Species Differences in the Organization of the Ventral Cochlear Nucleus

Abstract

The mammalian cochlear nuclei (CN) consist of two major subdivisions, the dorsal (DCN) and ventral (VCN) nuclei. We previously reported differences in the structural and neurochemical organization of the human DCN from that in several other species. Here we extend this analysis to the VCN, considering both the organization of subdivisions and the types and distributions of neurons. Classically, the VCN in mammals is composed of two subdivisions, the anteroventral (VCA) and posteroventral cochlear nuclei (VCP). Anatomical and electrophysiological data in several species have defined distinct neuronal types with different distributions in the VCA and VCP. We asked if VCN subdivisions and anatomically defined neuronal types might be distinguished by patterns of protein expression in humans. We also asked if the neurochemical characteristics of the VCN are the same in humans as in other mammalian species, analyzing data from chimpanzees, macaque monkeys, cats, rats and chinchillas. We examined Nissl- and immunostained sections, using antibodies that had labeled neurons in other brainstem nuclei in humans. Nissl-stained sections supported the presence of both VCP and VCA in humans and chimpanzees. However, patterns of protein expression did not differentiate classes of neurons in humans; neurons of different soma shapes and dendritic configurations all expressed the same proteins. The patterns of immunostaining in macaque monkey, cat, rat, and chinchilla were different from those in humans and chimpanzees and from each other. The results may correlate with species differences in auditory function and plasticity. Anat Rec, 301:862-886, 2018. © 2017 Wiley Periodicals, Inc.

Keywords: audition; chimpanzees; cochlea; human.

Figure 1. DCN, VCP and VCA in the human

A, C, E, G, I, K show low magnification photomicrographs of the Nissl-stained sections of the brainstem of Case180. The sections are about 600 µm apart; A is the most caudal. At that level, both the DCN and VCP are on the surface of the brainstem. Panels C, E, G show the VCP, and I, K show the VCA which is buried in the fibers of the middle cerebellar peduncle. The rectangle in each panel shows the location of each higher-magnification photomicrograph in the panel immediately below. A, C, E,G, I, K, scale bars = 1 mm. Panels B, D, F, H show higher magnification photomicrographs of Nissl-stained neurons in the VCP, and panels I and K show the VCA. The neurons in the VCP are have round, oval or polygonal somata but there is not much variation in size. In the VCA, the stained neurons appear more densely spaced. B, D, F, I, L, scale bars = 100 µm.

Figure 2. CR immunostaining in VCP and VCA, Case 176

A. Nissl-stained section showing the DCN dorsally and the VCP ventrally. B. CR immunostaining in the VCP on an adjacent section. The rectangle shows the location of the photomicrograph in C. C. Higher magnification photomicrograph showing the variety of shapes and sizes and spacing of immunostained neurons in the VCP. The arrow shows a neuron with dendrites emerging from one side of the soma. Note the staining of processes surrounding the stained neurons. D. Nissl-stained section of the VCA. The section is about 2 mm rostral to the sections in A and B. Note the close spacing and relatively regular soma size. E. Adjacent CR immunostained section. Note the relative similarity of the size and densities of stained somata in D and E. The rectangle shows the location of the photomicrograph in F. F. CR immunostained neurons in the VCA; most have round or oval somata. The arrow indicates a neuron with a single stained primary dendrite. A, B, D, E, scale bars = 500 µm. C, F scale bars = 50 µm.

