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. 2022 Apr;27(4):2068-2079.
doi: 10.1038/s41380-022-01439-4. Epub 2022 Feb 18.

Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects

Enrica Montalban  1   2   3   4 , Albert Giralt  1   2   3   5   6   7   8 , Lieng Taing  1   2   3   9 , Evelien H S Schut  10 , Laura F Supiot  10 , Laia Castell  11   12 , Yuki Nakamura  1   2   3 , Benoit de Pins  1   2   3   13 , Assunta Pelosi  1   2   3 , Laurence Goutebroze  1   2   3 , Pola Tuduri  11 , Wei Wang  14   15 , Katrina Daila Neiburga  16   17 , Letizia Vestito  16   18 , Julien Castel  4 , Serge Luquet  4 , Angus C Nairn  19 , Denis Hervé  1   2   3 , Nathaniel Heintz  20 , Claire Martin  4 , Paul Greengard  14 , Emmanuel Valjent  11 , Frank J Meye  10 , Nicolas Gambardella  21 , Jean-Pierre Roussarie  22   23 , Jean-Antoine Girault  24   25   26
Affiliations

Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects

Enrica Montalban et al. Mol Psychiatry. 2022 Apr.

Abstract

Forebrain dopamine-sensitive (dopaminoceptive) neurons play a key role in movement, action selection, motivation, and working memory. Their activity is altered in Parkinson's disease, addiction, schizophrenia, and other conditions, and drugs that stimulate or antagonize dopamine receptors have major therapeutic applications. Yet, similarities and differences between the various neuronal populations sensitive to dopamine have not been systematically explored. To characterize them, we compared translating mRNAs in the dorsal striatum and nucleus accumbens neurons expressing D1 or D2 dopamine receptor and prefrontal cortex neurons expressing D1 receptor. We identified genome-wide cortico-striatal, striatal D1/D2 and dorso/ventral differences in the translating mRNA and isoform landscapes, which characterize dopaminoceptive neuronal populations. Expression patterns and network analyses identified novel transcription factors with presumptive roles in these differences. Prostaglandin E2 (PGE2) was a candidate upstream regulator in the dorsal striatum. We pharmacologically explored this hypothesis and showed that misoprostol, a PGE2 receptor agonist, decreased the excitability of D2 striatal projection neurons in slices, and diminished their activity in vivo during novel environment exploration. We found that misoprostol also modulates mouse behavior including by facilitating reversal learning. Our study provides powerful resources for characterizing dopamine target neurons, new information about striatal gene expression patterns and regulation. It also reveals the unforeseen role of PGE2 in the striatum as a potential neuromodulator and an attractive therapeutic target.

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Conflict of interest statement

Conflict of interest

The authors declare no conflicts of interest.

