Advances in understanding the functions of native GlyT1 and GlyT2 neuronal glycine transporters

Accepted Manuscript Advances in understanding the functions of native GlyT1 and GlyT2 neuronal glycine transporters Cristina Romei, Luca Raiteri PII:

S0197-0186(16)30090-0

DOI:

10.1016/j.neuint.2016.07.001

Reference:

NCI 3892

To appear in:

Neurochemistry International

Received Date: 6 May 2016 Revised Date:

5 July 2016

Accepted Date: 5 July 2016

Please cite this article as: Romei, C., Raiteri, L., Advances in understanding the functions of native GlyT1 and GlyT2 neuronal glycine transporters, Neurochemistry International (2016), doi: 10.1016/ j.neuint.2016.07.001. 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.

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Advances in understanding the functions of native GlyT1 and GlyT2 neuronal glycine transporters Cristina Romeia and Luca Raiteria,b,* Department of Pharmacy, Pharmacology and Toxicology Section, University of Genoa, Genoa,

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a

Italy

Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy

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b

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* Corresponding author: Department of Pharmacy, Pharmacology and Toxicology Section, University of Genoa, Viale Cembrano 4, 16148 Genoa, Italy. Tel.: +39 010 3532093; fax: +39 010

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3993360. E-mail address: [email protected]

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Short title: On the functions of native glycine transporters

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Abbreviations: BoNT, botulinum toxin; GlyT1, Glycine transporter 1; GlyT2, Glycine transporter 2; HEK293, human embryonic kidney; NFPS (ALX 5407), N-[(3R)-3-([1,1′-biphenyl]-4-yloxy)-3(4-fluorophenyl)propyl]-N-methylglycine

hydrochloride;

Org25543,

N-[[1-(dimethylamino)

cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide hydrochloride.

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ABSTRACT Glycine can be substrate for two transporters: GlyT1, largely expressed by astrocytes but also by

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some non-glycinergic neurons, and GlyT2, most frequently present in glycine-storing nerve endings. In morphological studies, GlyT2 expression had been found to be restricted to caudal regions, being almost undetectable in neocortex and hippocampus. Here, we compared the uptake activities of GlyT1 and GlyT2 in synaptosomes purified from mouse spinal cord, cerebellum,

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neocortex and hippocampus. Although, as expected, [3H]glycine uptake was significantly lower in

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telencephalic than in caudal regions, selective GlyT2-mediated uptake could be evaluated in all areas. Appropriately, [3H]glycine selectively taken up into hippocampal synaptosomes through GlyT2 could be subsequently released by exocytosis. Native GlyT2, which did not contribute to basal release from cerebellum or spinal cord nerve terminals, could mediate release of [3H]glycine by transporter reversal in synaptosomes exposed to veratridine. Moreover, GlyT2 transporters could

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perform Na+-dependent homoexchange in response to externally added glycine. In conclusion, transporters of the GlyT2 type exhibited significant uptake also in telencephalic regions, probably

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because of the elevated driving force related to their stoichiometry. Although glycine release through GlyT2 had been predicted to be a very difficult process, GlyT2 expressed on isolated

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glycinergic nerve terminals can perform both release by transporter reversal and homoexchange.

Keywords: Glycine transporters; GlyT1; GlyT2; Glycine uptake; Transporter-mediated release; Glycine homoexchange

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1. Introduction Glycine behaves as an inhibitory neurotransmitter when it activates strychnine-sensitive receptors, especially in spinal cord, brainstem and cerebellum (Betz, 1992; Legendre, 2001). In addition, glycine exerts important excitatory functions throughout the CNS as an obligatory

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coagonist of glutamate at NMDA receptors (Johnson and Ascher, 1987). Glycine can be substrate for two transporters termed GlyT1 and GlyT2. GlyT1 is largely expressed by astrocytes (Zafra et al., 1995a,b), but significant GlyT1 pools are also present on non-glycinergic nerve endings (Cubelos et

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al., 2005, 2014; Yee et al., 2006; Raiteri and Raiteri, 2010; Harsing and Matyus, 2013). GlyT2 transporters are mostly localized on glycinergic nerve endings where they are critical for glycine

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uptake into the presynaptic cytosol for refilling synaptic vesicles with glycine (Zafra et al., 1995a,b; Poyatos et al., 1997; Gomeza et al., 2003; Aragón and López-Corcuera, 2005; Eulenburg et al., 2005).

Glycine transporters are differentially expressed throughout the CNS. In morphological studies

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GlyT1 was found to be present at the highest concentrations is spinal cord and brain stem, but also, in a lesser degree, in the brain hemispheres. As to GlyT2, its expression was reported to be restricted to spinal cord, brainstem and cerebellum, while it was essentially undetectable in cortex and

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hippocampus (Luque et al., 1995; Zafra et al., 1995a; Zeilhofer et al., 2005). On the other hand,

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hippocampal interneurons were shown to be immunopositive, although weakly, for GlyT2 (Danglot et al., 2004; Song et al., 2006), a finding compatible with the existence, yet poorly investigated, of functional glycinergic synapses also in the hippocampus. Voltage-clamped Xenopus oocytes expressing either GlyT1 or GlyT2 were used by Roux and Supplisson (2000) to evaluate the stoichiometry of the two transporters. It was found that GlyT1 has a stoichiometry of 2Na+/Cl-/glycine, while the stoichiometry of GlyT2 is 3Na+/Cl/glycine. Most neurotransmitter transporters are bidirectional devices that, under some conditions, can work in reverse to perform carrier-mediated release (Attwell et al., 1993; Belhage et al., 1993;

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Levi and Raiteri, 1993; Vizi, 1998; Vizi and Sperlágh, 1999; Jensen et al., 2000; Héja et al., 2009). In their study, Roux and Supplisson (2000) found that GlyT1 and GlyT2 have different reverse transport kinetics. In particular, following a comparable glycine load, the transmitter was easily

constraint for reverse transport that strongly limits glycine release.

