Aspects of Excitatory/Inhibitory Synapses in Multiple Brain Regions Are Correlated with Levels of Brain-Derived Neurotrophic Factor/ Neurotrophin-3

Biochemical and Biophysical Research Communications 509 (2019) 429e434

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Biochemical and Biophysical Research Communications

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Aspects of excitatory/inhibitory synapses in multiple brain regions are correlated with levels of brain-derived neurotrophic factor/ neurotrophin-3

* Yo Shinoda a, b, c, , Tetsushi Sadakata c, d, Kaori Yagishita b, Emi Kinameri c, ** Ritsuko Katoh-Semba b, c, Yoshitake Sano b, Teiichi Furuichi b, c, a Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan b Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan c Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama, 351-0198, Japan d Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan article info abstract

Article history: Appropriate synapse formation during development is necessary for normal brain function, and synapse Received 7 December 2018 impairment is often associated with brain dysfunction. Brain-derived neurotrophic factor (BDNF) and Accepted 14 December 2018 neurotrophin-3 (NT-3) are key factors in regulating synaptic development. We previously reported that Available online 26 December 2018 BDNF/NT-3 secretion was enhanced by calcium-dependent activator protein for secretion 2 (CADPS2). Although BDNF/NT-3 and CADPS2 are co-expressed in various brain regions, the effect of Cadps2-defi- Keywords: ciency on brain region-specific BDNF/NT-3 levels and synaptic development remains elusive. Here, we CADPS2 show developmental changes of BDNF/NT-3 levels and we assess disruption of excitatory/inhibitory BDNF NT-3 synapses in multiple brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex Excitatory synapse and prefrontal cortex) of Cadps2 knockout (KO) mice compared with wild-type (WT) mice. Compared Inhibitory synapse with WT, BDNF levels in KO mice were reduced in young/adult hippocampus, but increased in young hypothalamus, while NT-3 levels were reduced in adult cerebellum and young hippocampus, but increased in adult parietal cortex. Immunofluorescence of vGluT1, an excitatory synapse marker, and vGAT, an inhibitory synapse marker, in adult KO showed that vGluT1 was higher in the cerebellum and parietal cortex but lower in the hippocampus, whereas vGAT was lower in the hippocampus and parietal cortex compared with WT. Immunolabeling for both vGluT1 and vGAT was increased in the parietal cortex but vGAT was decreased in the cerebellum in adult KO compared with WT. These data suggest that CADPS2-mediated secretion of BDNF/NT-3 may be involved in development and maturation of synapses and in the balance between inhibitory and excitatory synapses. © 2018 Elsevier Inc. All rights reserved.

