Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The mitogen-activated protein kinase (MAPK) cascade is a ubiquitous serine/threonine kinase cascade that has been
classically studied as a critical biochemical pathway involved in cell proliferation and
differentiation[1]. MAPKs constitute a
superfamily of three related kinases that are activated by various extracellular stimuli including the extracellular signal
regulated kinases (ERKs), the Jun N-terminal kinases (JNKs), and p38 kinases
(p38)[2,3]. ERK, JNK, and p38 can all be
activated by a variety of stimuli, but these kinases are differentially affected by certain signals. For example, ERKs are most
highly activated in response to mitogenic stimulation, whereas JNKs and p38 show greater activation in response to cellular
stress[4,5]. The pathway leading to ERK activation by growth factors and other mitogens has been studied extensively. The
first step involves activation of membrane-associated tyrosine kinases, followed by the sequential
activation of Ras and Raf. Raf then phosphorylates the
mitogen-activated protein kinase kinase (MEK), which in turn activates
ERK[6,7]. Although this cascade is mainly studied in
mitotic cell regulation, its components are actually most abundantly expressed in postmitotic neurons of the
developed nervous system[8]. The hippocampus region, which is commonly used as a model to study synaptic plasticity, has highly
expressed ERK[9,10]. What are the physiologic roles of this cascade in mature neurons? PD98059, a specific inhibitor of MEK,
the enzyme that activates ERK[11], has been shown to block induction of LTP in area CA1 of the
hippocampus[12] and attenuates multiple forms of synaptic plasticity in rat dentate
gyrus in vitro[13]. However, the regulation mechanism or
physiological role of this cascade in the activity-dependent synaptic connections between neurons is not clear.
Calcium plays an important role in regulating a great
variety of neuronal processes, especially in the transmitter release and synaptic connection. Oscillations in cytoplasmic
calcium have been observed in a wide variety of neuronal cell types including cortical and hippocampal neu-
rons[14_16]. In primary cultured hippocampal neurons, after one week in culture, networks of interconnected neurons are
formed. At approximately 9 d in vitro, some networks show spontaneous synchronized
Ca2+ oscillations[15,17]. These
oscillations are believed to encode information in neural
circuits[18,19] and might play an important role during physiological
or pathological events[20,21]. Many studies have implied that the MAPK cascade might participate in
[Ca2+]i
regulation[22_24]. Here we used PD98059, a commercially available inhibitor of MEK, and SB202474, a negative control, to explore whether this
cascade participates in the regulation of spontaneous synchronized
Ca2+ oscillations.
Materials and methods
Drugs Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) media, neurobasal medium, fetal bovine serum, B27 supplements,
0.25% trypsin-EDTA, and poly-D-lysine for cell culture were from Invitrogen (Carlsbad, CA, USA). Equine serum and
L-glutamine were from Hyclone (Logan, UT, USA). PD98059 and SB202474 were purchased from Calbiochem (La Jolla, CA,
USA) and were dissolved in dimethyl sulfoxide. Fluo-4-AM was from Molecular Probes (Eugene, OR, USA). Other reagents
were purchased from Sigma (St Louis, MO, USA).
Hippocampal cell culture and experiment Hippocampal neurons from embryonic rats (E18) were obtained according to
the method previously described[25]. In brief, hippocampal tissues from 18-d-old fetal rats were dissected and treated with
0.25% trypsin in Ca2+-Mg2+-free HBSS at 37
oC for 15 min; they were then dissociated by trituration with a glass Pasteur pipette
and plated in 35 mm culture dishes with glass bottoms (MatTek, Ashland, MA) for culture and subsequent microscopy. The
glass surface in each dish (~15 mm diameter) was pretreated with
poly-D-lysine for 2 h (500 µg/mL in borate buffer), washed
three times, and air-dried before cell plating. Approximately 75 000 cells were plated in the glass area of each dish in DMEM
containing 5% fetal bovine serum and 5% horse serum. On the second day after plating, the culture medium was replaced by
serum-free Neurobasal medium containing 2% B27 supplement and
500 µmol/L glutamine for reduced glial growth. Cells were
maintained in a CO2 incubator at 37
oC, and one-half volume of the culture medium was replaced with fresh Neurobasal medium
every 3 d. The experiments were carried out on cultures after 7 d.
