Neuroscience Letters, 119 (1990) 19-22
19
Elsevier Scientific Publishers Ireland Ltd. NSL 07242
MK-801 blocked the functional N M D A receptors in identified cerebellar neurons Michisuke Yuzaki x, Yoshihisa K u d o 2, K y o k o Akita 2, Atsushi Miyawaki 1 and Katsuhiko Mikoshiba 1 Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Osaka (Japan) and aDepartment of Neuroscience, Mitsubishi-kasei Institute of Life Sciences, Tokyo (Japan) (Received 3 July 1990; Accepted 6 July 1990)
Keywords:
N-Methyl-D-aspartate receptor; MK-801; Cerebellum; Intracellular calcium; Fura-2 microfluorometry
In order to clarify the nature of N-methyl-D-aspartate (NMDA) receptors in cerebellum, where heterogeneity of the NMDA receptor has been suggested, we investigated the action of MK-801 on the NMDA-induced [Ca2+]~rise in cultured cerebellar neurons using video-assisted microfluorometry. MK-801 caused a potent and selective blockade of the NMDA-activated [Ca2+]i elevation. The blockade caused by MK-801 was dependent on the presence of NMDA, i.e., use-dependent. There was no difference in the mode of blockade between immtmocytochemically identified Purkinje and non-Purkinje cells, although the relative size of the NMDA-induced [Ca2+]i rise was significantly less in Purkinje cells. These results indicate that the NMDA receptors in cultured cerebellar neurons are coupled with the same channels as those in other brain regions.
The major excitatory neurotransmitter glutamate exerts its action through a number of receptor subtypes [17] including N-methyl-D-aspartate (NMDA), kainate, and quisqualate. The N M D A receptor-channel complex is putatively involved in the synaptic plasticity mechanism through its high permeability to Ca 2+ [11]. Autoradiographical [13] and electrophysiological [2-5, 7] studies have shown the presence of functional binding sites for N M D A in cerebellum. It is not clear, however, whether these binding sites in cerebellum constitute the same receptor-channel complex as those in other regions of brain, because N M D A receptors in cerebellar granule cells are reported to show only minimal interaction with Mg 2+ [7] and phencyclidine (PCP) analog [9, 12, 18], both of which are strong non-competitive antagonists in other brain regions. The action of PCP analogs on N M D A receptors in Purkinje cells is also unclear. MK-801, a more potent and selective non-competitive antagonist than PCP which shares the same binding site as PCP [18], is suggested to act at the channel portion of the NMDA receptor-channel complex. In order to clarify the nature of the N M D A receptor-channel complex in cerebellum, we investigated the action of MK-801
Correspondence: M. Yuzaki, Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
on the NMDA-induced [Ca2+]i increase in mouse cerebellar neurons using video-assisted microfluorometry with fura-2. Cerebellar neurons were dissociated from the cerebellum of 0-day-old ICR mice by trypsin treatment, resuspended in culture medium and plated at a density of 1520 x l04 cells/cm 2 onto a poly-L-lysine-coated glass coverslip with a silicon rubber wall. The culture was maintained for 10-14 days with serum-free defined medium [6]. Fura-2 loading and video-assisted Ca 2+ microfluorometry were performed as described [20]. The recording solution contained the following (mM): NaC1 130, KC1 5.4, CaC12 1.8, glucose 5.5, HEPES bufferNaOH (pH 7.3) 20. Tetrodotoxin (1 gM) was included in the solution unless otherwise noted. Neurons were discriminated from glial cells by their brighter fluorescence and the presence of long processes (Fig. 1A). For the identification of Purkinje cells, cells were fixed with 4% paraformaldehyde for 10 min and treated with 0.01% Triton X-100 for another 10 min. They were then incubated with monoclonal antibody 18A 10 against Purkinje cell-rich protein P400 [10] for 60 min, and stained by the avidin-biotin complex method. Purkinje cells cannot be identified by morphological criteria at this developmental stage. Most of the small non-Purkinje neurons were considered to be granule cells [6]. Data are expressed as mean + S.E.M. As shown in Fig. 1B, N M D A (100 aM), quisqualate
20
[Ca2~]i (nM)
400t NMDA
QUIS
KA ~/
NMDA+MK-801
QUIS+MK-801
KA÷MK-80~
100
sec
Fig. 2. Specific blockade by MK-801. MK-801 (I pM) blocked the [Ca2+]i elevation induced by 200/aM NMDA (A), without affecting [Ca2+]i elevation induced by 5/aM quisqualate (QUIS) (B) or 20/aM kainate (KA) (C). Presented as the mean value of 3 (A), 2 (B) and 2 (C) cells.
