DRR1 mRNA expression in the adult mouse brain
DRR1 mRNA is widely expressed in the whole adult mouse brain (Fig. 1; S1; S2; Table 1) with a distinct neuroanatomical expression intensity profile. From anterior to posterior relative to bregma, the strongest DRR1 mRNA expression was detected in the posterior part of the lateral septum; the subfornical organ; the cell bodies of the pyramidal CA3 region of the hippocampus, specifically the CA3a and initial part of CA3b subfield; and cells ensheathing the stalk of the habenular commissure and the Purkinje cell layer of the cerebellum.
Moderate DRR1 gene expression was visible, but not uniformly distributed, throughout the cortex. Especially high expression was detected in the primary somatosensory cortex and the retrosplenial cortex (both granular and agranular part). Interestingly, the cortical layer V (the inner pyramidal layer) displayed lower DRR1 mRNA expression compared to the other cortical layers. Moderate DRR1 expression was also found in the lateral part of the caudate putamen, the amygdala, the mammillary nuclei and several thalamic (ventromedial hypothalamic nucleus and the parafascicular thalamic nucleus) and hypothalamic nuclei.
Only very weak DRR1 mRNA expression was visible in the mesencephalon and the rhombencephalic medulla oblongata and pons.
Whereas weak to strong DRR1 expression was found in neuronal tissue throughout the brain, DRR1 mRNA expression was less observed in white matter structures like the anterior commissure, the corpus callosum, the cerebral peduncle and the optic tract. We can observe a punctate pattern in the high-resolution silver grain-stained DRR1 mRNA images; however, intensity of the signal provided by the autoradiograph is almost undetectable. Interestingly, the expression of DRR1 mRNA was also detected in the choroid plexus and in ependymal cells of the brain ventricles, similar to Fam107B (Masana et al. 2015).
Neuroanatomical distribution of DRR1 protein
The neuroanatomical distribution of DRR1 protein was assessed by immunofluorescence. DRR1 protein could be found throughout all the adult mouse brain, with high expression in specific regions (Table 1; Figs. 2, 3).
The DRR1 protein abundance in the septum nicely followed the pattern of mRNA expression with high DRR1 protein levels in the lateral septum. Moreover, also the caudate putamen, lateral globus pallidus, ventral pallidum as well as the nearby bed nucleus of the anterior commissure and the bed nucleus of the stria terminalis were DRR1 positive (Fig. 2a).
Within the cortex, DRR1 was found throughout all regions and layers of the cortex (Fig. 2b). Immunofluorescence outlines neuronal cell bodies of cortical neurons (arrows in Fig. 2b1) but was also located to the surrounding neuropil. Particularly high density of DRR1 signal could be observed in layer IV of the barrel field, where it appears in barrel like structures (Fig. 2b2). Whereas the mRNA signal was clearly reduced within layer V of the cortex, DRR1 protein is not. Comparable levels of DRR1 protein were also found in the amygdala (Fig. 2b4).
Moreover, strong DRR1 protein expression was present in the hippocampus and the adjacent habenular commissure (Fig. 2c1, c2). High protein levels could be found prominently in CA3 cell bodies but also in the dentate gyrus and the molecular layers. Additionally, cells covering the stalk of the habenular commissure were positive for both DRR1 mRNA and protein (Table 1; Fig. 2c2).
The cerebellum was one of the brain regions with the highest DRR1 protein level. DRR1 protein signal was strong in the Purkinje layer (cell bodies) and molecular layer of the cerebellum (Fig. 2c3). Because very strong mRNA expression of DRR1 was only visible in the Purkinje cell layer, it is likely that most of the DRR1 protein in the molecular layer belongs to Purkinje cell dendrites.
The distribution of DRR1 protein in the thalamus and hypothalamus is summarized in Table 1. Several nuclei showed moderate DRR1 protein levels as indicated. High DRR1 abundance could be observed in the mammillary nuclei while the paraventricular nucleus of the hypothalamus showed only basal levels of DRR1.
Interestingly, most of the circumventricular organs (CVOs) were strongly labeled by DRR1 immunofluorescence (Fig. 3). High levels of DRR1 could be observed in the vascular organ of the lamina terminalis, the area postrema, the subfornical organ and the subcommissural organ, but not the median eminence. A broader definition of CVOs includes also the choroid plexus, which showed prominent DRR1 mRNA and protein (Table 1; Fig. 3g). Also nuclei known to be highly interconnected with the CVOs, the supraoptic nucleus (data not shown) and the median preoptic nucleus (Fig. 2a3), do express DRR1.
