Characterization of the superior olivary complex of Canis lupus domesticus

Hearing Research 351 (2017) 130e140

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Research Paper

Characterization of the superior olivary complex of Canis lupus domesticus
n-Garciduen ~ as b, c, Randy J. Kulesza Jr. a, * Tatiana Fech a, Lilian Caldero a

Department of Anatomy, Lake Erie College of Osteopathic Medicine, Erie, PA, USA The University of Montana, Missouola, MT, USA c Universidad del Valle de Mexico, Mexico b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 April 2017 Received in revised form 10 June 2017 Accepted 13 June 2017 Available online 15 June 2017

The superior olivary complex (SOC) is a collection of brainstem auditory nuclei which play essential roles in the localization of sound sources, temporal coding of vocalizations and descending modulation of the cochlea. Notwithstanding, the SOC nuclei vary considerably between species in accordance with the auditory needs of the animal. The canine SOC was subjected to anatomical and physiological examination nearly 50 years ago and was then virtually forgotten. Herein, we aimed to characterize the nuclei of the canine SOC using quantitative morphometrics, estimation of neuronal number, histochemistry for perineuronal nets and immunofluorescence for the calcium binding proteins calbindin and calretinin. We found the principal nuclei to be extremely well developed: the lateral superior olive (LSO) contained over 20,000 neurons and the medial superior olive (MSO) contained over 15,000 neurons. In nearly all nonchiropterian terrestrial mammals, the MSO exists as a thin, vertical column of neurons. The canine MSO was folded into a U-shaped contour and had associated with the ventromedial tip a small, round collection of neurons we termed the tail nucleus of the MSO. Further, we found evidence within the LSO, MSO and medial nucleus of the trapezoid body (MNTB) for significant morphological variations along the mediolateral or rostrocaudal axes. Finally, the majority of MNTB neurons were calbindinimmunopositive and associated with calretinin-immunopositive calyceal terminals. Together, these observations suggest the canine SOC complies with the basic plan of the mammalian SOC but possesses a number of unique anatomical features. © 2017 Elsevier B.V. All rights reserved.

Keywords: Brainstem Superior olive Trapezoid body Calbindin Calretinin Perineuronal nets

1. Introduction The superior olivary complex (SOC) is a conglomerate of auditory nuclei in the mammalian brainstem and extends from the rostral medulla oblongata to the caudal pons. The SOC is an important relay station as it is the first level of the auditory pathway to receive a major convergence of ascending inputs from both the ipsilateral and contralateral cochlear nuclei (CN; reviewed by Malmierca and Hackett, 2010). The SOC additionally receives major descending inputs from the auditory cortex, thalamus and midbrain (reviewed by Schofield, 2010). Thus, the SOC is situated to provide a major influence onto both the ascending and descending auditory pathways. The SOC includes two well-characterized

* Corresponding author. Department of Anatomy, Lake Erie College of Osteopathic Medicine, 1858 West Grandview Blvd, Erie, PA, 16509, USA. E-mail address: [email protected] (R.J. Kulesza). http://dx.doi.org/10.1016/j.heares.2017.06.010 0378-5955/© 2017 Elsevier B.V. All rights reserved.

principal nuclei, the medial and lateral superior olives (MSO and LSO, respectively). These nuclei vary significantly in size and morphology according to the auditory sensitivity of the species, but function in the encoding of temporal features of sounds and localization of sound sources in azimuth (see reviews: Heffner and Masterton, 1990; Schwartz, 1992; Thompson and Schofield, 2000; Oliver, 2000; Malmierca and Hackett, 2010). The LSO varies according to the overall hearing range of the animal and is best developed in species with wide hearing ranges (mouse, rat; Glendenning and Masterton, 1998). Accordingly, the LSO is poorly developed in species with more limited hearing ranges (opossum, chinchilla; Glendenning and Masterton, 1998). The MSO is biased towards low frequency tones and is best developed in animals with excellent low-frequency hearing (gerbils, guinea pig, fox, monkey, cat, humans; Schofield and Cant, 1991; Smith et al., 1993; Grothe and Sanes, 1993; Glendenning and Masterton, 1998; Kulesza, 2007). Surrounding the MSO and LSO are a number of periolivary nuclei which vary significantly between species but contribute

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List of abbreviations AVCN CB CI CN CR D DMPO fn FN IR L LNTB LSO MNTB MSO

anterior ventral cochlear nucleus calbindin confidence interval cochlear nucleus calretinin dorsal dorsal medial periolivary nucleus facial nerve facial nucleus immunoreactive lateral lateral nucleus of the trapezoid body lateral superior olive medial nucleus of the trapezoid body medial superior olive

unique neuronal circuits and distinct functions within the auditory system (Schofield and Cant, 1991, 1992; Schofield, 2002). The medial nucleus of the trapezoid body (MNTB) is the largest and best characterized of the periolivary nuclei. The principal neurons of the MNTB receive input from the contralateral posteroventral cochlear nucleus (PVCN) via the calyx of Held (Held, 1893; Ramon y Cajal, 1909; Stotler, 1953). The MNTB sends glycinergic projections to the MSO, LSO, other periolivary nuclei and the ventral nucleus of the lateral lemniscus (VNLL) and plays essential roles in the localization of sound sources. Our current understanding of the canine SOC, compared to typical laboratory animals (rodents) and even rhesus and humans, is limited. Goldberg and Brown examined the canine SOC using morphological, degenerative and physiological techniques (Goldberg and Brown, 1968, 1969). The size and shape of the canine MSO appeared to be the motivation for their study, as these authors claimed to have had difficulty obtaining isolated recordings from the feline MSO. Indeed, they revealed the canine MSO as a peculiar U-shaped nucleus, with a dorsal limb, hilus and a ventral limb (see Goldberg and Brown, 1968, Fig. 3). Additionally, they identified an LSO, MNTB, medial periolivary nucleus (likely the ventral nucleus of the trapezoid body, VNTB) and lateral periolivary nucleus (likely the lateral nucleus of the trapezoid body, LNTB). Despite such a large and robust SOC, there have been very few studies of the auditory brainstem in this species since 1969. The few studies available focus on the distribution of perineuronal nets (PNNs) in the MSO and LSO (Atoji et al., 1989, 1990, 1995, 1997; Atoji and Suzuki, 1992). Collectively, the literature provides evidence for potentially interesting interspecies variations in the canine SOC. We therefore propose that exploration and characterization of these differences will contribute to our understanding of how the SOC trends between species to meet specific auditory needs. Herein, we aim to characterize the canine SOC using modern, quantitative morphometric techniques and immunofluorescence.