Figure 3. NPNFP immunostaining in the VCP and VCA, Case 158

A. Nissl-stained section at the level of the DCN and VCP. B. Adjacent section immunostained for NPNFP. Note the dense staining in both the DCN dorsally and the VCP ventrally. The arrow indicates the approximate border of the DCN and the VCP. The rectangle shows the location of the photomicrograph in C. C.NPNFP- immunostained neurons; the arrow indicates a cell with two dendrites emerging from one side of the soma, the black arrowhead a neuron with a single, thick primary dendrite. Neurons are embedded in a very dense meshwork of stained processes running in all directions. D. Nissl-stained section about 2 mm rostral to the ones in A, B. There is dense staining of round or oval somata and the higher density of stained neurons in VCA compared to the VCP (A). E. NPNFP immunostaining of neurons and processes in the VCA. The rectangle shows the location of the photomicrograph in F. F. Higher magnification photomicrograph of immunostained neurons embedded in a meshwork of immunostained fibers. The arrowhead indicates an immunostained neuron with a round soma and large primary dendrite. A, B, D, E, scale bars = 500 µm. C, F, scale bars = 50 µm.

Figure 4. CR and nNOS immunostaining in the VCP, Case 169

A, C, E, lower magnification photomicrographs of the VCP; the rectangles in each show the location of the photomicrographs in the panels to the right. A. Nissl-stained section in the VCP. B. Higher magnification photomicrograph showing large neurons with a variety of soma shapes. The black arrow indicates a neuron with a single primary dendrite, the arrowhead a neuron with a polygonal soma. C. CR immunostaining in VCP on a section about 200 µm caudal to the section in A. D. Larger magnification photomicrograph of immunostained neurons. The arrow indicates a neuron with a polygonal soma; other immunostained neurons have round or oval somata. The neurons are surrounded by stained processes, many of which run parallel to the long axis of the VCP. E. nNOS-ir section about 200 µm rostral to the section in A. There are scattered immunostained somata. F. Higher magnification photomicrograph of nNOS-ir neurons. The arrow indicates a neuron with an oval soma, the arrowhead a neuron with a polygonal soma. There is also punctate label and immunolabeled fibers. A, C, E, scale bars = 1 mm. B, D, F, scale bars = 50 µm.

Figure 5. nNOS immunoreactivity in VCP and VCA, Case 168

A–C. Low magnification photomicrographs of nNOS immunostained sections through the VCN. A is the most caudal. The rectangles in each show the location of the higher magnification photomicrographs just below (D–F). A. Caudal section at a level where the DCN and VCP are both present. B. More rostral section; the VCP has expanded. The white arrows indicate the region with a higher density of immunostained neurons. C. More rostral section with only the VCA present; there is a high density of neurons with round somata. A–C, scale bars = 500 µm. D. nNOS-ir in neurons, puncta and fibers of the caudal VCP. The arrow indicates a cell with an oval soma, the arrowhead a neuron with a polygonal soma. E. Immunostained neurons in the more rostral VCP with round, oval and polygonal somata. F. nNOS-ir in VCA neurons, puncta and fibers. The arrow indicates a neuron with a round soma and single dendrite. D–F, scale bars = 50 µm.

Figure 6. The VCP and VCA in chimpanzee WM

A–D. Nissl-stained sections about 1 mm apart; A is the most caudal. A. the DCN is bordered laterally by fibers and is not on the free surface of the brainstem; the VCP is ventral to it. A–D, scale bars = 500 µm. E. NPNFP- immunostaining of neurons and processes in the VCP on a section about 250 µm caudal to the one in A. The rectangle shows the location of the photomicrograph in F. F. Dense immunostaining of neurons and processes in VCP. The arrow indicates a neuron with a triangular soma. I. NPNFP immunostaining in the VCA. The rectangle shows the location of the photomicrograph in J. J. Immunostaining of neurons with round or oval somata (arrow) and a network of processes running in all directions. The arrow indicates a neuron with an oval soma and single proximal dendrite, the arrowhead shows a neuron with a polygonal soma. I. CR immunostaining in the VCA. The rectangle shows the location of the photomicrograph in J. J. CR immunostaining of neurons and processes in the VCA. The arrowhead indicates a neuron with a polygonal soma, the black and white arrows show neurons with oval soma and a single primary dendrites. E, G, I, scale bars = 500 µm. F, H, J, scale bars = 50 µm.

