1Graduate Institute of Life Sciences, National Defense Medical Center; and 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China
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ABSTRACT |
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Wang, Hai L., Li Y. Tsai, and Eminy H. Y. Lee. Corticotropin-Releasing Factor Produces a Protein Synthesis-Dependent Long-Lasting Potentiation in Dentate Gyrus Neurons. J. Neurophysiol. 83: 343-349, 2000. Corticotropin-releasing factor (CRF) was shown to produce a long-lasting potentiation of synaptic efficacy in dentate gyrus neurons of the rat hippocampus in vivo. This potentiation was shown to share some similarities with tetanization-induced long-term potentiation (LTP). In the present study, we further examined the mechanism underlying CRF-induced long-lasting potentiation in rat hippocampus in vivo. Results indicated that the RNA synthesis inhibitor actinomycin-D, at a concentration that did not change basal synaptic transmission alone (5 µg), significantly decreased CRF-induced potentiation. Similarly, the protein synthesis inhibitor emetine, at a concentration that did not affect hippocampal synaptic transmission alone (5 µg), also markedly inhibited CRF-induced potentiation. These results suggest that like the late phase of LTP, CRF-induced long-lasting potentiation also critically depend on protein synthesis. Further, prior maximum excitation of dentate gyrus neurons with tetanization occluded further potentiation of these neurons produced by CRF and vise versa. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed that CRF mRNA level in the dentate gyrus was significantly increased 1 h after LTP recording. Together with our previous findings that CRF antagonist dose-dependently diminishes tetanization-induced LTP, these results suggest that both CRF-induced long-lasting potentiation and tetanization-induced LTP require protein synthesis and that CRF neurons are possibly involved in the neural circuits underlying LTP.
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INTRODUCTION |
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We have previously demonstrated that the 41 amino
acid polypeptide corticotropin-releasing factor (CRF) produces a
long-lasting enhancement of synaptic efficacy in rat hippocampal
neurons (Wang et al. 1998). This CRF-induced enhancement
was shown to share some similar mechanisms with tetanization-induced
long-term potentiation (LTP) because pretreatment with the CRF receptor
antagonist dose-dependently diminishes the magnitude of LTP, and CRF
and tetanic stimulation have an additive effect on hippocampal neuron
excitation (Wang et al. 1998
). Further, the
N-methyl-D-aspartate (NMDA) receptor antagonist
MK801, known to prevent tetanization-induced LTP (Collingridge and Bliss 1987
), also partially blocks CRF-induced
potentiation. Morevoer, we have demonstrated that CRF-induced
potentiation, like the case of LTP, is also mediated through the
cyclase-adenosine-3,5-monophosphate (cAMP) signaling pathway because
the cAMP inhibitor Rp-cAMP significantly blocks CRF-induced
potentiation (Wang et al. 1998
).
Physiologically, CRF is known to stimulate the secretion of
adrenocorticotropin and -endorphin from the pituitary (Rivier et al. 1981
). CRF is also known to produce various behavioral activations (Dunn and Berridge 1990
). For example,
intraventricular injection of CRF enhances locomotion, rearing, and
sniffing responses in rats (Sutton et al. 1982
;
Veldhuis and DeWied 1984
). More related to the present
study, both our and other laboratories have found that direct central
injections of CRF improve acquisition and memory retention of rats in
various learning tasks (Koob and Bloom 1985
; Lee
et al. 1993
; Tershner and Helmstetter 1996
).
Further, CRF-induced memory facilitation is dependent on protein
synthesis because protein synthesis inhibitors prevent the
memory-enhancing effect of CRF (Lee et al. 1992
).
Because protein synthesis mediates CRF-induced memory facilitation and
that CRF and LTP share some similar mechanisms in producing hippocampal
neuron excitation, in the present study, we were aimed to investigate
the following. 1) Is protein synthesis also involved in the
cellular mechanism underlying CRF-induced long-lasting potentiation?
2) Does CRF-induced long-lasting potentiation share the same
mechanism with tetanization-induced LTP? To study this, we examined
whether maximum excitation of dentate gyrus neurons with tetanic
stimulation occludes further potentiation of these neurons produced by
CRF and vice versa. 3) Does tetanic stimulation activate CRF
neurons in the dentate gyrus?
