MICROSCOPY RESEARCH AND TECHNIQUE 41:224–233 (1998) Localization of Neurotrophin-3-Like Immunoreactivity in the Rat Cochlear Nucleus A. BURETTE,1 G. BELLIOT,2 E. ALBUISSON,3 AND R. ROMAND1* 1Laboratoire de Neurobiologie, Université Blaise Pascal, 63177 Aubière Cedex, France of Viral and Rickettsial Diseases, National Center for Infection Diseases, Centers for Disease Control and Prevention Atlanta, Georgia 30329 3Laboratoire de Biostatistiques, Faculté de Médecine, Université d’Auvergne, 63000 Clermont-Ferrand, France 2Division KEY WORDS neurotrophic factor; central auditory system; glial cells ABSTRACT Immunohistochemistry as well as immunohistofluorescence were used to investigate the distribution of the neurotrophin-3 (NT3) in the adult rat cochlear nucleus. We found a widespread distribution of NT3 immunolabeled neurons throughout the three divisions of this nucleus. NT3-like immunoreactivity was clearly population-specific, with some cell groups heavily (various small neurons and granule cells) or moderately (large neurons of the ventral cochlear nucleus) stained, while others remained negative (a major fraction of medium and large neurons of the dorsal cochlear nucleus). Double-labeling experiments were performed using antibody against the glial fibrillary acid protein, a classic marker for mature astrocytes. This colocalization study revealed that NT3 immunoreactivity was also present in a subpopulation of astrocytes, particularly in the glia limitans and their projections. Numerous small cells also colocalized NT3 together with the glial marker in the granule cell domain and in the molecular cell layer of the dorsal cochlear nucleus. These results suggest that NT3 may exist in widespread populations of adult cochlear nucleus neurons as well as in glial cells. This abundant distribution of NT3-like immunoreactivity implies that this neurotrophin may have an important role in the continued maintenance of mature cochlear nucleus and makes it an attractive candidate for playing a role in regulation or stabilization of neuronal circuits in this nucleus. Microsc. Res. Tech. 41:224–233, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION Neurotrophin-3 (NT3) is a member of the neurotrophin family including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-4/5 (NT4/5) (Hohn et al., 1990; Maisonpierre et al., 1990). The expression of NT3 is widely distributed throughout the central nervous system (CNS). In particular, mRNANT3 is found at the highest concentrations in hippocampus and cerebellum (Lauterborn et al., 1994). It is believed that NT3 controls survival, differentiation, and maintenance of specific populations of vertebrate cells (Davies, 1994; Skup, 1994; Snider, 1994). Besides these actions, there is increasing evidence that NT3, like the other neurotrophins, is involved in processes of neuronal plasticity. In particular, in adult CNS, NT3 may participate in synaptic plasticity in an activity-dependent manner (Kang and Schuman, 1995). NT3 mediates these effects by interacting with highand low-affinity receptors (Bothwell, 1995). The lowaffinity NGF receptor (LNGFR) binds all known neurotrophins with similar affinity. However, it may not be able to mediate the effect of neurotrophins (Chao, 1994). The high-affinity receptors are transmembrane tyrosine kinases encoded by the trk proto-oncogene family. Whereas TrkA is a receptor for NGF and TrkB is a receptor for BDNF and NT4/5, TrkC serves as a receptor for NT3. NT3 also binds to TrkA and TrkB, but at much lower efficiency than NGF and BDNF or NT4/5, respectively. r 1998 WILEY-LISS, INC. A recent study showed a widespread distribution of TrkC receptors throughout the central auditory system (Hafidi et al., 1996). This observation provides evidence for a possible role of this neurotrophin in auditory nucleus. In this context, the study of the expression of the NT3 in this system and particularly in the cochlear nuclei (CN) is of interest.As its cytoarchitecture as well as its pharmacological and physiological properties are well known (Merchan et al., 1993; Romand and Avan, 1997), the CN, which is the first relay station in the ascending auditory pathway, seems very appropriate for this study. Thus, the aim of the present study is to investigate, for the first time, the expression of NT3 protein in the CN, by using an affinity-purified polyclonal IgG. Furthermore, we tested the possibility that NT3-like immunoreactivity (NT3LI) could be present in glial cells by double-labeling experiments using