Distribution of glutamatergic, GABAergic, and glycinergic neurons in the auditory pathways of macaque monkeys

Neuroscience 310 (2015) 128–151

DISTRIBUTION OF GLUTAMATERGIC, GABAERGIC, AND GLYCINERGIC NEURONS IN THE AUDITORY PATHWAYS OF MACAQUE MONKEYS T. ITO, a,b* K. INOUE c AND M. TAKADA c

VGLUT1 and VGLUT2 in the medial geniculate, medial superior olive, and VNLL suggests that synaptic responses in the target neurons of these nuclei may be different between rodents and macaque monkeys. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Anatomy, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193, Japan b Research and Education Program for Life Science, University of Fukui, Fukui, Fukui 910-8507, Japan c Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan

Key words: vesicular glutamate transporter, GABA, glycine transporter 2, immunohistochemistry, in situ hybridization, primate.

Abstract—Macaque monkeys use complex communication calls and are regarded as a model for studying the coding and decoding of complex sound in the auditory system. However, little is known about the distribution of excitatory and inhibitory neurons in the auditory system of macaque monkeys. In this study, we examined the overall distribution of cell bodies that expressed mRNAs for VGLUT1, and VGLUT2 (markers for glutamatergic neurons), GAD67 (a marker for GABAergic neurons), and GLYT2 (a marker for glycinergic neurons) in the auditory system of the Japanese macaque. In addition, we performed immunohistochemistry for VGLUT1, VGLUT2, and GAD67 in order to compare the distribution of proteins and mRNAs. We found that most of the excitatory neurons in the auditory brainstem expressed VGLUT2. In contrast, the expression of VGLUT1 mRNA was restricted to the auditory cortex (AC), periolivary nuclei, and cochlear nuclei (CN). The co-expression of GAD67 and GLYT2 mRNAs was common in the ventral nucleus of the lateral lemniscus (VNLL), CN, and superior olivary complex except for the medial nucleus of the trapezoid body, which expressed GLYT2 alone. In contrast, the dorsal nucleus of the lateral lemniscus, inferior colliculus, thalamus, and AC expressed GAD67 alone. The absence of co-expression of

INTRODUCTION The use of auditory information is often highly speciesdependent, in particular, for the recognition of speciesspecific vocalizations (May et al., 1989; Gil-da-Costa et al., 2006), echo localization, and searching for prey (Covey and Carr, 2005). Accordingly, the organization of auditory neural circuits also shows significant interspecies differences (e.g. Winer and Larue, 1996; Kulesza and Grothe, 2015). Macaque monkeys form the genus Macaca of Old World monkeys (Cercopithecoidea), and they are widely used for studying brain functions as a model organism that is genetically close to humans (Gil-da-Costa et al., 2006). Macaque monkeys show complex social behaviors including several contact calls (May et al., 1989), suggesting that they should be useful for studying auditory function associated with complex sociality. However, the underlying neural circuitry is not well understood. Only a limited number of studies of neuronal organization in auditory pathways of the genus have been reported (e.g. Burton and Jones, 1976; Hashikawa et al., 1991; Bazwinsky et al., 2005; Hackett and de la Mothe, 2009). Indeed, the overall expression patterns of major neurotransmitters in the auditory system of the genus have not yet been provided. In the auditory system, the interaction of inputs from excitatory and inhibitory synapses is a key feature for coding auditory information (e.g. Casseday et al., 2000), and it is essential to know the organization of excitatory and inhibitory neurons in auditory neural circuitry. Glutamate, GABA, and glycine are used as neurotransmitters, and it is believed that most auditory neurons release one of these three neurotransmitters (e.g. Fredrich et al., 2009). Glutamate is a major excitatory neurotransmitter that is packed into synaptic vesicles by the vesicular glutamate transporter (VGLUT). VGLUT expression is

*Correspondence to: T. Ito, Department of Anatomy, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193, Japan. Tel: +81-776-61-8302; fax: +81-776-61-8132. E-mail address: [email protected] (T. Ito). Abbreviations: AC, auditory cortex; AVCN, anteroventral cochlear nucleus; CN, cochlear nuclei; DC, dorsal cortex of the inferior colliculus; DCN, dorsal cochlear nucleus; DEPC, diethylpyrocarbonate; DNLL, dorsal nucleus of the lateral lemniscus; DPO, dorsal periolivary nucleus; EDTA, ethylenediaminetetraacetic acid; GrC, granule cell domain; IC, inferior colliculus; ICC, central nucleus of the inferior colliculus; IHC, immunohistochemistry; ISH, in situ hybridization; LC, lateral cortex of the inferior colliculus; LNTB, lateral nucleus of the trapezoid body; LSO, lateral superior olive; MG, medial geniculate body; MGd, dorsal division of the MG; MGmc, magnocellular division of the MG; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; NLL, nuclei of the lateral lemniscus; NLS, N-lauroylsarcosine; PB, phosphate buffer; PVCN, posteroventral cochlear nucleus; RPO, rostral periolivary nucleus; SG, suprageniculate nucleus; SOC, superior olivary complex; VGLUT, vesicular glutamate transporter; VMPO, ventromedial periolivary nucleus; VNLL, ventral nucleus of the lateral lemniscus; VNTB, ventral nucleus of the trapezoid body. http://dx.doi.org/10.1016/j.neuroscience.2015.09.041 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 128

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sufficient for the glutamatergic phenotype (Fremeau et al., 2001, 2002), and typical glutamatergic neurons express VGLUT1 and/or VGLUT2 (Herzog et al., 2001; Takamori, 2006). GABA and glycine are major inhibitory neurotransmitters. GAD67 is an enzyme responsible for GABA synthesis and is widely used as a marker of GABAergic neurons. GLYT2 is a membrane transporter of glycine and is known to interact with the vesicular inhibitory amino acid transporter to determine glycinergic phenotype (Aubrey et al., 2007). In rodents, the colocalization of mRNAs for VGLUT1 and VGLUT2 was found in several nuclei of the auditory system including the ventral and dorsal divisions of the medial geniculate body (MG), medial superior olive (MSO), ventral cochlear nucleus (VCN), and some periolivary nuclei, whereas colocalization was rare or absent in the other auditory nuclei (Ito et al., 2011). The colocalization of mRNAs for GAD67 and GLYT2 is common in the ventral nucleus of the lateral lemniscus (VNLL), superior olivary complex (SOC), and cochlear nuclei (CN), whereas GLYT2 mRNA is not expressed in the dorsal nucleus of the lateral lemniscus (DNLL), inferior colliculus (IC), or forebrain (Tanaka and Ezure, 2004). Knowing the similarities and differences between rodents and monkeys will provide us with key insights into primate-specific auditory circuitry, particularly because both rodents and primates are members of a relatively close clade, Euarchontoglires (Murphy et al., 2001), and the auditory pathways of laboratory rodents (e.g. rats and mice) are less specialized than are those of many species. In this study, we examined the expression patterns of VGLUT1, VGLUT2, GAD67, and GLYT2 mRNA in the auditory system of Japanese macaque (Macaca fuscata). Furthermore, to validate the mRNA expression patterns, we examined the localization of VGLUT1, VGLUT2, and GAD67 proteins in the auditory system.

