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.

INTRODUCTION

The mammalian auditory nerve projects to the cochlear nuclei (CN) of the medulla (Feldman and Harrison, 1969; Cohen et al., 1972; Brawer and Morest, 1975; Fekete et al., 1984; Sento and Ryugo, 1989; Liberman, 1991; Ryugo and Sento, 1991; Ryugo, 1992; Liberman, 1993). Two subdivisions of the CN are recognized in most species, the dorsal (DCN) and the ventral cochlear nuclei (VCN; human, Olszewski and Baxter, 1954; cat, Berman, 1968; guinea pig, Hackney et al., 1990; chinchilla, Fleckeisen et al., 1991; rat, Paxinos and Watson, 1997; Paxinos, 1999; macaque monkey, Paxinos et al., 2000). Both receive direct input from the auditory nerve. We recently described differences in the laminar and neurochemical organization of the human DCN compared to that in several other species (Baizer et al., 2014). In the present study we asked whether are also be species differences in the structural and neurochemical organization of the VCN. We first addressed the organization of VCN subdivisions. In many species, the VCN is subdivided into the posterior (VCP) and anterior (VCA) nuclei, with the fibers of the cochlear nerve marking the boundary between them (reviews in Konigsmark et al., 1973; Brawer et al., 1974); also see studies of different species (cat, Berman, 1968; Brawer et al., 1974; chimpanzee, Strominger et al., 1977; guinea pig, Hackney et al., 1990; chinchilla, Fleckeisen et al., 1991; mouse, Franklin and Paxinos, 1997; rat, Paxinos, 1999; monkey, Paxinos et al., 2000; Paxinos and Franklin, 2004). In humans, however, the cochlear nerve does not separate the subdivisions (Moore and Osen, 1979). Some, but not all, reports further subdivide the VCP and VCA (Brawer et al., 1974; Tolbert and Morest, 1982). Descriptions of these subdivisions in humans are less consistent. Olszewski and Baxter (1954) show only the dorsal and ventral cochlear nuclei. Dublin (1982) divided the VCN in humans into superior (SVCN) and inferior (IVCN) nuclei. Paxinos and Huang (1995) show 3 subdivisions of the VC, the posteroventral cochlear nucleus (PVC), a ventral cochlear cap (VCC, dorsal to both PVC and AVC) and an anteroventral (AVC) cochlear nucleus. One goal of this study was to determine whether immunostaining could clarify the number and organization of VCN subdivisions in humans.

Our second goal was to attempt to distinguish different neuronal types in the VCN using immunostaining. Many electrophysiological and anatomical studies have attempted to define and classify VCN neurons. These studies have resulted in several classification schemes that reflect the different anatomical and electrophysiological methods used (review in Cant and Benson, 2003). Based on both soma shape and dendritic configuration, three major anatomically defined cell types are recognized: octopus cells, multipolar (stellate) cells and bushy cells (reviews in Konigsmark et al., 1973; Rouiller and Ryugo, 1984; Cant and Benson, 2003). Both multipolar cells and bushy cells are further subdivided. Two types of bushy cells, “globular and spherical” are recognized and these have very different response properties and connections (Warr, 1972; Rouiller and Ryugo, 1984; Sento and Ryugo, 1989; Ostapoff and Morest, 1991; Ryugo and Sento, 1991; Rhode, 2008). Multipolar/stellate cells are likewise subdivided into two classes (T stellate and D stellate; also called planar and radiate; Cant, 1981; Smith and Rhode, 1989; Cant and Benson, 2003; Doucet and Ryugo, 2006; Oertel et al., 2011). However, it is unclear if these different neuronal subtypes can also be distinguished on the basis of patterns of protein expression. To address this question, we have used immunohistochemistry to study the neurochemical characteristics of VCN neurons in humans. We then compared the immunostaining patterns in the human VCN with those in several other species: chimpanzees, macaque monkeys, cats, rats and chinchillas. The chimpanzee is one of the closest living primate relatives of humans. Therefore VCN features that are evolutionarily specialized in humans may be determined in comparison to data from chimpanzees. The macaque monkey has been used in studies of the organization of the auditory system and has tonotopic representation similar to that of humans (Brugge and Merzenich, 1973; Merzenich and Brugge, 1973; Hackett et al., 1998b, 1998a; Kaas and Hackett, 1998). While not widely used now, the cat was a major subject of auditory research for many years. The organization of the cat CN as well as anatomical and electrophysiological properties of neurons were well-described (Aitkin et al., 1970; Aitkin and Webster, 1971, 1972; Bock et al., 1972; Aitkin, 1973; Brawer et al., 1974; Aitkin and Boyd, 1975; Brawer and Morest, 1975; Boyd and Aitkin, 1976; Imig and Adrian, 1977; Imig and Brugge, 1978; Aitkin and Schuck, 1985; Clarey et al., 1994; Clarey et al., 1995; Barone et al., 1996; Imig et al., 2000). There are many behavioral, electrophysiological and anatomical studies of the CN in the rat (some examples: Harrison and Irving, 1965, 1966b, 1966a; Feldman and Harrison, 1969; Weedman et al., 1996; Weedman and Ryugo, 1996; Wright and Ryugo, 1996; Abbott et al., 1999; Milbrandt et al., 2000; Haenggeli et al., 2005; Turner et al., 2006; Ouda et al., 2012a; Ouda et al., 2012b; Ouda and Syka, 2012; Lobarinas et al., 2013; Baizer et al., 2015a). Further, the rat is often used as a model for auditory disorders such as noise-induced hearing loss, tinnitus and hyperacusis (Turner et al., 2006; Lobarinas et al., 2013; Baizer et al., 2015a). The chinchilla has also been used to study both the normal properties of the auditory system and changes with auditory damage (examples include: Saunders et al., 1987; Salvi et al., 1990; Fleckeisen et al., 1991; Josephson and Morest, 1998; Brozoski et al., 2002; Kraus et al., 2009; Zhou et al., 2009).

A brief report of some of these results was presented in an abstract and poster (Baizer et al., 2015b).

