Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate

Accepted Manuscript Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate Lindsey Foran, Kaitlyn Blackburn, Randy J. Kulesza PII: DOI: Reference:

S0306-4522(17)30010-6 http://dx.doi.org/10.1016/j.neuroscience.2017.01.004 NSC 17540

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Neuroscience

Received Date: Revised Date: Accepted Date:

10 June 2016 31 December 2016 3 January 2017

Please cite this article as: L. Foran, K. Blackburn, R.J. Kulesza, Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate, Neuroscience (2017), doi: http:// dx.doi.org/10.1016/j.neuroscience.2017.01.004

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Title:

Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate

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ABSTRACT Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and is stored and released by both neurons and astrocytes. Despite the important role of glutamate as a neurotransmitter, elevated extracellular glutamate can result in excitotoxicity and apoptosis. Monosodium glutamate (MSG) is a naturally occurring sodium salt of glutamic acid that is used as a flavor enhancer in many processed foods. Previous studies have shown that MSG administration during the early postnatal period results in neurodegenerative changes in several forebrain regions, characterized by neuronal loss and neuroendocrine abnormalities. Systemic delivery of MSG during the neonatal period and induction of glutamate neurotoxicity in the cochlea have both been shown to result in fewer neurons in the spiral ganglion. We hypothesized that an MSG-induced loss of neurons in the spiral ganglion would have a significant impact on the number of neurons in the cochlear nuclei and superior olivary complex. Indeed, we found that exposure to MSG from postnatal days 4 through 10 resulted in significantly fewer neurons in the cochlear nuclei and superior olivary complex and significant dysmorphology in surviving neurons. Moreover, we found that neonatal MSG exposure resulted in a significant decrease in the expression of both calretinin and calbindin. These results suggest that neonatal exposure to MSG interferes with early development of the auditory brainstem and impacts expression of calcium binding proteins, both of which may lead to diminished auditory function.

KEYWORDS: brainstem, cochlear nucleus, superior olivary complex, trapezoid body

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LIST OF ABBREVIATIONS 4V an aStell AVCN CB CR CI D DCN fn FN gc icp L LNTB LSO MNTB MSG MSO OC OCA PB pStell PVCN py RF SOC SPON STN stt tz VCN VN VNTB

fourth ventricle auditory nerve stellate neurons in AVCN anterior ventral cochlear nucleus calbindin calretinin confidence interval dorsal dorsal cochlear nucleus facial nerve facial nucleus granule cell area inferior cerebellar peduncle lateral lateral nucleus of the trapezoid body lateral superior olive medial nucleus of the trapezoid body monosodium glutamate medial superior olive octopus cell octopus cell area phosphate buffer stellate neurons in PVCN posterior ventral cochlear nucleus pyramid reticular formation superior olivary complex superior paraolivary nucleus spinal trigeminal nucleus spinal trigeminal tract trapezoid body ventral cochlear nucleus vestibular nuclei ventral nucleus of the trapezoid body

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INTRODUCTION Glutamate is the major excitatory neurotransmitter in the central nervous system (Fonnum, 1984; Robinson and Coyle, 1987; Michaelis, 1998) and the excitatory effects of glutamate on neurons have been known for many years (Hayashi, 1952; Curtis et al., 1959). Monosodium glutamate (MSG) is a common food additive that dissolves into L-glutamic acid and repeated exposure to MSG is associated with neurotoxic effects (Goldsmith, 2000; Bojanic et al., 2004; Stafstrom, 2004; Hashem et al., 2012). Certainly, administration of high levels of MSG (4-5 mg/g of body weight) results in persistent activation of glutamate receptors (Gill et al., 2000; Mattson, 2008). Further, repetitive and prolonged activation of NMDA receptors results in elevated intracellular Ca++ levels, disruption of mitochondrial membranes, DNA fragmentation and apoptosis (Hartley and Choi, 1989; Patel et al., 1996; Rivera-Cervantes et al., 2004; Fan et al., 2007; Jung et al., 2009; Ndountse and Chan, 2009; Shah et al., 2015).

It has been

established that the blood-brain barrier of neonates is immature and that glutamate can traverse this barrier and exert excitotoxic effects on adjacent brain regions (McCall et al., 1979; Shah et al., 2015). Thus, repeated exposure to MSG, especially during the neonatal period, results in neuronal damage and cell death in a number of different brain regions (Arees and Mayer, 1970; Burde et al., 1971; Everly, 1971; Oser et al., 1971; Quines et al., 2014). Specifically, MSG exposure has been shown to result in fewer neurons in the spiral ganglion (Carricondo et al., 2002), severe degeneration of retinal ganglion cells (Regan et al., 1981; van Rijn et al., 1986), smaller brains and lower body weights (Ureña-Guerrero, et al., 2003), fewer cerebellar Purkinje cells with deficits in motor coordination (Kiss et al., 2005; Prastiwi et al., 2015) and fewer neurons in the cerebral cortex (Chaparro-Huerta et al., 2002). Additionally,

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MSG exposure results in significant alterations in GABA and/or GAD activity in the forebrain, cerebellum and retina (Regan et al., 1981; Di Giorgio et al., 1985; Hashem et al., 2012). Beyond these direct neuronal effects of neonatal MSG exposure, it appears that astrocytes and microglia are susceptible to MSG-induced excitotoxicity (Martinez-Contreras et al., 2002; Chaparro-Huerta et al., 2002) and glial responses to excitotoxicity may in fact trigger neuronal death (Bronstein et al., 1994; Chaparro-Huerta et al., 2002).