2. Materials and methods 2.1. Animals This study is based on the examination of brainstems from four canines obtained from Instituto Nacional de Pediatría (INP) in Mexico City, Mexico. Included in this study were the brains of a 10 month-old female, a 25 month-old female, a 72 month-old male

PB PN PNN pt PVCN py RF SOC SPON STN stt t tz VCN VI VNLL VNTB

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phosphate buffer pontine nuclei perineuronal nets pyramidal tract posterior ventral cochlear nucleus pyramid reticular formation superior olivary complex superior paraolivary nucleus spinal trigeminal nucleus spinal trigeminal tract tail nucleus trapezoid body ventral cochlear nucleus abducens nerve ventral nucleus of the lateral lemniscus ventral nucleus of the trapezoid body

and a 120 month-old female. Nissl stained sections from these
n-Garciduen ~ as brains were included in a separate analysis (Caldero et al., submitted). All animal handling and care was approved by the Institutional Animal Care and Use Committee at the Instituto Nacional de Pediatría (INP). The procedures used were in accordance with the guidelines of the INP on the Use and Care of the Animals, the regulations of the NOM-062-ZOO-1999 Official Mexican Standard and the Guide for the Care and Use of Experimental Animals and the standard in the Guide for the Care and Use of Laboratory Animals (8th ed). The INP provided full veterinary care of the animals used in this study. The animals were housed in outdoor-indoor kennels and husbandry were in compliance with the Guide for the Care and Use of Laboratory Animals (8th ed). All animals had current vaccination status and underwent daily veterinary observation, regular anti-helmintic treatment for internal worms, daily clinical examinations, and weekly neurological examinations. Otoscopic examinations were performed by staff veterinarians to rule out any disease of the external ear and/or eardrum that could interfere with hearing. Animals were euthanized with an overdose of sodium pentobarbital (70 mg/kg; Pisabental PISA, Mexico) administered intravenously through the cephalic vein. Brains were removed from the skull within 3 min after death and preserved by submersion in formalin.

2.2. Sectioning and histology Approximately 1 week before frozen sectioning, whole brains were divided in the sagittal plane and the right side was trimmed to a block extending from the dorsal cochlear nucleus to the exit of the trigeminal nerve. These blocks were placed in a solution of 30% sucrose in 4% paraformaldehyde and 0.1 M phosphate buffer (PB; pH 7.2) at room temperature until they were saturated. Brainstem blocks were sectioned on a freezing microtome in the coronal plane at a thickness of 40 mm. For morphological studies, every sixth to eighth section was collected serially into 0.1 M PB. For staining of Nissl substance, tissue sections were mounted onto glass slides in caudal-to-rostral sequence from gelatin alcohol and air dried at room temperature. Mounted sections were rehydrated, stained for Nissl substance with Giemsa (Sigma-Aldrich, St Louis, MO), dehydrated through ascending alcohols, cleared and coverslipped with Permount (ThermoFisher Scientific, Waltham, MA). For staining of myelin sheaths, tissue sections were collected in deionized water. Free-floating sections were rinsed in water, incubated in 2.5% iron

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alum, rinsed in water, stained in hematoxylin-ethanol, rinsed again in water and mounted onto glass slides from gelatin alcohol. After drying, sections were stained for Nissl substance with neutral red and sealed under coverslips with Permount (ThermoFisher Scientific, Waltham, MA). 2.3. Neuronal morphology The morphology of SOC neurons was examined in each of the four brainstems used. Giemsa-stained tissue sections were examined with an Olympus BX45 microscope. Cell body contours were traced at a final magnification of 400
. Neuronal profiles were digitized and quantified using ImageJ (1.51 h). The number of neurons included in this analysis is provided in Table 1. A measure of the orientation angle of MSO somata was made from these tracings. For sections cut in the coronal plane, neurons with a long axis parallel to the dorsal-ventral axis had an orientation angle of ~90 while neurons with a long axis parallel to the medial-lateral axis had an orientation angle near 0 . See the orientation arrows in Fig. 2B for reference. For all cell body profiles, an index of circularity was calculated using the following equation: Circularity ¼ [4p * Area/Perimeter2] Classification of cell body morphology was made according to objective morphometric measures. Specifically, neuronal profiles were classified as fusiform if the major axis/minor axis was >3. If the circularity measure for a given neuronal profile was greater than 0.6, the soma was classified as ‘‘round/oval’’; all remaining profiles were classified as stellate. These criteria have previously been correlated with distinct cell body morphologies (Ruby et al., 2015; Foran et al., 2017). 2.4. Estimates of neuronal number The total number of neurons in each of the SOC nuclei was estimated in each of the four brainstems used in this study. For each nucleus, neuronal packing density was calculated by counting the number of neuronal profiles within the nuclear contour along the rostro-caudal extent of each nucleus. These counts were corrected using Konigsmark's (1970) formula (for recent application see Thompson and Brenowitz, 2005; Kulesza, 2008; Wagoner and Kulesza, 2009; Kulesza et al., 2011; Lukose et al., 2011, 2015; Foran et al., 2017) to account for profile splitting (rearranged): N ¼ n(t/[t þ 2a]) In this equation, N is the estimated number of neuronal profiles in a given nucleus, n is the total number of profiles counted, t is section thickness and “a” is the square root of r2 - (k/2)2. In this expression, r is the average radius of the nucleoli and k indicates the average minor axis of observed nucleoli. These corrected counts

Table 1 Number of Neurons and Axons Examined.

