Neuromedin U Neurons in the Edinger-Westphal Nucleus Respond to Alcohol Without Interfering with the Urocortin 1 Response

Abstract

The Edinger-Westphal nucleus (EW) is a midbrain nucleus composed of a preganglionic, cholinergic subpopulation and a densely clustered peptidergic subpopulation (EWcp). The EWcp is one of the few brain regions that show consistent induction of FOS following voluntary alcohol intake. Previous results in rodents point to urocortin 1 (UCN1) as one of the peptides most involved in the control of ethanol intake and preference. Notably, the functions described for UCN1, such as reward processing, stress coping or the regulation of feeding behavior are similar to those described for the neuropeptide neuromedin U (NMU). Interestingly, NMU has been recently associated with the modulation of alcohol-related behaviors. However, little is known about the expression and functionality of NMU neurons in alcohol-responsive areas. In this study, we used the recently developed Nmu-Cre knock-in mouse model to examine the expression of NMU in the subaqueductal paramedian zone comprising the EWcp. We delved into the characterization and co-expression of NMU with other markers already described in the EWcp. Moreover, using FOS as a marker of neuronal activity, we tested whether NMU neurons were sensitive to acute alcohol administration. Overall, we provided novel insights on NMU expression and functionality in the EW region. We showed the presence of NMU within a subpopulation of UCN1 neurons in the EWcp and demonstrated that this partial co-expression does not interfere with the responsivity of UCN1-containing cells to alcohol. Moreover, we proposed that the UCN1 content in these neurons may be influenced by sex.

Keywords: Alcohol; Edinger–Westphal nucleus; Knock-in mouse model; Neuromedin U; Urocortin 1.

Fig. 1

Neuroanatomical distribution of NMU-producing cells in adult mice brain slices. A Schematic representation of the protocol followed to prepare samples from Nmu-Cre:ZsGreen1 mice for imaging. Distribution of ZsGreen1-expressing cells in coronal slices showing endogenous ZsGreen1 expression in green. B Schematic representation of the intracerebral injection of the viral tracer AAV5-EF1a-DIO-RFP into the EW region of Nmu-Cre mice and the protocol followed to prepare samples for imaging. RFP fluorescent signal was amplified using an anti-mCherry primary antibody and a Cy3-conjugated secondary antibody. The amplified RFP signal is shown in red in coronal slices and C in a sagittal view. Representative images from 1 mouse brain; n = 3 brains were analyzed. Images were acquired with a confocal microscope with a 10 × objective lens. Numbers denote neuroanatomical coordinates relative to Bregma. Scale bar coronal view: 300 µm. Scale bar sagittal view: 1000 µm. EW: Edinger–Westphal nucleus (PrEW) pre-nucleus, RLi: rostral linear nucleus of the raphe, VTA: ventral tegmental area, PAG: periaqueductal grey matter (LPAG) lateral part, Aq: aqueduct, PH: posterior hypothalamus, RFP: red fluorescent protein

Fig. 2

Distribution of NMU-producing cells in the region comprising the PH, EW and DR. Schematic representation of the intracerebral injection of the viral tracer AAV5-EF1a-DIO-RFP into the EW region of Nmu-Cre mice (top). Schematic representations of coronal slices with the region of interest outlined in red (left). RFP fluorescent signal (red) was amplified using an anti-mCherry primary antibody and a Cy3-conjugated secondary antibody. An antibody against TPH2 was used to mark serotoninergic neurons (light blue). Representative images from 1 mouse brain; n = 3 brains were analyzed. Images were acquired with a confocal microscope with a 10 × objective lens. Numbers denote neuroanatomical coordinates relative to Bregma. Scale bar: 300 µm. DAPI (not shown) was used to determine regional boundaries. PH: posterior hypothalamus, EW: Edinger–Westphal nucleus (PrEW) pre-nucleus, RLi: rostral linear nucleus of the raphe, VTA: ventral tegmental area, PBP: parabrachial pigmented nucleus of the VTA, MA3: medial accessory oculomotor nucleus, 3N: oculomotor nucleus, DR: dorsal raphe nucleus (DRD) dorsal part (DRV) ventral part, RFP: red fluorescent protein, TPH2: tryptophan hydroxylase 2

