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Keywords:

  • superior olivary complex;
  • cochlea, Phodopus sungorus;
  • retrograde tracing;
  • oxytocin;
  • vasopressin;
  • VIP;
  • PACAP;
  • nitric oxide

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The present study was conducted to characterize the superior olivary complex (SOC) of the lower brain stem in the pigmented Djungarian hamster Phodopus sungorus. Using Nissl-stained serial cryostat sections from fresh-frozen brains, we determined the borders of the SOC nuclei. We also identified olivocochlear (OC) neurons by retrograde neuronal tracing upon injection of Fluoro-Gold into the scala tympani. To evaluate the SOC as a putative source of neuronal nitric oxide synthase (nNOS), arginine-vasopressin (AVP), oxytocin (OT), vasoactive intestinal polypeptide (VIP), or pituitary adenylate cyclase-activating polypeptide (PACAP) that were all found in the cochlea, we conducted immunohistochemistry on sections exhibiting retrogradely labeled neurons. We did not observe AVP-, OT-, or VIP-immunoreactivity, neither in OC neurons nor in the SOC at all, revealing that cochlear AVP, OT, and VIP are of nonolivary origin. However, we found nNOS, the enzyme responsible for nitric oxide synthesis in neurons, and PACAP in neuronal perikarya of the SOC. Retrogradely labeled neurons of the lateral olivocochlear (LOC) system in the lateral superior olive did not contain PACAP and were only infrequently nNOS-immunoreactive. In contrast, some shell neurons and some of the medial OC (MOC) system exhibited immunofluorescence for either substance. Our data obtained from the dwarf hamster Phodopus sungorus confirm previous observations that a part of the LOC system is nitrergic. They further demonstrate that the medial olivocochlear system is partly nitrergic and use PACAP as neurotransmitter or modulator. Anat Rec, 292:461–471, 2009. © 2009 Wiley-Liss, Inc.

The organ of Corti in the cochlea of the inner ear is the sensorineural organ for hearing. It includes supporting cells and sensory cells. Inner hair cells (IHC) are the true sensory cells that secrete glutamate upon sound-induced deflection of their stereocilia. This signal is transmitted to the cochlear nuclei by afferent spiral ganglion fibers. These peripheral dendrites, synapsing at IHC, are contacted by efferent fibers originating from the lateral superior olivary complex (SOC) of the brain stem (lateral olivocochlear (LOC) efferent fibers). These efferents may depolarize the afferents by releasing acetylcholine, dynorphin, or calcitonin gene-related peptide and thus enhance glutamate-driven activity, or they may use dopamine, enkephalin, or gamma-amino butyric acid to hyperpolarize and inhibit afferents. Outer hair cells (OHC), in contrast, enhance and modulate the function of IHC. They receive direct input from the SOC (by medial olivocochlear efferents) in addition to their minor spiral ganglion contacts. These projections, their transmitters, and effects have been described in several studies (cf. Eybalin,1993; Raphael and Altschuler,2003). In addition, other neuroactive substances were identified in the cochlea recently but there is only limited information on their origin.

One of these is the gaseous transmitter nitric oxide (NO). It is involved in the regulation of various physiological and pathological mechanisms in the mammalian body and is present at various sites of the auditory system. The neuronal isoform of its synthesizing enzyme, neuronal nitric oxide synthase (nNOS), has been demonstrated in the cochlea of rats, guinea pigs, and humans (Fessenden et al.,1994; Zdanski et al.,1994; Gosepath et al.,1997; Ruan et al.,1997; Fessenden and Schacht,1998). The substance has been shown to influence cochlear blood flow, endocochlear potentials and, in high doses, to induce degeneration of inner hair cell afferents (Brechtelsbauer et al.,1994; Chen et al.,1995; Kong et al.,1996). It was assumed that efferent neurons of the SOC might provide an additional source of NO within the organ of Corti. Indeed, nNOS-ir neurons were observed in the SOC of rat and golden hamster, and a nitrergic olivocochlear projection was identified in rats and guinea pigs (Riemann and Reuss,1999).