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exported from GlyT1 oocytes but not from GlyT2-bearing cells, indicating that GlyT2 has a kinetic

HEK293 cells expressing GlyT1 or GlyT2 were employed by Herdon et al. (2001) in release experiments. Transporter-mediated efflux of preloaded [3H]glycine from GlyT1 cells was increased

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by externally added glycine, indicating homoexchange, which was blocked by the GlyT1 selective inhibitor NFPS. In contrast, external glycine was unable to affect release from glycine-loaded

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GlyT2 cells. The absence of homoexchange at GlyT2 was explained by taking into account the peculiar kinetic characteristics of GlyT2 described by Roux and Supplisson (2000). Although glycine has been known to be involved in several important physiological and pathological conditions since decades, some aspects of glycinergic transmission, including glycine

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release and effects of glycine transporter activation in native nervous tissues under physiological and pathological conditions have started to be investigated only in recent years, also thanks to the availability of selective GlyT1 and GlyT2 inhibitors (Saransaari and Oja, 2001, 2009; Raiteri et al.,

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2002, 2008; Harsing et al., 2003; 2006; Luccini and Raiteri, 2007; Luccini et al., 2008; Oja and Saransaari, 2011, 2013; Lall et al., 2012; Harsing and Matyus, 2013; Milanese et al., 2014; Hanuska

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et al., 2016), These functional studies have produced substantial information on glycine uptake and on the transporter-mediated release of glycine occurring by reversal of GlyT1 transporters. Other functional aspects of glycine transport, particularly regarding GlyT2, merit to be better understood, considering that glycine transporters, both GlyT1 and GlyT2, have been related to many nervous system disorders (Eulenburg et al., 2006; Carta et al., 2012; Coyle, 2012; Harvey and Yee, 2013; Vandenberg et al., 2015). To improve the knowledge on the function of native glycine transporters, we here used purified nerve ending preparations from adult mouse CNS (i) to analyze the regional

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distribution of GlyT1- and GlyT2-mediated glycine uptake, with particular attention to neocortex and hippocampus; (ii) to evaluate the ability of native GlyT2 to perform glycine release by

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transporter reversal and glycine homoexchange.

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2. Materials and Methods

Animals

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2.1.

Adult Swiss mice (weighing 20–25 g; Charles River, Calco, Italy) were used. Animals were housed at constant temperature (22 ± 1°C) and relative humidity (50%) under a regular light dark

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schedule (lights 7.00 AM – 7.00 PM). Food and water were freely available. Experimental procedures and animal care complied with the European Communities Council Directive of 24

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November 1986 (86/609/EEC) and were approved by the Italian Ministry of Health in accordance with Decreto Ministeriale 116/1992. All experiments have been performed according to “ARRIVE” guidelines for reporting research. All efforts were made to minimize animal suffering and to use

Preparation of synaptosomes

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only the number of animals necessary to produce reliable results.

Animals were sacrificed by cervical dislocation and spinal cord, cerebellum, cortex and

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hippocampi were quickly removed. The tissues were homogenized in 10 volumes of 0.32 M sucrose, buffered at pH 7.4 with Tris-HCl, using a glass-teflon tissue grinder (clearance 0.25 mm). Purified synaptosomes were prepared using a discontinuous Percoll® gradient (Dunkley et al., 1988; Nakamura et al., 1993), with some modifications (Luccini and Raiteri, 2007). The homogenate was centrifuged (5 min, 1000×g at 4◦C) to remove nuclei and debris and the supernatant was gently stratified on a discontinuous Percoll® gradient (2, 6, 10 and 20%, v/v in Tris-buffered sucrose) and centrifuged at 33,500 × g for 5 min. The layer between 10% and 20% Percoll® (synaptosomal fraction) was collected, washed by centrifugation and resuspended in a

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physiological medium (standard medium) having the following composition (mM): NaCl, 140; KCl, 3; MgSO4, 1.2; CaCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 5; glucose, 10; HEPES, 10; pH adjusted to 7.4 with NaOH. All the above procedures were performed at 0–4°C. Protein was determined according

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to Bradford (1976) using bovine serum albumin as a standard.

Uptake experiments

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[3H]Glycine uptake was studied according to the following procedure. The synaptosomal pellet was resuspended in standard medium. Aliquots (500 µl) of the synaptosomal suspension (about 25

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µg protein) were incubated for 2 min at 37°C with [3H]glycine (0.3 µM) in the absence (control samples) or in the presence of drugs. At the end of the incubation period, samples were rapidly filtered on Whatman glass fibre filters (GF/B; VWR International, Milan, Italy). Each sample was washed three times with 5 ml aliquots of standard medium and filters were counted for radioactivity.

Release experiments

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2.4.

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Blank values were obtained by maintaining the samples in an ice water bath.