Figure 7. nNOS and NPNFP immunolabeling in the VCP and VCA of chimpanzee AN

A, C, E. Lower magnification photomicrographs of nNOS immunostaining in the VCP (A, E) and VCA (I). A is the most caudal. The rectangle in each shows the location of the higher magnification photomicrograph to the right. G, I, K. Lower magnification photomicrographs of NPNFP immunostaining in the VCP (G, I) and VCA (K). The rectangles show the location of the higher magnification photomicrographs to the right of each panel. A. There are scattered nNOS-immunostained somata in the VCP. B. The immunostained neurons have round or oval somata. The arrow indicates a neuron with a round soma and very lightly stained primary dendrite. There is very little staining of fibers. C. There are scattered immunostained somata and very little staining of processes or fibers. D. The immunostained somata are round (arrow) or oval and similar in size. nNOS immunostaining in the VCA; there are scattered somata with little stain of dendrites or fibers. F. The stained somata are round (arrow) or elongated. G. NPNFP immunostaining in the VCP. H. There are immunostained somata, dendrites and fibers (example at arrow). I. NPNFP-immunostaining of somata, processes and fibers (example at arrow) at a more rostral level in the VCP. J. The immunostained somata are of several different shapes; there is also staining of dendrites and fibers. The arrow shows a neuron with two dendrites emerging from one side of the soma. K. Scattered NPNFP-ir somata in the VCA. L. Immunostained neurons have round or oval somata. The arrow shows a neuron with a round soma and a single primary dendrite. There is also staining of processes and puncta. A, C, E, G, I, K, scale bars= 500 µm. B, D, F, H, J, L, scale bars = 50 µm.

Figure 8. The VCA in chimpanzee MT

A, C, E. The rectangles show the location of the photomicrographs in the panels to the right. A. CR immunostaining in the VCA. B. The immunostained somata are of a variety of shapes. The arrow shows a CR immunostained neuron with a round soma, the arrowhead a neuron with a round soma and single primary dendrite. C. NPNFP-immunostained section. The number, sizes, shapes and distribution of somata are similar to those illustrated in A and E. D. The higher magnification photomicrograph of NPNFP-ir shows neurons of different soma shapes. Dendrites, fibers and puncta are also immunostained. The arrowhead shows a multipolar cell with three dendrites visible, the arrow a neuron with a round soma and a single dendrite. E. Nissl-stained section showing neurons with round somata distributed throughout the VCA. F. Higher magnification photomicrograph showing somata of a variety of shapes including round (arrow) as well as elongated (arrowhead) and irregular. A, C, E, scale bars = 1 mm. B, D, F, scale bars = 50 µm.

Figure 9. The VCN in macaque monkey

A–C. Nissl-stained celloidin-embedded sections of the cochlear nuclei in a macaque monkey. A. Caudal section at the level of the DCN. B, C. More rostral sections show the VCP ventrally and the VCA dorsally, separated by fibers (arrow in C). A–C, scale bars = 500 µm. D–M show pairs of photomicrographs of immunostaining in the VCP and VCA. D–K show sections from a second macaque; L and M show sections from a third. The rectangles in the lower magnification photomicrographs (D, F, H, J, L) show the location of the higher magnification photomicrographs to the right of each (E, G, I, K, M). D. Low magnification photomicrograph of CAT-301 immunostaining in the VCP and VCA. The arrow shows the fibers dividing them. Immunostained somata are seen in both VCP and VCA. E. CAT-301 immunostaining outlines somata of several different shapes in the VCP. There is no immunostaining of proximal dendrites or of fibers. The arrow indicates an outlined neuron with an elongated, irregular soma. F. CAT-301 immunostaining more rostrally in the VCA; many small round somata are outlined by immunostaining. G. Higher magnification photomicrographs showing that the CAT-301 immunostained somata are small and round (example at arrow) or oval. There is no immunostaining outlining dendrites or fibers. H. CR immunostaining in the VCP. The section was counterstained with a Giemsa stain. I. The higher magnification photomicrograph shows that the CR immunostaining is comprised of fibers, puncta and scattered somata of different shapes (the example at arrow has an elongated soma). Some Giemsa-stained neurons are not CR-ir (example at arrowhead). J. nNOS immunostaining of somata in the VCP; they are similar in size, shape and density to the CR-ir somata in I. K. The higher-magnification photomicrograph shows scattered immunostained somata of different shapes (the arrow indicates a neuron with an elongated soma and staining of proximal dendrites).There is no immunostaining of puncta or fibers. L. NPNFP immunostaining in VCP. Immunostained somata are apparent even on the low magnification photomicrograph. M. The higher magnification photomicrograph shows many immunostained somata of different shapes; the arrow indicates a neuron with several proximal dendrites. Fibers running at all orientations are also NONFP-immunostained. D, F, G, J, L, scale bars = 500 µm; E, G, I, K, M, scale bars = 50 µm.

