Synergistic interactions between kainate and mGlu receptors regulate bouton Ca signalling and mossy fibre LTP

Abstract

It is currently unknown why glutamatergic presynaptic terminals express multiple types of glutamate receptors. We have addressed this question by studying both acute and long-term regulation of mossy fibre function in the hippocampus. We find that inhibition of both mGlu₁ and mGlu₅ receptors together can block the induction of mossy fibre LTP. Furthermore, mossy fibre LTP can be induced by the pharmacological activation of either mGlu₁ or mGlu₅ receptors, provided that kainate receptors are also stimulated. Like conventional mossy fibre LTP, chemically-induced mossy fibre LTP (chem-LTPm) depends on Ca²⁺ release from intracellular stores and the activation of PKA. Similar synergistic interactions between mGlu receptors and kainate receptors were observed at the level of Ca²⁺ signalling in individual giant mossy fibre boutons. Thus three distinct glutamate receptors interact, in both an AND and OR gate fashion, to regulate both immediate and long-term presynaptic function in the brain.

Figure 1. Antagonism of both mGlu₁ and mGlu₅ receptors inhibits the induction of mossy fibre LTP.

(a) The graph represents pooled data from 4 experiments to show that MCPG (200 μM) always blocked the induction of LTP (100 Hz, 1 s) in a reversible manner. In these, and subsequent experiments, D-AP5 (50 μM) was always present during each tetanus to ensure that only NMDA receptor-independent LTP was studied. Bars indicate the exposure time to various compounds. Insets at the top are representative fEPSPs taken at the indicated time on the plot. (b) Pooled data from 4 experiments showing the lack of effect of the selective mGlu₁ antagonist LY367385 (30 μM) [left panel] and mGlu₅ antagonist MPEP (30 μM) [right panel]. (c) Pooled data from 4 experiments showing reversible block of the induction of mossy fibre LTP by co-application of LY367385 (3 µM) plus MPEP (3 µM).

Figure 2. Chemically-induced mossy fibre LTP.

(a) Pooled data from 5 experiments showing that co-application of ATPA (1 µM) plus DHPG (3 µM), but neither agonist alone, induces a potentiation of mossy fibre synaptic transmission. In these and subsequent experiments D-AP5 (50 μM) was present throughout. Insets at the top are representative EPSPs taken at the indicated time on the plot. (b) Chem-LTPm can be induced by activation of GluK1-containing KARs plus activation of either mGlu₁ receptor subtype. Both panels illustrate pooled data from 4 experiments that show synaptic potentiation induced by ATPA (1 µM) plus DHPG (3 µM), in the presence of MPEP (30 µM) (left panel) or in the presence of LY367385 (30 µM) (right panel).

Figure 3. Properties of chem-LTPm.

(a) Chem-LTPm involves activation of PKA. Pooled data from 4 experiments to illustrate that KT5720 (3 µM) reversibly inhibits synaptic potentiation induced by ATPA (1 µM) plus DHPG (3 µM). (b) Chem-LTPm involves Ca²⁺ release from intracellular stores. Pooled data from 4 experiments to illustrate that ryanodine (10 µM) reversibly inhibits synaptic potentiation induced by ATPA (1 µM) plus DHPG (3 µM).

Figure 4. Both GluK1-containing KARs and group I mGlu receptors regulate Ca²⁺ signalling at individual mossy fibre boutons.

(a) Schematic illustration of technique (see methods for details) and typical fluorescence transient evoked by delivering a train of action potentials (5 at 20 Hz). (b) Representative single mossy fibre traced into CA3. Giant mossy fibre boutons were identified by their characteristic size (3 – 8 µm) and filopodial extensions. (c) Typical fluorescence transients recorded in line scanning mode before (black) and after (green) application of LY382884 (10 µM). Graph: Representative cell (red) and pooled data (blue). (d) Typical fluorescence transients recorded in line scanning mode before (black) and after (green) co-application of LY367385 (3 µM) and MPEP (3 µM). Graph: Representative cell (red) and pooled data (blue).

Figure 5. Chem-LTPm is associated with changes in bouton Ca²⁺ signalling.

(a) Schematic of technique: single action potentials were evoked in patched granule cells every 30 s. (b) A representative axon from this set of experiments. (c) Representative fluorescence transients and corresponding traces (averages of 10 successive line scans) before (t ∼ 80 min after obtaining a whole cell recording) and after co-application of ATPA (1 µM) and DHPG (3 µM) (t ∼ 100 min). White scale bar = 100 ms. (d) Quantified changes in basal calcium following the chem-LTPm protocol: representative bouton (red) and pooled data (blue).

Figure 6. Evidence for a role of bouton calcium stores in chem-LTPm.

(a) A representative experiment showing fluorescence transients in control conditions (t ∼ 70 min), following the chem-LTPm protocol in the presence of ryanodine (10 µM) (t ∼ 80 min), following washout of ryanodine (t ∼ 115 min) and after re-application of ATPA + DHPG (t ∼ 135 min). (b) The chem-LTPm protocol did not significantly affect basal Ca²⁺ in the presence of ryanodine. (c) The chem-LTPm protocol increased basal Ca²⁺ following washout of ryanodine. (d) Rate of decay of fluorescence transients. (e) Representative cell from this set of experiments.

Figure 7. A proposed mechanism for the induction of mossy fibre LTP.

See text for explanation.