Study of novel selective mGlu2 agonist in the temporo-ammonic input to CA1 neurons reveals reduced mGlu2 receptor expression in a Wistar substrain with an anxiety-like phenotype

Abstract

Group II metabotropic receptors (mGluRs) regulate central synaptic transmission by modulating neurotransmitter release. However, the lack of pharmacological tools differentiating between mGlu2 and mGlu3 receptors has hampered identification of the roles of these two receptor subtypes. We have used LY395756 [(1SR,2SR,4RS,5RS,6SR)-2-amino-4-methylbicyclo[3.1.0]-hexane2,6-dicarboxylic], an agonist at mGlu2 receptors and an antagonist at mGlu3 receptors in cell lines, to investigate the roles of these receptors in the temporo-ammonic path from entorhinal cortex to CA1-stratum lacunosum moleculare in rat hippocampal slices. Surprisingly, the degree of inhibition of the field EPSP induced by LY395756 fell into two distinct groups, with EC(50) values of <1 μm and >100 μm. In "sensitive" slices, LY395756 had additive actions with a mixed mGlu2/mGlu3 agonist, DCG-IV [(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine], whereas in "insensitive" slices, LY395756 reduced the effect of DCG-IV, with an IC(50) of ∼1 μm. This separation into sensitive and insensitive slices could be explained by LY395756 acting as an mGlu2 agonist and mGlu3 antagonist, respectively, a finding supported by data from mice lacking these receptors. The heterogeneity was correlated with differences in expression levels of mGlu2 receptors within our Wistar colony and other Wistar substrains. The initial search for a behavioral correlate indicated that rats lacking mGlu2 receptors showed anxiety-like behavior in open-field and elevated plus maze assays. These findings have implications for rat models of psychiatric disease and are especially pertinent given that mGlu2 receptors are targets for compounds under development for anxiety.

Figure 1.

Activation of group II mGluRs by DCG-IV causes a concentration-dependent inhibition of synaptic transmission in the TAP–SLM pathway. A, A representative single experiment showing the depression of the slope of the fEPSP in CA1–SLM after stimulation of the TAP by sequentially increasing concentrations of DCG-IV. The inhibition was reversed during washout. In this and subsequent figures, the application of drug and the concentration used are indicated by the bars and information above the plot. Each data point represents the average of four consecutive fEPSPs. Example traces, above, have been taken at the time points indicated. B, Pooled data (mean ± SEM) from six experiments as in A. C, Concentration–response curve for the effect of DCG-IV on fEPSP slope. Each data point is the mean ± SEM of 4–10 experiments. The curve has been plotted using a logistic four-parameter equation giving an EC₅₀ value of 80 nm. Statistically significant differences for individual concentrations of DCG-IV are given in Results. D, Histogram showing the lack of effect of LY341495 (300 nm) when administered alone (n = 4) and antagonism of DCG-IV (100 nm) when coadministered (n = 4).

Figure 2.

LY395756 has differential effects on synaptic transmission in the TAP–SLM pathway. A, Single example in which LY395756 produced almost complete depression of the TAP-evoked fEPSP in CA1–SLM at 10 μm. After washout and recovery, lower concentrations (0.1 and 1 μm) were also tested. B, Single example in which high concentrations of LY395756 (up to 300 μm) were required to depress the fEPSP response. C, Scatter plot of data in which each point represents the percentage depression induced by LY395756 at two concentrations (1 μm, n = 16; 10 μm, n = 16) in sensitive (filled circles) and insensitive (open circles) intact slices. D, Concentration–response curves for the effect of LY395756 in depressing the TAP–CA1 input in sensitive slices (filled circles) and insensitive slices (open circles). Each data point is the mean ± SEM of 4–10 experiments. Curves have been plotted using a logistic four-parameter equation. Fitted curves give EC₅₀ values of 0.63 and 117 μm for sensitive and insensitive slices, respectively. E, In sensitive slices, LY341495 (300 nm) fully reversed the large depressions of TAP evoked fEPSPs induced by LY395756 (10 μm; n = 4; filled bars). In contrast, in insensitive slices, it failed to reverse the small depressions induced by LY395756 (100 μm; n = 4; open bars).

Figure 3.

Pharmacological heterogeneity is maintained in the modified hippocampal slice preparation. A, Effects of LY395756 (10 μm) recorded from the cell body layer in CA1–stratum pyramidale in modified slices (Aa). Pooled data evoked by stimulation in the TAP–SLM (circles) and SC (triangles) from insensitive (open symbols; n = 3) and sensitive (filled symbols; n = 5) slices (Ab). Statistical differences are indicated in Results. B, Correlation between the depression of fEPSPs obtained by LY395756 (10 μm) evoked after stimuli in the distal and proximal SLM (Ba) in insensitive (open circles; n = 2) and sensitive (filled circles; n = 6) slices, respectively (Bb). C, In horizontal slices, the depression of the TAP–CA1 fEPSP with LY395756 (10 μm) also falls into two populations of sensitive (n = 4) and insensitive (n = 4) slices.

