Wisteria floribunda lectin is associated with specific cell types in the ventral cochlear nucleus of the gerbil, Meriones unguiculatus

Hearing Research

Hearing Research 216–217 (2006) 64–72

www.elsevier.com/locate/heares

Research paper

Wisteria floribunda lectin is associated with specific cell types in the ventral cochlear nucleus of the gerbil, Meriones unguiculatus Nell B. Cant *, Christina G. Benson Department of Neurobiology, Duke University Medical Center, P.O. Box 3209, 213 Bryan Research Building, Durham, NC 27710, United States Received 23 November 2005; received in revised form 9 January 2006; accepted 10 January 2006 Available online 23 February 2006

Abstract The cochlear nucleus is made up of a number of diverse cell types with different anatomical and physiological properties. A plant lectin, Wisteria floribunda agglutinin, that recognizes specific carbohydrate residues in the extracellular matrix binds to some cell types in the ventral cochlear nucleus but not to cells in the dorsal cochlear nucleus. In the ventral cochlear nucleus, the most intensely labeled cells are octopus cells, a subset of multipolar cells and cochlear root neurons. The multipolar cells that are labeled may correspond to the population that projects to the inferior colliculus. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Auditory system; Octopus cells; Multipolar cells; Perineuronal nets

1. Introduction The mammalian cochlear nucleus is populated by a variety of neuronal types that can be distinguished by differences in morphology, physiological properties, neurotransmitter chemistry, and connectivity (reviewed in Romand and Avan, 1997; Cant and Benson, 2003; Young and Oertel, 2004). The cell types first defined in cats (Osen, 1969; Brawer et al., 1974) and rats (Harrison and Irving, 1965, 1966) are also recognized in other species, including humans (reviewed by Cant, 1992; human: Moore and Osen, 1979; Richter, 1983; Adams, 1986, 1997). Many of the distinct cell populations in the cochlear nucleus, as well as the tracts formed by their axons, are segregated from one another, Abbreviations: AVCN, anteroventral cochlear nucleus; BDA, biotinylated dextran amine; cbm, cerebellum; cnr, cochlear nerve root; DCN, dorsal cochlear nucleus; ECM, extracellular matrix; gcl, granule cell layer; icp, inferior cerebellar peduncle; OCA, octopus cell area; PVCN, posteroventral cochlear nucleus; SCA, spherical cell area; scp, superior cerebellar peduncle; tb, trapezoid body; V, descending branch, trigeminal nerve root; VCN, ventral cochlear nucleus; vnr, vestibular nerve root; WFA, Wisteria floribunda agglutinin * Corresponding author. Tel.: +1 919 684 6555; fax: +1 919 684 4431. E-mail address: [email protected] (N.B. Cant). 0378-5955/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.01.008

making it possible to target different cell groups in physiological or anatomical studies. Because of this, the cochlear nucleus is one of the most well-understood parts of the brain with respect to its component neuronal types and their physiology and connections. However, segregation of the different cell populations is not complete. Some of them are intermingled, and the identification of specific markers for each type can aid in distinguishing them in neuroanatomical, neurophysiological and other types of studies. Lectins, proteins that bind to sugar residues in carbohydrate-containing macromolecules, have been shown to bind specifically to some cell groups in the central nervous system and not to others (reviewed by Spicer and Schulte, 1992). Because they can be conjugated to markers such as fluorescent tags or biotin that can be visualized histochemically, lectins can be used to detect the presence of specific carbohydrate-rich components of the extracellular matrix (ECM) in tissues throughout the body, including nervous tissue (see Section 4). In this paper, we describe the binding patterns of one such lectin, Wisteria floribunda agglutinin (WFA), which has been shown to bind to the ECM around some but not all neurons in the central nervous system (e.g., Celio et al., 1998). We suggest, based on the staining patterns in the ventral cochlear nucleus,

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

that WFA can be used in the gerbil to mark the locations of some specific cell types. 2. Materials and methods Young female gerbils were obtained from Charles River Laboratories and housed in Duke University animal quarters until use. The animals used in this study were between 9 and 17 weeks old at the time of sacrifice. All procedures involving animals were approved by the Duke University Institutional Animal Care and Use Committee and were in accord with guidelines established by the NIH. 2.1. Lectin histochemistry Animals were given an overdose of pentobarbital (Nembutal; >70 mg/kg). When withdrawal reflexes were absent and breathing ceased, the chest was quickly opened and the animal was perfused through the heart with a brief rinse of phosphate buffer (0.1 M, pH 7.6) followed by approximately 200 ml of buffered 4% paraformaldehyde. The brain was removed and frozen sections 40 lm thick were cut in one of the three standard planes. The sections were collected in serial order in phosphate buffer. Before they were processed for lectin binding, they were placed in a solution of 0.6% hydrogen peroxide for 30 min. They were then rinsed thoroughly in buffer and incubated in the following solution for 15–20 h at 4 °C: 0.5–1.0 lg/ml biotinylated Wisteria floribunda agglutinin (WFA, Sigma #L-1766 or Vector Laboratories #B-1355), 2% bovine serum albumin, phosphate buffered normal saline (PBS). At the end of the incubation period, the sections were rinsed in PBS, and placed in buffered Vector Elite ABC solution (Vector Laboratories; 1:100) for 1 h at room temperature. After rinses in PBS, the lectin was visualized by placing the sections in 0.05% diaminobenzidine in phosphate buffer with heavy metal intensification (Adams, 1981). The sections were mounted on glass slides, allowed to dry, dehydrated in a series of alcohols and sealed under coverslips with Permount. 2.2. Retrograde tracing study The spatial distribution of lectin binding was compared to the distribution of cells that project from the ventral cochlear nucleus to the inferior colliculus, as determined in an earlier study (Cant and Benson, 2006). Details of the experiments are provided in the description of that study. Briefly, biotinylated dextran amine (BDA) was injected iontophoretically into the central nucleus of the inferior colliculus. After a survival period of 5–11 days, the animals were perfused as above and sections were processed for the presence of BDA. Maps were made of the positions of labeled cells on alternate horizontal sections through the contralateral cochlear nucleus of 25 animals. Each section was then matched to a section at the comparable dorsal-to-ventral level in one horizontal series chosen