EXPERIMENTAL PROCEDURES

Humans

We studied eight human brainstems from the Witelson Normal Brain Collection (Witelson and McCulloch, 1991). Table 1 shows critical parameters of these cases including case number, age, sex, and post-mortem interval (PMI, in hours). Data on patterns of protein expression in other brainstem nuclei of these cases, including the DCN, have been reported (Baizer et al., 2007; Baizer and Broussard, 2010; Baizer et al., 2011a; Baizer et al., 2011b; Baizer et al., 2013b; Baizer et al., 2014). Methods for histology and immunohistochemistry with this tissue were described in those earlier reports. The VCN is present on many of the sections prepared for these projects and those sections were examined for this study.

TABLE 1.

Cases

CASE	AGE	SEX	PMI (hr)
155	50	f	9
158	51	m	1
164	45	f	3
166	65	f	3
168	69	m	3
169	70	m	2
176	71	f	3
180	54	m	2

Chimpanzees

We examined Nissl- and immunostained sections of the VCN from three chimpanzee brains (cases AN, MT and WM) that were provided by the National Chimpanzee Brain Resource (supported by NIH NS092988). These sections had been prepared for the analysis of several other brainstem structures and many sections were at levels where the VCN is present. The methods of tissue processing were described in those earlier reports (Baizer et al., 2011b; Baizer et al., 2013a; Baizer et al., 2013b).

Other species

We examined and photographed archival Nissl and immunostained slides that included the VCN from cat, macaque monkey, chinchilla and rat, again using slides had been prepared for several other projects (Baizer and Baker, 2004; Baizer and Baker, 2005, 2006a, 2006b; Baizer et al., 2010; Baizer et al., 2011a; Baizer et al., 2011b; Manohar et al., 2012; Baizer et al., 2013a; Baizer et al., 2013b; Baizer et al., 2014). The methods for histology and immunohistochemistry for these brains were described in those reports. For the rats, we used sections only from control, and not experimental animals (noise-exposed; Baizer et al., 2015a). We immunostained additional sections from cat, monkey, rat and chinchilla following published protocols as needed to be sure we had sections from both the VCP and VCA immunostained for critical markers.

Antibodies and Immunohistochemistry (IHC)

Table 2 shows the entire set of antibodies that were used for all species. Antibody specificity was described in earlier reports (Baizer et al., 2007; Baizer and Broussard, 2010; Baizer et al., 2013a; Baizer et al., 2013b; Baizer et al., 2014) and is summarized below.

Table 2.

Antibodies and dilutions

Antigen	Source, Catalogue #	Host	Dilution
Calbindin	Chemicon AB1778	Rabbit	1:2000
Calretinin	Chemicon AB5054	Rabbit	1:2000–1:3000
Chondroitin sulfate proteoglycan (CAT-301)	Chemicon MAB5284	Mouse	1:1000
GAD65/67	Chemicon AB1511	Rabbit	1:1000
nNOS	Cayman 160870	Rabbit	1:200
NPNFP	Covance; SMI;SMI-32	Mouse	1:1000
Parvalbumin	Sigma P3088	Mouse	1:2000

Calbindin (CB)

The immunogen was recombinant rat calbindin. The calbindin D28 antiserum (AB1778) was affinity purified and preadsorbed against calretinin. It recognizes a band of 28 kDa on a Western Blots of mouse brain (manufacturer’s data sheet). No immunostaining was seen on control sections on which the primary antiserum was omitted or on sections in which the antiserum was omitted.

Calretinin (CR)

The immunogen was recombinant rat calretinin. The calretinin antiserum (AB5054) recognizes a band of 31 kDa on Western Blot of rat brain (manufacturer’s data sheet). It recognizes both the calcium-bound and calcium-unbound conformations of calretinin by Western blots (data from manufacturer). No immunostaining was seen on control sections of the human brainstem in which the antiserum was omitted or in sections in which the antiserum was diluted (1:1,500 in 1.5 ml of antibody diluent) and preincubated with the calretinin protein (Swant, recombinant human calretinin produced in E. coli, 3 mg/ml) for 6 h at 4°C prior to immunostaining (according to manufacturer’s Swant instructions). It has also been used to label neurons in mouse thalamus (Delaunay et al., 2009) and interneurons in mouse cerebral cortex (Liodis et al., 2007). This antibody also showed the predicted labeling of hair cells in the chicken (Warchol and Speck, 2007), of interneurons in the rat striatum (Yang et al., 2008), and neurons in the rat amygdala (Rainnie et al., 2006).

CAT-301 (CPSG)

The immunogen was feline spinal cord fixed gray matter. The antiserum (Millipore, MAB5284) recognizes chondroitin sulfate proteoglycan (CSPG), a cell-surface protein that is expressed in subsets of neurons in several species including cats, monkeys and humans (McKay and Hockfield, 1982; Hockfield and McKay, 1983; Hendry et al., 1984; Hendry et al., 1988; DeYoe et al., 1990; Horn et al., 2008).

GAD65/67

The immunogen was a synthetic peptide with the amino acid sequence [C]DFLIEEIERLGQDL from rat glutamate decarboxylase (GAD65; C-terminus residues [Cys]1572–585). The GAD 65/67 antiserum (Millipore, AB1511) recognizes a doublet on Western Blot of mouse brain lysates at 65/68 kDa (manufacturer’s data sheet). Immunostaining was abolished by preincubation with the peptide (manufacturer’s data sheet). This antibody has previously been used for immunohistochemistry in rat brainstem (Belenky et al., 2008; Yeo et al., 2010) and in mouse cerebral cortex (Liguz-Lecznar et al., 2009).

NPNFP

The immunogen was rat brain lysate. The antiserum (Sternberger Monoclonals Inc.; Covance, SMI-32) recognizes a nonphosphorylated epitope on the 168 (M, medium) and 200 kDa (H, heavy) neurofilament subunits (Sternberger and Sternberger, 1983) of most mammalian species, and stains somas, dendrites and some thick axons (manufacturer’s data sheet). It lacks cross reactivity to microtubule-associated protein and to Alzheimer’s disease neurofibrillary tangles (Ksiezak-Reding et al., 1987; Lee et al., 1988). It has been widely used to characterize cortical pyramidal cells in several species (Hof et al., 1990; Hof and Morrison, 1995; Van der Gucht et al., 2007).

nNOS

The nNOS antiserum (Cayman Laboratories,160870) recognized a peptide of 155 kDa on Western blot of mouse brain (manufacturer’s quality control sheet). No immunostaining was seen on control sections in which the antiserum was omitted, or on sections in which the antibody was first incubated with the immunizing protein (Cayman Laboratories, Dallas, TX; Cat. No. 360871, 1 µg/ml) for 1 hour at room temperature prior to dilution (1:200 in 2 ml of antibody diluent; following manufacturer’s instructions) and immunostaining.