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METHODS |
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Animals
Adult male Sprague-Dawley rats weighing 250-350 g bred in the Animal Facility of the Institute of Biomedical Sciences, Academia Sinica were used. Animals were housed three per cage in a room maintained at 23 ± 2°C and a 12-h light/dark cycle (light on at 6:30 A.M.) with food and water continuously available.
Drugs and drug application
Corticotropin-releasing factor, actinomycin-D and emetine were
purchased from Sigma Company (St. Louis, MO). All other chemical reagents of the highest grade were purchased from Merck (Darmstadt, Germany) or Sigma. All drugs were dissolved in 0.9% saline. Thirty minutes after baseline recording, drugs were delivered to the dentate
gyrus through Hamilton syringe at a rate of 20 nl/min. At the end of
drug infusion, the Hamilton syringe was withdrawn, and the recording
electrode was placed into the dentate gyrus. Tetanic stimulation was
then given. The concentration of CRF, actinomycin-D, and emetine were
given in the dose of 100 ng, 5 µg, and 5 µg in a volume of 100 nl,
respectively. These concentrations were chosen because CRF at this
concentration was shown to produce a consistent long-lasting
potentiation in dentate gyrus neurons (Wang et al. 1998)
and actinomycin-D and emetine were shown to prevent the
memory-facilitating effect of CRF (Lee et al. 1992
). For
the drug interaction studies, actinomycin-D or emetine was given 90 min
before CRF or tetanic stimulation.
Stimulating and recording electrodes
The stimulating electrodes were platinum concentric bipolar
electrodes with a tip diameter of 25 µm. The stereotaxic coordinates were adjusted for variation in rat ages to maximize the monosynaptic responses of the excitatory postsynaptic potentials (pEPSP) produced by
the granule cells in response to stimulation of the dorsomedial perforant path. The correctness of the coordinates was verified by the
wave form that appeared on the oscilloscope on stimulation (McNaughton and Barnes 1977). The averaged stimulating
coordinates were AP
8.3 mm from bregma, ML +4.4 mm from the midline,
and 2.7 mm below the skull surface. The recording electrodes were prepared from single-barrel borosilicate glass micropipettes 1.2 mm
OD × 0.6 mm ID, pulled on a Narishige vertical puller and filled with 3 M NaCl. Resistance ranged from 1 to 3 M
. The electrodes were
positioned ~3.5 mm posterior to bregma and 2.0 mm from the midline.
The dentate gyrus was identified by single-unit activity characteristic
of granule cells, as well as by an electrode depth of 3.0-3.5 mm below
the brain surface. After the stimulating and recording electrodes were
positioned, 5% agar dissolved in 0.9% NaCl was applied over the
exposed brain and skull to prevent surface drying and reduce movement artifacts.
Electrophysiological recording
Approximately 1 h before the surgery, animals were
anesthetized with urethan (1.4 g/kg, 25% concentration ip). The rat
was then placed on a stereotaxic instrument for surgery. At the
beginning of the surgery, lidocaine was applied in small quantities to
the incision area. The surface of the skull was exposed, two holes were
made with a dental drill, and the dura matter was incised. Throughout
the surgery and experiments, body temperature was monitored and
maintained at 35 ± 1°C with a feedback control system.
Stimulation parameters for evoking population spikes in vivo were
adopted from the protocol of Chiba et al. (1992).
Briefly, stimulation consisted of 100 µs duration monophasic constant
current pulses delivered once per minute. The stimulus intensity was
set to a level that produced a population spike of ~50% of the
maximum amplitude. An input-output curve was obtained over the range of 100-600 µA. Stimulation parameters and electrophysiological
recording of in vivo LTP for pEPSPs were conducted according to the
method of Wayner et al. (1996)
. Briefly, stimulation
consisted of 50 µs duration monophasic constant current pulses
delivered once per minute. An input-output curve was obtained over the
range of 50-400 µA. Stimulus intensities for actual recording ranged from 50 to 200 µA and produced average pEPSP amplitudes of 3-5 mV.
Within this stimulus intensity, a population spike is not usually seen,
but the excitatory effect of tetanization and CRF can be clearly
observed. However, if the baseline stimulus intensity increased to
150-250 µA, a population spike of 7-8 mV in amplitude can always be
seen with tetanization, and most of the time seen with CRF injection.