antibody against the glial fibrillary acid protein (GFAP), a marker of mature astrocytes. Results indicated that NT3-LI was present in both neurons and glial cells throughout the three divisions of the CN. MATERIALS AND METHODS Tissue Fixation and Processing Four male Sprague-Dawley rats about 4 months old and raised in our facilities were used. Rats were *Correspondence to: R. Romand, Laboratoire de Neurobiologie, Université Blaise Pascal, 63177 Aubière Cedex, France. E-mail: email@example.com Received 20 June 1997; Accepted in revised form 2 January 1998 NEUROTROPHIN-3 IN THE MATURE COCHLEAR NUCLEUS anesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneally) and perfused transcardially with a flush of 0.1 M phosphate buffered saline (PBS), pH 7.4, containing 4% paraformaldehyde. The brainstems were then removed and postfixed at 4°C for 24 hours in the same fixative. After washing in PBS at 4°C overnight, all tissues were routinely embedded in paraplast (Sherwood Medical St. Louis, MO). Serial transverse sections through the CN were cut at 5 µm, and mounted onto gelatinized glass slides. In each series, one out of every 10 sections was Nissl stained. Thus, sections for immunohistochemistry were chosen in relation to the structures revealed on Nissl stained sections. Immunohistochemistry Immunodetection was carried out using a commercial antibody raised against amino acids 139–158 of human NT3 (Sc-547, Santa Cruz Biotechnology, Santa Cruz, CA). This sequence has no high sequence homology with any other known proteins. This antibody recognized the rat NT3 with no NGF, BDNF, or NT4 cross-reactivity. In rat, the signal detected by Western blot and immunohistochemistry can be competed off with the specific blocking peptide (Santa Cruz Biotechnology, personal communication). Brainstem sections containing CN were deparaffinized in xylene, hydrated, and washed for 5 minutes in distilled water. The sections were treated for 30 minutes at room temperature in methanol/0.3% H2O2 to exhaust endogenous peroxidase activity. Then a short Tris buffer saline (TBS) wash followed and the sections were incubated for 30 minutes at room temperature in 10% fetal bovine serum (FBS) and 0.1% Tweeny 20 in TBS to suppress background. Tissue sections were incubated overnight at 4°C in primary antibody diluted to 1:200 in TBS containing 5% FBS. After extensive washes in TBS, the sections were reacted for 1 hour at 37°C with secondary antibody (biotinylated horse antirabbit IgG, Vector Laboratories, Burlingame, CA) at 1:400. Sections were again washed thoroughly as above and reacted with avidin-biotin complex (Vectastain ABC-Elite, Vector) for 45 minutes at 37°C, following the manufacturer’s indications. The immunocomplexed sections were carried through several TBS washes. The resulting immune complexes have peroxidase activity that yields a brown reaction product when reacted with 0.3% hydrogen peroxide, 0.034 mg/ml diaminobenzidine in TBS, pH 7.8. After peroxidase reaction, the sections were dehydrated through graded ethanol solutions, dipped in xylene, and mounted with coverslips and Eukitty mounting medium (Kindler Gmbh fco). Finally, for each immunostained section (approximately one section out of every 40 µm), the adjacent section was Nissl stained to allow comparison with immunostained sections. Distribution of NT3 Immunostained Cells The CN complex of the rat is conventionally divided into ventral CN (VCN) with two divisions: the anteroventral CN (AVCN), the postero-ventral CN (PVCN), and the dorsal CN (DCN) (Merchan et al., 1993; Romand and Avan, 1997). For detailed parcellation and cell types, we refer to the work done on rats (Harrison and Irving, 1965, 1966), mice (Webster and Trune, 1982), and cats (Brawer et al., 1974; Osen, 1969). 225 For each of the three divisions of the CN (i.e., AVCN, PVCN, and DCN), three positions were mapped in the rostro-caudal axis: at 15, 50, and 85%. Percentages were determined by dividing the section number by the total number of sections per division. Only cells containing a nucleus were considered. The cartographic analysis of NT3-immunoreactive cells was performed using an image analysis workstation (Historag and Imaginia software by Biocom) coupled to a microscope by a video camera. Briefly, this system makes morphometric and cartographic measurements of objects selected by users at high magnification from a regional context, after first plotting the areas of interest at low magnification. All cells resulting from exploration of a cross-section