GlyT1 Expression Research Articles

Neurotransmitter content heterogeneity within an interneuron class shapes inhibitory transmission at a central synapse.

Neurotransmitter content is deemed the most basic defining criterion for neuronal classes, contrasting with the intercellular heterogeneity of many other molecular and functional features. Here we show, in the adult mouse brain, that neurotransmitter content variegation within a neuronal class is a component of its functional heterogeneity. Golgi cells (GoCs), the well-defined class of cerebellar interneurons inhibiting granule cells (GrCs), contain cytosolic glycine, accumulated by the neuronal transporter GlyT2, and GABA in various proportions. By performing acute manipulations of cytosolic GABA and glycine supply, we find that competition of glycine with GABA reduces the charge of IPSC evoked in GrCs and, more specifically, the amplitude of a slow component of the IPSC decay. We then pair GrCs recordings with optogenetic stimulations of single GoCs, which preserve the intracellular transmitter mixed content. We show that the strength and decay kinetics of GrCs IPSCs, which are entirely mediated by GABAA receptors, are negatively correlated to the presynaptic expression of GlyT2 by GoCs. We isolate a slow spillover component of GrCs inhibition that is also affected by the expression of GlyT2, leading to a 56% decrease in relative charge. Our results support the hypothesis that presynaptic loading of glycine negatively impacts the GABAergic transmission in mixed interneurons, most likely through a competition for vesicular filling. We discuss how the heterogeneity of neurotransmitter supply within mixed interneurons like the GoC class may provide a presynaptic mechanism to tune the gain of microcircuits such as the granular layer, thereby expanding the realm of their possible dynamic behaviors.

Withdrawn: Glycine transporter 1 (GlyT1) is a novel therapeutic target for Alzheimer’s disease

Withdrawal: Miran Yoo, Seong Muk Kim, Juho Lee, Hye‐Ju Kim, Seong Jeong Park “Glycine transporter 1 (GlyT1) is a novel therapeutic target for Alzheimer's disease” Alzheimer's & Dementia, Volume 18, Supplemental Issue 10.The above abstract, published online on 20th December 2022, on Wiley Online Library (https://alz‐journals.onlinelibrary.wiley.com/doi/abs/10.1002/alz.064468) has been withdrawn by agreement between the authors, the Senior Director of Scientific Programs and Outreach, Claire Sexton, the Director of Scientific Programs at the Alzheimer's Association, Ozama Ismail, and John Wiley & Sons Inc. The abstract has been withdrawn at the authors’ request because the authors did not have Amyloid Solution's authorization to publish the information contained in it.

Botulinum toxin type A counteracts neuropathic pain by countering the increase of GlyT2 expression in the spinal cord of CCI rats

Botulinum toxin type A (BoNT/A) is a potent toxin, acts by cleaving synaptosome-associated-protein-25 (SNAP-25) to regulate the release of the neural transmitter and shows analgesic effect in neuropathic pain. However, the mechanisms of BoNT/A actions involved in nociceptions remain unclear. Glycine transporter 2 (GlyT2) is an isoform of glycine transporters, which plays an important role in the regulation of glycinergic neurotransmission. Inhibition of GlyTs could decrease pain sensation in neuropathic pain, the role of GlyT2 in the analgesic effect of BoNT/A has not been studied yet. In our present study, we demonstrated that the protein levels of GlyT2 and SNAP-25 were upregulated in the spinal cord after the development of chronic constriction injury (CCI)-induced neuropathic pain. Intraplantar application of BoNT/A (20 U/kg) attenuated mechanical allodynia induced by CCI and downregulated GlyT2 expression in the spinal cord. The application of BoNT/A s also decreased the expression of GlyT2 in pheochromocytoma (PC12) cells. Moreover, intrathecal application of lentivirus-mediated GlyT2 reversed the antinociceptive effect of BoNT/A in CCI rats. These findings indicate that GlyT2 contributes to the antinociceptive effect of BoNT/A and suggest a novel mechanism underlying BoNT/A’s antinociception action.

