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Department of Neuropharmacology and Alcohol Research Center, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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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 (1S,3R)-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 (2S,3S,4S)-
-(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 mGluR1 in both the somatic and dendritic regions. All effects of the group I mGluR agonists were blocked by (+)--methyl-4-carboxyphenylglycine (1 mM), an mGluR antagonist. Furthermore, the phospholipase C inhibitor 1-[6-((17
-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-((17-3-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.
The excitatory amino acid glutamate has been shown to excite nearly all neuronal types in the CNS and appears to be the most prevalent excitatory neurotransmitter in the vertebrate CNS. Glutamate exerts its actions through a variety of receptor subtypes that can be broadly classified as either metabotropic or ionotropic. Metabotropic glutamate receptors (mGluRs) are membrane proteins that couple agonist binding to one of several second-messenger systems via guanosine 5 Culture techniques
Modified organotypic cultures containing Purkinje neurons were prepared from embryonic day 20 rat (Sprague-Dawley, Charles River) cerebellar cortices and maintained in vitro for 3 wk as described previously (Gruol 1983 Immunohistochemical techniques
Immunohistochemical staining of the cerebellar cultures was performed with an antibody to mGluR1 Electrophysiological techniques
Mature Purkinje neurons (i.e., Intracellular calcium measurement
Intracellular Ca2+ levels were determined for individual cells with the use of standard microscopic fura-2 digital imaging techniques (Gruol and Curry 1995 Pharmacology
All agents were prepared as stock solutions in water or 0.1 N NaOH (unless otherwise indicated) and then dissolved in bath saline (see above) at the appropriate concentrations. The mGluR agonists used were (1S,3R)-ACPD (the active stereoisomer oftrans-ACPD; 300 µM), quisqualate (5 µM), (R,S)-3,5-dihydroxyphenylglycine (DHPG, 50-500 µM), L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 200-1,000 µM), O-phospho-L-serine(L-SOP, 200-1,000 µM), and (2S,3S,4S)- Data analysis
Data acquired by the nystatin-patch method and during the first 30 min of whole cell recording were pooled for analysis. Over the first 15-30 min of whole cell recording (following membrane rupture), minimal changes occurred in resting membrane potential, passive and active membrane properties, or the membrane response of cells to (1S,3R)-ACPD or quisqualate. Cell rundown was often observed at times >30 min in the whole cell mode, but rarely with nystatin-patch recordings, even at 1-2 h.
Effects of group I mGluR agonists on Purkinje neurons
The message and protein for mGluR1, and in particular the mGluR1
Effects of group II and III agonists
Purkinje neurons have been shown to express mRNA for mGluR7 (Kinzie et al. 1995 Transduction pathway for mGluR1-mediated responses
mGluRs are coupled to effector pathways via G proteins. Group II and III mGluRs have been shown to be negatively coupled to adenylyl cyclase through pertussis toxin-sensitive Gi/Go proteins, whereas group I mGluRs have been reported to couple to phosphoinositide turnover via both pertussis toxin-sensitive (Gi/Go) and -insensitive (Gq) G proteins (Pin and Duvoisin 1995
We have found that the group I mGluR agonists (1S,3R)-ACPD, quisqualate, and DHPG elicit complex biphasic membrane responses, Ca2+ signals, and changes in the active membrane properties of cultured cerebellar Purkinje neurons. Several lines of evidence indicate that (1S,3R)-ACPD, quisqualate, and DHPG produced their effects through a group I mGluR. 1) All three agonists produced similar responses, suggesting that similar transduction mechanisms were activated. 2) DHPG is highly selective for the group I mGluRs over the group II and III mGluRs, suggesting that a group I mGluR was involved. 3) (1S,3R)-ACPD can act at group II and III mGluRs; however, group II and III mGluR agonists did not have an effect on the Purkinje neurons. Thus, although (1S,3R)-ACPD is nonselective for mGluRs, (rank order of potency: group II > group I > group III), it is likely that (1S,3R)-ACPD was acting predominantly through a group I mGluR in our studies. 4) Quisqualate, like DHPG, is selective for the group I mGluRs over the group II and III mGluRs, but also has actions on ionotropic AMPA receptors. In our studies, the actions of quisqualate on AMPA receptors were blocked with DNQX, a competitive antagonist of the AMPA receptor. Also, under conditions in which AMPA receptors are not blocked, application of quisqualate to the cultured Purkinje neurons produces significantly larger membrane depolarizations (20-30 mV) than observed in the present studies (5-8 mV) (Yool et al. 1992
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-triphosphate (GTP)-binding proteins (G proteins; reviewed in Bockaert et al. 1993
and Pin and Duvoisin 1995). In contrast, ionotropic glutamate receptors (iGluRs) consist of membrane proteins that form intrinsic ion channels. The iGluRs are referred to as -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors according to their selective agonists (Monaghan et al. 1989).
