Comparison of morphological features of fluorescent-labeled CRH neurons in three fluorescent-reporter mouse lines
By crossing CRH-IRES-Cre mice with Ai6, Ai14, and Ai32 reporter mice, in which the cassette containing ZsGreen1, td-Tomato, or CHR2-EYFP was expressed in a Cre-dependent manner, we obtained CRH-IRES-Cre;Ai6, CRH-IRES-Cre;Ai14, and CRH-IRES-Cre;Ai32 mice, respectively (Additional file 1, Figure S1. A). Then, we compared the distributions and morphologies of fluorescent-labeled CRH neurons in several brain regions, including the olfactory bulb (OB) (Fig. 1A, C), cortex (Fig. 1D, F), PVN (Fig. 1G, I), bed nucleus of the stria terminalis (BST) (Additional file 1, Figure S1. B and D), and central nucleus of the amygdala (CeA) (Additional file 1, Figure S1. E and G).
In the OB, the transgenic fluorescent proteins were mainly distributed in the glomerular layer (Gl), external plexiform layer (EPl), and the mitral cell layer (Mi) in all three mouse lines. CRH-IRES-Cre;Ai6 mice showed the brightest and largest number of fluorescent-labeled cells in all these layers (Fig. 1A); in particular, more fluorescent cells were labeled in the granule cell layer (GrO) in this mouse line compared to those in CRH-IRES-Cre;Ai14 and CRH-IRES-Cre;Ai32 mice. However, fluorescent-labeled neuronal fibers were short and their fluorescent distributions were not uniform; for example, the dendrites close to the cell bodies of mitral cells were strongly labeled (Fig. 1A, a’, indicated by the arrowheads), but the branches extending to the Gl were not clear (Fig. 1A, a, indicated by the dotted box). CRH-IRES-Cre;Ai14 mice were also labeled with bright cell bodies and dense fibers were labeled in the EPl (Fig. 1B, b’), but only a few dendritic structures were labeled in the Gl (Fig. 1B, b, indicated by the dotted box). By contrast, in each layer of CRH-IRES-Cre;Ai32 mice, the fluorescence distributed in cell bodies and fibers exhibited a uniform brightness (Fig. 1C), and the somata in these sections were organized in a ring-like structure (Fig. 1C, c’, indicated by the arrowhead). Unlike the former two mouse lines, the Gl showed a bushy spherical structure (Fig. 1C, c) that was comprised of mitral cells and/or peribulbar cells.
In the cortex, fluorescent-labeled cells were found in each layer in CRH-IRES-Cre;Ai6 mice (Fig. 1D). The cell bodies were strongly labeled, while fibers were rarely seen (Fig. 1D, d). The numbers of labeled cells in the medial prefrontal cortex (mPFC) of CRH-IRES-Cre;Ai14 (103.4 ± 7.9/mm2) and CRH-IRES-Cre;Ai32 (108.7 ± 5.1/mm2) mice were less than those in CRH-IRES-Cre;Ai6 (283.3 ± 27.8/mm2) mice (Fig. 1J, one-way ANOVA, P < 0.0001, F (2, 9) = 36.56), and the cells were mainly distributed in layer 2/3 (Fig. 1E, F). Neurons in CRH-IRES-Cre;Ai14 mice also showed clearer and brighter cell bodies (Fig. 1E, e), whereas more fibers were labeled (Fig. 1F) in CRH-IRES-Cre;Ai32 mice, especially in terms of a dense distribution in the first layer (Fig. 1F, f).