Once determined, stimulus current remained constant throughout the
experiment. After recording 30 min of baseline responses, four sets of
tetanic stimulation were administered to induce stable and long-lasting
potentiation. Each set contained 5 trains, 10 pulses per train at 400 Hz, delivered at a rate of 1 train per second for 5 s. The pulse
widths in the trains were 50, 100, 150, and 200 µs, respectively.
Tetanic stimulation was delivered at 10-min intervals for 40 min. LTP
as well as CRF-induced potentiations were measured as the increase in
slope during the first millisecond of pEPSP in millivolts. Percent
increase in the pEPSP was calculated as follows: the slope in
millivolts at each minute minus the mean baseline slope and then
divided by the mean baseline slope. The granule cell pEPSPs were
amplified by a conventional amplifier at a frequency band of 1.0 Hz to
10 kHz. All potentials were monitored on an oscilloscope. The pEPSP slope recorded at time point set at 1.52 ms was used for data analysis.
Total RNA extraction
Total RNA was prepared according to the method of
Chomczynski and Sacchi (1987). Briefly, the hippocampal
tissue from the recording site was dissected out at the end of
recording using a brain slicer. The dentate gyrus was further dissected
out using a punch with 1.5 mm diam (Ma et al. 1999
). The
dentate gyrus tissue was then homogenized with Ultraspec RNA isolation
kit. The homogenate was kept on ice at 4°C for 5 min, added with 0.2 ml chloroform, and centrifuged at 14,500 g for 15 min. The
aqueous phase was added with equal volume of isopropanol and kept on
ice for 10 min. The reaction mixture was centrifuged at 14,500 g for 10 min. The pellet RNA was washed twice with 75%
ethanol and precipitated by subsequent centrifugation at 11,500 g for 5 min. The pellet was briefly dried under vacuum and
dissolved in diethyl-pyrocarbonate (DEPC)-treated water. To avoid DNA
contamination, total RNA (20 µl) was further treated with DNase (0.1 U/µl) in the presence of RNase inhibitor (0.2 U/µl) for 30 min at
37°C in Taq buffer. After phenol/chloroform (3:1,
vol/vol) extraction, the aqueous phase was recovered by centrifugation
at 14,500 g for 15 min. It was then added with 1/10 volume of 2.5 M
sodium acetate and 2 volume of 95% ethanol. The mixed solution was
kept in a freezer (
20°C) for at least 2 h and centrifuged at
14,500 g for 15 min. The pellet was then washed with
75% ethanol and dried.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of CRF mRNA level
Because the total amount of RNA extracted from the dentate
gyrus was <20 µg, and because of the minute amount of CRF present in
the dentate gyrus, the quantitative RT-PCR method was adopted for the
present study. Briefly, 0.05 µg of total RNA was reverse transcribed
using avian myeloblastosis virus reverse transcriptase (AMVRT) with
oligo-dT (0.5 µg/µl) as primers in a 20-µl reaction buffer
containing 25 mM Tris-HCl (pH 8.3), 25 mM KCl, 5 mM
MgCl2, 5 mM dithiothreitol, 0.25 mM spermidine, 1 mM deoxy-nucleoside triphosphate (dNTPs), and 20 U RNase inhibitor for
1 h at 42°C. AMVRT was heat-inactivated by boiling for 5 min.
The endogenously expressed rat hypoxanthine phosphoribosyl transferase
(HPRT) mRNA was used as an internal control template, which was
coamplified with the CRF mRNA. One set of PCR primers was used for CRF
and HPRT, respectively. The oligonucleotide primer pairs for PCR were designed by using a computer program (Lowe et al. 1990).
The sequences for primer pairs of CRF were 5'(5'-CAG AAC AAC AGT GCG
GGC TCA-3') and 3'(5'-GGA AAA AGT TAG CCG CAG CCT-3'), and that for
HPRT were 5'(5'-CTC TGT GTG CTG AAG GGG GG-3') and 3'(5'-GGG ACG CAG
CAA CAG ACA TT-3'), which flanked a region of 365 and 625 bp for CRF and HPRT, respectively. In PCR amplification, specific oligonucleotide primer pairs of CRF and HPRT (0.5 and 0.2 µM, respectively) were incubated with 2 µl of the RT product and 2 U of Taq
polymerase in a 20-µl reaction mixture that included 1 × Taq buffer, 1.5 mM MgCl2 and 200 µM dNTPs.