Furthermore, the nonapeptides arginine-vasopressin (AVP) and oxytocin (OT), originally found in the hypothalamopituitary system, were detected in the rodent cochlea, and receptors for AVP and OT were demonstrated in the rat inner ear (Kitano et al.,1997). Application of AVP decreased evoked potentials of the guinea pig cochlea (Mori et al.,1986). It is thought that AVP plays a role in the regulation of ion and fluid balance in the cochlea, probably by increasing aquaporin-mRNA in the inner ear (Lohuis et al.,2001; Sawada et al.,2002). Aquaporins 1-6 are present and essential for inner ear sensory functions (cf. Beitz et al.,2003). OT is believed to possess a modulatory function in several parts of the auditory system (Kanwal and Rao,2002). Since the origin of these neuropeptides in the cochlea is unknown, it appears possible that their source is the efferent innervation originating in the SOC.

Another substance that may regulate inner ear fluid regulation is the neuropeptide vasoactive intestinal polypeptide (VIP). Protein and mRNA of VIP and its receptor were demonstrated in the rat inner ear (Kitanishi et al.,1998; Kitano et al.,1998). In addition, transcripts for another member of the same neuropeptide family, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, are present in the rat cochlea (Kawano et al.,2001; Abu-Hamdan et al.,2006). Although both substances were found in spiral ganglion cells, it is open whether the SOC may serve as an additional source. This is particularly the case with PACAP since a distribution study showed that the peptide is present in auditory nuclei of the rat (Hannibal,2002).

In the present study, we identified olivocochlear (OC) neurons by retrograde neuronal tracing upon injection of Fluoro-Gold (FG) into the cochlea and investigated these neurons with respect to immunofluorescence to the above-mentioned transmitters (AVP, OT, VIP, or PACAP) or to the NO-synthesizing enzyme, nNOS. Neurons capable of producing NO were found by NADPH-diaphorase histochemistry in the SOC of the dwarf hamster (Reuss et al.,2000), the species used also in the present study.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals and Treatment

Experiments were conducted with adult male Djungarian hamsters, Phodopus sungorus, held under a light:dark 16:8 hs (“summer”) regimen with food and water ad libitum. The procedures outlined in the following complied with German law for the protection of animals and were approved by the local government office (Bezirksregierung Rheinland-Pfalz).

Animals were killed by ether overdose. The brain was exposed, carefully taken out, and immediately frozen. The part containing the SOC was cut 14 μm thick on a cryostat in the frontal plane. This procedure avoids shrinking of tissue that is present with chemical fixation and embedding methods so that the calculated extents closely relate to in vivo conditions. Complete series of sections were then fixed in alcohol and Nissl-stained in cresyl violet.

Retrograde Tracing

Animals were anesthetized with tribromoethanol and received pressure-injections of 100 nL of a 5% FG (Fluorochrome, Englewood, CO; dissolved in distilled water) into the left scala tympani via the round window. The surgical approach was described previously (Riemann and Reuss,1998; Reuss et al.,1999). After 5 days, hamsters were killed by ether overdose at the middle of the light period and immediately perfused transcardially with phosphate-buffered 0.9% saline (PBS) to which 15,000 IU heparin/L were added, at room temperature (RT), followed by an ice-cold paraformaldehyde-lysine-periodate solution (McLean and Nakane,1974). The right atrium was opened to enable venous outflow.

Immunohistochemistry

The brains were removed, postfixed for 1 hr, and stored overnight at 4°C in phosphate-buffered 30% sucrose for cryoprotection. They were then sectioned serially at 40 μm thickness on a freezing microtome in the frontal plane. Sections were collected in PBS and, for immunohistochemistry, free-floating incubated overnight at RT in polyclonal rabbit-raised antibodies, to which 1% normal donkey serum and 0.1% Triton-X 100 were added.