Figure 10. The VCN of the cat

Photomicrographs of immunostained sections are presented in 7 pairs with a low magnification photomicrograph on the left (A, C, E, G, I, K, M) and a higher magnification photomicrograph directly to the right (B, D, F, H, J, K, L, N). For each pair, the rectangle in the low magnification photomicrograph shows the location of the photomicrograph in the panel to the right. Data from five different animals are shown (Cat 1, A, B; Cat 2, C, D, K–N; Cat 3 E, F; Cat 4, G, H; Cat 5, I, J). A. Low magnification photomicrograph of CAT-301 immunostaining in the VCP and DCN. Outlined somata are distributed throughout the VCP. B. Higher magnification photomicrograph showing several different immunostained somata. The arrow indicates an immunostained neuron with very large proximal dendrites. The arrowhead indicates another neuron with a small round soma. C. CR-immunostaining in a section with DCN dorsally and the VCP ventrally. D. Higher magnification photomicrograph showing immunostained somata of several shapes; the arrowhead indicates an oval soma with a single dendrite. There is also immunostaining of puncta, some surrounding somata (also seen at arrowhead), and of many fibers. E. nNOS immunostaining on a section with the DCN dorsally and the VCP ventrally. There are immunostained somata dendrites scattered throughout the VCP. F. Higher magnification photomicrographs illustrating nNOS-ir neurons in the VCP. The arrow shows a neuron with an irregularly shaped soma and two dendrites emerging from one side, the arrowhead a neuron with an oval soma and two dendrites emerging at opposite poles. G. The low-power photomicrograph shows NPNFP-immunostaining all over the VCP. H. The higher magnification photomicrograph shows immunostaining of somata of several shapes. The arrowhead shows a neuron with a round soma and a thick primary dendrite, the arrowhead a neuron with several large primary dendrites. Puncta and fibers running at all angles are also immunostained. I. CR immunostaining in the VCA on a section rostral to the VCP. There is CR immunostaining over the entire VCA. J. Higher magnification photo micrograph showing that there are immunostained somata, some surrounded by puncta, proximal dendrites and many stained puncta. K. Low magnification photomicrograph of nNOS immunostaining; the label appears uniform over the VCA. L. The higher magnification photomicrograph shows scattered immunostained somata. The arrow indicates a smaller darkly immunostained neuron with beaded process. M. The lower magnification photo micrographs shows that there is NPNFP-ir all over the VCA. N. A higher magnification photomicrograph shows that the immunoreactivity is composed of immunolabeled somata, proximal dendrites and fibers. The arrow indicates two immunostained neurons with single dendrites. A, C, E, G, I, K, M, scale bars = 500 µm. B, D, F, H, J, L, M, scale bars = 50 µm.

