Article Figures & Data
Figures
Visual Abstract
- Figure 1.
Distribution of ACh neurons in the mouse brain at E15 and E18. 50 µm horizontal brain sections of mice expressing tdTomato in ACh neurons at E15 and E18 were stained for DsRed. Sections were stained for DsRed to enhance tdTomato signal (magenta) and Hoechst 33342 (white). Brain regions were identified according to horizontal brain atlases of the prenatal mouse brain (Schambra, 2008). A, This dorsal E15 slice contains ventricular and subventricular zone (SVZ), lateral ganglionic eminence (LGE, striatal neuroepithelium), lateral ventricle (LV), caudate putamen (CPu), third ventricle (3 V), and cranial nerve 3 (CN III). Insets show ACh neurons within the CPU, and the lateral migratory stream (LMS), depicting potentially migratory ACh neurons. B, A more ventral slice contains medial septal and lateral septal nuclei (MS/LS), stria terminalis (st), 3 V, and CPu. As shown in the insets, ACh neurons were detected in the caudal CPu and MS. C, A more ventral slice contains ACh neurons in the globus pallidus (GP), striatum, and cranial nerve 5 (CN V). D, A more ventral slice contains ACh neurons in the GP/nucleus basalis of Meynert (NB), the vertical limb of the diagonal band of Broca (DBB), and the olfactory tubercle (OT). E, The most ventral slice contains ACh neurons in OT and basal forebrain nuclei (BF). F, A dorsal E18 slice contains brain regions VZ/SVZ, LV, corpus callosum (CC), and hippocampus (hc). G, More ventral, ACh neurons are cholinergic in the CPU and MS/LS. H, A more ventral slice contains cholinergic neurons in the MS/LS, GP/NB, and CPu. I, In a more ventral slice, additional cholinergic neurons are located in the DBB, nucleus accumbens (NAc), supramammillary nucleus (SuM), and CN V. The anterior commissure (ac) separates ventral and dorsal striatum. J, The most ventral slice contains additional cholinergic neurons in BF, OT, and the lateral hypothalamus (LH). See Extended Data Figures 1-2 and 1-3 for more details.
- Figure 2.
Several ACh neuron populations are cholinergic in the neonatal mouse brain. A–E, 100 µm sagittal brain sections of mice expressing tdTomato in ACh neurons were obtained at P1 (A), P6 (B), P12 (C), P50 (D), and 1 year of age (E). Sections were imaged for tdTomato (magenta) and DAPI (white) signals using a confocal laser scanning fluorescence microscope. These brain regions were identified: caudal brain nuclei in the brainstem and midbrain (left), striatum (second left), basal forebrain (second right), hypothalamus (right). Insets depict magnified ROIs outlined by the small white square. At P1, ACh neurons are present in the striatum, basal forebrain, hypothalamus, and caudal brain nuclei. Abbreviations: Ctx, cerebral cortex; cc, corpus callosum; NAc, nucleus accumbens; OT, olfactory tubercle; DBB, diagonal band of Broca; MS, medial septum; 3V, third ventricle; cbl, cerebellum; V, CN V; VII, CN VII; Amb, nucleus ambiguus; Cu, cuneate nucleus; PG, pontine gray; LDT, laterodorsal tegmentum; Thal, thalamus; PH, posterior hypothalamic nucleus; SUM, supramammillary nucleus; MM, medial mammillary nucleus.
- Figure 3.
Several ACh neuron populations are not cholinergic in the neonatal mouse brain but are found cholinergic at later ages. A–E, Brain sections of the mice from Fig. 1 were assessed for tdTomato fluorescence at P1 (A), P6 (B), P12 (C), P50 (D), and 1 year (E) in these brain regions: thalamus (left), cerebellum (second left), cerebral cortex (second right), and hippocampus (right). Insets depict magnified ROIs outlined by the small white square. At P1, ACh neurons are absent in the thalamus, cerebellum, cerebral cortex, and hippocampus. At P6, ACh neurons are present in the thalamus and cerebellum. At P12, P50, and 1 year, ACh neurons were present in all brain regions. Abbreviations: Ctx, cerebral cortex; cc, corpus callosum; 3V, third ventricle; hc, hippocampus; Thal, thalamus; mHab, medial habenula.
- Figure 4.