Figures

Fig.1:
Fig.1:. EGFP-L10a expression and differences in ribosome-associated mRNA expression in the PFC and striatum of D1-TRAP mice.
a. Brain sections from representative TRAP mice showing the location of the cells expressing EGFP-L10a (direct EGFP fluorescence). Upper panel, D1-TRAP mouse, left picture sagittal section (scale bar 1.5 mm), right picture higher magnification of the striatum (scale bar 50 μm) and blow up of a single neuron illustrating cytoplasmic and nucleolar labeling. Lower panel, D2-TRAP mouse, left picture, sagittal section, right picture, coronal section through the striatum (scale bars 1.5 mm). Images are stitched confocal sections. b. Collection of brain tissue samples. Brains were rapidly dissected and placed in a stainless steel matrix (lower left panel) with 0.5 mm coronal section interval, and two thick slices containing the PFC (cyan, 2 mm-thick) and the striatum (3 mm-thick) were obtained. The PFC was cut, and the dorsal striatum (DS, green) and the nucleus accumbens (NAc, light red) were punched out with a metal cannula on ice. Limits of the tissue samples are indicated on sagittal (left panel) and coronal (right panel) sections. c. PCA of RNAseq gene expression assessed in TRAP-purified mRNAs from PFC, DS and NAc of D1- or D2-TRAP mice. Each point corresponds to a sample of tissues from 1-3 mice. d. PCA of RNAseq from the DS and NAc of D1- and D2-TRAP mice. The same plot was differentially colored for DS and NAc samples (left panel) or D1 and D2 samples (right panel). e. Volcano plot showing differential mRNA expression between striatal D1 samples (blue) and D1 samples from PFC (cyan). Names of some top representative mRNAs are indicated (those with low expression levels are in grey). f-g. Main gene ontology (GO) pathways for genes more expressed in PFC than in striatum (f) or more expressed in striatum than in PFC (g). Only the most significant non-redundant pathways are shown. For complete results, see Supplementary Tables 3g, h.
Fig.2:
Fig.2:. Differential ribosome-associated mRNA expression in striatal regions of D1- and D2-TRAP mice.
mRNA was purified by BAC-TRAP from the DS and NAc of D1- or D2-TRAP mice and analyzed by RNAseq. a-b. Volcano plots of the differences in expression patterns between D1 (blue) and D2 (yellow) samples in the DS (a) or the NAc (b). c. Venn diagram of data in a and b showing the number of mRNAs differentially expressed in D1 vs. D2 samples in the NAc (light red) and DS (green). d. Main gene ontology (GO) pathways for genes more expressed in D1 or in D2 neurons in DS, NAc or both, as indicated. Only the most significant non-redundant pathways are shown. For complete results, see Supplementary Tables 7a–f. e, f. Volcano plot of the differences between DS (green) and NAc (red) in D1 (e) and D2 (f) samples. g. Venn diagram of the data in e and f showing the number of mRNAs differentially expressed in DS vs. NAc samples in the D1 (blue) and D2 (yellow) samples. h. Main gene ontology (GO) pathways for genes more expressed in DS or in NAc neurons in D1, D2, or both, as indicated. Only the most significant non-redundant pathways are shown (complete results in Supplementary Tables 12a–f). In a, b, e, and f, the names of top representative mRNAs are indicated (those with low expression levels are in grey). In a-c and e-g thresholds were Padj < 10−3, fold-change > 2 and mean baseMean ≥ 10.
Fig.3:
Fig.3:. Expression of PGE2 receptors in the striatum and cell population-specific effects of PGE2 receptor stimulation.
a-c. Single-molecule fluorescent in situ hybridization for PGE2 receptors in the DS. Sections through the DS of brains from wild-type C57/Bl6 male mice were processed for single molecule fluorescent in situ hybridization. Sections were labeled with probes for PGE2 receptor mRNAs, Ptger1 (a), Ptger2 (b), and Ptger4 (c) in red, and Drd1 (green), and Drd2 (cyan), as indicated, and counterstained with DAPI (gray scale). Ptger1 and Ptger2 are expressed in D1- and D2-SPNs, whereas Ptger4 is mostly in D1. Confocal microscope images, scale bar, 10 μm. d-f. RT-qPCR quantification of Ptger1 (d), Ptger2 (e), and Ptger4 (f) mRNA levels in ribosome-associated mRNA purified from the NAc or DS of D1- and D2-TRAP mice. Quantification by comparative ddCt method using Rpl19 as an internal control (arbitrary units, not comparable from one graph to the other). Note that because of gene overlap with Ptger1 we cannot exclude a contribution of Pkn1 transcripts. g. Examples of immunofluorescence of pSer235-236-rpS6 (blue) in DS sections of mice treated with vehicle (PBS) or misoprostol 30 min before sacrifice. Mice were transgenic for Drd1-tdTomato (red) and D2-TRAP (green) to identify D1- and D2-SPNs. Scale bar, 30 μm. h-i. Quantification of results as in g in D1 and D2-SPNs of NAc (h) and DS (i, n=12, 6 mice per group and 2 areas of interest per mouse). Statistical analysis, 2-way ANOVA (Supplementary Table 19), Holm-Sidak’s multiple comparisons tests, ** p<0.01, **** p<10−4.