Parvalbumin

The immunogen was rat muscle parvalbumin. The antiserum (Millipore, AB9312) recognizes both the calcium bound and calcium-free forms of parvalbumin. The specificity of this antibody for parvalbumin in the rodent brain was described by (Felch and Van Hooser, 2012). It was also shown to label neurons in cortex of rat and mouse (Salgado et al., 2007; Schmid et al., 2008) and also used in a study of cortical dysplasia in humans (André et al., 2008).

We did not have VCN sections immunostained with every antibody for every species. As detailed in the individual reports, we used two different visualization protocols for the immunohistochemistry. For the older experiments, we used a standard DAB protocol; visualizing immunoreactivity by incubating sections in 3,3’-diaminobenzidine (DAB, Sigma) with 0.0015–0.003% H₂O₂ in PBS (Baizer and Baker, 2005). For more recent experiments, we used a glucose oxidase (GO) modification of that protocol (Van der Gucht et al., 2006). The immunolabeling on sections processed for DAB is brown, that on the GO sections is gray-black; examples of both are illustrated. A few immunostained sections were counterstained with the Giemsa stain, resulting in blue-stained somata.

Data Analysis and Photography

Sections were examined with a Leitz Dialux 20 light microscope, and digital images (1200 × 1600 pixels) captured with a SPOT Insight Color Mosaic camera. Brightness and contrast of images were adjusted and figures assembled with Adobe Photoshop software (San Jose, CA). We also used Photoshop software to arrange all panels in each figure to show the same side of the brain to facilitate comparisons.

RESULTS

Human: organization of CN

Earlier reports found that the three-dimensional arrangement of the CN in relation to each other and to the surrounding brainstem varies among individuals (Moore and Osen, 1979). We therefore examined both Nissl and immunostained sections from each case to compare CN organization. The typical organization of the CN is shown in Figure 1 on Nissl-stained sections from one case. In humans, the DCN is typically found along the edge of the brainstem (Fig. 1A). Just ventral to it is a region with large, darkly stained, scattered neurons with somata of different shapes and sizes (illustrated in Fig. 1B). We consider this region to be the VCP, like the DCN it is found along the free edge of the brainstem (Fig. 1C, E, G). The sizes, shapes and density of the stained neurons are similar over the rostro-caudal extent illustrated (Fig. 1D, F, H). At more rostral levels (Fig.1I, K) the region labeled “VCA” is not on the free surface of the brainstem but embedded in fibers of the middle cerebellar peduncle. Compared to the VCP, the VCA has smaller, more densely packed neurons, most with round or oval somata (Fig 1J, L). We saw similar results in other cases in both Nissl and immunostained sections, supporting the conclusion that two VCN subdivisions distinguished by different cytoarchitecture are present in humans.

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.

Neurochemistry of human VCP and VCA

We first used Nissl-stained sections to identify the VCP and VCA. We next examined sections immunostained with markers that had been useful in delineating cell populations in the human DCN and other nuclei in the medulla (three calcium-binding proteins: calbindin (CB), calretinin (CR), and parvalbumin (PV); the synthetic enzyme for GABA (GAD65/67), the synthetic enzyme for nitric oxide (nNOS), and nonphosphorylated neurofilament protein (NPNFP); Baizer et al., 2007; Baizer and Broussard, 2010; Baizer et al., 2011a; Baizer et al., 2014). We found that antibodies to three proteins, CR, nNOS and NPNFP, immunolabeled somata in the human VCN. IHC with antibodies to the two other calcium-binding proteins, CB and PV, labeled elements in other brainstem regions of humans, but did not label somata or processes in the human VCN. GAD65/67 labeled puncta and not somata in the VCN. We therefore focus the description of results on the immunostaining patterns seen with antibodies to the three proteins expressed in VCN somata. With these antibodies, there were immunolabeled neurons in both VCN subdivisions. Each marker labeled several distinct neuronal populations as defined by soma shape and dendritic appearance and each immunolabeled somata in both the VCP and the VCA. Similar results were seen in multiple cases. Because the human cases varied in several critical parameters including age, sex, PMI and cause of death (Table 1) that could affect proteins and immunostaining, we illustrate the results with sections from more than one case. We also show photomicrographs of Nissl-stained sections to allow a qualitative comparison of sizes, shapes and numbers of immunostained neurons with the overall neuronal population.

We first illustrate CR immunostaining. Figure 2A, B show two adjacent sections in the VCP, a Nissl-stained section (Fig. 2A) and a CR-immunoreactive (-ir) section (Fig. 2B). At low magnification (Fig. 2B) somata and proximal dendrites are CR-ir. The CR-ir neurons are similar in size, shape and number to the neurons seen on the Nissl-stained section (Fig. 2A). At a higher magnification (Fig. 2C) immunostained somata, proximal dendrites and many stained fibers running at all angles are seen. The immunostained somata are not a uniform population; there is CR immunostaining of neurons with round, oval, and polygonal somata. CR may be expressed in octopus cells, one of the major cell types of the VCP. Figure 2C (arrow) shows a neuron with strong immunostaining of proximal dendrites extending from one side of the soma, a configuration characteristic of octopus cells. However, CR-ir is also seen in neurons with polygonal somata (Fig. 2C, arrowhead). There is also CR immunostaining more rostrally, in the VCA (Fig. 2C–F). On Nissl-stained sections in the VCA there are many round somata that are smaller and more densely packed than the neurons in the VCP (compare Figs. 2A and 2D). CR-immunostaining (Fig. 2E) shows a relatively uniform population of neurons with round or oval somata. At higher magnification (Fig. 2F), many neurons with a single dendrite are seen (Fig. 2F, example at arrow), a characteristic of bushy cells. In addition, there are stained processes running at all angles; these may represent either afferent or efferent axons. CR immunoreactivity is thus seen in both the VCP and the VCA and in several different neuronal types.

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.

