Calcium-binding protein, secretagogin, characterizes novel groups of interneurons in the rat striatum

Accepted Manuscript Title: Calcium-binding protein, secretagogin, characterizes novel groups of interneurons in the rat striatum Authors: Toshio Kosaka, Seiko Yasuda, Katsuko Kosaka PII: DOI: Reference:

S0168-0102(16)30195-X http://dx.doi.org/doi:10.1016/j.neures.2017.01.004 NSR 4009

To appear in:

Neuroscience Research

Received date: Revised date: Accepted date:

13-10-2016 28-12-2016 18-1-2017

Please cite this article as: Kosaka, Toshio, Yasuda, Seiko, Kosaka, Katsuko, Calciumbinding protein, secretagogin, characterizes novel groups of interneurons in the rat striatum.Neuroscience Research http://dx.doi.org/10.1016/j.neures.2017.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Calcium-binding protein, secretagogin, characterizes novel groups of interneurons in the rat striatum. Running title: novel secretagogin-containing striatal interneurons

Toshio Kosaka, Seiko Yasuda and Katsuko Kosaka

Department of Medical Science Technology, Faculty of Health and Welfare Sciences in Fukuoka, International University of Health and Welfare, 137-1 Enokizu, Okawa City, Fukuoka 831-8501

Corresponding author: Toshio Kosaka Department of Medical Science Technology, Faculty of Health and Welfare Sciences in Fukuoka, International University of Health and Welfare, 137-1 Enokizu, Okawa City, Fukuoka 831-8501 E mail: [email protected] Tel. 0944-89-2000

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Highlights: 

SCGN+ interneurons show vast species differences between rats and mice.



Rat striatum contains numerous SCGN+ neurons of various structural features.



Rat striatal SCGN+ neurons overlap with PV or CR or ChAT+ interneurons.



A population of SCGN+ neurons contain none of these interneuron markers.



There are some novel groups of interneurons in the rat striatum.

Abstract In the rat striatum numerous secretagogin (SCGN) positive neurons were scattered. They were heterogeneous in their morphological and chemical properties. We examined the colocalization of SCGN with known four interneuron markers, parvalbumin (PV), calretinin (CR), nitric oxide synthase (NOS) and choline acetyl transferase (ChAT). 6070 % of SCGN positive striatal neurons contained either PV or CR or ChAT, but none contained NOS. On the other hand the remaining 30-40 % expressed none of these markers, most of which were GAD positive. The present study indicates that there are hitherto unknown groups of striatal interneurons in the rat striatum.

Key words: calretinin; choline acetyl transferase; nitric oxide synthase; parvalbumin; GABA; heterogeneity.

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Telencephalic interneurons are heterogeneous and major groups are generally distinguished based on the expression of histochemical markers such as calcium binding proteins, neuroactive substances and transmitter receptor subtypes. Those chemically defined groups of interneurons were further characterized and/or correlated with morphological and physiological features. In the striatum four major classes of interneurons have been identified, cholinergic neurons and GABAergic neurons containing parvalbumin (PV), somatostatin/nitric oxide synthase (NOS) and calretinin (CR) (Kawaguchi et al. 1995; Tepper and Bolam, 2004; Tepper et al., 2010; Dudman and Gerfen, 2015). Recently, mainly based on the analyses of several transgenic mice expressing a fluorescent marker specifically and selectively in some neurons, additional GABAergic striatal interneurons were reported such as tyrosine hydroxylaseexpressing interneurons (Tepper et al., 2010; Silberberg and Bolam, 2015) and serotonin receptor 3a (5HT3a)-expressing interneurons (Silberberg and Bolam, 2015). Muñoz-Manchado et al. (2016) analyzed the striatum of the transgenic mouse 5HT3aEGFP and reported 5HT3aEGFP -positive (+) cells are novel major groups of GABAergic interneurons; according to Table 1 in Muñoz-Manchado et al. (2016), 5HT3aEGFP -positive (+) cells were about one-third of all striatal GABAergic interneurons, although about one-fourth of striatal 5HT3aEGFP -positive (+) cells were