Ca2+ imaging Hippocampal cells were loaded with 4
µmol/L Fluo-4-AM in Krebs-Ringer¡¯s saline (recording solution) (150
mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L
CaCl2, 1 mmol/L MgCl2, 10 mmol/L glucose, and 10 mmol/L HEPES, pH
7.4)[19] at 37 oC for 30 min, followed by three washes and a 15-min incubation period for further de-esterification of Fluo-4-AM before
imaging. Cells grown on the glass bottom in 35 mm dishes were directly imaged on a Nikon (Tokyo, Japan) TE300 inverted
microscope using a 40×numerical aperture, 1.30 oil immersion Plan Fluor objective. A Lambda DG-4 highspeed wavelength
switcher (Sutter Instruments, Novato, CA) was used for Fluo-4 excitation at 480 nm, and a cooled CCD camera (CoolSnap FX;
Roper Scientific, Princeton, NJ) was used for image acquisition. MetaFluor imaging software (Universal Imaging, Downingtown,
PA) was used for hardware control, image acquisition, and image analysis. The time-lapse recording of
Ca2+ signals in hippocampal neurons was carried out for a 2-min control period before and a 6-min period after the application of different
chemicals. The sampling rate was one frame every 2 s. The exposure time was 50 ms when CCD binning of 4×4 was used.
Quantitative analysis of synchronized
Ca2+ spikes Quantitative measurements of changes in intracellular
Ca2+ concentrations
([Ca2+]i) were done by obtaining the average
Fluo-4 fluorescence intensity of a 3×3 pixel analysis box placed at the center of the cell body; the intensity values were then
subtracted from the average background intensity measured in cell-free regions. Changes of
[Ca2+]i in each cell were then
represented by the changes of relative Fluo-4 fluorescence
(DF/F0) where
F0 was the baseline intensity obtained from the 2
min control period. Ca2+ spikes were defined as rapid elevation of
DF/F0 equal to or >20%. Under our imaging settings, fields
of 3_10 neurons were typically recorded and subsequently analyzed. To determine the frequency and amplitude of
Ca2+ spikes, we counted the number of
Ca2+ spikes and the average amplitude of these spikes over a 2-min period of the recording
as a defined time point. As a result, the 2-min control period yielded only one frequency and one amplitude value, whereas
the experiment period (6 min) resulted in three frequency and amplitude values at different time points after bath application
of a specific molecule. To the changes in the spike frequency and amplitude, these three frequency and amplitude values after
the drug application were normalized to the control frequency or amplitude values respectively and expressed as
per-centages, with a value of 100% indicating no change. We quantified and examined the changes in the spike frequency and
amplitude through the entire 6 min period after bath application. In order to assess the baseline changes after drug application,
we calculated the average
DF/F0 values of the rock bottom of each spike.
Bath application of different drugs To prevent adverse effects of high concentrations of drugs, a 2×working
concentration of the drug was made in Krebs-Ringer¡¯s solution and was applied to the cells to achieve the desired final concentration
through 1:1 dilution (v/v). Specifically, we first recorded
Ca2+ activities for a 2-min control period in 1 mL of Krebs-Ringer¡¯s
solution, removed 0.5 mL from the bath, added 0.5 mL of the 2×solution, and subsequently recorded for 6 min to examine the
effects on spontaneous Ca2+ spikes. For the control, we simply carried out the same procedure to apply Krebs-Ringer¡¯s
solution to determine that there was no artifact of this application method.
Statistical analysis Data from at least three dishes from different batches of cultures were pooled together and analyzed
for statistically significant differences using the paired
Student¡¯s t-test. Compiled data are expressed and graphed as
mean±SEM, with n denoting the number of neurons studied for each treatment. Differences were considered significant if a
P value was <0.05.