There are also NPNFP-ir neurons in both the VCP and the VCA (Fig. 3). Figure 3A shows a relatively caudal Nissl-stained section at the level where the DCN is present dorsally and the VCP ventrally. The adjacent section (Fig. 3B) was immunostained for NPNFP. There are scattered large Nissl-stained somata in the VCP (Fig. 3A); the arrow in Figure 3B indicates the location of the border between the DCN and the VCP. There is dense immunostaining in the VCP. The higher magnification photomicrograph (Fig. 3C) shows that there are many immunostained neurons embedded in a very dense meshwork of darkly stained fibers running in all directions. The arrow (Fig. 3C) shows a neuron with stained dendrites emerging from one side of the soma (octopus-like cell) and the arrowhead shows another neuron with a single large primary dendrite (characteristic of bushy cells). Figure 3D shows a more rostral Nissl-stained section with the VCA embedded in the fibers of the middle cerebellar peduncle. There are many small round somata. Figure 3E is a low-magnification photomicrograph of an NPNFP-stained section, as in the VCP there is dense NPNFP immunostaining. Figure 3F shows a higher-magnification photomicrograph of immunostained neurons. There are many immunostained smaller round somata that are embedded in a dense meshwork of stained processes. The arrowhead (Fig. 3F) indicates a neuron with a single primary dendrite (bushy cell). The appearance of the immunostained neurons is similar for CR and NPNFP, the main difference between the two markers is in the greater staining of fibers and processes with NPNFP immunostaining.

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.

The third marker that immunostained somata was nNOS. Nissl-stained neurons are similar in size and shape to the neurons immunolabeled with CR and nNOS (Fig. 4), suggesting that CR and nNOS might be expressed in the same neurons. Nissl-stained somata in the VCP are relatively large, and of a variety of shapes including round (Fig. 4B, arrow), oval, or polygonal (Figs. 4B, arrowhead). As in Case 176, neurons and fibers in the VCP are CR-ir (Fig. 4C, D); the immunostained neurons can have round, oval or polygonal somata (Fig. 4D, arrow). There are also CR-ir fibers; in this section they run roughly parallel to each other. The nNOS staining pattern (Fig. 4E) is similar to that with CR. At higher magnification (Fig. 4F) it is clear that nNOS-ir also labels neurons with round, oval (arrow) and polygonal somata (arrowhead). There is some immunostaining of proximal dendrites, but much more limited staining of processes running throughout the section than with CR. In this case, there is CR and nNOS immunolabeling of somata of similar sizes, shapes, density and distribution in the VCP but there is much more extensive staining of processes with CR than with nNOS.

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.

The pattern of nNOS immunoreactivity is similar in a second case (Case 168, Fig. 5, A, B, C). There are nNOS-ir in somata in the VCP (Fig. 5D, 5E). The immunostained neurons are not of a single type, with somata of different shapes including elongated (arrow) and polygonal (arrowhead). There is also immunostaining of processes and puncta. There is similarly dark immunostaining in the VCA (Fig. 5C) that is comprised of immunostained neurons with round (Fig. 5F, arrow) oval, and polygonal somata. While there is some immunostaining of proximal dendrites, and of puncta there is very little staining of fibers in the section.

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.

In summary, Figures 1–5 show that there are populations of neurons in both the human VCP and VCA that are immunoreactive for CR, nNOS and NPNFP. At low magnification, immunostaining with these markers appears similar in both the VCP and the VCA. Neurons of comparable sizes and shapes in both the VCP and the VCA are labeled by all three; we did not find a unique marker for any identified neuronal types. We next asked whether this pattern is the same as in other mammals.

Chimpanzee: organization of CN

In humans the DCN and caudal VCP are usually found at the same rostro-caudal level on the lateral edge of the medulla (Figs. 1–5; also see Olszewski and Baxter, 1954). The VCA is located more rostrally and is embedded in the fibers of the middle cerebellar peduncle (Figs.1, 2, 3, 5). The organization of the CN in the chimpanzee is illustrated on four Nissl-stained sections about 1 mm apart (Fig. 6, A–D, chimpanzee WM). Caudally, the DCN is bordered by fibers and is not on the lateral edge of the brainstem (Fig. 6A), unlike the arrangement in humans. At this level, the VCP, characterized by large, scattered darkly stained neurons, appears ventral to the DCN, as in humans. Also as in humans, the VCA is found rostrally embedded in fibers (Fig. 6D) with darkly stained neurons with round somata. A border between the VCP and VCA at intermediate levels (Fig. 6C) is difficult to distinguish. There are differences in the density of stained neurons along the dorso-ventral extent of the VCN. It may be that both the VCP and VCA are present at this level.

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.

Chimpanzee: neurochemistry of the VCN

The overall neurochemistry of the chimpanzee CN was very similar to that in humans. As in human, there are neurons immunoreactive to CR, nNOS and NPNFP in both the VCP and the VCA. Each marker appears to be expressed in multiple neuronal types. We show the critical features on sections from each of the three chimpanzee brains studied. Figure 6 shows immunostaining NPNFP and CR in a chimpanzee. There is NPNFP immunostaining of processes and somata in the VCP (Fig. 6E, F). There is a patch of dark staining at the ventral tip of the VCP (Fig, 6E, rectangle), comprised of staining of darkly stained somata and proximal dendrites. The somata include a number of shapes including triangular (arrow, Fig. 6F). Other neurons have oval and irregular somata. None of the examples in this section has sufficient staining of proximal dendrites to be clearly identified as an octopus cell; however the neuron at the arrowhead in Figure 6F is a candidate. The NPNFP-ir neurons are embedded in a dense meshwork of stained processes running in all directions. As in humans, NPNFP labels neurons in the VCA as well as in the VCP (Fig. 6G, H). At lower magnification (Fig. 6I) there is overall dark immunostaining and staining of somata. The higher magnification photomicrograph (Fig. 6H) shows that many stained somata are round or oval. There is staining of processes; the arrow indicates a neuron with a single prominent dendrite, fitting the description of a bushy cell. There is thus NPNFP-immunostaining of somata in both the VCP and the VCA in the chimpanzee. We were able to compare CR and NPNFP immunostaining in the VCA in this case. There are CR-ir somata in the VCA; at low magnification many densely- spaced stained somata are evident (Fig. 6I). At higher magnification (Fig. 6J) there are round or oval somata (the white arrow indicates two cells with round somata and single dendrites); as well as neurons with multiple proximal dendrites (Fig. 6J, arrowhead).The overall appearance of the immunostained somata in the CR-ir and NPNFP-ir sections in the VCA is very similar. Comparison with the Nissl-stained section in the VCA (Fig. 6D) suggests that about the same size, number and distribution of neurons are immunostained as were Nissl-stained.