are recorded on a map in highresolution cartography. Assembling maps were made with Vector drawing software. Immunofluorescence Colocalization of NT3 and GFAP Tissue Processing. The study was carried out in two 4-month-old male Sprague-Dawley rats. Animals were anesthetized with sodium pentobarbital (50 mg/ kg) and perfused transcardially with a flush of 25 ml physiological saline, followed by 800 ml of a fixative solution, containing 4% paraformaldehyde in PBS 0.1 M. After perfusion, brainstems were removed and postfixed for 2 hours at 4°C in the same fixative solution; then they were transferred for 3 hours in cold PBS. Brainstems were cryoprotected in 15% sucrose solution overnight and then sectioned in the transversal plane on a cryostat microtome at a thickness of 16 µm before being collected in cold PBS. Immunohistochemistry. All the following procedures were performed on free-floating sections. After washing, the sections were incubated overnight at 4°C with the mixture of antibodies, i.e., rabbit anti-NT3 diluted 1:200 and monoclonal anti-GFAP (clone GA5, Sigma, St. Louis, MO) diluted 1:400. In the second step, a mixture of an FITC goat anti-rabbit IgG and TRICT goat anti-mouse IgG, was applied overnight at 4°C. After a washing procedure, sections were mounted on gelatin-coated slides, air dried, and cleared with xylene before being coverslipped with Fluoromounty (Gurr, BDH Chemicals, Toronto, Canada). Controls were obtained by processing the tissue as described above except that an irrelevant IgG was substituted in place of each primary antibody. RESULTS NT3-like immunoreactivity (NT3-LI) was detected within the cerebellum (Fig. 1) and CN (Figs. 2–4). In contrast, no specific staining was observed in sections in which the primary antibody was deleted or substituted. In the cerebellum, intense NT3-LI was localized in the granular cell layer, while a moderate immunoreactivity was observed in Purkinje cells, as discrete punctate labeling in the cytoplasm (Fig. 1). In each division of the CN, numerous cells were stained with different intensities, showing an abundant and widespread distribution of NT3-LI (Figs. 2–4). By contrast with others brainstem areas, where intense NT3-IL was observed in the cell bodies as well as in dendrites and axons (Fig. 1), in the CN the immunoreactivity was 226 A. BURETTE ET AL. Fig. 1. Localization of NT3-LI in transversal sections of cerebellum and raphe pontis nucleus. A, B: Low- and high-power photomicrographs of cerebellum. An immunoreactivity in the granule cell layer is shown. Additionally, discrete punctate labeling is demonstrated in the cell bodies of the Purkinje neurons (arrows). Scale bar: A, 100 µm; B, 25 µm. C: Cells of the raphe pontis nucleus. Note that intense NT3-LI is visible in the cell bodies as well as in processes in the large neurons (arrows). Scale bar: 25 µm. mainly restricted to the cytoplasm. With prolonged incubation, reactive product could be detected in the beginning cell processes. fraction of the multipolar cell population appeared negative. Octopus cells of the PVCN do not seem to be labeled. By contrast, most, if not all, spherical and globular cells seem to be reactive. In fact, in line with this interpretation, in the very rostroventral pole of the AVCN where spherical cells occur as a homogeneous population in relative isolation (Harrison and Irving, 1965), nearly all large cells were immunoreactive (Fig. 2D,E). Just as in the region close to the nerve root, where globular cells could easily be distinguished (Tolbert and Morest, 1982), all cells of this type were labeled. Additionally, a group of distinctive immunolabeled cells, characterized by their large, oval soma and their eccentric nucleus, was found in the cochlear nerve root. From their distribution and morphological features, they were identified as root neurons (Merchan et al., 1988), classified as b cells by Harrison and Irving (1965). In addition to the principal neurons of the VCN, numerous small cells containing intense NT3-LI were evenly distributed throughout the VCN. These cells Neurotrophin-3-Like Immunoreactivity in the Adult Ventral Cochlear Nucleus A large number of NT3 immunoreactive cells was observed within both anterior and posterior parts of the VCN (Figs. 2, 3). A sample distribution map is shown in Figure 4. Positive cells were more or less homogeneously distributed throughout the nucleus, except in the granule cell domain where the highest density of NT3-LI could be seen. Inspection of NT3 immunostained