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overlapped with previously characterized interneurons. At any rate some new chemical markers could reveal novel groups of neurons in various brain regions. Secretagogin (SCGN) is a newly discovered calcium binding protein (Wagner et al., 2000) which characterizes some particular neuron groups in various regions of the nervous system (Mulder et al., 2009, 2010; Puthussery et al. 2009; Alpár et al., 2012; Maj et al., 2012; Shi et al., 2012; Gyengesi et al., 2013; Kosaka and Kosaka, 2013; Weltzien et al., 2014). Mulder et al. (2009) analyzed mouse and primate brains and reported the interspecies differences between them in the SCGN distribution. The striatum is one of the regions showing such interspecies differences. In the primate putamen approximately 50% of cholinergic interneurons were SCGN+, whereas in the mouse striatum SCGN+ neurons were rare and GABAergic with elaborate, spiny dendritic tufts (Mulder et al., 2009). Among rodents mice and rats are major targets in neuroscience researches. These two rodent species are generally similar in their distribution of various chemical markers, but in some cases show vast differences. Recently, Garas et al. (2016) reported the species differences in the striatal neurons expressing SCGN between mice and rats. They reported the colocalization of SCGN and PV in the striatum of rats and monkeys and the structural and functional details of PV+/SCGN+ and PV+/SCGN- neurons. Furthermore they showed that SCGN+ neurons in the rat striatum did not express the chemical marker of the striatal projection 4

neurons, Ctip2, and thus indicated that striatal SCGN+ neurons were interneurons. However, in their study Garas et al. (2016) did not analyzed the colocalization of SCGN with other striatal interneuron markers such as CR, NOS and choline acetyl transferase (ChAT). In the present study we examined the colocalization relationship of SCGN with those markers in the rat striatum and noticed that SCGN+ neurons in the rat striatum showed some peculiar colocalization relationship with those markers. Furthermore the present results suggest that some SCGN+ neurons in the rat striatum are presumed novel groups of interneurons. The preliminary results of this study have been reported (Yasuda, 2016). All experiments were carried out in accordance with “the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions” of the Ministry of Education, Culture, Sports, Science and Technology of Japan, the “Guide for the Care and Use of Laboratory Animals 8th edition (2011)” and the institutional guidance for animal welfare (the Guidelines for Animal Experiment in International University of Health and Welfare). Every experimental procedure was approved by the Committee of the Ethics on Animal Experiment in International University of Health and Welfare. All efforts were made to minimize the number of animals used and their suffering. In this study we used 8 male Wistar rats, 5-8weeks old, 110-180 g (Japan SLC, 5

Inc., Hamamatsu, Japan), and 5 male C57BL/6J mice, 8weeks old 22-25g (Japan SLC, Inc.). Animals were deeply anesthetized with 2.5-3.5 % isoflurane or with sodium pentobarbital (100 mg/kg body weight) and perfused transcardially with phosphatebuffered saline (PBS, pH7.4) followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH7.2-7.4). The brains were left in situ for 1-2 hours at room temperature and then removed from the skull. From each brain, coronal, parasagittal, or horizontal 50μm thick sections were cut serially on a vibratome (Leica VT1000S or Lancer 1000). The sections were incubated overnight with 1% bovine serum albumin in PBS containing 0.3% Triton X-100 and 0.05% sodium azide at 20 ºC. Then, they were incubated for 10 days at 20 °C in mixtures of primary antibodies raised in different species. The primary antibodies used are shown in Table 1. In the present study we used two anti-SCGN antibodies (Table1), one rabbit and the other sheep polyclonal antibodies. The rabbit anti-SCGN antibody is raised against recombinant human SCGN (Wagner et al. 2000), which also recognizes rat SCGN (Wagner et al. 2000) and mouse SCGN (Puthussery et al. 2009). The sheep anti-SCGN antibody is also raised against recombinant human SCGN, that is, recombinant protein containing a 276 amino acid sequence of human SCGN and 10 extra amino acids, N-terminal His-tag, which was reported to show identical staining patterns to the rabbit anti-SCGN antibody described above in the macaque retina (Weltzien et al., 2014). 6