Synaptosomes were incubated at 37°C for 15 min with [3H]glycine (0.3 µM). When

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appropriate, incubation with the radioactive tracer was performed in the presence of NFPS (0.1 µM). In a set of experiments, synaptosomes were incubated at 37°C for 90 min in the presence or in the absence of 10 nM Botulinum Toxin C1 (BoNT/C1); the radioactive tracer was present during the last 15 min of incubation. At the end of incubation, identical aliquots of the synaptosomal suspension (each corresponding to about 40 µg protein for spinal cord and cerebellum and 25 µg for hippocampus) were distributed on microporous filters placed at the bottom of a set of parallel superfusion chambers maintained at 37°C and superfused with standard medium at a rate of 0.5

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ml/min (Raiteri and Raiteri, 2000). In a group of experiments (experiments of depolarization-evoked [3H]glycine release), after 36 min of superfusion with standard medium, to equilibrate the system, fractions were collected as follows: two 3-min fractions (t = 36-39 min and t = 45-48 min; basal release) before and after one

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6-min sample (t = 39-45 min; evoked release). A 90-s period of depolarization was applied at t = 39 min. Synaptosomes were depolarized with high KCl (substituting for an equimolar concentration of NaCl) or veratridine. Org25543 was added 9 min before depolarization. When appropriate, Ca2+

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was omitted from the superfusion medium at t = 20 min of superfusion. The Ca2+-free medium contained 8.8 mM MgCl2 substituting for an isoosmotic amount of NaCl. Fractions collected and

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superfused filters were counted for radioactivity by liquid scintillation counting. In another set of experiments (experiments of glycine homoexchange), after 36 min of superfusion with standard medium, to equilibrate the system, four 3-min fractions were collected. Synaptosomes were exposed to different concentrations of glycine at the end of the first fraction

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collected (t = 39 min). When the dependence of glycine homoexchange on external Na+ was investigated, NaCl was omitted from min 30 of superfusion and was substituted with an isoosmotic amount of N-methyl-D-glucamine. In order to establish the involvement of GlyT1 or GlyT2 in

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glycine homoexchange, NFPS or Org25543 were introduced 9 min before glycine. Fractions

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collected and superfused filters were counted for radioactivity by liquid scintillation counting.

Calculations

Neurotransmitter released in each fraction collected was expressed as a percentage of the radioactivity content of synaptosomes at the start of the respective collection period (fractional rate x 100). In the experiments of depolarization-evoked [3H]glycine release, neurotransmitter overflow was calculated by subtracting the transmitter content of the basal release from the transmitter

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content in the 6-min fraction collected during and after the depolarization pulse (t = 39-45 min; evoked release). Effects of drugs were evaluated by calculating the ratio of the depolarizationevoked neurotransmitter release in the presence of the drugs versus that calculated in control conditions.

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In the experiments of glycine homoexchange, drug effects were evaluated by calculating the ratio between the efflux in the third fraction collected (in which the maximum effect of the externally added glycine was generally reached) and that of the first fraction. This ratio was

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compared to the corresponding ratio obtained under control conditions.

Appropriate controls were always run in parallel. In particular, the drugs used were tested

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alone in order to exclude that they could affect release on their own.

Statistics

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Statistical comparison of data was performed using the appropriate tests as indicated in the legends

Chemicals

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2.7.

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to the Figures and Tables. Differences were regarded as statistically significant for P < 0.05.

[3H]Glycine (specific activity: 1.65 · 1015 Bq/mol) was purchased from Perkin Elmer (Boston, USA). Percoll® was from Sigma Chemical Co. (St Louis, MO, USA). NFPS (ALX5407) was from Tocris Bioscience (Bristol, UK). Org25543 was a kind gift from Thijs de Boer (Organon Laboratories Ltd., Newhouse, Scotland).

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3. Results

3.1.

Regional distribution of GlyT1 and GlyT2 uptake activities in adult mouse brain nerve

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endings

We first performed [3H]glycine uptake assays with nerve endings isolated and purified from adult mouse spinal cord, cerebellum, neocortex and hippocampus, incubated in the presence or in

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the absence of selective GlyT1 and GlyT2 inhibitors. The aim of these uptake experiments was not to obtain the IC50 values of the different uptake inhibitors, already determined by several authors,

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but to establish the percentages of the total [3H]glycine uptake that occurred through GlyT1 and GlyT2 in the nerve terminals purified from the different regions under study. The contributions of GlyT1 and GlyT2 to the total [3H]glycine uptake were defined as the fractions of total uptake maximally sensitive to the different selective inhibitors.

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The [3H]glycine uptake performed by purified spinal cord nerve ending (93 pmol/mg protein; Table 1) was maximally prevented by about 25% in the presence of the selective GlyT1 inhibitor NFPS (Herdon et al, 2001). The selective inhibitor of GlyT2, Org25543 (Caulfield et al.,

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2001) blocked [3H]glycine uptake by about 75%. Table 1 also shows that the total [3H]glycine

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uptake was completely abrogated by the combination of NFPS and Org25543 added at their respective maximally effective concentrations. As reported in Table 2, the uptake of [3H]glycine carried out by purified cerebellar nerve terminals amounted to 15.5 pmol/mg protein. The selective GlyT1 inhibitor NFPS maximally prevented uptake by about 40%, suggesting that the relative contribution of GlyT1 to the total uptake in cerebellum could be higher than in spinal cord. As indicated by the experiments with Org25543, about 60% of the total [3H]glycine uptake occurred through GlyT2. The uptake was completely prevented by the mixture of NFPS and Org25543. Synaptosomes purified from cerebral cortex accumulated [3H]glycine somewhat less than cerebellar

