Modulation of intracellular calcium mobilization and GABAergic currents through subtype-specific metabotropic glutamate receptors in neonatal rat hippocampus

Brain Research Bulletin 81 (2010) 73–80

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Modulation of intracellular calcium mobilization and GABAergic currents through subtype-specific metabotropic glutamate receptors in neonatal rat hippocampus M. Taketo ∗ , H. Matsuda Department of Physiology 1, Faculty of Medicine, Kansai Medical University, 10-15 Fumizono-cho Moriguchi, Osaka 570-8506, Japan

a r t i c l e

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Article history: Received 30 May 2009 Received in revised form 13 July 2009 Accepted 14 July 2009 Available online 21 July 2009 Keywords: Metabotropic glutamate receptors Hippocampus Ca2+ mobilization GABAA ergic currents

a b s t r a c t Group I metabotropic glutamate receptors (mGluRs) are coupled to phosphoinositide hydrolysis, and are thought to modulate neuronal excitability, by mobilizing intracellular Ca2+ . Difference in Ca2+ mobilization among subclasses of the receptors has been reported, and regarded as a possible cause of variant neuronal modifications. In hippocampal interneurons, several subclasses of mGluRs including mGluR1 and mGluR5 have been immunohistochemically identified. The subclass-specific physiological effects of mGluRs on neuronal transmission in hippocampus, however, have not been fully elucidated. In the present study, effects of group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) on intracellular calcium concentration were examined in hippocampal interneurons. Application of DHPG increased fluorescence ratio in neonatal CA3 stratum oriens/alveus interneurons. The DHPG-induced calcium mobilization was markedly inhibited by mGluR1-specific antagonist, cyclopropan[b]chromen-1a-carboxylate (CPCCOEt). Inhibition of the calcium elevation by mGluR5-specific antagonist, 6-methyl-2-(phenylazo)-3-pyrindol (MPEP), was weaker than that of CPCCOEt. The fluorescence ratio was not significantly changed by application of mGluR5-specific agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). DHPG induced calcium responses in CA1 interneurons as in CA3, and the responses were partially inhibited by MPEP treatment. Effects of group I mGluR agonist and antagonist were also investigated, on GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in CA3 pyramidal neurons. The GABAergic sIPSCs were facilitated by DHPG perfusion, and the potentiation was reduced by CPCCOEt, and less distinctly by MPEP. The sIPSCs were not significantly potentiated by CHPG application. These results indicate that mGluR1 is functional in hippocampal interneurons, and DHPG exerts its effect mainly through this receptor at early developmental period. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Metabotropic glutamate receptors belong to the group of seven-transmembrane domain receptors. Eight mGluR subtypes (mGluR1–mGluR8) have been identified and classified into three groups (I–III), on the basis of their amino acid sequences as well as biochemical and pharmacological properties [7]. Group I receptors (consist of mGluR1 and mGluR5) couple to Gq protein and mobilize intracellular calcium. Group II (including mGluR2 and mGluR3) and III (consist of mGluR4, mGluR6, mGluR7 and mGluR8) receptors negatively couple to adenylyl cyclase. Immunocytochemical studies demonstrated heterogeneous distribution of the mGluR subtypes [5,19,25]. In central nervous system, activation of mGluRs modulates cell excitability and synaptic transmission [7,23]. In addition, involvement of mGluRs in regulation of cerebral development has been suggested [7,10]. Group I subtype of mGluRs receptors is

∗ Corresponding author. Tel.: +81 669 93 9422; fax: +81 669 92 1409. E-mail address: [email protected] (M. Taketo). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.07.011

also demonstrated to be important in induction of long-term plasticity in hippocampus [1,2]. Hippocampal interneurons regulate signal transduction, by modifying excitability of the principal cells. Immunocytochemical studies detected expression of the both subtypes of mGluR I receptors, in neonatal hippocampal interneurons [5,19]. Though studies of the mGluRs have been performed in young hippocampal CA1 interneurons [12,28] or pyramidal cells [20], function of mGluRs has not been revealed in neonate. In the present study, effect of group I mGluRs activation on intracellular calcium mobilization and GABAergic spontaneous IPSCs in interneurons was examined, and subtypes of the receptors concerned were determined in developing hippocampal CA3 and CA1 regions. 2. Materials and methods 2.1. Slice preparation All experimental protocols were performed in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals revised 1996, and the guidelines for animal research of the Physiological Society of Japan. All efforts were made to minimize animals suffering and to reduce the number of animals used.

