Chemical structure of mGlu1 receptor enhancers.

Effect of enhancers on rmGlu1a and hmGlu1a receptors. (A) Glutamate activated an inward current in a Chinese hamster ovary cell stably expressing GIRKs and transiently transfected with the rmGlu1a receptor cDNA. The cell was held at −70 mV, and the recording was made under conditions in which K⁺ currents would be inward. Ro 01-6128 alone elicited no response; however, glutamate application in the presence of Ro 01-6128 resulted in a greatly enhanced inward current. The effect of Ro 01-6128 was reversible. (B) Concentration-response curves for S-DHPG recorded from cells expressing rmGlu1a receptors in the absence and presence of Ro 01-6128. S-DHPG was applied for 20 sec locally to the cell with a rapid application system by using increasing concentrations. The interval between two successive applications was 90 sec. Ro 01-6128 was then added to the bath perfusion, and a second S-DHPG concentration-response curve was generated on the same cell in the presence of Ro 01-6128. The current amplitudes were normalized to the control responses and fitted individually for each cell. The pEC₅₀ values for S-DHPG in the absence and presence of Ro 01-6128 were 5.05 ± 0.03 and 5.78 ± 0.05, respectively (n = 4). The effect of enhancers on glutamate-induced currents in cells expressing rmGlu1a (C) and hmGlu1a (D) receptors is shown. Maximum current amplitudes obtained at different concentrations of Ro 01-6128, Ro 67-4853, and Ro 67-7476 are plotted as a function of concentration. The values are normalized to the control responses obtained with glutamate alone (3 μM, 100%). Each point represents the mean ± SEM of five cells.

The effect of enhancers in freshly dissociated CA3 neurons. (A) VGCCs were evoked at 5-sec intervals by 30-msec steps to 0 mV from a holding potential of −80 mV. The currents measured at the peak of the control response and their inhibition by S-DHPG in the presence and absence of Ro 01-6128 are plotted versus time. S-DHPG and Ro 01-6128 were applied for the times indicated by the bars. (Inset) Superimposed current traces generated in the absence and presence of S-DHPG and Ro 01-6128, represented by the open circles in the plot. (B) Concentration-response curves for the enhancement of the S-DPHG response by Ro 01-6128 and Ro 67-4853 in freshly dissociated CA3 neurons. VGCCs were evoked at 5-sec intervals by 30-msec steps to 0 mV from a holding potential of −80 mV. The currents measured at the peak of the control response and their inhibition by S-DHPG (5 μM) were recorded in the presence and absence of the different concentrations of the enhancers. The values for S-DHPG inhibition were normalized to the control responses and fitted individually for each cell. (C) Concentration-response curves for S-DHPG in freshly dissociated CA3 neurons in the absence and presence of 3 μM Ro 01-6128. S-DHPG was applied for 20 sec locally to the cell with a rapid application system by using increasing concentrations. The interval between two successive applications was 90 sec. Ro 01-6128 was then added to the bath perfusion, and a second S-DHPG concentration-response curve was generated on the same cell in the presence of Ro 01-6128. The current amplitudes were normalized to the control responses and fitted individually for each cell. The pEC₅₀ values for S-DHPG in the absence and presence of Ro 01-6128 were 5.05 ± 0.07 and 5.32 ± 0.05, respectively (n = 5).

Ro 67-7476 enhances glutamate-evoked increases in [Ca²⁺]_int. (A) Glutamate evoked a concentration-dependent increase in [Ca²⁺]_int in HEK293 cells transiently transfected with rmGlu1a assayed by single-cell fura-2 imaging (data are means ± SEM of three separate experiments with 15–20 cells per experiment). (B) Mean data from a single representative experiment (n = 15 cells). Applications of glutamate (1 μM, 30 sec; arrows) alone elicited no response; however, application in the presence of Ro 67-7476 (bar, 1 μM), which elicited no response when applied alone, resulted in a large increase in [Ca²⁺]_int.

A series of chimeric receptor constructs showing the fusion sites between rmGlu1a (R1, black) and rmGlu5a receptors (R5, white). (A) The numbering indicates amino acid fragments of wild-type rmGlu1a and rmGlu5a receptors. (B) Sequence alignment of the TMIII, TMV, and TMVII domains of the rmGlu1a and hmGlu1a receptors with rmGlu5a receptor, showing the nonconserved valine at position 757. The residues that were mutated in the current work are underlined. − indicates identical residues among rmGlu1a, hmGlu1a, and rmGlu5a receptors. Enhancement of the glutamate-induced current in GIRK-Chinese hamster ovary cells expressing chimeric (C) and mutated (D) rmGlu1a (R1), hmGlu1a (H1), and rmGlu5a (R5) receptors by 1 μM Ro 67-7476. To achieve a comparable activation of the receptors, glutamate concentrations approaching the EC₂₀ values were used (1 μM for rmGlu5a- and 3 μM for hmGlu1a- and rmGlu1a N-Terminal-containing receptors). Data are expressed as percentages of the maximum current amplitude induced by a control application of glutamate alone and represent the mean ± SEM of five to eight cells.

Potentiation of mGlu receptor responses in cerebellar slices by Ro 67-6747. (A) Mean (± SEM) population depolarizations evoked by the application of S-DHPG to cerebellar wedges in the presence or absence of Ro 67-7476 (3 μM), n = 6–22 (*, P < 0.05, ***, P < 0.001, paired Student's t test). (B) Mean mGlu receptor EPSC amplitude increased as a function of stimulation number and was potentiated after the application of Ro 67-7476 (3 μM), n = 5, but was stable after a 20-min placebo incubation, n = 4. Representative mGlu receptor EPSC was evoked in response to 1, 4, 8, and 15 stimulation pulses: C, control; D, in the presence of Ro 67-7476 (3 μM); E, in response to 15 pulses in the presence of Ro 67-7476 + 1 mM MCPG.