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nerve endings (9 pmol/mg protein; Table 3). NFPS was found to maximally prevent [3H]glycine uptake by about 45% (Table 3). Quantitatively similar inhibition was produced by the GlyT2 inhibitor Org25543. A combination of NFPS and Org25543 prevented almost completely the uptake of [3H]glycine. Synaptosomes purified from adult mouse hippocampus exhibited the lowest uptake

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activity (4 pmol/mg protein). As reported in Table 4, the relative contributions of GlyT1 and GlyT2 to the total [3H]glycine uptake seemed to differ in part from those in the cerebral cortex. The GlyT1 inhibitor NFPS maximally inhibited [3H]glycine uptake by about 30%. The contribution of GlyT2,

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evaluated in the presence of Org25543, amounted to about 70%, while the NFPS/Org25543

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combination blocked almost completely [3H]glycine uptake (Table 4).

Appropriate function of GlyT2 situated on nerve terminals purified from the hippocampus

The experiments of glycine uptake just described show that, although the ability of

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hippocampus nerve endings to take up the transmitter was quantitatively the lowest among the regions examined, transporters of the GlyT2 type contributed significantly to the total uptake in the hippocampus. Considering that GlyT2 transporters are crucial for the correct function of glycinergic

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nerve terminals, it was important to verify if hippocampal nerve endings, able to take up [3H]glycine through GlyT2, were also able to store and release glycine by vesicular exocytosis.

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Hippocampal synaptosomes were incubated with [3H]glycine, in the presence of NFPS to obtain preferential labelling through GlyT2, and depolarized with high-K+ under superfusion conditions. As illustrated in Fig. 1, the evoked release of [3H]glycine evoked by 25 mM KCl was almost completely abolished when Ca2+ ions were removed from the superfusion medium and was largely prevented in synaptosomes preincubated with the botulinum toxin BoNT/C1.

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Release of [3H]glycine mediated by GlyT2 reversal Synaptosomes purified from cerebellum or spinal cord were incubated with [3H]glycine in

the presence of NFPS to obtain preferential labelling through GlyT2 and then superfused with physiological solutions (experiments with Na+ gradient intact). In order to ascertain if GlyT2

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reversal contributed to the basal release, the selective GlyT2 inhibitor Org25543 was added to the superfusion solution. As illustrated in Fig. 2, Org25543 was unable to affect significantly the basal release of [3H]glycine from cerebellar synaptosomes. Identical results were obtained with spinal

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cord synaptosomes (not shown). Few experiments were carried out in which Na+ ions were removed during superfusion of cerebellar synaptosomes to cause Na+ gradient inversion. A modest,

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though not significant increase (about 10%) in [3H]glycine release could be observed (not shown). It is known that the alkaloid veratridine causes prolonged opening of Na+ channels with concomitant decreases in the Na+ gradient across the plasma membrane of nerve endings, a condition that should facilitate release of transmitters by transporter reversal. In order to investigate

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if GlyT2 reversal could mediate glycine release, synaptosomes purified from cerebellum were preincubated with [3H]glycine, in the presence of NFPS and then exposed in superfusion to 10 µM veratridine. The veratridine-evoked release of [3H]glycine, in the presence of external Ca2+, was

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strongly prevented by the GlyT2 selective inhibitor Org25543 (Fig. 3). Because carrier-mediated release of transmitters is known to be independent of external Ca2+, experiments were also

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performed in Ca2+-free solution. Removal of external Ca2+ inhibited by about 40% the veratridineevoked release and the Ca2+-independent release of [3H]glycine was almost abolished by Org25543 (Fig. 3). Release of glycine by GlyT2 reversal caused by veratridine was shown to occur also from spinal cord synaptosomes. As reported in Fig. 4, the release of [3H]glycine evoked by 10 µM veratridine, in the absence of external Ca2+, was largely prevented by the GlyT2 selective inhibitor Org25543.

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Homoexchange of glycine mediated by GlyT2

Synaptosomes purified from adult mouse cerebellum were preincubated with [3H]glycine (no GlyT1 inhibitor present) and then exposed in superfusion to glycine at 5, 10, 30 or 100 µM.

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Externally added glycine evoked release of [3H]glycine in a concentration-dependent manner (Fig. 5). The releasing effect of glycine was already significant when glycine was added to the superfusion solution at 5 µM. As transmitter homoexchange is expected to be dependent on the

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presence of Na+ in the external solution, experiments were carried out with no Na+ ions in the

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superfusion medium. Figure 6 shows that the glycine-evoked [3H]glycine releases from cerebellum and spinal cord synaptosomes were abolished when sodium ions were omitted from the external medium. Figure 7 illustrates that glycine, added at 100 µM to cerebellar synaptosomes, provoked a robust release of [3H]glycine (about 400% over basal). The releasing effect was almost abolished by the GlyT2 selective inhibitor Org25543. The [3H]glycine release evoked by 100 µM glycine from

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the same cerebellar nerve terminals superfused in parallel chambers remained unchanged when NFPS instead of Org25543 was added to the superfusion solution. Similarly to cerebellar

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synaptosomes, nerve terminals purified from spinal cord performed homoexchange sensitive to the

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GlyT2 inhibitor (Fig. 8).

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4. Discussion 4.1.