We tried

double-, triple- and quadruple-immunostaining of various combinations of primary antibodies as shown in Table 2. The sections were rinsed 3 times in PBS, and incubated overnight in a mixture of fluorochrome-conjugated donkey secondary antibodies (Jackson Immunoresearch) such as aminomethylcoumarin (AMCA)-conjugated antigoat IgG, which cross-reacts with sheep IgG (1:250), fluorescein isothiocyanate (FITC)conjugated anti-guinea pig IgG (1:250), indocarbocyanine (Cy3)-conjugated anti-rabbit IgG (1:1,000), and indodicarbocyanine (Cy5)-conjugated anti-mouse IgG (1:250). Other combinations of fluoroprobes were also applied and there appeared to be no appreciable differences among them. To examine whether SCGN+ neurons different from 4 groups of known striatal interneurons were GABAergic or not, sections were processed in Triton X-100 free solutions; sections were incubated in a mixture of 5 primary antibodies, that is, mouse anti-GAD67, rabbit anti-SCGN, goat anti-ChAT, goat antiCR, guinea pig anti-PV (No. 10 in Table 2), and then, after rinsing several times in PBS, incubated in a mixture of secondary antibodies composed with cy3-conjugated anti-mouse IgG, Alexa Fluor 488-conjugated anti-rabbit IgG, cy5-conjugated anti-goat IgG and cy5-conjugated anti-guinea pig IgG. After rinsing several times in PBS, the sections were mounted in the Vectashield (Vector). The fluorescent images of individual sections were examined and photographed with a fluorescence stereoscopic microscope (Leica MZ FL III) equipped with a color 7

CCD digital camera (Olympus DP70) and with a fluorescence microscope (Olympus BX53) equipped with a color CCD digital camera (Olympus DP73). The sections were also examined with a Neurolucida image analysis system (MBF Bioscience) composed of a fluorescence microscope (Nikon Eclipse 80i), a monochrome CCD digital camera (QIClick; QImaging), motorized digital imaging head (Nikon DIH-E) and motor-driven stage and focus drive (Ludl Electronic Products Ltd.). Image stacks of 3 or 4 channels were obtained using an objective lens x10 or x20. Montages of the whole striatum in coronal and parasagittal sections were made from the image stacks obtained with x10 objective lens. Fluorescent filter sets used were as follows; Semrock DAPI-1160B for AMCA, Semrock GFP-3035D for FITC and Alexa Fluor 488, Semrock TRIC-B for cy3 and Semrock cy5-4040C for cy5. Using the image analysis software ImageJ 1.50 the projection images of the SCGN channel were made from each frame, and montages were made from these projection images using the image-editing software Adobe Photoshop CS6 (Adobe Systems). The SCGN+ somata in the striatum were numbered on the montage images. The colocalization of 4 chemical markers (SCGN, PV, ChAT and CR) were examined with ImageJ 1.50. By comparing the montage images with the image stacks chemical properties of the numbered somata were determined and recorded in Excel files. We counted positive somata throughout the whole depth of sections without any systemic random sampling and disector, and thus, strictly 8

speaking, the present analyses are not quantitative and should be regarded as rough estimations. In the present study we focused on the SCGN+ cells in the caudate putamen, but did not touch the nucleus accumbens (although a part of the striatum) nor the globus pallidus, both of which were somewhat difficult to be differentiated clearly from the basal forebrain containing numerous SCGN+ cells. The approximate ventral borders of the caudate putamen in individual sections were determined according to the figures in the rat stereotaxic atlas (Paxinos and Watson, 2014) and the mouse stereotaxic atlas (Franklin and Paxinos, 2008). In mice a small number of SCGN+ neurons were scattered or clustered mainly at the peripheral regions of the striatum, that is, around the ventricle, rostral pole of the striatum and beneath the corpus callosum (Fig. 1). Most of these SCGN+ cells were small and occasionally spiny (Fig. 1 F) as reported previously (Mulder et al., 2009). In contrast a large number of SCGN+ cells were scattered in the rat striatum (Fig. 1). Although SCGN+ cells distributed throughout the striatum, they were apparently inhomogeneously distributed: they are more frequent in the dorsal, medial and ventral portions compared with the centrallateral portion (Figs. 1, 2). Furthermore in the rostro-caudal direction, SCGN+ cells appeared to distribute more densely in the caudal one-third as well as the rostral pole of the striatum (Fig. 1). In the rat striatum SCGN+ cells were apparently 9