LSO MSO MNTB DMPO VNTB LNTB Tail nucleus Axons measured

Giemsa

PNN

CB

CR

449 467 164 109 116 128 128 e

898 441 143 118 137 100 108 e

188 111 150 68 e e 65 85

580 541 44 101 e 66 50 95

were then divided by the tissue volume from which they were counted, yielding neuronal density. The total number of neurons in each nucleus was finally estimated by multiplying neuronal density by the total estimated volume of each nucleus (Thompson and Brenowitz, 2005). This method of estimating neuronal number has produced results statistically similar to the optical dissector (Thompson and Brenowitz, 2005; Kulesza, 2007). 2.5. Identification of perineuronal nets Perineuronal nets (PNNs) were identified by specific binding of biotinylated Wisteria floribunda agglutinin (WFA, catalog # B-1355; €rtig et al., 1992). After sectioning, every Vector Laboratories; Ha sixth to eighth free-floating tissue section was rinsed in 0.1 M phosphate buffer, endogenous peroxidase activity was quenched in a solution of 3% hydrogen peroxide, tissue was permeablized in a solution of 0.5% Triton X-100 and non-specific binding was blocked with a 1 h incubation in 1% normal donkey serum (NDS). Tissue sections were incubated overnight in a 20 mg/ml solution of biotinylated WFA. Tissue sections were then rinsed in PB and incubated for at least 1 h in ABC solution (Vector Laboratories, Elite Kit). Following this incubation, sections were rinsed in phosphate buffer, then 0.05 M Tris and the chromogenic reaction was developed by incubation in 0.05% diaminobenzidine, 0.01% hydrogen peroxide with heavy metal intensification (Adams, 1981). Tissue sections processed without biotinylated WFA failed to reveal any labeling. Sections were mounted onto glass slides from gelatin alcohol, dried, counterstained for Nissl substance with neutral red, dehydrated, cleared and coverslipped with Permount (ThermoFisher Scientific, Waltham, MA). 2.6. Immunofluorescence The expression patterns of the calcium binding proteins, calretinin and calbindin were investigated in all four brainstems. After sectioning as described above, every sixth to eighth tissue section was rinsed in 0.1 M PB and incubated for 1 h in a solution of 0.1 M PB, 0.5% Triton X-100 and 1% NDS (AbCam, Cambridge, MA). Free-floating sections were incubated overnight (~20 h) with 1% NDS and primary antisera (rabbit anti-calbindin, 1:1000, catalog # ab11426, AbCam or rabbit anti-calretinin, 1:100, catalog # ab 702, AbCam). Sections were then rinsed in 0.1 M PB and incubated for at least 2 h in secondary antisera (both goat anti-rabbit DyLight 488, 1:100, catalog # DI-1488, Vector Labs, Burlingame, CA). Sections were then rinsed in 0.1 M PB, counterstained with Neurotrace Red (a fluorescent Nissl stain; ThermoFisher Scientific, Waltham, MA), mounted onto glass slides from 0.1 M PB and coverslipped with Vectashield Hardset Antifade mounting medium (Vector Labs, Burlingame, CA). Tissue sections were studied with an Olympus CKX41 microscope equipped with Epifluorescence and a DP71 digital camera. Tissue sections processed as stated above, but with the omission of the primary antibody (anti-calbindin or anticalretinin), revealed no detectable fluorescent signal. The number of immunoreactive neurons was estimated by counting the number of CR-IR or CB-IR neurons in each nucleus and then dividing this number by the total number of neurons in each nucleus (identified with Neurotrace Red). The total number of immunoreactive neurons counted for each region is shown in Table 1. The diameter of CB or CR-IR axonal profiles was examined in the medial aspect of the trapezoid body (medial to the MNTB; see Table 1 for number of axons examined). 2.6.1. Statistical analysis Descriptive statistics were generated for all data sets using GraphPad Prism 7.02 (GraphPad Software, La Jolla, CA). All data sets

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were examined for a normal distribution using the D'Agostino & Pearson omnibus normality test. Data sets that met a normal distribution were compared using parametric tests (i.e. t-test) and results are presented in the text and figures as mean ± standard deviation (SD). When a normal distribution was not met, data sets were compared using non-parametric tests (e.g. Mann-Whitney [axon diameters] or Kruskal Wallis) and data are presented in the text as median and 95% confidence interval. For each of the SOC nuclei, a contingency table of cell body morphologies was constructed and the distribution of these morphologies was compared using a Chi-square test. Differences were considered statistically significant if p values were <0.05. 3. Results 3.1. Morphology of LSO neurons In the canine brainstem, the LSO first appeared in the ventral aspect of the medulla just rostral to the facial nucleus (Fig. 1A), at the level of the PVCN and extended rostrally over 3 mm. For most of the rostrocaudal course of the LSO, the nucleus appeared as a slightly wrinkled S-shape contour (Figs. 1 and 2). The canine LSO contained approximately 20,363 (±2127) neurons. An initial microscopic examination of the LSO revealed an apparent gradient of neuronal morphologies by medial to lateral location in the nucleus. The LSO was therefore divided into three limbs (medial, intermediate and lateral) for morphometric analyses (Fig. 3AeD). In the medial limb of the LSO (mLSO), 47% of the neurons were round/ oval, 26% were stellate and 27% were fusiform. In this region, cell bodies had a mean cross-sectional area of 195 mm2 (SD; ± 68.3 mm2). In the intermediate limb (iLSO), 80% of the neurons were round/oval, 14% were stellate and 6% were fusiform. In this region, cell bodies had a mean cross-sectional area of 219 mm2 (±72.2 mm2). In the lateral limb (lLSO), 60% of the neurons were round/oval, 32% were stellate and 8% were fusiform. These differences in the distribution of neuronal morphologies was statistically significant (Fig. 3D; Chi square, p < 0.0001). In the lLSO, cell bodies had a mean cross-sectional area of 245 mm2 (±75.1 mm2). Neurons in the lLSO