Taken together, there is overwhelming evidence that neonatal MSG exposure causes neuronal degeneration in many areas (but see Foran et al., 2016). The effects of MSG on the auditory brainstem have not been investigated, although creation of an excitotoxic environment in the cochlea leads to injury and/or death of neurons in the spiral ganglion and reduced auditory brainstem responses (Pujol et al., 1985; Janssen et al., 1991; Jiang et al., 2004; Hyodo et al., 2009; Lu et al., 2010). We therefore hypothesized that MSG exposure during the neonatal period would result in fewer neurons in the auditory hindbrain and that surviving neurons would be dysmorphic and have reduced expression of the calcium binding proteins calbindin (CB) and calretinin (CR). To examine this hypothesis, we exposed neonatal rat pups to MSG from P4 to P10 and examined the total number of neurons, neuron morphology and expression of CB and CR in the ventral cochlear nucleus (VCN) and nuclei of the superior olivary complex (SOC).

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MATERIALS AND METHODS ANIMALS All animal handling procedures were approved by the LECOM IACUC (protocol #14-04). Sprague Dawley rats were maintained on a 12 hours light/dark cycle with free access to food and water. Animals were mated and females were permitted to deliver litters without interference. On postnatal day 4 (P4), litters were culled to 4 or 6 male pups. This report is based on the study of saline and MSG-exposed animals (n=11 each) from 4 different litters. Between P4 and P10, the saline and MSG-exposed animals were weighed and then injected (subcutaneously, along the dorsal hindquarters) with MSG (4mg/g) or saline (equivalent volume of 0.9% NaCl). This paradigm has been shown previously to produce cytotoxic effects in the central nervous system (Chaparro-Huerta et al., 2002; Ureña-Guerrero, et al., 2003; Shah et al., 2015). Litters were weaned at P21 and saline and MSG-exposed animals were housed separately. On P28, animals were anesthetized with an intraperitoneal injection of pentobarbital (80 mg/kg); when they were unresponsive to toe pinch, they were perfused through the ascending aorta with normal saline followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PB; pH 7.2; fixative). Brains were dissected from the skull and the right side of the brainstem was marked with a register pin. Brains were then stored in 4% PFA-PB (at 4°C) for at least 24 hours.

SECTIONING AND HISTOLOGY Approximately 24 hours before frozen sectioning, brainstems were placed in a solution of 30% sucrose in fixative at 4°C until they were saturated. Brainstems were sectioned on a freezing microtome in the coronal plane at a thickness of 40 μm. For morphological studies, every third

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section was collected serially into 0.1 M PB. Tissue sections were mounted onto glass slides in caudal-to-rostral sequence from cresyl gelatin and dried at room temperature. Slides 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).

NEURONAL MORPHOLOGY The morphology of neurons in the VCN and SOC was examined in both saline and MSG-exposed animals (control, n=7; MSG, n=5). Giemsa-stained tissue sections were examined with an Olympus BX45 microscope. Cell body contours were traced by an observer blind to experimental condition using a 40x objective (final magnification of 1000x). Neuronal profiles were digitized and quantified using ImageJ (1.48v). 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 oriented along the dorsal-ventral axis had an orientation angle of ~90° while neurons with a long axis oriented along the medial-lateral axis had an orientation angle near 0°. See the orientation arrows in figure 3A for reference. For all cell body profiles, an index of circularity was calculated using the following equation: Circularity = [4π * Area/Perimeter2] Classification of cell body morphology (see Results, section 3.2) was made based on 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

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0.6, the soma was classified as ‘‘round/oval’’; all remaining profiles were classified as stellate. We have previously correlated these criteria with distinct cell body morphologies (Beebe et al., 2014; Kulesza, 2014; Lukose et al., 2015; Ruby et al., 2015).

ESTIMATES OF NEURONAL NUMBER The total number of neurons in the VCN and nuclei of the SOC was estimated in both saline and MSG-exposed animals (both right and left sides, n=3 animals each). For each nucleus, neuronal packing density was calculated by estimating the total number of neuronal cell bodies 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) to account for profile splitting (rearranged): N = n(t/[t + 2a]) In this equation, N is the estimated number of neuronal profiles in a given nucleus, n is the actual 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 were then divided by the tissue volume from which they were counted, yielding neuronal density. The 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).

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IMMUNOFLUORESCENCE The expression patterns of the calcium binding proteins, calretinin and calbindin were investigated in saline and MSG-exposed animals (both right and left sides; n=3 each). Brains were cryoprotected and frozen sectioned as described above. Tissue sections were collected into three wells - sections from wells 1 and 2 were rinsed in 0.1 M PB and incubated for 1 hour in a solution of 0.1 M PB, 0.5% Triton X and 1% NDS (AbCam, Cambridge, MA). Sections from well 3 were archived and not utilized in this study. Free-floating sections were incubated overnight (~20 hours) with 1% NDS and primary antisera (well 1: rabbit anti-calbindin, 1:1000, AbCam; well 2: goat anti-calretinin, 1:50, Santa Cruz, Dallas, TX). Sections were then rinsed in 0.1 M PB and incubated for at least 2 hours in secondary antisera (well 1: goat anti-rabbit DyLight 488; well 2: horse anti-goat DyLight 488; 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 or a Leica TCS SP 5 confocal microscope. Tissue sections processed as stated above, but with the omission of the primary antibody (anticalbindin or anti-calretinin), revealed no fluorescent signal from the secondary antibody.

