Distribution of Glutamatergic Neurons in the Central Nervous System of the Turtle(Pseudemys scripta elegans

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Title Distribution of Glutamatergic Neurons in the Central Nervous_{System of the Turtle(Pseudemys scripta elegans( 本文(Fulltext) )}

Author(s) SONJOY SARKAR

Report No.(Doctoral Degree) 博士(獣医学) 甲第523号 Issue Date 2019-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/77954 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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2018

The United Graduate School of Veterinary Sciences, Gifu University

(Gifu University)

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YHVLFOHV DWWKH SUHV\QDSWLF WHUPLQDOV WR UHOHDVH . In the synaptic cleft glutamate is released by exocytosis and binds to glutamate receptors on postsynaptic membranes

(Fig. 1.1).

Fig. 1.1. Schematic diagram of glutamatergic transmission in mammals. Glutamate is regulated by VGLUTs into synaptic vesicles of presynaptic terminals. This schematic diagram is adapted from Kanai and Hediger (62).

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Therefore, VGLUTs have a significant role in glutamatergic transmission. These glutamate transporters are found in rodents, humans, and birds (5, 60, 64, 91, 107, 115,

121)EXWQRWH[DPLQHGLQDQ\UHSWLOLDQVSHFLHV\HW.

Three isoforms of VGLUTs (VGLUT1-3) have been identified in mammals (5, 37,

38, 50, 91, 92) and the distribution of their mRNAs and proteins has been investigated thoroughly in the brain (Fig. 1.2).

Fig. 1.2. Schematic diagrams of frontal sections of the pallium and subpallium are represented in mammals (a) based on VGLUT1 expression (38, 39, 92) and in birds (b) based on VGLUT2 expression (60, 64). Cx, cerebral cortex; M, mesopallium; N, nidopallium. For other abbreviations: see the list.

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VGLUT1 and VGLUT2 are considered selective biomarkers to find glutamatergic neurons, and they show complementary distribution patterns in the mammalian brain

(56, 91, 92). Expression sites of VGLUT1 are primarily localized in the cerebral neocortex, hippocampus, and cerebellar cortex. The expression of VGLUT2 is found in

the thalamus, hypothalamus, amygdaloid nuclei, lower brainstem, and cerebellar nuclei (56). VGLUT3 is sparsely distributed in subpopulations of non-glutamatergic neurons

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acid (GABA), and also in the astrocytes of the brain (13, 37, 45, 51, 65, 113, 115). On the other hand, in birds two isoforms, VGLUT2 and VGLUT3, have been

identified and their mRNA distributions have been studied in the pigeon and zebra finch brain but VGLUT1 has not been identified yet (Fig. 1.2). Islam and Atoji (60) first

cloned a cDNA sequence for pigeon VGLUT2 and mapped its distribution in the pigeon brain. VGLUT2 mRNA is widely expressed in the brain of pigeon, chicken, and zebra

finch, including the pallium of telencephalon, diencephalon, mesencephalon, lower brainstem, and granule cells of the cerebellar cortex (2, 3, 4, 60, 64). The distribution

patterns of mammalian VGLUT1 and VGLUT2 mRNA are more or less similar with the distribution patterns of avian VGLUT2 and hence, it is widely accepted that avian

VGLUT2 is correspondence to mammalian VGLUT1 and VGLUT2 (64). VGLUT3 mRNA which is sparsely distributed in subpopulations of non-glutamatergic neurons in

mammals is expressed in the caudal linear nucleus, a serotonergic nucleus, in the pigeon brain (9).

Distribution of glutamatergic neurons in the brain of most vertebrates is still unknown especially in reptilian brains. However, in turtles, some ionotrophic glutamate

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1-PHWK\O-'-DVSDUWDWH (NMDA) receptors have been immunohistochemically observed in neurons of telencephalon and diencephalon (36). In addition, studies on freshwater

turtles showed that glutamate is involved in survival strategies under anoxic conditions by controlling ion channels (20, 98). Furthermore, cortical neurons of turtle can survive

glutamate exposures that are lethal to mammalian neurons (124). Likewise, glutamate is suggested to be a neurotransmitter in turtles, based on pharmacological experiments in

which antagonists for ionotropic glutamate receptors injection stopped firing of the

neurons (18, 69, 70, 86, 87). Moreover, pharmacological data suggest that glutamate is involved in learning and memory in the medial cortex of the turtle (86, 87). Therefore,

above evidence strongly suggests the presence of glutamatergic neurons in reptilian brains, including turtle, but other than the importance of glutamate as a neurotransmitter

in the turtle, distribution of glutamatergic neurons still remain unknown.

Reptilian brains become great models to explore questions related to the structural

and functional evolution of mammalian brains due to their diversity and evolutionary relationship to mammals (89). Basic structures of the turtle brain, a representative in

reptiles, are shown in Figure 1.3. Comparative studies need to identify homologies of particular brain regions between reptiles and mammals for their evolutionary

relationship (33). Conserved expression patterns of several transcription factors in the dorsal part of the embryonic reptilian, avian, and mammalian telencephalon suggest that

pallial regions are specified as the homologous territories in all amniotes (25). Adult mammalian and avian brains telencephalic pallium is the locus of glutamatergic neurons,

which has been proved recently by localization of VGLUTs mRNA by in situ hybridization (38, 60, 64, 92). Therefore, it is essential to do similar studies in adult

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However, investigation of VGLUTs in any reptilian species has not done yet. Therefore, the aim of the present study was to analyze the distributions of neurons

expressing VGLUT1-3 mRNAs in the brain of red-eared turtle by in situ hybridization (Fig. 2). The results reveal the specific distribution of glutamatergic neurons in the brain

of red-eared turtle and reflect comparisons with those of mammals and birds.