Cholinergic innervation of major brain regions changes substantially in the postnatal mouse brain between P3 and P50. A–E, Horizontal 50 µm brain sections were stained for DsRed to enhance tdTomato signal and better visualize cholinergic projections in the cerebral cortex (primary somatosensory cortex S1, A), prefrontal cortex (prefr. ctx, area 24, area 32, medial orbitofrontal cortex MO, B), hippocampus (C), CPu (D), and cerebellum (E). A, B, At P3, cholinergic fibers are dense in L6, sparser in L1, and sparsest in intermediate L2–5. At P6, cholinergic fibers are dense in deep and superficial cortical layers but sparse in intermediate cortical layers. At P9–P12, intermediate layers become more densely innervated but there remain gaps in innervation between individual layers. At P50, gaps in cholinergic innervation are closed. Borders of cortical layers L1, L2–4, L5, and L6 are indicated by dotted lines. C, Hippocampal cholinergic innervation is sparse at P3–P9 compared with P12–P50. D, In the dorsal striatum, there are areas which are avoided by cholinergic fibers. This compartmentalization becomes more pronounced from P12 to P50. E, In the cerebellum, cholinergic innervation is absent at P3 and at P6 sparse with little organization. Throughout development, fiber bundles and cholinergic terminals become better organized as well as structurally complex. Color coding corresponds to fluorescence intensity with red for low and white for high intensity, respectively. Insets depict magnified ROIs outlined by the small white square. Abbreviations: cc, corpus callosum. See Extended Data Figures 4-1–4-4 for more details.
- Figure 5.
ACh neuron populations become cholinergic at different times during development. Reconstructions of serial sectioned brains were collected at E12 (A), E15 (B), E18 (C), P1 (D), P3 (E), P6 (F), P9 (G), P12 (H), P50 (I), and >1 year of age (J). For better visibility, diagrams of ACh populations were added color-coded by brain regions: transparent dark purple, hippocampus; light blue, striatum; transparent light green, cerebral cortex; orange, thalamus; light gray, basal forebrain; transparent light pink, cerebellum; yellow, caudal brain nuclei; light purple, spinal cord; green, hypothalamus. Abbreviations: tel, telencephalon; MB, midbrain; OB, olfactory bulb. For clarity, the striatum, cerebral cortex, and hippocampus were outlined in only one hemisphere per brain. All brains viewed from the top except for E12 (side view). ACh neuron populations in the forebrain at E15 and E18 were added according to findings from tdTomato enhancement staining. See Movies 1–10 for rendered view from all sides. See Extended Data Figures 5-1 and 5-2 for more details.
- Figure 6.
Gross morphology of cholinergic nuclei in the forebrain and caudal brain change little during postnatal development. Reconstructions of serial sectioned brains collected at P1 and P50 were assessed for changes in individual nuclei in the forebrain and caudal brain during development. For better visibility, ACh populations were color-coded by individual brain nuclei in one hemisphere. A–D, Diagram of ACh neurons in forebrain and striatal nuclei (view from outside (A, B), view from midline (C, D). E–H, Diagram of ACh neurons in the midbrain, pons, and medulla [view from outside (E, F), view from midline (G, H)]. Abbreviations: CPu, caudate putamen; NAc, nucleus accumbens; OT, olfactory tubercle; GP, globus pallidus; VP, ventral pallidum; MS/LS, medial/lateral septal nuclei; DBB, diagonal band of Broca; NBM, nucleus basalis of Meynert; SI, substantia innominata; CN III/IV, cranial nerve III/IV; CN V, cranial nerve V; CN VI, cranial nerve VI; CN VII, cranial nerve VII; CN X, cranial nerve X; CN XII, cranial nerve XII; PBG, parabigeminal nucleus; Amb, nucleus ambiguus; Cu/Gr, cuneate/gracile nucleus; PG, pontine gray; PPN, pedunculopontine nucleus; LDT, laterodorsal tegmentum; NLL, nucleus of the lateral lemniscus. See Movies 11–12 for rendered view from all sides.
Tables
Target Donor species Dilution Vendor Conjugate Catalog # RRID ChAT Goat 1:100 Millipore N/A AB144P-200UL RRID:AB_90661 DsRed Rabbit 1:500 Rockland N/A 600-401-379 RRID:AB_2209751 NeuN Rabbit 1:500 Abcam N/A ab177487 RRID:AB_2532109 Goat IgG Donkey 1:500 Jackson ImmunoResearch Alexa Fluor 647 705-605-147 RRID:AB_2340437 Rabbit IgG Goat 1:500 Thermo Fisher Scientific Alexa Fluor Plus 647 A32733 RRID:AB_2633282 - Table 2.