B ~[Ca2+3i (nM)
J
I
NMDA
QUIS
KA
Fig. 1. Change in [Ca2+]i induced by glutamate agonists in cerebellar neurons loaded with fura-2. A: cerebellar neurons maintained in culture for 14 days were loaded with fura-2 and viewed through a siliconintensified target video camera. Arrows indicate Purkinje cells identified by Purkinje-specific monoclonal antibody 18A10. Note that Purkinje cells cannot be identified by morphological criteria at this developmental stage. B: reponses to 200 pM NMDA, 5/aM quisqualate (QUIS) and 20/aM kainate (KA) by Purkinje cells (shaded bar, n = 18) and non-Purkinje cells (open bar, n = 31). Each bar represents mean + S.D.
(5 pM) and kainate (10 pM) increased [Ca2+]i from the basal level (54 + 3 nM) to 103 _ 4, 603 _ 21 and 465 _ 5 nM, respectively, in Purkinje cells (n = 18). They caused an increase in [Ca2+]i from the basal level (48 + 2 nM) to 201 +__4, 3 6 4 _ 11 and 203 ___7 nM, respectively, in non-Purkinje neurons (n = 31). The basal [Ca2+]i level was slightly higher in Purkinje cells than in non-Purkinje neurons, but the difference was not significant (P > 0.05 by Student's t-test). Responses to non-NMDA agonists were, however, significantly larger and responses to N M D A were significantly smaller in Purkinje cells (P
19
Elsevier Scientific Publishers Ireland Ltd. NSL 07242
MK-801 blocked the functional N M D A receptors in identified cerebellar neurons Michisuke Yuzaki x, Yoshihisa K u d o 2, K y o k o Akita 2, Atsushi Miyawaki 1 and Katsuhiko Mikoshiba 1 Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Osaka (Japan) and aDepartment of Neuroscience, Mitsubishi-kasei Institute of Life Sciences, Tokyo (Japan) (Received 3 July 1990; Accepted 6 July 1990)
Keywords:
N-Methyl-D-aspartate receptor; MK-801; Cerebellum; Intracellular calcium; Fura-2 microfluorometry
In order to clarify the nature of N-methyl-D-aspartate (NMDA) receptors in cerebellum, where heterogeneity of the NMDA receptor has been suggested, we investigated the action of MK-801 on the NMDA-induced [Ca2+]~rise in cultured cerebellar neurons using video-assisted microfluorometry. MK-801 caused a potent and selective blockade of the NMDA-activated [Ca2+]i elevation. The blockade caused by MK-801 was dependent on the presence of NMDA, i.e., use-dependent. There was no difference in the mode of blockade between immtmocytochemically identified Purkinje and non-Purkinje cells, although the relative size of the NMDA-induced [Ca2+]i rise was significantly less in Purkinje cells. These results indicate that the NMDA receptors in cultured cerebellar neurons are coupled with the same channels as those in other brain regions.