heterogeneous in their structural features (Figs. 1, 2); some were small spiny neurons resembling to those in the mouse striatum (Fig. 1 G and inset), whereas others were multipolar neurons of various soma sizes and processes (Figs. 1 G, 2), which were not encountered in the mouse striatum. Taking those heterogeneous structural features of SCGN+ neurons into account, we examined the colocalization relationship of SCGN and other 4 known chemical markers of the striatal interneurons, that is, CR, NOS, PV and ChAT (Fig. 3). In addition we also confirmed that two different anti-SCGN antibodies labeled the same neurons (No. 11 in Table 2; Fig. 3 E1-3). In the rat striatum SCGN frequently colocalized with not only PV but also CR and ChAT, but not with NOS, so far examined (Figs. 3, 4 A1-4). SCGN+/CR+ neurons were usually most intensely SCGN+ and small neurons resembling to the SCGN+ neurons in the mouse striatum (Figs. 3, 4). They were located near the periphery of the caudate putamen, beneath the internal capsule, in the subventricluar region and at the rostral pole of the striatum, although they were sometimes encountered inner part of the striatum, mainly at the dorsal portion (Fig. 5). Importantly not all of small intensely SCGN+ neurons were CR+ and vice versa. As those SCGN+/CR+ neurons resembled SCGN+ neurons in the mouse striatum, we examined whether SCGN+ neurons in the mouse striatum also expressed CR or not. Similarly to the rat striatum SCGN and CR overlapped in those small neurons in the mouse striatum (not shown). SCGN+/ChAT+ neurons were usually 10

somewhat faintly SCGN+ and largest neurons with rather thick smooth processes (Figs. 3, 4). They were more frequently encountered in the ventral half of the striatum (Fig. 5). SCGN+/PV+ neurons were most numerous and distributed throughout the striatum (Figs. 3-5). To determine whether SCGN+ neurons with none of 3 markers, PV, CR and ChAT, exist or not, four fluorescently quadruple-stained sections were examined with a light microscope (No. 9 in Table 2; Fig. 4 A1-4, Fig. 5 A, B). These observations on the image stacks clearly showed that 60-70 % of SCGN positive striatal neurons contained either PV or CR or ChAT. On the other hand the remaining 30-40 % of SCGN positive neurons (33 %, 37 %, 39 %, 40 %, respectively) did express none of these markers of striatal interneurons, indicating that there are some novel populations of striatal neurons in rats. Then are those SCGN+ neurons without any known striatal interneuron markers GABAergic? Our observations indicated that is the case; most of SCGN+ neurons with none of known striatal interneuron markers were GAD67+ (No. 10 in Table 2; Fig. 4 B1-8). In the present study we confirmed the prominent species difference between mice and rats in the striatal SCGN+ neurons, and revealed their heterogeneity in the morphological and chemical properties in the rat striatum. They overlapped with known striatal interneurons to various extent, but some of the SCGN+ neurons were apparently novel groups different from 4 known striatal interneuron groups. Garas et 11

al. (2016) reported the details of the colocalization of SCGN and PV in the striatum of rats and monkeys and the structural and functional details of PV+/SCGN+ and PV+/SCGN- neurons in the rat striatum. Their stereological analysis revealed that about half of SCGN+ neurons in the rat dorsal striatum expressed PV. They also showed that the proportion of SCGN+ neurons expressing PV is not homogeneous throughout the rostro-caudal axis, and described as follows: "in the most caudal aspects where their density was around three times higher than that of PV+/SCGNinterneurons in any other plane." The coronal and parasagittal sections we showed in the present study did not contain the most caudal portions. Taking the striatal levels into consideration, our results on the colocalization of SCGN and PV correspond well to their results. However, as we showed in the present study, a matter of peculiar interest is that in the rat striatum SCGN+ neurons included some subpopulations of both GABAergic and cholinergic interneurons. That might be also the case in the striatum of primates when we combine the data of previous reports (Mulder et al., 2009; Garas et al., 2016). Furthermore our results revealed that SCGN characterizes not only subpopulations of known striatal interneuron groups but also presumed hitherto unrecognized interneuron groups in the rat striatum, whose structural and functional details remain to be revealed. As discussed by Garas et al. (2016), those SCGN+ presumed novel interneuron groups could correspond to those 5HT3aEGFP -positive 12