In addition, the cerebral cortex of reptilian brain is composed of medial, dorsomedial,

dorsal, and lateral cortical regions (Fig. 1.3). Among these regions the dorsal cortex is

generally considered as homologous to the mammalian isocortex (22, 79, 116). On the other hand, the lateral cortex is thought to be homologous to the mammalian piriform

cortex (22, 79, 116). Further, the medial cortex is supposed to be homologous to the mammalian hippocampal dentate gyrus and/or Ammon¶s horn (24, 77, 111). The

cytoarchitectures are analogous because neuronal cells of the reptilian medial cortex are similar to the granule cells of the dentate gyrus and pyramidal cells of the Ammon¶s

horn of the mammalian hippocampus (58). This hypothesis is also supported by the presence of similar kind of fiber connections; reciprocal connections with dorsomedial,

dorsal, and lateral cortices and projections to the septum (74, 97). This comparison is further supported by immunohistochemical findings; presence of zinc-containing fibers

(97, 99, 109). However, this hypothesis is not supported by any kind of gene expression data.

Prox1 is a prospero homeobox gene, requiredIRUWKHPDWXUDWLRQRIJUDQXOHFHOOVLQ WKH GHQWDWH J\UXV GXULQJ GHYHORSPHQW DQG IRU WKH PDLQWHQDQFH RI LQWHUPHGLDWH

SURJHQLWRUVGXULQJDGXOWQHXURJHQHVLVis expressed in granule cells of the dentate gyrus in the adult mammalian brain (42, 71). Therefore, expression of Prox1 in the medial

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hippocampal dentate gyrus of mammalian brain has been thoroughly characterized to be a typical VGLUT1 expression domain (16, 38, 50, 63, 120). VGLUT2 express strongly

in the granule cells of the dentate gyrus of the early stages of postnatal life express but decrease in the later stages (21, 31, 39, 46). Considering the localization of

zinc-containing presynaptic boutons described above (49, 108), VGLUT2 probably co-localizes in zinc-containing axon terminals. Therefore, Prox1 expressed in the

granule cells of the dentate gyrus and neurochemically neurons of the dentate gyrus are

glutamatergic in nature which is confirmed by VGLUT1 and VGLUT2 expression (16, 21, 31, 38, 39, 46, 50, 63, 120). Hence, expression of Prox1 and glutamatergic markers

in the medial cortex of the turtle brain will strongly support the above hypothesis (24, 77, 111). In the present study, I examined expression of the Prox1 gene in the turtle

telencephalon to determine the area of the turtle brain homologous to the mammalian dentate gyrus (Fig. 1.4).

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Chapter 2

Distribution of vesicular glutamate transporter 1 (VGLUT 1) mRNA

2.1. Introduction

One of the major excitatory neurotransmitter in the central nervous system (CNS) is glutamate, and its transmission is crucial for performing the normal neuronal activity.

The glutamate is loaded in the synaptic vesicles of the presynaptic terminals by

VGLUTs before its exocytotic release in the synaptic cleft. Three types of VGLUTs have been identified in mammalian brain: VGLUT1 (16, 91), VGLUT2 (38, 50), and

VGLUT3 (37, 45, 107, 114), and two types of VGLUTs have been identified in avian brain: VGLUT2 (2, 3, 4, 60, 64) and VGLUT3 (9). Avian VGLUT1 has not been

identified yet. VGLUT1 and VGLUT2 are present in majority of the glutamatergic neurons of the mammalian brain, whereas only VGLUT2 is present in similar types of

neurons of the avian brain. Therefore, VGLUT1 and VGLUT2 in the mammalian brain and VGLUT2 in the avian brain are considered as the selective gene markers of

glutamatergic neurons and avian VGLUT2 represents both VGLUT1 and VGLUT2 of mammals (60). The VGLUT1 is initially described as a brain specific Na+_-dependent

inorganic phosphate transporter I (BNPI) (91). There is considerable experimental evidence that demonstrates the pivotal role of BNPI in the exocytotic release of

glutamate at the presynaptic level, and accordingly, this protein is subsequently named VGLUT1 (17, 115). In mammalian brain, VGLUT1 expresses mostly in the cerebral

cortex and VGLUT2 in the thalamus (39). Besides cerebral cortex, the mammalian VGLUT1 also expresses in the hippocampal formation, cerebellum, amygdala, and

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and VI show highest expression (55). The pyramidal neurons of the hippocampus proper and the granular neurons of the dentate gyrus of the hippocampus showed the

high level of expression of the VGLUT1 (55, 72, 113). In the cerebellar cortex, VGLUT1 is contained in the parallel fibers (38, 57). The lateral and basolateral nuclei

of the amygdala of the mammalian brain show the expression of VGLUT1 (55). Glutamatergic neurons are also identified by the expression of VGLUT1 in some other

brain regions, such as; mitral cells of the olfactory bulb, lateral reticular nucleus,

external cuneate nucleus, and vestibular nucleus of the lower brainstem (57, 83). However in comparison with the localization of the VGLUT1 and VGLUT2 in

mammalian brain, it is evident that VGLUT1 is expressed mostly in the neurons of the cerebral cortex and VGLUT2 in the neurons of the thalamic regions (39). Therefore,

VGLUT1 is considered as a marker for the glutamatergic neurons of the cerebrum and VGLUT2 for thalamic regions (40, 41, 59, 68, 72). On the other hand, in birds,

VGLUT1 has not been identified yet and VGLUT2 denotes both VGLUT1 and VGLUT2 of mammals (60).

In reptiles, pharmacological and electrophysiological studies indicate a pivotal role of glutamate in the CNS including learning and memory by medial cortex (18, 69, 70,

86, 87), which designates the presence of the glutamatergic neurons in the reptilian brains. However, the distribution of the glutamatergic neurons in the brain of any

reptilian species has not been identified before. In the present study, I identified the presence of VGLUT1 mRNA in the red-eared turtle brain by reverse

transcription-polymerase chain reaction (RT-PCR) and then demonstrated the distribution of its mRNA-expressing glutamatergic neurons by in situ hybridization

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2.2. Materials and Methods Animals

Three adult red-eared turtles 3VHXGHP\V VFULSWD HOHJDQV of both sexes weighted 300~1,000 gm were used to conduct the present study. A young turtle, 50 gm, was used

for RT-PCR. The animal handling procedures were approved by the Animal Experimental Committee of the Faculty of Applied Biological Sciences, Gifu

University.