The major excitatory neurotransmitter glutamate exerts its action through a number of receptor subtypes [17] including N-methyl-D-aspartate (NMDA), kainate, and quisqualate. The N M D A receptor-channel complex is putatively involved in the synaptic plasticity mechanism through its high permeability to Ca 2+ [11]. Autoradiographical [13] and electrophysiological [2-5, 7] studies have shown the presence of functional binding sites for N M D A in cerebellum. It is not clear, however, whether these binding sites in cerebellum constitute the same receptor-channel complex as those in other regions of brain, because N M D A receptors in cerebellar granule cells are reported to show only minimal interaction with Mg 2+ [7] and phencyclidine (PCP) analog [9, 12, 18], both of which are strong non-competitive antagonists in other brain regions. The action of PCP analogs on N M D A receptors in Purkinje cells is also unclear. MK-801, a more potent and selective non-competitive antagonist than PCP which shares the same binding site as PCP [18], is suggested to act at the channel portion of the NMDA receptor-channel complex. In order to clarify the nature of the N M D A receptor-channel complex in cerebellum, we investigated the action of MK-801
Correspondence: M. Yuzaki, Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
on the NMDA-induced [Ca2+]i increase in mouse cerebellar neurons using video-assisted microfluorometry with fura-2. Cerebellar neurons were dissociated from the cerebellum of 0-day-old ICR mice by trypsin treatment, resuspended in culture medium and plated at a density of 1520 x l04 cells/cm 2 onto a poly-L-lysine-coated glass coverslip with a silicon rubber wall. The culture was maintained for 10-14 days with serum-free defined medium [6]. Fura-2 loading and video-assisted Ca 2+ microfluorometry were performed as described [20]. The recording solution contained the following (mM): NaC1 130, KC1 5.4, CaC12 1.8, glucose 5.5, HEPES bufferNaOH (pH 7.3) 20. Tetrodotoxin (1 gM) was included in the solution unless otherwise noted. Neurons were discriminated from glial cells by their brighter fluorescence and the presence of long processes (Fig. 1A). For the identification of Purkinje cells, cells were fixed with 4% paraformaldehyde for 10 min and treated with 0.01% Triton X-100 for another 10 min. They were then incubated with monoclonal antibody 18A 10 against Purkinje cell-rich protein P400 [10] for 60 min, and stained by the avidin-biotin complex method. Purkinje cells cannot be identified by morphological criteria at this developmental stage. Most of the small non-Purkinje neurons were considered to be granule cells [6]. Data are expressed as mean + S.E.M. As shown in Fig. 1B, N M D A (100 aM), quisqualate
20
[Ca2~]i (nM)
400t NMDA
QUIS
KA ~/
NMDA+MK-801
QUIS+MK-801
KA÷MK-80~
100
sec
Fig. 2. Specific blockade by MK-801. MK-801 (I pM) blocked the [Ca2+]i elevation induced by 200/aM NMDA (A), without affecting [Ca2+]i elevation induced by 5/aM quisqualate (QUIS) (B) or 20/aM kainate (KA) (C). Presented as the mean value of 3 (A), 2 (B) and 2 (C) cells.
B ~[Ca2+3i (nM)
J
I
NMDA
QUIS
KA
Fig. 1. Change in [Ca2+]i induced by glutamate agonists in cerebellar neurons loaded with fura-2. A: cerebellar neurons maintained in culture for 14 days were loaded with fura-2 and viewed through a siliconintensified target video camera. Arrows indicate Purkinje cells identified by Purkinje-specific monoclonal antibody 18A10. Note that Purkinje cells cannot be identified by morphological criteria at this developmental stage. B: reponses to 200 pM NMDA, 5/aM quisqualate (QUIS) and 20/aM kainate (KA) by Purkinje cells (shaded bar, n = 18) and non-Purkinje cells (open bar, n = 31). Each bar represents mean + S.D.
(5 pM) and kainate (10 pM) increased [Ca2+]i from the basal level (54 + 3 nM) to 103 _ 4, 603 _ 21 and 465 _ 5 nM, respectively, in Purkinje cells (n = 18). They caused an increase in [Ca2+]i from the basal level (48 + 2 nM) to 201 +__4, 3 6 4 _ 11 and 203 ___7 nM, respectively, in non-Purkinje neurons (n = 31). The basal [Ca2+]i level was slightly higher in Purkinje cells than in non-Purkinje neurons, but the difference was not significant (P > 0.05 by Student's t-test). Responses to non-NMDA agonists were, however, significantly larger and responses to N M D A were significantly smaller in Purkinje cells (P