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were significantly larger (Fig. 3C; ANOVA, p < 0.0001) than those in both iLSO (Tukey's, p ¼ 0.0049) and the mLSO (Tukey's, p < 0.0001). 3.2. Morphology of MSO neurons The MSO first appeared at the same rostro-caudal level as the LSO (Fig. 1) and extended rostrally nearly 4.5 mm. In the canine SOC, the MSO was folded into a U-shaped contour and included a ventral limb, a genu and a dorsal limb (Figs. 1 and 2). The canine MSO contained approximately 15,611 (±1238) neurons. The size and morphology of MSO neurons was examined in both the dorsal and ventral limbs of the nucleus (Fig. 3 EeH; dMSO and vMSO, respectively). Both limbs of the MSO contained 75e86% round/oval neurons, 10e19% stellate neurons and 4e6% fusiform neurons (Fig. 3H). There was no statistical difference in the distribution of neuronal morphologies between the dMSO and vMSO (Chi square, p ¼ 0.13). Neurons in the dMSO had a mean cross sectional area of 188 mm2 (±62.1 mm2) while those in the vMSO were 181 mm2 (±59.9 mm2). This difference was not significant (Fig. 3G; t-test, p ¼ 0.35). Somata in the dMSO (on the right side of the brain) had long axes that measured ~46 while the long axis of this segment of the nucleus measured 155 . Somata in the vMSO had long axes that measured 105 , while the long axis of this segment of the MSO measured ~15 . Thus, in both limbs of the MSO, the long axes of neuronal cell bodies were nearly perpendicular to the long axis of the nucleus. 3.3. Morphology of MNTB neurons The MNTB was situated medial and ventral to the MSO and first appeared at about the same rostrocaudal level as the MSO and LSO (Fig. 1B). The canine MNTB contained approximately 4128 (±937) neurons. Both the packing density and morphology of MNTB neurons appeared to vary along the rostrocaudal axis of the nucleus. Therefore, for morphological analyses the nucleus was divided into a caudal third (cMNTB), a middle third (mMNTB) and a rostral third (rMNTB; Fig. 3IeL). In the cMNTB, somata had a mean crosssectional area of 196 mm2 (±53.4 mm2). In this region of the

Fig. 1. Atlas of the canine SOC. Images A through H are from a series of tracings through the brainstem of the canine extending from the facial nucleus (A; FN) to the rostral end of the SOC and the VNLL (H). This series is based on tracings from every 16th section through the SOC. The heavy black line marks the ventral boundary of the brainstem. Neurons outside of clear nuclear boundaries are indicated by black circles. The LSO is indicated in red, the MSO is indicated in blue and the MNTB is shown in yellow. The tail nucleus (t) is composed of a small collection of neurons along the tail end of the MSO. The scale bar is equal to 500 mm. D, dorsal; M, medial. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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the distribution of neuronal morphologies between the three rostrocaudal regions was also significant (Fig. 3L; Chi square, p < 0.0001). 3.4. Morphology of periolivary neurons The dorsal medial periolivary nucleus (DMPO) was situated between the MSO and MNTB and consisted of few sparsely packed neurons (Figs. 1 and 2). The canine DMPO contained approximately 1163 (±268) neurons. Somata in the DMPO had a mean cross sectional area of 182 mm2 (±77 mm2) and the nucleus was composed of 65% round/oval neurons, 18% stellate and 17% fusiform neurons. The ventral nucleus of the trapezoid body (VNTB) was situated ventral to the MSO and lateral to the MNTB (Figs. 1 and 2). The canine VNTB contained approximately 1972 (±203) neurons. Somata in the VNTB had a mean cross sectional area of 160 mm2 (±68 mm2) and the nucleus was composed of 66% round/oval neurons, 22% stellate and 12% fusiform neurons. The lateral nucleus of the trapezoid body (LNTB) was situated ventral to the LSO among the fibers of the trapezoid body (Figs. 1 and 2). The canine LNTB contained approximately 2838 (±576) neurons. Somata in the LNTB had a mean cross sectional area of 122 mm2 (±60 mm2) and the nucleus was composed of 74% round/ oval neurons, 17% stellate and 9% fusiform neurons. Additionally, in each of the 4 brainstems there was a small collection of neurons situated along the tail of the vMSO (t in Figs. 1 and 2). These neurons (which we term the tail nucleus) do not appear to be part of the MSO as they are separated from this nucleus by a band of white matter (white arrow in Fig. 2A), demonstrate different myelin architecture (Fig. 2A), different neuropil staining (Fig. 2B) and the neurons are markedly smaller than those in the MSO (Fig. 3G). The tail nucleus of the MSO contained approximately 2356 (±550) neurons. The somata in the tail nucleus had a mean cross sectional area of 116 mm2 (±51 mm2) and the nucleus was composed of 79% round/oval neurons, 9% stellate and 12% fusiform neurons. 3.5. Calbindin