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Hippocampal slices were prepared from Wister rats aged 4–7 days (for calcium imaging) or 4–14 days (for current recording), as reported previously [27]. Following diethyl ether anesthesia, the animals were decapitated and the brains were removed, and cooled in artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 138.6, KCl 3.35, NaHCO3 21.0, NaH2 PO4 0.6, CaCl2 2.5, MgCl2 1.0, glucose 10.0) bubbled to equilibrium with 95% O2 /5% CO2 for 2 min. Then, the brains were dissected and the removed hippocampi were sliced to a thickness of 300–400 ␮m, using a vibrating tissue slicer (model DTK1000, Dosaka EM, Kyoto, Japan). Slices were placed in a storage chamber filled with ACSF, and kept at least for 1 h at room temperature. Before recording, the slices were individually transferred to a recording chamber and continuously perfused with ACSF during experiment. In specified cases, tetrodotoxin (TTX, final 0.5 ␮M) was added to ACSF. Each drug was applied by perfusion (agonist for 3 min; antagonist for 13–14 min). Sufficient time (20 min) was taken before the 2nd or 3rd application.

2.4. Data analysis To estimate intracellular Ca2+ mobilization, area between fluorescence ratio curve and base line was measured with a trapezoidal rule-based algorithm (Sigma Plot, Jandel Scientific). After current recording, average of inter-event interval and amplitude distributions of spontaneous IPSCs obtained in control and in test conditions were calculated (Mini Analysis, Synaptosoft) during 3 min. Statistical analyses were carried out using unpaired t-test with Welch’s correction (a level of significance of P ≤ 0.05). 2.5. Materials CPCCOEt and DHPG were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CHPG, CNQX, D-APV and MPEP were obtained from Tocris Cookson (Bristol, UK). TTX was purchased from Wako Pure Chemical Industries (Osaka, Japan).

2.2. Calcium imaging Calcium imaging was performed following an established method [13]. Cells were loaded with calcium indicator through incubation in fura-2 AM containing ACSF (10 ␮M × 20–90 min). The solution of Ca2+ indicator was prepared as follows: Fura-2 AM was dissolved in dimethyl sulfoxide (DMSO) containing chremophore. The DMSO solution was diluted by ACSF and sonicated to obtain final solution (10 ␮M Fura-2 AM, 0.001% chremophore). The slices were perfused with ACSF containing 0.5 ␮M TTX during the experiments. In particular cases, CaCl2 in ACSF was eliminated. Interneurons were visually identified under a differential interference contrast microscope with fluorescence imaging system, according to their localization of cell bodies in stratum oriens/alveus. Neurons having large shaped soma and horizontally spreading dendrite are selected. Fluorescence image was obtained through an objective lens (CFI Fluor, Nikon, Tokyo Japan) and a cooled-CCD camera (model C4742-95-12ER, Hamamatsu Photonics, Hamamatsu, Japan) under alternating excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. Back ground fluorescence was subtracted and images were stored and analyzed using a digital image processor (Aquacosmos; Hamamatsu Photonics). Intracellular calcium concentration ([Ca2+ ]i ) was expressed as the ratio of the fura-2 fluorescence intensities excited at 340 nm and 380 nm. The acquisition rate was usually 0.1 Hz. 2.3. Whole-cell recordings Whole-cell patch clamp recordings were performed in pyramidal neurons of the CA3 region, as described previously [26]. Each cell was identified morphologically, using CCD camera connected to a microscope (model E600-FN, Nikon). Patch electrodes (2–4 M, after filling with intrapipette solution) were pulled from borosilicate glass using a two-stage vertical puller (model PP-83, Narishige, Tokyo, Japan) and filled with symmetrical chloride-intracellular solution (composition in mM: CsCl 140, CaCl2 1, MgCl2 2, ethyleneglycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) 10, HEPES 10, ATP 2, lidocaine N-ethyl bromide 2, adjusted to pH 7.3 using CsOH). Under the experimental conditions, the GABAergic IPSCs were reversed at ∼0 mV equivalent to the ECl -predicted by Nernst equation. Cells were voltage-clamped at −70 mV, and the GABAergic IPSC was recorded as inward current. Electro-signals were measured with Axopatch 200B amplifier (MDS, Inc., Tronto, Canada) and filtered at 3 kHz. The signals were digitized at 10 kHz and stored, using DigiData 1200 with the pClamp8 data collection and analysis software (MDS, Inc.). Data recorded from cells with significant change in series resistance during experiment were discarded. All experiments were carried out at room temperature (∼23 ◦ C).

