Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor Crosstalk in the Prefrontal Cortex: Metamodulation of Glutamate Exocytosis

Abstract

The glutamatergic nerve endings of a rat prefrontal cortex (PFc) possess presynaptic 5-HT_2A heteroreceptors and mGlu2/3 autoreceptors, whose activation inhibits glutamate exocytosis, and is measured as 15 mM KCl-evoked [³H]D-aspartate ([³H]D-asp) release (which mimics glutamate exocytosis). The concomitant activation of the two receptors nulls their inhibitory activities, whereas blockade of the 5-HT_2A heteroreceptors with MDL11,939 (1 μM) strengthens the inhibitory effect elicited by the mGlu2/3 receptor agonist LY329268 (1 μM). 5-HT_2A receptor antagonists (MDL11,939; ketanserin; trazodone) amplify the impact of low (3 nM) LY379268. Clozapine (0.1-10 μM) mimics the 5-HT_2A agonist (±) DOI and inhibits the KCl-evoked [³H]D-asp overflow in a MDL11,939-dependent fashion, but does not modify the (±) DOI-induced effect. mGlu2 and 5-HT_2A proteins do not co-immunoprecipitate from synaptosomal lysates, nor does the incubation of PFc synaptosomes with MDL11,939 (1 μM) or clozapine (10 µM) modify the insertion of mGlu2 subunits in synaptosomal plasma membranes. In conclusion, 5-HT_2A and mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis. The mGlu2/3-5-HT_2A metamodulation could be relevant to therapy for central neuropsychiatric disorders, including schizophrenia, but also unveil cellular events accounting for their development, which also influence the responsiveness to drugs regimens.

Keywords: 5-HT2A receptor; clozapine; glutamate exocytosis; mGlu2/3 receptor; prefrontal cortex; synaptosomes; trazodone.

Figure 1

Effects of (±) DOI and of LY379268 on the 15 mM KCl-evoked release of preloaded [³H]D-aspartate ([³H]D-asp) from synaptosomes isolated from the prefrontal cortex (PFc) of adult rats. (a) (±) DOI (0.1–30 μM) inhibits the exocytosis of ([³H]D-asp) elicited by 15 mM KCl-enriched medium. Synaptosomes were exposed in superfusion to the depolarizing stimulus for 90 s in the absence or in the presence of the 5-HT_2A receptor agonist. When indicated, the 5-HT_2A antagonist MDL11,939 (0.1 μM) was added concomitantly to (±) DOI. The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.33 ± 0.02 nCi and represents the 0.90 ± 0.05% of the total tritium synaptosomal content. (b) PFc synaptosomes are endowed with the 5-HT_2A receptor protein. Synaptosomal lysates were immunoblotted and probed with anti-5-HT2A and anti-β-actin antibodies. (c) LY379268 concentration-dependently (0.003–3 μM) reduced the [³H]D-asp exocytosis elicited by high K⁺ from synaptosomes isolated from the PFc of adult rats. The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.49 ± 0.06 nCi and and represents the 0.77 ± 0.12% of the total tritium synaptosomal content. (d) Western blot analysis confirming the presence of mGlu2 receptor protein in the PFc lysates. Synaptosomal lysates were immunoblotted and probed with anti-mGlu2 and anti-β-actin antibodies. (a,c) results are expressed as induced overflow; data are the means ± SEM of four to six experiments run in triplicate (three superfusion chambers for each experimental conditions). * p < 0.05 vs. respective 15 mM KCl; ** p < 0.01 vs. respective 15 mM KCl; *** p < 0.001 vs. respective 15 mM KCl; ^^ p < 0.01 vs. 15 mM KCl/30 μM (±) DOI. The blots in (b,d) are representative of 5 to 7 blots run in different days using different samples.