Results
Synchronized spontaneous Ca2+ spikes and the mechanisms in cultured hippocampal
networks We prepared low-density hippocampal neurons culture as described by
Banker et al[25]. After at least one week in culture, many hippocampal
neurons formed local networks which usually contained 3_10 neurons (Figure 1A, left panel). Spontaneous synaptic
activities of these neurons were examined by
Ca2+ imaging using the calcium-sensitive dye
Fluo-4[26] (Figure 1A, right panel). We
observed periodic, spontaneous spike elevations of
[Ca2+]i and these spikes appeared to be primarily synchronized among
the local group of cells (Figure 1B) without removing or reducing
Mg2+ in medium.
The mechanisms underlying the synchronized spontaneous
Ca2+ spikes in hippocampal networks have been studied
extensively, but the results from different published
reports are confusing. This may be caused by the variety of preparations
used for experiments. For example, Leinekugel et
al reported that the synchronized spontaneous
Ca2+ spikes were mediated by the synergistic excitatory actions of gamma-aminobutyric acid
(GABAA) and N-methyl-D-aspartate (NMDA) receptors in
the neonatal hippocampus[27], whereas
Tanaka et al reported that the oscillation of
Ca2+ was mainly mediated by non-NMDA-type glutamatergic
transmission[15]. To confirm the
Ca2+ spikes observed in our culture were driven by particular receptors,
we applied different antagonists of these receptors to our cultures. We found that a non-selective antagonist of NMDA and
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors kynurenic acid 1 mmol/L completely
blocked the spikes immediately after application (Figure 2A). The NMDA receptor antagonist APV at 50
mmol/L only partially inhibited the spike amplitude (Figure 2B), whereas AMPA/kainate receptors antagonist 6,7-dinitro-quinoxaline-2,3-dione
(DNQX) at 20 mmol/L completely and immediately blocked the spikes (Figure 2B, 2C). The addition of the
GABAA receptor antagonist bicuculline had a mixed effect on the
Ca2+ oscillations, which caused an
increase in amplitude but a decrease in frequency (Figure 2D). Subsequently adding kynurenic acid or DNQX completely
blocked the spikes (Figure 2D_F). These findings suggest that the oscillations we observed are similar to those observed by
Tanaka et al, which were mainly mediated by non-NMDA-type glutamatergic transmission.
PD98059 acutely inhibited synchronized spontaneous
Ca2+ spikes at a higher concentration
To test whether the MAPK cascade is required to maintain
Ca2+ oscillations, we used the MEK inhibitor PD98059 to block MAPK activation in
hippocampal neurons. As reported by Dudley et
al, PD98059 exerts it inhibition effect on MEK at concentrations from 1 to 100
µmol/L, with the IC50 value at approximately 10
µmol/L[28]. We chose 10, 30, and 60 µmol/L concentrations to test the effect on
Ca2+ oscillations. We found that PD98059 at 10 µmol/L had no significant effect on
Ca2+ spike frequency and only slightly duced
decreased the Ca2+ spike amplitude (Figure 3A,3D,3E). PD98059 30 µmol/L significantly inhibited the
Ca2+ spikes immediately after application (Figure 3B). Six minutes after application, the spike frequency was decreased to 25.38%±7.40%
(mean±SEM, n=16) of that of the control period (Figure 3D) and the spike amplitude was 25.16%±
6.99% (n=16) of that of the control period (Figure 3E). Application of 60 µmol/L PD98059 caused a more rapid and severe
inhibition of the Ca2+ spikes (Figure 3C). Six minutes after application the spike frequency was decreased to 14.53%±
5.34% (n=16) of that of the control period (Figure 3D) and the
spike amplitude was 31.81%±6.27%
(n=16) of that of the control period (Figure 3E). No significant difference was found with the control period, indicating that no artifact was pro
by the bath application method. Overall, these results demonstrate that PD98059 rapidly inhibits the
Ca2+ spikes in a dose-dependent manner.
The effect of SB202474 on synchronized spontaneous
Ca2+ spikes SB202474 is an inactive structural analog of PD98059.