Regional distribution of glycine uptake by GlyT1 and GlyT2

Some of the characteristics of GlyT1- and GlyT2-mediated glycine uptake have been established

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in many laboratories, often using transfected cells. However, comparative studies of the uptake activities of native GlyT1 and GlyT2 in different regions of the adult CNS have not been reported. In the first part of the present work, the regional distribution of the GlyT1- and GlyT2-mediated

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uptake activities was investigated using purified nerve ending preparations of adult mouse spinal cord, cerebellum, cerebral cortex and hippocampus exposed to radiolabelled glycine in the presence

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or in the absence of selective GlyT1 and GlyT2 inhibitors.

The total uptake of glycine was highest in spinal cord and lowest in hippocampus, with intermediate values in cerebellum and neocortex. While these results are roughly in agreement with the regional distribution of glycine transporters determined by morphological techniques, the

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individual contribution to the total uptake of GlyT1 and GlyT2 merits to be discussed in some detail. Morphological studies had shown that expression of the neuronal GlyT2 could be hardly detected in neocortex and hippocampus (Zafra et al., 1995a; Luque et al., 1995; Zeilhofer et al.,

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2005). Based on the available information, one should have expected minimal, if any, GlyT2-

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mediated glycine uptake in hippocampus and cortex nerve endings. It was therefore somewhat surprising to observe that the cortical and hippocampal nerve ending preparations used in our study exhibited remarkable glycine uptake through GlyT2 transporters. Most important, depolarization of hippocampal nerve endings selectively prelabelled with [3H]glycine through GlyT2 provoked external Ca2+-dependent and botulinum toxin-sensitive exocytotic release, well compatible with the existence in the hippocampus of nerve terminals endowed with functional GlyT2 transporters. In our opinion, the apparent discrepancy between the above morphological results and the present uptake data could, at least in part, be explained as follows. As established by Roux and Supplisson (2000),

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GlyT1 has a stoichiometry of 2Na+/Cl-/glycine, whereas the stoichiometry of GlyT2 is 3Na+/Cl/glycine. According to the authors, a difference of one Na+ in ionic coupling implies that the driving force available for glycine uphill transport for GlyT2 is much larger than for GlyT1 (Supplisson and Roux,

2002).

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relatively few neuronal

GlyT2,

barely detectable

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immunocytochemical techniques, could perform relevant accumulation of glycine in experiments of functional uptake.

While GlyT2 transporters are generally believed to be present on glycine-storing nerve

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terminals, the localization of GlyT1 transporters deserves some consideration. At a first glance, the GlyT1-mediated uptake here observed could be attributed to astrocyte-derived particles (known as

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gliosomes; see Stigliani et al., 2006) produced during tissue homogenization and present as contaminants in the synaptosomal band of the Percoll gradient. Previous experiments of confocal microscopy (Luccini et al., 2008) had shown that about 10% of the particles present in purified preparations of hippocampal synaptosomes were positive for the astroglia protein GFAP. However,

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we found purified gliosome preparations to possess weak ability to take up and store glycine (unpublished results). Recent reports on GlyT1 transporters indicate that another possibility merits particular attention. There is increasing evidence that the ‘glial’ GlyT1 transporters can also exist on

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neurons, especially on glutamatergic nerve terminals (Cubelos et al., 2005, 2014; Raiteri and Raiteri, 2010; Musante et al., 2011). Mice selectively deprived of forebrain ‘neuronal’ GlyT1 have

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been developed. The GlyT1-mediated uptake of glycine in hippocampal synaptosomes prepared from these animals was about 30% lower than in wild-type mice, indicating a significant neuronal pool of GlyT1 (Yee et al., 2006). Interestingly, mice deprived of forebrain neuronal GlyT1 exhibited procognitive and antipsychotic phenotypic profiles (Yee et al., 2006) and, accordingly, inhibition of GlyT1-mediated glycine transport is a proposed approach to treatment of schizophrenia (see, for reviews, Javitt, 2012; Harvey and Yee, 2013). Thus, the contribution of GlyT1 to the uptake of glycine in the CNS regions examined in the present study may well include uptake into GlyT1-

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bearing glutamatergic nerve terminals present in the synaptosomal band of the Percoll gradient. Interestingly, this view is strongly supported by two recent reports. In one of these reports, using immunohistochemistry and electrophysiology techniques, Muller et al. (2013) show that, in mouse hippocampus, glycine accumulates in glutamatergic presynaptic terminals, is stored in presynaptic

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vesicles and is released during synaptic activity, possibly onto NMDA receptors. In the other study, Cubelos et al. (2014) present biochemical and immunohistochemical evidence indicating that, in rat hippocampus, GlyT1 is not only present in the plasma membrane of glutamatergic terminals

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(Cubelos et al., 2005), but also in intraterminal synaptic vesicles together with the vesicular

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glutamate transporter vGluT1.

Glycine release by GlyT2 transporter reversal

In their work with Xenopus oocytes, Roux and Supplisson (2000) did not observe current

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changes reflecting GlyT2-mediated glycine release when the Na+ gradient was intact. Glycine release by reversal of GlyT2 expressed in oocytes could only occur when the cytosol had received robust injections of glycine, Na+ and Cl-. The authors concluded that GlyT2 transporters are doubly

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unsuited to perform carrier-mediated release due to a kinetic constraint for reverse transport and to their particular stoichiometry. Differently from the release of glycine by GlyT1 reversal from