Fig. 2. Nuclei of the canine SOC. Shown in A is a tissue section stained for myelin and Nissl substance. The borders of the LSO and MSO are indicated with dashed lines. Along the fibers of the trapezoid body (tz) are the MNTB, VNTB and LNTB. The DMPO is situated between the MSO and MNTB. A band of axons (arrowhead) separated the tail nucleus (t) from the vMSO. Shown in B is a Nissl stained section from the same rostrocaudal level as shown in A. The orientation arrows in figure B are provided as reference for angle measurements of MSO neurons. Shown in C is a section from the same approximate rostro-caudal level as shown in A and B, showing the distribution of PNNs. The scale bar is equal to 500 mm.

nucleus, 93% of the neurons were round/oval, 5% were stellate and 2% were fusiform (Fig. 3L). In the mMNTB, somata had a mean cross-sectional area of 159 mm2 (±48.2 mm2). In the mMNTB, 66% of the neurons were round/oval, 14% were stellate and 20% were fusiform (Fig. 3L). In the rMNTB, neurons had a mean crosssectional area of 124 mm2 (±50.2 mm2). In the rMNTB, 66% of the neurons were round/oval, 16% were stellate and 18% were fusiform. The differences in somata size between the three rostrocaudal regions were significantly different (Fig. 3K; ANOVA, p < 0.0001; Tukey's multiple comparison test, all p < 0.0025). The difference in

Calbindin immunofluorescence revealed that the vast majority of neurons in the anteroventral cochlear nucleus were CBþ (Fig. 4B) and we additionally identified many CBþ axons coursing through the trapezoid body. CBþ axons in the trapezoid body had a median diameter of 2.1 mm (95% CI ¼ 2.1e2.4 mm; Fig. 6D). In the SOC there were relatively few CBþ neurons (Fig. 4A), but an abundance of perisomatic CBþ punctate profiles (Fig. 4D), which presumably represent synaptic boutons. CBþ cell bodies were readily apparent in the MNTB and 63% of all MNTB neurons were CBþ (Figs. 4C and 6A). In fact, 93% of all CBþ cell bodies in the canine SOC were within the boundaries of the MNTB. However, few CBþ neurons were identified in the LSO (1.5% of total LSO), MSO (5.4%), the tail nucleus (5%) and DMPO (7%). We also examined the number of perisomatic CBþ puncta in the SOC nuclei (Fig. 6B). In the mLSO there was a mean of 8.6 (±2) CBþ puncta per soma (Fig. 6B). However, in the iLSO and lLSO there were 4.5 (±2) and 3.4 (±3) CBþ puncta per soma, respectively. These differences were statistically significant (ANOVA, p < 0.0001; Dunn's multiple comparison test, mLSO-iLSO, p < 0.01; mLSO-lLSO, p < 0.0001). In the dMSO there were 5 (±2) CBþ puncta per soma and 4.9 (±1) in the vMSO and this difference was not significant (ANOVA, p ¼ 0.99). Notably, in the tail nucleus there were 10 (±3) CBþ puncta per soma. In terms of CBþ puncta, the tail nucleus was significantly different from both

T. Fech et al. / Hearing Research 351 (2017) 130e140

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Fig. 3. Morphology of SOC neurons. Shown in each horizontal series is morphometric data from the LSO (AeD), MSO (EeH) and MNTB (IeL). The coloring of the arrowheads in the photomicrographs correspond to the key provided in D. Comparisons of cell body size between nuclear regions are shown in C (LSO), G (MSO) and K (MNTB). Cell bodies in the lLSO were significantly larger than those in the iLSO or the mLSO. Likewise, neurons in the cMNTB were significantly larger than those in the rest of the nucleus. The distribution of cell body morphologies is shown in D (LSO), H (MSO) and L (MNTB). The distribution of cell body morphologies was significantly different amongst the regions of the LSO and MNTB (see text for details). Scale bar in J is equal to 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the dMSO (p ¼ 0.01) and the vMSO (p ¼ 0.04; Fig. 6B). Finally, neurons in the DMPO had 4.5 (±2) CBþ puncta per soma (not shown) and there were 1.6 (±2) per soma in the MNTB (Fig. 6B).

of MNTB neurons were CRþ. There were no CRþ somata in the DMPO.

3.6. Calretinin 3.7. Perineuronal nets Calretinin immunofluorescence revealed a robust population of CRþ neurons in the PVCN near the entry of the auditory nerve and there were abundant CRþ axons in the trapezoid body (Fig. 5B). CRþ axons in the trapezoid body had a median diameter of 3.3 mm (95% CI ¼ 3.2e3.6 mm; Fig. 6D). CRþ axons in the trapezoid body had significantly larger diameters than CBþ axons (Mann-Whitney, p < 0.0001). Throughout the rostrocaudal extent of the MNTB, there were large diameter CRþ axons that terminated as calyx terminals in close association to MNTB neurons (Fig. 5C). Further, there was an abundance of CRþ terminals in the LSO, MSO and LNTB. In the LSO and MSO, the CRþ terminals appeared as a perisomatic halo (Fig. 5D); individual punctate profiles could not be resolved. Such CRþ perisomatic halos were present around nearly all LSO and MSO neurons. LNTB somata were associated with relatively large CRþ puncta (Fig. 5E). There were few CRþ neurons in the SOC. In the LSO, there was a gradient of CRþ somata (Fig. 6C). In the mLSO only 2% of neurons were CRþ, while in the iLSO and lLSO 9% and 12% of LSO neurons were CRþ (respectively). In the MSO, there were few CRþ neurons (dMSO e 3% and vMSO e 8%). However, in the tail nucleus 24% of neurons were CRþ (Figs. 5A and 6C). In the LNTB, 66% of the neurons were CRþ (Figs. 5E and 6C) while only 5%