Time points at which ACh neurons are cholinergic in the mouse brain in brain anatomical structures
Brain region E12 (n = 3) E15a (n = 3) E18a (n = 5) P1 (n = 3) P3 (n = 3) P6 (n = 3) P9 (n = 3) P12 (n = 3) P50 (n = 3) >1 year (n = 3) Spinal cord ++ ++ ++ nd nd nd nd nd nd nd Hindbrain CN ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Midbrain CN − ++ ++ ++ ++ ++ ++ ++ ++ ++ Other hindbrain nuclei − + ++ ++ ++ ++ ++ ++ ++ ++ Other midbrain nuclei − + ++ ++ ++ ++ ++ ++ ++ ++ Striatum − +b ++ ++ ++ ++ ++ ++ ++ ++ Basal Forebrain − + ++ ++ ++ ++ ++ ++ ++ ++ Hypothalamus − − + + ++ ++ ++ ++ ++ ++ Thalamus − − − +c + ++ ++ ++ ++ ++ Cerebellumd − − − − − + ++ ++ ++ ++ Cerebral Cortex − − − − − − + ++ ++ ++ Hippocampus − − − − − − + ++ ++ ++ Estimation of cholinergic neuron distribution patterning: −, not cholinergic; +, cholinergic but different from adult; ++, adult-like.
↵a The results of the serial section brain reconstructions have been revised according to the tdTomato enhancement stains.
↵b High cell density in caudal and ventral areas but low density in rostral and dorsal areas of the CPu.
↵c All brains with low intensity tdTomato-positive population in some but not all brain slices.
↵d This structure does not contain ACh neuron soma, assessment reflects innervation.
See also Extended Data Figures 5-1 and 5-2.
Table 1-1
Animals used for experiments depicted in different figures. Download Table 1-1, DOC file.
Table 2-1
Data table for mean fluorescence intensity for cortical layers at P3, P6, and P9 (refers to Fig 4 and Extended Data Fig 7). Download Table 2-1, DOC file.
Fig 1-1
ACh neurons are reliably labeled throughout the brain using the ChAT-IRES-Cre x Ai14 tdTomato mouse model. A-I, 50 µm horizontal brain sections of mice expressing tdTomato in ACh neurons were obtained at P50 and immunostained for ChAT. Sections were imaged for tdTomato (green) and ChAT (magenta) signals using a confocal laser scanning fluorescence microscope. Maximum intensity projections of ∼50 µm z stacks of brain regions imaged with the confocal microscope include: caudate putamen (CPu, A), laterodorsal tegmentum and pedunculopontine nucleus (LDT/PPN, B), cranial nerve V (CN V, C), cranial nerve X and XII (CN X/XII, D), cerebral cortex (E), lateral septal complex (LS, F), medial septum and diagonal band of Broca (MS/DBB, G), and nucleus basalis of Meynert and globus pallidus external (NB/GP, H). Insets depict magnified ROIs outlined by the small white square. I, Mean ratio ± standard deviation of tdTomato- and ChAT-positive cell overlap per brain region. Numbers inside the bar graphs indicate individual z-stacks counted. Download Fig 1-1, TIF file.
Fig 1-2
Distribution of ACh neurons compared to neuron marker NeuN in the mouse brain at E15. A-H, 50 µm horizontal brain sections of mice expressing tdTomato in ACh neurons at E15 were immunostained for NeuN. Sections were imaged for tdTomato (green) and NeuN (magenta). Brain regions imaged include: CPu (A), LDT/PPN (B), CN V (C), cranial nerve III/IV (CN III/IV, D), NB/GP (E), cortical plate (F), cerebellum (cbl, G), and hippocampus (hc, H). Insets depict magnified ROIs outlined by the small white square. Download Fig 1-2, TIF file.
Fig 1-3
Distribution of ACh neurons compared to neuron marker NeuN in the mouse brain at E18. A-H, 50 µm horizontal brain sections of mice expressing tdTomato in ACh neurons at E18 were immunostained for NeuN. Sections were imaged for tdTomato (green) and NeuN (magenta). Brain regions imaged include: CPu (A), LDT/PPN (B), CN V (C), CN III/IV (D), NB/GP (E), cortical plate (F), cerebellum (G), and hippocampus (H). Insets depict magnified ROIs outlined by the small white square. Download Fig 1-3, TIF file.
Fig 4-1
Distribution of ACh neurons compared to neuron marker NeuN in the mouse brain at P3. A-H, Brain sections obtained at P3 were assessed as described in Figure 1-2. Imaged brain regions include: CPu (A), LDT/PPN (B), CN V (C), CN III/IV (D), NB/GP (E), S2/AuV (F), cerebellum (G), and hippocampus (H). Insets depict magnified ROIs outlined by the small white square. Download Fig 4-1, TIF file.