3. Results 3.1. DHPG-evoked [Ca2+ ]i elevation Calcium mobilization through activation of group I mGluRs was first investigated by fura-2 fluorimetry. The dye-loaded interneurons of stratum oriens/alveus were identified by their localization and morphology under fluorescent microscope. Because reduction of external K+ concentration to 1 mM or less has been reported to elicit Ca2+ transients in astrocytes but not in neurons [9,15], at the end of each recording, extracellular K+ was eliminated for 5 min to confirm that the images were recorded in neurons. Intracellular Ca2+ mobilization was evaluated as the area under the fluorescence ratio curve by integrating the fluorescence ratio over time. Application of DHPG (10 ␮M), a group I mGluR-specific agonist, increased fluorescence ratio in CA3 stratum oriens/alveus interneurons (area between ratio curve and base line = 0.620 ± 0.052 fluorescence ratio × min ± SE, n = 32 cells (Fig. 1A). Subsequent application of DHPG also increased fluorescence ratio (area of the 2nd response = 68.8 ± 3.1% of 1st response ± SE, area of the 3rd response = 57.4 ± 2.9% of 1st response ± SE, n = 32 cells (Fig. 1B). Preincubation with ionotropic glutamate receptor antagonists, CNQX and D-APV, did not prevent the increment of the ratio (data not shown). In a few neurons, cytoplasmic Ca2+ oscillatory response was observed. 3.2. Ca2+ mobilization through mGluR1 and mGluR5 receptors Receptor subtype-specific effects of DHPG on [Ca2+ ]i were investigated in the presence and absence of mGluR antagonists. The DHPG-induced calcium mobilization was partially inhibited

Fig. 1. DHPG-evoked intracellular calcium elevation in hippocampal CA3 interneuron. (A) Slices were loaded with fura-2 AM. Fluorescence ratio (340 nm/380 nm) during application of 10 ␮M DHPG was calculated and plotted. Repetitive application of the agonist increased fluorescence ratio in CA3 stratum oriens/alveus interneurons. (B) The calcium responses were evaluated by calculating area between fluorescence ratio curve and base line. Average of 32 responses was represented in a histogram.

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Fig. 2. Receptor-subtype specificity of the DHPG-induced calcium elevations in CA3 interneurons. Effects of DHPG (10 ␮M) on [Ca2+ ]i were investigated in the presence and absence of metabotropic glutamate receptor antagonists. DHPG-induced calcium mobilization was partially inhibited by perfusion of mGluR1 antagonist CPCCOEt (100 ␮M, Aa) or mGluR5 antagonist MPEP (10 ␮M, Ab). Co-application of MPEP and CPCCOEt almost completely prevented the increment of fluorescence ratio (Ad). Histograms show the average of DHPG-evoked calcium responses under preincubation with CPCCOEt or MPEP. *P < 0.05. (Ac) Areas under fluorescence ratio curve during the second DHPG application with (CPCCOEt and MPEP) or without (DHPG) mGluR antagonist were calculated and compared with each other. The area of 2nd response was normalized by area of the corresponding first DHPG response (control; see text) before comparison. Ad shows DHPG induced Ca2+ responses during co-application of CPCCOEt and MPEP. (B) Specific agonist of mGluR5, CHPG, did not significantly change [Ca2+ ]i , whereas subsequent application of DHPG increased fluorescence ratio.