1. Introduction

Synaptic development is the making of synapses between neurons and is critical for the formation of neural circuits. Improper 2þ Abbreviations: BDNF, brain-derived neurotrophic factor; CADPS2, Ca -depen- synaptic development is thought to cause synapse pathology, dent activator protein for secretion 2; Cb, cerebellum; Hip, hippocampus; h-Th, hypothalamus; KO, knock-out; NT-3, neurotrophin-3; PCx, parietal cortex; PFCx, which is linked to a risk of neurodevelopmental disorders such as prefrontal cortex; Str, striatum; vGAT, vesicular gamma-aminobutyric acid trans- autism [1,2] and schizophrenia [3,4]. However, the mechanisms of porter; vGluT1, vesicular glutamate transporter 1; WT, wild-type. synaptic development in different brain regions are not fully * Corresponding author. Department of Environmental Health, School of Phar- understood. macy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan. Neurotrophins constitute a family of secreted growth factors ** Corresponding author. Department of Applied Biological Science, Faculty of that regulate neuronal proliferation, development, survival and Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan. death, neuritogenesis and pruning, synaptic strength and plasticity E-mail addresses: [email protected] (Y. Shinoda), [email protected] [5,6]. Brain-derived neurotrophic factor (BDNF) is a neurotrophin (T. Furuichi). https://doi.org/10.1016/j.bbrc.2018.12.100 0006-291X/© 2018 Elsevier Inc. All rights reserved. 430 Y. Shinoda et al. / Biochemical and Biophysical Research Communications 509 (2019) 429e434 that is predominantly expressed in the brain and is involved in the were perfused transcardially with 4% paraformaldehyde (PFA) in proliferation, differentiation, maturation and survival of various 0.1 M phosphate buffer. Dissected brains were incubated in the types of neuron [6e10]. Neurotrophin-3 (NT-3) is also a neuro- same 4% PFA/0.1 M phosphate buffer overnight at 4 C. Fixative was trophin family member that is expressed in several organs then replaced with 30% sucrose in PBS and incubation continued including brain [11], and regulates proliferation, differentiation and overnight. Brains were embedded in O.C.T. compound (Sakura survival of neurons [12e14]. BDNF and NT-3 are highly associated Finetek Japan, Tokyo, Japan), and frozen at 80 C. Brains were with synaptic development and plasticity in several brain regions sagittally sectioned at a thickness of 14 mm with a cryostat [15e23]. Therefore, expression and secretion of BDNF and NT-3 are (CM1850; Leica Microsystems, Frankfurt, Germany) at 20 C. For thought to be key factors in regulating neural wiring and func- immunostaining, sections were washed three times with PBS. tioning during development. Washed sections were treated with blocking solution (1% Triton X- Calcium-dependent activator protein for secretion 2 (CADPS2) is 100, 2% donkey serum in 2 PBS) for 30 min at RT. Primary anti- associated with exocytosis of large dense-core vesicles [24e27] and bodies against vGluT1 (135311, Synaptic Systems, Gottingen,€ Ger- is known to enhance BDNF/NT-3 secretion in hippocampal and many) and vGAT (131002, Synaptic Systems) were applied in the cerebellar neurons [28e32]. BDNF/NT-3 and CADPS2 are co- same blocking solution for 1 h at RT. Sections were then washed localized in some but not all neuron types in mouse cerebral cor- three times with PBS and then incubated with Alexa 488- and 555- tex, hippocampus, cerebellum [30,33] and striatum [34e36]. Thus conjugated secondary antibodies (Thermo Fisher Scientific, Tokyo, we predicted that changes in the levels and secretion of BDNF/NT-3 Japan) for 1 h at RT. After washing, sections were mounted with in Cadps2 knock-out (KO) mice would influence synaptic develop- Fluoromount/Plus (CosmoBio, Tokyo, Japan). Immunofluorescence ment in various brain regions. Indeed, in Cadps2 mutant mice, was observed with a confocal Laser scanning microscope (LSM 510, synaptic connections and function are affected in the cerebellum Zeiss, Tokyo, Japan). Digital images were processed using Adobe [29,30,37] and hippocampus [28], and these alterations might be Photoshop software (Adobe, San Jose, CA, USA). Fluorescence in- associated with autistic-like behavior [32,33,38,39]. tensities and the numbers of vGluT1 and vGAT positive puncta were In the present study, we determined BDNF/NT-3 levels and digitally measured using ImageJ software [41]. At least three slices analyzed excitatory/inhibitory synapses in six different brain re- from each of three individuals were analyzed for quantification of gions (cerebellum, hypothalamus, striatum, hippocampus, parietal synapses. cortex and prefrontal cortex) in wild-type (WT) and Cadps2 KO mice. 2.4. Statistics

2. Materials and methods The levels of signiﬁcance for differences between data sets were assessed using Student's t-test. Results are expressed as the 2.1. Animals mean ± SEM.

