Metabotropic Glutamate Receptor Agonists Alter Neuronal Excitability and Ca²⁺Levels via the Phospholipase C Transduction Pathway in Cultured Purkinje Neurons

Abstract

Selective agonists for metabotropic glutamate receptor (mGluR) subtypes were tested on mature, cultured rat cerebellar Purkinje neurons (> or = 21 days in vitro) to identify functionally relevant mGluRs expressed by these neurons and to investigate the transduction pathways associated with mGluR-mediated changes in membrane excitability. Current-clamp recordings (nystatin/perforated-patch method) were used to measure the membrane response of Purkinje neurons to brief microperfusion pulses (1.5 s) of the group I (mGluR1/mGluR5) agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (300 microM), quisqualate (5 microM), and (R,S)-3,5-dihydroxyphenylglycine (50-500 microM). All group I mGluR agonists elicited biphasic membrane responses and burst activity in the Purkinje neurons. In addition, the group I mGluR agonists produced alterations in the active membrane properties of the Purkinje neurons and depressed the OFF response after hyperpolarizing current injection. In parallel microscopic Ca2+ imaging experiments, application of the group I mGluR agonists to fura-2-loaded cells elicited increases in intracellular Ca2+ in both the somatic and dendritic regions. The group II (mGluR2/mGluR3) agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (10 microM) and the group III (mGluR4/mGluR6/mGluR7/mGluR8) agonists L(+)-2-amino-4-phosphonobutyric acid (1 mM) and O-phospho-L-serine (200 microM) had no effect on the membrane potential or intracellular Ca2+ levels of the Purkinje neurons. The cultured Purkinje neurons, but not granule neurons or interneurons, showed immunostaining for mGluR1alpha in both the somatic and dendritic regions. All effects of the group I mGluR agonists were blocked by (+)-alpha-methyl-4-carboxyphenylglycine (1 mM), an mGluR antagonist. Furthermore, the phospholipase C inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (2 microM) blocked the group I mGluR agonist-mediated electrophysiological response and greatly attenuated the Ca2+ signal elicited by group I mGluR agonists, particularly in the dendrites. The inactive analogue 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidine-dione (2 microM) was relatively ineffective against the electrophysiological response and Ca2+ signal. These results indicate that functional group I mGluRs (but not group II or III mGluRs) can be activated on mature Purkinje neurons in culture and result in changes in neuronal excitability and intracellular Ca2+ mediated through phospholipase C. These data obtained from a defined neuronal type, the Purkinje neuron, confirm biochemical and molecular studies on the transduction mechanisms of group I mGluRs and show that this transduction pathway is linked to neuronal excitability and intracellular Ca2+ release in the Purkinje neurons.

L-Type Calcium Channels Mediate Calcium Oscillations in Early Postnatal Purkinje Neurons

“…Electrophysiological recordings were performed in combination with recordings of intracellular C a 2ϩ or in parallel with C a 2ϩ -imaging studies (see above). Current-clamp recordings were made from the somatic region of acutely isolated Purkinje neurons using the nystatin, perforated-patch method of whole-cell recording and the Axopatch-1C amplifier (Axon Instruments, Foster C ity, CA) following previously described methods (Netzeband et al, 1997). Briefly, patch pipettes (2-4 M⍀) were filled with a pipette solution containing 6 mM NaC l, 154 mM K ϩ -gluconate, 10 mM H EPES, and 200 g /ml nystatin (Sigma), pH adjusted to 7.3 with KOH.…”

“…The acutely dissociated cerebellar cells were immunostained according to standard methods (Netzeband et al, 1997). C ells were fixed for 15 min in 4% paraformaldehyde (or 4% paraformaldehyde and 0.1% glutaraldehyde for GABA immunostaining) and washed three times (10 min) with 5% sucrose in PBS and once with PBS (10 min).…”

Synaptic Control of Motoneuronal Excitability

“…Group I mGluR appear to be localized postsynaptically (71,762,800,1146) where they act to increase neuronal excitability (905,1105,1108). Group II and III receptors are predominantly localized in presynaptic terminals (135,638,735,1146,1147) where they inhibit transmitter release (70,373,419,773,1075,1107,1272,1314).…”

Presynaptic group I metabotropic glutamate receptors modulate synaptic transmission in the rat superior colliculus via 4‐AP sensitive K+ channels

“…Firstly, we used slices incubated with the PLC inhibitor U-73122 (2 mM, Figure 6a, n ¼ 8) for 2 h prior to DHPG application (the compound was also present throughout the experiment). In Purkinje neurons, this approach had been found to inhibit DHPG responses (Netzeband et al, 1997), but in SC slices no effect of U-73122 on DHPG responses was obtained (DHPGinduced inhibition in U-73122: 3272%, P40.05). Subsequently, we investigated whether PKC activation was necessary for, or involved in, the action of DHPG.…”