Article Figures & Data
Figures
- Figure 1.
Gene expression of a glutamatergic marker in LHb pv-positive neurons. A, The LHb neurons are classified into seven clusters by UMAP. The clusters are color coded as follows: LHb1 is red, LHb2 is yellow, LHb3 is yellowish green, LHb5 is light blue, LHb6 is purple, and LHb7 is pink. B, Scatter plots indicate normalized expressions of pv and vglut2, vglut1, vglut3, gad2, gat, gad1, vgat on the x- and y-axes, respectively (N = 1,538). Plot colors indicate the clusters. LHb, lateral habenular nucleus. pv, parvalbumin; gad1, glutamate decarboxylase 1; gad2, glutamate decarboxylase 2; vgat, vesicular GABA transporter; gat, GABA transporter; vglut1, vesicular glutamate transporter 1; vglut2, vesicular glutamate transporter 2.
- Figure 2.
Heterogeneity of PV neurons in the expression of glutamatergic and GABAergic markers. A, The expression of glutamatergic machinery in PV neurons in the LHb. vglut2 (cyan) and PV (magenta) are double stained (A1). A5–7, The dotted square area of A1. PV neurons (magenta in A2) and the expression of vglut1 (cyan in A3) are shown (merged in A4). PV neurons (A5) and the expression of vglut2 (A6) are shown (merged in A7). Arrows indicate double-positive for PV and vglut2. B, The expression of glutamatergic machinery in PV neurons in the LHb and other brain regions. Densities of PV neurons are compared between the LHb and other brain regions (B1). Percentages of vglut1- and vglut2-positive (B2 and B3, respectively) in PV neurons are compared between the LHb and other brain regions. C, The expression of GABAergic machinery in PV neurons in the LHb. gad2 (cyan) and PV (magenta) cells are double stained with HCR and IHC, respectively (C1). C2–4, The dotted square area of C1. PV neurons (C2) and the expression of gad2 (C3) are shown (merged in C4). Open arrowheads indicate PV neurons. Arrows indicate double positives for PV and gad2. Expressions of gad1 (C5) and vgat (C6) are shown; closed arrowheads indicate the positive neurons in the hippocampus, but not in the LHb. D, Percentages of the GABAergic machinery expression are compared between the LHb and other brain regions. Percentages of gad1-(D1), gad2-(D2), vgat-(D3), and gat-positive PV neurons (D4) are shown. C5, C6, Z-stack composite images with 10 confocal serial optical sections, whereas the others are single-optical sections of confocal images. Scale bars: 100 µm (A1, C1, C5,6), 50 µm (A2–7, C2–4). LHb, lateral habenular nucleus; cingulate Ctx, cingulate cortex; CA3, CA3 region of the hippocampus; BLA, basolateral amygdala; PV, parvalbumin; gad1, glutamate decarboxylase 1; gad2, glutamate decarboxylase 2; vgat, vesicular GABA transporter; gat, GABA transporter; vglut1, vesicular glutamate transporter 1; vglut2, vesicular glutamate transporter 2. N = 5 mice for each group. “A,” “B,” “C,” and “D” are significantly different (Tukey HSD test). For B1, p < 0.001 “A,” “B,” “A”–“C,” p < 0.05 “B,” “C.” For B2, p < 0.001 “A,” “B,” “A”–“C,” “A”–“D,” “B,” “C,” “B”–“D,” and “C,” “D.” For B3, ***p < 0.001. Welch's t test. For D1–4, p < 0.001 “A,” “B,” “A”–“C,” “A”–“D,” “B,” “C,” and “B”–“D.” p < 0.05 “C,” “D.”
- Figure 3.
Mediolateral distribution of glutamatergic and GABAergic PV neurons in the LHb. A, The expression of vglut2 (cyan) in the LHb PV neurons (magenta). PV and vglut2 are detected using IHC and HCR, respectively. The dotted square areas B and C are shown in B1–3 and C1–3, respectively. B, In the medial LHb PV neurons (magenta in B1), the expression of vglut2 (cyan in B2) is shown (merged in B3). C, In the lateral LHb PV neurons (magenta in C1), the expression of vglut2 (cyan in C2) is shown (merged in C3). Open and closed arrowheads indicate PV- and vglut2-positive neurons, respectively. Arrows indicate double-positive for PV and vglut2. All images are single-optical sections. Scale bars: 100 µm (A), 50 µm (B1–3, C1–3). D, Percentages of vglut2-(D1) and vgat-positive PV neurons (D2) are compared between the medial and lateral LHb. N = 5. **p < 0.01. Welch's t test. LHb, lateral habenular nucleus; PV, parvalbumin; vglut2, vesicular glutamate transporter 2; vgat, vesicular GABA transporter.
- Figure 4.
