Stimulation of Na-K-2Cl Cotransporter in Neurons by Activation of Non-NMDA Ionotropic Receptor and Group-I mGluRs

In a previous study, we found that Na+-K+-2Cl−cotransporter in immature cortical neurons was stimulated by activation of the ionotropic N-methyl-d-aspartate (NMDA) glutamate receptor in a Ca2+-dependent manner. In this report, we investigated whether the Na+-K+-2Cl−cotransporter in immature cortical neurons is stimulated by non-NMDA glutamate receptor–mediated signaling pathways. Expression of the Na+-K+-2Cl−cotransporter and metabotropic glutamate receptors (mGluR1 and 5) was detected in cortical neurons via immunoblotting and immunofluorescence staining. Significant stimulation of cotransporter activity was observed in the presence of both trans-(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ( trans-ACPD) (10 μM), a metabotropic glutamate receptor (mGluR) agonist, and (RS)-3,5-dihydroxyphenylglycine (DHPG) (20 μM), a selective group-I mGluR agonist. Both trans-ACPD and DHPG-mediated effects on the cotransporter were eradicated by bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid–AM, a Ca2+ chelator. In addition, DHPG-induced stimulation of the cotransporter activity was inhibited in the presence of mGluRs antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (1 mM) and also with selective mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (100 μM). A DHPG-induced rise in intracellular Ca2+ in cortical neurons was detected with Fura-2. Moreover, DHPG-mediated stimulation of the cotransporter was abolished by inhibition of Ca2+/CaM kinase II. Interestingly, the cotransporter activity was increased by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These results suggest that the Na+-K+-2Cl−cotransporter in immature cortical neurons is stimulated by group-I mGluR- and AMPA-mediated signal transduction pathways. The effects are dependent on a rise of intracellular Ca2+.

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

Journal of Neurophysiology ◽

10.1152/jn.1997.78.1.63 ◽

1997 ◽

Vol 78 (1) ◽

pp. 63-75 ◽

Cited By ~ 49

Author(s):

Jeffrey G. Netzeband ◽

Kathy L. Parsons ◽

Dan D. Sweeney ◽

Donna L. Gruol

Keyword(s):

Phospholipase C ◽

Glutamate Receptor ◽

Neuronal Excitability ◽

Metabotropic Glutamate Receptor ◽

Purkinje Neurons ◽

Transduction Pathway ◽

Electrophysiological Response ◽

Metabotropic Glutamate ◽

Group I ◽

Intracellular Ca

Netzeband, Jeffrey G., Kathy L. Parsons, Dan D. Sweeney, and Donna L. Gruol. Metabotropic glutamate receptor agonists alter neuronal excitability and Ca2+ levels via the phospholipase C transduction pathway in cultured Purkinje neurons. J. Neurophysiol. 78: 63–75, 1997. Selective agonists for metabotropic glutamate receptor (mGluR) subtypes were tested on mature, cultured rat cerebellar Purkinje neurons (≥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 (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (300 μM), quisqualate (5 μM), and ( R,S)-3,5-dihydroxyphenylglycine (50–500 μM). 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 (2 S,3 S,4 S)-α-(carboxycyclopropyl)-glycine (10 μM) and the group III (mGluR4/mGluR6/mGluR7/mGluR8) agonists l(+)-2-amino-4-phosphonobutyric acid (1 mM) and O-phospho-l-serine (200 μM) 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 mGluR1α in both the somatic and dendritic regions. All effects of the group I mGluR agonists were blocked by (+)-α-methyl-4-carboxyphenylglycine (1 mM), an mGluR antagonist. Furthermore, the phospholipase C inhibitor 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (2 μM) 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 analogue1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]2,5-pyrrolidine-dione (2 μM) 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.

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Metabotropic glutamate receptor agonist-induced hyperpolarizations in rat basolateral amygdala neurons: receptor characterization and ion channels

Journal of Neurophysiology ◽

10.1152/jn.1996.76.5.3059 ◽

1996 ◽

Vol 76 (5) ◽

pp. 3059-3069 ◽

Cited By ~ 28

Author(s):

K. H. Holmes ◽

N. B. Keele ◽

V. L. Arvanov ◽

P. Shinnick-Gallagher

Keyword(s):

Dicarboxylic Acid ◽

Glutamate Receptor ◽

Basolateral Amygdala ◽

Metabotropic Glutamate Receptor ◽

Outward Current ◽

Channel Blockers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Metabotropic Glutamate ◽

Outward Currents

1. Metabotropic glutamate receptor (mGluR)-agonist-induced hyperpolarizations and corresponding outward currents were analyzed in basolateral amygdala (BLA) neurons in rat brain slice preparations with current-clamp and single-electrode voltage-clamp recording to characterize the mGluR subtype(s) and the ion channel(s) mediating this response. 2. The mGluR agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) induced a membrane hyperpolarization or outward current in BLA neurons in a concentration-dependent manner (median effective concentration = 34 microM; range = 10-200 microM); the 1S,3R-ACPD hyperpolarizations are recorded in 89% of neurons that accommodate or cease firing in response to a 400-ms depolarizing current injection (0.5 nA). 3. mGluR agonists elicited hyperpolarizations or outward currents in a concentration-dependent manner in the following rank order of potency: (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) > 1S,3R-ACPD > (s)-4-carboxyphenylglycine = (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) > L-aminophosphonobutyric acid > (1S,3S)-1-amino-cyclopentane-1,3-dicarboxylic acid. In contrast, the mGluR agonists quisqualate and ibotenate induced only depolarizations in the presence of D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione in BLA neurons. 4. The 1S,3R-ACPD-induced outward current is mediated through a large-conductance calcium-dependent potassium (BK) conductance. The BK channel blockers iberiotoxin and charybdotoxin blocked the response, as did the potassium channel blockers tetraethylammonium and 4-aminopyridine; the small-conductance calcium-activated potassium channel blocker apamin did not affect the response. 5. The mGluR-agonist-induced hyperpolarization is blocked in amygdala slices from animals pretreated with pertussis toxin (PTX). 1S,3R-ACPD hyperpolarizations were recorded in neurons contralateral but not ipsilateral to the site of PTX injection. 6. The antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) reduced significantly the 1S,3R-ACPD-induced hyperpolarization. 7. In conclusion, the relative potency of L-CCG-I and 4C3HPG in evoking only hyperpolarizations (outward currents) in accommodating neurons, and the observation that MCPG (500 microM) reduces the hyperpolarization, suggest that a group-II-like mGluR underlies the hyperpolarizing response. The mGluR-induced response is sensitive to iberiotoxin and to pretreatment with PTX, suggesting activation of BK channels through a group II mGluR linked to a PTX-sensitive G protein in BLA neurons.

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