RNA isolation and cDNA synthesis for VGLUT1

For isolation of total RNA, the young turtle was deeply anaesthetized with an intraperitoneal injection of chloral hydrate (5.4 g/kg body weight). The brain was

dissected out quickly into pieces about 4mm3_{. The pieces were kept in RNA}

stabilization solution (RNAlater, Ambion, USA) and stored at -60°C until use. Total

RNA were collected from the telencephalon and brainstem of the turtle.

Complementary DNA (cDNA) syntheses were done according to Islam and Atoji

(60). Briefly, each brain sample was homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed for 5 min at room temperature. An appropriate volume

of chloroform was added and mixed vigorously, and the sample was centrifuged at J IRU PLQ DW Û& 7KH VXSHUQDWDQW IOXLG ZDV FROOHFWHG PL[HG ZLWK WKH VDPH

YROXPHRILVRSURSDQRODQGFHQWULIXJHGDWJIRUPLQDWÛ&WRSUHFLSLWDWHWRWDO RNA. After washing in 75% ethanol, the precipitate was dissolved into diethyl

pyrocarbonate (DEPC)-treated water. The concentration was checked with a Biophotometer plus (Eppendorf AG, Hamburg, Germany) and the dissolved precipitate

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First-strand cDNA was synthesized using a Superscript III First-Strand Synthesis 6\VWHP ,QYLWURJHQ 7RWDO 51$ ȝJ ZDV PL[HG ZLWK ȝ0 ROLJR-dT primer and

0.5mM 2ཛ-deoxyribonucleotide 5ཛ-WULSKRVSKDWHVG173PL[WXUHLQFXEDWHGDWÛ&IRU min, and placed on ice. Supplied reaction buffer of the enzyme, 5mM dithiothreitol, 2

units RNase out, and 10 units Superscript III reverse transcriptase were added to the PL[WXUHDQGLQFXEDWHGDWÛ&IRUPLQWKHUHDFWLRQZDVVWRSSHGE\KHDWLQJDWÛ&

for 15 min, and the synthesized product was preserved at -Û&XQWLOXVH

RT-PCR

Synthesized cDNA 500 ng was mixed with Takara Ex Taq (Takara Bio, Tokyo, -DSDQVXSSOLHGG173PL[WXUHDQG(;7DTEXIIHU7KHQȝ0RIDSSURSULDWHIRUZDUG

and reverse primers were added with the mixture. Primers for VGLUT1 DQG ȕ DFWLQ were designed based on turtle cDNA sequences registered in a GenBank database and

the information of the primer sequences is shown in Table 1. Beta actin was used as a positive control. PCR was carried out by 35 cycles of amplification (denaturation at

94 °C for 30 sec, annealing at 56 °C for 40 sec, extension at 72 °C for 1 min) and a final extension was done at 72 °C for 5 min. PCR products were measured in gel

electrophoresis (1.8% agarose) with lader markers.

Perfusion of animals for in situ hybridization

Adult turtles were anaesthetized deeply with an intraperitoneal injection of chloral

hydrate (5.4 g/kg body weigKW DQG SHUIXVHG ZLWK 5LQJHU¶V VROXWLRQ IROORZHG E\ paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Brains with an upper cervical

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then transferred to 30% sucrose in phosphate-buffered saline (PBS) at 4ÛC for until sink. 7KH EUDLQV ZHUH FXW WUDQVYHUVHO\ DW ȝP RQ D FU\RVWDW DQG ZHUH PRXQWHG RQWR JODVV

slides coated with aminosilane and poly-L-lysine hydrobromide. Some sections were FXWDWȝPDQGVWDLQHGZLWKcresyl violet.

Probe preparation

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In situ hybridization

Sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at room

temperature for 30 min and then treated with proteinase K (15 mg/ml; Kanto, Tokyo) in PBS at 37ÛC for 30 min. The sections were acetylated in water containing 1.35%

triethanol amine, 0.25% acetic anhydrite, and 0.058% HCl at room temperature for 10

min. After rinsing in PBS, the sections were hybridized with DIG-labeled antisense or sense RNA probes dissolved in a hybridization buffer consisting of 20% dextran sulfate,

50% formamide, 2% blocking solution, 0.01% N-lauroylsarcosine (NLS), and 0.01% sodium lauryl sulfate (SDS) in 5x standard saline citrate (SSC, pH 7.4). The information

of probe concentrations used in the present study is listed in Table 1. The sections were heated on a heat plate at 95ÛC for 4 min and then were incubated at 55ÛC overnight.

After hybridization, the sections were rinsed in a solution of 50% formamide and 0.01% NLS in 2x SSC at 65Û&IRUPLQDQGZHUHLQFXEDWHGZLWK51DVH$ȝJPO5RFKH

in NTE buffer (500mM NaCl, 10mM Tris, and 1mM EDTA, pH 8.0) at 37ÛC for 30 min. After rinsing in Tris buffered saline (pH 7.4) containing 0.025% Tween 20 (TBST), the

sections were incubated with a blocking solution containing 1% blocking reagent and 2% normal sheep serum in TBST at room temperature for 1h. After washing in TBST,

the sections were incubated with alkaline phosphatase conjugated sheep anti-DIG antibody (Roche, 1: 2,000) in TBST at 25ÛC overnight. After rinsing in TBST, the

alkaline phosphatase was visualized by incubation with a mixture of nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (Roche)

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25ÛC overnight. Finally, the sections were dehydrated by ascending grades of ethanols and mounted with a M.X. medium (Matsunami, Japan).

Image Processing

Photomicrographs were taken with a digital camera (Pro 600ES; Pixera Corporation, Los Gatos, CA, USA; or DS-Fi1, Nikon, Tokyo, Japan) mounted on a light microscope.