The number of immunoreactive neurons was estimated by counting the number of CR-IR or CBIR neurons in each region and then dividing this number by the total number of neurons in each region (identified with Neurotrace Red). The number of neurons counted for each region is

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shown in table 2. The diameter of CR-IR axonal profiles in the medial aspect of the trapezoid body (medial to the MNTB) was measured in saline and MSG-exposed animals (table 2). The number of CR-IR calyx terminals in the MNTB was estimated by counting the total number of MNTB neurons in each tissue section and then counting the CR-IR calyx terminals associated with each MNTB soma. The number of MNTB somata examined is provided in table 2. Finally, the MNTB was divided into five evenly spaced parasagittal columns (medial to lateral) and the optical density (OD) of CB-IR was measured using ImageJ. Thus, for each MNTB neuron, two pieces of data were collected: OD of CB immunofluorescence and medial to lateral location. This permitted OD values to be assigned to one of the five medial to lateral MNTB columns.

STATISTICAL ANALYSIS Descriptive statistics were generated for all data sets using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). All data sets 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 [saline versus MSG], Kruskal Wallis [subgroups of saline and MSG: neuronal morphology and optical density]) 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.

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RESULTS COCHLEAR NUCLEUS MSG EXPOSURE REDUCED THE NUMBER OF NEURONS IN THE COCHLEAR NUCLEUS In MSG-exposed animals, the dorsal and ventral divisions of the CN were readily apparent, but both the AVCN and PVCN were reduced in size (figure 1A [saline] and C [MSG]). Additionally, MSG exposure resulted in fewer neurons in the VCN. In saline-exposed animals the VCN contained 25,098 ± 1,483 neurons (mean ± SD). In MSG-exposed animals, the VCN contained 13,374 ± 1,830 neurons (figure 1E). This constitutes a decrease of nearly 47% and was statistically significant (t-test, p < .0001).

MSG EXPOSURE RESULTED IN SMALLER OC AND GBC SOMATA, BUT LARGER SBC SOMATA The major neuronal cell types of the VCN were evident in MSG-exposed animals, although the cell bodies were generally smaller than in saline-exposed animals (figure 2). Octopus cells (OC) are found clustered in the caudal-most aspect of the VCN and are characterized by large, round cell bodies with hazy cytoplasm (figure 1A and C, OCA). In saline-exposed animals, OC somata measured 235 µm2 (median, 95% CI [confidence interval]: 222-262 µm2), while these cells measured 197 µm2 (median, 95% CI – 197 - 234 µm2; figure 2C) in MSG-exposed animals. This difference in OC soma size was statistically significant (17% decrease with MSG exposure; Mann-Whitney, p = .03). Globular bushy cells (GBC) are characterized by large round/oval somata, eccentric nuclei and their location in the VCN along axons of the auditory nerve (figure 2A and B, white arrowheads). In saline-exposed animals, GBC had cell bodies which measured 184 µm2 (median, 95% CI: 184 - 205 µm2). In MSG-exposed animals, GBC somata measured 145

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µm2 (median, 95% CI: 149 - 168 µm2; figure 2A, B and C). This difference was statistically significant (22% decrease with MSG exposure; Mann-Whitney, p < .0001; figure 2C). Spherical bushy cells are characterized by round/oval somata and location in the AVCN. In saline-exposed animals, SBC had cell bodies which measured 128 µm2 (median, 95% CI: 131 - 143 µm2). In MSG-exposed animals, SBC somata were significantly larger and measured 143 µm2 (median, 95% CI: 141 - 158 µm2; 11% increase with MSG exposure; Mann-Whitney, p = .016; figure 2C). Cell bodies of stellate neurons in the PVCN measured 172 µm2 (median, 95% CI: 156 - 199 µm2) in saline-exposed animals and 114 µm2 (median, 95% CI: 102 - 157 µm2) in MSG-exposed animals (figure 2A and B, black arrowheads; figure 2C). This difference was statistically significant (Mann-Whitney, p = .006; figure 2C) and constituted a decrease of nearly 33% in cell body area. Cell bodies of stellate neurons in the AVCN measured 113 µm2 (median, 95% CI: 104 - 126 µm2) in saline-exposed animals and 113 µm2 (median, 95% CI: 103 - 142 µm2) in MSGexposed animals. This difference was not statistically significant (Mann-Whitney, p = .88; figure 2C). Fusiform neurons comprised approximately 1.5% of all VCN neurons in both saline and MSG-exposed animals (figure 2A and B, arrows).