by perfusion of mGluR1-specific antagonist CPCCOEt (100 ␮M, Fig. 2Aa) or mGluR5 antagonist MPEP (10 ␮M, Fig. 2Ab). The antagonist sensitivity was, however, different among individual cells. In Fig. 2 Ac, the second DHPG-evoked Ca2+ responses were normalized by the corresponding first (control) responses. Though the second Ca2+ responses without antagonists (Fig. 2Ac, “DHPG”)

were somewhat smaller than the first responses (68.8 ± 3.1% of the 1st response ± SE, n = 32), these responses were significantly larger than the second responses in the presence of CPCCOEt (10.7 ± 1.0% of the 1st response ± SE, n = 34) or MPEP (33.7. ± 2.0% of the first response ± SE, n = 50). Co-application of CPCCOEt and MPEP almost completely prevented the increment of fluorescence

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tion. Increment of fluorescence ratio induced by DHPG application was not significantly inhibited by La3+ perfusion (61.1 ± 3.0% of control ± SE, n = 9, Fig. 3B). 3.4. Calcium responses evoked by DHPG in CA1 region Effects of DHPG and mGluR subtype-specific antagonist on [Ca2+ ]i were determined also in CA1 oriens/alveus interneurons. DHPG induced Ca2+ responses, as in CA3 (area between ratio curve and base line = 0.577 ± 0.094  fluorescence ratio × min ± SE, n = 7, Fig. 4A). Oscillatory responses were also observed in CA1. The responses were partially inhibited by CPCCOEt (14.4 ± 3.7% of control ± SE, n = 5, Fig. 4Ba) or MPEP treatment (55.0 ± 6.7% of control ± SE, n = 4, Fig. 4Bb). Increment of fluorescence ratio induced by DHPG application was not significantly inhibited by La3+ application in CA1 (68.4 ± 12.0% of control ± SE, n = 3, Fig. 4C). 3.5. Metabotropic GluR-specific modulation of hippocampal inhibitory synaptic transmission Effect of mGluRs activation on spontaneous GABAA ergic IPSCs was determined in CA3 pyramidal neurons (4–14 days). The spontaneous GABAA ergic IPSCs were recorded while perfusing ACSF supplemented with excitatory amino-acid receptor blockers CNQX (10 ␮M) and D-APV (25 ␮M). Addition of 5 ␮M bicuculline to perfusing solution blocked all of the sIPSCs under these experimental conditions, indicating that the currents were mediated by GABAA receptor. The GABAergic IPSCs were facilitated by DHPG (10 ␮M) perfusion (the frequency of the IPSCs increased from 1.83 ± 0.18 Hz to 3.74 ± 0.24 Hz, n = 50, Fig. 5A). Temporal plot of the changes in frequency and amplitude of the spontaneous IPSCs revealed that frequency of the currents was transiently increased by DHPG application (Fig. 5B). Amplitude of the IPSC slightly increased during DHPG application (113.8 ± 4.43% of control amplitude, n = 50). Cumulative probability plot also indicated DHPG-induced facilitation of the IPSCs. DHPG was less effective on increasing amplitude than on shortening inter-event interval (Fig. 5Ca and Cb). Extracellular application of TTX (0.5 ␮M) abolished the DHPG-induced potentiation of the IPSCs (data not shown). Fig. 3. Extracellular Ca2+ -dependency and La3+ -insensitivity of [Ca2+ ]i elevation induced by DHPG (10 ␮M) in CA3 interneurons. (A) Effect of DHPG application on [Ca2+ ]i was determined with or without extracellular Ca2+ . The DHPG-evoked Ca2+ response was reduced by perfusion of nominally Ca2+ -free ACSF. (B) DHPG-induced Ca2+ responses in the presence of La3+ . Extracellular perfusion of 100 ␮M La3+ did not significantly reduce the DHPG-induced Ca2+ responses.