We also tested its effect on the Ca2+ spikes to confirm whether or not the inhibition effect of PD98059 on
Ca2+ spikes was through inhibition of the MAPK cascade. We found that 10 µmol/L SB202474 had no inhibition effect
on the Ca2+ oscillations frequency, but it had a small
enhance-ment effect on the frequency immediately after application. As a consequence of the
frequency increase, the amplitude of the spikes decreased (Figure 4A,4C,4D). Application of 60 µmol/L SB202474 had no
significant effect on frequency or amplitude of the
Ca2+ spikes (Figure 4B,4C,4D).
Discussion
We prepared the hippocampal culture based mainly on the method described by
Banker et al[25]. We used serum-free
Neurobasal medium with B27 supplement to reduce glia cell growth and increase neuron survival. As described previously,
in Neurobasal/B27 medium, glia growth is
reduced to less than 0.5%, resulting in a nearly pure population of
neurons[29]. We used the microtubule-associated
protein (MAP2) and 4¡¯,6-diamidino-2-phenylindole, dihydrochloride (DAPI) double-staining to detect the neuron proportion
in our culture. We found that there were more than 85% neurons in our culture, and the other cells were glia cells including
astrocytes and oligocytes. In this culture, glia cells were spread beneath the neuron network. The synchronized
spontaneous Ca2+ oscillations we observed were confined to the neuron networks. Although glia cells sometimes were observed
irregularly [Ca2+]i elevation, they did not directly participate in the
Ca2+ oscillation of neuron networks. We could deduce that
glia cells might act as a supporter in maintaining and modifying the synchronized spontaneous
Ca2+ oscillation in neuron networks. We detected the mechanism of the
Ca2+ oscillation in our culture, and found that it was mainly mediated by
non-NMDA-type glutamatergic transmission. NMDA-type glutamatergic transmission only partially inhibited the spike amplitude.
GABAergic synaptic transmission might act as a regulator of the
Ca2+ oscillation.
PD98059 has long been used as a specific MEK inhibitor to study the involvement of the ERK pathway on cellular events
as diverse as growth and differentiation, cell death and survival, and synaptic
plasticity[30]. Our results show that PD98059
acutely inhibits the synchronized Ca2+ oscillations in a dose-dependent manner. When the concentration (10 µmol/L) is too
low to inhibit MAPK activation[31], PD98059 has little effect on the
Ca2+ spikes. However, at higher concentrations (30
µmol/L and 60 µmol/L) PD98059 significantly and acutely inhibits synchronized
Ca2+ oscillations. We also showed that PD98059
inhibited the synchronized Ca2+ oscillations mainly through inhibition of MEK. This conclusion is based on the observation
that SB202474, a structural analog of PD98059, which is usually used as a negative control for MAPK inhibitor studies, has
no effect on the Ca2+ spike at a higher concentration (60 µmol/L). Although SB202474 has a strange effect on the
Ca2+ oscillations at a lower concentration (10 µmol/L), by slightly increasing the frequency of the
Ca2+ spikes and decreasing the amplitude of the spikes, it has no effect at a higher concentration (60 µmol/L). The effect of SB202474 on
Ca2+ spikes at a lower concentration might result from its nonspecific activation of the
Ca2+ channel, an idea we will try to explain in future studies.
The salient point of this study is that we can conclude that SB202474 does not inhibit
Ca2+ spikes, but activates
Ca2+ spikes at a lower concentration. As SB202474 is an inactive analog of PD98059, these observations indicate that PD98059 inhibits
Ca2+ oscillations mainly through the inhibition of MEK, but not its side-effect on
Ca2+ channel[30,32].