). Furthermore, mGluR activation can be both neuroprotective or neurotoxic (Patel and Zinkand 1994), and thus may be involved in the etiology of neurological disease. Eight mGluR subtypes have been cloned (designated mGluR1-mGluR8), and these have been subdivided into three main subfamilies on the basis of agonist selectivity and signal transduction mechanisms (Pin and Duvoisin 1995). Thus multiple pathways exist by which mGluRs could alter neuronal excitability. The differential distributions of mGluRs (Abe et al. 1992; Duvoisin et al. 1995; Fotuhi et al. 1993; Hampson et al. 1994; Kinzie et al. 1995; Nakajima et al. 1993; Ohishi et al. 1993, 1995; Shigemoto et al. 1992; Tanabe et al. 1993), G proteins (Brann et al. 1987; Mailleux et al. 1992), and the multiple isoforms of transducer elements (e.g., phospholipase C, adenylyl cyclase) (Fotuhi et al. 1993; Ross et al. 1989; Yamada et al. 1991) in the brain (as shown by in situ hybridization or immunohistochemistry) suggest that the neuronal responses mediated through a specific mGluR subtype could vary dramatically between brain regions and neuronal types.
,5-cyclic monophosphate levels as markers of mGluR activity (Bockaert et al. 1993; Pin and Duvoisin 1995). Much less is known, however, about the transduction pathways involved in mGluR-mediated changes in neuronal excitability in defined neuronal types. Thus in the current study we have investigated the transduction pathway(s) and changes in neuronal excitability linked to mGluR activation in an identified neuronal type, the cerebellar Purkinje neuron.
; Masu et al. 1991) and protein (Baude et al. 1993; Fotuhi et al. 1993; Hampson et al. 1994; Martin et al. 1992; Nusser et al. 1994; Shigemoto et al. 1994) for mGluR1, as well as for phospholipase C (Ross et al. 1989; Yamada et al. 1991) and 1,4,5-inositol trisphosphate (IP3) receptors (Fotuhi et al. 1993). Activation of mGluR1 is thought to activate phospholipase C, which hydrolyzes membrane phospholipids into IP3 and diacylglycerol (Bockaert et al. 1993). Purkinje neurons also express low levels of mRNA transcript for mGluR7 (Kinzie et al. 1995; Ohishi et al. 1995), which is thought to be negatively linked to adenylyl cyclase. The message for other mGluR subtypes (mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, and mGluR8) has not been detected in Purkinje neurons (Abe et al. 1992; Duvoisin et al. 1995; Nakajima et al. 1993; Nakanishi 1992; Ohishi et al. 1993; Tanabe et al. 1993), although it is possible that the abundance of the message is too low for detection.
; Linden et al. 1994; Lingenhohl et al. 1993; Staub et al. 1992; Vranesic et al. 1993; Yool et al. 1992) and to increase intracellular Ca2+ levels (Gruol et al. 1996; Linden et al. 1994; Staub et al. 1992; Yuzaki and Mikoshiba 1992) in Purkinje neurons. These results indicate that Purkinje neurons express functional mGluRs and that mGluR activation produces a complex physiological response. Furthermore, recent studies suggest that mGluRs contribute to glutamatergic transmission at the parallel fiber-Purkinje neuron synapse under conditions of high-frequency stimulation (Batchelor and Garthwaite 1993; Batchelor et al. 1994). mGluRs have also been implicated in the acquisition of long-term depression in Purkinje neurons (Linden et al. 1991; Shigemoto et al. 1994), a mechanism that may underlie motor learning in the cerebellum.
; Gruol and Crimi 1988; Gruol and Franklin 1987), including sensitivity to iGluR and mGluR agonists (Franklin and Gruol 1991; Joels et al. 1989; Yool et al. 1992). Our current results demonstrate that mGluR agonists elicit changes in membrane excitability and intracellular calcium in cultured Purkinje neurons via a group I mGluR (presumably mGluR1). Furthermore, these studies show that the group I mGluR agonist-mediated effects were transduced through phospholipase C via a pertussis toxin-insensitive G protein, consistent with studies in which cloned mGluRs were used in nonneuronal cells. Some of these data have appeared in abstract form (Netzeband et al. 1995).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Gruol and Crimi 1988). Briefly, cerebella were isolated, minced, and triturated without enzymatic treatment. The resultant cell suspension was plated on Matrigel (Collaborative Biochemical)-coated tissue culture dishes or coverglasses containing minimal essential medium with Earle's salts and L-glutamine (MEM, Gibco), 10% fetal calf serum (heat inactivated, Gibco), 10% horse serum (heat inactivated, Gibco), and was supplemented with D-glucose to a final concentration of 5.0 g/l. Cultures were incubated at 37°C in a 5% CO2 humidified atmosphere. Medium was changed twice weekly. Brief treatment with 5-fluorodeoxyuridine (20 µg/ml, 3 days, Sigma) was begun at the first medium change (day 3 in vitro) to retard the growth of nonneuronal cells. No antibiotics were used.
(Pharmingen; antisera used at 1:250 dilution) and with the use of techniques reported previously (Gruol and Crimi 1988; Gruol and Franklin 1987). In brief, cultures were rinsed with serum-free MEM and fixed with a solution of 3% paraformaldehyde in phosphate-buffered saline (100 mM, pH 7.3) for 15 min. The cultures were then rinsed free of paraformaldehyde and treated with 0.05% Triton X-100 in phosphate-buffered saline for 30 min. The fixed cells were incubated overnight (4°C) in phosphate-buffered saline containing primary antibody and 0.5% bovine serum albumin. Immunoreactivity was detected the following day by an immunoperoxidase reaction with the use of the materials and procedures provided in the Vectastain kit (Vector Laboratories). No staining was observed when the entire procedure was performed in the absence of primary antibody.