The antibodies used in this study are well characterized. The one directed against nNOS from rat cerebellum (nNOS, 1:1,000 in PBS; Laboserv, Giessen, Germany, cat.no. B 220-1; see Alm et al.,1993) has been used in our laboratory in a variety of studies (e.g., Reuss,1998; Riemann and Reuss,1999; Reuss and Reuss,2001; Schaeffer et al.,2003). We also used antibodies directed against synthetic AVP (1:2,000 in PBS, Chemicon, Temecula, USA, cat.no. AB937), synthetic OT (1:5,000 in PBS, Chemicon, cat.no.AB911), purified natural porcine VIP (1:100 in PBS, Paesel & Lorei, Hanau, Germany, cat.no.X 24887), or synthetic pituitary adenylate cyclase-activating peptide-38 (PACAP; 1:1,000 in PBS, Progen, Heidelberg, Germany, cat.no.16063).

After three rinses in PBS, the reaction was visualized using Cy3 conjugated to an F(ab)2 fragment of a donkey anti-rabbit IgG (1:200 in PBS; Jackson Immuno-Research). Sections were mounted on gelatinized glass slides, dried, cleared in xylene, and covered. The material was analyzed using an Olympus BX51 research microscope equipped with an epifluorescence unit and a digital color camera. After removing the coverslips from selected sections, these were counterstained by cresyl violet, covered, and photographed again.

Cell Counting

From each section, FG-labeled cells of the brainstem were quantified with regard to their location within the SOC. Neurons exhibiting immunoreactivity to the antibodies tested were counted when immunoreactivity was clearly over the background level. Counts were corrected according to Abercrombie (1946) to prevent double counting of cells. Single- and double-labeled neurons were quantified separately and the respective percentages were calculated for each nucleus.

Brainstem regions were identified using the stereotaxic brain atlases for rat and mouse (Paxinos and Watson,1998; Paxinos and Franklin,2001), and the overviews given by Schwartz (1992), Warr (1992), Kulesza et al. (2002), Malmierca and Merchan (2004). According to Vetter and Mugnaini (1992), FG-labeled neurons were divided into three subgroups: LOC neurons in the lateral superior olive (LSO), shell-neurons (surrounding the LSO, including periolivary regions) and medial OC (MOC) neurons in the ventral nucleus of the trapezoid body (VNTB), and rostral periolivary region (RPO).

Control Studies

Specificity studies, carried out by omitting primary or secondary antibodies or by preabsorption of the antibodies with the respective immunogen, showed the absence of the fluorescent signal. Middle ear application of FG without injection into the inner ear did not label SOC neurons.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The localization of different SOC regions investigated in this study was carried out using fresh-frozen material cut at 14 μm thickness that was afterwards fixed and Nissl-stained. The sections were then analyzed in high magnification. Additionally, the appearance of SOC regions in thicker sections from our immunohistochemical studies was used for comparison. We found that the data obtained from serial Nissl-stained sections stemming from seven male hamsters showed relatively little variation, and determined the approximate borders of the SOC from representative sections. Figure 1 demonstrates representative Nissl-stained frontal sections from a 14-week-old hamster of 48 g body weight. Figure 2 shows a section in which SOC nuclei have been delineated.

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Figure 1. Frontal sections of the Phodopus sungorus brain stem showing the location of the superior olivary nuclei (Nissl-stain; medial is to the left). The arrangement of selected sections is AE rostral to caudal; the coordinates relative to interaural 0 are A +120 μm, B +80 μm, C −50 μm, D −180 μm, E −240 μm. Plotted in B are the delineations of the nuclei. 7n, facial nerve; A5, A5 noradrenaline cell group; bas, basilary artery; DPO, dorsal periolivary region; LNTB, lateral nucleus of the trapezoid body; LSO, lateral superior olive; ml, medial lemniscus; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; py, pyramidal tract; rs, rubrospinal tract; SPO, superior paraolivary nucleus; VNTB, ventral nucleus of the trapezoid body.