We also found that, as in humans, nNOS is expressed in neurons in the VCP and VCA of chimpanzees. Figure 7A–F illustrates both nNOS and NPNFP immunolabeling in the VCP and VCA of chimpanzee AN. There are scattered nNOS-ir somata in the caudal VCP (Fig. 7A). The somata are of several shapes; there is very little staining either of proximal dendrites or fibers (Fig. 7B). nNOS-ir somata are found more rostrally in the VCP (Fig. 7C) and again the somata are round or oval (Fig. 7D) with little staining of proximal dendrites and almost no staining of fibers. Finally, more anteriorly in the VCA there are scattered nNOS-ir somata (Fig. 7E). As in the VCP, nNOS labels somata, but not fibers or dendrites (Fig. 7F). We also immunostained sections from this case for NPNFP allowing a comparison of the immunostaining with the two markers in sections from the same case. Caudally in the VCP (Fig. 7G) there are many NPNFP-ir somata and these are embedded in a dense network of stained processes and fibers (Fig. 7H). The same pattern is seen more rostrally in the VCP (Fig. 7I). The immunostained somata are of a variety of shapes and include neurons that resemble octopus cells (Fig. 7J, arrow). NPNFP-ir is also seen in many neurons over the extent of the VCA (Fig. 7K). The higher magnification image (Fig. 7J) shows that NPNFP-ir is also seen in fibers and puncta (Fig. 7L). Labeled somata are primarily round or oval and in many a single proximal dendrite is seen (Fig. 7L, example at arrow). Comparing the nNOS and NPNFP immunolabeling suggests that the numbers, sizes and distribution of labeled somata for the two is similar in the VCP and VCA.

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.

NPNFP and CR are also expressed in comparable neuronal populations in the VCA of chimpanzees (Fig. 8). Figure 8A, B show a CR-immunostained section in the VCA. There are immunostained somata of a variety of shapes, (round, arrow; polygonal, arrowhead) and staining of proximal dendrites. There is some punctate staining but very little staining of fibers. Figure 8C, D shows NPNFP-immunostaining in VCA; both somata and proximal dendrites are labeled. Immunostained somata are of a variety of shapes, round (arrow) and multipolar (arrowhead) and are similar in size, shape and distribution to the CR-ir neurons. In contrast to the CR staining with NPNFP there is also staining of fibers running in all directions and some staining of puncta. Figure 8E, F show a Nissl-stained section in the VCA; there are large neurons with round (arrow), polygonal and elongated cell bodies. Comparison of the immunostaining in the VCA with CR and NPNFP with the Nissl staining (Fig. 8E, F) at the same level suggests a very similar number, shape and distribution of stained neurons. Again, there is no suggestion that CR and NPNFP are expressed in different populations of neurons.

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.

Macaque monkey

We used published data as a guide to the location and extent of the VCP and the VCA in macaques (Strominger and Strominger, 1971; Paxinos et al., 2000). In the monkey, unlike the human or chimpanzee, the DCN is shown caudal to the VCP (Paxinos et al., 2000, see plates 107 and 108) which in turn is caudal to the VCA. We examined the borders and cytoarchitectural characteristics of the CN using Nissl-stained sections and sections immunostained for the protein CAT-301. (The CAT-301 antibody did not yield reliable immunostaining in humans or chimpanzees, but it was very useful in indicating CN subdivisions and outlining the somata and proximal dendrites of individual neurons in both macaque monkeys and cats). On our sections the DCN, located caudally, has a distinctive laminar structure (Fig. 9A). More rostrally, however, we found the VCP and VCA to overlap in rostrocaudal extent. Figure. 9B, C show the VCA is present dorsally and the VCP ventrally. Figure 9C clearly shows both nuclei and the fibers dividing the two. The immunolabel for CAT-301 outlines somata of different shapes, sizes and distributions (Fig. 9D–G). On the most caudal section illustrated (Fig. 9D) both the VCA and VCP are seen, more rostrally (Fig. 9F) only the VCA is present. Immunostained somata are distributed throughout both the VCP and the VCA. At higher magnification (Fig. 9E, G) somata ringed by immunostaining are seen. In the VCP (Fig. 9E) somata are round, oval or multipolar. In the VCA (Fig. 9G), somata are smaller, round or oval and more closely spaced. We then analyzed immunostaining patterns with CR, nNOS and NPNFP, the three markers that immunolabeled somata in VCP and VCA of humans and chimpanzees. For macaques, we had sections that were immunostained for CR and counter-stained with a Giemsa cell stain. At low magnification there is relatively uniform staining; it is difficult to identify immunostained elements in the VCP (Fig. 9H). At higher magnification, (Fig. 9I) scattered immunostained somata with minimal staining of proximal dendrites are apparent (example in Fig. 9I, arrow). The immunostained somata are surrounded by many immunostained puncta and fibers. All elements are distributed across the entire VCP. There are also neurons that are stained only by the Giemsa counterstain (example at Fig. 9I, arrowhead), suggesting that only a subset of VCP neurons expresses CR. Similar CR-ir is seen in the VCA (not shown) with CR-ir in somata and fibers. Again, on a Giemsa-counterstained section it was clear that not all somata were CR-ir. For nNOS, at low magnification immunolabeling in the VCP and VCA is relatively pale (Fig. 9J). Scattered labeled somata in the VCP (Fig. 9K, example at arrow) are seen at higher magnification, with very little staining of puncta or fibers. The immunostaining for NPNFP in macaque monkey is similar to that seen in humans and chimpanzees. Immunostaining of somata in the VCP is clearly seen even at low magnification (Fig. 9L); dense staining of somata, dendrites and processes is dramatic at higher magnification (Fig. 9M). The density of immunostained somata on the NPNFP immunostained section (Fig. 9M) is much higher than that seen with either CR (Fig. 9I) or nNOS (Fig. 9K), in contrast to the human and chimpanzee in which immunostaining was similar for all three.

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.