Bupivacaine reduces GlyT1 expression by potentiating the p-AMPK\u03b1/BDNF signalling pathway in spinal astrocytes of rats

Bupivacaine, a local anaesthetic, is widely applied in the epidural or subarachnoid space to clinically manage acute and chronic pain. However, the underlying mechanisms are complex and unclear. Glycine transporter 1 (GlyT1) in the spinal cord plays a critical role in various pathologic pain conditions. Therefore, we sought to determine whether bupivacaine exerts its analgesic effect by regulating GlyT1 expression and to determine the underlying mechanisms of regulation. Primary astrocytes prepared from the spinal cord of rats were treated with bupivacaine. The protein levels of GlyT1, brain-derived neurotrophic factor (BDNF) and phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase α (p-AMPKα) were measured by western blotting or immunofluorescence. In addition, 7,8-dihydroxyflavone (7,8-DHF, BDNF receptor agonist) and AMPK shRNA were applied to verify the relationship between the regulation of GlyT1 by bupivacaine and the p-AMPKα/BDNF signalling pathway. After treatment with bupivacaine, GlyT1 expression was diminished in a concentration-dependent manner, while the expression of BDNF and p-AMPK was increased. Moreover, 7,8-DHF decreased GlyT1 expression, and AMPK knockdown suppressed the upregulation of BDNF expression by bupivacaine. Finally, we concluded that bupivacaine reduced GlyT1 expression in spinal astrocytes by activating the p-AMPKα/BDNF signalling pathway. These results provide a new mechanism for the analgesic effect of intrathecal bupivacaine in the treatment of acute and chronic pain.

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain.

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of ‘patches’ of higher expression, interspersed within a less immunoreactive ‘matrix’, which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

Regulation of the Glycine Transporter GLYT1 by microRNAs.

The glycine transporter GLYT1 participates in inhibitory and excitatory neurotransmission by controlling the reuptake of this neuroactive substance from synapses. Over the past few years, microRNAs have emerged as potent negative regulators of gene expression. In this report, we investigate the possible regulation of GLYT1 by microRNAs. TargetScan software predicted the existence of multiple targets for microRNAs within the 3' UTR of the human GLYT1 (miR-7, miR-30, miR-96, miR-137 and miR-141), and as they are all conserved among mammalian orthologues, their effects on GLYT1 expression were determined experimentally. Dual reporter bioluminescent assays showed that only miR-96 and miR-137 down-regulated expression of the Renilla reporter fused to the 3' UTR of GLYT1. Mutations introduced into the target sequences blocked this inhibitory effect. Consistently, these two microRNAs downregulated the uptake of [3H]glycine into glial C6 cells, a cell line where GLYT1 is the main carrier for glycine. Moreover, the expression of endogenous GLYT1 in primary mixed cultures from rat spinal cord was decreased upon lentiviral expression of miR-96 and miR-137. Although the bulk of GLYT1 is glial, it is abundantly expressed in glycinergic neurons of the retina and in smaller amounts in glutamatergic neurons though the brain. Since miR-96 in the retina is strongly downregulated by light exposure, when rats were maintained in darkness for a few hours we observed a concomitant increase of GLYT1 expression, suggesting that at least miR-96 might be an important negative regulator of GLYT1 under physiological conditions.

Sarcosine Suppresses Epileptogenesis in Rats With Effects on Hippocampal DNA Methylation.

Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.

E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may cause sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that E3 ubiquitin ligase Ligand of Numb proteins X1/2 (LNX1/2) modulate the ubiquitination status of GlyT2. The N-terminal RING-finger domain of LNX1/2 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791), and this process regulates the expression levels and transport activity of GlyT2. The genetic deletion of endogenous LNX2 in spinal cord primary neurons causes an increase in GlyT2 expression and we find that LNX2 is required for PKC-mediated control of GlyT2 transport. This work identifies, to our knowledge, the first E3 ubiquitin-ligases acting on GlyT2, revealing a novel molecular mechanism that controls presynaptic glycine availability. Providing a better understanding of the molecular regulation of GlyT2 may help future investigations into the molecular basis of human disease states caused by dysfunctional glycinergic neurotransmission, such as hyperekplexia and chronic pain.