Figure 2

Presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT_2A heteroreceptors functionally crosstalk in synaptosomes from the prefrontal cortex of adult rats. (a) Effects of (±) DOI (1 μM) and of LY379268 (0.1–1 μM) alone or concomitantly added on the 15 mM KCl-evoked [³H]D-asp exocytosis. The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.39 ± 0.02 nCi and represents the 0.88 ± 0.12% of the total tritium synaptosomal content. (b) Effects of 1 μM MDL11,939 on the release of tritium elicited by the 15 mM KCl-containing medium in the absence or in the presence of LY379268 (0.1–1 μM). The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.46 ± 0.03 nCi, the 1.03 ± 0.08% of the total tritium synaptosomal content. The results are expressed as induced overflow; data are the means ± SEM of five to six experiments run in triplicate. ** p < 0.01 vs. 15 mM KCl; *** p < 0.001 vs. 15 mM KCl; ^^^ p < 0.01 vs. 15 mM KCl/1 μM LY379268; ### p < 0.001 vs. 15 mM KCl/1 μM (±) DOI; °° p < 0.01 vs. 15 mM KCl/1 μM MDL11,939; § p < 0.05 vs. 15 mM KCl/0.1 μM LY379268.

Figure 3

Presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT_2A heteroreceptors do not physically interact in synaptosomes from the prefrontal cortex (PFc) of adult rats. mGlu2 receptor proteins (a) and 5-HT_2A receptor proteins (b) were immunoprecipitated from lysates of synaptosomes isolated from the PFc of adult rats. Synaptosomal lysates (L), immuno-precipitates (IP) and negative control (B), here used as a negative control, were analyzed for the presence of mGlu2 and of 5-HT_2A immunopositivity. (c) mGlu2 receptor proteins were immunoprecipitated (IP) from PFc homogenates and analyzed for the presence of mGlu2 and 5-HT_2A receptor proteins (H); negative control (B). The figures in panels (a–c) are representative of the analysis of five different samples for each experimental condition. (d) The cartoon illustrates the colocalization but not the physical association of the mGlu2/3 receptors and the 5-HT2A receptors in PFc synaptosomes.

Figure 4

Clozapine inhibits the 15 mM KCl-evoked [³H]D-aspartate in synaptosomes from the PFc of adult rats: reversal by MDL11,939. (a) Concentration dependent clozapine (0.1–10 μM) inhibits the 15 mM KCl-evoked [³H]D-asp exocytosis from PFc synaptosomes in a MDL11,939-dependent fashion. The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.56 ± 0.03 nCi and represents the 1.01 ± 0.06% of the total tritium synaptosomal content. (b) Effects of 10 μM (±) DOI and 10 μM clozapine alone or concomitantly added on the 15 mM KCl evoked [³H]D-asp exocytosis from prefrontal cortex synaptosomes. The release of [³H]D-asp in the first 3 min fraction collected (b1) amounted to 0.57 ± 0.06 nCi and represents the 1.12 ± 0.09% of the total tritium synaptosomal content. Results are expressed as induced overflow; data are the means ± SEM of five to six experiments run in triplicate. * p < 0.05 vs. respective 15 mM KCl; °° p < 0.001 vs. 15 mM KCl/10 μM clozapine.

Figure 5

Preincubation of PFc synaptosomes with MDL11,939 or with clozapine does not modify the surface expression of the mGlu2 receptor subunits. (a) Representative Western blot analysis of the density of the mGlu2 receptor subunits in PFc synaptosomal plasma membranes following preincubation of synaptosomes with MDL11,939 (1 μM) or with clozapine (10 μM). The blot compares the total synaptosomal lysate (L) and the synaptosomal membranes of particles that were not exposed to biotin, but were subjected to streptavidin pulldown (B), the synaptosomal membranes of untreated particles that were incubated with biotin and then were subjected to streptavidin pulldown (C), the synaptosomal membranes from MDL11,939-treated particles that were incubated with biotin and then were subjected to streptavidin pulldown MDL11,939 (1 μM), and the synaptosomal membranes of clozapine-treated particles that were incubated with biotin and then were subject to streptavidin pulldown (10 μM). The figure in panel (a) is representative of five different experiments carried out on different days with samples from different animals. (b) Changes in the mGlu2 receptor protein surface density in MDL11,939 (grey bar) and in clozapine (black bar) treated synaptosomal plasma membranes when compared to untreated particles (here indicated as a dotted line). Data are expressed as percent changes versus respective controls.