Adjustment of photographs for contrast, brightness and sharpness, layout, and lettering

were performed in Adobe Photoshop 7.0J and Adobe Illustrator 10.0J.

Nomenclature

The nomenclature in the present study was accorded by ten Donkelaar (116). In some

cases, Cruce and Nieuwenhuys (30), Kiehn et al. (66), and Powers and Reiner (102, 103) were also followed.

2.3. Results

Initially VGLUT1 expression in the brain was utilized reverse transcription of RNA followed by DNA amplification (RT-PCR). In situ hybridization with VGLUT1

oligonucleotides probe was subsequently used for identification of VGLUT1-expressing neurons in the different regions of the red-eared turtle brain.

RT-PCR

RT-PCR by using the cDNA of telencephalon was performed. A VLQJOHFOHDUEDQG in the second lane of Figure 2.1 proves the existence of 9*/87 P51$ LQ WKH

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are highly similar to the VGLUT1 localization in the mammalian cerebrum.

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terminals originating from granule cells are VGLUT1-positive (53, 63). In the present study, granule cells strongly express VGLUT1 mRNA but not VGLUT2 mRNA. These

results suggest that, as in mammals, the granule cells of the reptilian cerebellum are glutamatergic in nature, expressing the VGLUT1 isoform.

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Chapter 3

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30 DOVRVKRZHGWKHH[SUHVVLRQ

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pharmacological and electrophysiological studies specify the presence of glutamate and its pivotal function in the CNS (18, 69, 70, 86, 87), which indicates the presence of the

glutamatergic neurons in the reptilian brains. In the present study, I resolute the presence of VGLUT2 mRNA in the red-eared turtle brain by reverse

transcription-polymerase chain reaction (RT-PCR) and then demonstrated the

distribution of its mRNA-expressing glutamatergic neurons by in situ hybridization histochemistry.

Three adult red-eared turtles 3VHXGHP\V VFULSWD HOHJDQV of both sexes weighted

300~1,000 gm were used to conduct the present study. A young turtle, 50 gm, which was previously used for RT-PCR of VGLUT1, was also used for this study. The animal

handling procedures were approved by the Animal Experimental Committee of the Faculty of Applied Biological Sciences, Gifu University.

The procedure for the preparation of RNA isolation and the cDNA synthesis was similar with chapter 2. Basically, the cDNA which was synthesized for the study of

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31

RT-PCR

RT-PCR was done according to the description of the chapter 1. Briefly, 500 ng of

previously synthesized cDNA was mixed with Takara Ex Taq (Takara Bio, Tokyo, -DSDQVXSSOLHGG173PL[WXUHDQG(;7DTEXIIHU7KHQȝ0RIDSSURSULDWHIRUZDUG

and reverse primers of VGLUT2 were added with the mixture. Primers for VGLUT 2 DQG ȕ DFWLQ ZHUH GHVLJQHG EDVHd on turtle cDNA sequences registered in a GenBank

database and the information of the primer sequences are shown in Table 3. Beta actin

used as a positive control. PCR was carried out by 35 cycles of amplification (denaturation at 94 °C for 30 sec, annealing at 56 °C for 40 sec, extension at 72 °C for

1 min) and a final extension was done at 72 °C for 5 min. PCR products were measured in gel electrophoresis (1.8% agarose) with lader markers.

Three adult turtles were used for the present study. The procedure for the DQLPDO¶V perfusion was similar with the description of previous chapter.

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In situ hybridization procedure was same as described in chapter 2.

DS-Fi1, Nikon, Tokyo, Japan a digital camera mounted on a light microscope was used for taken photomicrographs. In some cases Pro 600ES; Pixera Corporation, Los

Gatos, CA, USA another digital camera mounted on a light microscope was also used. Adjustment of photographs for contrast, brightness and sharpness, layout, and lettering

were performed in Adobe Photoshop 7.0J and Adobe Illustrator 10.0J. *HQHV$FFHVVLRQ1R )RUZDUGSULPHUVIRU3&5 )RUZDUGSULPHUVIRUSUREH 5HYHUVHSULPHUVIRU3&5 5HYHUVHSULPHUVIRUSUREH 3UREHFRQFHQWUDWLRQV 9*/87/& ȕ-DFWLQ)- *****$&$*$77*&$*$777 *****$&$*$77*&$*$777 7*&*7*$&$7&$$$*$*$$* 77&$&77*7&7*77&$***7&$ 77&$&77*7&7*77&$***7&$ *7$&77*&*&7&$**$**$* ȝJȝO

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33

Nomenclature

The suggestion of ten Donkelaar (116) about turtle brain was adopted in the present

study. The proposals of Cruce and Nieuwenhuys (30), Kiehn et al. (66), and Powers and Reiner (120, 103) were also followed in some cases.

3.3. 5HVXOWV

The initial analysis of VGLUT2 expression in the different brain regions utilized

reverse transcription of RNA followed by DNA amplification (RT-PCR). In situ hybridization with VGLUT2 oligonucleotides probe was subsequently used for

identification of VGLUT2-expressing neurons in the different regions of the red-eared turtle brain.