interneurons reported in the transgenic mice. However, previous in situ hybridization study (Morales and Bloom, 1997) and immunocytochemical study using specific antibody against 5HT3aR (Morales et al. 1998) reported that the rat striatum contains a few 5HT3aR expressing neurons, indicating that those SCGN+ neurons and 5HT3aR expressing neurons could be somewhat different, although direct comparisons between them are needed. Furthermore even in the mouse striatum previous in situ hybridization data (Tecott et al. 1993; Allen Brain Atlas Htr3a 70593142, 74724760) show that the adult mouse striatum contains a few 5HT3aR mRNA-expressing neurons; thus there are some discrepancies between those in situ hybridization data and the data obtained from 5HT3aEGFP -transgenic mice. In the cortex of the transgenic mice the 5HT3aEGFP -positive neurons were confirmed to be 5HT3aR mRNA positive (Lee et al., 2010), but not yet in the striatum. Even if neurons corresponding to the mouse 5HT3aEGFP -positive neurons exist in the adult rat striatum, they may not express 5HT3aR mRNA or 5HT3aR proteins. When this is the case, it might be difficult to detect such neurons in the animals other than transgenic mice.

Funding : This work was supported by JSPS KAKENHI Grant 26430039 and Research funds from IUHW.

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Conflict of Interest: The authors declare there are no known conflicts of interest associated with this publication

Author contributions: T. K designed research; T. K. and S. Y. performed research; T. K., S. Y. and K. K. analyzed data and wrote the paper.

Acknowledgements The authors thank Dr. P. C. Emson, Dr. C. W. Heizmann and Dr. L. Wagner for their gifts of primary antibodies.

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olfactory bulb. Neurosci Res. 77, 16-32. Lee, S. H., Hjerling-Leffler, J., Zagha, E., Fishell, G., Rudy, B., 2010. The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors. J. Neurosci. 30, 16796-16808. Maj, M., Milenkovic, I., Bauer, J., Berggård, T., Veit, M., Ilhan-Mutlu, A., Wagner, L., Tretter, V., 2012. Novel insights into the distribution and functional aspects of the calcium binding protein Secretagogin from studies on rat brain and primary neuronal cell culture. Front. Mol. Neurosci. 5, article 84. Morales, M., Bloom, F. E., 1997. The 5-HT3 receptor is present in different subpopulations of GABAergic neurons in the rat telencephalon.

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Yanagawa, Y., Aujard, F., Hökfelt, T., Hurd, Y. L., Harkany, T., 2010. Secretagogin is a Ca2+-binding protein identifying prospective extended amygdala neurons in the developing mammalian telencephalon. Eur. J. Neurosci. 31, 2166-2177. Muñoz-Manchado, A. B., Foldi, C., Szydlowski, L., Farries, M., Wilson, C., Silberberg, G., Hjerling-Leffler, J., 2016. Novel Striatal GABAergic interneuron populations labeled in the 5HT3aEGFP mouse. Cereb. Cortex 26, 96-105. Paxinos, G., Watson, C., 2014. The rat brain in stereotaxic coordinates, 7th edition. Elsevier, Academic Press, San Diego. Puthussery, T., Gayet-Primo, J., Taylor, R., 2010. Localization of the calcium-binding protein secretagogin in cone bipolar cells in the mammalian retina.

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Tepper, J. M., Bolam, J. P., 2004. Functional diversity and specificity of neostriatal interneurons. Curr. Opin.Neurobiol. 14, 685-692. Tepper, J. M., Tecuapetla, F., Koós, T., Ibanez-Sandoval, O., 2010. Heterogeneity and diversity of striatal GABAergic interneurons. Front. Neuroanat. 4, 150. Wagner, L., Oliyarny, O., Gartner, W., Nowotny, P., Groeger, M., Kaserer, K., Waldhäusl, W., Pasternack, M. S., 2000. Cloning and expression of secretagogin, a novel neuroendocrine- and pancreatic islet of Langerhans-specific Ca2+-binding protein. J. Biol. Chem. 275, 24740-24751. Weltzien, F., Dimarco, S., Protti, D. A., Daraio, T., Martin, P. R., Grunert, U., 2014. Characterization of secretagogin-immunoreactive amacrine cells in marmoset retina. J. Comp. Neurol. 522, 435-455. Yasuda, S., 2016. Secretagogin, a newly found Ca2+ binding protein, containing neurons in the striatum. IFBLS2016, PE-16. The 32nd World Congress of Biomedical Laboratory Science, Kobe.