65

as a reference; all of the labeled cells from every case were re-plotted onto these reference sections. Because the BDA injections included the entire topographic (tonotopic) axis of the inferior colliculus, this procedure provided an estimate of the spatial distribution of all cells in the cochlear nucleus that project to it. 3. Results In the gerbil, the lectin Wisteria floribunda agglutinin (WFA) binds selectively to the ECM around some but not all of the cell populations in the cochlear nucleus (Figs. 1–6). The dorsal cochlear nucleus (DCN) and the granule cell layer covering the surface of much of the ventral cochlear nucleus (VCN) are almost unstained after incubation with WFA, although occasional cell bodies in the DCN may sometimes be lightly outlined (e.g., Fig. 1F, arrowhead). In the VCN, octopus cells, some but not all multipolar cells, and cochlear root neurons are intensely stained compared to other cell groups. The octopus cells in the PVCN are the most intensely stained cells in the cochlear nucleus. The lectin binds to the ECM around both the cell body and also the large primary and secondary dendrites (Fig. 1F and G, large black arrows; Fig. 2A–C, OCA; Fig. 3A and B, small black arrow). Staining around the cell body has the characteristic reticular appearance often seen with lectin binding (Fig. 3A, inset). The differential staining in the caudal PVCN allows visualization of the ‘‘octopus cell area’’ (Osen, 1969), made up exclusively of this cell type. However, some of the labeled octopus cells on the boundaries of this region appear to intermingle with unstained cells (Figs. 1F and 2C, lower boxed-in area; Fig. 3B). The octopus cells in the gerbil extend dorsally and medially along the path of the intermediate acoustic stria (Fig. 1G, arrow; Fig. 2, panel A, OCA). Most of the other neurons in the VCN labeled by the WFA are multipolar cells located in both the PVCN and AVCN (Figs. 1–6). These cells are smaller than the octopus cells, and the lectin staining is intense only around the cell bodies; dendrites are rarely outlined (Fig. 3B and C, black arrows; Figs. 5 and 6). The lectin-labeled multipolar cells are not distributed homogeneously throughout the VCN. A few are found scattered among large numbers of unlabeled spherical bushy cells in the rostral AVCN (Fig. 1A and B, small arrows; Fig. 5, SCA, arrows). Most of the labeled multipolar cells lie in the middle of the VCN, straddling the border between the AVCN and PVCN (Figs. 4 and 5). In the middle of the PVCN, between the labeled multipolar cells and the octopus cell area, lies a small area that is almost devoid of WFA-binding (Fig. 1F, white arrow; Fig. 2C, lower boxed-in area; Fig. 3B, white arrows). Most of the unlabeled cells in this area are probably multipolar cells, as the globular bushy cells located in the PVCN are situated more ventrally (unpublished observations). We could not distinguish the globular bushy cells from multipolar cells based on lectin-staining patterns. In the

66

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

Fig. 1. Evenly spaced transverse sections through the cochlear nucleus processed for WFA histochemistry. Panel A is the most rostral section shown; panel G is the most caudal. The sections are separated by 280 lm. Octopus cells in the PVCN are intensely stained (large black arrows, panels F and G). On the boundaries of the octopus cell area, labeled cells (F, small black arrows) intermingle with unstained cells. The cell bodies of multipolar cells in the VCN are also intensely stained. Most of these lie in the middle regions of the VCN, but some are scattered among unlabeled spherical cells in the rostral AVCN (A and B, small black arrows). In the ventral PVCN, where globular bushy and multipolar cells are intermingled, almost all the cells are labeled (E, arrows). In the central PVCN, a small almost completely unstained area lies between the octopus cell area and labeled multipolar cells (F, white arrow). Occasional cell bodies in the DCN are lightly stained (F, arrowhead).

Fig. 2. Evenly spaced parasagittal sections through the cochlear nucleus processed for WFA histochemistry. Panel A is the most medial section shown; panel D is the most lateral. The sections are separated by 120 lm. The boxed-in areas in panels C and D are shown at higher magnification in Fig. 3.