Spinal Cord Glycine Transporter 2 Mediates Bilateral ST35 Acupoints Sensitization in Rats with Knee Osteoarthritis.

The concept of “acupoint sensitization” refers to the functional status of acupoint switches from silent to active under pathological conditions. In clinic, acupoint sensitization provides important guidance for acupoints selection in different diseases. However, the mechanism behind this phenomenon remains unclear. We generated a model of knee osteoarthritis (KOA) by intra-articular injection of monosodium iodoacetate (MIA) into the left knee of rats. The paw withdrawal mechanical threshold (PWMT) and the total number of mast cells as well as mast cell degranulation rate (MCDR) of acupoint tissue were used to test whether the acupoints were sensitized. The results showed that KOA resulted in a reduced mechanical threshold and elevated total number of mast cell as well as mast cell degranulation rate at bilateral ST35 (Dubi) but not GB37 (Guangming) or nonacupoint area. The acupoint sensitization was accompanied by upregulation of glycine transporter 2 (GlyT2) and reduction of extracellular glycine levels in the bilateral dorsal horns of the spinal cord at L3-5. Selective inhibition of GlyT2 or intrathecal administration of glycine attenuated ST35 acupoint sensitization. The sensitization of bilateral ST35 was blocked after intraspinal GlyT2 short hairpin (sh) RNA (GlyT2-shRNA) microinjection to specifically downregulate GlyT2 expression in the left side (ipsilateral) L3-5 spinal cord dorsal horn before MIA injection. Moreover, electroacupuncture (EA) stimulation at ST35 ameliorated articular pathological lesions and improved KOA-related pain behaviors. GlyT2-shRNA injection reversed EA-induced pain relief but not EA-induced reduction of joint lesions. Overall, this study demonstrated that spinal GlyT2, especially elevated GlyT2 expression in the ipsilateral dorsal horn of the spinal cord, is a crucial mediator of ST35 acupoint sensitization in KOA rats.

P2X receptors up-regulate the cell-surface expression of the neuronal glycine transporter GlyT2

Glycinergic inhibitory neurons of the spinal dorsal horn exert critical control over the conduction of nociceptive signals to higher brain areas. The neuronal glycine transporter 2 (GlyT2) is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft and its activity modulates intra and extracellular glycine concentrations. In this report we show that the stimulation of P2X purinergic receptors with βγ-methylene adenosine 5′-triphosphate induces the up-regulation of GlyT2 transport activity by increasing total and plasma membrane expression and reducing transporter ubiquitination. We identified the receptor subtypes involved by combining pharmacological approaches, siRNA-mediated protein knockdown, and dorsal root ganglion cell enrichment in brainstem and spinal cord primary cultures. Up-regulation of GlyT2 required the combined stimulation of homomeric P2X3 and P2X2 receptors or heteromeric P2X2/3 receptors. We measured the spontaneous glycinergic currents, glycine release and GlyT2 uptake concurrently in response to P2X receptor agonists, and showed that the impact of P2X3 receptor activation on glycinergic neurotransmission involves the modulation of GlyT2 expression or activity. The recognized pro-nociceptive action of P2X3 receptors suggests that the fine-tuning of GlyT2 activity may have consequences in nociceptive signal conduction.

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

Glycine can be substrate for two transporters: GlyT1, largely expressed by astrocytes but also by 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, neocortex and hippocampus. Although, as expected, [3H]glycine uptake was significantly lower in 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 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 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 glycinergic nerve terminals can perform both release by transporter reversal and homoexchange.

