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First published online 13 May 2003
doi: 10.1242/jcs.00481

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Glutamate-mediated [Ca²⁺]_c dynamics in spontaneously firing dopamine neurons of the rat substantia nigra pars compacta

Medical Research Center for Regulation of Neuronal Cell Excitability and Department of Physiology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong Jangan-ku, Suwon 440-746, Korea

View larger version (42K): [in a new window]   Fig. 1. Identification of dopaminergic neurons. (A) Transmitted images. Among the three neurons isolated, two large multipolor cells (marked a), were dopaminergic, whereas a small bipolar cell (marked b) was not (stained with tyrosine hydroxylase (TH) antibodies and FITC-conjugated secondary antibodies). Bar, 20 µm. (B) Classification of acutely isolated cells from the SNc according to their shapes.

View larger version (25K): [in a new window]   Fig. 2. Electrical activities of acutely isolated SNc dopamine neurons. Spontaneous firing activities were recorded from the cell-attached current-clamp cells (A,Ba). Their firing activities were converted into the frequency-time graph (A,Bb). The [Ca2+]c using fura-2 was measured from the same cells (A,Bc). (A) The spontaneous firing activities were completely blocked by 500 nM TTX. Note that the change in [Ca2+]c was mirrored by the firing rate. (B) The spontaneous firing was initially blocked by a nonspecific Ca2+ channel antagonist, 100 µM CdCl2. But it recovered after some time even in the presence of CdCl2. Note the persistent decreased [Ca2+]c level after a full recovery of the spontaneous electrical activities.

View larger version (22K): [in a new window]   Fig. 3. KCl-elicited [Ca2+]c rises through voltage-operated calcium channels. After application of 60 mM KCl for 12 seconds (), the KCl solution was reapplied under the presence of L-type Ca2+ channel (10 µM nifedipine, A), N-type Ca2+ channel (1 µM -conotoxin GVIA, B), and P/Q-type Ca2+ channel (1 µM -agatoxin IVA, C) antagonists, a cocktail of the above three antagonists (D), or 100 µM CdCl2 (E). (F) Summary of the inhibitions of the KCl-elicited [Ca2+]c rises by VOCC antagonists; nifedipine (36.0±3.0% of control, n=10), -conotoxin GVIA (69.5±8.8%, n=5), -agatoxin IVA (92.3±6.6%, n=4), cocktail of the above three antagonists (23.2±4.1%, n=3), and CdCl2 (15.0±2.7%, n=4). *Significant difference (paired student t-test, P<0.05). Nif, nifedipine; -cono GVIA, -conotoxin GVIA; -aga IVA, -agatoxin IVA.

View larger version (15K): [in a new window]   Fig. 4. Dose dependence of glutamate-induced [Ca2+]c rises. (A) The [Ca2+]c rises in response to different glutamate concentrations in the same cell. (B) The concentration-response curve of the glutamate-induced [Ca2+]c rises. The concentration of the half-maximum [Ca2+]c rise (EC50) is 3.9±1.0 µM. All the solutions contain 1 µM glycine. Each point represents the average of 4-20 cells (mean ± s.e.m.).

View larger version (32K): [in a new window]   Fig. 5. Glutamate-induced [Ca2+]c increases. (A) Effects of antagonists for ionotropic glutamate receptors (a,b) and VOCCs (c-g) on the 100 µM glutamate-elicited [Ca2+]c rises. The black curves are [Ca2+]c rises in response to 100 µM glutamate and the red curves are [Ca2+]c rises in the presence of specific antagonists. (a) 30 µM CNQX; (b) 50 µM AP-5; (c) 10 µM nifedipine; (d) 1 µM -conotoxin GVIA; (e) 1 µM -agatoxin IVA; (f) cocktail of the above VOCC antagonists; (g) 200 µM CdCl2. (B) Summary of a-f experiments (means ± s.e.m., n). CNQX, 39.5±6.7% of control, n=16; AP-5, 93.3±3.9%, n=13; nifedipine, 82.3±3.9%, n=7; -conotoxin GVIA, 93.9±0.3%, n=5; -agatoxin IVA, 100%, n=3; cocktail of the above three antagonists, 67.5±2.4%, n=3; CdCl2, 27.7±4.8%, n=6. All the solutions contain 1 µM glycine. *Significant difference (paired student t-test, P<0.05).