The outlines of nuclei were clearly visible in the fluorescent labeling of CRH neurons in the PVN (Fig. 1G, I, indicated by the dotted line), BST (Additional file 1, Figure S1. B and D, indicated by the dotted box), and CeA (Additional file 1, Figure S1. E and G, indicated by the dotted box) of the three mouse lines. Similarly, CRH-IRES-Cre;Ai6 mice showed the highest number of labeled neurons in the PVN (1702 ± 238.9/mm2, one-way ANOVA, P = 0.2606, F (2, 9) = 1.57), BST (714.6 ± 125.3/mm2, one-way ANOVA, P < 0.005, F (2, 9) = 11.38), and CeA (1085 ± 67.35/mm2, one-way ANOVA, P = 0.0778, F (2, 9) = 3.44) (Fig. 1J), as well as the strongest fluorescent labeling within these cell bodies (Fig. 1G; Additional file 1, Figure S1. B, a and E, d). In CRH-IRES-Cre;Ai14 mice, distinguishable cell bodies and dense fibers were labeled in the BST (Additional file 1, Figure S1. C, b) and CeA (Additional file 1, Figure S1. F, e), while only the cell bodies were clearly seen in the PVN (Fig. 1H, indicated by the dotted line). By contrast, CRH-IRES-Cre;Ai32 mice showed dense fibers in all of these regions, and the fluorescent signals of the cell bodies were distinguishable (Fig. 1I, Additional file 1, Figure S1. D, c and G, f); especially in the PVN, neuronal fibers extending to the lateral and third ventricle (Fig. 1I, i, fibers indicated by the arrowheads) were visible, and there was a uniform fluorescent intensity distributed in the nearby cell bodies and fibers.
In summary, among the three reporter mouse lines, CRH-IRES-Cre;Ai6 and CRH-IRES-Cre;Ai14 mice showed clearer and brighter cell bodies of CRH neurons. CRH-IRES-Cre;Ai6 mice had the largest number of labeled CRH cells in each tested brain region, but almost no neuronal fibers were visible. CRH-IRES-Cre;Ai14 mice showed clear but incomplete fibers. Only CRH-IRES-Cre;Ai32 mice showed the most complete fibrous structures, especially in terms of distributions in neuronal terminals (e.g., the bushy spherical structures in the glomerular layer of the OB; the extended fibers in the cortex and PVN); regardless of their weaknesses in distinguishing single-cell bodies, the fluorescent distributions in the whole cell were uniform, which is conducive to the adjustment of exposure and the collection of complete morphologies of neurons during imaging.
Whole-brain distributions of CRH neurons at high resolution in the CRH-IRES-Cre;Ai32 mouse line
Since the single-cell morphology of CRH neurons was most clearly visible in CRH-IRES-Cre;Ai32 mice, we used this mouse line to image EYFP-labeled CRH neurons throughout the brain at a resolution of 0.2 × 0.2 × 1.0 μm via an fMOST system. First, 100-μm down-sampled coronal projection sections (Fig. 2a) were provided to show the overall distributions of CRH neurons in various brain regions. EYFP-labeled cells were distributed in many regions that have not previously been reported, such as in vascular organ of the lamina terminalis (VOLT), ventromedial preoptic nucleus (VMPO), caudate putamen (CPu), bed nucleus of the anterior commissure (BAC), triangular septal nucleus (TS), suprachiasmatic nucleus (SCN), Kölliker-Fuse nucleus (KF), and nucleus X (X) (Fig. 2a). We analyzed the co-localization of EYFP expression with CRH immunoreactivity in these brain regions (Additional file 1, Figure S2. A) and found that most of the EYFP-labeled “novel” CRH neurons coexisted with CRH-immunoreactive cells in all of the above brain regions (Additional file 1, Figure S2. A, indicated by arrows), but low ratio of co-labeling was observed in some regions such as in the BAC, CPu, SCN, and TS, and some CRH-immunoreactive neurons did not express EYFP. Furthermore, bundles of CRH projection fibers were visible in accumbens nucleus, shell (AcbSh), interstitial nucleus of the posterior limb of the anterior commissure (IPAC), anterior commissure, posterior (acp), corpus callosum (cc), and inferior cerebellar peduncle (icp) (Fig. 2a, indicated by arrowheads and Fig. 2b). The movies of serial sections showed that the fibers in the AcbSh, IPAC, and acp were projections from neurons in the OB (Additional file 2, Movie 1) and that fibers in the icp were projections from IO CRH neurons (Additional file 1, Figure S2. B and Additional file 3, Movie 2). Moreover, we found novel populations of CRH-positive neurons in some brain regions, such as a sparsely distributed group in the CPu (Fig. 2c) that had dendrites that were radially distributed (with the maximum radius from the terminals to the somata being 40–70 μm). Neurons gathered in the BAC (Fig. 2d) had round cell bodies and two short processes. The average number of CRH-positive neurons found in the SCN was 45.67 ± 0.88, and nearly every neuron had two thick primary dendrites with few branches (Fig. 2e). Neurons in the dorsal cochlear nucleus (DC) (Fig. 2f) had dense apical dendrites distributed in the superficial glial zone, and chandelier cells with apical dendrites vertically distributed were labeled in the cerebellum (Fig. 2g). A cluster of swollen structures (Fig. 2h, indicated by arrowheads) presenting transparent smooth surfaces was visible around the third ventricle (3 V) and always extended to the 3 V border. Densely labeled vascular-like structures and terminals of CRH neurons were found in VOLT (Fig. 2i) and ME (Fig. 2j). The distributions and morphologies of CRH neurons in other brain regions are shown in Additional file 1, Figure S2. B.