SUPERIOR OLIVE MSG EXPOSURE RESULTED IN FEWER, SMALLER AND IRREGULARLY ORIENTED MSO NEURONS The MSO of saline-exposed animals contained 1,290 ± 228 neurons. In MSG-exposed animals there were significantly fewer neurons (770 ± 184 neurons; 42% decrease with MSG exposure; t-test, p < .0001, figure 1F). However, the MSO contained statistically similar (p > .05) distributions of cell body morphologies in both saline-exposed (55% round, 20% stellate and

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25% fusiform neurons) and MSG-exposed animals (41% round, 26% stellate and 33% round; Chi-square, p = .14, figure 3A and B). There were no differences in the size of the neuronal cell bodies between saline and MSG-exposed animals (Kruskal-Wallis, Dunn’s multiple comparison test, p > .05). Finally, in saline-exposed animals MSO neurons had an orientation angle of 20° (right side; median, 95% CI: 68 - 96°), while in MSG-exposed animals MSO neurons had an orientation angle of 31° (right side; median, 95% CI: 46 - 70°; figure 3C). These populations were highly asymmetric (saline: skewness = .24, kurtosis = -1.9; MSG: skewness = 1.5, kurtosis = -.54) and significantly different (Kolmogorov-Smirnov test, p = .003; figure 3C).

MSG EXPOSURE DID NOT IMPACT THE LSO The LSO of saline-exposed animals contained 2,273 ± 309 neurons. In MSG-exposed animals there were 1,952 ± 494 neurons, but this decrease was not significant (14% decrease with MSG exposure; t-test, p = .07; figure 1B, D and F). The LSO contained statistically similar distributions of cell body morphologies in both saline-exposed (55% round, 30% stellate and 15% fusiform neurons) and MSG-exposed animals (67% round, 21% stellate and 12% round; Chi-square, p = .21). There were no differences in the size of the neuronal cell bodies between saline and MSGexposed animals (Kruskal-Wallis, Dunn’s multiple comparison test, p > .05).

MSG EXPOSURE RESULTED IN FEWER MNTB NEURONS The MNTB of saline-exposed animals contained 6,501 ± 696 neurons. In MSG-exposed animals there were significantly fewer neurons (4,947 ± 840 neurons; 24% decrease with MSG exposure; t-test, p < .0001; figure 1B, D and F and figure 3D and E). In saline-exposed animals,

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the MNTB was composed of 98% round/oval neurons and 2% stellate neurons. In MSG-exposed animals the MNTB was composed of 78% round/oval and 22% stellate neurons and this distribution of neuronal morphologies was significantly different (Chi-square, p < .0001; figure 3F). There were no differences in the size of the neuronal cell bodies between saline and MSGexposed animals (Kruskal-Wallis, Dunn’s multiple comparison test, p > .05; figure 3D and E).

MSG EXPOSURE RESULTED IN FEWER SPON NEURONS The SPON of saline-exposed animals contained 2,226 ± 360 neurons. In MSG-exposed animals there were significantly fewer neurons (1,608 ± 181 neurons; 28% decrease with MSG exposure; t-test, p = .0007; figure 1B, D and F). The SPON contained statistically similar distributions of cell body morphologies in both saline-exposed (34% round, 50% stellate and 17% fusiform neurons) and MSG-exposed animals (25% round, 62% stellate and 14% round; Chi-square, p = .22). Lastly, there were no differences in the size of the neuronal cell bodies between saline and MSG-exposed animals (Kruskal-Wallis, Dunn’s multiple comparison test, p > .05).

MSG EXPOSURE RESULTED IN REDUCED CR EXPRESSION IN GBCS AND CALYX TERMINALS The expression of CR is known to be associated with specific neuronal populations in the rat auditory brainstem. The most prominent CR-IR population in the rat auditory hindbrain are the GBCs in the PVCN and CR is localized to the soma, dendrites, axons and calyx terminals of these neurons (Arai et al., 1991; Rogers and Resibois, 1992; Felmy and Schneggenburger, 2004; Por et al., 2005). In saline-exposed animals, approximately 77 ± 5% of GBC were CR-IR (figure 4A, 5C).

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However, in MSG-exposed animals only 56 ± 9% of GBCs were CR-IR and this difference was statistically significant (figure 4B, 5C; t-test, p < .0001). GBC axons traverse the trapezoid body to reach the contralateral MNTB (Warr, 1972; figure 5A and B, black arrows). The diameter of CR-IR axons in the trapezoid body was 0.93 µm (median, 95% CI: .92 – 1.01 µm) in salineexposed animals (figure 5D). However, in MSG-exposed animals these axons measured only 0.82 µm (median, 95% CI: .76 – .86 µm) in diameter (figure 5D). This difference in axon diameter was statistically significant (12% decrease with MSG exposure; Mann-Whitney, p < .0001). Finally, in saline-exposed animals 87 ± 5% of MNTB neurons were associated with CR-IR calyx terminals (figure 5A and E). In MSG-exposed animals, only 66 ± 9% of MNTB neurons were associated with CR-IR calyx terminals and this difference was statistically significant (figure 5B and E; t-test, p < .0001).