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nervous tissue preparations, which has been described by several authors (Tsen et al., 2000; Huang et al., 2004; Harsing et al., 2012; Harsing and Matyus, 2013), the release of the transmitter mediated by GlyT2 reversal in native nervous tissues has remained largely unexplored. Here we show that purified mouse cerebellar and spinal cord synaptosomes, when superfused in conditions of Na+ gradient intact, did not exhibit GlyT2 transporter-mediated glycine release (Fig. 2). In a few experiments, in which Na+ ions were removed during superfusion to cause inversion of the Na+ gradient, a modest increase in release was observed, which however was not significant, possibly

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because the Na+ concentration in the cytosol was insufficient to satisfy the requests of the intraterminal Na+ sites on GlyT2 (data not shown). Synaptosomes purified from both cerebellum and spinal cord did exhibit Org25543-sensitive GlyT2-mediated glycine release when the terminals were exposed to the alkaloid veratridine to cause persistent Na+ influx and decrease of the Na+

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gradient (Ulbricht, 2005), a condition known to facilitate transmitter release by reverse transport. To conclude, although carried out with different experimental approaches, the experiments with Xenopus oocytes (Roux and Supplisson, 2000) and with isolated brain nerve endings produced

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apparently similar results. The finding that, under conditions of ionic dysregulation, native GlyT2 can mediate release of glycine by transporter reversal may have pathophysiological implications.

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Veratridine has been reported to represent a model of ischemia (Tretter and Adam-Vizi, 2002; Fekete et al., 2009). Thus release of glycine mediated by GlyT2 onto NMDA receptors during ischemic insults should be taken into consideration.

GlyT2-mediated glycine homoexchange

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4.3.

To our knowledge, glycine homoexchange mediated by GlyT2 transporters has not been

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investigated. We here report that mouse cerebellar and spinal cord isolated nerve terminals preloaded with [3H]glycine released the tritiated neurotransmitter when exposed in superfusion to

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glycine. As illustrated in Fig. 5, the effect of glycine was concentration-dependent. Because the major aim of the present investigation was to get information about the functions of native GlyT2, we tested the ability of the selective GlyT2 inhibitor Org25543 to affect the glycine-evoked [3H]glycine release. The robust effects caused by 100 µM glycine in both cerebellar and spinal cord synaptosomes was completely prevented by the GlyT2 inhibitor, whereas the selective GlyT1 inhibitor NFPS had no effect. The findings clearly indicate that GlyT2 transporters are implicated in glycine homoexchange. However, the lack of inhibition by NFPS does not indicate that

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homoexchange mediated through GlyT1 can not occur. In fact, GlyT1 works near the equilibrium potential (Roux and Supplisson, 2000; Aubrey et al., 2005) and can perform release by transporter reversal and homoexchange in conditions and systems different from those here employed to investigate GlyT2-mediated glycine transport (Tsen et al., 2000; Huang et al., 2004; Harsing et al.,

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2012; Harsing and Matyus, 2013)

The release of intraterminal glycine provoked by the externally added glycine could be already observed when nerve terminals were exposed to concentrations of glycine in the low

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micromolar range (see Fig. 5). Moreover, in pathological conditions, such as CNS ischemia, epilepsy and glycine encephalopathy, extracellular glycine concentrations in the tens to hundreds of

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micromolars have been observed (Danysz and Parsons, 1998; Nong et al., 2003) which could participate in homoexchange processes. Of note, a recent work (Romei et al., 2015) shows that GABA homoexchange does not consist exclusively in release mediated by GABA GAT1 transporter reversal. A portion of the process occurred through previously unsuspected mechanisms including

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reverse plasmalemmal Na+/Ca2+ exchangers (Blaustein and Lederer, 1999; Annunziato et al., 2004), mitochondrial Na+/Ca2+ exchangers (Palty et al., 2012) and anion channels (Franco et al., 2000; Lee et al., 2010). The unexpected results obtained from the study on GABA homoexchange (Romei et

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al., 2015) suggest that the mechanisms involved in the GlyT2-mediated homoexchange of glycine

5.

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also should be analyzed in detail.

Conclusions

Glycine transporters of the GlyT2 type, reported to be practically undetectable in cortex and hippocampus in morphological studies, exhibited significant signals also in telencephalic regions when studied in functional uptake tests, probably because of the elevated driving force due to their

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19

stoichiometry. The glycine taken up through GlyT2 in hippocampus can be stored into vesicles from which the amino acid can subsequently be released by exocytosis. Native GlyT2 could release glycine by transporter reversal and perform homoexchange. The pathophysiological significance of GlyT2 transporters, including those present in telencephalic CNS regions, so far unexplored, awaits

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further consideration.

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Conflict of interest No conflict of interest

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Acknowledgements

The work was supported by Grants from the Italian MIUR. The authors wish to thank Mrs. Maura

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Agate for her expert editorial assistance.

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Legends to the Figures

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Fig. 1. Release of [3H]glycine evoked by 25 mM KCl from mouse hippocampal synaptosomes and effect of Ca2+ deprivation and of BoNT/C1. Synaptosomes were exposed to a 90-s pulse of 25 mM KCl at t = 39 min of superfusion; when appropriate, Ca2+ was omitted 19 min before

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depolarization. In the experiments with BoNT/C1 synaptosomes had been incubated for 90 min in the presence or in the absence of the toxin; labelling with [3H]glycine was performed during the

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last 15 min of incubation. Neurotransmitter overflow was calculated by subtracting the basal release from the neurotransmitter content in the 6-min fraction collected during and after the depolarization pulse. Data are means ± SEM of 3-5 experiments in triplicate (three superfusion chambers for each experimental condition). *P < 0.01 vs the respective control value (one-way

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ANOVA followed by post hoc Dunnett’s-test).

