E. Aronica

Desensitization of metabotropic glutamate receptors in neuronal cultures

Abstract: Preexposure of cultured cerebellar neurons to glutamate reduced the stimulation of polyphosphoinositide (PPI) hydrolysis induced by subsequent addition of glutamate without affecting the response to the muscarinic receptor agonist carbamylcholine. Desensitization of glutamate‐stimulated PPI hydrolysis developed rapidly and persisted up to 48 h after removal of glutamate from the incubation medium. Stimulation of PPI hydrolysis by quisqualate was abolished in cultures pretreated with quisqualate or glutamate, but not with N‐methyl‐D‐aspartate (NMDA). In contrast, pretreatment with NMDA reduced the stimulation of PPI hydrolysis induced by a subsequent addition of NMDA, leaving the action of quisqualate intact. The lack of cross‐desensitization between NMDA and quisqualate supports the existence of two distinct subtypes of glutamate receptors coupled to PPI hydrolysis. Desensitization induced by a 30‐min (but not by a 6‐h) exposure to glutamate was attenuated or prevented by putative protein kinase C inhibitors, including mono‐ and trisialogangliosides, sphingosine, and polymyxin B, but not by inhibitors of arachidonic acid metabolism, nor by the nonselective calpain inhibitor leupeptin, nor by the lectin concanavalin A. These results suggest that desensitization of metabotropic glutamate receptors involves, at least in its rapid component, activation of protein kinase C.

Enhanced Sensitivity of “Metabotropic” Glutamate Receptors After Induction of Long-Term Potentiation in Rat Hippocampus

Stimulation of [3H]inositol monophosphate <[3H]InsP) formation by ibotenate or trans‐1‐aminocyclopentyl‐l, 3‐dicarboxylic acid (t‐ACPD) in rat hippocampal slices was enhanced after tetanic stimulation of die Schaffer collaterals projecting to the CA1 region (in vitro) or the perforant pathway projecting to the dentate gyms (in freely moving animals). This effect was observed 5 h (but not 2 h) after long‐term potentiation (LTP) induction and was abolished if tetanic stimulation was performed in the presence of specific antagonists of N‐methyl‐D‐aspartate receptors. The delayed increase in excitatory amino acid‐induced polyphosphoinositide (PPI) hydrolysis was accompanied by an enhanced responsiveness to norepinephrine, whereas the basal and carbamylcholine‐stimulated [3H]InsP formation were unchanged. These results suggest that an increased activity of „metabotropic” glutamate receptors may contribute to the synaptic mechanisms enabling the late expression and or maintenance of LTP. Accordingly, LTP decayed more rapidly (within 5 h) in rats repeatedly injected with LiCl (60–120 mg/kg, i.p., for 10 days), a treatment that led to a reduced efficacy of ibotenate and norepinephrine in stimulating PPI hydrolysis in hippocampal slices.

Metabotropic Glutamate Receptors and Neuronal Toxicity

Specific glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis have been described in brain slices, cultured neurons, and astrocytes, and in amphibian oocytes injected with rat brain mRNA (Sladeczek et al., 1985; Nicoletti et al., 1986a,b; Sugiyama et al., 1987). In most of the systems, metabotropic receptors are activated by lS,3R-aminocyclopentandicarboxylic acid (ACPD), quisqualate, ibotenate, and L-glutamate, but not by α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) (Nicoletti et al., 1986a; Schoepp and Johnson, 1988; 1989; Palmer et al., 1989). Trans-ACPD has been described as the most selective agonist of metabotropic receptors (Palmer et al., 1989), although it is less potent than quisqualate in stimulating inositolphosphate formation. In brain slices, stimulation of PPI hydrolysis by metabotropic receptor agonists is extremely high at the earlier stages of postnatal development (within the first 2 weeks after birth) and progressively declines during maturation (Nicoletti et al., 1986a). In adult tissue, the activation of metabotropic receptors is amplified in response to deafferentation (Nicoletti et al., 1987), as well as after induction of long-term potentiation (Aronica et al., 1991) or electrical kindling (Iadarola et al, 1986; Akiyama et al., 1987). Hence, it is likely that metabotropic receptors contribute to the synaptic events involved in the regulation of neuronal plasticity. However, based on the toxic effects of quisqualate in hippocampal slices (Garthwaite and Garthwaite, 1989) and cultured cortical neurons (Patel et al., 1990), a role for metabotropic receptors in the mechanism of neuronal degeneration has been suggested. We have addressed this problem in primary cultures of cerebellar neurons.

Melittin enhances excitatory amino acid release and AMPA-stimulated 45Ca2+ influx in cultured neurons

Melittin, a potent activator of phospholipase A2, enhanced both spontaneous and depolarization-induced release of D-[3H]aspartate in primary cultures of cerebellar granule cells. The action of melittin was concentration-dependent (EC50 value = 300 ng/ml) and did not require the presence of extracellular Ca2+. Melittin also stimulated the release of glutamate and aspartate, in addition to other endogenous amino acids (taurine, alanine and gamma-aminobutyric acid). These effects were accompanied by an enhanced influx of 45Ca2+, which was in part mediated by the activation of excitatory amino acid receptors by endogenous agonists. Low concentrations of melittin (50 ng/ml) potentiated the efficacy of AMPA in stimulating 45Ca2+ influx without affecting stimulation by kainate or by glutamate added in the absence of extracellular Mg2+ (a condition that favors the activation of NMDA receptors). These results indicate that activation of phospholipase A2 evokes both an enhanced glutamate release and an increased sensitivity of AMPA receptors, two events that may support synaptic facilitation and LTP formation.