EXPERIMENT PROCEDURES Animals Nine Japanese macaques (M. fuscata) were obtained from breeding colonies at the Primate Research Institute, Kyoto University. All experiments were done in accordance with institutional guidelines at the University of Fukui and the Kyoto University. All efforts were made to minimize the number of animals used and their suffering. Monkeys were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.v.) and perfused transcardially with 0.1 M phosphate-buffered saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The fixed brains were stored in a mixture of 30% sucrose, 0.2% sodium azide, and 0.1 M PB for one month to nine years at 4 °C. Forty micron thick serial coronal sections were cut with a freezing microtome, and every 12th section was collected in a different bottle, and stored in antifreeze solution, consisting of 30% (v/v) glycerol, 30% (v/v) ethylene glycol, 3.4% (w/v) NaCl, and 0.04 M PB, at
20 °C until use. Before the histological experiments, sections were briefly washed with diethylpyrocarbonate-treated (DEPC-treated) 0.1 M PB twice. For each animal, eight series of every 12th section

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were used, and eight different histochemical experiments were performed (one for Nissl stain, four for in situ hybridization (ISH), and three for immunohistochemistry (IHC)) in this study. For Nissl staining, sections were mounted on gelatin-coated glass slides, and stained with a mixture of Cresyl Violet and Thionin. ISH Both digoxigenin (DIG)- and fluorescein (FL)-labeled antisense riboprobes were made from mouse cDNAs of VGLUT1 (nucleotides of 152–1085, GenBank accession number NM_182993.2, Watakabe et al., 2006), VGLUT2 (848–2044, NM_080853.2, Nakamura et al., 2007), and GAD67 (276–894, NM_008077.4, Li et al., 2005), and human cDNAs of VGLUT1 (349–1200 and 2163–2881, NM_020309, Komatsu et al., 2005), VGLUT2 (693–1888, NM_020346, Takahata et al., 2010), GAD67 (422–1051, BC037780, Komatsu et al., 2005), and GLYT2 (427–1481 and 1542–2461, NM_004211.3). The cDNA sequences of mouse VGLUT1, VGLUT2, GAD67, human VGLUT1, VGLUT2, GAD67, and GLYT2 probes are highly similar (mouse VGLUT1, 90%; mouse VGLUT2, 88%; mouse GAD67, 88%; human VGLUT1, 98% and 94%; human VGLUT2, 98%; human GAD67, 94%; human GLYT2, 98% and 97%) to the corresponding cDNAs of rhesus monkey, which is phylogenetically close to Japanese macaque. The cDNAs of VGLUT1 and VGLUT2 riboprobes had low similarity (less than 75%) to each other and to the cDNAs of VGLUT3 of rhesus monkey. We used two riboprobes for each gene and found identical staining patterns. Sections reacted with sense riboprobes exhibited no signal at all. Therefore, the riboprobes used seem to be highly specific in Japanese macaque. The procedure for non-radioactive ISH was described previously (Liang et al., 2000; Ito et al., 2007, 2011). Freefloating sections were washed in 0.1 M PB (pH 7.0) for 5 min twice, immersed in PB containing 0.3% Triton X-100, and washed in 0.1 M PB. Proteinase K treatment (1–5 lg/ml; 30 min at 37 °C) was performed to enhance signals. Then sections were acetylated for 10 min at room temperature with 0.003% acetic acid anhydrate, 1.3% (v/v) triethanolamine, and 6.5% (w/v) HCl diluted in DEPC-treated water. After being washed in 0.1 M PB twice, the sections were incubated for 1 h at 60 °C in a prehybridization buffer containing 50% (v/v) formamide, 5 SSC buffer (a 5 concentration of SSC buffer; 1 SSC contains 16.65 mM sodium chloride and 16.65 mM sodium citrate buffer, pH 7.0), 2% blocking reagents (Roche Diagnostics, Mannheim, Germany), 0.1% N-lauroylsarcosine (NLS), and 0.1% sodium dodecyl sulfate. Then, the sections were hybridized with 1 lg/ml sense or antisense RNA probes labeled with DIG or FL in freshly prepared prehybridization buffer for 20 h at 60 °C. After two washes in 2 SSC, 50% formamide, and 0.1% NLS for 20 min at 60 °C, the sections were incubated with RNase A (20 lg/ml; Sigma–Aldrich, St. Louis, MO, USA) in 0.01 M Tris–HCl (pH 8.0), 1 mM EDTA, 0.5 M NaCl for 30 min at 37 °C. The sections were washed in 2 SSC with 0.1% NLS for 20 min twice at 37 °C, and in 0.2 SSC with 0.1% NLS for 20 min twice at 37 °C. These sections were

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incubated with 1% blocking reagent (Roche) diluted in 0.1 M Tris–HCl (pH 7.5) and 0.15 M NaCl (TS7.5) for 1 h at room temperature. For bright field ISH, the sections were incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody Fab fragment (1:2000; Roche) in 1% blocking reagent (Roche) diluted in TS7.5 at room temperature overnight. The bound phosphatase was visualized by a reaction with nitro blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate toluidine salt (Roche) for 4 h at 37 °C in 0.1 M Tris–HCl (pH 9.5), 0.15 M NaCl, and 10 mM MgCl2. Sections were mounted on glass slides, dehydrated, cleared with xylene, and coverslipped. For fluorescent ISH, the sections were incubated with alkaline phosphatase-conjugated sheep anti-FL Fab fragment (1:2000; Roche) and peroxidase-conjugated sheep anti-DIG antibody Fab fragment (1:2000; Roche) in 1% blocking reagent (Roche) diluted in TS7.5 at room temperature overnight. The sections were washed three times, and the bound peroxidase was reacted with dinitrophenol-tyramide signal amplification (Perkin–Elmer, Waltham, MA, USA). After three more washes, the sections were incubated with AlexaFluor 488-conjugated rabbit anti-dinitrophenol (1:250; Invitrogen, Carlsbad, CA, USA) in 1% blocking reagent diluted in TS7.5. To visualize bound alkaline phosphatase by fluorescent microscopy, sections were developed with 0.005% (w/v) 4-chloro-2-methylbenzenediazonium hemi-zinc chloride (Fast Red TR, Roche), 1% (v/v) 2-hydroxy-3-naphtoic acid-20 -phenylanilide phosphate (HNPP, Roche) diluted in 0.1 M Tris–HCl (pH8.0), 0.15 M NaCl, 10 mM MgCl2, for 30 min at room temperature. The sections were mounted on glass slides with CC/Mount (DBS, Pleasanton, CA, USA).

absorption test (Raju et al., 2008). Western blot analysis identified a single band at 65 kDa, as predicted for the VGLUT2 protein in monkey. Preabsorption of the antibody with control peptide abolished immunolabeling. The immunogen used for anti-GAD67 antibody was a recombinant whole protein of rat GAD67. In a previous study (Ito et al., 2007), we confirmed the specificity of the anti-GAD67 by western blot and an absorption test. In the western blot, a single band around 67 kDa was detected (which is also described in manufacturer’s sheet). No signal was detected in rat brain sections incubated with the antibody (1:3000) preabsorbed with recombinant rat GAD67 protein (180 lg/ml). Furthermore, the staining pattern of the antibody was consistent with that in a previous study (Mugnaini and Oertel, 1985). Sections were incubated in 0.01 M sodium citrate (pH 6.0) at 80 °C for 2 h to enhance immunoreactivity. After a brief wash, the sections were incubated with primary antibodies for 48 h at 4 °C. For bright field microscopy, the sections were then incubated with a biotinylated antimouse, rabbit, or guinea-pig secondary antibody (1:200; Jackson Immunoresearch, West Grove, PA, USA) for 3 h, followed by incubation with avidin-biotinylatedperoxidase complex (ABC Elite, Vector Laboratories, Burlingame, CA, USA). Bound peroxidase was visualized by a nickel-enhanced diaminobenzidine reaction. Sections were mounted on glass slides, dehydrated, cleared with xylene, and coverslipped. For fluorescent microscopy, Cy3-conjugated donkey anti-guinea-pig IgG (1:200; Jackson Immunoresearch), AlexaFluor 488conjugated donkey anti-rabbit IgG (1:200; Invitrogen), and Cy5-conjugated donkey anti-mouse IgG (1:200; Jackson) were used as secondary antibodies. Sections were mounted on glass slides, air dried, rehydrated, and cover-slipped with 1,4-diazabicyclo[2.2.2]octane.

