Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles Beatriz Cubelos a , Cristiana Leite a,b , Cecilio Giménez a,b , Francisco Zafra a,b,⇑ a Centro de Biología Molecular ‘‘Severo Ochoa’’ and Departamento de Biología Molecular, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain b Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain article info Article history: Available online xxxx Keywords: Glycine Transporter GLYT1 Synaptic vesicles Glutamate abstract We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the pres- ence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate trans- porter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Glycine is a neurotransmitter that serves an inhibitory function in the spinal cord and brain stem, but also is an obligatory co-ago- nist at excitatory glutamate receptors of the NMDA type (Kemp and Leeson, 1993). While it was initially believed that the concen- tration of glycine in the synaptic cleft would be high enough to saturate the glycine site on the NMDA receptor, later pharmacolog- ical and electrophysiological studies indicate that this might be not the case due to the action of the glycine transporter GLYT1 (Zafra and Gimenez, 2008). Similarly to glycine, GLYT1 also seems to accomplish a dual role in neurotransmission. First, it is highly ex- pressed in glycinergic areas of the nervous system where it localize predominantly in glial cells (Zafra et al., 1995a), and mice lacking GLYT1 showed an impaired glycinergic neurotransmission that was attributed to an increase in the extracellular glycine close to strychnine sensitive glycine receptors (Gomeza et al., 2003). Second, GLYT1 also has been identified in neuronal elements throughout all the brain closely associated to glutamatergic pathways. GLYT1 is enriched in presynaptic boutons, where it lar- gely colocalize with the vesicular glutamate transporter vGLUT1, and it is also present in the postsynaptic densities of asymmetric synapses (Cubelos et al., 2005). Immunoprecipitation assays showed the existence of immunoprecipitable complexes contain- ing both NMDA and GLYT1 (Cubelos et al., 2005). These anatomical and biochemical evidence support a role of GLYT1 in controlling the glycine concentration in the microenvironment of the NMDA receptor and are consistent with functional studies showing that N[3-(4’-fluorophenyl)-3-(4’-phenylphenoxy)propyl]sarcosine (NFPS), a specific inhibitor of GLYT1, potentiates the NMDA-medi- ated responses both in vitro and in vivo (Bergeron et al., 1998; Chen et al., 2003; Kinney et al., 2003). The possible role of GLYT1 in glutamatergic neurotransmission was also confirmed in hetero- zygous Glyt1 +/À animals that expressed only 50% of GLYT1 and had an enhanced hippocampal NMDA receptor function and better memory retention (Tsai et al., 2004). While this evidence indicate that the glycine concentration in the microenvironment of NMDA receptors is regulated by reuptake through GLYT1, it is unclear how the levels of glycine in forebrain glutamatergic synapses oscillate in response to neuronal activity. Glycine release from hippocampal synaptosomes (Galli et al., 1993; Luccini et al., 2008) and cultured neurons (Fatima-Shad and Barry, 1998) has been reported. Additionally, a recent study showed the existence of a vesicular release of glycine in glutamatergic terminals of the hippocampus in response to neuronal depolarization, and this might modulate NMDA receptor function (Muller et al., 2013). However, the source of this glycine must be other than classical glycinergic terminals since boutons containing both the plasma 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.09.002 ⇑ Corresponding author. Address: Centro de Biología Molecular ‘‘Severo Ochoa’’, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Tel.: +34 914964630. E-mail address: fzafra@cbm.uam.es (F. Zafra). Neurochemistry International xxx (2013) xxx–xxx Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci Please cite this article in press as: Cubelos, B., et al. Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles. Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.002