sections showed that the large neurons in the VCN, which presumably represent spherical, globular, and multipolar cells contained NT3-LI in their cell bodies as discrete punctate labeling (Figs. 2C,D, 3C,D). However, some large cells remained negative (Fig. 3D). As judged by comparing adjacent NT3 and Nissl-stained sections, a significant Fig. 2. Localization of NT3-LI in transversal sections of anterior ventral cochlear nucleus. A–C: Section throughout the middle of the AVCN. One can observe a large and homogeneous distribution of labeled cells (A, B). However, a larger number of immunostained cells in the granule cell domain (arrows) can be observed (A, B). NT3-LI is visible in the cell bodies as discrete punctate labeling (C). Scale bar: A, 200 µm; B, 100 µm; C, 10 µm. D, E: Section through the very rostroventral pole of the VCN. Most spherical cells appeared immunopositive (D) with discrete punctate labeling in their cell bodies (E). Scattered throughout this area small, highly reactive cells can be observed (arrows). Numerous cells are labeled in the small cell cap (D, arrowheads). Scale bar: D, 50 µm; E, 25 µm. 228 A. BURETTE ET AL. Fig. 3. Localization of NT3-like immunoreactivity in a transversal section of posterior ventral cochlear nucleus. A: Low-power photomicrograph illustrating the overall distribution of NT3-LI in the middle of the PVCN. Scale bar: 200 µm. B: Higher magnification of the granule cell domain. Note that intense NT3-LI is observed in the granule cell domain (arrows). Scale bar: 50 µm. C: Higher magnification of central region of the PVCN. Several immunopositive large cells can be observed (large arrows). More or less homogeneously distributed throughout this area, a large number of highly reactive small cells can be also seen (small arrows). Scale bar: 100 µm. D: Two large cells containing NT3-LI in their cell bodies as discrete punctate labeling. Scale bar: 10 µm. NEUROTROPHIN-3 IN THE MATURE COCHLEAR NUCLEUS 229 Fig. 4. Localization of NT3-like immunoreactivity in dorsal cochlear nucleus. A: Low-power photomicrograph illustrating the overall distribution of NT3-LI throughout the three layers of the DCN. Numerous small cells, evenly distributed, are highly reactive (small arrows). Some larger size cells are lightly stained (large arrows). m: molecular cell layer; f: fusiform cell layer; p: polymorphic cell layer. Scale bar: 100 µm. B: Two immunopositive cells from the fusiform cell layer (arrows). Scale bar: 10 µm. C: A lightly labeled large cell (arrow) from the fusiform cell layer. Scale bar: 10 µm. D: An immunonegative large cell (arrows) from the polymorphic cell layer. Scale bar: 10 µm. could represent small neurons as well as glial cells (see Colocalization Studies). A part of these immunoreactive small cells could correspond to granule cells as suggested by the large number of stained cells in the granule cell domain where they represent the main population (Fig. 3A,B) (Mugnaini et al., 1980). Lastly, in the small cell cap, numerous cells also appeared NT3 positive (Fig. 2D). 230 A. BURETTE ET AL. Fig. 5. Distribution of NT3 immunoreactive cells throughout a transversal sections of the three parts of the cochlear nucleus. A large number of immunopositive cells can be observed within the three subnuclei, showing a more or less homogeneous distribution. However, a higher concentration of labeled cells can be seen in the granule cells domain. The three maps were made through the middle of the rostro-caudal axis of each subnucleus. Neurotrophin-3-Like Immunoreactivity in the Adult Dorsal Cochlear Nucleus NT3-LI was observed in cells within all layers of the DCN (Figs. 4, 5), but unlike the VCN, where numerous large cells were positive, only scattered medium and large cells appeared immunolabeled. At the transition between the molecular and the fusiform cell layer, rounded medium-size cell bodies were immunolabeled. From their distribution and morphological features, they may represent cartwheel neurons (Mugnaini et al., 1987; Ryugo and Willard, 1985). In the fusiform cell layer some large cells containing NT3-LI were observed. The distribution of these cells as well as their size and shape lead us to think that they may correspond to pyramidal cells. In the polymorphic cell layer, large cells that presumably represent giant, turberculoventral and radiate stellate cells appeared most of the time negative or occasionally containing only some rare immunoreactive puncta (Fig. 4B–D). As observed in the VCN, numerous small cells were