Cat

We used the atlas of Berman (1968) as well as our own Nissl and immunostained-stained sections to identify the DCN, VCP and VCA. As with monkeys, we found that immunoreactivity to CAT-301 clearly defines the CN components. At caudal levels both the DCN and the VCP are present (Fig. 10A, E), more rostrally both the VCP (ventrally) and the VCA (dorsally) are present and still further rostrally only the VCA is present (Fig. 10I). With CAT-301, somata ringed by immunostaining are distributed across the VCP (Fig. 10E). The immunolabeled somata are of several shapes; for some proximal dendrites were also labeled (Fig. 10B). The arrow in Figure 10B shows a large neuron with dendrites emerging from one side, a pattern characteristic of octopus cells. The arrowhead indicates a neuron with a small round soma. There is no label of puncta or fibers.

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.

Again, our goal was to compare the pattern of immunolabeling in cats with the three markers that labeled neurons in the human VCN (CR, nNOS, NPNFP) with the patterns in other species. In the VCP, CR immunolabeled elements that were distributed over the entire extent of the nucleus (Fig. 10C), including somata, fibers and puncta (Fig. 10D). The CR-immunolabeled somata were of a variety of shapes (Fig. 10D; the arrowhead marks a neuron with a single dendrite). There were darkly labeled puncta, presumably terminals of afferent fibers, surrounding many of the labeled somata (Fig. 10D, arrowhead). nNOS also labeled neurons of different shapes in the VCP distributed over its entire extent (Fig. 10E). There was some label of proximal dendrites (Fig. 10F) but not of puncta or fibers. Neurons resembling octopus cells (Fig. 10F, arrow) as well as neurons of other soma shapes (Fig. 10F, arrowhead) were nNOS-ir. As with the other species described so far, NPNFP immunolabeling defines somata, proximal dendrites, and a dense network of fibers distributed over the entire VCP (Fig. 10G). Immunostained somata are of a number of shapes including oval (Fig. 10H, arrowhead) and multipolar (Fig. 10H, arrow). There were also immunolabeled elements with each marker in the VCA. With CR, immunolabeling was found throughout the VCA (Fig. 10I), comprised of immunolabeled somata, puncta and fibers (Fig. 10J). nNOS-ir was also seen over the entire VCA (Fig. 10K) and included immunolabeled scattered small neurons with beaded dendrites (Fig. 10L arrow) as well as larger somata. Finally, there is dense NPNFP label all over the VCA (Fig. 10M) that is composed of somata (Fig. 10N, arrow shows two neurons with round somata and single dendrites) dendrites and fibers.

Rat

We followed the atlas of Paxinos (1999) to identify VCN subdivisions and to determine the Bregma levels of our sections. Again the goal was to compare the patterns of immunostaining for CR, nNOS and NPNFP in rats with humans and the other species studied. Figure 11 compares CR (Fig. 11A–D), nNOS (Fig. 11E–F) and NPNFP (Fig. 11 I–L) immunostaining in both the VCP and VCA. CR is expressed in some large somata in the VCP (Fig.11B, arrow). There are also a few labeled small somata (Fig. 11D, example at arrow). As in all other species, CR immunostaining is also seen in fibers and puncta in both subdivisions. nNOS also immunolabeled somata of different shapes in the VCP (Fig. 11F, arrow, large labeled multipolar cell), other examples in that photomicrograph have smaller round or oval somata. In the VCA (Fig. 11H) only small somata are immunolabeled. There was a dramatic difference in NPNFP expression in rats compared to the other species. While low magnification images (Fig. 11, I, K) in the VCP and VCA show uniform staining, higher magnification images show NPNFP immunoreactivity in fibers (Fig. 11J, L, arrows) and puncta but not in somata in either the VCA or VCP.

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.

Chinchilla

The general organization of the VCN in chinchillas is similar to that in rats and we assessed whether there might be similarities in neurochemical characteristics of the VCN as well. Figure 12 shows immunolabeling for CR (A–D), nNOS (E–H) and NPNFP (I–M) in the VCP and the VCA. The chinchilla differed from the rat in the pattern of CR expression. Caudally, there are immunolabeled somata in the DCN, (Fig. 12 A) but in the VCP ventrally on the same section both fibers and puncta are CR-ir but somata are not (Fig. 12B). On a more rostral section, at a level with the VCA dorsally and the VCP ventrally (Fig. 12C) scattered immunostained somata are seen in the VCP. In the VCA, (Fig. 12D) there is dense CR immunolabeling of puncta and fibers, but again no somata. The expression of CR in the chinchilla VCP and VCA is thus different from what was seen in all other species.

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.

With the second marker, nNOS, there is immunolabeling of somata in the VCP and the VCA. At low magnification caudally the DCN is seen dorsally and the VCP ventrally; the border between them is very clearly defined with much darker staining dorsally (Fig. 12E). In the VCP there is immunostaining of somata of different sizes, from relatively large (Fig. 12F, arrow) to small (Fig. 12 F, arrowhead). There are also labeled fibers and puncta. More rostrally there is nNOS immunolabeling evenly distributed over the VCA (Fig. 12G); many small round somata are labeled (example at arrow, Fig. 12H).

Finally, the pattern of immunolabeling with NPNFP is unlike that in human, chimpanzee, cat, and monkey but similar to that in rat. Caudally (Fig. 12I) NPNFP immunolabeling is prominent in puncta and fibers but not somata in the VCP (Fig. 12J). More rostrally, low magnification shows the immunolabeling is uniformly distributed over the VCA (Fig. 12K) but again reflects labeled fibers and puncta but not somata (Fig. 12L).

DISCUSSION

Our earlier analysis of the DCN showed differences in laminar organization and neurochemical properties of different classes of neurons between humans and several other species (Baizer et al., 2014). We have now compared immunostaining for three proteins (CR, nNOS, NPNFP) in the VCN of six species: humans, chimpanzees, macaque monkeys, cats, rats and chinchillas. Table 3 summarizes the similarities and differences in immunostaining among species. For humans and chimpanzees, immunostaining was remarkably similar with all three markers. In those species, we did not identify either separate neuronal populations or VCN subdivisions on the basis of immunostaining. There were different immunostaining patterns with these markers in the other species. We shall first discuss the results for humans and chimpanzees and then compare those results with the data from the other species.

Table 3.

Summary of CR, nNOS and NPNFP immunolabel.