The parameters for PCR amplification were as follows: denaturation at
94°C for 1 min, annealing at 55°C for 1.5 min, and polymerization
at 72°C for 2 min. The reactions were performed for 30 cycles and
followed by 10-min incubation at 72°C for complete elongation. Ten
microliters of each PCR product was investigated by 2% agarose gel
electrophoresis. The gels were stained with ethidium bromide solution
at a concentration of 1 µg/ml for 30 min and destained in distilled
water for 15 min. For quantification, the gels were illuminated by
ultraviolet lamp, and the intensity of each CRF cDNA band was
normalized with the corresponding HPRT cDNA band and quantified with a
densitometer (Eagle Eye II, Stratagene).
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RESULTS |
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Effects of actinomycin-D on CRF-induced potentiation
To study whether RNA synthesis is involved in CRF-induced long-lasting potentiation, we have examined the effects of actinomycin-D, a transcription inhibitor, on CRF-induced potentiation. Results in Fig. 1A show that actinomycin-D at a dose of 5 µg/µl, which by itself did not markedly affect basal synaptic transmission in dentate gyrus neurons [t(1,8) = 0.61, P > 0.05], significantly prevented CRF-induced long-lasting potentiation [t(1,8) = 13.74, P < 0.001; Fig. 1B] in a manner similar to actinomycin-D-induced impairment in tetanization-induced LTP in vivo [t(1,8) = 20.25, P < 0.001; Fig. 1C]. Figure 1D illustrates the patterns of a single response evoked by CRF (1) and actinomycin-D + CRF (2), respectively.
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Effects of emetine on CRF-induced potentiation
To investigate whether CRF-induced potentiation also depends on protein synthesis, we have examined the effects of emetine, a protein synthesis inhibitor, on CRF-induced potentiation. Similar to the effects observed with actinomycin-D, emetine at 5 µg/µl, which did not markedly affect the baseline activity of dentate gyrus neurons [t(1,8) = 0.53, P > 0.05; Fig. 2A], markedly decreased the magnitude of CRF-induced long-lasting potentiation [t(1,8) = 10.93, P < 0.001; Fig. 2B] in a way similar to emetine-induced inhibition of tetanization-induced LTP in vivo [t(1,8) = 18.25, P < 0.001; Fig. 2C]. Figure 2D illustrates the patterns of a single response produced by CRF (1) and emetine + CRF (2), respectively.
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Effects of prior tetanization on CRF-induced potentiation
We have previously demonstrated that tetanic stimulation and CRF,
when both were not given at the maximum intensity and concentration, produced an additive effect on hippocampal neuron potentiation (Wang et al. 1998). If tetanic stimulation and CRF do
share a common mechanism, then prior maximum excitation of dentate
gyrus neurons should occlude further excitation of these neurons
produced by the other stimulus. The present experiment examined this
hypothesis. Results in Fig. 3A
show that prior maximum excitation of dentate gyrus neurons with four
trains of tetanic stimulation effectively prevented further excitation
of these neurons produced by 100 ng of CRF
[t(1,8) = 0.78, P > 0.05]. Figure 3B illustrates the patterns of a single
response produced by tetanic stimulation alone (1) and
tetanic stimulation followed by CRF administration (2).
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Effects of CRF pretreatment on tetanization-induced potentiation
To further test the above hypothesis, we have also examined the
effects of tetanic stimulation and CRF on hippocampal neuron potentiation in an opposite way. Results in Fig.
4A indicate that if 100 ng CRF
was given to produce a maximum excitation of dentate gyrus neurons
(Wang et al. 1998), the following tetanic stimulation did not produce further consistent excitation on these neurons [t(1,8) = 1.53, P > 0.05], but a transient increase in the amplitude of pEPSP was
observed. Figure 4B illustrates the patterns of a single
response produced by CRF alone (1) and CRF followed by tetanic stimulation (2).