Glycine transporter 1 is a target for the treatment of epilepsy

Glycine is the major inhibitory neurotransmitter in brainstem and spinal cord, whereas in hippocampus glycine exerts dual modulatory roles on strychnine-sensitive glycine receptors and on the strychnine-insensitive glycineB site of the N-methyl-d-aspartate receptor (NMDAR). In hippocampus, the synaptic availability of glycine is largely under control of glycine transporter 1 (GlyT1). Since epilepsy is a disorder of disrupted network homeostasis affecting the equilibrium of various neurotransmitters and neuromodulators, we hypothesized that changes in hippocampal GlyT1 expression and resulting disruption of glycine homeostasis might be implicated in the pathophysiology of epilepsy. Using two different rodent models of temporal lobe epilepsy (TLE) – the intrahippocampal kainic acid model of TLE in mice, and the rat model of tetanic stimulation-induced TLE – we first demonstrated robust overexpression of GlyT1 in the hippocampal formation, suggesting dysfunctional glycine signaling in epilepsy. Overexpression of GlyT1 in the hippocampal formation was corroborated in human TLE samples by quantitative real time PCR. In support of a role of dysfunctional glycine signaling in the pathophysiology of epilepsy, both the genetic deletion of GlyT1 in hippocampus and the GlyT1 inhibitor LY2365109 increased seizure thresholds in mice. Importantly, chronic seizures in the mouse model of TLE were robustly suppressed by systemic administration of the GlyT1 inhibitor LY2365109. We conclude that GlyT1 overexpression in the epileptic brain constitutes a new target for therapeutic intervention, and that GlyT1 inhibitors constitute a new class of antiictogenic drugs. These findings are of translational value since GlyT1 inhibitors are already in clinical development to treat cognitive symptoms in schizophrenia

Glycine transporter GlyT1, but not GlyT2, is expressed in rat dorsal root ganglion—Possible implications for neuropathic pain

Glycinergic inhibitory neurotransmission plays a pivotal role in the development of neuropathic pain. The glycine concentration in the synaptic cleft is controlled by the glycine transporters GlyT1 and GlyT2. GlyT1 is expressed throughout the central nervous system, while GlyT2 is exclusively located in glycinergic neurons. Aim of the present study was to investigate whether GlyTs are also expressed in the peripheral sensory nervous system and whether their expression is modulated in experimental neuropathic pain.Neuropathic pain was induced in male Wistar rats by Chronic Constriction Injury (CCI) and verified by assessment of mechanical allodynia (von Frey method). Expression patterns of GlyTs and the glycine binding subunit NR1 of the N-methyl-d-aspartate (NMDA) receptor in the spinal cord and dorsal root ganglia (DRG) were analyzed by Western blot analysis, PCR and immunohistochemistry.While both GlyT1 and GlyT2 were detected in the spinal cord, only GlyT1, but not GlyT2, was detected in DRG. Immunofluorescence revealed a strictly neuronal localization of GlyT1 and a co-localization of GlyT1 and NR1 in DRG. Compared to sham procedure, spinal cord and DRG expression of GlyT1 was not altered and NR1 was unchanged in DRG 12 days after CCI.GlyT1, but not GlyT2, is expressed in the peripheral sensory nervous system. The co-expression of GlyT1 and NMDA receptors in DRG suggests that GlyT1 regulates glycine concentration at the glycine binding site of the NMDA receptor. Differential regulation of GlyT1 expression in the spinal cord or DRG, however, does not seem to be associated with the development of neuropathic pain.

Na+/K+-ATPase is a new interacting partner for the neuronal glycine transporter GlyT2 that downregulates its expression in vitro and in vivo.