All experimental protocols were evaluated and approved by the 3. Results Regulation for Animal Research at RIKEN and Tokyo University of Science, which follows the National Institutes of Health guide for 3.1. Levels of BDNF and NT-3 are altered in multiple brain regions of the care and use of Laboratory animals (NIH Publications No. 8023, Cadps2 KO mice revised 1978). The generation of Cadps2 KO mice was described previously [33]. Male mice were housed in home cages, with at Neurotrophins are expressed at different levels in multiple brain most four per cage, and maintained under a 12:12-h lightedark regions and during different developmental stages. Therefore, we cycle, with ad libitum access to water and food. All efforts were investigated changes in age- and region-dependent levels of brain made to minimize the number of animals used and their suffering. BDNF and NT-3 proteins. We dissected six different brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex 2.2. Two-site enzyme immunoassay and prefrontal cortex) from WT and Cadps2 KO mice at postnatal weeks 1, 3 and 8, and measured the content of BDNF and NT-3 A two-site enzyme immunoassay was performed as described protein in each brain region by two-site enzyme immunoassay. In previously [40]. To determine BDNF and NT-3 contents, WT and Cadps2 KO mice, BDNF levels were significantly decreased in the Cadps2 KO male mice at postnatal weeks 1, 3 and 8 were deeply hippocampus especially in the older developmental stages (Fig. 1). anesthetized and six brain regions (cerebellum, hypothalamus, Other brain regions showed no changes (except for a small increase striatum, hippocampus, parietal cortex and prefrontal cortex) were in hypothalamus at 1 week) between WT and KO. NT-3 levels were quickly dissected, weighed, frozen and stored at 80 C until use. significantly decreased in the cerebellum and hippocampus at 8 Dissected brain regions were sonicated with guanidine-HCl in and 3 weeks, respectively. In contrast, a small but significant in- phosphate buffer and the supernatants after centrifugation were crease of NT-3 was observed in the parietal cortex at 8 weeks incubated with polystyrene beads (Immunochemical, Okayama, (Fig. 2). Therefore, the levels of BDNF/NT-3 were different at each Japan) bearing immobilized BDNF/NT-3 specific antibodies. Beads stage and in each brain region, and were influenced by CADPS2 were washed and treated with beta-galactosidase-labeled neuro- deficiency. trophin antibody Fab’ fragments. Galactosidase activity was detected using 4-methylumbellifery-beta-D-galactosidase (Sigma- 3.2. The development of excitatory and inhibitory synapses in some Aldrich, Tokyo, Japan) as the substrate. Fluorescence of the brain regions is affected by CADPS2 deficiency and shows moderate metabolite, 4-methylumbelliferone, was measured using a FP-6300 correlation with BDNF/NT-3 levels spectrofluorometer (JASCO International, Tokyo, Japan). Next we analyzed excitatory and inhibitory synapses in selected 2.3. Immunohistochemistry brain regions in adult (P56) WT and Cadps2 KO mice using anti- vGluT1 and vGAT antibodies (Fig. 3). Immunofluorescence was Immunohistochemistry was performed as described previously processed and measured digitally. Integrated fluorescence in each [28], with small modifications. On postnatal day 56, male animals punctum was analyzed to estimate the development of excitatory Y. Shinoda et al. / Biochemical and Biophysical Research Communications 509 (2019) 429e434 431

Fig. 1. BDNF levels in selected brain regions from WT and Cadps2 KO mice at postnatal weeks 1, 3 and 8. (A) Cerebellum [Cb], (B) hypothalamus [h-Th], (C) striatum [Str], (D) hippocampus [Hip], (E) parietal cortex [PCx] and (F) prefrontal cortex [PFCx]. *p < 0.05, **p < 0.01; n ¼ 7. Data were analyzed with Student's t-test. Data are presented as the mean ± SEM.