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CRF mRNA expression on tetanic stimulation
If CRF neurons are involved in the neural circuits underlying LTP, it is expected that CRF neurons are activated on tetanic stimulation. Results in Fig. 5 reveal that there was ~30%, but a significant increase in CRF mRNA level in the dentate gyrus 1 h after tetanic stimulation [t(1,18) = 4.63, P < 0.05].
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DISCUSSION |
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In a previous study, we have demonstrated that direct injections
of CRF into the dentate gyrus produce a long-lasting enhancement of
synaptic efficacy in rat hippocampal neurons. This CRF-induced potentiation shares some similarities with tetanization-induced late
phase of LTP (L-LTP) in that both potentiations are blocked by the cAMP
inhibitor Rp-CAMP, and CRF-induced potentiation and tetanization-induced potentiation have an additive effect on dentate neuron excitation (Wang et al. 1998). However, one
important cellular mechanism underlying L-LTP is that it requires
protein synthesis (Frey et al. 1996
; Krug et al.
1984
). We have therefore examined whether protein synthesis is
also involved in CRF-induced potentiation. Actinomycin-D and emetine
are known to prevent RNA synthesis and protein synthesis, respectively
(Frey et al. 1996
; Stanton and Sarvey
1984
; Stryer 1988
). In the present study, we
have demonstrated that both inhibitors effectively blocked CRF-induced
potentiation. More interestingly, this effect mainly occurred in the
late phase of CRF-induced potentiation, similar to that observed with
tetanization-induced LTP. Although actinomycin-D and emetine also
slightly decreased the pEPSP slope in the early phase of LTP (Figs.
1C and 2C), this effect was far less than that
observed in the late phase of LTP. In both studies, ~5-10% of
potentiation in pEPSP slope was still observed at the end of recording.
This is probably because the dose chosen for the present study was
moderate because higher concentration of both inhibitors was known to
produce cell toxicity (Lindenboim et al. 1995
;
Wenzel and Cosma 1984
). In another study, Huang
and Kandel (1995)
have similarly reported that dopamine D1/D5
receptor activation also produces a protein synthesis-dependent long-lasting potentiation of hippocampal neurons. Further, DA receptor
activation-induced potentiation was also suggested to be mediated
through the cAMP signaling pathway. Because CRF receptor activation is
known to stimulate adenylyl cyclase activity (Dave et al.
1985
) and increase hippocampal cAMP level (Wang et al. 1998
), CRF also increases protein phosphorylation associated
with memory facilitation in rats (Hung et al. 1992
). It
is possible that CRF may activate the cAMP and cAMP responsive element
binding protein (CREB) phosphorylation pathway and turn on the
downstream genes for gene expression and protein synthesis. The
specific genes that are turned on on CRF administration require further investigation.
To address the issue that CRF and L-LTP may share similar mechanisms in
producing hippocampal neuron potentiation, we have first maximally
excited hippocampal neurons with four trains of tetanic stimulation
followed by CRF injection or vise versa. Our results that the second
stimulation could not produce further excitation of these neurons
suggest that CRF and tetanization do act additively and synergistically
to activate hippocampal neurons. But in the case of CRF followed by
tetanization, a transient increase in the magnitude of LTP immediately
after tetanic stimulation was still observed (Fig. 3A).
This is probably due to the fact that the induction of LTP is mainly
dependent on NMDA receptor activation (Collingridge and Bliss
1987; Morris et al. 1986
), although NMDA
receptor blockade only partially prevents CRF-induced potentiation
(Wang et al. 1998
). Therefore the transient increase in
pEPSP following tetanization possibly reflects a transient activation
of NMDA receptor and Ca2+ influx, rather than protein
synthesis. This differential effect of NMDA receptor blockade on CRF
and tetanization-induced potentiations also suggests that the
mechanisms underlying the induction of these two potentiations are not
completely the same.