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

67

Fig. 4. Horizontal sections through the cochlear nucleus. (A–D) WFAstained sections at approximately the same dorsal-to-ventral level from four gerbils. (E) Composite map of the locations of labeled cells after BDA injections in the IC of 25 gerbils (data from the study reported in Cant and Benson (2006)). Each small injection resulted in labeled cells in a restricted part of the cochlear nucleus. Collapsing the results of 25 cases onto one section provides an estimate of the distribution of all cells that project to the IC. Dotted line on panel E indicates the approximate border between the PVCN and the most ventral part of the DCN. The boxed-in area on panel D is shown at higher magnification in Fig. 5. For all sections, rostral is toward the right of the figure and lateral is toward the top. Scale bar equals approximately 1.0 mm for all sections. Fig. 3. WFA-binding to specific cell types in the PVCN. Each panel is a higher magnification view of the boxed-in areas in panels C and D of Fig. 2. (A) Octopus cells and (B) labeled multipolar cells (large black arrows); unlabeled multipolar cells (white arrows); labeled octopus cell (small black arrow). (C) Labeled cells in the part of the PVCN that contains both multipolar cells and globular bushy cells. Some cells appear to be more lightly labeled (white arrows) than others (black arrow), but the different cell types cannot be distinguished from one another.

gerbil, most of these cells lie caudal to the cochlear nerve root in the ventral part of the PVCN, although a few lie rostral to the nerve root in the caudal part of the AVCN (similar to the distribution in the guinea pig, Moore, 1986). In both cases, they are intermingled with multipolar cells. In the ventral PVCN where most of the globular

68

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

Fig. 5. WFA-labeled cells at the junction of the PVCN and AVCN. This is a higher magnification view of the boxed-in area on Fig. 4, panel D. The multipolar cells in the central part of the VCN stand out because of the lectin binding. Scattered labeled cells are visible in the SCA (arrows) but most of the cells in that part of the AVCN are not labeled. Scale bar = 200 lm.

ular bushy cells are marked by WFA staining to some degree. However, it is not possible to distinguish them from neighboring multipolar cells using this method. A third neuronal population that is labeled with WFA is the small group of large neurons known as cochlear root neurons that lie among the fibers of the cochlear nerve as they enter the brain (Fig. 6). The lectin histochemistry yielded highly consistent results in the cochlear nucleus in all of the cases we examined. Panels A–D in Fig. 4 illustrate horizontal sections located at approximately the same level through the cochlear nuclei of four gerbils. In each case, multipolar cells in the caudal AVCN and rostral PVCN stand out because of the lectin binding in the ECM that surrounds them. The rostral AVCN, where the spherical bushy cells are located, contains only scattered labeled cells (Figs. 4 and 5, arrows). The octopus cell area (OCA), with its heavily labeled neurons, is separated from the labeled multipolar cells by a region where labeling is absent (Fig. 4B, arrow). The DCN (the most ventral part of which is seen in the sections in Fig. 4) is also unstained. As noted, some but not all of the multipolar cells in the VCN are stained by the WFA histochemical procedure (e.g., Fig. 3B). We were struck by the similarity of the pattern of WFA-binding to multipolar cells and the distribution of multipolar cells in the VCN that project to the inferior colliculus (IC) as determined in a previous study (Cant and Benson, 2006) (Fig. 4E compared to Fig. 4A– D). Most of the multipolar cells that project to the IC are concentrated in the caudal part of the AVCN and the rostral part of the PVCN. In addition, scattered multipolar cells that project to the IC lie among the spherical bushy cells in the rostral AVCN. This distribution is the same as that seen for the lectin-labeled multipolar cells (e.g., Fig. 4A–D). Therefore it appears that WFA labels some, and perhaps all, of the multipolar cells that project to the IC. Not all of the multipolar cells in the VCN are labeled, however, as there are unlabeled multipolar cells located in the middle of the PVCN (Fig. 1F, white arrow; Fig. 2C, lower boxed-in area; Fig. 3B, white arrows and Fig. 4B, arrow). In contrast to those in the VCN, the cells in the DCN that project to the inferior colliculus are not labeled with WFA. 4. Discussion

Fig. 6. Two horizontal sections (separated by 40 lm) through the most ventral part of the cochlear nucleus and the cochlear nerve root. Upper inset: Labeled cells in the ventral PVCN, which contains both multipolar and globular cells. Lower inset and small arrows: Cochlear root neurons. The peripheral part of the vestibular nerve (vnr) and Scarpa’s ganglion (arrowhead) are intensely labeled. Rostral is toward the right of the figure; lateral is toward the top.

bushy cells are found (e.g. Fig. 1E, black arrows; Fig. 2D, boxed-in area; Figs. 3C and 6) most, if not all, neurons are outlined by the lectin, although some of them only lightly so (Fig. 3C, white arrows). Thus, it appears that the glob-

4.1. WFA recognizes a component of the extracellular matrix Although the extracellular space between the nerve cells and glia that make up the central nervous system is narrow and appears relatively non-descript in conventional electron micrographs, it contains a complex variety of macromolecules that collectively make up an extracellular matrix (ECM) (reviewed by Venstrom and Reichardt, 1993; Celio and Blu¨mcke, 1994; Rauch, 1997; Bandtlow and Zimmermann, 2000; Yamaguchi, 2000; Kleene and Schachner,