IHC

Imaging

Primary antibodies used in this study were anti-VGLUT1 raised from rabbit and guinea pig (0.5 mg/ml, Fujiyama et al., 2001), anti-VGLUT2 raised from rabbit (1:500; VGT2-6, MAb technologies, Stone Mountain, GA, USA), anti-GAD67 raised from mouse (1:1000; Millipore, Billerica, MA, USA). The immunogen used for anti-VGLUT1 antibody was a synthetic peptide to the C-terminal amino acid residues CGATHSTVQPPRPPPPVRDY of rat VGLUT1 (aa. 552–560). The sequence has very high similarity (95%) to C-terminal region of rhesus monkey VGLUT1. Specificity for the antibody was characterized by western blot and an absorption test (Fujiyama et al., 2001). In the western blot, a single band around 62 kDa was detected. No signal was detected in rat brain sections incubated with antibody preabsorbed with 10,000-fold (in mol) excess amounts of the synthetic peptide used for the immunization. The immunogen used for anti-VGLUT2 antibody was a synthetic peptide to C-terminal amino acid residues KKEEFVQGEVQDSHSYKDR of human VGLUT2 (aa. 560–578). The sequence has very high similarity (95%) to the C-terminal region of rhesus monkey VGLUT2. The specificity of the VGLUT2 antibody on monkey tissue was determined by western blots and an

Bright-field specimens were examined with an AX80 microscope (Olympus, Tokyo, Japan) and photomicrographs were acquired with a digital camera (PDMC II, Polaroid, Minnetonka, MS). In the following bright-field figures, representative data obtained from one monkey were shown for convenience of comparison. Fluorescent micrographs were acquired with a laser scanning confocal microscope (TCS AOBS SP 2, Leica, Wetzlar, Germany). AlexaFluor 488 was excited by a 488 nm Ar laser, and emitted fluorescence was filtered with a 500– 530 nm band-pass filter. Cy3 and FastRed were excited by a 543 nm He–Ne laser, and emitted fluorescence was filtered with a 565–615 nm band-pass filter. Cy5 was excited by a 633 nm He–Ne laser, and emitted fluorescence was filtered with a 650 nm low-pass filter. Minimal adjustments of levels were made in Photoshop CS3 (Adobe Systems, San Jose, CA, USA).

RESULTS In each auditory structure (i.e. CN, SOC, nuclei of the lateral lemniscus (NLL), IC, MG, and auditory cortex (AC)), we will first describe mRNA expression of VGLUT1, VGLUT2, GAD67, and GLYT2 (summarized in Table 1), and then protein distribution of VGLUT1,

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T. Ito et al. / Neuroscience 310 (2015) 128–151 Table 1. The pattern of mRNA expression of VGLUT1, VGLUT2, GAD67, and GLYT2 in the auditory pathways Nucleus

mRNA expression pattern Excitatory

Inhibitory

V1+/V2


V1
/V2+

V1+/V2+

GAD+/GLYT


GAD
/GLYT+

GAD+/GLYT+

CN

VCN DCN GrC


++ +++


++


+++



+ + +

++ ++ ++

+ + +

SOC

MSO LSO MNTB LNTB VNTB RPO VMPO DPO











+++ ++
+ + + ++ +




+
+














++ +++ ++ ++ ++ + ++


++
+ + ++
+

NLL

DNLL VNLLd VNLLv







++ +






+++




+ +


+ +++





+++ +++





++ ++










++ +++







+


++ ++ ++











IC MG A1

Layer I Layers III & IV Other layers

The relative density of cells in each category was expressed as – (very low or zero), + (low), ++ (moderate), and +++ (high). Abbreviations: A1, primary AC; V1, VGLUT1; V2, VGLUT2; GAD, GAD67; GLYT, GLYT2.

Fig. 1. Nissl staining (A) and mRNA signals for VGLUT1 (B), VGLUT2 (C), GAD67 (D), and GLYT2 (E) in the cochlear nuclei. In the ventral cochlear nucleus (VCN), VGLUT1- and VGLUT2-expressing neurons were found throughout the nuclei. GLYT2-expressing neurons were sparsely distributed, and GAD67-expressing neurons were very few (insets in D). Boxes in D indicate loci of insets. In caudal sections (bottom row), small VGLUT1-expressing neurons were found in the granule cell domain (GrC) and layer 1 of the DCN, while larger VGLUT2-expressing neurons were found in the deeper layer of the DCN. GAD67- and GLYT2-expressing neurons were more numerous in the deeper layer than surface. L, lateral; V, ventral. Scale bar = 1 mm.

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Fig. 2. Fluorescent ISH for VGLUT1 (1st column) and VGLUT2 (2nd column) in the auditory brainstem nuclei. In the DCN (D) and granule cell domain (E), colocalization was not found, while in the VCN (A–C), RPO (F), and LNTB (G), colocalization of two molecules (arrows) was common. Note the presence of double-labeled cochlear root neurons (CRN; C). Scale bar = 40 lm.

VGLUT2, and GAD67. The terms ‘‘express” and ‘‘expression” refer to mRNA expression. ‘‘Positive” and ‘‘+” refer to ‘‘immunopositive” (immunoreactive), whereas ‘‘negative” and ‘‘
” refer to ‘‘immunonegative.” CN The cytoarchitecture of the CN was in good agreement with a previous study (Moore, 1980). The anteroventral cochlear nucleus (AVCN), especially the more anterior part, mainly consisted of large oval-shaped cells, while the posteroventral cochlear nucleus (PVCN) contained a mixture of various shapes of cell bodies. In both AVCN and PVCN (Fig. 1A1, A2), the densities of VGLUT1- and

VGLUT2-expressing neurons were high (Fig. 1B, C). These neurons had large cell bodies and appeared to co-express VGLUT1 and VGLUT2 (Fig. 2A, B). Based on the size and location of cell bodies, we concluded that the cells expressing both VGLUT1 and VGLUT2 corresponded to glutamatergic projection neurons, i.e. bushy, octopus, and T-stellate cells. We also found a few large cells that expressed both VGLUT1 and VGLUT2 in the cochlear nerve root (Fig. 2C) and identified them as cochlear root neurons (CRN), which have been described in rodents (Merchan et al., 1988; Lopez et al., 1999). Cells expressing mRNAs for GAD67 or GLYT2 were sparsely distributed in the AVCN and PVCN (Fig. 1D, E). These neurons had small, irregular-shaped cell bodies. Some

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neurons co-expressed GAD67 and GLYT2 mRNAs (arrows in Fig. 3A, B). In the dorsal cochlear nucleus (DCN; Fig. 1A2), the expression of the molecules tested was different among the layers. VGLUT1 mRNA was expressed in granule cells (Fig. 2D, E). Granule cells were especially densely distributed in the granule cell domain (GrC), but packed more sparsely in the DCN layers (Fig. 1B2). VGLUT2expressing cells were large and were mainly located in layer 2 of the DCN (Fig. 1C2), and identified as fusiform and giant neurons (Ito et al., 2011). Double fluorescent ISH showed no overlap of VGLUT1 and VGLUT2 expression (Fig. 2D, E). GAD67-expressing cells were sparsely distributed in the DCN (Fig. 1D2). The density of GAD67-expressing cells in the DCN was higher than that in the PVCN. Like GAD67, GLYT2-expressing cells were sparsely distributed in the DCN (Fig. 1E2). Some cells co-expressed GAD67 and GLYT2 mRNAs (Fig. 3C). In the GrC, a few cells expressing GLYT2 alone were found (Fig. 3D). In summary, VGLUT1 and VGLUT2 mRNAs were strongly expressed in the CN. The expression of GLYT2 mRNA was more predominant than that of GAD67 mRNA. VGLUT1, VGLUT2, and GAD67 immunoreactivity were present in the CN (Fig. 4). VGLUT1 and VGLUT2 immunoreactivity were stronger in the DCN (Fig. 4A2, B2) than in the AVCN or PVCN (Fig. 4A, B). This pattern was most prominent with VGLUT2 immunoreactivity (Fig. 4B). At high magnification, most of the axosomatic terminals on AVCN and PVCN cells were positive for VGLUT1, but not for VGLUT2 (Fig. 5A, B). There were more VGLUT2+ terminals in the DCN than in the VCN, and some of the terminals were also positive for VGLUT1 (Fig. 5C, D). We observed numerous mossy fiber endings positive for VGLUT1 in the GrC (Fig. 5E). Colocalization of the two molecules was not common in GrC (Fig. 5E), in contrast to that seen the granule cell layer of the cerebellum (Fig. 5F), where some of the large terminals were positive for both VGLUT1 and VGLUT2 (arrows). GAD67 immunoreactivity was equally strong in the AVCN, PVCN, and DCN (Fig. 4C). SOC