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Figure 2. Magnification from a frontal section of the Phodopus sungorus brain stem showing location and approximate borders of the superior olivary nuclei (Nissl-stain; medial is to the left). See legend of Fig. 1 for abbreviations.

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Because of their distinct morphology and arrangement of neurons and their higher cell density in relation to neighboring structures, the boundaries of the medial nucleus of the trapezoid body (MNTB), LSO, and superior paraolivary nucleus (SPO) are relatively distinct. The MNTB is the most medially located SOC structure. It is found dorsal to the pyramidal tract, and extends ventrally with its lateral part (see Fig. 2). It has the largest rostrocaudal extent (∼600 μm) and is composed of mainly large neurons (up to 30 μm soma-diameter). Lateral to the rostralmost part of the MNTB, the RPO is located, just caudal to the anterior part of the trigeminal motor nucleus. Without a clear boundary, the RPO continues into the LSO.

The LSO consists of tightly packed bipolar, predominantly small neurons (10–15 μm soma-diameter). In frontal sections, it shows the typical N-like shape that is observed in many rodents (cf. Schwartz,1992) and extends ∼360 μm rostrocaudally. The caudal periolivary region follows the LSO in sections of the more posterior SOC.

The medial superior olive (MSO) is located between LSO and MNTB, and extends less than 300 μm in the anterior–posterior direction. It consists basically of a conspicuous small vertical column of middle-sized, horizontally oriented bipolar neurons. These neurons seem to be accompanied by small neurons and/or glial cells that contribute to the “globular” appearance of the MSO. The SPO, a triangular structure located medial to the LSO, extends ∼450 μm.

The dorsal and ventral periolivary regions are not well defined. This is especially the case for their rostral and caudal ends and for their peripheral extensions, whereas the borders to the LSO were well seen. The region dorsal to the LSO was defined as dorsal periolivary region (DPO). The region ventral to SPO and MSO was assigned as VNTB that accommodates mainly small neurons. It continues laterally (without a clear separation) into the lateral nucleus of the trapezoid body (LNTB; see Figs. 1 and 2) that contains large multipolar neurons.

Identification of Olivocochlear Neurons by Retrograde Tracing

Following unilateral FG injection into the hamster scala tympani, the tracer was consistently found in cell bodies and processes of the bilateral SOC. An average of 526 cells per animal was labeled. Their distribution within the SOC is given in Table 1, and examples are depicted in Figs. 3A,C, 4C, and 5A,C,E. They were seen in three topographical aspects as typical for OC neurons. LOC neurons in the LSO made up to an average of 44% of all retrogradely labeled neurons and showed a clear ipsilateral predominance. Shell-neurons, located around the LSO and in periolivary regions, amount to 23% of OC neurons, two-thirds of which were located ipsilateral to the injection site. MOC neurons were seen predominantly in the VNTB with a contralateral dominance. A relatively small number of MOC neurons were observed in the MNTB, where labeling was often found to be weak in comparison to LSO neurons. These neurons were concentrated in the ventral aspect of the posterior part of the nucleus.

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Figure 3. Frontal sections of the Phodopus brain. A–D: Superior olivary complex (medial is to the right), showing Fluoro-gold (FG)-labeled neurons upon intracochlear application of the tracer (A) and neuronal nitric oxide synthase (nNOS)-immunofluorescence (B) in the same section. The higher magnifications (C, D) of the boxed region in A, B show the lateral part of the LSO and demonstrate that many FG-labeled neurons are also nNOS-ir. Examples of double-labeled neurons are marked by arrows in C, D; the arrowhead in F depicts nNOS-ir neurons that are not containing FG. The panels E–G demonstrate immunoreactivity to arginine-vasopressin (AVP) and oxytocin (OT) in the hypothalamic paraventricular nucleus (PVN), and to vasoactive intestinal polypeptide (VIP) in the hypothalamic suprachiasmatic nucleus (SCN) in sections from the Phodopus brain.