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Figure captions Fig. 1.

SCGN+ neurons in the striatum (caudate-putamen) of mouse (A1, 2, E, F) and

rat (B1, 2, C, D, G). Stereomicroscopic images of immunofluorescent labeled coronal (A1, B1), horizontal (C) and parasagittal (D) sections. A2 and B2 are drawings based on A1 and B1, showing the distributions of SCGN+ somata in A1 and B1, respectively. In C and D the left is rostral. In the mouse only a small number of SCGN+ cells were scattered mainly at the peripheral portion of the striatum, which were occasionally clustered (E). In contrast, in the rat a large number of SCGN+ cells were scattered all over the striatum, although they were some inhomogeneity in their distribution. Inset in G is a higher magnification image of the neuron indicated with arrow. F and inset in G show small neurons with spiny processes. CA, hippocampus. CPu, caudate putamen. GP, globus pallidus. Rt, reticular thalamic nucleus. ac, anterior commissure. cc, corpus callosum. fr, fornix. ic, internal capsule. st stria terminalis. A1 and A2 correspond to Fig. 25 (Bregma 0.74mm) of Franklin and Paxinos' mouse brain atlas. B1 and B2 correspond to Fig. 26 (Bregma 0.84mm) of Paxinos and Watson's rat brain atlas. C and D correspond to Fig. 197 (Bregma -5.60mm) and Fig. 173 (Lateral 2.90mm) of Paxinos and Watson's rat brain atlas, respectively. Scale bars in A1, A2, B1, B2, C, D are 1 mm. Scale bars in E and G are 100 m. Scale bars in F and inset in G are 10 m.

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Fig. 2.

Montage of photomicrographs obtained with a light microscope using X10

objective lens. Part of the rat striatum in a coronal section shown in Fig. 1 B1, which is also shown in inset. The area outlined by rectangle in inset corresponds to this photomontage. Pseudocolor image. Heterogeneous SCGN+ neurons are scattered. This photomontage shows the SCGN channel of a quadruple stained section (No. 9 in Table 2). The area outlined by rectangle is also shown in Fig. 4 A1. Scale bar is 100 m. Scale bar in inset is1mm.

Fig. 3. Montages of photomicrograpes of fluorescently double-immunostained sections of the rat striatum. Images of individual channels are shown in the second and third columns. In A-C arrows indicate double-stained neurons. A1-3 (No. 1 in Table 2); SCGN (A1 green, A2) and PV (A1 red, A3). B1-3 (No. 4 in Table 2); SCGN (B1 green, B2) and ChAT (B1 red, B3). C1-3 (No. 2 in Table 2); SCGN (C1 green, C2) and CR (C1 red, C3). D1-3 (No. 3 in Table 2); SCGN (D1 green, D2) and NOS (D1 red, D3). No neurons are double-stained. E1-3 (No. 11 in Table 2); SCGN + neurons stained with rabbit antiSCGN (E1 green, E2) and sheep anti-SCGN (E1 red, E3). Rabbit and sheep antibodies stain the same neurons. Scale bars are 100 m.

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Fig. 4. A1-4: Montages of photomicrographs of a coronal section quadruple immunostained for SCGN (A1), PV (A2), ChAT (A3) and CR (A4). This montage corresponds to the area outlined by rectangle in Fig. 2. Among SCGN+ cells 1-22, cell1 is CR+, cells 2, 4-12, 14, 15, 19, 21 and 22 are PV+, cells 13, 16, 17 and 20 are ChAT+, cell 3 is PV negative, ChAT negative and CR negative. The intensities of either one or both immunostainings of cells 4, 8, 10, 14, 21 and 22 are faint. Scale bar in A4 is100 m, which is appreciable to A1-A4. B1-8: Montages of photomicrographs of sections multiple immunostained (No. 10 in Table 2) for GAD67 (red in B1and B5, B2, B6), SCGN (green in B1 and B5, B3, B7) and a mixture of ChAT, CR and PV (blue in B1and B5, B4, B8). SCGN+ cells 1, 3, 4, 10 and 11 are GAD 67+ and positive for some of the markers. SCGN+ cells 7, 8 and 9 are GAD67+ but negative for ChAT, CR and PV. SCGN+ cell 2 is GAD67 negative but positive for some of the markers (presumably ChAT). SCGN+ profile 5 is supposed to be a peripheral portion of a cell whose immunoreactivities for GAD67 and others are not determined. Cell 12 is postive for some of the markers (presumably ChAT) but SCGN negative and GAD67 negative. Cell 13 is SCGN negative but positive for some of the markers (presumably PV) and GAD67+. Scale bar in B1 is100 m, which is appreciable to B1-B4. Scale bar in B5 is100 m, which is appreciable to B5-B8.