Fig. 3

Characterization of NMU neurons in the EW and VTA using IHC. A–D, top: Schematic representation of a sagittal view of the brain of a Nmu-Cre:ZsGreen1 mouse and Nmu-Cre mouse injected with the viral tracer AAV5-EF1a-DIO-RFP into the EW region. A–D left: Schematic representations of coronal slices with the region of interest outlined in red. Numbers denote neuroanatomical coordinates relative to Bregma. A, B Expression of NMU-producing neurons in the region comprising the EW nucleus. A In green, endogenous ZsGreen1 fluorescence; in red, ChAT, TH and CCK8 (secondary antibody coupled to Cy5). B In red, RFP fluorescent signal amplified with an anti-mCherry primary antibody and a Cy3-conjugated secondary antibody; in green, ChAT, TH and CCK8 (secondary antibody coupled to Alexa Fluor 488). Representative images from 1 mouse brain; n = 3 brains were analyzed. Images were acquired with a confocal microscope with a 20× objective lens. Areas of higher magnification (40×) are outlined in grey (left-bottom). C, D Expression of NMU-producing neurons in the dorsal VTA. C In green, endogenous ZsGreen1 fluorescence; in red, TH (secondary antibody coupled to Cy5). The enlarged images (from left to right) are enclosed in a dashed box. Scale bar: 100 µm (left, middle) and 50 µm (right). (D) In red, RFP fluorescent signal amplified with an anti-mCherry primary antibody and a Cy3-conjugated secondary antibody; in green, TH (secondary antibody coupled to Alexa Fluor 488. Images were acquired with a confocal microscope with a 20× objective lens. The enlarged images (from left to right) are enclosed in a dashed box. Scale bar: 100 µm (left) and 50 µm (right). DAPI (not shown) was used to determine regional boundaries. PrEW: Pre-Edinger–Westphal nucleus, RLi: rostral linear nucleus of the raphe, IF: interfascicular nucleus, VTA: ventral tegmental area, RFP: red fluorescent protein, TH: tyrosine hydroxylase, CCK8: cholecystokinin-8, ChAT: choline acetyltransferase

Fig. 4

Characterization of NMU neurons in the EW using single-molecule fluorescent in situ hybridization (RNAscope®). A RNAscope® for Slc17a6 (blue, VGLU2), Slc32a1 (green, VIAAT) and Nmu (red) mRNA. B RNAscope® for Slc17a6 (blue, VGLU2), Cck (green) and Nmu (red) mRNA. C RNAscope® for Tac1 (blue), Cartpt (green) and Nmu (red) mRNA. Schematic representations of coronal slices with the region of interest outlined in red (A–C, left). Representative images from 1 mouse brain; n = 2 brains were analyzed. Images were acquired with a confocal microscope with a 40× objective lens. Areas of higher magnification are outlined in grey (right-bottom). Numbers denote neuroanatomical coordinates relative to Bregma. Scale bar: 50 µm. DAPI (not shown) was used to determine regional boundaries

Fig. 5

Assessment of Ucn1 mRNA and UCN1 peptide content of NMU neurons in the EWcp area by immunofluorescence and RNAscope® in situ hybridization. Immunofluorescence for UCN1 peptide (red in A and B) in the EWcp of male (A) and female (B) Nmu-Cre:ZsGreen1 mice revealed that a subpopulation of UCN1-immunoreactive cells contained ZsGreen1 also in both sexes. RNAscope® for Ucn1 mRNA (green in C and D) and Nmu mRNA (red in C and D) in male (C) and female (D) wild type mice confirmed the partial co-existence of these at the mRNA level. We saw also some Nmu-expressing cells that were negative for Ucn1 (see insets). In line with this, immunofluorescence for the UCN1 peptide (green in E and F) combined with RNAscope® for Nmu mRNA (red in E and F) revealed that the Nmu mRNA might be present, but it is not restricted to UCN1-containing neurons of the male and female EWcp. Nuclear counterstaining (blue in C–F) was performed with DAPI. Scale bars: 50 µm