Anteroposterior distribution of glutamatergic and GABAergic PV neurons in the LHb. A, The expression of vglut2 (cyan) in the anterior LHb PV neurons (magenta; A1). PV and vglut2 are detected using IHC and HCR, respectively. The dotted square area of A1 is shown in A2–4. In PV neurons (magenta in A2), the expression of vglut2 (cyan in A3) is shown (merged in A4). B, The expression of vglut2 (cyan) in the posterior LHb PV neurons (magenta; B1). The dotted square area of B1 is shown in B2–4. In the PV neurons (magenta in B2), the expression of vglut2 (cyan in B3) is shown (merged in B4). Open and closed arrowheads indicate PV- and vglut2-positive neurons, respectively. Arrows indicate double positive for PV and vglut2. All images are single-optical sections. Scale bars: 100 µm (A1, B1), 50 µm (A2–4, B2–4). C, Percentages of glutamatergic and GABAergic machinery expression are compared between the anterior and posterior LHb. Percentages of vglut2-(C1), vgat-(C2), gad1-(C3), gad2-(C4), and gat-positive PV neurons (C5) are compared between the anterior and posterior LHb. N = 5. *p < 0.05; ***p < 0.001. Welch's t test. LHb, lateral habenular nucleus; PV, parvalbumin; vglut2, vesicular glutamate transporter 2; vgat, vesicular GABA transporter.
- Figure 5.
Coexpression of glutamatergic and GABAergic machinery in the PV neurons. A, PV (blue), vglut2 (cyan), and gad2 (magenta) are triple stained in the LHb using IHC and HCR (A1). The dotted square area of A1 is shown in A2–5. In PV neurons (blue in A2), the expression of vglut2 (cyan in A3) and gad2 (magenta in A4) is shown (merged in A5). Arrows indicate triple positive for PV, vglut2, and gad2. B, PV (blue), vglut1 (cyan), and gad1 (magenta) are triple stained in the cingulate cortex using IHC and HCR (B1). The dotted square area of B1 is shown in B2–5. In PV neurons (blue in B2), the expression of vglut1 (cyan in B3) and gad1 (magenta in B4) is shown (merged in B5). Arrows indicate triple positive for PV, vglut1, and gad1. All images are single-optical sections. Scale bars: 100 µm, (A1, B1). 50 µm, (A2–5, B2–5). C, Percentage of vglut2, gad2, and double-positive PV neurons in the LHb. N = 4. LHb, lateral habenula; PV, parvalbumin; vglut1, vesicular glutamate transporter 1; vglut2, vesicular glutamate transporter 2; gad1, glutamate decarboxylase 1; gad2, glutamate decarboxylase 2.
Tables
Numbers Probes Target gene HCR amplifier Fluorophore 1 vglut1 Slc17a7 B1 Alexa 594 B4 Alexa 488 2 vglut2 Slc17a6 B1 Alexa 594 B4 Alexa 488 3 vglut2(set1) Slc17a6_set1 B1 Alexa 594 4 vglut2(set2) Slc17a6_set2 B4 Alexa 488 5 vglut3 Slc17a8 B4 Alexa 488 6 gad1 Gad1 B1 Alexa 594 7 gad2 Gad2 B4 Alexa 488 8 gad2(set1) Gad2_set1 B1 Alexa 594 9 gad2(set2) Gad2_set2 B4 Alexa 488 10 vgat Slc32a1 B1 Alexa 594 11 parvalbumin Pvalb B4 Alexa 488 12 serotonin transporter Slc6a4 B1 Alexa 594 13 gat Slc6a1 B1 Alexa 594 14 enhanced green fluorescent protein EGFP B1 Alexa 594 15 choline acetyltransferase ChAT B4 Alexa 488 16 tyrosine hydroxylase Th B4 Alexa 488 The DNA probe set size, HCR amplifier, fluorophore, and for each target are shown.
Figure 2-1
Heterogeneity of PV neurons in the expression of glutamatergic markers in the cingulate cortex, the hippocampus CA3, and the basolateral amygdala. A The expression of glutamatergic machinery in PV neurons in the cingulate cortex. vglut1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (A1). A2-7 shows the dotted square area of A1. PV neurons (magenta in A2) and expression of vglut1 (cyan in A3) are shown (merged in A4). PV neurons (magenta in A5) and expression of vglut2 (cyan in A6) are shown (merged in A7). Arrows indicate double-positive for PV and vglut1. B The expression of glutamatergic machinery in PV neurons in the hippocampus (CA3 area). vglut1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (B1). B2-7 shows the dotted square area of B1. PV neurons (magenta in B2) and expression of vglut1 (cyan in B3) are shown (merged in B4). PV neurons (magenta in B5) and expression of vglut2 (cyan in B6) are shown (merged in B7). Arrows indicate double-positive for PV and vglut1. C Expression of glutamatergic machinery in PV neurons in the basolateral amygdala. vglut1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (C1). C2-7 shows the dotted square area of C1. PV neurons (magenta in C2) and expression of vglut1 (cyan in C3) are shown (merged in C4). PV neurons (magenta in C5) and expression of vglut2 (cyan in C6) are shown (merged in C7). Arrows indicate double-positive for PV and vglut1. All images are single optical sections of confocal images. Scale bars: 100 µm (A1, B1, C1), 50 µm (A2-7, B2-7, C2-7). PV, parvalbumin. vglut1, vesicular glutamate transporter 1. vglut2, vesicular glutamate transporter 2. Download Figure 2-1, TIF file.