Many studies have implied that the MAPK cascade might participate in
[Ca2+]i
regulation[22,23]. The synchronized
spontaneous Ca2+ spikes in networked neurons represent the periodic firing of action potentials, which are believed to play a major
role in the development and plasticity of neuronal
circuitry[19], and the encoded information of the spontaneous
Ca2+ oscillations was reported to lie in their frequency or
amplitude[33]. Therefore, the inhibitory effect of PD98059 on the frequency and
amplitude of spontaneous Ca2+ oscillations reported here implied that the MAPK cascade was required to maintain the
spontaneous Ca2+ oscillations in developing hippocampal
neurons. We know that MAPKs are a family of serine/threonine
protein kinases which have classically been studied as regulators of cell proliferation and differentiation. The most important
and well-known member of the MAPK family is ERK, which is initiated by growth factor receptor signaling. ERKs are
extensively expressed in dendrites and somas of pyramidal neurons of the adult nervous system and can be activated by
several neurotransmitters in neuronal culture
system[34]. These points suggest that MAPKs might be excellent
candidates for regulation of synaptic plasticity in post-mitotic neurons. MAPKs have been reported to regulate glutamate release and
participate in the introduction of
LTP[12]. Previous findings have shown that the MAPK cascade regulates synaptic
transmission, and our work substantiates this by providing the time-course of PD98059 actions on synaptic transmission (the
synchronized Ca2+ transients). It is also well known that
Ca2+ plays an important role in the epileptiform
discharge[35,36]. The synchronized spontaneous
Ca2+ oscillations in the neuron
network[37] are usually considered a kind of spontaneous
epileptiform activity. Zhao et al reported that ERK1/2 was required for the induction of group I metabotropic glutamate
receptor-mediated epileptiform discharges.
Murray et al reported that PD98059 protected hippocampal neurons from seizure-like
events[38]. Our results provide further evidence for the effect of PD98059 on the hippocampal network. The inhibitory effect
of PD98059 on synchronized spontaneous
Ca2+ oscillations through MAPK might be used to develop drugs for epileptiform
therapy.
References
1 Seger R, Krebs EG. The MAPK signaling cascade. FASEB J 1995; 9: 726_35.
2 Lazou A, Sugden PH, Clerk A. Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the
G-protein-coupled receptor agonist phenylephrine in the perfused rat heart. Biochem J 1998; 332: 459_65.
3 Haq SE, Clerk A, Sugden PH. Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by adenosine in the
perfused rat heart. FEBS Lett 1998; 434: 305_8.
4 Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ, Ben-Levy R, Ashworth
A, et al. Stimulation of the stress-activated
mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun
N-terminal kinases are activated by ischemia/reperfusion. Circ Res 1996; 79: 162_73.
5 Stofega MR, Yu CL, Wu J, Jove R. Activation of extracellular signal-regulated kinase (ERK) by mitogenic stimuli is repressed in
v-Src-transformed cells. Cell Growth Differ 1997; 8: 113_9.
6 Marais R, Marshall CJ. Control of the ERK MAP kinase cascade by Ras and Raf. Cancer Surv 1996; 27: 101_25.
7 Harrisingh MC, Perez-Nadales E, Parkinson DB, Malcolm DS, Mudge AW, Lloyd AC. The Ras/Raf/ERK signalling pathway drives Schwann
cell dedifferentiation. EMBO J 2004; 23: 3061_71.
8 Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul
1991; 2: 357_71.
9 Thomas KL, Hunt SP. The regional distribution of extracellularly regulated kinase-1 and -2 messenger RNA in the adult rat central nervous
system. Neuroscience 1993; 56: 741_57.
10 Fiore RS, Bayer VE, Pelech SL, Posada J, Cooper JA, Baraban JM. p42 mitogen-activated protein kinase in brain: prominent localization
in neuronal cell bodies and dendrites. Neuroscience 1993; 55: 463_72.
11 Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein
kinase kinase in vitro and in vivo. J Biol Chem 1995; 270: 27489_94.
12 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol
Chem 1997; 272: 19103_6.
13 Coogan AN, O'Leary DM, O'Connor JJ. P42/44 MAP kinase inhibitor PD98059 attenuates multiple forms of synaptic plasticity in rat
dentate gyrus in vitro. J Neurophysiol 1999; 81: 103_10.