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

2004). The composition of the ECM in brain is quite different from that of other tissues (Rouslahti, 1996). Many proteins that are common in other parts of the body are missing, but some ubiquitous components of cell surfaces, the large and varied family of macromolecules known as proteoglycans, are abundant in nervous tissue (Rouslahti, 1996; Yamaguchi, 2000). The various components that make up the ECM are not distributed homogeneously throughout the extracellular space in the brain. Rather, distinct populations of cells are surrounded by specific matrix components that differ from those surrounding other populations (e.g., Nakagawa et al., 1986a; Spicer et al., 1996; Lander et al., 1997; Hilbig et al., 2001; Matthews et al., 2002). Of particular relevance in the context of the present study is the fact that various plant lectins have been shown to bind selectively to specific sugars in the family of proteoglycans known as lecticans, a major component of the ECM (Spicer and Schulte, 1992; Yamaguchi, 2000). The lectin that we used, WFA, has been used in many parts of the central nervous system to distinguish populations of neurons (e.g., Nakagawa et al., 1986a; Bru¨ckner et al., 1994; Seeger et al., 1994; Preuss et al., 1998). A number of other markers for specific components of the ECM are found in the same or similar locations as the epitopes that bind to WFA (Lander et al., 1997; Gray et al., 1999; Bru¨ckner et al., 1994, 2000; Matthews et al., 2002). The structures that are visualized have come to be called ‘‘perineuronal nets’’ and have been related to similar structures first described by Camillo Golgi (reviewed by Celio and Blu¨mcke, 1994; Celio et al., 1998). Whether cells that are not surrounded by the WFA-binding sites also possess such nets is not completely clear, although the results of Matthews et al. (2002) demonstrate that distinct proteoglycans, distinguished by modifications in their carbohydrate composition, are expressed on the surfaces of different sets of neurons. It may be that markers specific for different sugar residues would recognize the ‘‘nets’’ associated with other groups of cells (Spicer et al., 1996; Bertolotto et al., 1996). A variety of functions, including roles in development, regeneration and plasticity, and maintenance of local ion homeostasis, especially in areas with high levels of spike activity, have been ascribed to the ECM components in general and to the perineuronal nets in particular (e.g., Celio et al., 1998; Bandtlow and Zimmermann, 2000; Morris and Henderson, 2000; Yamaguchi, 2000; Ha¨rtig et al., 2001; Kleene and Schachner, 2004; Morawski et al., 2004; Laabs et al., 2005). However, it is not known why some populations of neurons are surrounded by WFA-binding nets and others are not. Attempts to relate the WFA-binding to specific neurotransmitter profiles or to other functional characteristics have not proven to be generalizable (e.g., Nakagawa et al., 1986b; Seeger et al., 1994; Ha¨rtig et al., 1999; Horn et al., 2003). Species differences in the types of neurons that are surrounded by the WFA-binding nets further confuse the issue of the functional significance of the chemical composition of the nets (Bru¨ckner et al.,

69

1998; Halpern et al., 1998; Salazar and Sa´nchez Quinteiro, 1998; Gray et al., 1999). Although we do not understand the functional significance of the WFA binding to the ECM surrounding particular populations of neurons, it appears to serve as a reliable marker for those cell groups in a given species. The pattern of WFA labeling in the gerbil brain, in both the cochlear nucleus and other brain regions, was highly consistent in every animal we examined. Therefore, the lectins provide a marker specific for certain populations of cells in the ventral cochlear nucleus that may be useful in identifying them in a variety of experimental situations. 4.2. Markers of specific neuronal populations in the cochlear nucleus The different cell types in the cochlear nuclear complex, first defined on the basis of morphological and physiological criteria (reviewed by Young and Oertel, 2004), also differ in their biochemical make-up. Among the cell types in the dorsal and ventral cochlear nuclei, there is differential expression of proteins related to excitatory (Hunter et al., 1993; Bilak et al., 1996; Wright et al., 1996; Caicedo and Eybalin, 1999; Korada and Schwartz, 2000; McInvale et al., 2002) and inhibitory (Adams and Mugnaini, 1987; Roberts and Ribak, 1987; Wenthold et al., 1987; Wickesberg et al., 1991) neurotransmission. Cell types can be further distinguished by their content of calcium-binding proteins (Arai et al., 1991; Lohmann and Friauf, 1996), specific ion channel proteins (Perney and Kaczmarek, 1997; Rosenberger et al., 2003), and proteins associated with neuronal signaling pathways (Ryugo et al., 1995; Burette et al., 2001). Differential staining of the ECM has also been noted in previous studies. Immunocytochemical staining for proteoglycans in the developing cochlear nucleus of the gerbil demonstrated an increase during the first three postnatal weeks after which an adult-like pattern was established (Lurie et al., 1997). The antibody used, Cat301, like WFA, stained the octopus cells in the PVCN and multipolar cells throughout the VCN but stained the DCN lightly or not at all. In contrast to our study, Lurie et al. (1997) found that Cat-301 was most intense around spherical busy cells, which were not stained by the WFA. This difference may mean that the carbohydrate composition of the proteoglycan coat associated with the bushy cells is different from that associated with multipolar cells and octopus cells (cf. Matthews et al., 2002). In a previous study in the gerbil, Gleich (1994), using a lectin from soybeans that binds to the same carbohydrate moiety recognized by WFA, reported heavy staining of all cell types throughout the DCN and VCN. A combination of factors may explain the difference in his results and ours. First, Gleich used a horseradish peroxidaseconjugated lectin, whereas the WFA that we used was biotinylated. He also used a concentration of the lectin 10–40 times higher than ours. As a histochemical stain, the peroxidase conjugated lectins may produce a higher, non-specific