Three-dimensional distributions and single-cell reconstructions of CRH neurons in several brain regions
We reconstructed EYFP-labeled CRH neurons in several brain regions (Fig. 3a, h), including the OB (Fig. 3a), dorsal part of lateral septal nucleus (LSD) (Fig. 3b), BST (Fig. 3c), CeA (Fig. 3d), VMPO (Fig. 3e), hippocampus (Hip) (Fig. 3f), SCN (Fig. 3g), and DC (Fig. 3h). We found that the reconstructed neurons in several brain regions (e.g., mPFC, BST, VMPO, anterior parvicellular part of paraventricular hypothalamic nucleus (PaAp), periventricular hypothalamic nucleus (Pe), and SCN) shared similar morphological characteristics consistent with bipolar neurons (Fig. 3i). CRH neurons in the LSD had the largest average volume of somata (1632 ± 159.6 μm3) (Fig. 3j) and the longest dendritic length (1.9 ± 0.2 mm) (Fig. 3k). Dendritic length significantly increased as a function of somatic volume (R2 = 0.597, P = 0.0032) (Fig. 3n). CRH neurons in the VMPO also had a larger cell bodies (1172 ± 228.1 μm3) (Fig. 3j), but the number of dendritic branches (15.2 ± 3.7) (Fig. 3l) and dendritic length (1.1 ± 0.1 mm) (Fig. 3k) was less than those of neurons in the LSD. Dendritic length was also positively correlated with somatic volume in the VMPO (Fig. 3o). Sholl analysis showed that neurons in the LSD had the largest maximum number of intersections, while the VMPO had the least maximum number of intersections. For all of these regions, the maximum numbers of intersections were located at radial distances of 50–100 μm from the somata (Fig. 3m). The more complex dendrites of CRH neurons in the LSD, compared to those in other areas, suggested that CRH neurons in the LSD may receive comparatively more inputs. Most of the dendritic morphologies of CRH neurons in the hippocampus exhibited a similar pattern of an umbrella shape of upward dendrites (Fig. 3f). CRH neurons in the SCN were scattered throughout the nucleus and the dendrites were interlaced with one another (Fig. 3g). We next compared the parameters of all reconstructed neurons (Additional file 1, Table S1) in different brain regions and found that CRH neurons in hypothalamic regions—including the PaAp (640.1 ± 60.4 μm3), Pe (951.2 ± 108.3 μm3), and SCN (636.0 ± 55.4 μm3)—had smaller somatic volumes (Fig. 3j and Additional file 1, Table S1). Similarly, there were also shorter dendritic lengths of CRH neurons in the PaAp (0.5 ± 0.02 mm), Pe (0.5 ± 0.06 mm), and SCN (0.6 ± 0.05 mm) (Fig. 3k and Additional file 1, Table S1). The simpler morphologies of hypothalamic CRH neurons may be related to their endocrine and other conserved functions.