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Fig. 2. Spontaneous release of [3H]glycine, amounting to 1.66 ± 0.13 % (n = 13) of the total tritium content per minute, from mouse cerebellar synaptosomes, and effect of Org25543. Synaptosomes were loaded with [3H]glycine and superfused as described in Materials and Methods. Org25543 was added at t = 30 min of superfusion. Data are expressed as a percentage of

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control and are means ± SEM of 5-7 experiments in triplicate (three superfusion chambers for each experimental condition).

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Fig. 3. Release of [3H]glycine evoked by 10 µM veratridine from mouse cerebellar synaptosomes and effect of Ca2+ deprivation and of Org25543. Synaptosomes were depolarized with a 90-s pulse

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of veratridine at t = 39 min of superfusion; when appropriate, Ca2+ was omitted 19 min before depolarization. Org25543 was added 9 min before depolarization. Neurotransmitter overflow was calculated by subtracting the basal release from the neurotransmitter content in the 6-min fraction collected during and after the depolarization pulse. Data are means ± SEM of 4-6 experiments in

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triplicate (three superfusion chambers for each experimental condition). * P < 0.01 vs the respective control value in standard medium; ° P < 0.01 vs the respective control value in Ca2+-

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free medium (one-way ANOVA followed by post hoc Dunnett’s-test).

Fig. 4. Release of [3H]glycine evoked by 10 µM veratridine from mouse spinal cord

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synaptosomes in the absence of external Ca2+ and effect of Org25543. Synaptosomes were depolarized with a 90-s pulse of veratridine at t = 39 min of superfusion; Ca2+ was omitted 19 min before depolarization. Org25543 was added 9 min before depolarization. Neurotransmitter overflow was calculated by subtracting the basal release from the neurotransmitter content in the 6-min fraction collected during and after the depolarization pulse. Data are means ± SEM of 3-5 experiments in triplicate (three superfusion chambers for each experimental condition). * P < 0.01 vs the respective control value in Ca2+-free medium (one-way ANOVA followed by post hoc

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Dunnett’s-test).

Fig. 5. Concentration-dependent release of [3H]glycine evoked by exogenous glycine from mouse cerebellar synaptosomes. Synaptosomes were exposed to glycine at t = 39 min of superfusion.

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Results are expressed as per cent potentiation with respect to the basal release. Data are means ± SEM of 4-5 experiments in triplicate (three superfusion chambers for each experimental

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condition).

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Fig. 6. Release of [3H]glycine evoked by 100 µM glycine from mouse cerebellum (white bars) and spinal cord (black bars) synaptosomes and effects of Na+ deprivation. Synaptosomes were exposed to glycine at t = 39 min of superfusion. Na+ was omitted 9 min before. Results are expressed as per cent potentiation with respect to the basal release. Data are means ± SEM of 4 experiments in

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triplicate (three superfusion chambers for each experimental condition). *P < 0.01 vs the respective control value (two-tailed Student's t-test).

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Fig. 7. Release of [3H]glycine evoked by 100 µM glycine from mouse cerebellar synaptosomes and effects of NFPS and Org25543. Synaptosomes were exposed to glycine at t = 39 min of

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superfusion. NFPS or Org25543 was added 9 min before glycine. Results are expressed as per cent potentiation with respect to the basal release. Data are means ± SEM of 3-5 experiments in triplicate (three superfusion chambers for each experimental condition). * P < 0.01 vs the respective control value (one-way ANOVA followed by post hoc Dunnett’s-test).

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Fig. 8. Release of [3H]glycine evoked by 100 µM glycine from mouse spinal cord synaptosomes and effects of Org25543. Synaptosomes were exposed to glycine at t = 39 min of superfusion. Org25543 was added 9 min before glycine. Results are expressed as per cent potentiation with respect to the basal release. Data are means ± SEM of 4-5 experiments in triplicate (three

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(one-way ANOVA followed by post hoc Dunnett's-test).

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superfusion chambers for each experimental condition). * P < 0.01 vs the respective control value

ACCEPTED MANUSCRIPT Table 1 Uptake of [3H]glycine mediated by GlyT1 and GlyT2 into purified mouse spinal cord synaptosomes. ______________________________________________________________________________ [3H]glycine uptake percent inhibition (pmol/mg protein) ______________________________________________________________________________

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Drug

93.00 ± 2.67

NFPS (0.3 µM)

68.70 ± 7.09 *

NFPS (1 µM)

66.53 ± 6.16 *

Org25543 (1 µM)

24.00 ± 5.19 *

Org25543 (10 µM)

19.20 ± 6.03 *

79.4

1.80 ± 0.90 *

98.1

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NFPS (0.3 µM) + Org25543 (1 µM)

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Control (no drugs)

26.1

28.5 74.1

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______________________________________________________________________________

Synaptosomes were incubated 2 min at 37°C in standard medium containing 0.3 µM [3H]glycine in the absence (control samples) or in the presence of the drugs. Data are means ± SEM of 3–6

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experiments run in triplicate. *P < 0.01 vs the respective control value (one-way ANOVA

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followed by post hoc Dunnett’s-test).

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Table 2 Uptake of [3H]glycine mediated by GlyT1 and GlyT2 into purified mouse cerebellar synaptosomes.