70

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

background staining than do the biotinylated versions (unpublished observations; literature from Vector Laboratories). In addition, the gerbils in Gleich’s study were older than those used in our study, and staining with the lectins appears to increase with age (Lurie et al., 1997). It may well be that the ECM around all cells in the cochlear nucleus contains at least a small amount of the proteoglycans recognized by WFA and that the differences seen in staining are a matter of degree. Nevertheless, under the conditions used in our study, the distinction among cell types is quite reliable. The octopus cells are the most intensely labeled of all of the neuron types in the VCN. They stand out particularly because not only is the cell body surrounded by the lectin-bound matrix, so are the primary and secondary dendrites. Octopus cells differ from other cell types in the cochlear nucleus in a number of striking ways (e.g., Golding et al., 1999; Bal and Oertel, 2000, 2001; Ferragamo and Oertel, 2002). However, it is not currently possible to relate specialization of the extracellular matrix surrounding the octopus cells to any particular aspect of their functional properties. It is of interest that only some of the multipolar cells in the VCN are labeled with the WFA. The cells classified as multipolar cells (Osen, 1969) can be further subdivided based on anatomical and physiological criteria (e.g., Ostapoff et al., 1994; Doucet and Ryugo, 1997; Doucet et al., 1999; Palmer et al., 2003; Smith et al., 2005), but the different subclasses are often difficult to identify because they are not clearly segregated from one another in different parts of the nucleus. Although our results are not definitive, it appears that the multipolar cells that project to the inferior colliculus are labeled by WFA binding. Whether the multipolar cells that project to the contralateral cochlear nucleus are also labeled is not possible to determine since they are sparsely distributed throughout the ventral cochlear nucleus (e.g, Cant and Benson, 2003; Smith et al., 2005). There is one region containing multipolar cells in the PVCN that is not labeled. Because they are clustered together (unlike the neurons that project to the contralateral cochlear nucleus) and are not labeled with WFA (unlike the more rostrally located neurons that project to the inferior colliculus), the unlabeled multipolar cells in this region (e.g., Fig. 1F, white arrow) may project to other targets. One possibility is that they participate in olivocochlear reflexes. It has been demonstrated that some neurons in the PVCN project to regions in the superior olivary complex that contain olivocochlear neurons (Thompson and Thompson, 1991) and that lesions of the PVCN (but not other parts of the cochlear nucleus) result in disruption of olivocochlear reflexes (Brown et al., 2003). Further, the PVCN cells that participate in these reflexes are classified as chopper units based on their physiological response properties (Brown et al., 2003) and, morphologically, these neurons belong to the multipolar cell class (Feng et al., 1994; Ostapoff et al., 1994; Palmer et al., 2003). Our results suggest, but do not prove, that multipo-

lar neurons in the PVCN that have different patterns of projections may have different lectin-binding properties. Tracing studies in combination with lectin histochemistry could be used to further investigate the correlations between lectin binding patterns and projection patterns. The ability to distinguish different cell types in the cochlear nucleus facilitates correlations of structure and function and has led to a detailed understanding of the multiple, parallel pathways arising in the cochlear nucleus and projecting to other parts of the auditory brainstem (reviewed, e.g., by Romand and Avan, 1997; Cant and Benson, 2003; Young and Oertel, 2004). Identification of cell-specific markers that can be used to distinguish functional populations has a number of potential applications for further study. For example, vital staining with lectins or other markers can be used to distinguish among cell types in slice cultures through various parts of the brain (Bru¨ckner et al., 2004). New ways of studying the differences among cell populations at the molecular and genetic level, such as laser-capture techniques combined with gene expression analyses are facilitated by the ability to distinguish easily among cell types (e.g., Luo et al., 1999; Mojsilovic-Petrovic et al., 2004; Ginsberg and Che, 2005). The use of such methods to develop ‘‘molecular fingerprints’’ for the various cell types in the cochlear nucleus and other parts of the brain may lead to the development of new techniques for studying their anatomical and physiological properties at levels of specificity that are not currently possible (e.g., Braz et al., 2002; Miesenbo¨ck and Kevrekidis, 2005). Finally, knowledge of the distribution of the different cell types is of critical importance in designing and situating auditory prostheses for stimulation of the cochlear nucleus (Møller, 2001). Acknowledgements It is a pleasure to dedicate this paper to Dr. Aage Møller in appreciation of his many important contributions to hearing research. We thank Dr. Ken Hutson for his assistance with this study. The work was supported by a grant from the National Institute on Deafness and Other Communication Disorders, DC00135. References Adams, J.C., 1981. Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 29, 775. Adams, J.C., 1986. Neuronal morphology in the human cochlear nucleus. Arch. Otolaryngol. Head Neck Surg. 112, 1253–1261. Adams, J.C., 1997. Projections from octopus cells of the posteroventral cochlear nucleus to the ventral nucleus of the lateral lemniscus in cat and human. Audit. Neurosci. 3, 335–350. Adams, J.C., Mugnaini, E., 1987. Patterns of glutamate decarboxylase immunostaining in the feline cochlear nuclear complex studied with silver enhancement and electron microscopy. J. Comp. Neurol. 262, 375–401. Arai, R., Winsky, L., Arai, M., Jacobowitz, D.M., 1991. Immunohistochemical localization of calretinin in the rat hindbrain. J. Comp. Neurol. 310, 21–44.