Multiple morphological types of CRH neurons form distinct dendritic connections in the medial prefrontal cortex (mPFC)
Recent studies have shown that CRH neurons in the mPFC play a critical role in higher cognitive functions [29, 30]. In the prelimbic cortex (PrL) within the mPFC, we reconstructed the entire somata (Fig. 4A, purple bodies) and dendrites (Fig. 4A, color lines) of EYFP-labeled CRH neurons within a column that had a volume of 350 × 500 × 500 μm. The cell bodies of these neurons were mostly distributed within layers 2–4 and most of their dendrites were vertically distributed. There were dendritic branches in both the upper and lower parts of the somata. The apical dendrites that branched in the first layer formed a dense dendritic network, and most of them reached the pia mater (Fig. 4A, reconstructed fibers indicated in layer 1); furthermore, the basal dendrites extended and branched into layer 4 at a distance of approximately 500 μm from the cortical surface (Fig. 4A, reconstructed fibers indicated in layer 4). Individual reconstructed neurons were classified according to the distances (50–100, 100–150, 150–200, 200–250, and > 250 μm) between their somata and the surface of the cortex (Fig. 4B), and the percentages of neurons in these categories were 19%, 31%, 35%, 10%, and 5%, respectively (Fig. 4B, indicated in the pie chart). We found that there was significant correlation between the somatic depth (distance from the cortical surface) and both the total dendritic length (Fig. 4C, upper half, r = 0.4440) and total Euclidean distance (the straight-line distance from the soma to the given point of the dendrite) (Fig. 4C, bottom half, r = 0.5399). Sholl analyses showed that the number of intersections with a radial distance from the soma being less than 50 μm was larger in neurons with a somatic depth of 50–100 μm than that of neurons with a somatic depth of 100–150 μm; in contrast, the number of intersections with a radial distance from the soma being more than 50 μm was smaller and ended at approximately 100 μm of the radial distance from the soma in neurons with a somatic depth of 50–100 μm. Interestingly, the maximum numbers of intersections were similar between these two types of neurons (Fig. 4D). Sholl analyses of CRH neurons with different somatic depths are shown in additional file 1, Figure S3. J. We also found that the total dendritic length (Fig. 4E, left half, one-way ANOVA, P = 0.0008, F (2, 60) = 8.100) and the total Euclidean distance (Fig. 4E, right half, one-way ANOVA, P = 0.0002, F (2, 60) = 9.915) of neurons with a somatic depth of less than 100 μm were significantly smaller than those with somatic depths of 100–150 μm (P = 0.0285) and more than 150 μm (P = 0.0005), while there were no significant differences in the total number of dendritic branches or the total number of dendritic terminal points (Additional file 1, Figure S3, K). The average dendritic lengths of neurons at somatic depths of less than 100 μm, 100–150 μm, and more than 100–150 μm were 0.82 ± 0.26, 1.33 ± 0.54, and 1.48 ± 0.68 mm, respectively; furthermore, their total Euclidean distances were 14.65 ± 5.19, 38.77 ± 25.49, and 48.67 ± 30.38 mm, and their total numbers of dendritic branches were 23.23 ± 8.65, 32.86 ± 20.28, 33.32 ± 19.44, respectively.
To investigate local CRH-CRH connection patterns within the cortex, we divided CRH-CRH connections into three types (Fig. 4F, H). Type I consisted of basal-to-apical connections. Here, somata in layer 2 sent dendrites downward (the green cell of Fig. 4F, a) that contacted with the upward dendrites (the purple cell of Fig. 4F, a) from the somata in layer 3 (as shown in the red dotted box of Fig. 4F, a, b). Type II consisted of basal-to-somatic connections (as shown in the yellow-dotted box of Fig. 4Gc, d). In layer 2–3, a soma in the upper layer sent dendrites downward (as shown in Fig. 4G, c, red cell), and the end of one branch (Fig. 4G, d red arrows) was in contact with an adjacent lower cell body (Fig. 4G, c, orange cell; Fig. 4G, d, orange arrows). Type III consisted of basal-to-basal connections (Fig. 4G, c, e; Fig. 4H, f, green-dotted box). Two cell bodies in layers 2–3 sent dendrites downward, and the end of one branch (Fig. 4G, e, red arrows; Fig. 4H, g, yellow arrows) from the upper soma and the branch (Fig. 4G, e, orange arrows; Fig. 4H, g, blue arrows) from the lower soma formed a connection. A common feature of the three types of connections was that the fluorescent intensity increased at the contact point, indicating a possible connection of structures (Fig. 4F, b, G, d and e, and H, g, purple arrows). Examples of type-II and type-III connections are demonstrated in Additional file 4, Movie 3.