Fig. 3. Fluorescent ISH for GLYT2 (1st column) and GAD67 (2nd column) in the auditory brainstem nuclei. In the CN (A–D), part of GAD67- or GLYT2-expressing neurons coexpressed both molecules (arrows). In the SOC (E–J) and NLL (K and L), a subpopulation of GLYT2-expressing neurons expressed GAD67. Scale bar = 40 lm.

According to Nissl cytoarchitecture (Fig. 6A), the monkey SOC can be subdivided into three principal nuclei and a number of periolivary cell groups (Moore and Moore, 1971). In this study, we identified the medial and lateral superior olivary nuclei (MSO and LSO, respectively), lateral, ventral, and medial nuclei of the trapezoid body (LNTB, VNTB, and MNTB), and dorsal, rostral, and ventromedial periolivary nuclei (DPO, RPO, and VMPO). The MSO can be easily identified as a dense cluster of cells extending along the dorsoventral axis (Fig. 6A2). Almost all MSO neurons expressed VGLUT2 mRNA (Fig. 6C2, C3). The LSO is located in the caudolateral part of the SOC, and medium-sized cells were densely packed therein (Fig. 6A3). No VGLUT1 expression was detected in the LSO (Fig. 6B3), whereas VGLUT2-expressing neurons were abundant (Fig. 6C3). GAD67 and GLYT2

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Fig. 4. Immunoreactivity for VGLUT1 (A), VGLUT2 (B), and GAD67 (C) in the cochlear nuclei. VGLUT1, VGLUT2, and GAD67 immunoreactivities were present in the VCN, and VGLUT2 immunoreactivity was weaker than VGLUT1. In the DCN (bottom row), VGLUT1 immunoreactivity was stronger in the upper layer, while such a gradient is not obvious in case of VGLUT2 and GAD67 immunoreaction. Scale bar = 1 mm.

were expressed in many LSO neurons (Figs. 3E, 6D3, E3). GLYT2-expressing neurons were more numerous than GAD67-expressing ones, and a subpopulation of GLYT2-expressing neurons co-expressed GAD67 mRNA (Fig. 3E). The MNTB is located in the rostromedial part of the SOC, and large, oval-shaped cells formed a cluster (Fig. 6A1, A2). The vast majority of MNTB neurons expressed GLYT2 mRNA alone (Fig. 6E1, E2). The remaining nuclei showed a somewhat lower packing density of cells (Fig. 6A). Among them, the VMPO, VNTB, and RPO displayed a relatively high density of cells, whereas the DPO showed the lowest cell density. The LNTB showed intermediate packing density. These nuclei contained mixed cell types (Figs. 2F, G, 3F–J). The majority of VMPO neurons expressed VGLUT2 (Fig. 6C2), whereas others expressed GLYT2 mRNA (Figs. 3J, 6E2). VGLUT1 mRNA was not detected in the VMPO (Fig. 6B2). GAD67 and GLYT2 were expressed in many VNTB neurons (Fig. 6D, E). The number of GLYT2-expressing neurons was more numerous than the number of GAD67-expressing neurons, and a subpopulation of GLYT2-expressing neurons co-expressed GAD67 mRNA

(Fig. 3G). VGLUT2-expressing neurons were sparsely distributed throughout the VNTB (Fig. 6B). Neurons expressing GAD67 and GLYT2 were more common than those expressing VGLUT2. Many RPO neurons co-expressed GAD67 and GLYT2 (Fig. 3H). Expression of VGLUT1 and VGLUT2 was observed in a few RPO neurons (Fig. 2F). In the DPO, the majority of neurons expressed GLYT2, and some of them co-expressed GAD67 as well (Fig. 3I). Few VGLUT2-expressing neurons were present in the DPO (Fig. 6C2, C3). In the LNTB, the expression of the molecules is similar to that in the VNTB (Fig. 3F), although some of VGLUT2-expressing neurons co-expressed VGLUT1 (Fig. 2G). In short, the vast majority of SOC glutamatergic cells expressed VGLUT2, but not VGLUT1. Many inhibitory neurons in the SOC, except for those in the MNTB, co-expressed GAD67 and GLYT2, but the expression of GLYT2 was stronger than that of GAD67. VGLUT1, VGLUT2, and GAD67 immunoreactivity were present in the SOC (Fig. 7). Here, we emphasize the pattern of VGLUT1 immunoreactivity because it labels the terminals of the axons projecting to the SOC

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Fig. 5. Fluorescent IHC for VGLUT1 (1st column, red in 4th column), VGLUT2 (2nd column, green in 4th column), and GAD67 (3rd column, blue in 4th column) in the cochlear nuclei. In the AVCN (A) and PVCN (B), large VGLUT1+ terminals encircled the cell bodies (presumably bushy and octopus cells in A and B, respectively). Most of these terminals were negative for VGLUT2 although few terminals showed weak VGLUT2 immunoreactivity (arrows). In the DCN (C and D), terminals were smaller, and colocalization of VGLUT1 and VGLUT2 was more common. In layer 1 of the DCN (C), VGLUT1+ terminals were small and densely packed. In the deeper layer of the DCN (D), VGLUT1+ terminals were larger and sparser. In the granule cell domain (E), mossy fiber endings exhibited strong VGLUT1 and very weak (or absence of) VGLUT2 immunoreactivities. In granule cell layer of the cerebellum (F), some mossy fibers colocalized VGLUT1 and VGLUT2. Scale bar = 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

from the VCN. VGLUT1 immunoreactivity was strongest in the MSO (Fig. 7A2, A3). The somata of MSO neurons were densely covered by terminals strongly positive for VGLUT1, and some of them were weakly positive for VGLUT2 (arrows in Fig. 8E). A few GAD67+ terminals

were found in the MSO. The MNTB and LSO were the nuclei with the second strongest VGLUT1 immunoreactivity (Fig. 7A). MNTB neurons were covered by axosomatic terminals positive for both VGLUT1 and VGLUT2 and a few GAD67+ axosomatic

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Fig. 6. Nissl staining (A) and expression of mRNA for VGLUT1 (B), VGLUT2 (C), GAD67 (D), and GLYT2 (E) in the superior olivary complex (SOC). The 1st row indicates most rostral level, while the 3rd row indicates most caudal level. Few VGLUT1-expressing cells were found in the RPO (B1) and LNTB (B2). MNTB neurons expressed almost exclusively GLYT2 (E1, E2), while MSO neurons expressed almost exclusively VGLUT2 (C2, C3). In the VMPO, GLYT2 and VGLUT2 expression was observed (C2, E2). In the other nuclei, VGLUT2, GAD67, and GLYT2 were expressed. Of these nuclei, the LSO contained more VGLUT2-expressing neurons than others. The signal intensity of GLYT2 is slightly stronger than GAD67. Scale bar = 1 mm.

