M. V. Catania

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.

Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes

Abstract: Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate ([3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12‐h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl‐3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12‐tetradecanoate 13‐acetate. Glutamate and ibotenate reduced [methyl‐3H]‐thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2‐amino‐4‐phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective. Inhibition of [methyl‐3H]thymidine incorporation by quisqualate required a lag time of about 4 h and, in cells synchronized to proliferate, occurred when the drug was added during the transition between G0 and G1, but not during the S phase of the mitotic cycle. This suggests that an inducible factor may be involved in the antiproliferative effect of excitatory amino acids. Accordingly, activation of quisqualate receptors led to a rapid and transient increase in mRNA levels of the early inducible gene, c‐fos. These results suggest that activation of a specific class of “quisqualate‐preferring”excitatory amino acid receptors reduces proliferation of astrocytes in primary cultures.

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.

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate‐type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)‐ and calretinin (CR)‐positive cells, which represent different populations of non‐principal cells. To this end, non‐radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double‐immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV‐containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR‐A and GluR‐D mRNAs, moderate levels of GluR‐C and low levels of GluR‐B mRNAs in all the examined regions of hippocampus. The vast majority of CR‐containing cells have a much lower expression of GluR‐A, ‐C and ‐D mRNA than PV‐positive neurons, although similarly featuring low levels of GluR‐B mRNA. Only a subpopulation of CR‐containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR‐A and ‐D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR‐B subunit expression, PV‐ and CR‐containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

Phorbol Esters Attenuate Glutamate‐Stimulated Inositol Phospholipid Hydrolysis in Neuronal Cultures

Abstract: The phorbol diesters 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and phorbol‐12,13‐dibutyrate, but not 4–α‐phorbol‐didecanoate, inhibited the stimulation of inositol phospholipid hydrolysis by excitatory amino acids and carbamylcholine in primary cultures of cerebellar neurons. This inhibition was mimicked by the synthetic diacylglycerol 1,2‐dioleoyl‐rac‐glycerol (DOG) and was selective for a specific glutamate‐phosphoinositide receptor subtype (GP2 receptor) activated by glutamate and quis‐qualate. TPA was nearly inactive in inhibiting the stimulation of inositol phospholipid hydrolysis by N‐methyl‐d‐aspartate, a selective agonist of the GP1 receptor. Phorbol diesters and DOG attenuated the stimulation of inositol phospholipid hydrolysis by glutamate and quisqualate also in cerebellar slices from 9–15‐day‐old rats; however, using this preparation, their action was weak and required high concentrations (> 1 μM). The inhibition of signal transduc‐tion by phorbol diesters was not consequent to a reduced binding of glutamate to its membrane recognition sites. In fact, TPA induced only a small increase in the KD but no change in the Bmax of [3H]glutamate binding in cerebellar membranes. Phorbol diesters may act to inhibit specific GTP‐binding proteins or particular molecular forms of phosphoinositidase C associated with GP2 or muscarinic cholinergic receptors.

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.

Adenosine deaminase increases release of excitatory amino acids through a mechanism independent of adenosine depletion.

Addition of adenosine deaminase to cultured cerebellar neurones, led to large increases in the influx of 45Ca2+ and hydrolysis of polyphosphoinositide. These effects were inhibited or attenuated by glutamate receptor antagonists (AP5 or MK-801) and were not observed in cells stimulated by maximum concentrations of glutamate or quisqualate. Stimulation of the influx of 45Ca2+ and hydrolysis of phosphoinositide by adenosine deaminase may be secondary to an enhanced release of endogenous glutamate that in turn activates specific excitatory amino acid receptors. Accordingly, adenosine deaminase potently increased release of D-[3H]aspartate, an effect that requires the presence of extracellular Na+ and is insensitive to inhibition by MK-801. None of the effects of adenosine deaminase may be simply related to a fall in endogenous adenosine. In fact, the action of adenosine deaminase was neither reversed by agonists (L-PIA or NECA), nor mimicked by antagonists (IBMX or theophylline) of adenosine receptors. It is speculated that adenosine deaminase stimulates release of neurotransmitter through a mechanism independent of depletion of adenosine. A possible direct action of adenosine deaminase should be taken into account when the enzyme is used to unmask the effects of endogenous adenosine.

Thyrotropin releasing hormone (TRH) and its analog, RGH-2202, accelerate maturation of cerebellar neurons in vitro

We have studied the trophic action of thyrotropin releasing hormone (TRH) in cultured cerebellar granule cells, a pure and homogeneous population of glutamatergic neurons. As an index of neuronal maturation, we have measured the uptake of D-[3H]aspartate (a non-metabolizable analog of glutamate) at different days of maturation in vitro (DIV). In control cultures, D-[3H]Aspartate increased linearly during maturation reaching plateau values between 7 and 9 DIV; daily addition of TRH tartrate (TRH-t) or RGH-2202 (a TRH analog) accelerated in a concentration-dependent manner the maturation profile of D-[3H]aspartate uptake. This effect was more pronounced for RGH-2202: in cultures treated daily with RGH-2202, D-[3H]aspartate uptake was fully expressed after 3 DIV. Neither TRH-t nor RGH-2202 significantly increased D-[3H]aspartate uptake in mature cells, excluding a direct action on the glutamate transport system. Both compounds specifically potentiated the increase in [3H]inositol monophosphate formation (but not the stimulation of 45Ca2+ influx) induced by N-methyl-D-aspartate (NMDA) receptor agonists, without affecting the stimulation of inositol phospholipid hydrolysis by quisqualate or carbamylcholine. We suggest that, in cultured cerebellar granule cells, TRH and RGH-2202 enhance the trophic action of endogenous glutamate by amplifying some of the intracellular events that follow the influx of extracellular Ca2+ through NMDA-gated ion channels.