21 days in culture) were used for these studies. Purkinje neurons in culture were identified by size and morphology; this method of identification has been verified with a specific immunohistochemical marker (Gruol and Franklin 1987). Before recording, the culture medium was replaced with physiological saline that consisted of (in mM) 140 NaCl, 3.5 KCl, 0.4 KH2PO4, 1.25 Na2HPO4, 2.2 CaCl2, 2 MgSO4, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)-NaOH, pH 7.3. In most experiments, bicuculline (30 µM, methiodide salt, Sigma) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 50 µM, Tocris Cookson) were added to the bath saline and drug solutions (see below) to block synaptic input. This treatment completely suppressed synaptic potentials in the Purkinje neurons.
. The tips of patch pipettes (2-4 M
) were first filled with nystatin-free solution, and then the pipettes were backfilled with the nystatin-containing solution. The recording pipette solution contained (in mM) 6 NaCl, 154 K+-gluconate, 2 MgCl2, 10 HEPES-KOH, 10 glucose, 0.5 CaCl2, pH 7.3, and 1 bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid. Stock solutions of nystatin (Sigma) were made fresh the day of the experiment. Nystatin was prepared in dimethyl sulfoxide (DMSO, 50 mg/ml) and then diluted in the pipette solution to a final concentration of 200 µg/ml. For whole cell recordings, microelectrodes were filled with the same solution as above, except that the solution contained 2 mM Mg2+-ATP and 0.3 mM Na+3-GTP (and no nystatin). The free Ca2+ concentration of these solutions was calculated to be 0.1 µM. Recordings were made at room temperature (21-23°C).
62 mV. Active and passive membrane properties were measured under control conditions and during the agonist-induced response. Voltage responses elicited by a standardized series of hyperpolarizing and depolarizing current pulses (500 ms in duration) were used to assess membrane properties. Measurements were made immediately before agonist application and during the hyperpolarizing phase (typically 1-2 min after agonist application) of the agonist-induced response. Recordings were monitored on a polygraph and oscilloscope. For better resolution of fast events, selected data were recorded on frequency-modulated tape (Racal Store 4DS recorder) for playback at reduced tape speed onto a polygraph recorder (Gould). Polygraph recordings were used for manual measurement of agonist-induced responses.
) based on the methods of Grynkiewicz et al. (1985). In brief, Purkinje neurons were loaded for 30 min with the Ca2+-sensitive dye fura-2/AM (3 µM, Molecular Probes) in physiological saline (see above) containing 0.02% pluronic F-127 (Molecular Probes). The fura-2 solution was then removed and cells were incubated in dye-free saline solution for an additional 45 min to allow for cleavage of the acetoxymethyl ester. Coverglasses were mounted in a chamber attached to the stage of an inverted microscope (Nikon Diaphot) and fields of neurons were selected for study under phase-contrast or bright-field optics.
Rmin)/Rmax R)]Fo/Fs, where R is the ratio value, Rmin is the ratio for a Ca2+-free solution, Rmax is the ratio for a saturated Ca2+ solution, Kd = 135 (the dissociation constant for fura-2), Fo is the intensity of a Ca2+-free solution at 380 nm, and Fs is the intensity of a saturated Ca2+ solution at 380 nm. Calibration was performed with the use of fura salt (100 µM) in solutions of known calcium concentration.
-(carboxycyclopropyl)-glycine (L-CCG-I, 10-100 µM), all from Tocris Cookson. mGluR agonists were applied by rapid microperfusion (1.5 s) from glass micropipettes (tip 1-2 µm) placed under visual control near target neurons. Fast Green (Sigma) was included in all agonist solutions so that neuronal exposure could be monitored. Fast Green (0.03%) by itself had no effect on neuronal responses.
-methyl-4-carboxyphenylglycine [(+)-MCPG, 1 mM, Tocris], 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122, 1-5 µM, Calbiochem), and 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidine-dione (U-73343, 2 µM, Calbiochem). Stock solutions of U-73343 and U-73122 (3.3 mM) were prepared in DMSO and diluted in bath saline (final concentration of 0.03-0.06% DMSO); a corresponding concentration of DMSO was added to the bath saline for control recordings in these experiments.
-aminobutyric acid-B (GABAB) receptor agonist baclofen (100 µM, Sigma) on granule neurons in culture. We have previously found that overnight pertussis toxin treatment blocks baclofen-mediated hyperpolarizations in the granule neurons (unpublished observations).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
splice variant, have been shown to be expressed in abundance in cerebellar Purkinje neurons in vivo (Baude et al. 1993; Fotuhi et al. 1993; Hampson et al. 1994; Martin et al. 1992; Masu et al. 1991; Nusser et al. 1994; Shigemoto et al. 1994). We also found strong immunostaining for mGluR1 in both the somatic and dendritic regions of the cultured Purkinje neurons (Fig. 1). Immunostaining for mGluR1 was detected in the Purkinje neurons as early as 9 days in culture (earlier times not examined). In contrast, few of the cultured granule neurons and inhibitory interneurons showed immunoreactivity for the mGluR1 antibody. These results are in general agreement with studies examining the expression and localization of mGluR1 in the cerebellum in vivo (Baude et al. 1993; Martin et al. 1992; Masu et al. 1991).