Figure 11. CR, nNOS and NONFP immunostaining in the VCP and VCA of the rat

Data are from four different animals (Rat 1, A–D; Rat 2, E, F; Rat 3, G–J; Rat 4, K, L). Each row shows two pairs of low and higher magnification photomicrographs of immunostaining in the VCP and VCA; immunostaining with a different antibody is illustrated in each row. The rectangles in the higher magnification photomicrographs (A, C, E, G, I, K) show the locations of the photomicrographs in the panels to the right (B, D, F, H, J, L). A–D. CR immunostaining in the VCP. A. Caudal section (Bregma −11.2) on which both the DCN and VCP are present. At lower magnification CR-ir in the VCP is uniformly dark. B. Higher magnification photomicrograph showing that the immunostaining in the VCP is composed of scattered somata of different shapes as well as puncta and fibers. C. Uniform CR immunostaining more rostrally (Bregma −10.2) in the VCA. D. The higher magnification photomicrograph shows immunostaining of scattered somata puncta and fibers. The arrow indicates an immunostained neuron with an elongated soma. E. Low magnification photomicrograph showing uniformly dark nNOS immunostaining in the VCP (Bregma −11.2). F. The immunostaining includes large, multipolar somata and proximal dendrites in the VCP. The arrow indicates a large immunostained neuron with multiple proximal dendrites. G. nNOS immunostaining on a section through the VCA (Bregma −10.14). H. Immunostaining of somata in the VCA, example at arrow). The somata are smaller than the immunostained somata in the VCP; they are round or oval, and with little staining of dendrites. I. Immunostaining for NPNFP in the VCP (Bregma −11.20). The low magnification image shows uniform staining. J. The higher magnification image shows that there is no immunostaining of somata, but that there is staining of fibers and puncta. The arrow indicates an immunostained fiber. The section is at about Bregma −11.20. K. A low magnification photomicrograph showing uniform NPNFP-immunostaining in the VCA (section at Bregma −9.98). L. The higher magnification image shows no immunostaining of somata but there is immunostaining of fibers and puncta. A, C, E, G, I, K, scale bars = 500 µm; B, D, F, H, J, L, scale bars = 50 µm.

Figure 12. Immunostaining for CR, nNOS and NPNFP in the chinchilla

All data are from a single chinchilla. A–D. CR immunostaining in the VCP (A, B) and VCA (C, D) on low (A, C) and high (B, D) magnification photomicrographs. The rectangles in A, C, show the location of the higher magnification photomicrograph. A. CR immunostaining on a caudal section; the DCN is dorsal and the VCP ventral; the arrow shows the border between them. There is dense, uniform immunostaining over the VCP. B. Fibers and puncta are immunolabeled; there is no immunolabeling of somata. C. More rostral section with the VCA dorsally and the VCP ventrally; the large arrow shows the location of the border between them. At this level there is immunostaining of scattered somata in the VCP. D. Higher magnification photomicrograph of CR immunostaining in the VCA. There is immunolabeling of fibers and puncta but not of somata. E–H. nNOS immunostaining in the VCP and VCA, format as for A–D. E. The immunostaining is much darker in the DCN than in the VCP. Scattered immunolabeled are seen throughout the VCP. F. There is immunostaining of somata of different sizes and shapes. The arrow shows a large soma and proximal dendrite, the arrowhead a smaller soma. There are also immunostained fibers and puncta. G. nNOS Immunostaining on a more rostral section with the VCA dorsally with more uniform staining and the VCP ventrally with scattered immunostained profiles visible. H. There are scattered small round, immunolabeled soma (example at arrow). There are also a few immunolabeled fibers. I–L. NPNFP immunostaining in the VCP and VCA, format as for A–D. I. NPNFP immunostaining in the VCP; at low magnification is appears uniform. J. The immunostaining in the VCP is composed of fibers and puncta and not somata. The arrowhead indicates a longer fiber; many shorter fiber segments are seen throughout, many running in a dorso-ventral direction. K. More rostral section showing the VCP ventrally and the VCA dorsally. The immunostaining appears uniform over the entire VCA. L. Higher magnification photomicrograph showing immunostaining of fibers and many puncta but not somata. A, C, E, G, I, K, scale bars = 500 µm. B, D, F, H, J, L, scale bars = 50 µm.