MSG EXPOSURE RESULTED IN REDUCED CB EXPRESSION IN THE MNTB BUT NOT OCS The expression of CB is known to be associated with specific neuronal populations in the rat auditory brainstem. Specifically, OCs and principal neurons in the MNTB are characteristically CB-IR (Friauf, 1993; 1994; figure 4C and E). In saline-exposed animals, 76 ± 5% of OCs were CBIR and in MSG-exposed animals there were 70 ± 12% CB-IR OC cells (figures 4C and D; 6C). This difference was not significant (p = .29). In saline-exposed animals, 91 ± 4% of MNTB neurons were CB-IR but in MSG-exposed animals significantly fewer neurons were CB-IR (figures 4E and F, 6A, B and D; 70 ± 13%; t-test, p = .0004). Consistent with previous reports (Bazwinsky et al., 2005), there was a clear gradient of CB-IR in the MNTB. Specifically, CB labelling intensity was lowest in the medial region of the MNTB, was higher in the central regions and was highest in

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the lateral region of the nucleus (Kruskal-Wallis, Dunn’s post hoc, p=.02; figure 6A and E). Such a gradient of CB-IR was not evident in MSG-exposed animals (figure 6B and E). Notably, in MSGexposed animals the lowest CB-IR levels were found in the medial-central region and the highest levels were observed in the lateral-central region (figure 6B and E). In each of the five medial to lateral regions of the MNTB, CB OD was significantly lower in MSG-exposed animals (figure 6E; Mann-Whitney, p < .05). Finally, in saline-exposed animals, CB immunofluorescence was characteristically observed in the cytoplasm of MNTB neurons. However, in MSG-exposed animals, MNTB neurons were often observed with CB-IR restricted only to the cell nucleus (figure 6B, black arrows).

DISCUSSION GENERAL CONSIDERATIONS This report provides the first evidence that neonatal exposure to MSG has a widespread impact on neurons in the auditory hindbrain. Neonatal MSG exposure has been shown to result in drastic atrophy of the optic nerves (Regan et al., 1981; van Rijn et al., 1986) and we have found similar changes in the optic nerves (at P28) of the MSG-exposed animals used in this study (Foran et al., 2016). Despite our findings being consistent across animals and highly significant, the experimental design might be hampered by a low number of animals. Regardless, our results indicate that MSG exposure during the early neonatal period results in a significant loss of neurons in the CN and SOC and that surviving neurons have smaller cell bodies and reduced expression of CB or CR. Our findings are in accordance with the observations of a marked decrease in the number of neurons in the spiral ganglion after MSG exposure (4mg/g/day from

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P9-12; Carricondo et al., 2002). In fact, excitotoxic events induced by intracochlear exposure (kainic acid, glutamate) are known to result in loss of neurons in the spiral ganglion (Pujol et al., 1985; Janssen et al., 1991; Jiang et al., 2004; Lu et al., 2010). Furthermore, normal development of both the CN and SOC require an intact cochlea and auditory nerve (Kitzes et al., 1995; Kotak and Sanes, 1997; Russell and Moore, 1995; 1999; Godfrey et al., 2014). Therefore, the changes observed in the VCN are most likely due to significant loss of type I afferents from the spiral ganglion. Together, these results support a particular sensitivity of primary sensory neurons to excitotoxic events during the neonatal period. In particular, our observations indicate that loss of neurons in the spiral ganglion significantly impacts the structure and function of second and third order neurons in the CN and SOC.

In rats, the external auditory meatus is blocked by a meatal plug until about P12 and adult-like auditory thresholds are not present until P22 (Geal-Dor et al., 1993). Therefore, our paradigm of MSG exposure (from P4 to P10) occurs during a developmental window of significant synaptic and cellular maturation (Holcomb et al., 2013), much of which is occurring in the absence of stimulation by airborne sounds. Damage or removal of the cochlea during this early neonatal period results in severe loss of neurons in the cochlear nucleus (Hashisaki and Rubel, 1989; Mostafapour et al., 2000) and significantly smaller cell bodies in the MNTB (Pasic et al, 1994). The changes we have observed following MSG exposure are consistent with these studies of neonatal injury to the cochlea and provide further support for the inner ear as major target of the excitotoxic effects of MSG.

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IMPACT OF MSG EXPOSURE ON THE GBC/MNTB CIRCUIT GBCs in the VCN are contacted by modified endbulbs derived from type I afferents in the spiral ganglion (Feldman and Harrison, 1969; Ryugo and Rouiller, 1988; Smith and Rhode, 1987). Rat GBC are immunopositive for both CR+ and parvalbumin (Arai et al., 1991; Rogers and Resibois, 1992; Lohmann and Friauf, 1996; Felmy and Schneggenburger, 2004; Por et al 2005). GBCs large caliber axons cross the midline in the trapezoid body to reach the contralateral MNTB where they terminate as the calyces of Held. Each MNTB neuron receives only a single calyx of Held (Friauf and Ostwald, 1988; Hoffpauir et al., 2006). The GBC-MNTB circuit precisely encodes temporal features of sounds, which is required for localization of sound sources.