The neuronal glycine transporter GlyT2 plays a fundamental role in the glycinergic neurotransmission by recycling the neurotransmitter to the presynaptic terminal. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is absolutely necessary to preserve quantal glycine content in synaptic vesicles. Alterations in GlyT2 activity modify glycinergic neurotransmission and may underlie several neuromuscular disorders, such as hyperekplexia, myoclonus, dystonia, and epilepsy. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans and produce congenital muscular dystonia type 2 (CMD2) in Belgian Blue cattle. GlyT2 function is strictly coupled to the sodium electrochemical gradient actively generated by the Na+/K+-ATPase (NKA). GlyT2 cotransports 3Na+/Cl-/glycine generating large rises of Na+ inside the presynaptic terminal that must be efficiently reduced by the NKA to preserve Na+ homeostasis. In this work, we have used high-throughput mass spectrometry to identify proteins interacting with GlyT2 in the CNS. NKA was detected as a putative candidate and through reciprocal coimmunoprecipitations and immunocytochemistry analyses the association between GlyT2 and NKA was confirmed. NKA mainly interacts with the raft-associated active pool of GlyT2, and low and high levels of the specific NKA ligand ouabain modulate the endocytosis and total expression of GlyT2 in neurons. The ouabain-mediated downregulation of GlyT2 also occurs in vivo in two different systems: zebrafish embryos and adult rats, indicating that this NKA-mediated regulatory mechanism is evolutionarily conserved and may play a relevant role in the physiological control of inhibitory glycinergic neurotransmission.

Synthesis and evaluation of 2-chloro N-[(S)-{(S)-1-[11 C]methylpiperidin-2-yl} (phenyl)methyl]3-trifluoromethyl-benzamide ([11 C]N-methyl-SSR504734) as a PET radioligand for glycine transporter 1

BackgroundDysfunction of the glycine transporter 1 (GlyT1) has been suggested to be involved in psychiatric disorders such as schizophrenia. GlyT1 inhibitors have therefore been considered to have antipsychotic therapeutic potential. Positron emission tomography (PET) imaging probes for GlyT1 are, consequently, expected to be useful for investigating the mechanism of such disease conditions and for measuring occupancy of GlyT1 inhibitors in vivo. The aim of this study was to assess the potential of 2-chloro N-[(S)-{(S)-1-[11 C]methylpiperidin-2-yl} (phenyl)methyl] 3-trifluoromethyl-benzamide ([11 C]N-methyl-SSR504734) as a PET imaging agent for GlyT1.Methods[11 C]N-methyl-SSR504734 was synthesized by N-[11 C]methylation of SSR504734 via [11 C]CH3OTf. In vitro brain distribution of [11 C]N-methyl-SSR504734 was tested in whole-hemisphere autoradiography (ARG) on human brain slices. Initial PET studies were performed using a cynomolgus monkey at baseline and after pretreatment with 0.1 to 1.5 mg/kg of SSR504734. Then, PET studies using rhesus monkeys were performed with arterial blood sampling at baseline and after pretreatment with 1.5 to 4.5 mg/kg SSR504734. Distribution volumes (VT) were calculated with a two-tissue compartment model, and GlyT1 occupancy by SSR504734 was estimated using a Lassen plot approach.Results[11 C]N-methyl-SSR504734 was successfully synthesized in moderate radiochemical yield and high specific radioactivity. In the ARG experiments, [11 C]N-methyl-SSR504734 showed specific binding in the white matter and pons. In the initial PET experiments in a cynomolgus monkey, [11 C]N-methyl-SSR504734 showed high brain uptake and consistent distribution with previously reported GlyT1 expression in vivo (thalamus, brainstem > cerebellum > cortical regions). However, the brain uptake increased after pretreatment with SSR504734. Further PET studies in rhesus monkeys showed a similar increase of brain uptake after pretreatment with SSR504734. However, the VT of [11 C]N-methyl-SSR504734 was found to decrease after pretreatment of SSR504734 in a dose-dependent manner. GlyT1 occupancy was calculated to be 45% and 73% at 1.5 and 4.5 mg/kg of SSR504734, respectively.Conclusions[11 C]N-methyl-SSR504734 is demonstrated to be a promising PET radioligand for GlyT1 in nonhuman primates. The present results warrant further PET studies in human subjects.