We next performed immunofluorescent staining to determine the specificity of EYFP-labeled neurons in CRH-IRES-Cre;Ai32 mice. The results showed that most of the EYFP-labeled neurons in the mPFC were CRH-immunoreactive cells (Additional file 1 Figure S3, A–C, indicated by white arrowheads). We further identified that these CRH interneurons were GAD67-GFP-positive neurons (Additional file 1, Figure S3, D-F, indicated by white arrowheads) by using CRH-IRES-Cre;Ai14;GAD67-GFP mice. Interestingly, in adult mouse brains, EYFP-labeled pyramidal neurons were visible in layer 3 or layer 5 of the cortex (Additional file 1 Figure S3, H), but there were no EYFP-labeled pyramidal neurons on the 21st day after birth (Additional file 1, Figure S3, G). These fluorescently labeled pyramidal neurons were not CRH-immunoreactive cells (Additional file 1, Figure S3, I), including within their dendrites and spines (Additional file 1, Figure S3, a, indicated by arrowheads). We also observed that some EYFP-labeled neurite swellings in layer 1 were also labeled with CRH antibodies (Additional file 1, Figure S3, b, indicated by arrowheads).
Reconstructions and morphological features of CRH neurons in the PaAp and Pe
Hypothalamic neuroendocrine CRH neurons play an important role in stress responses, but neurons within different subregions require more detailed morphological analysis. We chose EYFP-labeled neurons in the PaAP and Pe to reconstruct their somata and processes (Fig. 5A, C; Additional file 1, Figure S4, A and B). There was a noteworthy co-localization pattern (Additional file 1, Figure S4, E–G) for the EYFP-labeled signals and CRH immunoreactivity in the PaAP. There were vesicular fluorescent labels (dendritic varicosities) (Fig. 5B, D, gray reconstructed structures) on the neurites of neurons in both the PaAP and Pe, and they also co-labeled with CRH immunopositive-structures (Additional file 1, Figure S4, H, indicated by arrowheads). In terms of their 3D patterns, the somata of some neurons (Fig. 5A, B, purple reconstructed cell bodies) distributed in the PaAP sent out fibers rostrally (Fig. 5A, B, red lines), and there were spaced and small dendritic varicosities (Fig. 5B, b–d, gray bodies) on these fibers. An example of these reconstructed cells is shown (Fig. 5B) according to the primary branches and number of dendritic varicosities, and there were four distribution patterns of these neurons. Pattern 1 (Fig. 5B, a) consisted of cells that had two primary branches with the shortest dendritic length and no varicosities. Pattern 2 (Fig. 5B, b) consisted of cells that had two primary branches with similar dendritic lengths at both ends of the somata and were distributed almost vertically, and the fibers extending rostrally had varicosities. Pattern 3 (Fig. 5B, c) consisted of cells with both ends of the dendrites having varicosities and the one dendritic branch that was distributed horizontally was longer and extended rostrally, whereas the other short branch was distributed vertically. Finally, pattern 4 (Fig. 5B, d) consisted of dendrites of multipolar cells extending rostrally having varicosities and being distributed horizontally. The locations of the reconstructed somata in the PaAP are shown in Additional file 1, Figure S4 A (purple bodies indicated by red circles).
In the Pe, the reconstructed somata were located in the lower part of the PaAp and around the 3V (Fig. 5C, purple bodies; Additional file 1, Figure S4, B, indicated by red circles). These cells sent out fibers and one or two of them extended close to the 3V (Fig. 5C, D, red line). There were large varicosities on the dendrites (Fig. 5D, gray reconstituted bodies; Fig. 5E, indicated by white arrowheads) and they terminated (Fig. 5G, indicated by the yellow arrowhead) near the ependymal cell (Fig. 5G, indicated by the white arrowhead) layer adjacent to the 3V. Most of the reconstructed cells within the Pe were bipolar neurons (Fig. 5D, e, f), and the fibers extending downward to the 3V had more varicosities (Fig. 5D, f, g). We further identified the immunopositive substances contained in these varicosities and found that there were extentive MAP 2 -immunopositive signals (a marker of dendrite) in the varicosities (Fig. 5E, indicated by white arrowheads; Additional file 1, Figure S4, I). Chromogranin B (ChgB) immunoreactivity (associated with large dense core vesicles) was also found in the dendritic varicosities in PaAp (Fig. 5F) and Pe (Additional file 1, Figure S4, J-L) indicating that these varicosities contain a large amount of dense core vesicles. Interestingly, the ependymal cells adjacent to the 3V were also found to be ChgB immunopositive (Fig. 5G). The dendritic varicosities were also stained with Kinesins (molecular motors used for intracellular transport and trafficking) in PaAp (Additional file 1, Figure S4, M-O) and Pe (Additional file 1, Figure S4, P-R).