Our paradigm of MSG exposure resulted in a loss of nearly 50% of the neurons in the VCN and this change is most likely attributable to loss of neurons on the spiral ganglion. Surviving GBCs had significantly smaller cell bodies and less likely to be express CR. CR+ axons in the trapezoid body had smaller diameters and there were fewer MNTB neurons associated with CR+ calyx endings. Although there was no change in the size of MNTB somata after MSG exposure, MSGexposed animals had significantly fewer round neurons and significantly more stellate neurons in the MNTB. These stellate or non-principal cells in the MNTB are known to be devoid of calyx terminals (Banks and Smith, 1992). We interpret these findings to suggest that normal-sized, round MNTB neurons in MSG-exposed animals each receive a calyx terminal. However, it appears as though many of these terminals (and their respective GBCs) may be devoid of CR after MSG exposure. Additionally, we observed a discrepancy in the loss of neurons between the VCN and MNTB (47% in VCN compared to 25% in MNTB). Since each GBC provides a single

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calyx terminal to an MNTB neuron, our results might indicate that a proportion of GBC axons give rise to multiple calyx terminals. Indeed such a phenomenon has been observed in the ipsilateral MNTB after neonatal deafferentation (Kitzes et al., 1995; Hsieh and Cramer, 2006).

The vast majority of rat MNTB neurons are CB+ (Fredrich et al., 2009). Our MSG exposure paradigm resulted in fewer CB+ MNTB neurons and a dispersion of the medial to lateral gradient of CB expression. Similar changes in CB expression have been observed in cerebellar Purkinje cells after pre or neonatal stressors (Wierzba-Bobrowicz et al., 2001; Hatano et al., 2009; Wallace et al., 2010; Main and Kulesza, 2017). We believe that reduced CR and CB expression after MSG exposure is due to aberrant neuronal activity and our findings further support the activity dependent nature of CR and CB expression. Finally, the structural deficits that we have identified in this circuit after MSG exposure will likely impact the brainstems ability to encode temporal features of sound and localization of sound sources.

IMPACT OF MSG EXPOSURE ON SBC/MSO AND LSO CIRCUIT SBC in the VCN are contacted by up to four endbulbs of Held which are derived from type I afferents in the spiral ganglion (Gentshev and Sotelo, 1973; Nicol and Walmsley, 2002). SBC in the rat are immunonegative for CB and CR, express parvalbumin (Resibois and Rogers, 1992; Rogers and Resibois, 1992; Por et al 2005) and project to the ipsilateral LSO and both the ipsilateral and contralateral MSO (Cant and Casseday, 1986). This SBC-MSO and LSO circuit also precisely encodes temporal features of sounds, which is required for localization of sound sources.

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Following our paradigm of MSG exposure, we found that surviving SBCs were significantly larger than those in control animals. It is unlikely that SBCs are spared the effects of MSG-induced loss of spiral ganglion neurons as a single type I afferent innervates both globular and spherical bushy cells (Feldman and Harrison, 1969; Ryugo and Rouiller, 1988; Brown and Benson, 1992; Redd et al., 2000; Nicol and Walmsley, 2002). The increase in size of SBC might be attributed to a number of factors. First, MSG may have resulted in selective loss of neurons in the small spherical cell area, preserving SBCs with larger somata (Webster and Trune, 1982). Second, the surviving SBCs may have gained additional end bulb terminals or developed more extensive axonal projections or dendritic arbors in an effort to overcome neuronal loss. Indeed, larger and more expansive dendrites are associated with larger cell bodies (Friede, 1963). We believe that expression of parvalbumin can be ruled out as a factor since both GBCs and SBCs express this calcium binding protein (Por et al., 2005). We found that MSG exposure also resulted in 42% fewer neurons in the MSO, this was similar to the loss of neurons in the VCN (47% decrease) and nearly twice as much as observed in the MNTB (24% decrease). This loss of neurons in the MSO is likely attributable, at least in part, to loss of neurons in the spiral ganglion and VCN. However, there is evidence that MSO neurons may be uniquely susceptible to certain neurodevelopmental disorders, teratogens and air pollution (Kulesza and Mangunay, 2008; Kulesza et al., 2010; Calderón-Garcidueñas et al., 2011; Lukose et al., 2015; Ruby et al., 2015). The reason for this is unclear, but might be attributable to alterations in blood supply and/or metabolic activity in MSO neurons. We found that the number of LSO neurons was not affected by MSG exposure. This finding is peculiar given the significant loss of neurons in the VCN, MSO

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and MNTB. The reason for preservation of the LSO after MSG exposure is unclear, but protection against excitotoxicity might be provided by the extensive inhibitory inputs to the LSO by the MNTB; such inputs are mature as early as P7 (Altieri et al, 2014). Furthermore, we have previously observed preservation of neuronal number in the LSO after in utero exposure to the antiepileptic and teratogen, valproic acid (Lukose et al., 2011). Regardless, the structural deficits that we have identified in SBC-MSO/LSO circuits after MSG exposure will likely impact encoding of temporal features of sound and the localization of sound sources.