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72 Bal, R., Oertel, D., 2000. Hyperpolarization-activated, mixed-cation current (Ih) in octopus cells of the mammalian cochlear nucleus. J. Neurophysiol. 84, 806–817. Bal, R., Oertel, D., 2001. Potassium currents in octopus cells of the mammalian cochlear nucleus. J. Neurophysiol. 86, 2299–2311. Bandtlow, C.E., Zimmermann, D.R., 2000. Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol. Rev. 80, 1267–1290. Bertolotto, A., Manzardo, E., Guglielmone, R., 1996. Immunohistochemical mapping of perineuronal nets containing chondroitin unsulfate proteoglycan in the rat nervous system. Cell Tissue Res. 283, 283–295. Bilak, M.M., Bilak, S.R., Morest, D.K., 1996. Differential expression of N-methyl-D-aspartate receptor in the cochlear nucleus of the mouse. Neuroscience 75, 1075–1097. Brawer, J.R., Morest, D.K., Kane, E.C., 1974. The neuronal architecture of the cochlear nucleus of the cat. J. Comp. Neurol. 155, 251–300. Braz, J.M., Rico, B., Basbaum, A.I., 2002. Transneuronal tracing of diverse CNS circuits by Cre-mediated induction of wheat germ agglutinin in transgenic mice. Proc. Natl. Acad. Sci. USA 99, 15148– 15153. Brown, M.C., de Venecia, R.K., Guinan Jr., J.J., 2003. Responses of medial olivocochlear neurons. Specifying the central pathways of the medial olivocochlear reflex. Exp. Brain Res. 153, 491–498. Bru¨ckner, G., Seeger, G., Brauer, K., Ha¨rtig, W., Kacza, J., Bigl, V., 1994. Cortical areas are revealed by distribution patterns of proteoglycan components and parvalbumin in the Mongolian gerbil and rat. Brain Res. 658, 67–86. Bru¨ckner, G., Ha¨rtig, W., Seeger, J., Ru¨bsamen, R., Reimer, K., Brauer, K., 1998. Cortical perineuronal nets in the gray short-tailed opossum (Monodelphis domestica): a distribution pattern contrasting with that shown in placental mammals. Anat. Embryol. 197, 249–262. Bru¨ckner, G., Grosche, J., Schmidt, S., Ha¨rtig, W., Margolis, R.U., Delpech, B., Seidenbecher, C.I., Czaniera, R., Schachner, M., 2000. Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R. J. Comp. Neurol. 428, 616–629. Bru¨ckner, G., Kacza, J., Grosche, J., 2004. Perineuronal nets characterized by vital labelling, confocal and electron microscopy in organotypic slice cultures of rat parietal cortex and hippocampus. J. Mol. Histol. 35, 115–122. Burette, A., Petrusz, P., Schmidt, H.H.H.W., Weinberg, R.J., 2001. Immunohistochemical localization of nitric oxide synthase and soluble guanylyl cyclase in the ventral cochlear nucleus of the rat. J. Comp. Neurol. 431, 1–10. Caicedo, A., Eybalin, M., 1999. Glutamate receptor phenotypes in the auditory brainstem and mid-brain of the developing rat. Eur. J. Neurosci. 11, 51–74. Cant, N.B., 1992. The cochlear nucleus: neuronal types and their synaptic organization. In: Webster, D.B., Popper, A.N., Fay, R.R. (Eds.), The Mammalian Auditory Pathways: Neuroanatomy. Springer Verlag, New York, pp. 66–116. Cant, N.B., Benson, C.G., 2003. Parallel auditory pathways: projection patterns of the different neuronal populations in the dorsal and ventral cochlear nuclei. Brain Res. Bull. 60, 457–474. Cant, N.B., Benson, C.G., 2006. Organization of the inferior colliculus of the gerbil (Meriones unguiculatus): differences in distribution of projections from the cochlear nuclei and the superior olivary complex, J. Comp. Neurol., in press. Celio, M.R., Blu¨mcke, I., 1994. Perineuronal nets – a specialized form of extracellular matrix in the adult nervous system. Brain Res. Rev. 19, 128–145. Celio, M.R., Spreafico, R., De Biasi, S., Vitellaro-Zuccarello, L., 1998. Perineuronal nets: past and present. Trends Neurosci. 21, 510–515. Doucet, J.R., Ryugo, D.K., 1997. Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats. J. Comp. Neurol. 385, 245–264. Doucet, J.R., Ross, A.T., Gillespie, M.B., Ryugo, D.K., 1999. Glycine immunoreactivity of multipolar neurons in the ventral cochlear

