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

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Highlights

  • CADPS2 KO perturbs BDNF levels in hypothalamus and hippocampus.

  • CADPS2 KO changes NT3 levels in cerebellum, hippocampus and parietal cortex.

  • CADPS2 KO alters synapse growth in cerebellum, hippocampus and parietal cortex.

Abstract

Appropriate synapse formation during development is necessary for normal brain function, and synapse impairment is often associated with brain dysfunction. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are key factors in regulating synaptic development. We previously reported that 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-deficiency on brain region-specific BDNF/NT-3 levels and synaptic development remains elusive. Here, we show developmental changes of BDNF/NT-3 levels and we assess disruption of excitatory/inhibitory synapses in multiple brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex and prefrontal cortex) of Cadps2 knockout (KO) mice compared with wild-type (WT) mice. Compared 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.

Introduction

Synaptic development is the making of synapses between neurons and is critical for the formation of neural circuits. Improper synaptic development is thought to cause synapse pathology, which is linked to a risk of neurodevelopmental disorders such as autism [1,2] and schizophrenia [3,4]. However, the mechanisms of synaptic development in different brain regions are not fully understood.

Neurotrophins constitute a family of secreted growth factors that regulate neuronal proliferation, development, survival and death, neuritogenesis and pruning, synaptic strength and plasticity [5,6]. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is predominantly expressed in the brain and is involved in the proliferation, differentiation, maturation and survival of various types of neuron [[6], [7], [8], [9], [10]]. Neurotrophin-3 (NT-3) is also a neurotrophin family member that is expressed in several organs including brain [11], and regulates proliferation, differentiation and survival of neurons [[12], [13], [14]]. BDNF and NT-3 are highly associated with synaptic development and plasticity in several brain regions [[15], [16], [17], [18], [19], [20], [21], [22], [23]]. Therefore, expression and secretion of BDNF and NT-3 are thought to be key factors in regulating neural wiring and functioning during development.

Calcium-dependent activator protein for secretion 2 (CADPS2) is associated with exocytosis of large dense-core vesicles [[24], [25], [26], [27]] and is known to enhance BDNF/NT-3 secretion in hippocampal and cerebellar neurons [[28], [29], [30], [31], [32]]. BDNF/NT-3 and CADPS2 are co-localized in some but not all neuron types in mouse cerebral cortex, hippocampus, cerebellum [30,33] and striatum [[34], [35], [36]]. Thus we predicted that changes in the levels and secretion of BDNF/NT-3 in Cadps2 knock-out (KO) mice would influence synaptic development in various brain regions. Indeed, in Cadps2 mutant mice, synaptic connections and function are affected in the cerebellum [29,30,37] and hippocampus [28], and these alterations might be associated with autistic-like behavior [32,33,38,39].

In the present study, we determined BDNF/NT-3 levels and analyzed excitatory/inhibitory synapses in six different brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex and prefrontal cortex) in wild-type (WT) and Cadps2 KO mice.

Section snippets

Animals

All experimental protocols were evaluated and approved by the Regulation for Animal Research at RIKEN and Tokyo University of Science, which follows the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The generation of Cadps2 KO mice was described previously [33]. Male mice were housed in home cages, with at most four per cage, and maintained under a 12:12-h light–dark cycle, with ad libitum access to water and food. All

Levels of BDNF and NT-3 are altered in multiple brain regions of Cadps2 KO mice

Neurotrophins are expressed at different levels in multiple brain regions and during different developmental stages. Therefore, we investigated changes in age- and region-dependent levels of brain BDNF and NT-3 proteins. We dissected six different brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex 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 protein in each brain region by two-site enzyme

Discussion

In the present study, Cadps2 KO mice showed (1) a decrease of BDNF levels in 3–8 week-old hippocampus, but an increase in 1 week-old hypothalamus, (2) a decrease of NT-3 levels in 8 week-old cerebellum and 3 week-old hippocampus, but an increase in 8 week-old parietal cortex, (3) excitatory synaptic vesicles in a presynapse were increased in cerebellum and parietal cortex synapses, but decreased in hippocampus synapses, (4) inhibitory synaptic vesicles were decreased in hippocampus and parietal

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgement

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network), MEXT KAKENHI (21790219) and JSPS KAKENHI (17H03563), the Takeda Science Foundation, and the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics. We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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