Species	VCPVCA	CR	nNOS	NPNFP
Human	VCP	fibers		fibers –very dense
		somata	somata	somata
	VCA	fibers		fibers- very dense
		somata	somata	somata
				proximal dendrites
Chimpanzee	VCP			fibers –very dense
		somata	somata	somata
				proximal dendrites
	VCA			fibers
		somata	somata	somata
		proximal dendrites		proximal dendrites
Macaque monkey	VCP	fibers	fibers	fibers
		somata (not all)	somata	somata
	VCA	fibers		fibers
		somata (not all)	somata	somata
Cat	VCP	fibers		fibers
		somata	somata	somata
		proximal dendrites	proximal dendrites	proximal dendrites
	VCA	fibers		fibers
		somata	somata	somata
				proximal dendrites
Rat	VCP	fibers		fibers
		somata	somata	NO SOMATA
		proximal dendrites
	VCA	fibers		fibers
		somata- very few	somata	NO SOMATA
Chinchilla	VCP	fibers	fibers	fibers
		caudal-NO SOMATA	somata	NO SOMATA
		rostral-somata
	VCA	fibers		fibers
		NO SOMATA	somata	NO SOMATA

Humans and chimpanzees: subdivisions of the VCN

Our results for both humans and chimpanzees support the division of the VCN into two subdivisions, VCP and VCA. However, these nuclei are clearly distinguished by cytoarchitecture rather than by neurochemistry. In both humans and chimpanzees, the VCP is characterized by more heterogeneity in cellular organization than the VCA; its neurons in general are larger and less densely packed. The differences in cytoarchitecture are clearest between the caudal VCP and the rostral VCA. The boundary between them at intermediate levels is not as obvious; the ambiguity of a clear border in humans was also noted by Moore and Osen (1979). The difference in cytoarchitecture between VCP and VCA is in agreement with observations in other species (e.g. Brawer et al., 1974) and also with earlier reports on human VCN organization (Bacsik and Strominger, 1973; Moore and Osen, 1979). Immunolabeled neurons were distributed over the entire extent of both the VCP and the VCA, there was no evidence of segregation of expression of particular proteins into one nucleus or compartments within nuclei as has been reported for seen in other brainstem and thalamic nuclei (Moreno-Lopez et al., 1996; Moreno-Lopez et al., 2001; Moreno-Lopez et al., 2002; Baizer and Baker, 2005, 2006a, 2006b; Baizer and Broussard, 2010; Baizer et al., 2013a).

Neurons in the VCP and VCA of humans and chimpanzees

In both humans and chimpanzees, there were three proteins, CR, nNOS and NPNFP, that were expressed in neurons in both the VCP and VCA. The labeled neurons were similar in size, shape and density with all three markers. One interpretation of these data is that CR, nNOS and NPNFP may all be expressed in the same neurons; double-labeling experiments are needed to confirm this suggestion. Another possibility is that all neurons in the nuclei are immunostained. This idea is also supported by a comparison of the immunostained sections with adjacent Nissl-stained sections; similarities in size, shape and density of Nissl compared to immunostained neurons also suggests that the entire population of VCN neurons is immunolabeled. Again, this is a tentative conclusion based on qualitative comparisons and requires rigorous quantitative analysis to be established.

The three markers did differ in the nature of the immunolabel of puncta, fibers, and processes. CR is expressed in dendrites and some fibers as well as in somata. With nNOS there is virtually no label of fibers, very limited label of dendrites, and some punctate label. There is NPNFP immunoreactivity in a network of stained fibers, the network is sometimes so dense that the characteristics of individual neurons were difficult to discern. There is much more expression of NPNFP than CR in fibers. This could reflect either more extensive immunolabel of neuronal processes or immunolabel of selective populations of afferent fibers.

Neuron types and neurochemistry, humans and chimpanzees

As none of the markers used resulted in extensive label of dendritic trees, we could only use soma shape and staining of proximal dendrites to compare the neurons we labeled with established cell types. We were able to identify three major neuron types in the human and chimpanzee VCN: octopus cells, bushy cells and multipolar cells; all of which have been described in many species (review in Cant and Benson, 2003) including humans (Bacsik and Strominger, 1973; Richter, 1983; Adams, 1986). With each marker there were examples of immunolabeled neurons that fit the criteria of bushy, multipolar and octopus cells. We classified labeled neurons into only three major types. We were not able to subdivide bushy cells or multipolar cells into subclasses based on their appearance in immunostained sections. However, we used a limited set of antibodies to investigate the VCN. It is certain that there are differences in neurochemistry among the different neuron populations identified by other anatomical and electrophysiological criteria since they vary in connections (affecting receptor types and distributions) and transmitters (affecting many key proteins critical for transmitter synthesis and reuptake). Such differences presumably could be detected with additional markers, but a thorough search would be extremely difficult given the number of candidate proteins.

These markers have been used in many other brain regions to categorize different classes of neurons. For example, in cerebral cortex NPNFP identifies a subset of projection neurons, while calcium-binding proteins are expressed in different classes of GABAergic interneurons (Van Brederode et al., 1990; Hendrickson et al., 1991; van Brederode et al., 1991; Hof and Morrison, 1995; Hof et al., 1995; Hof et al., 1999; DeFelipe et al., 2013).

VCN organization, neurochemistry and neuron types in other species

Our goal was to compare the neurochemical characteristics of major cell types in other species with the results from humans and chimpanzees. Our results showed species differences in immunolabeling with the three markers that labeled somata in the VCP and VCA of humans and chimpanzees (summary in Table 3).

Macaques

Both the Nissl and CAT-301-immunostained sections show the differences of cytoarchitecture between the VCP and the VCA. The immunostaining with each marker is uniformly distributed with no suggestions of neurochemically defined subregions. In macaques, as in chimpanzee and human, somata in the VCP were immunolabeled with all three markers and examples of multipolar cells and neurons with round somata were found with each. However, in contrast to the data from human, several observations suggest that not all somata are immunolabeled with CR, nNOS, or NPNFP. Sections immunostained for CR and counterstained with a Giemsa cell stain showed examples of Giemsa-stained neurons that were not CR-ir. We found somata in VCA that were immunolabeled with an antibody to the calcium-binding protein parvalbumin (data not shown); somata in human and chimpanzee were not immunoreactive for this protein.