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Figure 4. Pituitary adenylate cyclase-activating peptide (PACAP)-immunofluorescence in the superior olivary complex (SOC) of a hamster (medial is to the left). A: The low magnification demonstrates that the peptide is present in the lateral and medial superior olivary nuclei (LSO, MSO), in the medial nucleus of the trapezoid body and in periolivary regions. The MSO (box in A) is shown in higher magnification in B. The superior paraolivary region (SPO) is shown in C and D. The pictures taken from the same section demonstrate that some periolovary neurons retrogradely labeled by Fluoro-Gold (FG) (C) are also PACAP-ir (D). Arrows in C and D exemplarily point to double-labeled cells. The arrowhead in D shows a PACAP-ir neuron not labeled by FG.

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Figure 5. Pituitary adenylate cyclase-activating peptide (PACAP)-immunofluorescence in the superior olivary complex (SOC) of a hamster in which Fluoro-Gold (FG) was applied to the cochlea (medial is to the left). A, B: Location of FG neurons (A) and of PACAP-ir neurons (B) in the same SOC section showing the SPO (in the left third of the panel) and the LSO. The higher magnifications (C, D) demonstrate that there is no colocalization of both substances in the LSO. Panels E and F taken from the same section demonstrate that some MNTB/VNTB neurons (in the lower third) show colocalization of FG and PACAP-immunoreactivity. A large neuron outside the lateral boundaries of the nucleus labeled by FG but not exhibiting PACAP-ir is depicted by arrowhead in E.

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Table 1. Distribution of neurons retrogradely labeled by Fluoro-gold (FG) into the cochlea and of FG neurons immunoreactive to nNOS or PACAP in the hamster SOC
 FG-neuronsFG + nNOS-neuronsFG + PACAP-neurons
  • The numbers given are the means of five animals ± standard deviations.

  • a

    Percent of total FG-neurons.

  • b

    Percent of FG-neurons of the respective group.

LOC intrinsic ipsilateral218 ± 54 (41%)a29 ± 8 (13%)b0
LOC intrinsic contralateral15 ± 4 (3%)4 ± 2 (26%)0
Shell ipsilateral82 ± 22 (16%)30 ± 4 (37%)58 ± 18 (70%)
Shell contralateral37 ± 16 (7%)13 ± 3 (35%)34 ± 13 (92%)
MOC ipsilateral64 ± 8 (12%)10 ± 4 (16%)33 ± 9 (52%)
MOC contralateral110 ± 21 (21%)15 ± 6 (14%)86 ± 28 (78%)
SOC total526 ± 121 (100%)101 ± 23 (19%)a211 ± 76 (40%)a

Neuronal NOS-Immunoreactivity in the SOC

Neuronal NOS-immunoreactivity was observed in many cell bodies and fibers within the hamster SOC (see Fig. 3B). The fraction was relatively high in the nuclei of the trapezoid body where up to half of the neurons exhibited nNOS-immunoreactivity. The proportions were about 10% in periolivary regions and less than 20% in the LSO, respectively. In addition to perikaryal staining, dense accumulations of punctate nNOS-stained structures were observed in the SPO and LSO. While only few MOC neurons expressed nNOS-immunoreactivity, the amount of double-labeled LOC neurons was larger. Neurons that were labeled by both, the retrograde tracing and immunoreaction were only observed in the LSO (Fig. 3C,D) and anterior part of the VNTB. While in the LSO the amount was rather low, some few MOC neurons located in the MNTB were also nNOS-ir.

AVP-, OT-, and VIP-Immunoreactivity in the SOC

We did not detect neurons in the SOC that exhibited immunoreactivity to vasopressin, OT, or VIP. In simultaneously incubated control sections containing hypothalamus from the same animals, we observed AVP-ir neurons in the dorsomedial suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), and hypothalamic paraventricular nucleus (PVN; Fig. 3E). OT-ir neurons were present in the SON and PVN (Fig. 3F), and VIP was found in the ventrolateral SCN (Fig. 3G), according to reports in the literature (cf. Reuss,1996; Armstrong,2004).