Then how might CRF neurons involved in the neural circuits
underlying LTP? To address this issue, we have analyzed CRF mRNA expression in the dentate gyrus on tetanic stimulation by quantitative RT-PCR analysis. Our results indicated that there was a significant increase in CRF mRNA level 1 h after tetanization. In an earlier study, we have similarly reported that hippocampal CRF mRNA level is
increased in rats showing good retention performance on learning (Lee et al. 1996). These results together suggest that
perforant path stimulation may directly or indirectly activate local
CRF neurons in the hippocampus. This suggestion is also supported by
the observation that in the paraventricular nucleus of the hypothalamus, ~70% of the CRF-positive neurons also express the NMDA
receptor NR1R mRNA, and ~20-45% of these neurons also express other
types of the glutamate receptors (Aubry et al. 1996
). In a more recent study, we have demonstrated that intra-dentate CRF injection dose-dependently increases brain-derived neurotrophic factor
(BDNF) mRNA level (Ma et al. 1999
), while BDNF mRNA
expression was also increased on LTP stimulation (Castren et al.
1993
). However, the extent of CRF mRNA elevation was ~30%,
indicating that the number of CRF neurons in the dentate gyrus is
limited. Further, the present results indicated that actinomycin-D and
emetine inhibited tetanization-induced LTP to a greater extent than
CRF-induced long-lasting potentiation (Figs. 1, B and
C, and 2, B and C). These
results also suggest that, although CRF neurons are possibly involved
in the neural circuits underlying LTP, the mechanisms underlying these
two processes may not be completely the same. Tetanic stimulation may
activate more neurons and involve more complicated neural networks. It
would be ideal to examine CRF mRNA level after actinomycin-D and
emetine treatments. Alternatively, CRF may activate the noradrenergic
innervation to the hippocampus to produce potentiation. This suggestion
is based on the findings that norepinephrine (NE) also produces
long-lasting potentiations in dentate gyrus neurons in a protein
synthesis-dependent manner (Stanton and Sarvey 1984
)
and that CRF facilitates hippocampal NE release (Lee et al.
1994
) and activates noradrenergic neurons in the rat brain
(Emoto et al. 1993
). Further, NE receptor blockade prevents the memory-facilitating effect of CRF injected into the hippocampus (Lee et al. 1993
). Otherwise, CRF may exist
in GABAergic neurons in the hippocampus because
-aminobutyric acid
(GABA) neuron constitutes the major type of interneurons in the
hippocampus (Freund and Buzsaki 1996
) and
CRF-immunoreactive neurons were found to be co-immunolabeled with
glutamate decarboxylase (GAD) in this area (Yan et al.
1998
). Further, amygdala kindling was found to increase the
expression of CRF and CRF-binding protein in GABAergic neurons in the
dentate hilus (Smith et al. 1997
). Thus concurrent
activation of CRF and GABA on tetanic stimulation may therefore reduce
the GABAergic inhibition that then leads to excitation. However, the
exact synaptic relationship among CRF neuron, GABA neuron, glutamate
neuron, and the neural circuits underlying LTP requires further
investigation. On another topic, CRF has been shown to result in
seizure activity in hippocampal CA3 layers in immature rats
(Hollrigel et al. 1998
). Whether it also contributes to
the potentiation observed in the present study is not known.
Electroencephalogram (EEG) monitoring should help to delineate this point.
In conclusion, the present results demonstrate that CRF and tetanic
stimulation share some similar mechanisms in producing long-lasting
potentiation of dentate gyrus neurons, and protein synthesis appears to
be one of the underlying mechanisms. Tetanic stimulation also seems to
activate CRF neurons in the dentate gyrus because CRF mRNA level was
increased on tetanization. In conjunction with our previous observation
that RNA synthesis and protein synthesis inhibitors also effectively
block the memory-facilitating effect of CRF (Lee et al.
1992), these results together suggest that CRF plays an
important role in modulating synaptic efficacy of hippocampal neurons
and behavioral plasticity in rats. Further, these processes critically
depend on protein synthesis.
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ACKNOWLEDGMENTS |
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This work was supported by Research Grant NSC88-2314-B-001-012 from the National Science Council of Taiwan, Republic of China.
Present address of H. L. Wang: Dept. of Medical Technology, Yuan-Pei Institute of Medical Technology, Hsin-Chu, Taiwan, Republic of China.
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FOOTNOTES |
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Address for reprint requests: E.H.Y. Lee, Institute of Biomedical Sciences, Academia Sinica, Taipei (115), Taiwan, Republic of China.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 June 1999; accepted in final form 23 September 1999.
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REFERENCES |
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