DRD2 probes was used to detect receptors in mFC (left panel) and STR (right panel). For each time point, a representative antisense slice was placed next to a sense slice to show background levels of hybridization. Boxes indicate the regions in which in situ densitometry was quantitated. Scale bar is 500μm.

Figure 4. DR mRNA expression measured by in situ hybridization and QPCR

Densitometric analyses of in situ hybridization results for DRD1 and DRD2 reveals significant increases in mFC (A) and STR (B) in a preliminary statistical analysis. Shown are the levels of intensity of antisense probes, normalized to the background signal generated by sense probes from an adjacent section. N=12-17 area measurements in 2 slices per time point. QPCR was used to measure mRNA transcript levels of DRD1 and DRD2 in samples from mFC (C) and STR (D). Values were normalized to the control genes beta actin (ACTB) and 18S rRNA. Inserts: Magnification of expression levels at E15. n=5 samples/time point. *p<0.05, **p<0.01, *** p<0.001 in the comparison of DRD1 to DRD2 at individual time points; mean ± SEM. Brackets around asterisks denote that these data are semiquantitative. Paired t-tests were used in (C) and (D).

Figure 5. DR mRNA expression in mFC and STR neuronal cultures increases over time

The developmental trajectory of mRNA transcript levels of DRD1 and DRD2 was examined in primary culture from mFC (A) and STR (B) plated at E15, and primary culture from mFC at E18 (C). DIV = day in vitro starting 24 hours after plating as DIV 1. Values were normalized to the control genes beta actin (ACTB) and 18S rRNA. Data mean ± SEM; paired t-tests: *p<0.05, **p<0.01; n=3 per time point. Expression levels for both DRs were significantly altered over time in all experiments (see ‘Results’).

Figure 6. DRD1-mediated activation of signal transduction pathways in embryonic neuronal cultures from the rat mFC and STR

Phosphorylation of CREB (A, B), ERK1/2 (C, D), and GSK3β (E, F) was measured in embryonic neurons from mFC (A, C, E) or STR (B, D, F) at E15 and E18 in response to 15-minute treatments with the DRD1 agonist SKF82958. All neurons were cultured for 3 days. Bands were normalized to total protein on the membrane. Representative blots are shown beneath each histogram. Data mean ± SEM; t-tests: * = p<0.05, ** = p<=0.01. N=10-14 per group.

Figure 7. DRD2-mediated activation of signal transduction pathways in embryonic neuronal cultures from the rat mFC and STR

Phosphorylation of CREB (A, B), ERK1/2 (C, D), and GSK3β (E, F) was measured in embryonic neurons from mFC (A, C, E) or STR (B, D, F) at E15 and E18 in response to 15-minute treatments with the DRD2 agonist PPHT. All neurons were cultured for 3 days. Bands were normalized to total protein on the membrane. Representative blots are shown beneath each histogram. Data mean ± SEM; t-tests: * = p<0.05, ** = p<=0.01, *** = p<= 0.001. N=4-12 per group.

Figure 8. PPHT mediated activation of GSK3β is specific for DRD2

Co-treatment of E18 mFC neuronal cultures for 15 minutes with the potent DRD2 agonist PPHT and the DRD1 antagonist SCH23390. Antagonism of DRD1 in conjunction with PPHT did not affect PPHT-mediated activation of GSK3β. Bands were normalized to ß-actin. Representative blots are shown beneath histogram. Data mean ± SEM; t-tests: * = p<0.05. N=3 per group.