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FIG. 1.
Localization of metabotropic glutamate receptor (mGluR)1 immunoreactivity to Purkinje neurons in mature, cultured rat cerebellar neurons. Cultures were immunostained with antisera to mGluR1 and observed under Hoffman optics to visualize mGluR1 immunoreactivity. Both the soma (P) and dendrites (open arrows) of the Purkinje neuron were immunoreactive for mGluR1 antibody. However, note that the antibody did not label neighboring granule neurons (g) and inhibitory interneurons (i).
21 days in vitro) to assess the changes in neuronal excitability produced by these agents. DHPG and quisqualate are selective for group I mGluRs, whereas (1S,3R)-ACPD is nonselective for the three mGluR groups (order of potency: group II > group I > group III). Standard test doses of (1S,3R)-ACPD (300 µM), quisqualate (5 µM), and DHPG (200 and 500 µM) were chosen on the basis of the reported median effective concentrations (EC50s) for these agonists (Ito et al. 1992; Pin and Duvoisin 1995), as well as the ability of the agonists to produce robust electrophysiological responses in the Purkinje neurons. Recordings were made in the presence of the AMPA and GABAA receptor antagonists DNQX (50 µM) and bicuculline (30 µM), respectively, to block synaptic events and thus isolate the direct actions of the mGluR agonists on Purkinje neurons. The synaptic events produced in Purkinje neurons by the non-Purkinje neuronal types in culture are mediated almost exclusively by GABAA and AMPA receptors; Purkinje neurons from mature animals or in culture do not express functional NMDA receptors (Joels et al. 1989; Krupa and Crepel 1990). The DNQX served the additional purpose of blocking the direct activation of AMPA receptors by quisqualate.
51 ± 0.5 (SE) mV (range 43 to 58 mV, n = 47), which is comparable with the resting membrane potential recorded in the presence of 500 nM TTX to block spontaneous spike activity (52 ± 1.0 mV, range 48 to 54 mV, n = 7). A few cells (n = 7) displayed spontaneous burst activity (i.e., 2 action potentials superimposed over a plateau potential) in addition to the single spikes or doublets. (1S,3R)-ACPD, DHPG, and quisqualate all elicited prolonged biphasic changes in the spike activity and membrane potential of Purkinje neurons consisting of an initial membrane depolarization and increase in firing rate followed by a small hyperpolarization and a decrease or complete block of activity (Fig. 2). The responses to (1S,3R)-ACPD, DHPG, and quisqualate were similar in general form, duration, and amplitude, although there was some cell-to-cell variability in the responses to all three agonists. Responses for each agonist ranged from 1 to 3 min in duration, with the hyperpolarizing phase typically longer than the depolarizing phase. In addition, the increase in spike activity was accompanied by an increase in the appearance of burst events in ~50% of the cells tested with the group I mGluR agonists (Fig. 2).
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FIG. 2.
Group I mGluR agonist-mediated responses in Purkinje neurons. A-C: mGluR1 agonists (R,S)-3,5-dihydroxyphenylglycine (DHPG, 500 µM, A), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD, 300 µM, B], and quisqualate (5 µM, C) all elicited complex, biphasic voltage responses in cultured Purkinje neurons at resting membrane potential ( 50 to 55 mV). Agonists were applied by rapid microperfusion (1.5 s in duration, 
). For each agonist-induced response shown, selected portions of response are shown at faster time scales (a-d). Spontaneous spike activity under baseline conditions is labeled a. Biphasic responses consisted of an initial excitatory phase characterized by a membrane depolarization, an increase in single-spike firing frequency (b), and appearance of burst activity (c). Excitation was followed by an inhibitory phase consisting of a membrane hyperpolarization and loss of spike activity. Recovery is labeled d. Recordings were made with the use of either whole cell (A and C) or nystatin-patch techniques (B). In this and all figures of spike activity, amplitudes of spikes are not fully reproduced.
; Linden et al. 1994; Staub et al. 1992; Yuzaki and Mikoshiba 1992). Agonist-evoked Ca2+ signaling events provide an additional method for establishing the presence of group I mGluRs and for examining the effect of pharmacological manipulations on the group I mGluR pathway. Thus Ca2+ imaging studies in which the Ca2+-sensitive dye fura-2 was used were performed in parallel with electrophysiological studies on Purkinje neurons from the same culture sets. As previously described (Gruol et al. 1996), (1S,3R)-ACPD and DHPG produced increases in intracellular Ca2+ in both the somatic and dendritic regions of the cultured Purkinje neurons (Figs. 3, 7, and 9). Somatic increases in Ca2+ were slightly larger than dendritic changes in Ca2+. The Ca2+ responses were 20-40 nM in amplitude, with the maximum occurring 6-12 s after agonist application. Ca2+ levels recovered to baseline concentrations over the next 30-40 s. A comparison of the Ca2+ signals and electrophysiological responses indicated that the Ca2+ signals correlated with the agonist-mediated depolarization (Figs. 3 and 7). These data show that functional group I mGluRs are expressed by the cultured Purkinje neurons and mediate prolonged changes in membrane excitability and intracellular Ca2+ levels.