ratio (Fig. 2Ad). Involvement of mGluR5 in DHPG-evoked Ca2+ responses was further examined, using specific agonist CHPG. The fluorescence ratio was not significantly changed by CHPG (100 ␮M) application, whereas the ratio was increased by succeeding DHPG perfusion (Fig. 2B, n = 4). 3.3. Extracellular Ca2+ -dependency and La3+ -insensitivity of DHPG-induced [Ca2+ ]i elevation Dependency of DHPG-induced Ca2+ elevation on extra cellular Ca2+ was subsequently determined (Fig. 3A). DHPG-induced response was reduced during perfusion of nominally Ca2+ freesolution (22.3 ± 3.9% of the first response ± SE, n = 4). Cytoplasmic Ca2+ oscillation became obvious during perfusion of Ca2+ -free solution. Several Ca2+ permeable channels including transient receptor potential (TRP) channels are reported to be activated after stimulation of group I mGluR. To examine contribution of the Ca2+ influx through La3+ -sensitive TRP channels to DHPG-indused Ca2+ mobilization, La3+ (100 ␮M) was perfused during DHPG applica-

3.6. Effect of subtype-specific mGluR antagonist on spontaneous IPSC DHPG-induced facilitation of the IPSCs was determined in the presence of the mGluR subtype-specific antagonists. In the presence of CPCCOEt, facilitation of IPSCs induced by DHPG perfusion was reversibly reduced at 2nd postnatal week (11–14 days) (Fig. 6Aa, 29.4 ± 9.8% of control ± SE, n = 4). On the contrary, the effect of DHPG application on the IPSCs was still observed in the presence of MPEP (Fig. 6Ab, 72.7 ± 14.3% of control ± SE, n = 5). The subtype specificity of DHPG-induced [Ca2+ ]i elevation was similar in neurons during the 1st postnatal week (4–7 days). The facilitation of IPSCs was reversibly decreased by CPCCOEt as in 2nd week (20.4 ± 6.3% of control ± SE, n = 5). MPEP was also less effective to prevent the DHPG-induced facilitation of IPSCs than CPCCOEt (35.0 ± 9.7% of control ± SE, n = 7). Contribution of mGluR5 to DHPG-induced potentiation of the IPSCs was further examined by agonist application. Neither frequency nor amplitude of the IPSCs was significantly increased by CHPG application (Fig. 6B). 4. Discussion In the present experiments, DHPG, group I mGluR agonist, elevated [Ca2+ ]i in interneurons and facilitated GABAergic IPSCs in neonatal hippocampus. The experimental results of subtypespecific agonists or antagonists, showed that DHPG exerted its

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Fig. 4. DHPG-induced calcium responses in CA1 interneuron. Application of DHPG also induced intracellular calcium elevation, in CA1 interneurons as well. [Ca2+ ]i oscillations were discernible (A). DHPG-induced calcium response in CA1 was inhibited by perfusion of CPCCOEt (Ba). MPEP is less effective on inhibition of the DHPG-evoked Ca2+ response (Bb). DHPG-induced Ca2+ responses were insensitive to La3+ , also in CA1 (C).