highly reactive (Fig. 4A). They were evenly distributed throughout the DCN and represented the principal fraction of immunostained cells. These cells could correspond to interneurones like granule cells and glial cells (see Colocalization Studies). with GFAP antigens (Fig. 6). This cytoskeletal protein is a specific marker for mature astroglial cells (Bignami et al., 1972) and has been used with success in the CN (Jalenques et al., 1995). The immunofluorescence procedure demonstrated a NT3-LI similar to the immunohistochemical staining with the exception that the reactivity was more intense and processes of numerous small cells were labeled. In both VCN and DCN, a significant fraction of GFAP-positive astrocytes colocalized also NT3-LI. In particular, NT3-LI was present in the glia limitans and their projections. Furthermore, numerous small cells colocalized NT3 together with the glial marker in the granule cell domain and in the molecular cell layer of the DCN (Fig. 6). In the others part of the CN, double staining cells were less frequently observed but were evenly distributed. Colocalization Studies The distinction between small neurons on the one hand and glial cells on the other hand was difficult in our immunostained sections (morphology and size of small neurons and glial cells can be very similar). Thus, we examined the biochemical phenotype of small cells using a double-fluorescence procedure colocalizing NT3 DISCUSSION In the present study, we report on the regional and cellular distribution of NT3-LI in the mature CN of the rat. We found a widespread distribution of NT3-LI, present in both glia and neurons, throughout the three divisions of this nucleus. Significance of the Present Results With Respect to the Previous Investigation on NT3 Distribution In the cerebellum, we found an intense level of NT3-LI in granule cells and a lower one in the Purkinje cells, with discrete punctate labeling. This observation appears to conflict with previous investigations, where NT3-LI was only found in Purkinje cells (Zhou and Rush, 1994). The discrepancy may result from the NEUROTROPHIN-3 IN THE MATURE COCHLEAR NUCLEUS 231 Fig. 6. Localization of NT3-like immunoreactivity in GFAP positive glial cells. Arrows point toward two glial cells, located in the molecular cell layer of the DCN, which showed GFAP (A) and NT3 (B) colocalization. The two photomicrographs were taken from the same field using TRITC (A) or FITC (B) filter cubes. Scale bar: 5 µm. difference of the titer of the antibodies used. However, previous in situ localization of NT3 mRNA showed that in the cerebellum, NT3 transcripts were present in granule cells (Lauterborn et al., 1994; Rocamora et al., 1993). One hypothesis is that NT3 could act in Purkinje cells and granule cells itself by an autocrine route (Lauterborn et al., 1994; Rocamora et al., 1993; Segal et al., 1992). The localization of NT3-LI observed in the present study within the cerebellum mimics quite well the distribution of NT3 mRNA and was consistent with the potential function of this neurotrophin in this part of the CNS. Immunoreactivity of Neuronal Cell Bodies Our results showed that NT3-LI found in neuronal cells was predominantly distributed throughout the cytoplasm as small punctate accumulations (Figs. 2B,E, 3D, 4B,C). However, we could not eliminate the possibility that some of the punctate labeling was present in presynaptic boutons at the surface of neuron cell bodies. Further electron microscopic evaluation will be needed to clarify this hypothesis. This distribution of immunoreaction products may correspond to a vesicular localization of NT3-like antigen. A discrete cytoplamic punctate labeling was reported for other neurotrophins (i.e., NGF and BDNF) particularly in neurotrophin-responsive neurons (Nishio et al., 1994; Wetmore et al., 1991). In PC12 cells, the most extensively studied and well-understood NGF-responsive cells, the same immunoreactive pattern for TrkA was shown (Grimes et al., 1996). They also demonstrated that these punctae represented an endosomal localization of active TrkA-NGF complex after internalization. The same phenomena might apply for the TrkC-NT3. Significance of NT3-LI Cells: NT3-Synthesizing Cells or NT3-Responsive Cells The NT3-LI-positive neurons could be NT3-synthesizing cells or, alternatively, NT3-responsive cells. Unfortunately, to our knowledge the expression of mRNANT3 has not yet been studied in the central auditory system, so the localization of NT3-synthesizing cells remains