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FIG. 3.
Dose-dependent effects of DHPG. This series of experiments was performed in the presence of 500 nM tetrodotoxin (TTX) to block synaptic input and spike firing. A, left: examples of dose-dependent effects of DHPG (50 and 200 µM, 1.5-s application at ) on Ca2+ signals. Each dose of DHPG was tested on a different cell. A, right: mean values for somatic and dendritic Ca2+ signals to a range of doses of DHPG (50-500 µM). B, left: examples of dose-dependent effects of DHPG (50 and 200 µM, 1.5-s application at ) on membrane response of a single Purkinje neuron. Same time scale as in A. B, right: mean values for amplitude and duration of depolarizing phase of DHPG-mediated membrane response at different concentrations of DHPG (range from 50 to 500 µM). All results obtained at resting membrane potential. Values in parentheses: number of cells tested. C: example of changes in conductance produced by application of DHPG (200 µM, 1.5-s application at ). Downward deflections are voltage responses of cell to repetitive pulses of constant hyperpolarizing current (150 pA). Conductance increase during peak of depolarization was not mimicked by manual depolarization (
) to same potential. Membrane potential was adjusted to baseline during hyperpolarization () to show that there was little change in conductance during the hyperpolarizing phase.
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FIG. 7.
Lack of effect of the group III mGluR agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) on Purkinje neurons. A: representative voltage response of a Purkinje neuron to (1S,3R)-ACPD (300 µM, 1.5-s application at ) and lack of effect to L-AP4 (1 mM, 1.5-s application at ) in same neuron. B: representative examples of (1S,3R)-ACPD-elicited increases in Ca2+ levels in somatic and dendritic regions of a neuron. In another neuron, L-AP4 did not alter Ca2+ levels. (1S,3R)-ACPD and L-AP4 were applied as in A.
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FIG. 9.
Involvement of phospholipase C in group I mGluR agonist-mediated Ca2+ response. A: typical examples of somatic Ca2+ response induced by DHPG (200 µM, 1.5-s application at ) under control conditions and in the presence of the phospholipase C inhibitor U-73122 or its inactive analogue U-73343 in the bath. Each response is from a different cell. Similar findings were observed in the dendritic region. B: mean peak amplitude of somatic and dendritic Ca2+ responses induced by DHPG under conditions listed above. Analysis was performed by analysis of variance, followed by the Scheffe post hoc test for multiple comparisons. Asterisk: P < 0.05; significant difference compared with control. Double dagger: P < 0.05; significant difference between U-73343 and U-73122. Number of cells tested with each treatment is listed in respective bar of graph.
20 to 105 pA, 500 ms) given before application of DHPG (200 µM, 1.5 s) and during the DHPG-mediated membrane response (Fig. 3C). Near the peak of the DHPG-mediated depolarization, the apparent conductance was increased to 174 ± 23% of the baseline conductance (n = 4). When the membrane potential was changed manually to a similar potential, the apparent conductance increased to 124 ± 11% of the baseline conductance, which was significantly smaller than the DHPG-induced conductance change (P < 0.05, paired t-test, n = 4). There was no significant change in conductance at the peak of the DHPG-mediated hyperpolarizing phase (data not shown).
62 mV. At this potential, spontaneous spike activity was considerably reduced, which allowed for more accurate measurement of agonist-induced changes in membrane potential and the active/passive membrane properties. Neurons were chosen for study if they 1) were stable at 62 mV, 2) had action potentials that overshot zero potential, and 3) had active and passive membrane properties indicative of uninjured cells.
62 mV, the group I mGluR agonists elicited prominent biphasic membrane responses (38 of 49 cells) and increases in spike activity, including burst activity in 60% (27 of 45) of the cells tested. Figure 4A shows a typical example of the (1S,3R)-ACPD-mediated response of a Purkinje neuron at 62 mV; the inset shows the burst activity. Similar responses were obtained with quisqualate and DHPG. The actions of (1S,3R)-ACPD were stereoselective, because similar concentrations of (1R,3S)-ACPD, the inactive stereoisomer of trans-ACPD, did not elicit changes in the membrane potential of Purkinje neurons (n = 5, Fig. 4A). Even high concentrations of the inactive isomer (1 mM) failed to elicit any response (n = 2, data not shown).
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FIG. 4.
Group I mGluR agonist-mediated effects on Purkinje neurons at a standardized membrane potential of 62 mV. A: example of biphasic membrane response produced by (1S,3R)-ACPD (300 µM) in a Purkinje neuron at 62 mV. Inset: burst activity elicited by (1S,3R)-ACPD. In same cell, inactive isomer (1R,3S)-ACPD (300 µM) had no effect on membrane potential. Agonists were applied at arrows by a 1.5-s rapid microperfusion pulse. Recordings were made with the use of nystatin-patch techniques. 6,7-Dinitroquinoxaline-2,3-dione (DNQX, 50 µM) and bicuculline (30 µM) were present in the bath. B: mean values for peak amplitude and duration of depolarizations elicited by group I mGluR agonists (1S,3R)-ACPD (300 µM), quisqualate (5 µM), and DHPG (500 µM) at standardized potential of 62 mV. Depolarizations elicited by the 3 agonists were similar in amplitude and duration at the doses tested. Bars: means ± SE.