Ca2+ -mobilizing function mainly through mGluR1 receptors in hippocampal interneurons at this age. DHPG-evoked [Ca2+ ]i elevation was TTX insensitive, because the fluorescence imaging was performed in the presence of this blocker. Conversely, DHPG-induced facilitation of IPSC was prevented by TTX. Group I mGluRs are reported to play a substantial role in development of central nervous system [7,32]. Our functional study of group I mGluRs demonstrated that most of DHPG-induced Ca2+ responses and IPSC facilitation were mediated by mGluR1. In immature cerebellar Purkinje neurons, fast hyperpolarization and Ca2+ signal induced by DHPG were blocked by CPCCOEt [21], concordantly with the high expression of mGluR1s [5,25]. Developmental studies however have shown relative abundance of mGluR5 protein or mRNA in neonatal hippocampus. According to an immunocytochemical study in hippocampus, hybridization signals for mGluR1 were not yet clearly detected or very weak on embryonic day 18, and were weak on postnatal days 0 and 4 [25]. Labeling of this region then gradually increased to reach the adult level on the postnatal day 11. Similar hybridization results were also reported [19]. In the case of mGluR5, expression increases perinatally, peaking around the 2nd postnatal week [5]. Because expression of mGluR5 decreases during postnatal development, it is not probable that effect of mGluR5 activation on hippocampal signal transduction is larger in adult than in neonate. Functional studies revealed that in young hippocampal CA1 region, most DHPG-induced calcium mobilization was mediated by mGluR1 in both pyramidal cells [20] and interneurons

[12,28]. Expression of group I mGluRs has been reported to show region-specific distribution within hippocampus [10,19,25]. Function of mGluRs in CA3 is possibly dissimilar to that in CA1, whereas our results suggested no difference in mGluR-subtype specificity between CA3 and CA1. Relatively small contribution of mGluR5 to DHPG-induced Ca2+ responses might originate in subcellular localization of this subtype. A previous immunochemical study demonstrated that during late prenatal development, mGluR1␣ was localized in neuropil, whereas mGluR5 was localized in neuronal somata. In contrast, during postnatal development, mGluR1␣ was found in somata, whereas mGluR5 was found in neuropil [19]. Stimulation of mGluRs induces inhibition of K+ channels and/or direct activation of cationic channels, as well as Ca2+ mobilization from internal stores [7,11,18]. Since synaptic currents can be affected by not only somatic depolarization but also dendritic mGluRs or the receptors at presynaptic terminals, subtype specificity of IPSC facilitation may differ from that of Ca2+ mobilization which may be affected by subcellular localization pattern of the receptors. Activation of mGluRs at presynaptic terminal was not main cause of the DHPG-induced facilitation of the IPSCs, because miniature IPSCs recorded in the presence of TTX were not potentiated by DHPG in our experiments. The results suggest that DHPG facilitated the IPSCs through direct excitation of inhibitory interneurons, as in frontal cortex [6] or CA1 [20]. Our results demonstrated that both Ca2+ mobilization and IPSC facilitation were predominantly mediated by mGluR1. Another possible reason is that the two subtypes may couple to different

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Fig. 5. Effect of DHPG on GABAergic synaptic transmission in hippocampus. Spontaneous GABAA ergic IPSCs were recorded during DHPG application in CA3 pyramidal neurons. Ionotropic glutamate receptors were blocked by CNQX and D-APV, to isolate GABAA ergic currents. Application of 10 ␮M DHPG facilitated the IPSCs (A). (B) Representative DHPG-induced temporary changes of IPSCs recorded from single neuron are shown. Both amplitude (circles) and frequency (squares) of the spontaneous IPSCs were increased by the agonist application. Cumulative probability plotting of the IPSC amplitude and inter-event interval also revealed DHPG-induced facilitation of the IPSCs. Solid and dashed lines indicate control and DHPG-facilitated events, respectively. DHPG increased frequency of the spontaneous IPSCs (Cb). Increment of the IPSC amplitude was not remarkable (Ca).