PACAP-Immunoreactivity in the SOC

Neurons exhibiting PACAP-immunoreactivity were found in all regions of the Phodopus superior olivary nucleus. Approximately one-third of all SOC cells exhibited the respective immunofluorescence. An overview is given in Fig. 4A, higher magnifications are shown in Figs. 4B,D and 5B,D,F. Many large ir neurons were found in the MSO (Fig. 4B) and MNTB. In other regions such as the LSO and periolivary regions, the proportions of ir neurons roughly were up to one-fourth of all neurons. Double-labeled neurons were not found in the LSO (Fig. 5A–D). Relatively high proportions of FG-labeled neurons that were also PACAP-ir are present in the ventrolateral edge of the MNTB and the VNTB (Fig. 5E,F).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The present data characterize the SOC of the auditory brain stem in the dwarf hamster, Phodopus sungorus, with regard to composition and extent of SOC nuclei, location of OC neurons, and distribution and possible contribution to the efferent pathway of five different neuroactive substances.

The analysis of fresh-frozen, Nissl-stained sections revealed that location, composition, and morphological appearance of the Phodopus SOC highly resemble that of other rodent species (cf. Schwartz,1992; Paxinos and Watson,1998; Paxinos and Franklin,2001; Kulesza et al.,2002). In general, it is slightly larger than the mouse SOC but clearly smaller compared to the rat complex. Most medially located is the MNTB that is the SOC nucleus exhibiting the largest rostrocaudal extent. Its dimensions of ∼600 μm are similar to that seen in mouse and less extending than the rat SOC, but proportionally similar to it. The LSO of Phodopus is laterally located, large in all extents and exhibits the typical N-like shape as seen in mouse, rat, and other rodents. The MSO is relatively small and appears as an oval structure with blurred boundaries.

Peri- and paraolivary regions such as the triangular SPO and the dorsal DPO are not well defined. While their borders to the LSO were well seen in the sections, the peripheral extensions are diffuse. Ventral to the MSO/LSO, the VNTB is located and continues laterally into the LNTB, without the clear separation that is apparent in rats (Paxinos and Watson,1998).

Location of Olivocochlear Neurons

We identified efferent cells by FG injection into the scala tympani of Phodopus sungorus and found retrogradely labeled neurons in some nuclei of the SOC, in general accordance with data obtained from other rodent species (Strutz,1981; White and Warr,1983; Guinan et al.,1984; Strutz and Bielenberg,1984; Robertson et al.,1987c; Aschoff and Ostwald,1988; Vetter and Mugnaini,1992; Warr et al.,1997; Maison et al.,2003; Sanchez-Gonzalez et al.,2003; Kraus and Illing,2005; Brown and Levine,2008). More than 500 neurons per animal were labeled on average.

LOC neurons were seen in the LSO where nearly half of the labeled cells were found (predominantly ipsilateral), distributed more or less regularly throughout the N-shaped structure. Shell neurons made up to approximately one-fourth and consisted of labeled neurons on the margins of the LSO (“peri-LSO cells” according to Robertson et al.,1987c). Shell neurons also include labeled cells in dorsal (DPO) and superior (SPO) peri and paraolivary regions and in the LNTB. The latter is known to project to the third and fourth turns of the cochlea (Robertson et al.,1987a).

None were found in the MSO (in agreement to literature data), strengthening the view that this SOC nucleus does not contribute to the efferent innervation of the cochlea.

MOC neurons were found in the VNTB, in its rostral aspects anterior to the LSO and, in more caudal sections, ventral to the MSO region. The VNTB projects to both cochleae and to the contralateral LSO (Warr and Beck,1996).