Fig 4-2
Distribution of ACh neurons compared to neuron marker NeuN in the mouse brain at P6. A-H, Brain sections obtained at P6 were assessed as described in Figure 1-2. Imaged brain regions include: CPu (A), LDT/PPN (B), CN V (C), CN III/IV (D), NB/GP (E), S2/AuV (F), cerebellum (G), and hippocampus (H). Insets depict magnified ROIs outlined by the small white square. Download Fig 4-2, TIF file.
Fig 4-3
Distribution of ACh neurons compared to neuron marker NeuN in the mouse brain at P12. A-H, Brain sections obtained at P12 were assessed as described in Figure 1-2. Imaged brain regions include: CPu (A), LDT/PPN (B), CN V (C), CN III/IV (D), NB/GP (E), S2/AuV (F), cerebellum (G), and hippocampus (H). Insets depict magnified ROIs outlined by the small white square. Download Fig 4-3, TIF file.
Fig 4-4
Quantification of cholinergic innervation of S1 layers at P3, P6, and P9. A-C, Box plots depicting the average fluorescence intensity as a measure for cholinergic innervation. Cholinergic innervation of S1 L1 (orange) and L6 (blue) is significantly higher than L2-4 (yellow) and L5 (green) from P3-P9. L2-4 and L5 are not significantly different from each other from P3-P9. A Brown-Forsythe and Welch ANOVA with correction for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli method was used to assess differences. Only statistically significant comparisons (q<0.05, p<0.05) are shown. Download Fig 4-4, TIF file.
Fig 5-1
All reconstructed mouse brains from E12 to P3. A-E, Reconstructions of serial sectioned brains were collected at E12 (A), E15 (B), E18 (C), P1 (D), P3 (E). For better visibility cartoons of ACh populations were added color-coded by brain regions: transparent dark purple – hippocampus, light blue – striatum, transparent light green – cerebral cortex, orange – thalamus, light gray – basal forebrain, light pink – cerebellum, yellow – caudal brain nuclei, light purple – spinal cord, green - hypothalamus. Download Fig 5-1, TIF file.
Fig 5-2
All reconstructed mouse brains from P6 to >1 yr. A-E, Reconstructions of serial sectioned brains were collected at P6 (A), P9 (B), P12 (C), P50 (D), and >1 year of age (E). For better visibility cartoons of ACh populations were added color-coded by brain regions: transparent dark purple – hippocampus, light blue – striatum, transparent light green – cerebral cortex, orange – thalamus, light gray – basal forebrain, transparent light pink – cerebellum, yellow – caudal brain nuclei, light purple – spinal cord, green - hypothalamus. Download Fig 5-2, TIF file.
Movie 1
Animated View at the cholinergic ACh neuron populations in E12 brain reconstruction depicted in Fig 5 A. Download Movie 1, MP4 file.
Movie 2
Animated View at the cholinergic ACh neuron populations in E15 brain reconstruction depicted in Fig 5 B. Download Movie 2, MP4 file.
Movie 3
Animated View at the cholinergic ACh neuron populations in E18 brain reconstruction depicted in Fig 5 C. Download Movie 3, MP4 file.
Movie 4
Animated View at the cholinergic ACh neuron populations in P1 brain reconstruction depicted in Fig 5 D. Download Movie 4, MP4 file.
Movie 5
Animated View at the cholinergic ACh neuron populations in P3 brain reconstruction depicted in Fig 5 E. Download Movie 5, MP4 file.
Movie 6
Animated View at the cholinergic ACh neuron populations in P6 brain reconstruction depicted in Fig 5 F. Download Movie 6, MP4 file.
Movie 7
Animated View at the cholinergic ACh neuron populations in P9 brain reconstruction depicted in Fig 5 G. Download Movie 7, MP4 file.
Movie 8
Animated View at the cholinergic ACh neuron populations in P12 brain reconstruction depicted in Fig 5 H. Download Movie 8, MP4 file.
Movie 9
Animated View at the cholinergic ACh neuron populations in P50 brain reconstruction depicted in Fig 5 I. Download Movie 9, MP4 file.
Movie 10
Animated View at the cholinergic ACh neuron populations in >1 year brain reconstruction depicted in Fig 5 J. Download Movie 10, MP4 file.
Movie 11
Animated View at individual cholinergic nuclei in the fore- and hindbrain in P1 brain reconstruction depicted in Fig 6 A, C, E, G. Download Movie 11, MP4 file.
Movie 12
Animated View at individual cholinergic nuclei in the fore- and hindbrain in P50 brain reconstruction depicted in Fig 6 B, D, F, H. Download Movie 12, MP4 file.
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