Fig. 2. NT-3 levels in selected brain regions from WT and Cadps2 KO mice at postnatal weeks 1, 3 and 8. (A) Cb, (B) h-Th, (C) Str, (D) Hip, (E) PCx and (F) PFCx. *p < 0.05, **p < 0.01; n ¼ 7. Data were analyzed with Student's t-test. Data are presented as the mean ± SEM. and inhibitory synapses in each brain region (Fig. 4AeF). The in- Cadps2 KO mice were partly correlated with altered levels of BDNF/ tegrated fluorescence signal was calculated by multiplying the area NT-3 in the cerebellum, hippocampus and parietal cortex. There- of fluorescence by the intensity of fluorescence of each fluorescent fore, these data indicate that synaptic development is regulated, in punctum, which represents the total number of excitatory and part, by BDNF/NT-3 levels. inhibitory synaptic vesicles in each presynaptic terminal. The in- fl tegrated uorescence of vGluT1-positive excitatory synapses was 4. Discussion significantly increased in the cerebellum and parietal cortex but decreased in the hippocampus. In vGAT-positive inhibitory synap- In the present study, Cadps2 KO mice showed (1) a decrease of ses, the integrated fluorescence was significantly decreased in the BDNF levels in 3e8 week-old hippocampus, but an increase in 1 hippocampus and parietal cortex. week-old hypothalamus, (2) a decrease of NT-3 levels in 8 week-old The numbers of excitatory and inhibitory synapses were cerebellum and 3 week-old hippocampus, but an increase in 8 compared between WT and Cadps2 KO mice (Fig. 4G-L). The week-old parietal cortex, (3) excitatory synaptic vesicles in a pre- fi number of excitatory synapses was signi cantly increased in the synapse were increased in cerebellum and parietal cortex synapses, parietal cortex, while the numbers of inhibitory synapses were but decreased in hippocampus synapses, (4) inhibitory synaptic fi signi cantly decreased in the cerebellum but increased in the pa- vesicles were decreased in hippocampus and parietal cortex syn- rietal cortex. apses, (5) the number of excitatory synapses was increased in the Together, these data show that the synaptic alterations in parietal cortex, and (6) the numbers of inhibitory synapses were 432 Y. Shinoda et al. / Biochemical and Biophysical Research Communications 509 (2019) 429e434

Fig. 3. Representative pictures of excitatory (vGluT) and inhibitory (vGAT) synapses in selected brain regions. (A) Cb, (B) h-Th, (C) Str, (D) Hip, (E) PCx and (F) PFCx.

Fig. 4. Integrated immunofluorescence (AeF) and the numbers of excitatory and inhibitory synapses (GeL). (A and G) Cb, (B and H) h-Th, (C and I) Str, (D and J) Hip, (E and K) PCx and (F and L) PFCx. *p < 0.05, ***p < 0.001; n ¼ 9. Data were analyzed with Student's t-test. Data are presented as the mean ± SEM. increased in the parietal cortex, but decreased in the cerebellum. expression in the visual cortex [42,43], whisker sensory stimulation BDNF levels in each brain region are regulated by a multitude of increases BDNF expression in the somatosensory barrel cortex neuronal events. For example, light stimulation induces BDNF [44,45], osmotic stimulation enhances BDNF expression in the Y. Shinoda et al. / Biochemical and Biophysical Research Communications 509 (2019) 429e434 433 paraventricular nucleus [46], exercise induces BDNF expression in formation. the hippocampus [10,47], and high frequency neural activity, such as seizure/kindling, increases BDNF expression in multiple brain Conflicts of interest regions, including the hippocampus, cortex and amygdala [48,49]. Therefore, the expression level of BDNF is principally regulated by The authors declare no conflicts of interest. neural activity. We previously reported that Cadps2 KO mice show a significant reduction in the frequency and amplitude of mEPSCs in Acknowledgement the hippocampus [28] and paired-pulse facilitation in the cerebellum [37]. In this context, it is possible that reduced synaptic This work was supported by a Grant-in-Aid for Scientific activity in Cadps2 KO mice results in lower BDNF expression levels Research on Innovative Areas (Comprehensive Brain Science in several brain regions. Moreover, BDNF secretion and activation of Network), MEXT KAKENHI (21790219) and JSPS KAKENHI a signaling cascade enhances BDNF mRNA levels in neocortical (17H03563), the Takeda Science Foundation, and the Kawano neurons [50]. CADPS2 is known as a positive regulator of activity- Masanori Memorial Public Interest Incorporated Foundation for dependent BDNF secretion [28e32]; therefore, it is also possible Promotion of Pediatrics. We thank Jeremy Allen, PhD, from Edanz that the reduced BDNF expression levels in some brain regions of Group (www.edanzediting.com/ac) for editing a draft of this Cadps2 KO mice was caused by impairment of activity-dependent manuscript. BDNF secretion during development. Secreted BDNF is a regulator of several biological events, such as References changes in axon and dendrite morphology, including synapse formation [51,52]. Activation of TrkB, a BDNF receptor, is associated [1] D. Ebrahimi-Fakhari, M. 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