Article Figures & Data
Figures
- Figure 1.
Experimental timeline depicting treatment and locomotor activity assessment schedules. NAc, nucleus accumbens; K, ketamine; IP, immunoprecipitation, PRO, proestrus; DI, diestrus.
- Figure 2.
Sex-dependent sensitivity to the locomotor-activating effects of intermittent ketamine. A–C, Activity counts in males (panel A; n = 26 0.0 mg/kg; n = 45 10.0 mg/kg; n = 20 20 mg/kg), proestrus females (panel B; n = 21 0.0 mg/kg; n = 21 10.0 mg/kg; n = 14 20.0 mg/kg), and diestrus females (panel C; n = 23 0.0 mg/kg; n = 21 10.0 mg/kg; n = 14 20.0 mg/kg) across the six intermittent ketamine injections show development of sensitization to ketamine in females—but not males—at 10.0 and 20.0 mg/kg doses. D, Dose-response comparisons within male and female groups of rats show similar dose-dependent increases in activity following a single ketamine injection. E, F, Within-dose activity comparisons after the first (panel E) and sixth (panel F) injections demonstrate greater locomotor stimulant response in females compared with males following ketamine injections yet similar baseline activity after vehicle administration. Data are depicted as means ± SEMs; (A–C) *p < 0.05; **p < 0.005; ***p < 0.0005 versus first injection using Šídák's post hoc; (D) ***p = 0.0003 versus 0.0 mg/kg (diestrus); **p < 0.005; ****p < 0.0001 versus 0.0 or 10.0 mg/kg ketamine using Tukey's post hoc. E, F, *p = 0.0135; **p < 0.003; ***p = 0.0001; ****p < 0.0001 versus males using Tukey's post hoc. All statistical analyses corresponding to this figure are presented in Extended Data Figure 2-1.
- Figure 3.
Temporal patterns of locomotor response to repeated ketamine across sex and estrous cycle. A, Only males treated with 20.0 mg/kg ketamine exhibited increased locomotor activity 5 min after the sixth compared with first injection, with activity levels otherwise similar between first and last injections in all male treatment groups. B, C, Increased activity during the sixth compared with first sessions was most notable from 5 to 30 min post-treatment in proestrus (panel B) and diestrus (panel C) females treated with 10.0 mg/kg (middle) or 20.0 mg/kg (bottom) ketamine. D–F, AUC values generated from time-binned activity plots confirm the absence of locomotor sensitization in males (panel D) regardless of dose and sensitized locomotor response by the sixth injection in proestrus (panel E) and diestrus (panel F) females treated with either 10.0 or 20.0 mg/kg ketamine. Data are expressed as means ± SEMs; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus Injection/Session 1 using Šídák's post hoc. All statistical analyses corresponding to this figure are presented in Extended Data Figure 2-1. Between-dose comparisons of time-binned activity from sessions 1 and 6 are shown in Extended Data Figure 3-1.
- Figure 4.
Validation of RiboTag viral expression and isolation of NAc D1- and D2-MSNs in female rats. A, Representative image of EYFP (AAV-DIO-RiboTag) expression in the NAc of D1-Cre and D2-Cre rats. B, Cell-type-specific enrichment of ribosome-associated mRNA for known MSN subtype genes. Data represent the fold change of IP relative to input samples isolated from the NAc of D1-Cre (n = 3) and D2-Cre (n = 2) rats, where all values were normalized to Actb. C, Normalized read coverage of the Drd1 and Drd2 gene loci in IP samples from D1-Cre and D2-Cre rats. D, Heatmap of the rlog-transformed read counts of the 200 genes with the most reads counts. Genes are depicted in rows, whereas samples (D1-Cre or D2-Cre IP) are depicted in columns. E, A volcano plot depicting the log2 fold change (x-axis) against the −log10 of the uncorrected p value (y-axis) for each gene. Differentially expressed genes are shown in red, and the 15 genes with the lowest adjusted p values biased in D1- or D2-Cre IP samples are labeled. Data are depicted as means + SEMs; *p < 0.05; **p < 0.01 D2 versus D1, unpaired t test. a.c., anterior commissure. A full list of results for the differential expression analysis is available in Extended Data Figure 4-1.