RT-PCR

Complementary DNA of telencephalon was used for the RT-PCR of VGLUT2. The existence of VGLUT2 mRNA was identified by the high level expression of VGLUT2

mRNA which was observed by the presence of a VLQJOHFOHDUEDQG in the second lane of Figure 3.1 9*/ 8 7 ȕ D FW LQ )LJ57-3&5RIP51$VIRU9*/87DQGȕ DFWLQ LQ WKH WXUWOH FHUHEUXP ȕ DFWLQ ES LV XVHGDVDFRQWURO

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,Q PDPPDOV WKH QXPEHU RI 9*/87-H[SUHVVLQJ QHXURQV LQ WKH FHUHEUDO FRUWH[ LV VLPLODUWRWKHQXPEHURI9*/87-H[SUHVVLQJQHXURQVEXW9*/87H[SUHVVLRQLVOHVV

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ZHUH ZLGHO\ GLVWULEXWHG LQ WKH WHOHQFHSKDORQ DQG PRVW 9*/87-SRVLWLYH DUHDV DOVR H[SUHVVHG9*/87+RZHYHUWKHLQWHQVLW\RI9*/87H[SUHVVLRQLQWKHVHDUHDVZDV

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VGLUT2 mRNA is highly expressed in nuclei of the diencephalon in human and rat brains. HLJK 9*/87 P51$ H[SUHVVLRQ LV IRXQG LQ WKH WKDODPXV HJ DQWHURGRUVDO

DQG DQWHURPHGLDO WKDODPLF QXFOHL SDUDYHQWULFXODU WKDODPLF QXFOHXV SDUDIDVFLFXODU WKDODPLFQXFOHXVODWHUDODQGPHGLDOJHQLFXODWHQXFOHLDQGVXEWKDODPLFQXFOHXV

9*/87H[SUHVVLRQZDV ORZHU LQWKH hypothalamic nuclei than in the thalamic nuclei. 9*/87 H[SUHVVLRQ ZDV REVHUYHG LQ WKH ODWHUDO DQG PHGLDO KDEHQXODU QXFOHL

GRUVRPHGLDO WKDODPLF QXFOHXV GRUVRODWHUDO WKDODPLF QXFOHXV GRUVRODWHUDO JHQLFXODWH QXFOHXVQXFOHXV UHXQLHQVURWXQGDOQXFOHXVSHULYHQWULFXODUK\SRWKDODPLF QXFOHXVDQG

(48)

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9*/87-H[SUHVVLQJ QHXURQV DUH D SULQFLSDO JOXWDPDWHUJLF VXESRSXODWLRQ LQ WKHWXUWOH GLHQFHSKDORQ

In the rat cerebellum, no VGLUT2 expression was detected in the cortical region, but the deep cerebellar nuclei showed high expression levels (38, 57). The lower brainstem

contained many nuclei expressing VGLUT2 at various degrees of intensity (e.g., dorsal cochlear nucleus, vestibular nuclei, inferior olivary nucleus, lateral reticular nucleus,

gigantocellular reticular nucleus, paramedian reticular nucleus, dorsal

paragigantocellular nucleus, and the spinal nucleus of the trigeminal nerve) (57). The spinal cord also exhibited VGLUT2 expression (55). In the present study, VGLUT2 was

expressed in the cerebellar nuclei but not in the cerebellar cortex. Many brainstem nuclei exhibited VGLUT2 expression, including the reticular, vestibular, and cochlear

nuclei.

&RPSDULVRQRI9*/87P51$LQELUGVDQGWXUWOHV

As 9*/87P51$KDVQRW\HWEHHQLGHQWLILHGLQELUGVWhe distribution of VGLUT2

expression in the avian brain appears to correspond with both VGLUT1 and VGLUT2 expression in mammalian brains (60, 64).,QWKHROIDFWRU\EXOEPLWUDOFHOOVVKRZHGKLJK

9*/87H[SUHVVLRQLQDGXOWSLJHRQVDQG]HEUDILQFKHV(60, 64) and chick embryos (2, 4). In the present study, VGLUT2 was moderately expressed in mitral cells, whereas

VGLUT1 showed the strongest expression. In birds, all regions of the Wulst, mesopallium, nidopallium, arcopallium, and hippocampal formation expressed

VGLUT2 (60, 64). These results suggest that glutamatergic neurons in the telencephalon are widely distributed in the avian pallium. In the present study,

(49)

44

parts of the cortex, thickening pallium, DVR, central nucleus of the DVR, and amygdala (except the central nucleus)), which is consistent with findings in birds.,QWKHVXESDOOLDO

UHJLRQWKHPHGLDOVHSWDOQXFOHXVLQWKHDGXOWUDWH[SUHVVHV9*/87,QPRXVH DQG FKLFNHQ HPEU\RV D UHJLRQ FRUUHVSRQGLQJ WR WKH PHGLDO VHSWDO QXFOHXV H[SUHVVHV

P51$IRU9*/87DQGVRPHSDOOLDOPDUNHUVHJ/K[/K[DQG7EU- ,QWKHSUHVHQWVWXG\WKHPHGLDOVHSWDOQXFOHXVVKRZHGZHDN9*/87H[SUHVVLRQ

7KHUHIRUH LW VHHPV WKDW JOXWDPDWHUJLF QHXURQV LQ WKH PHGLDO VHSWDO QXFOHXV RI WXUWOHV

RULJLQDWHIURPWKHSDOOLXPDQGLPPLJUDWHLQWRWKHVHSWXP

7KH VXSUDRSWR-SDUDYHQWULFXODU GRPDLQ 639 LQ FKLFN HPEU\RV VKRZV KLJKHU

9*/87 H[SUHVVLRQ WKDQ WKH WKDODPXV +RZHYHU LQ DGXOW UDWV DQG SLJHRQV 9*/87H[SUHVVLRQLVZHDNHULQWKHK\SRWKDODPLFQXFOHLWKDQLQWKHWKDODPLFRQHV

,QWKHSUHVHQWVWXG\LQDGXOWWXUWOHVa large number of nuclei in the diencephalon of the turtle brain (i.e., ODWHUDODQG PHGLDO KDEHQXODUQXFOHLGRUVRPHGLDODQGGRUVRODWHUDO

WKDODPLF QXFOHL GRUVRODWHUDO JHQLFXODWH QXFOHXV DQG URWXQGDO QXFOHXV expressed VGLUT2, and their expression LQWHQVLW\ZDVVWURQJHUWKDQWKDWLQWKHK\SRWKDODPXV,W