71

nucleus which project to the dorsal cochlear nucleus. J. Comp. Neurol. 408, 515–531. Feng, J.J., Kuwada, S., Ostapoff, E.-M., Batra, R., Morest, D.K., 1994. A physiological and structural study of neuron types in the cochlear nucleus. I. Intracellular responses to acoustic stimulation and current injection. J. Comp. Neurol. 346, 1–18. Ferragamo, M.J., Oertel, D., 2002. Octopus cells of the mammalian ventral cochlear nucleus sense the rate of depolarization. J. Neurophysiol. 87, 2262–2270. Ginsberg, S.D., Che, S., 2005. Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons. J. Comp. Neurol. 487, 107–118. Gleich, O., 1994. The distribution of N-acetylgalactosamine in the cochlear nucleus of the gerbil revealed by lectin binding with soybean agglutinin. Hear. Res. 78, 49–57. Golding, N.L., Ferragamo, M.J., Oertel, D., 1999. Role of intrinsic conductances underlying responses to transients in octopus cells of the cochlear nucleus. J. Neurosci. 19, 2897–2905. Gray, D., Gutierrez, C., Cusick, C.G., 1999. Neurochemical organization of inferior pulvinar complex in squirrel monkeys and macaques revealed by acetylcholinesterase histochemistry, calbindin and cat-301 immunostaining, and Wisteria floribunda agglutinin binding. J. Comp. Neurol. 409, 452–468. Halpern, M., Jia, C., Shapiro, L.S., 1998. Segregated pathways in the vomeronasal system. Micro. Res. Technol. 41, 519–529. Harrison, J.M., Irving, R., 1965. The anterior ventral cochlear nucleus. J. Comp. Neurol. 124, 15–42. Harrison, J.M., Irving, R., 1966. The organization of the posterior ventral cochlear nucleus in the rat. J. Comp. Neurol. 126, 391–402. Ha¨rtig, W., Derouiche, A., Welt, K., Brauer, K., Grosche, J., Ma¨der, M., Reichenbach, A., Bru¨ckner, G., 1999. Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations. Brain Res. 842, 15–29. Ha¨rtig, W., Singer, A., Grosche, J., Brauer, K., Ottersen, O.P., Bru¨ckner, G., 2001. Perineuronal nets in the rat medial nucleus of the trapezoid body surround neurons immunoreactive for various amino acids, calcium-binding proteins and the potassium channel subunit Kv3.1b. Brain Res. 899, 123–133. Hilbig, H., Bidmon, H.-J., Blohm, U., Zilles, K., 2001. Wisteria floribunda agglutinin labeling patterns in the human cortex: a tool for revealing areal borders and subdivisions in parallel with immunocytochemistry. Anat. Embryol. 203, 45–52. Horn, A.K., Bru¨ckner, G., Ha¨rtig, W., Messoudi, A., 2003. Saccadic omnipause and burst neurons in monkey and human are ensheathed by perineuronal nets but differ in their expression of calcium-binding proteins. J. Comp. Neurol. 455, 341–352. Hunter, C., Petralia, R.S., Vu, T., Wenthold, R.J., 1993. Expression of AMPA-selective glutamate receptor subunits in morphologically defined neurons of the mammalian cochlear nucleus. J. Neurosci. 13, 1932–1946. Kleene, R., Schachner, M., 2004. Glycans and neural cell interactions. Nat. Rev. Neurosci. 5, 195–208. Korada, S., Schwartz, I.R., 2000. Calcium binding proteins and the AMPA glutamate receptor subunits in gerbil cochlear nucleus. Hear. Res. 140, 23–37. Laabs, T., Carulli, D., Geller, H.M., Fawcett, J.W., 2005. Chondroitin sulfate proteoglycans in neural development and regeneration. Curr. Opin. Neurobiol. 15, 116–120. Lander, C., Kind, P., Maleski, M., Hockfield, S., 1997. A family of activity-dependent neuronal cell-surface chondroitin sulfate proteoglycans in cat visual cortex. J. Neurosci. 17, 1928–1939. Lohmann, C., Friauf, E., 1996. Distribution of the calcium-binding proteins parvalbumin and calretinin in the auditory brainstem of adult and developing rats. J. Comp. Neurol. 367, 90–109. Luo, L., Salunga, R.C., Guo, H., Bittner, A., Joy, K.C., Galindo, J.E., Xiao, H., Rogers, K.E., Wan, J.S., Jackson, M.R., Erlander, M.G.,