WFA histochemistry revealed a rich network of PNNs in the canine SOC (Fig. 2C). Indeed, the majority of SOC neurons in the canine were associated with WFAþ PNNs, although there appeared to be gradients of PNNs in the LSO and MSO. As stated above, we examined PNNs in the three limbs of the LSO (Fig. 7A, B and E). In the mLSO, 68% (±16%) of neurons were associated with PNNs, while 84% (±6%) of neurons in the iLSO and 83% (±6%) of neurons in the lLSO were associated with PNNs. Despite this gradient, the difference in number of PNNs across the limbs of the LSO was not statistically significant (ANOVA, p ¼ 0.07). In the dMSO, 35% (±8%) of neurons were associated with PNNs, while in the vMSO 78% (±5%) of neurons had PNNs (Figs. 2C and 7C, D and E). This difference was statistically significant (ANOVA, p < 0.001, Sidak's multiple comparison test, p ¼ 0.0013). In the MNTB, 80% (±15%) of neurons had PNNs. In the DMPO, LNTB and VNTB, 58% (±22%), 59% (±22%) and 65% (±19%) of neurons were associated with PNNs. In the tail nucleus, 72% (±27%) were associated with PNNs and this was significantly different from the dMSO (ANOVA, p < 0.001, Sidak's multiple comparison test, p ¼ 0.0072).

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Fig. 4. Calbindin expression in the SOC. Shown in A is CB expression in the SOC. The majority of neurons in the MNTB were CBþ. Throughout the rest of SOC, there were abundant CBþ punctate profiles. Shown in B is the AVCN where the majority of neurons were also CBþ. Shown in C is the MNTB where many neurons were CBþ (white arrowheads; CB immunonegative neurons are indicated with red arrowheads). Shown in D is a section through the LSO. CB immunonegative cell bodies with numerous CBþ perisomatic profiles are indicated by white arrowheads. The scale bars are as follows: A ¼ 500 mm, B ¼ 100 mm and C & D ¼ 40 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion 4.1. General considerations This report provides the first quantitative morphometric analyses of the nuclei and neurons of the canine SOC and the first quantitative analyses of calbindin and calretinin expression in this region. To the best of the authors knowledge, this is the first demonstration that canine MNTB neurons are CBþ and are contacted by CRþ calyx terminals. This report confirms earlier observations that the medial limb of the canine LSO contains smaller neurons than the lateral two limbs and that the MSO exists as a Ushaped contour (Goldberg and Brown, 1968). Additionally, this report confirms earlier reports on the distribution of PNNs in the canine SOC and provides quantitative confirmation of PNN gradients in the MSO and LSO (Atoji et al., 1997).

4.2. Comparative features of the principal nuclei The results provided above suggest that the canine SOC is hypertrophied compared to common laboratory species (mouse, rats, gerbils, guinea pigs, cat, rhesus), and even humans. This observation supports previous findings. Specifically, Glendenning and Masterton (1998) examined the subcortical auditory nuclei in a series of 53 mammalian species, including lemur, macaque and human. While no canine species were included in their study, they did include the red fox (Vulpes vulpes; both the red fox and

domestic dog are classified within the Canidae family). The red fox had the largest (by volume) LSO and MSO of all mammals included in their study and the fifth largest MNTB. In fact, the LSO/MSO/ MNTB comprised a remarkably large proportion (11.49%) of the red fox's subcortical auditory system. Our cell counts from the canine SOC parallel these findings (discussed further below).

4.2.1. Lateral superior olive Our results indicate the canine LSO contains over 20,000 neurons and also that there are marked differences in neuronal morphology across the regions/limbs of the nucleus. Specifically, in the lLSO, neurons were larger (Goldberg and Brown, 1968), were more likely to be CRþ and were more likely to have a PNN (compared to the iLSO and mLSO). However, neurons in the mLSO had significantly more perisomatic CBþ puncta. Notably, the rat LSO appears to lack a significant medial-to-lateral gradient in neuronal size (Lukose et al., 2011) and appeared to lack any such gradient in PNNs (Myers et al., 2012). However, a gradient of perisomatic CBþ inputs to the LSO has been observed in the feline (Matsubara, 1990). These topographic morphological differences most likely result from a gradation of synaptic inputs from the VCN and MNTB. According to the tonotopic axis of the LSO of the cat (Guinan et al., 1972), it would appear that the largest neurons of the canine LSO are associated with lower characteristic frequencies and more PNNs. On the contrary, the smallest neurons are associated with more perisomatic CBþ puncta, fewer PNNs and higher characteristic frequencies. LSO neurons in the canine appear to receive

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Fig. 5. Calretinin expression in the SOC. Shown in A is a section through the mid-SOC demonstrating CR immunoreactivity. There were few CRþ neuronal cell bodies in the SOC. Shown in B is CR expression in axons of the trapezoid body. Shown in C are large CRþ axons in the MNTB (white arrowheads). The axons were often observed to give rise to large, calyx terminals (open white arrowhead; MNTB soma indicated by a white asterisks). The yellow arrowhead gives rise to a clear calyx terminal associated with an MNTB soma (white asterisk). The axon indicated by the yellow arrowhead measured 3.83 mm in diameter. Shown in D are LSO somata (asterisks) associated with CRþ halos (open white arrowheads). A CRþ axon is seen traversing the nucleus (closed arrowhead). Shown in E are CRþ LNTB neurons (asterisks). These cell bodies were associated with larger CRþ puncta. The scale bars are as follows: A ¼ 500 mm, B ¼ 100, C & D ¼ 40 mm, E ¼ 60 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