LVOLNHO\WKDWLQDGXOWDPQLRWHVWKHWKDODPXVLVWKHSUHIHUUHG9*/87-H[SUHVVLQJDUHD FRPSDUHGWRWKHK\SRWKDODPXV

In pigeons, layer 13 of the optic tectum and the rotundal nucleus showed strong VGLUT2 expression (8, 38). In turtles, the periventricular cellular layer of the optic

tectum and rotundal nucleus displayed VGLUT2 expression. Atoji (8) found high VGLUT2 expression in the parvocellular part of the isthmic nucleus of the pigeon,

whereas the magnocellular part of the isthmic nucleus expressed GAD 65. In the turtle, the magnocellular part of the isthmic nucleus expressed VGLUT2. The underlying

(50)

45

the pigeon and the turtle remains unclear. However, a hypothesis has been proposed regarding the homology of the isthmic nuclei in birds and reptiles. ,QUHSWLOLDQLVWKPLF

QXFOHL the magnocellular part of the isthmic nucleus VKRZV LPPXQRUHDFWLYLW\ IRU FKROLQHDFHW\OFKROLQHWUDQVIHUDVH&K$7ZKHUHDVWKHparvocellular part LVQHJDWLYH

,QFRQWUDVWWKHparvocellular part of the avian isthmic nucleusUHYHDOV&K$7 LPPXQRUHDFWLYLW\ ZKHUHDV WKH magnocellular part LV QHJDWLYH 7KHVH &K$7

LPPXQRUHDFWLYLW\ILQGLQJVVXJJHVWWKHKRPRORJ\EHWZHHQWKHLVWKPLFQXFOHLRIUHSWLOHV

DQG ELUGV WKH UHSWLOLDQ magnocellular part ZLWK WKH DYLDQ parvocellular part DQG WKH UHSWLOLDQ parvocellular part with WKH DYLDQ magnocellular part 7KHUHIRUH 9*/87

ORFDOL]DWLRQLQWKHLVWKPLFQXFOHLDSSHDUVWREHVLPLODULQELUGVDQGUHSWLOHV 6XPPDU\ ,QWKHSUHVHQWVWXG\,PDSSHGWKHGLVWULEXWLRQRI9*/87P51$LQWKH&16RIWKH UHG-HDUHGWXUWOHE\LQVLWXK\EULGL]DWLRQ7KHUHVXOWVVKRZHG9*/87P51$H[SUHVVHG H[FOXVLYHO\LQWKHSDOOLXPRIWHOHQFHSKDORQDQGQRH[SUHVVLRQLQVXESDOOLXP9*/87 P51$ZDVPRUHDEXQGDQWLQWKDODPXVWKDQK\SRWKDODPXV7KHGLIIHUHQWLDOH[SUHVVLRQ SDWWHUQV RI WKH 9*/87 ZHUH IRXQG LQ WKH SHULYHQWULFXODU FHOOXODU OD\HU RI WKH RSWLF

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(51)

46

Chapter 4

Glutamate is a ubiquitous amino acid that is act as a signaling molecule in the brain, stored in the presynaptic vesicles by means of VGLUTs (113). Three types of VGLUTs

have been identified in mammalian brain (5, 37, 38, 50, 91, 92) and two in avian brain

(60, 64). VGLUT3 is one of them and is present both in mammalian and avian brains. In contrast to other two VGLUTs isoforms that are expressed in the glutamatergic neurons;

the distribution of the VGLUT3 in the brain is somewhat astonishing. VGLUT3 is sporadically distributed throughout the brain and co-localizes with other

neurotransmitters, such as GABA, serotonin, and acetylcholine (113). Therefore, VGLUT3 is capable to co-release both glutamate and other classical neurotransmitter

(37, 51, 107, 113).

In the mammalian brain, VGLUT3 is expressed in the different nuclei of the

cerebrum and brainstem. VGLUT3 is expressed in neocortical layer 2, hippocampus, entorhinal cortex, caudate-putamen, and nucleus accumbens of the cerebrum; substantia

nigra pars compacta, interpeducular nucleus, ventral tegmental area, and raphe nuclei of the brainstem (21, 37, 45, 51, 55, 100, 107). The raphe nuclei of the midbrain consists

of dorsal, median, and caudal linear raphe nuclei, all of those are the main source of serotonergic neurons and 80% neurons of the median raphe nucleus are serotonergic in

nature and express VGLUT3 (28, 52, 54, 61, 84).

In the avian brain, VGLUT3 is expressed only in the caudal linear nucleus of the

(52)

47

expression and the caudal linear nucleus is serotonergic in natures (9)

As previously noted, glutamate is involved in different activities in CNS including

learning and memory of the medial cortex of reptiles, which is determined by some previous pharmacological and electrophysiological studies (18, 69, 70, 86, 87). The

above evidence indicates the presence of the glutamatergic neurons in the reptilian brains. However, the distribution of the glutamatergic neurons in the brain of any

reptilian species has not been identified before. In the present study, I examined the

presence of VGLUT3 mRNA in the red-eared turtle brain by RT-PCR and then demonstrated the distribution of its mRNA-expressing neurons by in situ hybridization

histochemistry.

Two adult red-eared turtles 3VHXGHP\VVFULSWDHOHJDQVof both sexes weighted 567 gm and 914 gm were used to conduct the present study. A young turtle, 50 gm, which

was previously used for RT-PCR of VGLUT1, was also used for this study. The animal handling procedures were approved by the Animal Experimental Committee of the

Faculty of Applied Biological Sciences, Gifu University.

The procedure for the preparation of RNA isolation and the cDNA synthesis was

similar with chapter 2. Basically, the cDNA which was synthesized for the study of VGLUT1 of the chapter 2 was kept in -Û& and used for the study of VGLUT3 also.

(53)

48

RT-PCR

RT-PCR was done according to the description of the chapter 1. Briefly, 500 ng of

previously synthesized cDNA was mixed with Takara Ex Taq (Takara Bio, Tokyo, -DSDQVXSSOLHGG173PL[WXUHDQG(;7DTEXIIHU7KHQȝ0RIDSSURSULDWHIRUZDUG

and reverse primers of VGLUT3 were added with the mixture. Primers for VGLUT3 DQG ȕ DFWLQ ZHUH GHVLJQHG EDVHG on turtle cDNA sequences registered in a GenBank

database and the information of the primer sequences are shown in Table 5. Beta actin

used as a positive control. PCR was carried out by 35 cycles of amplification (denaturation at 94 °C for 30 sec, annealing at 56 °C for 40 sec, extension at 72 °C for

1 min) and a final extension was done at 72 °C for 5 min. PCR products were measured in gel electrophoresis (1.8% agarose) with lader markers.