IMPACT OF MSG EXPOSURE ON OC/SPON CIRCUIT OCs in the VCN have broad tuning curves, well-timed spikes in response to pure tone stimuli and phase locking capabilities (Godfrey et al., 1975; Rhode and Smith, 1986; Golding et al., 1995). Rat OCs are characteristically CB+ (Celio, 1990; Friauf, 1994; Rogers and Resibois, 1992; Por et al 2005) and project to the contralateral SPON and VNLL, the later via endbulb-like terminals (Stotler, 1953; Thompson and Thompson, 1991; Schofield, 1995; Adams, 1997). MSG exposure resulted in smaller OC somata and this is most likely a direct impact of diminished input from type I afferent axons. However there was no difference in the percentage of CB+ OCs. We did not directly count the number of OCs in MSG-exposed animals and the relatively small number of OCs in rodents (Willott and Bross, 1990) made drawing any conclusion about loss of OCs difficult. Regardless, the broad tuning of OCs due to input from a large number of primary afferents may lessen the impact of such drastic loss of such inputs. SPON neurons have narrow tuning curves, well-timed spikes timed to the stimulus offset and phase locking capabilities (Kulesza et al., 2003). The SPON receives its major inputs from OCs, GBCs and

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stellate neurons in the VCN (Friauf and Ostwald, 1988; Banks and Smith, 1992; Schofield, 1995). SPON neurons appear to function in encoding temporal features of sounds, especially the sound offset. MSG exposure resulted in fewer SPON neurons, although the surviving neurons were the same size as those in saline exposed animals. We attribute the loss of neurons in the SPON to diminished input from the VCN. The preserved size of SPON neurons might be attributed to these neurons having additional/aberrant inputs from the VCN. The decrease in size of the OCs and the reduced number of SPON neurons is most likely an effect of loss of stellate and/or GBC in the VCN or MNTB. The structural deficits that we have identified in the OC/SPON circuit will likely impact the encoding of temporal features of sound, especially cues related to the stimulus onset and offset.

IMPACT OF MSG EXPOSURE ON EXPRESSION OF CALBINDIN AND CALRETININ Both CB and CR are believed to confer some degree of neuroprotection to neurons (D'Orlando et al., 2002). After removal of the cochlea, CR expression is decreased in the ipsilateral cochlear nucleus and SOC (Winsky and Jacobowitz, 1995; Alvarado et al., 2004; Fuentes-Santamaria et al., 2005). In chicks, removal of the cochlea results in a marked decrease in CR expression by 3 days postsurgery (Stack and Code, 2000). Although, surgical removal of both cochlea did not alter CR expression patterns (Stack and Code, 2000); this suggests that a symmetric pattern of input may be important for CR expression in auditory neurons. Further, cochlear ablation during the neonatal period has been shown to result in decreased expression of CB in the MNTB without neuronal loss (Hatano et al., 2009) and cochlear ablation at P30-40 (in ferrets) also results in decreased CR expression in the LSO (Alvarado et al., 2004). Our results indicate

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reduced expression of CR in GBCs and CB in the MNTB after neonatal MSG exposure. Such decreased expression of these calcium binding proteins in brainstem neurons might render them susceptible to subsequent insults or excitotoxic events and/or disrupt the ability of these neurons to fire rapid trains of action potentials.

In the rat, only 18% of calcyes of Held are CR+ at P14, but by P31 more than 74% of calyces are CR+ (Felmy and Schneggenburger, 2004). Therefore, it seems that by P28-31 the majority of GBC are expressing CR. However, expression of CB by OCs and neurons in the MNTB reaches adult-like patterns earlier. Specifically, most OCs are CB+ by P9 and MNTB neurons begin to express CB between P8 and P12 (Friauf, 1993; 1994). Based on these observations, our MSG exposure paradigm would have overlapped with the onset of CB expression in OCs and the MNTB. However, we found that CB expression was only depressed in the MNTB. We interpret this to suggest that the effect of MSG on CB expression is not related to the timing of the exposure, but rather some other feature of OCs such as number/strength of synaptic inputs at the time of the MSG exposure.

SUMMARY AND CONCLUSIONS We have demonstrated that neonatal exposure to MSG results in significant atrophy of the auditory hindbrain and anomalous expression of CR and CB. We believe that our findings in the CN can be attributed to loss of neurons in the spiral ganglion, while the impact on the nuclei of the SOC are most likely due to reduced innervation, abnormal neuronal activity and/or aberrant growth of axonal inputs from surviving neurons in the VCN.

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ACKNOWLEDGEMENTS The authors would like to thank the Lake Erie College of Osteopathic Medicine Research Collective, Jeffrey Esper, Swati Laroia and Jerome McGraw (Penn State Behrend) for his technical assistance with confocal microscopy. This work was supported by the Lake Erie College of Osteopathic Medicine and the Lake Erie Consortium for Osteopathic Medical Training.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors declare that they have no conflict of interest.

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FIGURE LEGENDS FIGURE 1. Neonatal MSG exposure results in fewer neurons in the CN and SOC. Serial reconstructions are shown of the CN (A – saline, C – MSG) and SOC nuclei (B – saline, D - MSG). The PVCN, AVCN and MNTB are noticeably smaller after MSG exposure. The total estimated number of neurons in the VCN is shown in E. MSG exposure results in a nearly 50% decrease in VCN neurons. The estimated number of neurons in each of the SOC nuclei is shown in F and there are significantly fewer neurons in the MSO, MNTB and SPON after MSG exposure. The scale bar for A and C is equal to 200 µm; the scale bar for B and D is equal to 100 µm. The whiskers in E and F demonstrate the standard deviation. Key to symbols: *** p < .001, **** p < .0001. The orientation arrows refer to dorsal (D) and lateral (L).