abundant input from the ipsilateral VCN and a less dense input from the contralateral VCN (Goldberg and Brown, 1968). Only four LSO units were recorded by Goldberg and Brown (1968) and 75% (3 neurons) were monaural and inhibited by the ipsilateral ear and 1 neuron (25%) was excited by the ipsilateral ear and inhibited by the contralateral ear (EI). The LSO has been extensively studied in other species and it seems that the vast majority of LSO neurons are EI (Guinan et al., 1972). Unfortunately, this discrepancy in the canine LSO cannot be rectified from any of the results provided above. Additionally, the canine LSO appears to receive a heavy CRþ input and a gradient of perisomatic CBþ input. The canine VCN appears to include populations of CBþ and CRþ neurons and both CBþ and CRþ axons were observed in the trapezoid body. Furthermore, the majority of MNTB neurons were CBþ. Thus, potential sources of CBþ puncta in the canine LSO could be the VCN and/or the MNTB while CRþ puncta most likely arise from the VCN and/or the LNTB. Finally, there appears to be a significant population of CRþ cell bodies in the LSO. In fact, in the lLSO approximately 12% of the neurons were CRþ. Such a population has not been described in other species (rat e Fredrich et al., 2009; rhesus e Bazwinsky et al., 2005; Human e Kulesza, 2014). 4.2.2. Medial superior olive Our results indicate that the canine MSO is composed of a largely homogenous population of neurons with their long axes perpendicular to the long axis of the nucleus, confirming previous

observations (Goldberg and Brown, 1968). Further, Goldberg and Brown (1968) indicated that the canine MSO receives bilateral input from the VCN, consistent with contemporary models of the nucleus (Smith et al., 1993; Goldwyn et al., 2014; Myoga et al., 2014). The canine MSO appears to include over 15,000 neurons. In comparison, the feline MSO includes approximately 4600 neurons and that of rhesus includes 3100 neurons (unpublished observations). This seems especially remarkable given very similar hearing ranges between felines and canines (~125 Hze60 kHz; Poncelet et al., 2002; Malmierca and Hackett, 2010). Furthermore, cell counts from a total of 24 brainstems indicates that the human MSO includes approximately 14,000 neurons (Kulesza, 2007; Kulesza et al., 2011; Lukose et al., 2015). These results are consistent with reported volume measurements of the MSO (Glendenning and Masterton (1998). Thus, the canine MSO appears to be one of the largest of terrestrial mammals, independent of brain size. A unique feature of the canine MSO is the U-shaped contour throughout the rostrocaudal extent of the nucleus. Goldberg and Brown (1968) found that neurons in the dMSO trended towards low best frequencies (1e2 kHz) while those in the vMSO trended towards higher best frequencies (5e12 kHz). Notably, the MSO of the rabbit and cat each appear to have a very slight lateral curvature along the dorsal aspect of the MSO column (Batra et al., 1997; Berman, 1968; unpublished observations). Additionally, in some species of bats the MSO shows a complex contour (Covey, 2005; see

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Fig. 6. Distribution of CB and CR in SOC neurons. Shown in A is the total number of CBþ neurons in each of the SOC nuclei. The majority of MNTB neurons are CBþ. Shown in B are the number of CBþ perisomatic puncta in SOC nuclei. There were significantly more CBþ puncta in the mLSO compared to the rest of the LSO. There were more CBþ puncta in the tail nucleus compared to the adjacent MSO. Whiskers in B represent the 10th and 90th percentiles. Shown in C are the total number of CRþ neurons in the SOC. There were more CRþ neurons in the tail nucleus, compared to the rest of the MSO and the majority of LNTB neurons were CRþ. Shown in D are the diameters of axons in the trapezoid body that were either CBþ or CRþ. CRþ axons had significantly larger diameters than those that were CBþ. The asterisks in B and D are as follows: * ¼ p < 0.05, **** ¼ p < 0.0001.

especially Pteronotus). Regardless, the etiology for the U-shaped appearance of the canine MSO is unclear. We hypothesize that the U-shaped contour is a consequence of developmental processes (i.e. neuronal migration), the large size and neuronal density and/or the afferent inputs reaching the nucleus. Goldberg and Brown (1968) observed axons from the contralateral VCN terminating along the convex (medial-facing) border of the MSO and axons from the ipsilateral VCN terminating along the concave (lateralfacing) border. However, there were also axons originating from the ipsilateral VCN, that coursed dorsal to the MSO to terminate along the dorsal convex surface of the nucleus (i.e. medial facing dendrites). These ipsilateral axons coursing over the dorsal edge of the nucleus and terminating on the contralateral dendrites could contribute to the orientation of neurons in the dMSO (rotating them laterally) and thereby the peculiar orientation of the dorsal aspect of the nucleus. A similar pattern of termination of ipsilateral VCN axons has been observed in the cat (Warr, 1966, 1969; Beckius et al., 1999), but appears to be much more prominent in canine (Goldberg and Brown, 1968). Such a pattern of inputs to the lateral-