terminals (Fig. 8G). LSO neurons made contact with large VGLUT1+ terminals, and many of them were positive for VGLUT2 as well (Fig. 8F). Both somata and terminals immunopositive for GAD67 were sparsely found in the LSO (Figs. 7C3, 8F). In the VNTB, the density of VGLUT1-immunopositive terminals was lower in the caudal part than in the rostral part (Fig. 7A), which may simply reflect the density of cells in the VNTB (Fig. 6A). The VNTB also contained terminals positive for VGLUT2 or GAD67 (Fig. 8C). The density of VGLUT2+terminals was higher in the VNTB than in anywhere else in the SOC (Fig. 7B). In the VNTB, most of the VGLUT1+, VGLUT2+, or GAD67+ terminals likely originated from different neurons, because colocalization of these molecules was rare (Fig. 8C). In the LNTB and VMPO, VGLUT1+ terminals were sparser than in other nuclei, and many of them were weakly positive for VGLUT2 (Fig. 8A, B). VGLUT2+/VGLUT1
terminals were more commonly found in the LNTB (Fig. 8B) than in the VMPO (Fig. 8A). In the DPO, the density of VGLUT1+ terminals was lowest among the SOC nuclei

(Fig. 7A2, A3), and it was lower than that of VGLUT2+ terminals (Fig. 7B2, B3). Colocalization of VGLUT1 and VGLUT2 was infrequent in the DPO (Fig. 8D). NLL Two nuclei were identified in the lateral lemniscus based on Nissl cytoarchitecture (Fig. 9A); the large ventrodorsally elongated ventral nucleus (VNLL) and the circular dorsal nucleus (DNLL). Further, complementary mRNA expression patterns of VGLUT2 and GLYT2 revealed two subdivisions of the VNLL, a small dorsal part and a large ventral part (VNLLd and VNLLv): VGLUT2 mRNA was expressed by many VNLLd neurons and very few VNLLv neurons (Fig. 9C). GLYT2 mRNA was intensely expressed by the majority of VNLLv neurons, and weakly expressed by a few VNLLd neurons (Fig. 9E). Clusters of intensely GLYT2-positive cells were found throughout the VNLLv (Fig. 9E1), and the pattern resembled the spatial organization of rat VNLL neurons (Malmierca et al., 1998). In the VNLLv

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Fig. 7. Immunoreactivity for VGLUT1 (A), VGLUT2 (B), and GAD67 (C) in the SOC. VGLUT1 immunoreactivity was strong in most nuclei except for the caudal VNTB (A3) and DPO (A2, A3). VGLUT2 immunoreactivity was present in all nuclei, and slightly weaker in the MSO and center of the LSO. GAD67 immunoreactivity was present in all nuclei, and weaker in the MSO and center of the LSO. Scale bar = 1 mm.

and VNLLd, co-expression of GAD67 and GLYT2 was frequently seen (Fig. 3K, L). VGLUT1 mRNA was not detected in the NLL (Fig. 9B). GAD67 mRNA was expressed in the NLL (Fig. 9D). Staining intensity for GAD67 was highest in the DNLL and lowest in the VNLLd (Fig. 9D2). VGLUT2 and GLYT2 mRNA were absent from the DNLL (Fig. 9C2, E2). In summary, most VNLLv neurons co-expressed GAD67 and GLYT2. The majority of VNLLd neurons expressed VGLUT2 and almost all DNLL neurons expressed GAD67. Immunoreactivity for VGLUT1 and VGLUT2 was similar in the VNLLd and VNLLv (Fig. 10A, B). VGLUT1+ terminals were large and many of them showed weak immunoreactivity for VGLUT2 (Fig. 11D, E). Based on the density of terminals positive for VGLUT1 and VGLUT2, the DNLL was subdivided into peripheral and

central parts (DNLLp and DNLLc, respectively; Fig. 10). In the DNLLc (Fig. 11B), VGLUT1+ terminals were sparse, whereas VGLUT2+ terminals were dense. In the DNLLp, the density of VGLUT1 and VGLUT2 exhibited a relationship that was opposite to that seen in DNLLc (Fig. 11C). In the DNLL, colocalization of VGLUT1 and VGLUT2 was rare (Fig. 11B, C), suggesting that these terminals have different origins. GAD67 immunoreactivity was strong throughout the NLL (Fig. 10C). We found both terminals and somata positive for GAD67 (Fig. 11C3–E3). IC In the IC, three major subdivisions, the central nucleus (ICC), lateral cortex (LC), and dorsal cortex (DC), were

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Fig. 8. Fluorescent IHC for VGLUT1 (1st column, red in 4th column), VGLUT2 (2nd column, green in 4th column), and GAD67 (3rd column, blue in 4th column) in the SOC. The majority of VGLUT1+ terminals were positive for VGLUT2 (arrows). In several nuclei especially in the VNTB (C), small VGLUT2+/VGLUT1
terminals were found. Large terminals positive for both VGLUT1 and VGLUT2 were common in the MSO (E), LSO (F), and MNTB (G). Scale bar = 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

identified based on Nissl cytoarchitecture (Fig. 12A). In the more rostral sections, bundles of unstained commissural fibers were found, and the region is likely

to correspond with the intercollicular tegmentum of cats (Morest and Oliver, 1984) and the rostral cortex of rats (Malmierca et al., 2011). In coronal sections, the ICC

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Fig. 9. Nissl staining (A) and expression of mRNA for VGLUT1 (B), VGLUT2 (C), GAD67 (D), and GLYT2 (E) in the nuclei of the lateral lemniscus. Two nuclei were identified in the lateral lemniscus; dorsal and ventral nucleus of the lateral lemniscus (DNLL and VNLL). The VNLL was further subdivided into dorsal and ventral parts (VNLLd and VNLLv). No VGLUT1-expressing neurons were found in these nuclei. In the DNLL (bottom row), only GAD67-expressing neurons were found. In the VNLLd (both rows) most neurons expressed VGLUT2, and a few neurons expressing GAD67 or GLYT2 were found in the ventral end of the VNLLd (D1 and E1). In the VNLLv (top row), GAD67- and GLYT2-expressing neurons were found throughout the nucleus, while only a few VGLUT2-expressing neurons were found. Scale bar = 1 mm.

contained numerous flat cell bodies that aligned in a direction from dorsomedial to ventrolateral, which likely reflect the isofrequency laminae. The ICC had an ovoid shape, suggesting enlarged low frequency laminae compared with the rodent ICC. Neither VGLUT1 nor GLYT2 mRNA was expressed in the IC (Fig. 12B, E). The ICC stood out because of intense labeling for VGLUT2 and GAD67 mRNAs (Fig. 12C, D). The density of neurons expressing VGLUT2 or GAD67 was lower in the IC cortex, namely LC, DC, and rostral cortex, than in the ICC. In the ICC, GAD67-expressing neurons had large cell bodies (compare Fig. 12C, D). We concluded that excitatory IC neurons expressed VGLUT2, whereas inhibitory neurons expressed GAD67. Immunoreactivity for VGLUT1, VGLUT2, and GAD67 was equally strong across the IC subdivisions (Fig. 13). Colocalization of VGLUT1 and VGLUT2 was restricted to a subpopulation of large terminals (arrows in Fig. 11A). GAD67+ soma often received dense axosomatic contacts with VGLUT2+ terminals (asterisks in Fig. 11A). Such GAD67+ cells tended to have larger cell bodies than the GAD67+ cells without dense axosomatic terminals.