DRR1 was prominently but not exclusively located to neurons, as it is found in virtually all neurons but also in non-neuronal tissue like in the cells of the choroid plexus and the ependyma (Fig. 3g, h).
Cellular localization of DRR1 protein
DRR1 protein appeared in a punctate pattern independent of the neuroanatomical region and the intracellular localization (Figs. 2, 3). DRR1 was found in the neuropil as well as in the neuronal somata including the nuclei, where it shows denser signal. DRR1 was also located to cellular specializations as it was found in cilia of ependymal cells (Fig. 3h).
Furthermore DRR1 might be located to both presynaptic and postsynaptic structures (Figure S1). DRR1 co-localize with some of the synapsin and PSD-95 labeled structures. Thus, DRR1 appears to be a putative but not integral part of synaptic structures.
Glucocorticoids increase DRR1 mRNA in grey and white matter of the adult mouse brain
The GR agonist dexamethasone (10 mg/Kg s.c.) increased DRR1 mRNA throughout the whole adult mouse brain, as shown in the representative autoradiographs of [35S]-labeled DRR1 mRNA (Fig. 4a) and densitometric analysis (Fig. 4b).
Under basal conditions, DRR1 mRNA showed maximum expression (in arbitrary units, a.u.) in the hippocampal CA3 region (79 ± 6), followed by the lateral septum (48 ± 3) and the primary somatosensorial cortex-Barrel field (49 ± 6), motor cortex (40 ± 5), striatum (25 ± 2) and with low (almost undetectable) expression in white matter fiber tracts such as the anterior commissure (12 ± 3) and corpus callosum (12 ± 3).
After glucocorticoid receptor activation, DRR1 mRNA levels increased significantly (Student’s t test) in all DRR1-rich regions but also in white matter regions. In detail, dexamethasone treatment increased mRNA expression in grey matter approximately 136% in the hippocampal CA3 region, 186% in the lateral septum, 145% in the primary somatosensorial cortex-Barrel field, 152% in the motor cortex and 195% in the striatum; while in fiber tracts increased 436% in the anterior commissure and 401% in the corpus callosum, reaching similar levels to neighboring areas such as striatum and cortex. Within the hippocampus, we could observe qualitatively increases in all CA3, DG and CA1 pyramidal neurons, and also in the str. Lac/mol and Str. Moleculare, and hilus, but this increase was less evident in the str. Radiatum. Within the cortex, DRR1 mRNA expression increased in all areas, with the somatosensory barrel field area showing the maximum expression. Moreover, the differential expression intensity pattern through the different cortical layers was maintained after dexamethasone treatment, with higher expression in layer IV and low expression in layer V.
Interestingly, DRR1 was also strongly increased in the epithelium surrounding hippocampus, around ventricles and in the choroid plexus.
Glucocorticoids increase DRR1 in the membrane fraction
DRR1 protein levels were increased by dexamethasone (10 mg/kg s.c.) treatment compared to vehicle in the mouse hippocampus, and at greater extent in membrane-rich fractions. Dexamethasone increased total protein expression of DRR1 in the hippocampus (Fig. 5a) (p < 0.0001 vs actin; p < 0.05 vs tubulin) but not in the cerebellum (data not shown) when compared to vehicle-treated mice (n = 6 mice/group).
Subcellular fractioning of hippocampal dissected tissue showed that DRR1 is expressed ubiquitously in the cell and dexamethasone increased DRR1 protein expression significantly in the membrane (F2) (p < 0.05), but not cytosol (F1), nuclei (F3) or cytoskeleton fractions (F4) (Fig. 5b) compared to vehicle-treated mice (n = 6 mice/group). Second subcellular fractioning showed that dexamethasone increased DRR1 in the crude membrane fraction (P2) (p < 0.05), cytosol (S2) (p < 0.05), postsynaptic membrane fraction (Triton-insoluble fraction, TIF) (p < 0.05) and Triton-soluble fraction (TSF) (p < 0.05) [when normalized to tubulin; all of them when normalized to actin (not shown)] (Fig. 5c). In all experiments corticosterone levels were measured and were significantly reduced in dexamethasone-treated mice compared to vehicle: 46.8 ± 10.6 and 0.7 ± 0.3 ng/ml for the total protein/subcellular fraction experiment in Veh and DEX-treated mice, respectively (n = 11–12 mice/group); and 66.6 ± 10.6 and 1.2 ± 1.3 ng/ml for the alternative subfraction procedure in Veh and DEX-treated mice, respectively (n = 16 mice/group). Corticosterone levels were measured and were significantly reduced in dexamethasone-treated mice compared to vehicle (Figure S4).