Electrophysiological characteristics of inhibitory neurons of the prepositus hypoglossi nucleus as analyzed in Venus-expressing transgenic rats

The identification and characterization of excitatory and inhibitory neurons are significant steps in understanding neural network functions. In this study, we investigated the intrinsic electrophysiological properties of neurons in the prepositus hypoglossi nucleus (PHN), a brainstem structure that is involved in gaze holding, using whole-cell recordings in brainstem slices from vesicular GABA transporter (VGAT)-Venus transgenic rats, in which inhibitory neurons express the fluorescent protein Venus. To characterize the intrinsic properties of these neurons, we recorded afterhyperpolarization (AHP) profiles and firing patterns from Venus-expressing [Venus⁺] and Venus-non-expressing [Venus⁻] PHN neurons. Although both types of neurons showed a wide variety of AHP profiles and firing patterns, oscillatory firing was specific to Venus⁺ neurons, while a firing pattern showing only a few spikes was specific to Venus⁻ neurons. In addition, AHPs without a slow component and delayed spike generation were preferentially displayed by Venus⁺ neurons, whereas a firing pattern with constant interspike intervals was preferentially displayed by Venus⁻ neurons. We evaluated the mRNAs expression of glutamate decarboxylase (GAD65, GAD67) and glycine transporter 2 (GlyT2) to determine whether the recorded Venus⁺ neurons were GABAergic or glycinergic. Of the 67 Venus⁺ neurons tested, GlyT2 expression alone was detected in only one neuron. Approximately 40% (28/67) expressed GAD65 and/or GAD67 (GABAergic neuron), and the remainder (38/67) expressed both GAD(s) and GlyT2 (GABA&GLY neuron). These results suggest that most inhibitory PHN neurons use either GABA or both GABA and glycine as neurotransmitters. Although the overall distribution of firing patterns in GABAergic neurons was similar to that of GABA&GLY neurons, only GABA&GLY neurons exhibited a firing pattern with a long first interspike interval. These differential electrophysiological properties will be useful for the identification of specific types of PHN neurons.

Synthesis and characterization of [125I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [3H]glycine uptake (IC50=2.4nM). Saturation assay in rat cortical membranes revealed that [125I]5 had a single high affinity binding site with a Kd of 1.54nM and a Bmax of 3.40pmol/mg protein. In vitro autoradiography demonstrated that [125I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [125I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [125I]5 showed high blood–brain barrier permeability (1.68–2.17% dose/g at 15–60min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [125I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.

Pharmacological characterisation of the GlyT-1 glycine transporter using two novel radioligands

Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [ 3H]-SB-733993 and [ 3H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. K d and B max values for the two radioligands were fairly similar, with K d values of 1–2 nM and B max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [ 3H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [ 3H]-GSK931145. Dissociation was also much slower for [ 3H]-GSK931145 than for [ 3H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [ 3H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [ 3H]-GSK931145, though with much lower density ( B max 570 fmol/mg protein) and slightly lower affinity (K d 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [ 3H]-SB-733993 binding was reduced by >90% in the absence of added Na + ions, whilst [ 3H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B max values, suggesting that both [ 3H]-SB-733993 and [ 3H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [ 3H]-SB-733993 and [ 3H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [ 3H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology.

Glial glycine transporter 1 function is essential for early postnatal survival but dispensable in adult mice

The glycine transporter 1 (GlyT1) is expressed in astrocytes and selected neurons of the mammalian CNS. In newborn mice, GlyT1 is crucial for efficient termination of glycine-mediated inhibitory neurotransmission. Furthermore, GlyT1 has been implicated in the regulation of excitatory N-methyl-D-asparate (NMDA) receptors. To evaluate whether glial and neuronal GlyT1 have distinct roles at inhibitory synapses, we inactivated the GlyT1 gene cell type-specifically using mice carrying floxed GlyT1 alleles GlyT1((+)/+)). GlyT1((+)/(+)) mice expressing Cre recombinase in glial cells developed severe neuromotor deficits during the first postnatal week, which mimicked the phenotype of conventional GlyT1 knock-out mice and are consistent with glycinergic over-inhibition. In contrast, Cre-mediated inactivation of the GlyT1 gene in neuronal cells did not result in detectable motor impairment. Notably, some animals deficient for glial GlyT1 survived the first postnatal week and did not develop neuromotor deficits throughout adulthood, although GlyT1 expression was efficiently reduced. Thus, glial GlyT1 is critical for the regulation of glycine levels at inhibitory synapses only during early postnatal life.