Cat

CAT-301 immunoreactivity is useful in showing the diversity of soma shapes in the VCP and the overall neuronal density (Fig. 10A, B). Cats were similar to the macaques in that there were examples of somata in the VCP that were immunoreactive for CR, nNOS and NPNFP and these included more than one soma shape. The label of fibers and puncta with CR is very heavy; CR-ir puncta are seen surrounding neurons. However, the cat VCN may be more neurochemically diverse than that of humans or chimpanzees; we found both PV-ir and CB-ir neurons in the VCP of cats and not in humans. In the VCA there are also CR-ir, nNOS-ir, and NPNFP-ir somata.

In both cats and macaques the CAT-301 antibody labeled somata of several different sizes and shapes in both the VCP and the VCA. Its apparent lack of selectivity in the VCN of these species is in contrast to the findings in cerebral cortex, superior colliculus and thalamus where specific cell populations are CAT-301-ir (Hendry et al., 1984; Mize and Hockfield, 1989; Hockfield and Sur, 1990; Crabtree and Kind, 1993).

Rat and chinchilla

While the four species described so far have basic neurochemical similarities in VCN neurons, the two rodents, the rat and the chinchilla, have different immunolabeling with the same markers and are were also different from each other. This may not be surprising since the rat is an Old World rodent and the chinchilla is from the New World and are separated from each other by approximately 70 million years of independent evolution (Fabre et al., 2012). CR immunolabeling in rats reflects very heavy label of fibers and puncta in both the VCP and VCA; there are also some CR-ir somata. These data again suggest that CR-ir does not define a single neuron population. This is in contrast to the results for the vestibular ganglion where calcium-binding proteins are expressed in subpopulations of neurons (Raymond et al., 1993). The pattern in the chinchilla is different. In caudal VCP, CR is expressed in fibers and puncta but not somata. There are labeled somata more rostrally in the VCP, but in the VCA there are no somata, just puncta and fibers. These data strongly suggest that in contrast to the rat, in chinchilla CR expression is limited to a restricted set of neurons. The CR-ir fibers and puncta probably reflect the expression of this protein in fibers of the auditory nerve (rat, Arai et al., 1991; Résibois and Rogers, 1992; mouse, Idrizbegovic et al., 2001; gerbil, Bazwinsky et al., 2008). Species differences in the expression of CR have also been reported for the dentate gyrus of the hippocampus (Murakawa and Kosaka, 1999).

In the rat, nNOS also is expressed in neurons in both the VCP and the VCA, with the labeled neurons in the VCA smaller than those in the VCP, suggesting its expression in more than one neuronal type. In chinchilla, nNOS-ir, labeled somata in VCP and a population of very small somata in the VCA. It should be noted, however, that as nNOS may also be expressed in glial cells some of the smaller profiles could be glia (Galea et al., 1992; Park et al., 1994; Simic et al., 2000). The immunolabeling with NPNFP in both and chinchilla is very different from that in all other species in that there is dense immunolabeling of fibers and puncta but not of somata.

What is the significance of the absence of NPNFP expression in neurons of the rodent VCN? NPNFP immunoreactivity is seen in axons and puncta, presumably axons of neurons in afferent structures. Neurofilaments are essential components of axons (Rao et al., 2012). The difference of NPNFP expression in neurons of difference species could be related to differences in the composition of axons as a function of length; axon lengths obviously vary greatly among the different species. It could also correlate with differences in mechanisms of plasticity or response to injury among species (Yang et al., 1995; Siman et al., 2009; Johnson et al., 2016).

How do our results compare with earlier studies of neurochemical properties in the VCN? Interpreting differences in immunohistochemical results among laboratories and species is complicated by possible differences in the antibodies, tissue history and preparation, many details of immunostaining protocols, and strains and ages of the animals used. Our results are not in total agreement with other reports. There are differences among studies in descriptions of CR expression in VCN neurons. Arai et al. (1991) found CR-ir neurons in both the VCP and the VCA of the rat, in agreement with our observations. They also noted immunostained endbulbs of Held. Résibois and Rogers (1992) found that CR-ir in large globular neurons and octopus neurons in the VCP but not in “spherical cells” (bushy cells) in rostral VCA. In contrast to our observations of lack of NPNFP-ir somata, an earlier study that found about 30% of neurons in the VCN of the rat were NPNFP-ir (Ouda et al., 2012b). Again, the reason for the discrepancy is not clear.

Regardless of the differences among reports, our data clearly support the idea that there are species differences in the expression of several proteins that have key roles in neuronal function. There is one important limitation to this study. The data from humans and chimpanzees was from relatively old animals, the other species were on average much younger. There are suggestions that expressions of different proteins decrease with age (Iacopino and Christakos, 1990; Kishimoto et al., 1998), differences in relative age could affect neurochemical diversity in the older humans compared to the younger animals. However, whatever the time course, it is still important to consider the implications of the differences for understanding the auditory system. The human auditory system is very vulnerable to an age-related loss of receptor cells resulting in hearing loss and, in some patients, associated pathological conditions like tinnitus and hyperacusis (Katzenell and Segal, 2001; Baguley, 2003; Eggermont, 2005; Roberts et al., 2010; Shargorodsky et al., 2010a; Shargorodsky et al., 2010b; Auerbach et al., 2014). There is thus much interest in understanding the plastic responses of the auditory system after receptor loss, especially those resulting in auditory pathology. One approach to understanding auditory plasticity is to use animal models, most commonly the rat and chinchilla, and study changes in auditory structures after damage to the periphery either through noise exposure (Abbott et al., 1999) or the use of ototoxic drugs (Hofstetter et al., 1997; Zhou et al., 2009). Many studies have shown cellular, molecular and electrophysiological changes at different sites in the auditory system in animals after hair cell loss (Wang et al., 1996; Benson et al., 1997; Abbott et al., 1999; Taggart et al., 2001; Wang et al., 2002; Norena and Eggermont, 2005; Valentine et al., 2005; D’Sa et al., 2007; Kraus et al., 2009; Fuentes-Santamaria et al., 2012; Fuentes-Santamaria et al., 2013; Baizer et al., 2015a). The differences of neurochemical profiles of neurons in humans and the experimental species suggest that the neurochemical correlates of the mechanisms of plasticity discovered in animals may not always translate directly to humans.