Two adult turtles were used for the present study. The procedure for the DQLPDO¶V perfusion was similar with the description of chapter 2.

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(54)

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PL[WXUH[WUDQVFULSWLRQEXIIHU51DVHLQKLELWRUSRO\PHUDVH7IRUPDNLQJVHQVHRU SRO\PHUDVH 6S IRU PDNLQJ DQWLVHQVH 51$ SUREH DQG '(3& WUHDWHG ZDWHU $IWHU

LQFXEDWLRQDW & IRU K 0/L&ODQGHWKDQROZHUHDGGHGDQGWKH PL[WXUH ZDV LQFXEDWHG DW í & RYHUQLJKW 6\QWKHVL]HG 51$ WUDQVFULSW ZDV FROOHFWHG E\

FHQWULIXJDWLRQDQGDOLTXRWHGLQWR QJȝOZLWK'(3&WUHDWHGZDWHU

In situ hybridization procedure was same as described in chapter 2.

DS-Fi1, Nikon, Tokyo, Japan a digital camera mounted on a light microscope was

used for taken photomicrographs. In some cases Pro 600ES; Pixera Corporation, Los Gatos, CA, USA another digital camera mounted on a light microscope was also used.

Adjustment of photographs for contrast, brightness and sharpness, layout, and lettering were performed in Adobe Photoshop 7.0J and Adobe Illustrator 10.0J.

Nomenclature

The suggestion of ten Donkelaar (116) about turtle brain was adopted in the present study. The proposals of Cruce and Nieuwenhuys (30), Kiehn et al. (66), and Powers and *HQHV$FFHVVLRQ1R )RUZDUGSULPHUVIRU3&5 )RUZDUGSULPHUVIRUSUREH 5HYHUVHSULPHUVIRU3&5 5HYHUVHSULPHUVIRUSUREH 3UREHFRQFHQWUDWLRQV 9*/87/& ȕ-DFWLQ)- $&777*&7777**7**77** $*&$**&7**&$$&&$&*7& 7*&*7*$&$7&$$$*$*$$* &&*&$&$7&$77*7777*$& $77*&&&$&&$$7***$$&$$ *7$&77*&*&7&$**$**$* ȝJȝO

(55)

50

Reiner (102, 103) were also followed in some cases.

5HVXOWV

RT-PCR analysis of VGLUT3 was first done. In situ hybridization with VGLUT3

oligonucleotides probe was subsequently carried out for identification of VGLUT3-expressing neurons in the different regions of the red-eared turtle brain.

RT-PCR

Complementary DNA of brainstem was used for the RT-PCR of VGLUT3. The

existence of VGLUT3 mRNA was observed by the presence of a VLQJOHFOHDUEDQG in the second lane of Figure 4.1

9*/87P51$ZDVOLPLWHGO\H[SUHVVHGLQQXFOHLRIWKHORZHUEUDLQVWHP7DEOH

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H[SUHVVLRQ)LJDE,QWKHUKRPEHQFHSKDORQWKHVXSHULRUUDSKH QXFOHXV VKRZHG ES ES g 9*/ 8 7 ure ȕ DFW LQ )LJ57-3&5RIP51$VIRU9*/87 DQGȕDFWLQLQWKHWXUWOHEUDLQVWHPȕDFWLQ ESLVXVHGDVDFRQWURO

(56)

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

(57)

52 7DEOH&RQWLQXHG 5HJLRQV 6LJQDOLQWHQVLW\ 0HVHQFHSKDORQ 3HULYHQWULFXODUFHOOXODUOD\HU 7RUXVVHPLFLUFXODUFHQWUDOQXFOHXV 0DJQRFHOOXODUSDUWRILVWKPLFQXFOHXV 3DUYRFHOOXODUSDUWRILVWKPLFQXFOHXV 5KRPEHQFHSKDORQ *UDQXODUOD\HURIFHUHEHOODUFRUWH[ &RFKOHDUQXFOHXV 7DQJHQWLDOYHVWLEXODUQXFOHXV 9HQWURODWHUDOYHVWLEXODUQXFOHXV 'HVFHQGLQJQXFOHXVRIWKHWULJHPLQXV /DWHUDOFHUHEHOODUQXFOHXV 0HGLDOFHUHEHOODUQXFOHXV 6XSHULRUYHVWLEXODUQXFOHXV 6XSHULRUUHWLFXODUQXFOHXV 3ULQFLSDOVHQVRU\QXFOHXVRIWKHWULJHPLQXV 6XSHULRUUDSKHQXFOHXV ,QIHULRUUDSKHQXFOHXV 0HGLDQUHWLFXODUQXFOHXV 6SLQDOFRUG 8SSHUFHUYLFDOVHJPHQW - - - - - - - - - - - - - -

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(58)

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'LVFXVVLRQ

,QPDPPDOVWKHGLVWULEXWLRQRIP51$VIRU9*/87DQGSURWHLQVZHUHH[WHQVLYHO\

VWXGLHGLQPDPPDOLDQEUDLQV,QELUGV9*/87ZHUHFRQILUPHG LQ FDXGDO OLQHDU QXFOHXV RI SLJHRQ EUDLQV . The present study identified

VGLUT3-expressing neuronal cells in the turtle brain. The findings indicate the existence of VGLUT3-expressing neuronal cells in the turtle brain and provide evidence

to compare its distribution with other mammalian and avian brains on the basis of

evolutionary point. I discuss important points in the turtle brain and compare the findings with those in mammals and birds.