14 Wang X, Gruenstein EI. Mechanism of synchronized
Ca2+ oscillations in cortical neurons. Brain Res 1997; 767: 239_49.
15 Tanaka T, Saito H, Matsuki N. Intracellular calcium oscillation in cultured rat hippocampal neurons: a model for glutamatergic
neurotransmission. Jpn J Pharmacol 1996; 70: 89_93.
16 Liu XH, Lu GW, Cui ZJ. Calcium oscillations in freshly isolated neonatal rat cortical neurons. Acta Pharmacol Sin 2002; 23: 577_81.
17 Koizumi S, Inoue K. Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones. Br J Pharmacol 1997; 122: 51_8.
18 Lisman JE. Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci 1997; 20: 38_43.
19 Bacci A, Verderio C, Pravettoni E, Matteoli M. Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons
in culture. Eur J Neurosci 1999; 11: 389_97.
20 Traub RD, Wong RK. Cellular mechanism of neuronal synchronization in epilepsy. Science 1982; 216: 745_7.
21 Miles R, Wong RK. Single neurones can initiate synchronized population discharge in the hippocampus. Nature 1983; 306: 371_3.
22 Ansari HR, Husain S, Abdel-Latif AA. Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha,
ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isopro-terenol. J Pharmacol Exp
Ther 2001; 299: 178_86.
23 Kupzig S, Walker SA, Cullen PJ. The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and
the ERK/MAPK cascade. Proc Natl Acad Sci USA 2005; 102: 7577_82.
24 Doherty P, Williams G, Williams EJ. CAMs and axonal growth: a critical evaluation of the role of calcium and the MAPK cascade. Mol
Cell Neurosci 2000; 16: 283_95.
25 Banker GA, Cowan WM. Rat hippocampal neurons in dispersed cell culture. Brain Res 1977; 126: 397_42.
26 Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I. Chemical and physiological characterization of Fluo-4
Ca(2+)-indicator dyes. Cell Calcium 2000; 27: 97_106.
27 Leinekugel X, Medina I, Khalilov I, Ben-Ari Y, Khazipov R.
Ca2+ oscillations mediated by the synergistic excitatory actions of GABA(A)
and NMDA receptors in the neonatal hippocampus. Neuron 1997; 18: 243_55.
28 Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl
Acad Sci USA 1995; 92: 7686_9.
29 Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new
serum-free medium combination. J Neurosci Res 1993; 35: 567_76.
30 Pereira DB, Carvalho AP, Duarte CB. Non-specific effects of the MEK inhibitors PD098,059 and U0126 on glutamate release from
hippocampal synaptosomes. Neuropharmacology 2002; 42: 9_19.
31 Gould MC, Stephano JL. MAP kinase, meiosis, and sperm centrosome suppression
in Urechis caupo. Dev Biol 1999; 216: 348_58.
32 Gould MC, Stephano JL. Inactivation of Ca(2+) action potential channels by the MEK inhibitor PD98059. Exp Cell Res 2000; 260:
175_9.
33 Gu X, Spitzer NC. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous
Ca2+ transients. Nature 1995; 375: 784_7.
34 English JD, Sweatt JD. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 1996;
271: 24329_32.
35 Pisani A, Bonsi P, Martella G, De Persis C, Costa C, Pisani
F, et al. Intracellular calcium increase in epileptiform activity: modulation by
levetiracetam and lamotrigine. Epilepsia 2004; 45: 719_28.
36 Shang ZC, Sun BZ, Chen ZN, Wang J, Feng Q, Wang W,
et al. Effect of PD98059 on Ras-MAPK signal transduction pathway of chronic
myelogenous leukemia. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2003; 19: 54_5.
37 Yaari Y, Konnerth A, Heinemann U. Spontaneous epileptiform activity of CA1 hippocampal neurons in low extracellular calcium
solutions. Exp Brain Res 1983; 51: 153_6.
38 Murray B, Alessandrini A, Cole AJ, Yee AG, Furshpan EJ. Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in
a cell-culture model of seizure activity. Proc Natl Acad Sci USA 1998; 95: 11975_80.
|