most low-frequency tip of the MSO would most likely result in a bias towards ipsilateral stimuli. Unfortunately, physiological recordings from the canine MSO do not support this anatomical observation. Nearly 65% of canine MSO neurons are EE, 11% are driven by the contralateral ear and 15% were EI (Goldberg and Brown, 1968). Recordings from feline MSO neurons also fail to clarify this point as the majority of units recorded by Guinan and coworkers (Guinan et al., 1972) received symmetric inputs (64% EE and 18% EI-EI). The canine MSO appears to receive a comparatively sparse perisomatic CBþ input but a heavy CRþ input. The canine VCN includes populations of CBþ and CRþ neurons and we observed both CB and CRþ axons in the trapezoid body. Again, the majority of canine MNTB neurons were CBþ. The sources of CBþ puncta in the MSO likely arise from the MNTB, but the VCN cannot be ruled out. The CRþ puncta likely arise from the VCN and/or the LNTB. Interestingly, a previous study failed to identify any perisomatic CBþ puncta in the cat MSO or CBþ axons in the trapezoid body (Matsubara, 1990). Our observations of the canine SOC are markedly different, especially concerning CBþ input. Such differences are likely attributed to interspecies differences and/or antibody differences. Finally, there were significantly more PNNs in the vMSO compared to the dMSO confirming previous observations (Atoji et al., 1989). This pattern of PNNs in the MSO suggests that, contrary to the LSO, neurons with lower characteristic best frequencies are less likely to be associated with PNNs. Notably, the MSO of the rat, cat and rhesus show no such gradients of PNNs (Myers et al., 2012; unpublished observations). PNNs are believed to play a role in the stabilization of synapses, synaptic plasticity and neuronal protection (Pizzorusso et al., 2002; Dityatev and Schachner, 2003). The gradation of PNNs in the canine LSO and MSO of the canine SOC might contribute to an elevated level of plasticity in low-frequency circuits. Additionally, PNNs are associated with fast-spiking neu€rtig et al., 1999; Balmer, 2016) and the paucity of PNNs in rons (Ha low-frequency regions of the LSO and MSO might simply reflect firing patterns in these regions. 4.3. Comparative features of the periolivary nuclei The majority of canine MNTB neurons were CBþ, as in nearly all mammals (mouse e O'Neill et al., 1997; gerbil/opossum e Bazwinsky-Wutschke et al., 2016; rat e Friauf, 1993; chinchilla e Kelley et al., 1992; rhesus - Bazwinsky et al., 2005; cat - Matsubara, 1990; human - Kulesza, 2014), and were contacted by large, CRþ calyx terminals (Lohmann and Friauf, 1996). In line with these similarities, 97% of canine MNTB neurons received excitation from the contralateral ear (Goldberg and Brown, 1968). However, the canine MNTB demonstrated a rostrocaudal gradient of principal cells. In the caudal-most sections, the MNTB appears very typical: it is composed of a dense collection of round, principle neurons (as is seen throughout the MNTB in rat, cat, rhesus). Such a change in the nucleus is not apparent in other lab species and the cause/effect of this alteration is not clear. No rostrocaudal gradient of CBþ puncta were readily apparent in the LSO. Although, there were significantly more CBþ puncta in the mLSO. It may be that the cMNTB projects mainly to the mLSO and the rMNTB projects mainly to the lLSO, where the density of CBþ puncta was significantly less. In this paradigm, besides a tonotopic projection, there would be some degree of rostro-caudal mapping of projections from the MNTB to the LSO. The canine LNTB was composed of mainly round/oval neurons and approximately two-thirds of these neurons were CRþ and associated with larger CRþ terminals. We suggest that these CRþ terminals correspond with inputs from globular bushy cells in

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Fig. 7. PNNs in the SOC. Shown in A through D are WFA-positive PNNs in the LSO and MSO, respectively. Cell bodies with PNNs are indicated with white arrowheads, ensheathed dendritic processes are indicated by white arrows and a cell body lacking a PNN is indicated by a red arrowhead (B). Shown in E are counts of the total number of neurons associated with PNNs in each of the nuclei. There was no difference among the regions of the LSO, although there were more neurons in the vMSO with PNNs compared to the dMSO. The scale bar in C is equal to 20 mm. Whiskers in E represent the standard error of the mean. The asterisks in E represent a p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the ipsilateral VCN as seen in cat (Spirou and Berrebi, 1996). We propose that at least some of the CRþ terminals in the MSO arise from the LNTB, as has been demonstrated in other species (Cant and Hyson, 1992; Kuwabara and Zook, 1992). Goldberg and Brown (1968) found that LNTB neurons (their LPO) received a topographic and strictly ipsilateral projection from the VCN. An additional unique feature of the canine SOC was the population of neurons identified along the lateral edge of the vMSO. Despite the proximity to the MSO, these neurons were significantly smaller than neurons in the dMSO and vMSO. Neurons in the tail nucleus received more than twice as many perisomatic CBþ puncta compared to the MSO, and had more PNNs than the dMSO and more than three times as many CRþ somata than the MSO. We believe that these features distinguish neurons in the tail nucleus from the MSO. Regardless, the function of this collection of neurons is not intuitive and unknown. A similarly situated nucleus does not appear within the feline SOC and such a collection of neurons was not discussed by Goldberg and Brown (1968). However, this group was included within the MSO contour in Atoji et al. (1989). Nonetheless, it is possible that the tail nucleus of the MSO in canines is a displaced cell group. In the feline SOC, neurons of the pvLNTB often abut the ventral MSO (Spirou and Berrebi, 1996). Indeed, the pvLNTB shares some features with the tail nucleus. For example, the neurons in the tail nucleus and pvLNTB both appear to receive an abundant innervation from the CN and both are mainly populated by round/oval neurons. Finally, it may be that the curving of the dorsal aspect of the MSO pulled the medial dorsal periolivary nucleus ventrally (Thompson and Schofield, 2000). Regardless, determination of the function of this cell group awaits further histochemical classification. 5. Conclusion/summary The canine SOC appears to be particularly noteworthy among terrestrial mammals. First, the LSO and MSO appear to be hypertrophied in comparison to many mammals and especially the feline. The canine LSO appears to contain more than 20,000 neurons. Further, the canine MSO appears to contain more than three times as many neurons as the feline and five times as many as rhesus MSOs. Indeed, the canine MSO includes even more neurons than the human MSO. Given such a large contingency of LSO and MSO

neurons, it is unclear why this species was largely disregarded in studies of the SOC. The neurons of the canine LSO display a number of differences that appear to vary along a medial to lateral topographic map. We believe such differences in the LSO can be attributed, at least in part, to gradations along the tonotopic axis. Additionally, the canine MSO is arranged in a U-shaped contour which is in stark contrast to the vast majority of mammals. Finally, the MNTB appears to be arranged along a rostrocaudal gradient. These alterations in the MSO and MNTB are likely related to specific auditory needs/abilities of the species as might be required for listening to vocalizations and environmental sounds, Such an enlarged auditory brainstem is suggestive of excellent hearing over a wide range of sound frequencies, a pronounced ability in sound source localization and fine discrimination of temporal features of sound.

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