- Figure 5.
Estrous cycle influence on the cell-type-specific pattern of gene expression in the NAc at baseline. A, The number of DEGs between D2- and D1-Cre IP samples common or distinct between proestrus (Pro) and diestrus (Di) is depicted. In the common genes, the direction of change was consistent between cycle stages: upregulated (D2-bias, blue arrows) or downregulated (D1-bias, red arrows). B, The functional enrichments in the common set of genes (Pro & Di) reveal differences in the ontologies of the biological processes biased in each MSN subtype. C, D, The DEGs specific to each estrous cycle stage are enriched in distinct KEGG terms (panel C) and biological process ontologies (panel D), highlighting the uniqueness in subtype-specific regulations by the estrous cycle. In all panels, the circle size depicts the number of associated genes. ***p < 0.001, hypergeometric test for the overlap between two sets of genes. A full list of results for the differential expression analysis is available in Extended Data Figure 4-1. Functional enrichment analyses are reported for all comparisons related to this figure in Extended Data Figure 5-1.
- Figure 6.
Estrous cycle influence on the cell-type-specific effects of ketamine. A, B, Intermittent ketamine treatment results in a greater number of DEGs in proestrus (Pro, panel A) than diestrus female rats (Di, panel B), and primarily involves D1-MSNs. Volcano plots (bottom) depict the log2 fold change (x-axis) against the −log10 of the uncorrected p value (y-axis) for each gene. Differentially expressed genes are shown in red, and up to five genes with the lowest adjusted p values up- or downregulated by ketamine are labeled. C, Most genes regulated by ketamine in D1-Cre IP samples in proestrus are distinct from those affected in diestrus (left side); their functional enrichment is displayed on the right side, with enriched terms depicted as pink diamonds, and genes as green circles. *p < 0.05, hypergeometric test for the overlap between two sets of genes. All results from differential expression and functional enrichment analyses related to this figure are available in Extended Data Figures 6-1 and 6-2, respectively.
Extended Data
Figure 2-1
Behavioral statistical analysis tables. Download Figure 2-1, XLS file.
Figure 3-1
Dose comparison of temporal locomotor response patterns for first and last ketamine treatments within sex and estrous cycle stage. A, Males (top), proestrus females (middle), and diestrus females (bottom) exhibit dose-dependent increases in activity most notably during the first 25 min after a single 10.0 (light blue, pink asterisks) or 20.0 (dark blue, red asterisks) mg/kg ketamine injection compared to vehicle. B, Between-dose comparisons after the sixth ketamine injection in males (top), proestrus females (middle) and diestrus females (bottom) demonstrate extended increases in activity compared to saline across the hour-long session. Data are expressed as means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0.0 mg/kg; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, 20.0 mg/kg vs. 10.0 mg/kg; Tukey’s multiple comparisons. Download Figure 3-1, TIF file.
Figure 4-1
Differential expression analysis of genes isolated from D1- versus D2-MSNs in the nucleus accumbens of either all saline-treated females (combined), proestrus females only, or diestrus females only. Download Figure 4-1, XLS file.
Figure 5-1
Functional enrichment analyses of genes differentially expressed in nucleus accumbens D1- versus D2-MSNs in either proestrus or diestrus female rats. Download Figure 5-1, XLS file.
Figure 6-1
Differential expression analysis for cell-type-specific effects of repeated ketamine treatment on gene expression in D1- and D2-MSNs in the nucleus accumbens of female rats treated either during proestrus or diestrus stages of the estrous cycle. Download Figure 6-1, CSV file.
Figure 6-2
Functional enrichment analysis of genes differentially expressed in D1-MSNs following repeated ketamine treatment in proestrus female rats. Download Figure 6-2, CSV file.
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