View larger version (18K): [in a new window]   Fig. 6. AMPA-induced [Ca2+]c increases. 100 µM AMPA was applied at each point indicated by an arrowhead for 12 seconds. (A) Nifedipine (10 µM) inhibited the 100 µM AMPA-elicited [Ca2+]c rises (n=4). In the Ca2+-free solution, AMPA did not rise [Ca2+]c at all (n=3). (B) 1 µM -agatoxin IVA (n=2; b) but not 1 µM -conotoxin GVIA (n=4; a) slightly inhibited the AMPA-induced [Ca2+]c rises. (C) Summary of the above data. Nifedipine, 67.7±12.5%, n=4; -conotoxin GVIA, 95.0±0.5%, n=4; -agatoxin IVA, 88.8±0.5%, n=2; *Significant difference (paired student t-test, P<0.05).

View larger version (26K): [in a new window]   Fig. 7. The [Ca2+]c increases by the activation of metabotropic glutamate receptors. (A) 100 µM glutamate-mediated [Ca2+]c rises in the absence/presence of ionotropic glutamate receptor antagonists (30 µM CNQX + 50 µM AP-5, n=6). (B) 100 µM glutamate-mediated [Ca2+]c rises with and without calcium in the bath solution (n=5). (C) 100 µM glutamate- and DHPG (mGluR agonist)-mediated [Ca2+]c increases (n=10). (D) Summary of the above data. 100% (control) means the peak values of [Ca2+]c increases to 100 µM glutamate. CNQX and AP-5, 31.5±3.5% of control, n=6; glutamate in Ca2+-free solution, 23.7 ± 3.5%, n=5; DHPG in Ca2+-free solution, 19.3±4.2%, n=5; DHPG (31.3±1.9%, n=10. All the solutions contain 1 µM glycine.

View larger version (21K): [in a new window]   Fig. 8. Two phases of the [Ca2+]c rise by activation of the metabotropic glutamate receptors. (A) Application of 10 µM DHPG (a) elicited characteristic [Ca2+]c rises: the initial transient Ca2+ rise and long-sustained [Ca2+]c elevation (n=7). Disappearance of the later persistent [Ca2+]c elevation (b) under the Ca2+-free solution (n=7). (B) Effects of glutamate receptor antagonists on the late phase of glutamate-induced [Ca2+]c increases. All the solutions contain 1 µM glycine. Note the glutamate concentration.

View larger version (24K): [in a new window]   Fig. 9. Inhibition of [Ca2+]c rises by antagonists for AMPA/kainate, NMDA, and metabotropic glutamate receptors at different glutamate concentrations in SNc dopamine neurons. The black curves indicate the glutamate-induced [Ca2+]c rises at the indicated concentrations, and the red curves indicate the glutamate-induced [Ca2+]c rises in the presence of 30 µM CNQX (A), 50 µM AP-5 (B), 100 µM CPCCOEt (C), respectively. (D) Different contributions of AMPA/kainate, NMDA, and metabotropic glutamate receptors to the [Ca2+]c rises according to glutamate concentrations. Each point was calculated by analysis of the data from 4-20 cells. All the solutions contain 1 µM glycine.

View larger version (31K): [in a new window]   Fig. 10. Glutamate at submicromolar concentrations increases the frequency of spontaneous firing and [Ca2+]c in SNc dopamine neurons. (A) Spontaneous firing activities of the dopamine cell were recorded in the cell-attached current-clamp configuration. The concentration of KCl was gradually increased and thereafter the glutamate concentration increased too. (B) Frequency plot of the data in A. When stimulated with KCl, a maximum frequency was obtained at 10-12 mM. Glutamate more dramatically raised the firing frequency dose-dependently but the spontaneous firing suddenly disappeared after exposure to 10 µM glutamate. (C) The [Ca2+]c changes from the same cell. The increased spontaneous firing reflects the increase in [Ca2+]c. Glutamate has a much stronger effect on the spontaneous firing and [Ca2+]c than KCl. The solution contains 1 µM glycine.