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______________________________________________________________________________

Control (no drugs)

15.5 ± 1.93

NFPS (0.01 µM)

13.2 ± 1.50

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percent inhibition [3H]glycine uptake (pmol/mg protein) ______________________________________________________________________________

Drug

15.0

9.88 ± 2.00 *

NFPS (0.3 µM)

8.95 ± 1.39 **

42.3

Org25543 (0.1 µM)

8.01 ± 0.75 **

48.3

Org25543 (1 µM)

5.84 ± 0.61 **

62.3

Org25543 (3 µM)

5.35 ± 0.51 **

65.5

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NFPS (0.1 µM)

NFPS (0.3 µM) + Org25543 (1 µM)

0.42 ± 0.03 **

36.3

97.3

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Synaptosomes were incubated 2 min at 37°C in standard medium containing 0.3 µM [3H]glycine

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in the absence (control samples) or in the presence of the drugs. Data are means ± SEM of 3–6 experiments run in triplicate. *P < 0.05; **P < 0.01 vs the respective control value (one-way ANOVA followed by post hoc Dunnett’s-test).

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Table 3 Uptake of [3H]glycine mediated by GlyT1 and GlyT2 into purified mouse cortical synaptosomes. ______________________________________________________________________________ percent inhibition [3H]glycine uptake (pmol/mg protein) ______________________________________________________________________________

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Drug

Control (no drugs)

9.00 ± 0.63

NFPS (0.1 µM)

5.96 ± 0.64 *

NFPS (0.3 µM)

5.20 ± 0.73 *

Org25543 (1 µM)

4.94 ± 0.73 *

Org25543 (3 µM)

4.80 ± 0.71 *

46.7

NFPS (0.3 µM) + Org25543 (1 µM)

0.59 ± 0.07 *

93.5

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33.7 42.2

45.1

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Synaptosomes were incubated 2 min at 37°C in standard medium containing 0.3 µM [3H]glycine in the absence (control samples) or in the presence of the drugs. Data are means ± SEM of 3–5

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experiments run in triplicate. *P < 0.01 vs the respective control value (one-way ANOVA

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followed by post hoc Dunnett’s-test).

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Table 4 Uptake of [3H]glycine mediated by GlyT1 and GlyT2 into purified mouse hippocampal synaptosomes.

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______________________________________________________________________________ percent inhibition [3H]glycine uptake (pmol/mg protein) ______________________________________________________________________________ Drug

4.00 ± 0.35

NFPS (0.1 µM)

2.70 ± 0.24

NFPS (0.3 µM)

2.67 ± 0.37

Org25543 (1 µM)

1.48 ± 0.41 *

63.0

Org25543 (3 µM)

1.20 ± 0.21 *

70.0

NFPS (0.1 µM) + Org25543 (1 µM)

0.16 ± 0.02 *

95.6

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Control (no drugs)

32.5 33.3

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______________________________________________________________________________

Synaptosomes were incubated 2 min at 37°C in standard medium containing 0.3 µM [3H]glycine

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in the absence (control samples) or in the presence of the drugs. Data are means ± SEM of 3–5 experiments run in triplicate. *P < 0.01 vs the respective control value (one-way ANOVA

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followed by post hoc Dunnett’s-test).

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KCl-evoked [ H]glycine release (% overflow)

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KCl (mM)

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Figure 1

*

25 CaCl2 (1.2 mM) BoNT/C1 (nM)

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8

6

4

2

0 + + 10

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100 80 60

20

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0

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40

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[3H]glycine release (% of control)

Figure 2

1

3

Org 25543 (µM)

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25

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30

*

20 *

15

*

TE D

10 5

EP

0

+

+

AC C

Veratridine-evoked [3H]glycine release (% overflow)

Figure 3

+

3

1

°

° Veratridine (µM)

10

CaCl2 (1.2 mM) 1

3

Org 25543 (µM)

ACCEPTED MANUSCRIPT

RI PT SC

30

20 15

*

5

EP

0

TE D

10

M AN U

25

AC C

Veratridine-evoked [3H]glycine release (% overflow)

Figure 4

*

10

Veratridine (µM)

1

CaCl2 (1.2 mM) Org25543 (µM)

3

ACCEPTED MANUSCRIPT

RI PT

Figure 5

SC M AN U

400

300

100

TE D

200

EP

3 Glycine-evoked [ H]glycine release (% potentiation)

500

0

AC C

5

10

30

100

Glycine concentration (µM)

ACCEPTED MANUSCRIPT

SC M AN U

500

400

300

TE D

200

100

0

EP

*

AC C

Glycine-evoked [3H]glycine release (% potentiation)

RI PT

Figure 6

146.2

-

* Glycine (µM)

100 146.2

-

Na+ (mM)

ACCEPTED MANUSCRIPT

SC M AN U

400

300

TE D

200

0

EP

*

100

*

Glycine (µM)

100

-

AC C

Glycine-evoked [3H]glycine release (% potentiation)

RI PT

Figure 7

1

-

-

-

1

10

NFPS (µM) Org 25543 (µM)

ACCEPTED MANUSCRIPT

SC M AN U

500

400

300

TE D

200

*

0

EP

100

-

AC C

Glycine-evoked [3H]glycine release (% potentiation)

RI PT

Figure 8

*

Glycine (µM)

100 1

10

Org 25543 (µM)

ACCEPTED MANUSCRIPT

Morphologically undetectable glycine GlyT2 transporters can perform glycine uptake into nerve endings in telencephalic areas Glycine release by GlyT2 reversal can occur in nerve endings

AC C

EP

TE D

M AN U

SC

RI PT

Glycine GlyT2-mediated homoexchange can occur in nerve endings