62 mV before measurements were made.
; Gruol et al. 1992). At the termination of the hyperpolarizing current pulse, the membrane potential overshot baseline (62 mV), often producing a burst event (OFF response). Depolarizing current pulses typically initiated an initial burst event that was followed by repetitive single spikes (Fig. 5). An afterhyperpolarization occurred at the termination of the depolarizing current pulse. For the response to depolarizing current pulses, measurements were made of the total spike number, sustained voltage level (measured at the end of the current pulse), and amplitude of the afterhyperpolarization. For the response to hyperpolarizing current pulses, measurements were made of the peak and sustained voltage levels. Input-output curves were constructed from the peak and sustained voltages.
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FIG. 5.
Effects of group I mGluR agonists on membrane properties. A: characteristic membrane responses (see text for details) elicited by constant hyperpolarizing or depolarizing current injections (±90 pA in amplitude, 500 ms in duration) under baseline conditions (left) and during hyperpolarizing phase of quisqualate-induced response (right). B: comparison of single spikes evoked by constant depolarizing current pulses under control conditions (A) and in presence of quisqualate. Spikes are same data as shown in A and were selected at times indicated by closed arrowheads. Note presence of the afterdepolarizing potential following the action potential evoked during the quisqualate response, but not during the control response. Peaks of action potentials were aligned for comparison. C: comparison of burst events occurring at termination of constant hyperpolarizing current under control conditions (A) and in presence of quisqualate. Spikes are same data as shown in A and were taken at times indicated by open arrowheads. Note that the plateau potential of the burst was shortened during the quisqualate-mediated response. Initial action potential of each burst was aligned for comparison. All recordings were from the same cell at a standardized potential of 62 mV. Similar effects were observed when the other group I mGluR agonists, (1S,3R)-ACPD or DHPG, were used. Electrophysiological recordings were made as described in Fig. 4A. D: effect of DHPG (200 µM, 1.5 s) on OFF response in presence of 500 nM TTX. These experiments were similar to those described above, except that they were performed at resting membrane potential. Recovery of all effects occurred within 5 min of agonist application (not shown). AHP, afterhyperpolarization.
). Therefore we tested the effect of this antagonist to determine whether it could block the electrophysiological and Ca2+ responses to the mGluR agonists. (+)-MCPG (1 mM) reduced the mean peak depolarizations induced by (1S,3R)-ACPD (n = 6, Fig. 6), DHPG (n = 2), and quisqualate(n = 14) by 92%, 100%, and 69%, respectively. The effects of (+)-MCPG were reversible in four cells that were tested under control, (+)-MCPG, and washout conditions (Fig. 6). A lower concentration of (+)-MCPG (500 µM) was also tested against (1S,3R)-ACPD (n = 3) and quisqualate (n = 3), and produced ~50% inhibition of responses. In parallel Ca2+ imaging experiments, 1 mM (+)-MCPG almost completely antagonized (1S,3R)-ACPD-mediated Ca2+ signals. The mean peak (1S,3R)-ACPD-mediated Ca2+ signals in the somata and dendrites were 34 ± 5 nM (n = 12) and 25 ± 2 nM (n = 27), respectively, under control conditions, and 1 ± 1 nM (n = 8) and 3 ± 1 nM (n = 19), respectively, in the presence of (+)-MCPG. A comparison of control and (+)-MCPG-treated cells showed that (+)-MCPG did not affect resting Ca2+ levels, resting membrane potential, input resistance, or the passive or active membrane properties of the neurons studied (data not shown). These data indicate that there is little or no activation of mGluRs under basal conditions.
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FIG. 6.
Effect of (+)- -methyl-4-carboxyphenylglycine [(+)-MCPG] on membrane response to (1S,3R)-ACPD. Membrane responses elicited by (1S,3R)-ACPD (300 µM, 1.5-s application at ) under control conditions, in presence of 1 mM (+)-MCPG and after washout of (+)-MCPG. (+)-MCPG resulted in complete antagonism of (1S,3R)-ACPD-induced membrane response. All responses are from same cell. For this and all subsequent figures, electrophysiological recordings were made as described in Fig. 4A.
; Ohishi et al. 1995) in addition to mGluR1. To determine whether mGluR7 (or other group III mGluRs) is involved in the responses to mGluR agonists, we tested the group III mGluR-specific agonists L-AP4 and L-SOP. Applications of L-AP4 (200 µM, n = 11) and L-SOP (200 µM, n = 6) had no effect on the membrane potential of Purkinje neurons. Higher concentrations of L-AP4 (1 mM, n = 5) also failed to elicit a membrane response, and in Ca2+ imaging studies did not produce a Ca2+ signal in either the soma (n = 13) or dendrites (n = 21, Fig. 7). Finally, the effects of L-AP4 or L-SOP on the passive and active membrane properties were also examined as for the group I mGluR agonists (see above). Neither L-AP4 nor L-SOP (200 µM, n = 11; 1 mM, n = 2) produced significant effects on input resistance, the OFF response elicited at the termination of a hyperpolarizing current pulse, or the number of spikes evoked by depolarizing current pulses in any of the cells tested; nor did these agents affect the shape of current-induced spikes.