Fig.4:
Fig.4:. Effects of PGE2 receptor stimulation on electrophysiological properties of DS D1-SPNs and D2-SPNs neurons.
Male Drd1-Cre x Ai14 tdTomato reporter mice were injected i.p. with vehicle or misoprostol (0.1 mg.kg−1). Thirty minutes later mice were sacrificed, and brain slices were made for patch clamp electrophysiological experiments. D1- and putative D2-SPNs in the dorsomedial striatum were identified based on red fluorescence and morphology and patched. a. In current clamp, incrementally increasing depolarizing currents were injected into the cell, while action potential output was monitored. In D1-SPNs no differences occurred between cells from animals pretreated with vehicle (Veh, ncells=12; nmice=5) or with misoprostol (Miso, ncells=12; nmice=6). Left: representative examples of action potential profiles in response to a depolarizing current injection of 200 pA. Right: Average current-action potential number relationship across cells from the vehicle or misoprostol condition. Two-way repeated measures-ANOVA (RM-ANOVA), misoprostol effect not significant. b. In D2-SPNs misoprostol pre-treatment (ncells=12; mmice=5) compared to vehicle (ncells=12; nmice=5), resulted in a reduction of action potential output (RM-ANOVA, misoprostol effect, p=0.04). c. The rheobase (i.e., the minimal injected current into a neuron required to make it fire an action potential) was not affected by misoprostol pretreatment in D1-SPNs, but was significantly increased by it in D2-SPNs (2-way ANOVA, interaction, p=0.001). d. The resting membrane potential was unaltered by misoprostol in D1-SPNs, but reduced in D2-SPNs (2-way ANOVA interaction, p=0.002). e. Misoprostol reduced the membrane resistance of D2-SPNs (2-way ANOVA misoprostol effect, p=0.037). c-e, multiple comparisons with Holm-Sidak’s test, *p<0.05, ***p<0.001. See Supplementary Table 19 for detailed statistical results.
Fig.5:
Fig.5:. Effects of PGE2 receptor stimulation on DS neurons activity and mouse behavior.
a-c. Misoprostol pretreatment does not alter D1 neurons Ca2+ activity during exploration of a novel environment (new cage). The activity was evaluated by fiber photometry in the DS of Drd1-Cre mice stereotactically injected with an AAV GCaMP6f (Supplementary Fig.10a–c). Each mouse was recorded twice with an interval ≥ 1 day, 30 min after receiving either vehicle (Veh) or misoprostol (Miso, 0.1 mg.kg−1, i.p.). a. Average traces of mice injected with vehicle and placed for 1 min in a novel environment. b. Same as in a for mice injected with misoprostol. c. Plot of the area under the curve (AUC) in a and b during the novel environment exploration (60 s) minus the AUC during baseline (50 s), 10 mice per group. Mann-Whitney test, p = 0.39. d-f. Misoprostol decreases Ca2+ responses to change in environment in D2 neurons. Same experiment as in a-c but in Drd2-Cre mice injected with vehicle (d, n = 10) or misoprostol (e, n = 9). Mann-Whitney test, p = 0.043. g. The effects of PGE2 receptors stimulation on DRD2 function were investigated by evaluating the immobility 45-180 min after haloperidol injection (0.1 mg.kg−1, i.p.), in mice pretreated 15 min before haloperidol with misoprostol (0.1 mg.kg−1, i.p.) or vehicle (9 mice per group). The same experiment was run twice on different groups of mice with results similar to the one shown here. 2-way repeated measures-ANOVA (RM-ANOVA), misoprostol and time effects, both p<10−4. h, i. Effects of chronic misoprostol on procedural learning and reversal. (h) Wild-type male mice were implanted with an i.p. osmopump delivering vehicle (20 mice) or misoprostol (24 mice). Acquisition and reversal of the food-rewarded arm choice in a Y maze was tested 20-25 days later. RM-ANOVA, misoprostol effect, learning phase, not significant, reversal, p=2.10−4. (i) Same as h except that osmopump infusion bilaterally delivered into the DS vehicle (10 mice) or misoprostol (9 mice). 2-way RM-ANOVA, misoprostol effect, learning phase, p=0.003, reversal, p=0.002. g-i, multiple comparison Holmes-Sidak’s tests, *p < 0.05, **p < 0.01, ***p<0.001, **** p<10−4. See Supplementary Table 19 for detailed statistical results.
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