Fig. 6

Analysis of FOS expression after acute alcohol exposure in NMU neurons in the EW region. A Schematic representation of the protocol followed to prepare samples from Nmu-Cre:ZsGreen1 for imaging. Coronal slices showing endogenous ZsGreen1 expression in green and FOS in red (secondary antibody coupled to Cy5). A-a Comparison of the FOS+cell count upon vehicle (0,9% NaCl) or ethanol (2,4 mg/kg) injection. Mann Whitney test, U = 0, **p = 0.0022. A-b Nested graph showing rostral, midline and caudal EW FOS+counts as technical replicates per mouse, considering male (M1-6) and female (F1-6) mice separately (nested t test, F = 43.93, ****p < 0.0001). A-c Percentage of ZsGreen1 and FOS co-expressing cells upon vehicle or ethanol injection (Mann–Whitney test, U = 2, **p = 0.0087). A-d The percentage of ZsGreen1 and FOS double-positive cells was influenced by the effect alcohol treatment (F_1,8 = 12.64, p = 0.0075). A-e No differences between groups were observed in the amount of ZsGreen1 + cells (Mann Whitney test, U = 14, p = 0.5563). B Schematic representation of the intracerebral injection of the viral tracer AAV5-EF1a-DIO-RFP into the EW region of Nmu-Cre mice and the protocol followed to prepare samples for imaging. Coronal slices showing RFP fluorescent signal in red (amplified using an anti-mCherry primary antibody and a Cy3-conjugated secondary antibody) and FOS in green (secondary antibody coupled to Cy2). B-a Comparison of the FOS+cell count upon vehicle (0,9% NaCl) or ethanol (2,4 mg/kg) injection. Mann Whitney test, U = 0, **p = 0.0012. B-b Nested graph rostral, midline and caudal EW FOS + counts as technical replicates per mouse, considering male (M1-6) and female (F1-7) mice separately (nested t test, F = 40.98, ****p < .0001). B-c Percentage of RFP and FOS co-expressing cells upon vehicle or ethanol injection (Mann–Whitney test, U = 4, *p = 0.014). B-d The percentage of RFP and FOS double-positive cells was influenced by the effect of sex (F_1,9 = 9.254, p = 0.014) and alcohol treatment (F_1,9 = 22.65, p = 0.001). B-e No differences between groups were observed in the amount of RFP + cells (Mann Whitney test, U = 10, p = 0.1265). B-f Total number of RFP and ZsGreen1 cells confirmed the widespread patter of ZsGreen1 at the level of the EW (Mann–Whitney test, p = 0.0003). EW: Edinger–Westphal nucleus, 3N: oculomotor nucleus, RFP: red fluorescent protein. (A, B) Representative images from 1 mouse brain; n = 3–4 brains per experimental group and 3–4 slices per mouse were analyzed. Images were acquired with a confocal microscope with a 20× objective lens. The region of interest is outlined in white. Numbers denote neuroanatomical coordinates relative to Bregma. Scale bar: 100 µm. DAPI (not shown) was used to determine regional boundaries

Fig. 7

Analysis of UCN1 and FOS content of NMU cells upon alcohol treatment in the EW. Confocal images of triple immunofluorescence preparations A–H illustrate the nuclear FOS immunoreactivity (green) in UCN1-immunopositive (white) neurons that were sometimes positive for red fluorescent protein (RFP, red) referring to AAV5-EF1a-DIO-RFP virus infection upon EW injection in male and female Nmu-Cre mice. Panels E–H are higher magnification images of areas boxed in panels A–D. Histogram I illustrates the number of UCN1 and FOS positive cells in the EWcp. The ratio of UCN1 cells that were also positive for FOS is shown in panel J. Panel K illustrates that the ratio of RFP-expressing UCN1 neurons was not affected by sex or alcohol treatment. The effect of sex influenced the UCN1 specific signal density (SSD) in the EWcp is shown in L. Histogram M illustrates the UCN1 SSD in cells that contained NMU (RFP positive) or were negative for NMU (RFP negative). Scale bars: 50 µm in A–D, 10 µm in E–H