). Thus, although L-CCG-I can have actions on all three mGluR subgroups, the group II mGluR effects of L-CCG-I can be pharmacologically isolated. L-CCG-I at 10 µM failed to alter membrane potential (n = 3) at the standard holding potential of 62 mV, whereas (1S,3R)-ACPD (300 µM) elicited either depolarizing or biphasic changes in membrane potential in these same cells. Also, L-CCG-I (10 µm) did not elicit Ca2+ signals in 16 of 17 somata or 20 of 27 dendrites tested. The remaining soma and dendrites showed only small increases (<10 nM) of intracellular Ca2+ with L-CCG-I application, perhaps because of activation of a group I mGluR. A higher concentration of L-CCG-I (100 µM) did elicit biphasic changes in membrane potential that were similar to but smaller than those elicited by (1S,3R)-ACPD (300 µM, n = 2), but these effects can be attributed to activation of group I mGluRs. Thus results from our studies with selective mGluR agonists show that under the conditions tested only group I mGluRs produce detectable membrane and Ca2+ signals in the cultured Purkinje neurons.
). To determine whether a pertussistoxin-sensitive G protein was involved in the response of the cultured Purkinje neurons to mGluR agonists, we measured the Ca2+ signals to DHPG in cultures incubated overnight in pertussis toxin (200 ng/ml) to inactivate Gi/Go proteins. Control cultures were treated similarly with denatured toxin. The pertussis toxin treatment did not alter the Ca2+ signal to DHPG (200 µM, 1.5-s-duration microperfusion pulse) in either the somata or the dendrites. The mean peak amplitudes of the DHPG-mediated Ca2+ signals in the somata and dendrites were 34 ± 3 nM (n = 47) and 22 ± 1 nM (n = 71), respectively, in control neurons, and 30 ± 3 nM (n = 33) and 20 ± 2 nM (n = 59), respectively, in pertussis toxin-treated neurons. In parallel electrophysiological studies, pertussis toxin treatment did not block the membrane responses to DHPG (200 µM, n = 3) or quisqualate (5 µM, n = 3). We verified the effectiveness of the pertussis toxin treatment in inhibiting Gi/Go proteins by testing the pertussis toxin treatment on the response of granule neurons in the culture to the GABAB receptor agonist baclofen, because it is known that the GABAB-receptor-mediated responses are mediated through the pertussis toxin-sensitive Gi protein. Pertussis toxin treatment completely blocked the hyperpolarizations induced by 1-s applications of 100 µM baclofen in the cultured granule neurons (n = 7, data not shown). Treatment of cultures with denatured pertussis toxin did not block the baclofen-induced responses in granule neurons.
). Therefore blockade of phospholipase C should inhibit both the electrophysiological and Ca2+ responses to mGluR1 agonists. Incubation of cultures with the membrane permeable phospholipase C inhibitor U-73122 (1-2 µM) (Chen et al. 1994) for 30-60 min blocked the voltage responses elicited by both DHPG (200 µM, n = 3, Fig. 8) and (1S,3R)-ACPD (300 µM, n = 3). In addition, incubation of cells with U-73122 (2 µM) for more than 20 min greatly attenuated the Ca2+ response to DHPG, particularly in the dendrites (Fig. 9). The inactive analogue U-73343 (2 µM, n = 3) (Chen et al. 1994) had little effect on the electrophysiologic response to (1S,3R)-ACPD or DHPG (Fig. 8), but U-73343 (2 µM) did produce some attenuation of the Ca2+ response to DHPG (Fig. 9). The mean peak amplitude of the Ca2+ responses to DHPG in both the somata and dendrites are shown in Fig. 9B.
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FIG. 8.
Involvement of phospholipase C in group I mGluR agonist-mediated electrophysiological response. Presence of the phospholipase C inhibitor 1-[6-((17 -3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]1H-pyrrole-2,5-dione (U-73122) in the bath blocked voltage response (left) of Purkinje neurons to DHPG (200 µM, 1.5-s application at ), whereas similar application of the inactive analogue 1-[6-((17-3-methoxyestra1,3,5(10)-trien-17-yl)amino)hexyl]-2,5pyrrolidine-dione (U-73343) had no effect. Examples of current-evoked (±120 pA) voltage responses under various conditions are also reproduced (right) to show that U-73343 and U-73122 had only minor effects on active and passive electrical properties of the cell. All responses are from same cell.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). Furthermore, AMPA does not produce burst activity in the cultured Purkinje neurons (Yool et al. 1992). Again, these data imply that quisqualate is acting through a group I mGluR. Thus our results show that group I mGluRs are the predominant functional mGluR subtype expressed in the cultured Purkinje neurons, and that activation of these receptors can produce effects on multiple neuronal functions.
in cerebellar Purkinje neurons (Baude et al. 1993; Fotuhi et al. 1993; Hampson et al. 1994; Martin et al. 1992; Masu et al. 1991; Nusser et al. 1994; Shigemoto et al. 1994), without detectable levels of mRNA for mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, and mGluR8 (Abe et al. 1992; Duvoisin et al. 1995; Nakajima et al. 1993; Nakanishi 1992; Ohishi et al. 1993; Tanabe et al. 1993). In agreement with these data, and consistent with a prominent role for mGluR1 in producing the effects observed in our studies, we found that the cultured Purkinje neurons showed strong immunoreactivity for mGluR1. At this time, however, we cannot rule out "multiple" agonist effects through other isoforms of the group I mGluRs (mGluR1 and mGluR5), because pharmacological tools are not available to distinguish these receptors from one another.
