The glycine transporter type 1 inhibitor N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior

Abstract

Glycine acts as a necessary coagonist for glutamate at the NMDA receptor (NMDAR) complex by binding to the strychnine-insensitive glycine-B binding site on the NR1 subunit. The fact that glycine is normally found in the brain and spinal cord at concentrations that exceed those required to saturate this site has led to the speculation that glycine normally saturates NMDAR-containing synapses in vivo. However, additional lines of evidence suggest that synaptic glycine may be efficiently regulated in synaptic areas by the glycine transporter type 1 (GlyT1). The recent description of a potent and selective GlyT1 inhibitor (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine [NFPS]) provides a tool for evaluation of the hypothesis that inhibition of GlyT1 may increase synaptic glycine and thereby potentiate NMDAR function in vivo. In the present study, we found that (+)-NFPS demonstrated >10-fold greater activity in an in vitro functional glycine reuptake assay relative to the racemic compound. In vivo, (+/-)-NFPS significantly enhanced long-term potentiation in the hippocampal dentate gyrus induced by high-frequency electrical stimulation of the afferent perforant pathway. Furthermore, (+)-NFPS induced a pattern of c-Fos immunoreactivity comparable with the atypical antipsychotic clozapine and enhanced prepulse inhibition of the acoustic startle response in DBA/2J mice, a strain with low basal levels of prepulse inhibition. Collectively, these data suggest that selective inhibition of GlyT1 can enhance NMDAR-sensitive activity in vivo and also support the idea that GlyT1 may represent a novel target for developing therapeutics to treat disorders associated with NMDAR hypofunction.

Figure 1.

Chemical structure of NFPS (N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl] sarcosine).

Figure 2.

Competition experiments revealed that (+/-)-NFPS and its enantiomers fully antagonized [¹⁴C]glycine (10 μm) uptake in HEK-293 cells recombinantly expressing rat GlyT1a with IC₅₀ values shown in Table 1. Similar experiments with rat GlyT2 revealed the subtype selectivity of these compounds. Data are the mean IC₅₀ values ± SEMs from three experiments. Nonspecific uptake was determined in the presence of 10 mm cold glycine. Error bars represent SEMs.

Figure 3.

Photomicrographs illustrating the expression of c-Fos immunoreactivity in nucleus accumbens (NAcc) (left column) and prefrontal cortex (PFC) (right column) of rats treated with vehicle (A, B), (+)-NFPS (10 mg/kg, i.p.) (C, D), and clozapine (20 mg/kg, i.p.) (E, F).

Figure 4.

Group data depicting the effect of (+/-)-NFPS (3 mg/kg, i.v.) administration on LTP in the hippocampal dentate gyrus produced by delivery of a high-frequency electrical stimulation of the perforant path in anesthetized rats. An injection of vehicle or (+/-)-NFPS was administered 30 min after the initiation of each experiment and 30 min before the delivery of the tetanic stimulation. After stimulation of the perforant pathway, NFPS-treated rats displayed a significantly greater magnitude of LTP that was maintained for the duration of the testing period. Data are grouped in 10 min bins, and asterisks represent a significant difference from vehicle-treated rats: ^*p < 0.05, ^**p < 0.01. Error bars represent SEMs; n = 10 per group.

Figure 5.

Group data depicting basal PPI in three mouse strains at four prepulse intensities (5-20 dB above background). DBA/2J mice showed significantly lower levels of PPI relative to the 129S6 and C57BL/6 strains. Asterisks represent a significant difference from DBA/2J mice: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Error bars represent SEMs; n = 8 per group.

Figure 6.

A, The effect of vehicle, two doses of NFPS (1 and 10 mg/kg, i.p.), and clozapine (6 mg/kg, i.p.) on PPI in DBA/2J mice at four prepulse intensities (5-20 dB above background). Vehicle and NFPS were administered 120 min before placement in the testing apparatus, whereas clozapine was administered 20 min before testing. Asterisks represent a significant difference from the vehicle group: ^**p < 0.01, ^***p < 0.001. Error bars represent SEMs. B, The effect of vehicle, NFPS, and clozapine on startle amplitude during pulse-alone trials in the same mice represented in A. The asterisk represents a significant difference from the vehicle group: ^*p < 0.05. Error bars represent SEMs.