unknown. However, much information is available on NT3-responsive cells. These cells should express the NT3 receptors: a low-affinity receptor, LNGFR, and a high-affinity receptor, TrkC. Previous reports have shown that LNGFR is present in the CN during development (Després et al., 1991) as well as in adult nucleus (Pioro et Cuello, 1990). The neurons expressing LNGFR may be the neurons responsive to NT3 (Dechant et al., 1997). However, the role of LNGFR in neurotrophin responsiveness is currently controversial (Kaplan and Miller, 1997). An abundant and widespread distribution of the high-affinity receptor (TrkC) has been observed in the adult CN (Burette et al., in press; Hafidi et al., 1996), predicting that NT3 may exert significant effects on a large proportion of neurons. Additionally, from our observations, at least some neuronal populations may be immunoreactive to both NT3 and TrkC. For instance, in the granule cell domain, many granule cells appear immunoreactive in both NT3 and TrkC stained sections. Therefore, it seems reasonable to speculate that this cell type colocalized both proteins. The same observations could be made for spherical and globular cells in the very rostroventral pole of the VCN and in the region close to the nerve root, respectively. For the other cell populations of the VCN and in the DCN, where the different cell types could not be observed in relative isolation, colocalization study will be required to address this issue. All of these observations together with the immunoreactive pattern (i.e., labeling as discrete punctate) lead us to think that many NT3-LI neurons observed in the CN may be NT3-responsive neurons. Implications for NT3 Functions in the CN NT3-LI is clearly population-specific, with some cell groups heavily (various small cells and granule cells) or moderately (large neurons of the VCN) labeled, while others remain negative (a large fraction of large neu- 232 A. BURETTE ET AL. rons of the DCN). The explanation for selectivity in NT3 distribution across neuronal groups is not immediately obvious. However, the presence of NT3-LI in granule, globular, and spherical cells, together with the limited number of immunoreactive large cells in the DCN, could lead us to think that this selectivity may be related to the use by these cells of an excitatory amino-acid as a neurotransmitter. It is possible that NT3 supports the survival of these specific neurons and stabilizes neural homeostasis by regulating calcium concentration. This neurotrophin could also be involved in synaptic plasticity in an activity-dependent manner as suggested in the hippocampus (Kang and Schuman, 1995). However, destruction of the cochlea does not seem to affect NT3-LI in the ipsilateral CN at any survival time from 24 hours to 30 days (personal observations). These findings could indicate that continued expression of NT3 is not controlled by afferent activity. This raises the possibility that NT3 could act by an autocrine route (Davies and Wright, 1995). By analogy with the granule cells of the cerebellum, this hypothesis is particularly tempting for granule cells of the CN. These last cells have some features in common with the granule cells of the cerebellum including ontogenetic origin (Mugnaini et al., 1980). Localization of NT3-IR in Glia Our results indicate that a subpopulation of GFAPpositive glial cells were stained with NT3 antibody. A similar result was also reported by Zhou and Rush in other parts of the CNS (Zhou and Rush, 1994). The significance of NT3-LI in astrocytes is not clear. First, astrocytes could be considered as sites of action for NT3. It is well known that astrocytes could express, in vivo and in vitro, the truncated forms of Trk receptors (Rudge et al., 1992, 1994; Zhou et al., 1994). However, the functional status of these receptors on astrocytes is not fully understood, but it has been proposed that truncated Trks may have important functions such as sequestration or presentation of neurotrophins within the CNS (Rudge et al., 1994). Second, astrocytes could synthesize NT3 and may have the capacity to provide their partner neurons with this neurotrophin. Whether or not NT3 is also produced by astrocytes in vivo is not known, but in vitro mRNANT3 have been detected in hippocampal astrocytes (Rudge et al., 1992). In addition, glial cells express mRNANT3 following colchicine treatment (Ceccatelli et al., 1991). Thus, it is possible that glial cells in vivo can also produce NT3. 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