; Ohishi et al. 1995), but our results would suggest that activation of this receptor does not directly alter Purkinje neuron activity, because mGluR7 agonists had no effect on membrane potential or intracellular Ca2+ levels. However, mGluR7 could play a role during conditions of increased adenylyl cyclase activity, as could occur during synaptic transmission involving receptors linked to adenylyl cyclase (e.g., norepinephrine) (Siggins et al. 1971). In agreement with our findings, Inoue et al. (1992) found that L-AP4 did not produce a membrane response in mouse Purkinje neurons. Interestingly, in most regions in the CNS, L-AP4-sensitive receptors have been shown to inhibit transmitter release through presynaptic mechanisms (Monaghan et al. 1989). Furthermore, the distribution of mGluR7 mRNA correlates well with the distribution of the presynaptic L-AP4 receptors in the rat (Kinzie et al. 1995). Thus it is likely that the mGluR7 protein is expressed in the axonal terminals of Purkinje neurons located in the deep cerebellar nuclei.
). For example, in the studies of Chen et al. (1994), U-73343 was tested on NR6 cells transfected with the 1 isoform of phospholipase C, whereas Purkinje neurons are known to express both and isoforms of phospholipase C (Ross et al. 1989; Yamada et al. 1991).
found that pertussis toxin treatment (1-10 µg/ml, 20-22 h) had no effect on the Ca2+ signals elicited by trans-ACPD, quisqualate, glutamate, or ibotenate in cultured mouse Purkinje neurons. Thus mGluR-mediated responses in Purkinje neurons do not appear to be mediated through the Gi or Go classes of G proteins, which are both known to be sensitive to inhibition by pertussis toxin. A likely candidate, then, would be Gq, a G protein that is insensitive to pertussis toxin (Pin and Duvoisin 1995) and that has been shown to be expressed by cerebellar Purkinje neurons in adult rats (Mailleux et al. 1992).
). Furthermore, Ca2+ channel antagonists did not alter the Ca2+ signal to group I mGluR agonists, indicating that Ca2+ influx via Ca2+ channels is not involved (Gruol et al. 1996).
; Staub et al. 1992), whereas the hyperpolarization is due to inhibition of a tonic inward current (Vranesic et al. 1993). Future studies will determine whether these mechanisms mediated the changes in excitability observed in our studies.
), suggesting that group I mGluR activation of protein kinase C would also attenuate this K+ current. However, it is unlikely that such an action would explain the effects of the group I mGluR agonists on the OFF response or single-spike afterdepolarization observed in our studies, because the delayed rectifier would not be active in the potential range (60 to 40 mV) at which we observed our effects. Charybdotoxin, a blocker of the large-conductance, Ca2+-activated K+ channel (BK channel), also unmasks single-spike afterdepolarizations in the cultured Purkinje neurons (unpublished observations), similar to that observed with group I mGluR agonists. This similarity suggests that the group I mGluR agonists may induce an afterdepolarization by depressing the function of BK channels.
) and sensory neurons (Schroeder et al. 1990). We have shown previously that the cultured Purkinje neurons express a low-threshold, T-type Ca2+ conductance and a high-threshold, P-type Ca2+ conductance (Gruol et al. 1992). In the presence of TTX, the OFF response is largely resistant to the P-type Ca2+ channel antagonist 
-agatoxin-IVA (unpublished observation), suggesting that the OFF response is due to activation of the T-type Ca2+ channel. Thus the group I mGluR-mediated reduction in the OFF response could be due to reduction of T-type Ca2+ channels due to activation of protein kinase C. Future studies will address this hypothesis.
). However, (+)-MCPG has been shown to inhibit trans-ACPD-induced phosphoinositide hydrolysis in the cerebellum with an IC50 of 410 µM (Littman and Robinson 1994), whereas 1 mM (±)-MCPG was necessary to fully block (1S,3R)-ACPD-induced currents in Purkinje neurons in vivo (Lingenhohl et al. 1993). Also, IC50s of 60-1200 µM have been found for MCPG on other trans-ACPD-mediated actions elsewhere in the CNS (Littman and Robinson 1994; Watkins and Collingridge 1994). The reason for the wide range of IC50s for MCPG is unknown, but it may relate to the parameter under study, the multiplicity and differential distribution of mGluR isoforms (mGluR1, mGluR1, mGluR1c, mGluR5a, and mGluR5b) in the CNS, or to posttranslational modifications of mGluRs in specific neuronal types that do not occur in nonneuronal cells transfected with cloned mGluRs.
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ACKNOWLEDGEMENTS |
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The authors thank J. Caguioa for the immunohistochemical work and assistance with the preparation of cultures.
This study was supported by National Institute on Alcohol Abuse and Alcoholism (NIAAA) Grant AA-05421 to J. G. Netzeband and NIAAA Alcohol Research Center Grant ARC 06420.
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FOOTNOTES |
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Address for reprint requests: D. L. Gruol, Dept. of Neuropharmacology, CVN-11, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.
Received 6 January 1997; accepted in final form 4 March 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.
Neuron
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