72

N.B. Cant, C.G. Benson / Hearing Research 216–217 (2006) 64–72

1999. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat Med. 5, 117–122. Lurie, D.I., Pasic, T.R., Hockfield, S.J., Rubel, E.W., 1997. Development of cat-301 immunoreactivity in auditory brainstem nuclei of the gerbil. J. Comp. Neurol. 380, 319–334. Matthews, R.T., Kelly, G.M., Zerillo, C.A., Gray, G., Tiemeyer, M., Hockfield, S., 2002. Aggrecan glycoforms contribute to the molecular heterogeneity of perineuronal nets. J. Neurosci. 22, 7536–7547. McInvale, A.C., Staudinger, J., Harlan, R.E., Garcia, M.M., 2002. Immunolocalization of PICK1 in the ascending auditory pathways of the adult rat. J. Comp. Neurol. 450, 382–394. Miesenbo¨ck, G., Kevrekidis, I.G., 2005. Optical imaging and control of genetically designated neurons in functioning circuits. Ann. Rev. Neurosci. 28, 533–563. Mojsilovic-Petrovic, J., Nesic, M., Pen, A., Zhang, W., Stanimirovic, D., 2004. Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses. J. Neurosci. Meth. 133, 39–48. Møller, A.R., 2001. Neurophysiologic basis for cochlear and auditory brainstem implants. Am. J. Audiol. 10, 68–78. Moore, J.K., 1986. Cochlear nuclei: relationship to the auditory nerve. In: Altschuler, R.A., Hoffman, D.W., Bobbin, R.P. (Eds.), Neurobiology of Hearing. The Cochlea. Raven Press, New York, pp. 283–301. Moore, J.K., Osen, K.K., 1979. The cochlear nuclei in man. Am. J. Anat. 154, 393–418. Morawski, M., Bru¨ckner, M.K., Riederer, P., Bru¨ckner, G., Arendt, T., 2004. Perineuronal nets potentially protect against oxidative stress. Exp. Neurol. 188, 309–315. Morris, N.P., Henderson, Z., 2000. Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/ diagonal band complex. Eur. J. Neurosci. 12, 828–838. Nakagawa, F., Schulte, B.A., Spicer, S.S., 1986a. Selective cytochemical demonstration of glycoconjugate-containing terminal N-acetylgalactosamine on some brain neurons. J. Comp. Neurol. 243, 280–290. Nakagawa, F., Schulte, B.A., Wu, J.-Y., Spicer, S.S., 1986b. GABAergic neurons of rodent brain correspond partially with those staining for glycoconjugate with terminal N-acetylgalactosamine. J. Neurocytol. 15, 389–396. Osen, K.K., 1969. Cytoarchitecture of the cochlear nuclei in the cat. J. Comp. Neurol. 136, 453–484. Ostapoff, E.-M., Feng, J.J., Morest, D.K., 1994. A physiological and structural study of neuron types in the cochlear nucleus. II. Neuron types and their structural correlation with response properties. J. Comp. Neurol. 346, 19–42. Palmer, A.R., Wallace, M.N., Arnott, R.H., Shackleton, T.M., 2003. Morphology of physiologically characterized ventral cochlear nucleus stellate cells. Exp. Brain Res. 153, 418–426. Perney, T.M., Kaczmarek, L.K., 1997. Localization of a high threshold potassium channel in the rat cochlear nucleus. J. Comp. Neurol. 386, 178–202. Preuss, T.M., Gray, D., Cusick, C.G., 1998. Subdivisions of the motor and somatosensory thalamus of primates revealed with Wisteria floribunda agglutinin histochemistry. Somatosen. Motor Res. 15, 211–219.

Rauch, U., 1997. Modeling an extracellular environment for axonal pathfinding and fasciculation in the central nervous system. Cell Tissue Res. 290, 349–356. Richter, E., 1983. The ventral cochlear nucleus in human brains. Adv. Oto-Rhino-Laryng. 31, 59–71. Roberts, R.C., Ribak, C.E., 1987. GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil. J. Comp. Neurol. 258, 267–280. Romand, R., Avan, P., 1997. Anatomical and functional aspects of the cochlear nucleus. In: Ehret, G., Romand, R. (Eds.), The Central Auditory System. Oxford University Press, New York, pp. 97–191. Rosenberger, M.H., Fremouw, T., Casseday, J.H., Covey, E., 2003. Expression of the Kv1.1 ion channel subunit in the auditory brainstem of the big brown bat, Eptesicus fuscus. J. Comp. Neurol. 462, 101–120. Rouslahti, E., 1996. Brain extracellular matrix. Glycobiology 6, 489–492. Ryugo, D.K., Pongstaporn, T., Wright, D.D., Sharp, A.H., 1995. Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus. J. Comp. Neurol. 358, 102–118. Salazar, I., Sa´nchez Quinteiro, P., 1998. Lectin binding patterns in the vomeronasal organ and accessory olfactory bulb of the rat. Anat. Embryol. 198, 331–339. Seeger, G., Brauer, K., Ha¨rtig, W., Bru¨ckner, G., 1994. Mapping of perineuronal nets in the rat brain stained by colloidal iron hydroxide histochemistry and lectin cytochemistry. Neuroscience 58, 371–388. Smith, P.H., Massie, A., Joris, P.X., 2005. Acoustic stria: anatomy of physiologically characterized cells and their axonal projection patterns. J. Comp. Neurol. 482, 349–371. Spicer, S.S., Schulte, B.A., 1992. Diversity of cell glycoconjugates shown histochemically: a perspective. J. Histochem. Cytochem. 40, 1–38. Spicer, S.S., Naegele, J.R., Schulte, B.A., 1996. Differentiation of glycoconjugates localized to sensory terminals and selected sites in brain. J. Comp. Neurol. 365, 217–231. Thompson, A.M., Thompson, G.C., 1991. Posteroventral cochlear nucleus projections to olivocochlear neurons. J. Comp. Neurol. 375, 267–285. Venstrom, K.A., Reichardt, L.F., 1993. Extracellular matrix 2: role of extracellular matrix molecules and their receptors in the nervous system. FASEB J. 7, 996–1003. Wenthold, R.J., Huie, D., Altschuler, R.A., Reeks, K.A., 1987. Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex. Neuroscience 22, 897–912. Wickesberg, R.E., Whitlon, D., Oertel, D., 1991. Tuberculoventral neurons project to the multipolar cell area but not to the octopus cell area of the posteroventral cochlear nucleus. J. Comp. Neurol. 313, 457–468. Wright, D.D., Blackstone, C.D., Huganir, R.L., Ryugo, D.K., 1996. Immunocytochemical localization of the mGluR1a metabotropic glutamate receptor in the dorsal cochlear nucleus. J. Comp. Neurol. 364, 729–745. Yamaguchi, Y., 2000. Lecticans: organizers of the brain extracellular matrix. Cell. Mol. Life Sci. 57, 276–289. Young, E.D., Oertel, D., 2004. Cochlear nucleus. In: Shepherd, G.M. (Ed.), The Synaptic Organization of the Brain. Oxford University Press, New York, pp. 125–163.