Fig 2: Identification of imprinted gene controls in molecular subtypes of monoaminergic hypothalamic and adrenal cells(A) The schematic summarizes the sympathetic-adrenal-medullary (SAM) and hypothalamic-pituitary-adrenal (HPA) components of the brain-adrenal axis and Ddc allelic expression.(B) The schematic graph shows the logic of the Imprinting-Expression Network (IEN) analysis that matches variance in cell-type marker gene expression to the magnitude of Ddc imprinting effects across bulk RNA-seq replicates to reveal major cell types with imprinting effects.(C) The mosaic plot shows the results of an IEN analysis for linkage of Ddc hypothalamic imprinting effects to the expression of GABAergic (Gaba) versus glutamatergic (Glu) neuron marker genes. Ddc imprinting effects are significantly dependent on neuron class (**p = 0.005, chi-square test), and maternal allele expression is associated with GABA neurons (dashed black line indicates the null hypothesis).(D) The mosaic plot shows the results of an IEN analysis for linkage of Ddc imprinting effects to gene markers of hypothalamic neuron subtypes from published single-cell RNA-seq. Ddc imprinting effects depend significantly on cell type (*p = 0.01, chi-square test). GABA subtypes (orange text) are more associated with maternal allele expression than Glu types (black text).(E) Co-immunolabeling of GABA, EGFP, and V5 in DdcV5/EGFP preoptic area confirms that subsets of hypothalamic GABAergic neurons exhibit maternal Ddc allele expression (white arrow, V5 expression, box indicates magnified cell in right-side images). Other GABA+ cells express both alleles (yellow arrow). Size bars are 10 µm.(F) Unsupervised hierarchical clustering of hypothalamus single-cell RNA-seq (scRNA-seq) data from 17,000 cells shows sets of co-expressed MEGs (red) and PEGs (blue). A set of imprinted genes co-expressed with Ddc reveal parental controls over monoaminergic cells. See Figure S7 for full data.(G and H) Immuno-labeling of DBH (G) and PNMT (H) in the adrenal medulla of DdcV5/EGFP mice reveals subsets of NE- and E-synthesizing cells, respectively, that exhibit paternal allele (blue arrow) versus biallelic expression (orange arrow). All DDC+ cells express DBH.(I) The bar plot shows the percentage of PNMT+ (dark bars) and PNMT- (light bars) adrenal cells that exhibit paternal Ddc allele expression (blue bar) versus expression of both alleles (orange bar).

Fig 3: LC neuronal activity during social interaction with a singing tutor regulates NCM auditory responses and song selectivity to the tutor song.a Schematic drawings of the experimental design and timeline. b Top left: Parasagittal sections of the LC showing that LC neurons expressing Arch-GFP are dopamine beta-hydroxylase (DBH) positive (magenta). Parasagittal section in the NCM showing LC axonal terminals expressing Arch-GFP are DBH positive (magenta; top right) or tyrosine hydroxylase (TH) positive (red) but not GABA positive (blue; bottom left) (scale bars 100 um: top left, 20 um top right, and bottom left). Bottom right: Proportion of Arch-GFP axon terminals that are double-positive to DBH, TH or GABA. Mean response strength (RST) of all BS NCM (c) or TUT selective BS neurons (d) to tutor song playbacks (Playback 1 and 2) or tutor singing with optogenetic inactivation of LC inputs (Opto+LIVE TUT). e Mean d-prime values for TUT over other song stimuli of TUT selective BS neurons before (Playback 1) and after (Playback 2) hearing tutor singing with optogenetic inactivation of LC inputs. The boxes show the 25–75%, the center lines are defined by the median and open squares by the mean. The whiskers include all data points within 1.5 IQR (Interquartile range) and the ‘outsider’ dots are the data points that fall outside the whisker line. Gray areas indicate non-selective responses (-0.5 < d’ value < 0.5). f Raster plots of spiking activity in a TUT selective BS NCM neuron to tutor song playback 1 and 2 or Opto+LIVE TUT (scale bar: 1 s). Inset: spike waveform of the same TUT selective BS neuron (mean ± s.d., scale bars: 0.5 ms horizontal, 0.5 mV vertical). g Proportion of BS NCM neurons that show selectivity to one song or no selectivity (Non-selec.) before (Playback 1) and after (Playback 2) Opto+LIVE TUT. N = 6 (b–e, g), n = 83 (c), n = 7 (d, e), n = 110 (g). BS: broad-spiking neuron, TUT: tutor song, CON1: conspecific song 1, CON2: conspecific song, HET heterospecific song, N: number of birds, n: number of neurons. Mean ± s.e.m. b–d Two-sided Student T-test (c), Two-sided Mann–Whitney Rank Sum Test (d). Source data are provided as a Source Data file. The bird drawings in a were created by Nicolas Baudoin.