FIGURE 2. Neonatal MSG exposure results in smaller neuronal cell bodies in the VCN. Giemsastained sections from the PVCN are shown in A (saline) and B (MSG). GBC are indicated by white arrowheads, stellate neurons are indicated by yellow arrowheads and fusiform neurons are indicated by red arrows. Shown in C are boxplots of cell body areas from saline and MSGexposed animals. The whiskers indicate the 5th and 95th percentile and the cross indicates the data mean, symbols represent data points above or below the 5th and 95th percentiles. The scale bar for A and B is equal to 300 µm. Key to symbols: * p < .05, ** p < .01, **** p < .0001. The orientation arrows refer to dorsal (D) and lateral (L).

FIGURE 3. Neonatal MSG exposure results in dysmorphology in the SOC. Giemsa-stained sections from the MSO (A – saline, B - MSG) and MNTB (D – control, E - MSG) are shown. The orientation

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of the long axis of MSO neurons was measured (see orientation arrows in A; degrees) and results are plotted as a histogram in C. The MNTB was noticeably smaller after MSG exposure. The cell bodies of MNTB neurons were classified as round or stellate and the results are shown in F. There were significantly more stellate neurons in the MNTB after MSG exposure. The scale bar for A, B, D and E is equal to 200 µm. Key to symbols: ** p < .01, **** p < .0001. The orientation arrows refer to dorsal (D) and lateral (L).

FIGURE 4. MSG exposure results in reduced expression of CR and CB in auditory nuclei. CR-IR (green) and Nissl staining (red) is demonstrated in the PVCN of saline (A) and MSG-exposed animals (B) where GBCs are abundant. CB-IR (green) and Nissl staining (red) is shown in the PVCN (C – saline, D - MSG) and SOC (E – saline, F – MSG). There was no difference in the CB-IR pattern in the OCA (yellow dashed line, C and D) between saline and MSG-exposed animals. There was an obvious decrease in the number of CB-IR MNTB neurons between saline and MSG-exposed animals (E and F). The scale bars are both equal to 400 µm. The orientation arrows refer to dorsal (D) and lateral (L).

FIGURE 5. MSG exposure resulted in fewer CR-IR terminals in the MNTB. CR-IR is shown in the MNTB (saline – A, MSG – B). There was a marked decrease in the number of CR-IR axons in the trapezoid body (black arrowheads) and surrounding MNTB somata (white arrowheads) in MSGexposed animals. CR-IR neurons were present in the dorsal medial wedge (white arrows), between the MNTB and SPON. The mean number of CR-IR GBC are shown in C (whiskers show the standard deviation). The diameter of CR-IR axons in the trapezoid body are shown in D

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(whiskers show the 10th and 90th percentile, the mean is indicated by the cross). The mean number of MNTB neurons surrounded by a CR-IR terminal is shown in E (whiskers indicate the standard deviation). The scale bar for A and B equal to 250 µm. Key to symbols: **** p < .0001. The orientation arrows refer to dorsal (D) and lateral (L).

FIGURE 6. MSG exposure resulted in fewer CB-IR MNTB neurons. CB-IR is shown in the MNTB (saline – A, MSG – B). In saline-exposed animals, CB-IR was typically distributed throughout the somatic cytoplasm of MNTB neurons (arrow). In MSG-exposed animals, CB-IR was commonly observed throughout the cytoplasm of the soma (arrow), but in many neurons CB-IR was limited to the nucleus (black arrowheads). The mean number of CB-IR is shown (C – OCs; D – MNTB). The whiskers represent the standard deviation. The OD of CB-IR in five parasagittal divisions of the MNTB is shown in E for saline and MSG-exposed animals. The whiskers in E represent the 10th and 90th percentile; the cross represents the data mean. The scale bar for A and B equal to 250 µm. Key to symbols: * p < .05, ** p < .01, *** p < .001, **** p < .0001. The orientation arrows refer to dorsal (D) and lateral (L).

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Table 1: Number of Neurons included in Morphology

Saline MSG

OC 76 66

GBC 185 105

SBC Stell 181 83 116 45

MSO 60 59

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LSO 102 89

MNTB SPON 181 59 101 65

Table 2: Immunofluorescence for Calretinin or Calbindin Somata and axonal profiles analyzed with Calretinin immunofluorescence GBC CR-IR axons MNTB neurons Saline 623 148 713 MSG 697 123 1273 Somata analyzed with Calbindin immunofluorescence OC MNTB Saline 220 1142 MSG 201 1791

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MSG is known to be excitotoxic to the brain during the neonatal period. Neonatal exposure results in fewer neurons in the spiral ganglion. Neonatal MSG exposure resulted in significantly fewer neurons in the VCN and SOC. Neonatal MSG exposure resulted in significantly reduced expression of CR and CB.

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Title: Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate Authors: Lindsey Foran Kaitlyn Blackburn Randy J Kulesza Jr, PhD Affiliations: Department of Anatomy Lake Erie College of Osteopathic Medicine Erie, PA Corresponding Author: Randy J Kulesza Jr., PhD Department of Anatomy Lake Erie College of Osteopathic Medicine 1858 West Grandview Blvd Erie, PA 16504 814-866-8423 [email protected]

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