MG The MG of the thalamus was subdivided into dorsal, ventral, magnocellular, and suprageniculate divisions (MGd, MGv, mc, and SG, respectively; Fig. 14A) based on Nissl staining in accordance with previous studies (Burton and Jones, 1976; Hashikawa et al., 1991), although we did not subdivide the MGd into the anterodorsal and posterodorsal divisions because the labeling patterns of the molecules tested was very similar. In the MGv, medium-sized cells were densely packed. The density of cells was lower in the MGd than in the MGv. However, the density of cells in the MGd was not uniform, and the cell density was high in the rostral and peripheral part of the MGd (asterisks in Fig. 14). The mc clearly stood out due to the presence of very large cells. The SG was identified as the nucleus with the highest density of cells among all MG subdivisions. Neither VGLUT1 nor GLYT2 mRNA was detected in the MG (Fig. 14B, E). VGLUT2-expressing cells varied in size, ranging from small to large (Fig. 14C). The density and size of VGLUT2-expressing cells highly corresponded with those identified with Nissl staining (compare Fig. 14A, C). Hence, it is likely that the most

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Fig. 10. Immunoreactivity for VGLUT1 (A), VGLUT2 (B), and GAD67 (C) in the nuclei of the lateral lemniscus. VGLUT1, VGLUT2, and GAD67 immunoreactivities were present in VNLLd and VNLLv (top row) with a similar pattern. In the DNLL (bottom row), VGLUT1 immunoreactivity was weaker in the center (DNLLc) than periphery (DNLLp), while VGLUT2 immunoreactivity was stronger in the center than periphery. Scale bar = 1 mm.

of the cytoarchitectonic features of MG subdivisions come from a variety of VGLUT2-expressing cells. GAD67expressing cells were small- or medium-sized and present in all divisions (Fig. 14D), as shown previously (Winer and Larue, 1996). The distribution of GAD67expressing cells was not uniform, but rather clustered. In the MGd, the clusters of GAD67-expressing cells corresponded to those identified with Nissl staining (compare Fig. 14A, D). Immunoreactivity for VGLUT1, VGLUT2, and GAD67 was present in the MG (Figs. 15 and 16). VGLUT1 and VGLUT2 immunoreactivity was restricted to terminals, whereas GAD67 immunoreactivity was found in both cell bodies and terminals (Fig. 17). The density of VGLUT1+ terminals was high and uniform in most parts of the MG (Fig. 15A). On the other hand, the distribution of VGLUT2- and GAD67-immunoreactive structures was not uniform but patchy (Fig. 15B, C). There was some overlap of patchy clusters positive for

GAD67 and VGLUT2, particularly in the periphery of the MGd (Fig. 16). Similar to ISH, GAD67-immunoreactive cell bodies formed clusters, and the density of GAD67immunoreactive terminals was higher in the clusters. In the MGd, the region in which densities of both VGLUT2+ and GAD67+ terminals were high corresponded to clusters of cells identified with Nissl staining (asterisks in Figs.15 and 16). At high magnification, VGLUT1+ terminals appeared to be small- to medium-sized (Fig. 17A1–F1). VGLUT2+ terminals were either small or large (Fig. 17A2–F2). Large VGLUT2+ terminals were especially rich in the MGv and the periphery of the MGd (asterisks in Figs. 13 and 14; Fig. 17A2, C2). Co-localization of VGLUT1 and VGLUT2 was not found in the MG. Large GAD67+ neuropils that formed clusters with glutamatergic terminals were commonly found in the MG (Fig. 17A, D); these clusters were likely synaptic glomeruli (Jones and Rockel, 1971; Morest, 1975).

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Fig. 11. Fluorescent IHC for VGLUT1 (1st column, red in 4th column), VGLUT2 (2nd column, green in 4th column), and GAD67 (3rd column, blue in 4th column) in the IC (A) and nuclei of the lateral lemniscus (B–E). In these nuclei, some large VGLUT1+ terminals were positive for VGLUT2 (arrows). In the IC (A), some GAD67+ neurons (asterisks) received dense axosomatic contacts from VGLUT2+ terminals. In the DNLL (B, C), the distribution of VGLUT1
and VGLUT2+ terminals was somewhat complementary: VGLUT2+ terminals were denser in the center (B) and VGLUT1+ terminals were more numerous in the periphery (C). Distribution patterns of VGLUT1+ and VGLUT2+ terminals were similar in the VNLLd (D) and VNLLv (E). In the DNLL, numerous GAD67+ cell bodies were found, while they were rare in the VNLLd. Scale bar = 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

AC Next, we examined the expression of the VGLUT1, VGLUT2, GAD67, and GLYT2 in the primary AC. Six layers of the neocortex were identified with Nissl staining (Fig. 18D), and the expression patterns of each molecule in each layer of neighboring sections were analyzed. Numerous VGLUT1-expressing cells were present in layers II–VI (Fig. 18E). The density of VGLUT1-expressing cells was lower in the upper part of layer V. VGLUT2 mRNA was expressed in some cells in layers III and IV (Fig. 18F, I). The intensity of labeling for the VGLUT2 riboprobe was lower in the cortex than in the brainstem. GAD67-expressing cells were found in all layers (Fig. 18G), and the density of GAD67-expressing cells was highest in layer II. GLYT2 mRNA was not expressed in the cerebral cortex (Fig. 18H).

The density of VGLUT1+ terminals was high in all layers (Fig. 18A). Across all layers, layer I showed the highest density, and layer VI showed a slightly higher density than layers II–V. In layer I, large VGLUT1+ terminals were sparsely present (Fig. 19A). VGLUT2+ terminals were concentrated in a deeper part of layer III and layer IV (Fig. 18B). At high magnification, small VGLUT2+ terminals were found in all layers, whereas larger terminals were frequently found in layers I, III, and IV (Fig. 19A, C, D). The colocalization of VGLUT1 and VGLUT2 was found in subpopulation of terminals (arrows in Fig. 19). GAD67+ terminals and cell bodies were present in all layers (Figs. 18C, 19A3–F3).

DISCUSSION In the present study, we examined the expression of molecules that are associated with glutamatergic,

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Fig. 12. Nissl staining (A) and expression of mRNA for VGLUT1 (B), VGLUT2 (C), GAD67 (D), and GLYT2 (E) in the inferior colliculus (IC). In the IC, neither VGLUT1 nor GLYT2 expression was found. VGLUT2-expressing neurons were more numerous than GAD67-expressing ones. Density of GAD67-expressing neurons was higher in the central nucleus (ICC) than lateral or dorsal cortices (LC, DC). Scale bar = 1 mm.

Fig. 13. Immunoreactivity for VGLUT1 (A), VGLUT2 (B), and GAD67 (C) in the IC. VGLUT1, VGLUT2, and GAD67 immunoreactivities were equally strong in the IC with a slight variation of staining pattern. VGLUT1 immunoreactivity was especially strong in the cortical regions. Scale bar = 1 mm.

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Fig. 14. Nissl staining (A) and expression of mRNA for VGLUT1 (B), VGLUT2 (C), GAD67 (D), and GLYT2 (E) in the medial geniculate body (MG). MGv cells were medium-sized and densely packed. MGd cells were smaller and sparser. Cells in the mc were large and sparsely distributed. SG cells were densely packed. In the MGd, the density of neurons was higher in the rostral and caudolateral part (asterisks). Expression of VGLUT2 was overwhelming in all subdivisions. GAD67-expressing neurons showed patchy distribution in all subdivisions. Neither VGLUT1 nor GLYT2 expressing neurons were found. Scale bar = 1 mm.

GABAergic, and glycinergic phenotypes in the auditory system of macaque monkeys (Fig. 20; Table 1). Most glutamatergic neurons in the brainstem and thalamus expressed VGLUT2 mRNA except for granule cells in the CN. Co-expression of VGLUT1 and VGLUT2 was common in the VCN, and less frequent in SOC subnuclei and layers III and IV of AC. Protein distribution of VGLUT1 and VGLUT2 confirmed this result. GLYT2

mRNA was expressed in the VNLLd, VNLLv, SOC, and CN. Co-expression of GAD67 and GLYT2 was common in these nuclei except in the MNTB and VMPO, where no GAD67-expressing cells were found. In the higher order nuclei such as the DNLL, IC, MG, and AC, inhibitory neurons expressed GAD67 alone. Our results provide new information for studying the organization of neural circuitry in monkey brain.