Fig 4: Loss of vigilance-dependent BG Ca2+ activation does not contribute to ethanol-induced ataxic motor coordination.a Time course of plasma ethanol concentration following i.p. injection of 1 or 2 g/kg in Slc1a3-CreERT;Ai95, Aldh1l1-CreERT2;Ai95, and Dbh-Cre;Lck-GCaMP6fflox mice (n = 8, respectively). For statistical analysis, Friedman test was used followed by Tukey–Kramer correction. b Upper, representative CatWalk XT raw data where any contact area of the mouse with the track appears green. Lower, paw prints with color-coded paw identity under baseline conditions or at indicated amounts of time following 2 g/kg i.p. ethanol. c Quantification of paw swing speed in Aldh1l1-CreERT2 mice, dependent on time and dosage of ethanol exposure (left, time: F(5, 110) = 4.834; ethanol dosage: F(10,110) = 3.911) and precision of paw positioning (right, time: F(5, 110) = 28.871; ethanol dosage: F(10, 110) = 22.108), mean ± SEM from 8 mice (saline), 8 mice (1.0 g/kg), and 9 mice (2.0 g/kg), three between-subjects groups repeated measures ANOVA followed by Tukey–Kramer correction. d as c; however, the effect of 2 g/kg i.p. ethanol in combination with 0.03 mg/kg i.p. MK912 on paw swing speed (F(3, 18) = 10.226) and on precision of paw positioning (F(3, 18) = 31.480) was tested, mean ± SEM from 7 mice, repeated measures ANOVA followed by Tukey–Kramer correction. e as c; however, the effect of deletion of a1A-adrenergic receptor on sensitivity to 2 g/kg i.p. ethanol was tested, mean ± SEM from 7 mice (Adra1a+/+) and from 6 mice (Adra1a-/-); mixed between (genotype) and within (baseline versus 15 min) subjects ANOVA. n.s. not significant. Source data are provided as a Source Data file.

Fig 5: Ethanol inhibition of vigilance-dependent Ca2+ elevations in NE terminals accounts for loss of BG Ca2+ responsiveness.a Confocal image of 6-week-old Dbh-Cre;Lck-GCaMP6fflox mouse LC stained for eGFP (Lck-GCaMP6f, green) and dopamine ß-hydroxylase (Dbh) (magenta). Three independent experimental repetitions were obtained with similar results. b Upper, In vivo image of Dbh-Cre;Lck-GCaMP6fflox mouse cerebellar molecular layer. Lower, Locations of ROIs. Nineteen independent experimental repetitions were obtained with similar results. c Upper, Pseudocolour plot of ROIs’ Ca2+ dynamics. Green bars, enforced locomotion. Lower, Average Ca2+ response of all ROIs (dark red) and Ca2+ response traces within number-defined ROIs. d Respective pairwise linear Pearson correlation coefficient plots between individual ROIs’ Ca2+ change traces during indicated experimental episodes. e–g Population data representing mean ?F/F10s (e), mean time to peak (f), and mean correlation coefficient (r) (g) among ?F/F traces of individual ROIs. Red symbols, mean ± SEM. Lines between dots support readability. n = 10 mice; repeated measures ANOVA (e F(2, 18) = 29.575; f F(2, 18) = 27.698; g F(2, 18) = 49.194) was followed by Tukey–Kramer correction; n.s. not significant. Source data are provided as a Source Data file.