Figure 4.

LY395756 antagonizes the actions of DCG-IV only in insensitive slices. A, Pooled data from five insensitive slices showing the antagonist action of LY395756 (10 μm) versus depression induced by DCG-IV (100 nm). B, Pooled data showing the effect of LY395756 during the application of DCG-IV (100 nm) in insensitive (open circles; n = 11) and sensitive (filled circles; n = 5) slices. Note how it acts as an antagonist and an agonist, respectively. Note also the lack of significant difference between the effects of DCG-IV in the two types of slices. C, Concentration–response curve for the agonist effect of LY395756 in sensitive slices (filled circle; dotted line) overlaps with the bell-shaped curve of the antagonist effect of LY395756 in insensitive slices (open circles; solid line). Each data point is the mean ± SEM of 4–10 experiments.

Figure 5.

Actions of LY395756 in group II mGluR knock-out mice. A–D, Effect of LY395756 (1–10 μm) in mGlu2 KO (A, B) and mGlu3 KO (C, D) mice (pooled data, n = 3–5). LY395756 (1–10 μm) is without effect in mGlu2 KO mice (A) but depresses the fEPSP response in mGlu3 KO mice (C). LY395756 (10 μm) also antagonizes the action of DCG-IV (300 nm) in mGlu2 KO (B) but not in mGlu3 KO (D) mice.

Figure 6.

Effects of LY395756 on substrains of Wistar rats. A, Scatter grams showing the effect of LY395756 on TAP-evoked fEPSPs in five different substrains of Wistar rats and one strain of Sprague Dawley (SD) rats. Open circles indicate an effect similar to insensitive preparations and filled circles to sensitive ones. Gray circles represent the data from Sprague Dawley rats. B, Simplified scheme of the relationship and apparent derivation of the different substrains of Wistar rats.

Figure 7.

Expression of mGlu2 and mGlu3 in tissues from knock-out mice and different substrains of Wistar rats. A, Specificity of mGlu2 and mGlu3 receptor antibodies determined by Western blots using tissue from adult hippocampal slices from two WT, two mGlu2 KO, and two mGlu3 KO mice. These antibodies were used at 1 μg/ml concentration and are selective for mGlu2 and mGlu3 proteins. The same blots were probed with anti-actin antibody (0.2 μg/ml) to ensure equal loading. B, Western blots performed using the same antibodies on a parasagittal slice (Whole) and the cortical (Cortex), hippocampal (Hippo), cerebellar (Cereb), and striatal (Striat) regions of adjacent slices from postnatal day 14 Crl:Wi sensitive and B&K:Wi insensitive rats. The sensitivity to LY395756 was verified in hippocampal slices from the same rats. C, D, Histogram of densitometric analysis of the protein expression of mGlu2 (C) and mGlu3 (D) receptors, respectively, as shown in B using NIH ImageJ. IB, Immunoblot. ***p < 0.001 compared with control (Student's t test).

Figure 8.

Behavioral studies in Crl:Wi and B&K:Wi rats. A, B, Open-field assay; C–E, elevated plus maze assay. A, Histogram showing total locomotor activity over 20 min in the open field expressed as the number of line crossings in 5 min epochs made by Crl:Wi (filled bar) and B&K:Wi (open bar) rats. B, Histograms showing greater tendency of Crl:Wi rats (filled bar) to explore the inner areas of the open-field arena. Line crossings in the inside area of the open field are normalized by expressing as a percentage of total number of line crossings in the 5 min test. C, Histograms showing that Crl:Wi (filled bar) rats spend more time in the open arms of the elevated plus maze expressed as percentage of total 5 min compared with B&K:Wi rats (open bar). D, Histograms showing that Crl:Wi rats (filled bar) make more entries into the open arm from the central area of the elevated plus maze than B&K:Wi rats (open bar). E, Histograms showing that the Crl:Wi rats (filled bar) enter the open arms of the elevated plus maze more quickly than B&K:Wi rats (open bar), expressed as the latency to the first open arm entry. F, Actions of LY395756 (10 μm) on synaptic transmission in the TAP–SLM pathway of Crl:Wi (filled circles) and B&K:Wi (open circles) rats used previously for the above behavioral assays. The sample traces are taken from one of the experiments as indicated on the graph.