pathway of calcium influx or release in neonatal hippocampus. Both mGluR1 and mGluR5 are coupled to phospholipase C which induces phosphoinositide hydrolysis and mobilization of Ca2+ from IP3 sensitive stores in many systems [7,10,11]. Other resources of type I mGluR-evoked [Ca2+ ]i elevation are ryanodine sensitive Ca2+ stores and influx through Ca2+ permeable channels [3,4,10,11], but subtype specificity of these pathways have not been established. Since DHPG-induced [Ca2+ ]i was reduced by elimination of extracellular Ca2+ , influx seems to be an important way of [Ca2+ ]i elevation in our experiment. La3+ (100 ␮M) blocks voltage-gated Ca2+ channels and either facilitates or inhibits TRP channels [14,29]. Because modulation of DHPG-induced calcium mobilization by La3+ was feeble, calcium influx through La3+ -sensitive TRP channels voltage-gated Ca2+ channels does not explain the large mGluR1-mediated calcium mobilization observed in this study. La3+ -insensitive TRP channels

or other non-selective cationic channels may amplify function of mGluR1. The results that MPEP partially inhibited the neuronal responses to DHPG, but CHPG did not induce significant change of both [Ca2+ ]i or IPSCs, suggested that the mGluR5-mediated responses became manifest by activation of mGluR1. A little change of membrane voltage or [Ca2+ ]i which was induced by DHPG in the presence of mGluR1 agonist may potentiate mGluR5-mediated signals. It is reported that oscillatory or non-oscillatory patterns of [Ca2+ ]i increase depend on receptor identity [16]. Glutamate induces single-peaked [Ca2+ ]i mobilization in mGluR1␣transfected cells, but elicits Ca2+ oscillations in mGluR5atransfected cells, though prolonged receptor stimulation induce mGluR1 mediated oscillation [17]. Because a few neurons showed cytosolic Ca2+ oscillation, we could not decide which subtypes of

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Fig. 6. Inhibitory effect of receptor-subtype-specific antagonists on DHPG-evoked facilitation of the IPSCs. DHPG-induced potentiation of the IPSCs was determined in the presence of the mGluR subtype-specific antagonists. (Aa) DHPG application was performed in the presence of CPCCOEt. The effect of DHPG application on amplitude (circles) and frequency (squares) of the IPSCs was reversibly reduced by the mGluR1 antagonist CPCCOEt. (Ab) DHPG application was performed in the presence of MPEP. Increment of frequency of the IPSCs was still observed. (B) the spontaneous IPSCs were not facilitated by mGluR5-specific agonist, CHPG (100 ␮M), but were raised by DHPG.

the mGluRs mediated the oscillation. The oscillatory Ca2+ responses seem to be independent of extracellular Ca2+ and usually inhibited or masked by Ca2+ influx, as the oscillation became obvious during perfusion of CPCCOEt or Ca2+ -free ACSF in several cases. In hippocampus, phosphatidylinositol (PI) hydrolysis induced by glutamate is very high at postnatal days 6 and 8, but it decreases and reaches adult values between 19 and 24 days after birth [22]. The period when high glutamate-induced PtdIns hydrolysis was observed corresponds with that of intense hippocampal synaptogenesis which occurs during the first postnatal week, and slows down after 10th day of life. Other study using low-specific mGluR agonist, trans 1-aminocyclopnntane-trans-1,3-dicarboxylic acid (trans-ACPD) showed similar results. Trans-ACPD stimulated PI hydrolysis was much lager at 8 days of postnatal life than at 1 day or 30 days [8]. In medial vestibular nuclei, induction of LTD requires activation of mGluR5, at early developmental stages [24]. DHPG-induced LTD in hippocampal dentate gyrus also shows age-dependency, and high frequency stimulation-induced and ageindependent LTD in that region is mainly mediated by mGluR5 [30]. Possible reason for relatively small effect of mGluR5 on neonatal hippocampal signal transduction is that mGluR5-induced developmental changes and synaptic plasticity may not be mediated by [Ca2+ ]i elevation. Otherwise, particular source of Ca2+ elevation may important in mGluR-mediated changes. It has been reported that mGluR5-induced Ca2+ release from internal stores was required by LTP in dentate gyrus [33]. Our results might indicate that release of Ca2+ from intracellular Ca2+ store by mGluR5, rather than mGluR1mediated Ca2+ influx, is necessary for regulation of hippocampal development or plasticity.

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