We also observed a few MOC neurons in the MNTB, located in the ventral posterior aspect directly neighboring the VNTB. It is thus difficult to distinguish their exact location in the VNTB or MNTB, and we may not exclude the possibility that they actually are ectopic VNTB cells. However, a few MOC neurons in the rodent MNTB were previously observed in some studies (Robertson,1985; Robertson et al.,1987c; Riemann and Reuss,1999) but not in others (White and Warr,1983; Vetter and Mugnaini,1992; Sanchez-Gonzalez et al.,2003). Interestingly, these cells, some of which project to both cochleae (Robertson et al.,1987b), were labeled only after application of the tracer into basal turns (Robertson et al.,1987a). Thus, technical parameters (e.g., tracer application site and spread) may contribute to their sporadic observation.

Neuronal NOS-Immunoreactivity in the SOC

Our present findings in the dwarf hamster show that approximately one-fourth of all neurons exhibit immunoreactivity to the NO-synthetizing enzyme, neuronal NOS. The data, suggesting that these neurons produce NO, support earlier observations that about 25% of the SOC neurons are nitrergic in rat, guinea pig, and golden hamster (Reuss,1998; Riemann and Reuss,1999). In the present study, the amounts of immunoreactive neurons ranged from ∼10 (MSO and DPO) to nearly 50% (MNTB), which is in accord to data obtained from rats and guinea pigs (Fessenden et al.,1999; Riemann and Reuss,1999; Schaeffer et al.,2003). Our previous demonstration that the nNOS-ir subpopulation is, in part, identical with those neurons that project to the cochlea, was substantiated presently by the finding that 19% of all OC neurons in Phodopus are nitrergic.

In accordance with our previous tracing study (Riemann and Reuss,1999), neurons labeled by both, retrograde tracing from the cochlea and nNOS-antibodies were observed consistently in the LSO. Fourteen percent of the LOC neurons were nNOS-ir. It is conceivable that their axons branch to provide collaterals to many hair cells, and the gaseous substance NO diffuses out of the terminals and may well reach and influence multiple hair cells. Nitrergic influence on IHC may further be added by shell neurons that were to one-third nNOS-ir in the present study. Since nNOS-ir terminals were found at the bases of both, inner and OHC in a previous study (Riemann and Reuss,1999), nitrergic IHC terminals may stem from dendrites of the primary auditory neurons that are nNOS-immunoreactive as well (Franz et al.,1996; Fessenden et al.,1999; Riemann and Reuss,1999).

Finally, out of the MOC cells that project to the OHC on both sides, 14% were labeled by the nNOS-antibody. These were located in the VNTB that may provide NO to OHC via this path. Few additional MOC neurons were located presumably in the MNTB where many neurons were nNOS-ir. This nucleus sends fibers to the ipsilateral LSO and SPO (Spangler et al.,1985; Helfert et al.,1989) where dense accumulations of nNOS terminals were found (Reuss,1998; this study). Efferent fibers at the OHC originate, however, also in periolivary regions (cf. Schwartz,1992; Guinan,1996).

In total, only a relatively small portion of nNOS-ir neurons in the SOC project to the cochlea of Phodopus sungorus. Similar results were obtained in rats and guinea pigs (Riemann and Reuss,1999). Taking into account that also less than 10% of the nitrergic neurons are olivocollicular neurons (Schaeffer et al.,2003), the projection target of more than 80% of the nNOS-ir neurons in the SOC is still unknown. The cochlear nuclei and the nuclei of the lateral lemniscus, other well-known major targets of ascending SOC projections, will be tested in future studies.

Nitric neurons of the SOC, however, may additionally have local actions. Evidence is provided by strong immunostaining for the NO-receptor, guanylate cyclase, and for its product, cyclic guanosine monophosphate in neuronal somata and processes of the SOC (Southam and Garthwaite,1993; Fessenden et al.,1999). In cochlear processing, the substance may cause a loss of the auditory gross neural response, and damage electrical and morphological parameters of the cochlea (Ohlsen et al.,1993; Nakashima et al.,1994; Kong et al.,1996). Previous work showed that NO is involved in glutamate-mediated neurotoxicity (cf. Garthwaite,2008). L-glutamate acts via AMPA receptor subtypes that have been implicated in glutamate excitotoxicity at afferent synapses at IHC. Indeed, overstimulation conditions, such as those occurring during acoustic trauma, lead to an excessive release of glutamate accompanied by swelling of dendrites within the cochlea (Puel,1995). It is also interesting here that some forms of presbyacusis have been attributed to long-term excitotoxic damage (Pujol et al.,1993).