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Fig. 5.

Distributions of SCGN only(×), SCGN+/PV+(●), SCGN+/ChAT+ (∆) and

SCGN+/CR+ (□) neuronal somata in the rat caudate putamen. A: Drawings of a quadruple immunostained coronal shown in Fig. 1 B1and B2. Coronal section at the rostral level corresponding to Fig. 26 (Bregma 0.84mm) of Paxinos and Watson's rat brain atlas. Among 381 SCGN+ neurons, 140 (37%) are PV+, 51 (13%) are ChAT+ and 43 (11%) are CR+, whereas 147 (39%) show none of these markers. B: Drawings of a quadruple immunostained section shown in Fig. 1 D. Parasagittal section corresponding to Fig. 173 (Lateral 2.90mm) of Paxinos and Watson's rat brain atlas. Among 449 SCGN+ neurons, 145 (32%) are PV+, 80 (18%) are ChAT+ and 46 (10%) are CR+, whereas178 (40%) show none of these markers. ac, anterior commissure. cc, corpus callosum. GP, globus pallidus. Scales bars are 1mm.

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Fig.1

23

Fig.2

24

Fig.3

25

Fig.4

26

Fig.5

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Table 1. Primary antibodies used in this study Host Antigen

Dilution

Source / references

animal calretinin CR

mouse

1: 5,000

Transduction Lab., clone 34

calretinin CR

rabbit

1: 5,000

Swant, 7699/4

calretinin CR

goat

1: 5,000

Swant, CG1

choline acetyl transferase ChAT

goat

1: 500

Chemicon, AB144P

mouse

1: 5,000

Chemicon, MAB5406, clone 1G10.2

sheep

1: 10,000

glutamic acid decarboxylase 67 GAD67 gift from Dr. P.C. Emson (Herbison et al., nitric oxide synthase NOS

1996, J. Neuroendocrinol. 8, 211–216.) parvalbumin PV

guinea pig

1: 5,000

Frontier Institute Co. Ltd, AB2571615 gift from Dr. C. W. Heizmann (Kägi et al.

parvalbumin PV

rabbit

1: 5,000 1987, J. Biol. Chem. 262, 7314-7320) gift from Dr. L. Wagner (Wagner et al., 2000, J. Biol. Chem. 275, 24740-24751;

secretagogin SCGN

rabbit

1: 20,000 Puthussery et al., 2010, J. Comp. Neurol. 518, 513-525.)

secretagogin SCGN

sheep

1: 5,000

28

BioVendor, RD184120100

Table 2. Combinations of primary antibodies and secondary antibodies. All secondary antibodies were fluorochrome-conjugated donkey affinity-purified antibodies against rabbit, goat, mouse or guinea-pig IgG and, according to the products specifications sheet, show minimally cross-reaction to serum proteins of several animals. host animals combinations of primary antibodies

rabbit

goat/sheep

mouse

guinea pig

No. 1

SCGN

-

-

PV

No. 2

SCGN

CR

-

-

No. 3

SCGN

NOS

-

-

No. 4

SCGN

ChAT

-

-

No. 5

SCGN

-

CR

-

No. 6

PV

SCGN

CR

-

No. 7

CR

SCGN

-

PV

No. 8

SCGN

NOS

CR

PV

No. 9

SCGN

ChAT

CR

PV

No. 10

SCGN

ChAT+CR

GAD67

PV

No. 11

SCGN

SCGN

29