Next, we compared the somatic and dendritic parameters of the neurons in the PaAP and Pe and found that the average volume of the soma in the Pe (951.2 ± 108.3 μm3) was larger than that in the PaAP (640.1 ± 60.4 μm3) (Fig. 5H, P = 0.0119, t = 2.697), and the number of dendritic varicosities in the Pe (19.4 ± 2.5) was significantly greater than that in the PaAP (7.9 ± 1.6) (Fig. 5I, P = 0.0005, t = 3.974). The total dendritic varicosities volume (2678 ± 652.3 μm3) (Fig. 5J, P = 0.0003, t = 4.348) per cell and the average volume of dendritic varicosities (153.4 ± 43.1 μm3) (Fig. 5K, P = 0.0047, t = 3.148) in the Pe were significantly larger than those (468.2 ± 79.2 μm3 and 47.7 ± 5.3 μm3) in the PaAP. We also found that there was a negative correlation between the number of dendritic varicosities and the soma volume in the PaAP (r = − 0.4519, P = 0.0455) (Fig. 5L). There was no significant difference in the total dendritic length (PaAP: 455.4 ± 24.1 μm, Pe: 537.5 ± 57.6 μm) (Fig. 5M) or the total number of dendritic branches (PaAP: 5.4 ± 0.9, Pe: 6.3 ± 1.6) (Fig. 5N) of neurons in the PaAP and Pe. We found that most (75% in the PaAP and 70% in the Pe) (Fig. 5O, right half, indicated in the pie chart) of the reconstructed neurons were bipolar neurons, which were characterized by the number of primary dendritic branches (Fig. 5O, left half).
Collectively, these 3D reconstructions of hypothalamic CRH neurons may be indicative of the transport and storage of CRH peptides in hypothalamic neurons, as well as the possible their release sites, such as the third ventricle. These findings provide a structural basis for further elucidating the neural circuits and functions of CRH neurons.
Arborization-dependent dendritic spine characteristics of CRH neurons
We further detected and analyzed the characteristics of dendritic spines of CRH neurons. In general, CRH neurons with sparse dendritic branches had less spines. Consistent with previous studies, mushroom-like and thin dendritic spines were found in the cortex, hippocampus, BST, and CeA (Fig. 6A, B). There were also several areas containing CRH neurons with dendritic spines that have not previously been reported. CRH neurons in the VMPO and SCN were aspiny with few strong filopodia-like spines (Fig. 6A, VMPO and SCN, indicated by arrowheads), the maximum lengths of which reached 5 μm. Furthermore, CRH neurons in the LSD had mushroom-like spines (Fig. 6B, LSD, indicated by arrowheads). CRH neurons with few dendritic branches appeared to be aspiny, such as bipolar CRH neurons in the cortex, VMPO, SCN (Fig. 6A), and BST (Fig. 6E), while CRH neurons with many branches (CRH neurons showed in Fig. 6B) were spiny. In the BST and CeA, we further calculated the densities of dendritic spines and found that CRH neurons in the CeA had more spines than those in the BST (Fig. 6C, P < 0.0001, t = 5.467, 11 different lengths of dendrites from three mice were calculated). Most oGAD67-GFP-positive CRH neurons in both the BST (Fig. 6D, a) and CeA (Fig. 6F, b) were spiny, while aspiny CRH neurons (Fig. 6E) and GAD67-GFP-negative spiny CRH neurons (Fig. 6c) were also found in the BST and CeA. Interestingly, by injecting fluorogold into the mPFC (Fig. 6G) in CRH-IRES-Cre;Ai32 mice, we found that long-range-projecting CRH neurons that were co-labeled with fluorogold (Fig. 6H, d) in the anteromedial thalamic nucleus were aspiny (Fig. 6e, thin spines indicated by the arrows).