Attention should again be drawn to the results of the aging studies mentioned earlier, which revealed the age-related increase of NO-production in cochlea and MNTB (Kimura et al.,1998; Reuss et al.,2000). Since inner hair cell loss is minimal in the aged guinea pig cochlea (Ingham et al.,1999), age-related changes may occur rather in functional parameters of sensory cells. It is conceivable that nNOS-immunoreactive terminals at the base of IHC provide more NO to sensory cells in the aging inner ear that also would result in altered function. However, the mechanisms underlying the augmented production of NO in the senile auditory system and its functional implications require elucidation.

Since NO is an important factor in pathophysiological mechanisms of the inner ear, its sources are of high interest. The present data, in accordance with our previous studies on rat and guinea pig, suggest that lateral and medial OC neurons of the dwarf hamster are sources of NO shown to be released in the cochlea (Shi et al.,2001).

AVP-, OT-, and VIP-Immunoreactivity in the SOC

Another aim of the present study was to investigate whether the SOC is a source of fibers containing the peptides AVP, OT, or VIP. Experimental evidence previously suggested that the substances might influence cochlear physiology (see introduction). These neuroactive substances or their receptors, respectively, were found in the cochlea (Kitano et al.,1997,1998; Kitanishi et al.,1998) but their origin remained unknown. A few lightly labeled OT-ir perikarya were described for the bat SOC (Kanwal and Rao,2002) but it is not known whether these are OC neurons. Our data, however, reveal that AVP, OT, and VIP are not present in OC neurons (and not at all in the Phodopus SOC) and suggest another (probably local cochlear) source that should be further investigated. Control incubations of hypothalamic sections showed that all antibodies yielded convincing immunolabeling in this species (see Figs. 3A–G).

PACAP-Immunoreactivity in the SOC

Another peptide belonging to the VIP family, that is, PACAP, was found in considerable number of SOC neurons. Approximately one-third of all neurons were immunoreactive revealing that the neuropeptide is produced in the SOC of, at least, the Phodopus brain, although data from other species are not yet available. We observed the peptide in MNTB neurons and in the MSO suggesting that it is involved in the neural projections of these nuclei to LSO, periolivary regions and to the inferior colliculi (cf. Schwartz,1992). Interestingly, the large PACAP-ir neurons of the MSO were similarly stained for nNOS (present study; Riemann and Reuss,1999) indicating that both substances are colocalized.

Our findings further suggest that PACAP is transported into the cochlea since PACAP-ir neurons also include identified OC neurons. Since 40% of all FG-neurons exhibited PACAP-immunoreactivity, the SOC may serve as a source of PACAP recently found in the cochlea (Kawano et al.,2001). However, since PACAP-mRNA was also detected in spiral ganglion neurons (Kawano et al.,2001) it is conceivable that both afferent and efferent fibers contribute to its putative effects on hair cells. It is well known that in the brain the substance acts synergistically to glutamate, the primary amino acid neurotransmitter in the cochlea (Eybalin,1993). The effects of PACAP are mediated by the activation of adenylate cyclase and phospholipase C signaling pathways (Spengler et al.,1993) that play important roles in inner ear physiology. Since PACAP presently was not found in lateral but in medial olivocochlear efferents, it is conceivable that OHC rather than IHC are contacted by PACAP-ir terminals and are influenced by the substance. This was recently substantiated by Drescher et al. (2006) who found PACAP colocalized with ChAT in the intraganglionic spiral bundle and hypothesized that PACAP acts as an efferent neuromodulator.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED