Transport mechanism and pharmacology of the human GlyT1.

The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.

Beyond dopamine: Novel strategies for schizophrenia treatment.

Despite extensive research efforts aimed at discovering novel antipsychotic compounds, a satisfactory pharmacological strategy for schizophrenia treatment remains elusive. All the currently available drugs act by modulating dopaminergic neurotransmission, leading to insufficient management of the negative and cognitive symptoms of the disorder. Due to these challenges, several attempts have been made to design agents with innovative, non-dopaminergic mechanisms of action. Consequently, a number of promising compounds are currently progressing through phases 2 and 3 of clinical trials. This review aims to examine the rationale behind the most promising of these strategies while simultaneously providing a comprehensive survey of study results. We describe the versatility behind the cholinergic neurotransmission modulation through the activation of M1 and M4 receptors, exemplified by the prospective drug candidate KarXT. Our discussion extends to the innovative approach of activating TAAR1 receptors via ulotaront, along with the promising outcomes of iclepertin, a GlyT-1 inhibitor with the potential to become the first treatment option for cognitive impairment associated with schizophrenia. Finally, we evaluate the 5-HT2A antagonist paradigm, assessing two recently developed serotonergic agents, pimavanserin and roluperidone. We present the latest advancements in developing novel solutions to the complex challenges posed by schizophrenia, offering an additional perspective on the diverse investigated drug candidates.

[3H]-NFPS binding to the glycine transporter 1 in the hemi-parkinsonian rat brain.

L-3,4-dihydroxyphenylalanine (L-DOPA) is the main treatment for Parkinson's disease (PD) but with long term administration, motor complications such as dyskinesia are induced. Glycine transporter 1 (GlyT1) inhibition was shown to produce an anti-dyskinetic effect in parkinsonian rats and primates, coupled with an improvement in the anti-parkinsonian action of L-DOPA. The expression of GlyT1 in the brain in the dyskinetic state remains to be investigated. Here, we quantified the levels of GlyT1 across different brain regions using [3H]-NFPS in the presence of Org-25,935. Brain sections were chosen from sham-lesioned rats, L-DOPA-naïve 6-hydroxydopamine (6-OHDA)-lesioned rats and 6-OHDA-lesioned rats exhibiting mild or severe abnormal involuntary movements (AIMs). [3H]-NFPS binding decreased in the ipsilateral and contralateral thalamus, by 28% and 41%, in 6-OHDA-lesioned rats with severe AIMs compared to sham-lesioned animals (P < 0.01 and 0.001). [3H]-NFPS binding increased by 21% in the ipsilateral substantia nigra of 6-OHDA-lesioned rats with severe AIMs compared to 6-OHDA-lesioned rats with mild AIMs (P < 0.05). [3H]-NFPS binding was lower by 19% in the contralateral primary motor cortex and by 20% in the contralateral subthalamic nucleus of 6-OHDA-lesioned rats with mild AIMs animals compared to rats with severe AIMs (both P < 0.05). The severity of AIMs scores positively correlated with [3H]-NFPS binding in the ipsilateral substantia nigra (P < 0.05), ipsilateral entopeduncular nucleus (P < 0.05) and contralateral primary motor cortex (P < 0.05). These data provide an anatomical basis to explain the efficacy of GlyT1 inhibitors in dyskinesia in PD.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro.

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

Synthesis of [3 H]Org24598 using in-house prepared [3 H]MeI.

The synthesis of tritium-labelled glycine transporter 1 inhibitor Org24598 is reported. Because of the instability of the Org24598 skeleton under hydrogenation conditions, a synthetic approach using an in-house prepared tritium-labelled alkylating agent ([3 H]MeI, SA = 26.2 Ci/mmol) was employed. Alternative methods of labelling are discussed.

Development of the novel GlyT1 inhibitor, iclepertin (BI 425809), for the treatment of cognitive impairment associated with schizophrenia.

Schizophrenia is a psychiatric disorder characterised by symptoms in three domains: positive (e.g. delusions, hallucinations), negative (e.g. social withdrawal, lack of motivation) and cognitive (e.g. working memory and executive function impairment). Cognitive impairment associated with schizophrenia (CIAS) is a major burden for patients and negatively impacts many aspects of a patient's life. Antipsychotics are the standard-of-care treatment for schizophrenia but only address positive symptoms. So far there are no approved pharmacotherapies for the treatment of CIAS. Iclepertin (BI 425809) is a novel, potent and selective glycine transporter 1 (GlyT1) inhibitor, under development by Boehringer Ingelheim for the treatment of CIAS. Phase I studies have shown it to be safe and well tolerated in healthy volunteers, and central target engagement (inhibition of GlyT1) was achieved in a dose-dependent manner from 5 to 50 mg in healthy volunteers. A Phase II study has demonstrated that iclepertin is safe and well tolerated in patients with schizophrenia and improves cognition at doses of 10 mg and 25 mg. Phase III studies are ongoing to confirm these initial positive safety and efficacy findings with the 10 mg dose, and if successful, iclepertin could become the first approved pharmacotherapy used to treat CIAS.

Carbon 14 synthesis of glycine transporter 1 inhibitor Iclepertin (BI 425809) and its major metabolites.

Carbon 14 labeled Iclepertin (BI 425809, 1) and its major metabolites were needed for ADME and several other studies necessary for the advancement of this drug candidate in clinical trials. Iclepertin is composed of two main chemical blocks, (R)-5-(methylsulfonyl)-2-([1,1,1-trifluoropropan-2-yl]oxy)benzoic acid (2), and 3-[(1R,5R)-3-azabicyclo[3.1.0]hexan-5-yl]-5-(trifluoromethyl)isoxazole (3) linked to each other via an amide bond. In the first synthesis of carbon 14 labeled 1, 2-fluorobenzoic acid, carboxyl-14 C was converted to [14 C]-2 in three steps and then coupled to 3 to provide [14 C]-1a in 45% overall yield. In the second synthesis, [14 C]-3 was prepared in six radioactive steps and coupled to the acid 2 to furnish [14 C]-1b in 20% overall yield. Both synthetic routes provided [14 C]-1a and [14 C]-1b with specific activities higher than 53 mCi/mmol and radiochemical, chemical, and enantiomeric purities above 98%. Two major metabolites of 1, BI 761036 and BI 758790, were also prepared labeled with carbon 14 using intermediates already available from the synthesis of [14 C]-1.

Stable isotope synthesis of glycine transporter 1 inhibitor Iclepertin (BI 425809) and its major metabolites.

Stable isotope labeled Iclepertin (BI 425809, 1) and its major metabolites are needed as internal standards in bioanalytical studies. BI 425809 consists of two main building blocks, 5-methylsulfonyl-2-[(1R)-2,2,2-trifluoro-1-methyl-ethoxy]benzoic acid (2) and 3-[(1R,5R)-3-azabicyclo[3.1.0]hexan-5-yl]-5-(trifluoromethyl)isoxazole (3) linked to each other via an amide bond. We used fluoro[13 C6 ]benzene as the starting material in the preparation of [13 C6 ]-2. This intermediate was then employed to access carbon 13 labeled Iclepertin ([13 C6 ]-1) and other metabolites. The major metabolite BI 761036 (6), which resulted from cytochrome P450 oxidation and amide hydrolysis of BI 425809, was prepared labeled with carbon 13 and nitrogen 15 via two synthetic routes. In the first route, diethyl [13 C3 ]malonate, [13 C]methyl iodide, and hydroxyl[15 N]amine were used to provide [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6a) in 13 steps in 6% overall yield, whereas in the second route, [13 C3 ]propargyl alcohol, potassium [13 C]cyanide, and [15 N]ammonia were used to furnish [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6b) in 11 steps in 1% overall yield. The detailed stable isotope synthesis of 1 and its major metabolites is described.

Maternal Separation Alters Ethanol Drinking and Reversal Learning Processes in Adolescent Rats: The Impact of Sex and Glycine Transporter Type 1 (GlyT1) Inhibitor.

Adverse early life experiences are associated with an enhanced risk for mental and physical health problems, including substance abuse. Despite clinical evidence, the mechanisms underlying these relationships are not fully understood. Maternal separation (MS) is a commonly used animal model of early neglect. The aim of the current study is to determine whether the N-methyl-D-aspartate receptor (NMDAR)/glycine sites are involved in vulnerability to alcohol consumption (two-bottle choice paradigm) and reversal learning deficits (Barnes maze task) in adolescent rats subjected to the MS procedure and whether these effects are sex dependent. By using ELISA, we evaluated MS-induced changes in the NMDAR subunits (GluN1, GluN2A, GluN2B) expression, especially in the glycine-binding subunit, GluN1, in the prefrontal cortex (PFC) and ventral striatum (vSTR) of male/female rats. Next, we investigated whether Org 24598, a glycine transporter 1 (GlyT1) inhibitor, was able to modify ethanol drinking in adolescent and adult male/female rats with prior MS experience and reversal learning in the Barnes maze task. Our findings revealed that adolescent MS female rats consumed more alcohol which may be associated with a substantial increase in GluN1 subunit of NMDAR in the PFC and vSTR. Org 24598 decreased ethanol intake in both sexes with a more pronounced decrease in ethanol consumption in adolescent female rats. Furthermore, MS showed deficits in reversal learning in both sexes. Org 24598 ameliorated reversal learning deficits, and this effect was reversed by the NMDAR/glycine site inhibitor, L-701,324. Collectively, our results suggest that NMDAR/glycine sites might be targeted in the treatment of alcohol abuse in adolescents with early MS, especially females.

Analysis of Binding Determinants for Different Classes of Competitive and Noncompetitive Inhibitors of Glycine Transporters.

Glycine transporters are interesting therapeutic targets as they play significant roles in glycinergic and glutamatergic systems. The search for new selective inhibitors of particular types of glycine transporters (GlyT-1 and GlyT-2) with beneficial kinetics is hampered by limited knowledge about the spatial structure of these proteins. In this study, a pool of homology models of GlyT-1 and GlyT-2 in different conformational states was constructed using the crystal structures of related transporters from the SLC6 family and the recently revealed structure of GlyT-1 in the inward-open state, in order to investigate their binding sites. The binding mode of the known GlyT-1 and GlyT-2 inhibitors was determined using molecular docking studies, molecular dynamics simulations, and MM-GBSA free energy calculations. The results of this study indicate that two amino acids, Gly373 and Leu476 in GlyT-1 and the corresponding Ser479 and Thr582 in GlyT-2, are mainly responsible for the selective binding of ligands within the S1 site. Apart from these, one pocket of the S2 site, which lies between TM3 and TM10, may also be important. Moreover, selective binding of noncompetitive GlyT-1 inhibitors in the intracellular release pathway is affected by hydrophobic interactions with Ile399, Met382, and Leu158. These results can be useful in the rational design of new glycine transporter inhibitors with desired selectivity and properties in the future.

GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.

Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

Bicyclo((aryl)methyl)benzamides as inhibitors of GlyT1.

A series of isoquinuclidine benzamides as glycine uptake inhibitors for the treatment of schizophrenia are described. Potency, lipophilicity, and intrinsic human microsomal clearance were parameters for optimization. Potency correlated with the nature of the ortho substituents of the benzamide ring, and reductions in lipophilicity could be achieved through heteroatom incorporation in the benzamide and pendant phenyl moieties. Improvements in human CLint were achieved through changes in ring size and the N-alkyl group of the isoquinuclidine itself, with des-alkyl derivatives (40-41, 44) demonstrating the most robust microsomal stability. Dimethylbenzamide 9 was tested in a mouse MK801 LMA assay and had a statistically significant attenuation of locomotor activity at 3 and 10 µmol/kg compared to control.

Glycine release from astrocytes via functional reversal of GlyT1.

It was previously reported that functional glycine receptors were expressed in neonatal prefrontal cortex; however, the glycine-releasing cells were unknown. We hypothesized that astrocytes might be a major glycine source, and examined the glycine release properties of astrocytes. We also hypothesized that dopamine (DA) might be a trigger for the astrocytic glycine release, as numerous DA terminals localize in the cortex. We combined two different methods to confirm the glycine release from astrocytes. Firstly, we analyzed the supernatant of astrocytes by amino acid analyzer after DA stimulation, and detect significant glycine peak. Furthermore, we utilized a patch-clamp biosensor method to confirm the glycine release from astrocytes by using GlyRα1 and Glyß-expressing HEK293T cells, and detected significant glycine-evoked current upon DA stimulation. Thus, we clearly demonstrated that DA induces glycine release from astrocytes. Surprisingly, DA caused a functional reversal of astrocytic glycine transporter 1, an astrocytic type of glycine transporter, causing astrocytes to release glycine. Hence, astrocytes transduce pre-synaptic DA signals to glycine signals through a reversal of astrocytic glycine transporter 1 to regulate neuronal excitability. Cover Image for this issue: doi: 10.1111/jnc.13785.

Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK-6577 in baboons.

Glycine transporter type-1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [(11) C]GSK931145, and [(18) F]MK-6577, in baboons. Two baboons were imaged with [(11) C]GSK931145 and [(18) F]MK-6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [(11) C]GSK931145 and [(18) F]MK-6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ∼ 30% of parent at 30 min post-injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [(18) F]MK-6577 displayed higher uptake and faster kinetics than [(11) C]GSK931145. Time activity curves were well described by the two-tissue compartment model. Regional volume of distribution (VT ) values were higher for [(18) F]MK-6577 than [(11) C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of VT estimates between tracers indicated higher specific binding for [(18) F]MK-6577 than [(11) C]GSK931145, consistent with higher regional binding potential (BPND ) values of [(18) F]MK-6577 calculated using VT from the baseline scans and non-displaceable distribution volume (VND ) derived from blocking studies. [(18) F]MK-6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [(11) C]GSK931145.

Preclinical Reproductive and Developmental Toxicity Profile of a Glycine Transporter Type 1 (Glyt1) Inhibitor.

Bitopertin is a glycine type 1 (GlyT1) inhibitor intended for the treatment of psychiatric disorders. The principle adverse effect in the regulatory reproductive toxicity studies was peri-natal pup death when rat dams were treated during parturition at a dose resulting in five-times the human therapeutic exposure (AUC). Cessation of dosing two days before parturition prevented the pup deaths. Investigatory experiments and pharmacokinetic modelling suggested that the neonatal mortality was related to transplacental passage of bitopertin leading to high systemic levels in the newborn pups. Brain levels of bitopertin in the rat fetus and neonate were two-fold higher than in the mother. As illustrated by knock-out mice models, GlyT1 function is essential for neonatal pup survival in rodents, but is not necessary for normal prenatal morphological development. The glycine transport systems are immature at birth in the rat, but are functionally well-developed in the human newborn. While the relevance to humans of the neonatal mortality seen in rats following late gestational exposure is unknown, bitopertin would not be recommended for use during late pregnancy unless the anticipated benefit for the mother outweighs the potential risk to the newborn.

Characterization of the novel GlyT1 PET tracer [18F]MK-6577 in humans.

Decreased glutamatergic neurotransmission is hypothesized to be involved in the pathophysiology of schizophrenia. Inhibition of glycine transporter Type-1 (GlyT1) reuptake is expected to increase the glutamatergic neurotransmission and may serve as treatment for cognitive and negative symptoms of schizophrenia. In this article, we present human data from a novel GlyT1 PET tracer, [(18) F]MK-6577. In the process of developing a GlyT1 inhibitor therapeutic, a PET tracer can assist in determining the dose with a high probability of sufficiently testing the mechanism of action. This article reports the human PET studies with [(18) F]MK-6577 for measuring GlyT1 receptor availability at baseline in normal human subjects and occupancy with a GlyT1 inhibitor, MK-2637. Studies were also performed to measure radiation burden and the baseline test-retest (T-RT) variability of the tracer. The effective dose from sequential whole-body dosimetry scans in three male subjects was estimated to be 24.5 ± 2.9 µSV/MBq (mean ± SD). The time-activity curves from T-RT scans modeled satisfactorily using a two tissue compartmental model. The tracer uptake was highest in the pons (VT = 6.7 ± 0.9, BPND = 4.1 ± 0.43) and lowest in the cortex (VT = 2.1 ± 0.5, BPND = 0.60 ± 0.23). VT T-RT variability measured in three subjects was <12% on average. The occupancy scans performed in a cohort of 15 subjects indicated absence of a reference region. The in vivo potency (Occ50 ) of MK-2637 was determined using two methods: A: Lassen plot with a population input function (Occ50 = 106 nM, SE = 20 nM) and B: pseudo reference tissue model using cortex as the pseudo reference region (Occ50 = 141 nM, SE = 21 nM).

Forebrain glycine transporter 1 deletion enhances sensitivity to CS-US discontiguity in classical conditioning.

The deletion of glycine transporter 1 (GlyT1) in forebrain neurons can apparently strengthen Pavlovian aversive conditioning, but this phenotype is not expressed if conditioning followed non-reinforced pre-exposures of the to-be-conditioned stimulus (CS). To examine whether GlyT1 disruption may only enhance aversive associative learning under conditions that most favour the formation of CS-US excitatory link, we evaluated the impact of GlyT1 disruption on the trace conditioning procedure whereby a trace interval between a tone CS and a shock US was introduced during conditioning. CS and US occurrences were thus rendered discontiguous, which was expected to impede conditioning compared with contiguous CS-US pairing. Conditioned freezing to the CS was measured in a retention test conducted 48 h after conditioning. The genetic disruption significantly modified the temporal dynamics of the freezing response over the course of the 8-min presentation of the CS, although the immediate conditioned response to the CS was unaffected. The separation between "trace" and "no-trace" conditions was augmented in the mutant mice, but this only became apparent in mid-session; and the augmentation can be attributed to the combined effects of (i) weaker conditioned freezing in the mutant relative to control subjects in the "trace" condition, and (ii) stronger conditioned freezing in mutants relative controls in the "no-trace" condition. The demonstrated increased sensitivity to the effect of CS-US temporal discontiguity further highlights the importance of GlyT1-dependent mechanisms in the regulation of associative learning.