Figure 2-2
Heterogeneity of PV neurons in the expression of GABAergic markers in the cingulate cortex, hippocampus CA3, and basolateral amygdala. A The expression of GABAergic machinery in PV neurons in the cingulate cortex. gad1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (A1). A2-4 shows the dotted square area of A1. PV neurons (magenta in A2) and expression of gad1 (cyan in A3) are shown (merged in A4). PV neurons (magenta in A5) and expression of gad2 (cyan in A6) are shown (merged in A7). PV neurons (magenta in A8) and expression of vgat (cyan in A9) are shown (merged in A10). Open arrowheads indicate PV neurons. Arrows indicate positive for gad1, gad2, or vgat in PV neurons. Closed arrowheads indicate vgat positive neurons. B The expression of GABAergic machinery in PV neurons in the hippocampus (CA3 area). gad1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (B1). B2-4 shows the dotted square area of B1. PV neurons (magenta in B2) and expression of gad1 (cyan in B3) are shown (merged in B4). PV neurons (magenta in B5) and expression of gad2 (cyan in B6) are shown (merged in B7). PV neurons (magenta in B8) and expression of vgat (cyan in B9) are shown (merged in B10). Open arrowheads indicate PV neurons. Arrows indicate positive for gad1, gad2, or vgat in PV neurons. Closed arrowheads indicate gad1 or gad2 positive neurons. C The expression of GABAergic machinery in PV neurons in the basolateral amygdala. gad1 (cyan) and PV (magenta) are double-stained with HCR and IHC, respectively (C1). C2-4 shows the dotted square area of C1. PV neurons (magenta in C2) and expression of gad1 (cyan in C3) are shown (merged in C4). PV neurons (magenta in C5) and expression of gad2 (cyan in C6) are shown (merged in C7). PV neurons (magenta in C8) and expression of vgat (cyan in C9) are shown (merged in C10). Open arrowheads indicate PV neurons. Arrows indicate positive for gad1 or vgat in PV neurons. All images are single optical sections of confocal images. Scale bars: 100 µm (A1, B1, C1), 50 µm (A2-10, B2-10, C2-10). PV, parvalbumin. gad1, glutamate decarboxylase 1. gad2, glutamate decarboxylase 2. vgat, vesicular GABA transporter. Download Figure 2-2, TIF file.
Figure 2-3
A CSV file of extended data table supporting Figure 2. Download Figure 2-3, XLSX file.
Figure 3-1
Mediolateral distribution of GABAergic pv mRNA positive neurons in the LHb. A PV-protein (magenta), pv-mRNA(green), and vgat (cyan) are triple-stained with IHC and HCR in the LHb (A1). The dotted square area of A1 is shown in A2-5. In PV-protein positive neurons (magenta in A2), pv-mRNA positive neurons (green in A3), and expression of vgat (cyan in A4) is shown (merged in A5). Open arrowheads indicate PV-protein positive neurons. Arrows indicate triple-positive for PV-protein, pv-mRNA, and vgat. Closed arrowheads indicate pv-mRNA positive neurons. B Percentages of vgat expression in the pv-mRNA positive neurons are compared between the medial and lateral LHb (B1). The pv-mRNA positive neurons are counted, and the vgat positive neurons among them are counted in the medial (B2) and lateral LHb (B3). The percentages of vgat positive neurons in the pv-mRNA positive neurons were significantly higher in the lateral LHb than those in the medial (vgat: 22.03 ± 3.53% in lateral and 0 ± 0% in medial, p = 0.02, Welch's t-test, HCR, B1). In the medial LHb, 102 pv-mRNA positive neurons were observed; among them, no neurons expressed vgat (B2). In contrast, 63 pv-mRNA positive neurons were observed; among them, the 14 neurons expressed vgat in the lateral LHb (B3). All images are single optical sections. Scale bars: 100 µm (A1), 50 µm (A2-5). LHb, lateral habenular nucleus. PV, parvalbumin (protein). pv, parvalbumin (mRNA). vgat, vesicular GABA transporter. N = 3 mice. *p < 0.05. Welch's t-test. Download Figure 3-1, TIF file.
Figure 3-2
A CSV file of extended data table supporting Figure 3. Download Figure 3-2, XLSX file.
Figure 4-1
A CSV file of extended data table supporting Figure 4. Download Figure 4-1, XLSX file.
Figure 5-1
A CSV file of extended data table supporting Figure 5. Download Figure 5-1, XLSX file.
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