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Localization patterns of mRNA and protein VGLUT1 and VGLUT2 proteins are localized in axonal terminals but not cell bodies (Fremeau et al., 2001; Fujiyama et al., 2001; Herzog et al., 2001). On the other hand, mRNAs are mainly localized in cell bodies. Combining these facts with current knowledge of VGLUT expression (Ito and Oliver, 2010) and connections within the auditory pathways of other species, we can speculate about the synaptic organization of VGLUT1- and VGLUT2-containing neurons in the auditory system. As most axosomatic terminals on VCN neurons were positive for VGLUT1 but not VGLUT2 (Fig. 5A, B of this study, Zhou et al., 2007), neurons in the spiral ganglion should express VGLUT1 but not VGLUT2. In the DCN, it is likely that VGLUT2+/VGLUT1
terminals are derived from the IC (Caicedo and Herbert, 1993; Malmierca et al., 1996), that VGLUT1+/VGLUT2
terminals are from granule cells, and that VGLUT1+/VGLUT2+ terminals are from T-stellate cells (Doucet and Ryugo, 1997). The VGLUT1+/VGLUT2
identity of mossy fibers in the GrC was unexpected because most GrC mossy fibers are VGLUT1
/VGLUT2+ in guinea pig (Zhou et al., 2007; Zeng et al., 2012). Mossy fibers originate from cuneate and spinal trigeminal nuclei (Wright and Ryugo, 1996; Zeng et al., 2012), and the expression patterns of VGLUT1 and VGLUT2 in these nuclei may be different in guinea pig and monkeys. Double ISH for VGLUT1 and VGLUT2 combined with retrograde tracing will clarify this issue. In the SOC, many medium-sized terminals were positive for both VGLUT1 and VGLUT2, and these

Fig. 15. Immunoreactivity for VGLUT1 (A), VGLUT2 (B), and GAD67 (C) in the MG. Immunoreactivity for VGLUT1 was strong in all subdivisions with relatively uniform intensity. VGLUT2 immunoreactivity was present in all subdivisions with clear biased distribution of signal intensity; stronger immunoreactivity in the MGv and periphery of MGd (asterisks) in a patchy manner. Immunoreactivity for GAD67 was strong in all subdivisions with a patchy staining pattern. Note the overlapping of some GAD67 and VGLUT2 patches. Scale bar = 1 mm.

Fig. 16. Composite images of MG sections that contained MGd immunostained for VGLUT2 (red) and GAD67 (green). Image levels in Fig. 15 were inverted, and pseudocolor, composite images were made. In the periphery of the MGd (asterisks), clusters of strong immunoreactivities for VGLUT2 and GAD67 overlapped. Scale bar = 1 mm. (In the printed version of this article, overlap of VGLUT2 and GAD67 immunoreactivities was extracted and shown in gray scale.)

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Fig. 17. Fluorescent IHC for VGLUT1 (1st column), VGLUT2 (2nd column), and GAD67 (3rd column) in the MG. In common, density of VGLUT1+ terminals was high in all subdivisions of the MG. The size of VGLUT1+ terminals was always small, while the size of VGLUT2+ terminals ranged from small to large. Density of VGLUT2+ terminals varied between subdivisions; higher in the MGv (A) and periphery of the MGd (C), and lower in the central part of the MGd (B). Clusters of VGLUT+ endings and GAD67+ structures, presumably synaptic glomeruli, were found (arrows in D). Colocalization of VGLUT1 and VGLUT2 was not observed. Scale bar = 10 lm.

terminals likely originated from the VCN. A high incidence of VGLUT2+/VGLUT1
terminals in the VNTB is consistent with the fact that this region is the main target of the descending pathway from the IC to the SOC (Caicedo and Herbert, 1993). In the VNLLv and VNLLd, large VGLUT1+/VGLUT2+ terminals likely originate from octopus cells, bushy cells, and T-stellate cells because these cells express both VGLUT1 and VGLUT2 and target these regions (Glendenning et al., 1981; Friauf and Ostwald, 1988; Malmierca et al., 1998; Malmierca, 2015). In the DNLL,

VGLUT1+ terminals are likely from the contralateral VCN (Glendenning et al., 1981), whereas VGLUT2+ terminals likely originate from the SOC (Glendenning et al., 1981) and ipsilateral IC (Caicedo and Herbert, 1993). Because VGLUT1+ terminals were mainly concentrated in the periphery, which is a high-frequency region (Merchan et al., 1994), it is suggested that VCN axons mainly target the high frequency region of the DNLL. Further detailed study is needed to clarify this topic. In the IC, large VGLUT1+/VGLUT2+ terminals likely originate from VCN T-stellate cells (Ito and Oliver, 2010).

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Fig. 18. Bright-field immunohistochemistry and ISH in the primary auditory cortex. VGLUT1 immunoreactivity (A) was strong in the all layers, and strongest in layers I and VI. VGLUT2 immunoreactivity (B) was strong in deep layer III and upper part of layer IV. GAD67 immunoreactivity (C) was strong in all layers, and intensity tended to stronger in upper layers. Numerous cells expressing VGLUT1 mRNA (E) were distributed except for layer I. Staining intensity was weaker in deeper part of layer IV. VGLUT2 mRNA (F) was weakly expressed in cells lying in deeper part of layer III (box, I) and upper part of layer IV. GAD67-expressing cells (G) were distributed in all layers and density was highest in layer II. No GLYT2-expressing cell was found (H). Scale bar = 0.5 mm.

VGLUT2+ terminals originate from various brainstem sources. Small VGLUT1+ terminals may arise from the

AC because layer V and VI neurons, which are responsible for descending projection (Winer and Lee, 2007),

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Fig. 19. Fluorescent IHC for VGLUT1 (1st column, red in 4th column), VGLUT2 (2nd column, green in 4th column), and GAD67 (3rd column, blue in 4th column) in layers I–VI (A–F) of the primary AC. (A) In layer I, VGLUT1+ terminals were densely distributed. Small VGLUT2+ terminals were also found, and some of them colocalized VGLUT1 (arrows). Note the presence of large VGLUT1+ terminals. (B) In layer II, terminals positive for VGLUT1 terminals were densely found, while those positive for VGLUT2 were few. (C) In layer III, intensity of VGLUT1 immunostaining was weaker than that of layer I. On the other hand, large terminals intensely labeled for VGLUT2 were found. (D) In layer IV, VGLUT1+ terminals were sparse, while large VGLUT2+ terminals were densely distributed. (E) In layer V, weakly labeled VGLUT1+ terminals were found. (F) In layer VI, VGLUT1+ terminals were more numerous than layer IV. VGLUT2+ terminals were sparsely found and some of them colocalized VGLUT1. Scale bar = 5 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

express VGLUT1 but not VGLUT2. Because the immunostaining patterns of VGLUT1 and VGLUT2 are very similar to those in rat (Altschuler et al., 2008; Ito et al., 2009), large GAD67+ cells that receive dense

VGLUT2+ axosomatic terminals (Fig. 11A) are most likely to be large GABAergic cells, which receive converging inputs from the IC, VNLLd, SOC, and DCN (Ito and Oliver, 2010, 2014; Ito et al., 2015) and are responsible

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Fig. 20. Schematic diagrams of mRNA expression of VGLUT1, VGLUT2, GAD67, and GLYT2 in the auditory nuclei. (A) In most of the auditory nuclei, VGLUT2 expression is dominant; VGLUT1 expression is common only in the cochlear nuclei and AC. Note that in the RPO and LNTB, only a few cells express VGLUT1. VGLUT1-expressing neurons co-express VGLUT2 mRNA in most nuclei except for DCN and AC. The DNLL and MNTB do not contain glutamatergic cells. (B) In the AC, MG, IC, and DNLL, GAD67 but not GLYT2 is expressed, while GLYT2 but not GAD67 is expressed in the MNTB and VMPO. The MSO does not express GAD67 or GLYT2. In the other nuclei, both GAD67 and GLYT2 are expressed.