&RPSDULVRQRI9*/87P51$LQPDPPDOVDQGWXUWOHV

,QPDPPDOV9*/87 is expressed in layer II of some neocortices, the hippocampus, entorhinal cortex, caudate-putamen, and nucleus accumbens of the cerebrum, as well as

in the substantia nigra pars compacta, interpeduncular nucleus, ventral tegmental area, and raphe nuclei of the brainstem (37, 51, 107). VGLUT3 expression is co-localized in

GABAergic neurons of the hippocampus and neocortex (51)FKROLQHUJLFQHXURQVRIWKH VWULDWXPDQGVHURWRQHUJLFQHXURQVRIWKHUDSKHQXFOHL. In the present study,

VGLUT3 expression was strong in the superior raphe nucleus, moderate in the parvocellular part of the isthmic nucleus, and weak in the cochlear nucleus and inferior

raphe nucleus. The raphe nuclei of the turtle are serotonergic in nature (66). Some FKROLQHUJLFperikarya were found in the parvocellular part of the isthmic nucleus and the

(60)

55

&RPSDULVRQRI9*/87P51$LQELUGVDQGWXUWOHV

$WRML DQG .DULP IRXQG WKDW LQ WKH SLJHRQ 9*/87 LV H[SUHVVHG RQO\ LQ WKH

FDXGDOOLQHDUQXFOHXVZKLFKLVVHURWRQHUJLFLQQDWXUHIn the turtle brain, VGLUT3 was expressed in the parvocellular part of the isthmic nucleus, cochlear nucleus, superior

raphe nucleus, and inferior raphe nucleus. In the red-eared turtle, the parvocellular part of the isthmic nucleus is cholinergic in nature, and the superior and inferior raphe nuclei

are serotonergic (66, 103).

4.5. Summary

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9*/87 P51$ GLG QRW H[SUHVVHG LQ WKH WHOHQFHSKDORQ DQG GLHQFHSKDORQ 7KH SDUYRFHOOXODU SDUW RI LVWKPLF QXFOHXV RI PHVHQFHSKDORQ VXSHULRU DQG LQIHULRU UDSKH

QXFOHL DQG FRFKOHDU QXFOHXV RI UKRPEHQFHSKDORQ RQO\ VKRZHG WKH H[SUHVVLRQ RI 9*/877KHVHUHVXOWVVXJJHVWWKDWWKHGLVWULEXWLRQVRI9*/87P51$LQWKHWXUWOH

EUDLQDUHYHU\VLPLODUWRWKRVHRIPDPPDOLDQDQGDYLDQ9*/87DQGco-localizes with other neurotransmitters.

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56

Chapter 5 Distribution of Prox1 mRNA

The reptilian cerebral cortex composed of three definite layers based on different

density of neurons. The outer layer 1, which is also known as superficial plexiform or molecular layer, composed of few scattered cells and bounded dorsally by pia matter.

The intermediate layer 2, the cellular layer, composed of a large number of densely packed neurons. It forms a continuous sheet of cells extending from the medial to the

lateral edge of the cortex. The inner layer 3, the deep plexiform or subcellular layer, contains a moderate number of loosely arranged neurons (116). The cortex of the

red-eared turtle is divided into four areas: the medial, dorsomedial, dorsal, and lateral cortices (119). The dorsal cortex is generally considered as homologous with the

mammalian isocortex and the lateral cortex is with mammalian piriform cortex (22, 79, 116). The medial cortex which is situated above the septum, occupies a considerable

part of the caudal wall of the cerebral hemispheres, and characterized by the presence of a few and scattered neurons in layers 1 and 3 and densely packed many small neurons in

layer 2 is considered to homologous to the mammalian hippocampal dentate gyrus and/or Ammon`s horn (24, 77, 112). This hypothesis is further supported by findings

obtained from studies on cytoarchitectures- neurons similar to the granule cells of the dentate gyrus and pyramidal cells of the Ammon¶s horn, fiber connections with

dorsomedial, dorsal, and lateral cortices and projections to the septum, and histochemical characteristics- presence of zinc-containing fibers and glutamatergic

(62)

57 is not supported by any gene expression data.

The homeobox gene Prox1 is originally cloned in mouse by homology to the drosophila melanogaster gene prospero (23, 96). Prox1 homologues have been

LGHQWL¿HGLQ RWKHUYHUWHEUDWHVLQFOXGLQJ ;HQRSXV]HEUD¿VKFKLFNHQDQGKXPDQ (44, 95, 117). The mammalian hippocampal dentate gyrus is composed of three layers -

molecular, granule, and polymorphic and Prox1 is selectively expressed in the granular cells. Prox1 is not expressed in the pyramidal cells of $PPRQ¶VKRUQof the adult and

embryonic mammalian brain (42, 71). In chick embryos, a part of the V-shaped layer of hippocampal formation expresses Prox1 (1, 48) and adult pigeon hippocampal V-shaped

layer shows the expression of Prox1 which becomes moderate to weak towards the triangular region (10). Therefore, it is evident that the mammalian hippocampal dentate

gyrus is homologous with avian V-shaped layer of hippocampal formation, but similar kind of gene expression data is not present to support that the reptilian medial cortex is

homologous with mammalian hippocampal dentate gyrus or avian V-shaped layer. Therefore Prox1 can be used as a convenient marker to identify a similar structure in

reptiles. In the present study, I examined the presence of Prox1 mRNA in the red-eared turtle brain by reverse transcription-polymerase chain reaction (RT-PCR) and then

demonstrated its distribution in situ hybridization histochemistry to identify the mammalian dentate gyrus homologous part in reptilian brain.

Two adult red-eared turtles 3VHXGHP\VVFULSWDHOHJDQVof both sexes weighted 567

Distribution of Glutamatergic Neurons in the Central Nervous System of the Turtle(Pseudemys scripta elegans

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RIWKHTurtle (Pseudemys scripta elegans)

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2018

The United Graduate School of Veterinary Sciences, Gifu University

(Gifu University)

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RIWKHTurtle (Pseudemys scripta elegans)

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Contents

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