for tectothalamic inhibition (Winer et al., 1996; Peruzzi et al., 1997; Ito and Oliver, 2012). In the MG, VGLUT2+ terminals are likely from the IC, whereas VGLUT1+ terminals are from the AC. Some GAD67+ terminals may arise from large GABAergic neurons in the IC (Ito et al., 2009). Differences in the size

and density of these terminals among MG subdivisions suggest they have different roles. GAD67-expressing cells showed more clustered distribution than VGLUT2expressing cells. Interestingly, VGLUT2+ terminals also showed clustered distribution that overlapped with GAD67+ structures, where VGLUT1+ terminals were

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distributed more uniformly. Because GABAergic interneurons in the MG have short, poorly branched axons but have dendrodendritic synapses (Morest, 1975), the overlapping distribution of VGLUT2+ terminals and GAD67+ structures suggested that ascending terminals tend to form glomerular synapses together with dendrites of GABAergic interneurons and principal neurons, and VGLUT1+ corticothalamic axons do not show such a preference. In the AC, VGLUT1+/VGLUT2
terminals are highly likely to be from layer II, V, and VI of neocortex, VGLUT1+/ VGLUT2+ terminals are from neurons in layers III and IV, and VGLUT2+/VGLUT1
terminals are from the MG. This view is consistent with the data showing that the pattern of distribution of VGLUT1+ and VGLUT2+ terminals was very similar to those in other species (Nakamura et al., 2007; Hackett and de la Mothe, 2009). The origin of large VGLUT1+ terminals in layer I is unknown because they have not been reported in previous studies. Interspecies comparison There are three studies that described VGLUT1 and VGLUT2 expression patterns in mammalian species (marmoset, Hackett et al., 2010; Japanese monkey, this study; rodents, Ito et al., 2011). Because all of these studies used the same techniques and riboprobes developed in one laboratory (Yamamori Laboratory, National Institute for Basic Biology, Japan), any major difference between studies must arise from species differences. In all three studies, excitatory neurons, except granule cells, in the auditory brainstem expressed VGLUT2. VCN neurons co-expressed VGLUT1 and VGLUT2. VGLUT1 was weakly expressed in some periolivary nuclei. In the AC, excitatory neurons expressed VGLUT1, and some excitatory neurons in layers III and IV co-expressed VGLUT2 (Ito and Oliver, 2010). These patterns likely reflect basic features of the mammalian auditory system. On the other hand, the incidence and spatial pattern of VGLUT1 expression in the brainstem and thalamic nuclei are different among rodents (rat and mouse), New World monkeys (marmoset), and Old World monkeys (Japanese macaque). In rodents, many neurons co-expressed VGLUT1 and VGLUT2 in the MSO and VNLLd, and almost all MGV neurons co-expressed VGLUT1, suggesting the importance of VGLUT1 in these nuclei. In marmoset, it seems that most VGLUT2-expressing neurons in brainstem and thalamus weakly express VGLUT1. In the Japanese macaque, VGLUT1 expression was restricted to the RPO, LNTB, and VCN. The absence of strong VGLUT1 expression in the MGV of Japanese macaques is strikingly different from the VGLUT1 expression seen in rodents. Based on these species differences, properties of excitatory synaptic transmission in the IC (receiving inputs from SOC and VNLLd), and layer IV of the AC (receiving inputs from the MGV) may exhibit differences among the species (discussed below). The distribution of inhibitory neurons was similar to that of rodents (Tanaka and Ezure, 2004; Ito et al., 2011) with some minor differences. We did not identify the superior paraolivary nucleus, in which most neurons are GABAergic (Kulesza and Berrebi, 2000). The superior

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paraolivary nucleus may be a rodent-specific nucleus (Malmierca, 2015), although a small number of neurons that show a similar projection pattern are present in cat (Adams, 1983). Further studies will be needed to test for the presence of this nucleus in monkeys. In the VNTB, a few VGLUT2-expressing cells were present in monkeys, whereas no glutamatergic cells were found in rodents, suggesting some functional differences in this descending control pathway. The presence of GABAergic interneurons in the MG is another feature and was already discussed above and in another study (Winer and Larue, 1996). Functional considerations Although the three VGLUT subtypes have similar properties for transporting glutamate into synaptic vesicles (Bellocchio et al., 2000; Takamori et al., 2000; Fremeau et al., 2002), they are expressed in different, sometimes overlapping populations of neurons, and differences in other physiological properties have been reported. The C-terminal amino acids of VGLUT1, but not VGLUT2 or VGLUT3, have the ability to interact with endophilin A1, which is involved in clathrin-mediated vesicular recycling, suggesting that the vesicular recycling mechanism is different between VGLUT1 and other VGLUTs (De Gois et al., 2006; Vinatier et al., 2006). Indeed, by interacting with endophilin A1, VGLUT1containing terminals have a lower release probability than VGLUT2-containing ones, and show short-term depression (Weston et al., 2011). Synaptic terminals that require high reliability and reproducibility, e.g. endbulb terminals on bushy cells and calyx terminals on MNTB cells, express high levels of VGLUT1 protein which should limit depression during repetitive firing, and have large active zones that should compensate for the low release probability of VGLUT1. The large terminals originating from the VCN that terminate in the MNTB can also be considered to reflect a compensatory mechanism. Furthermore, colocalization of VGLUT1 and VGLUT2 in VCN-originating terminals may also compensate for a low release probability. Small cortical terminals contain VGLUT1, but they likely have different functions. On the other hand, glutamatergic terminals that do not contain VGLUT1, which is the most prevalent glutamatergic terminal type in brainstem nuclei, show short-term depression during repetitive firing (e.g. more than half of MGv neurons showed shortterm depression after IC stimulation, Bartlett and Smith, 2002), suggesting modulation of auditory information at synapses. This may be one cause of degradation of phase-locking ability in higher centers (Langner, 1992). The most striking difference between rodents and macaque monkeys is the absence of VGLUT1 expression in the MSO and MGv of monkeys. We expect that in rodents, but not monkeys, the temporal pattern of discharge in MSO neurons is reproduced as EPSPs in IC neurons with high fidelity. Likewise, in rodents but not in monkeys, the temporal discharge pattern of neurons in the core pathway (MGv) may be reproduced reliably in AC neurons, whereas this may not be true for neurons from other MG subdivisions. Future studies will clarify whether or not this view is

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accurate. Taken together, the differences among the tested species that show different sound associated behaviors, suggest a diversity of properties of glutamatergic terminals in auditory pathways among mammals, reflecting species-specific behaviors to sounds.

AUTHOR CONTRIBUTIONS T.I. designed the study, and collected the data for the paper. K.I. and M.T. prepared specimens. All authors wrote the paper, and approved the final version of the manuscript.

CONFLICT OF INTEREST The authors declare no conflict of interest. Acknowledgements—Authors gratefully acknowledge the gift of VGLUT1 antibody from Dr. Takeshi Kaneko (Kyoto University), and the plasmids for riboprobes from Drs. Akiya Watakabe and Tetsuo Yamamori (National Institute for Basic Biology), and Dr. Kouichi Nakamura (Kyoto University) for proof reading. This work was supported by grants from Japan Society for the Promotion of Science (KAKENHI Nos. 22700365 and 25430034), The Ichiro Kanehara Foundation, and The Uehara Memorial Foundation, NOVARTIS Foundation for the Promotion of Science, Research and Education Program for Life Science of University of Fukui to T.I.

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(Accepted 14 September 2015) (Available online 29 September 2015)