,
100-120 g, Grade II, Certificate No 26-001 conferred by Medical Animal
Management Committee, Guangdong Province) were obtained from the Experimental
Animal Center of Sun Yat-sen University of Medical Sciences.
He J et al / Acta Pharmacol Sin 2003 May; 24 (5): 398-402
HE Jin, DENG Chun-Yu, ZHU Xiao-Nan, YU Jian-Ping, CHEN Ru-Zhu2
Department of Pharmacology, Zhongshan Medical School, Sun Yat-sen University, Guangzhou 510089, China
1 Project supported by Ministry of Public Health Foundation (No 98-1-086).
2 Correspondence to Prof CHEN Ru-Zhu. Phn 86-20-8733-0561. E-mail yujian@gzsums.edu.cn
Received 2002-09-17 Accepted 2002-01-16
KEY WORDS nicotine; long-term potentiation; hippocampus; MK-801; L-NAME
ABSTRACT
AIM: To investigate whether long-term potentiation (LTP) induced by nicotine and tetanic stimulation in the hippocampal CA1 region shares different mechanisms. METHODS: Extracellular population spikes of the pyramidal cell layer in the hippocampal CA1 region were recorded in vitro. RESULTS: LTP induced by the tetanic stimulation could be facilitated by nicotine 10 µmol/L, meanwhile, the tetanic stimulation did the same effect on LTP induced by nicotine 10 µmol/L. MK-801 10 µmol/L or NG-nitro-L-arginine methyl ester (L-NAME) 20 µmol/L blocked LTP induced by tetanic stimulation, but did not inhibit LTP induced by nicotine. CONCLUSION: LTP induced by nicotine shares different synaptic mechanisms with LTP induced by tetanic stimulation.
INTRODUCTION
The roles of cholinergic system on learning and memory have been widely studied mainly due to the cholinergic hypothesis of cognitive dysfunction in Alzheimer's disease (AD)[1]. Clinical data also revealed that nicotine ameliorated learning and short-term memory in AD patients[2,3].
Long-term potentiation (LTP), an activitydependent strengthening of synaptic efficacy evoked by tetanic stimulation, is widely used as a model to investigate the cellular machanisms of learning and memory[4]. LTP elicited by tetanic stimulation requires the release of glutamate from the glutamergic fibres and the activation of the postsynaptic NMDA receptor consequently resulting in Ca2+ entry into the postsynaptic neurons[5].
We previously reported that nicotine induced LTP in the hippocampal CA1 region with Ca2+-dependence[6]. It is important to study whether or not LTP induced by nicotine shares different synaptic mechanisms from that by tetanic stimulation, because it would give some clues in studying the mechanisms of nicotine to improve the function of learning and memory.
MATERIALS AND METHODS
Materials Nicotine, MK-801 (an antagonist specific for NMDA receptor)
and NG-nitro-L-arginine methyl ester (L-NAME)
(one of inhibitors specific for NO synthase) were purchased from Sigma. Sprague-Dawley
rats (,
100-120 g, Grade II, Certificate No 26-001 conferred by Medical Animal
Management Committee, Guangdong Province) were obtained from the Experimental
Animal Center of Sun Yat-sen University of Medical Sciences.
Methods The rats (110-120 g) were anesthetized
by inhaling ether. Hippocampal slices
(400-
m thickness) were prepared from rats at 0 ºC, then
incubated in artificial cerebrospinal fluid (ACSF) at 28 ºC
for at least 90 min. ACSF (in mmol/L: NaCl 124, KCl
3.4, KH2PO4 1.2,
MgSO4·7H2O 1.7,
NaHCO3 25, CaCl2 2.4, glucose 10; pH 7.4) was saturated with 95 %
O2 and 5 % CO2 gas mixture. Before recording, the slices
were transferred to an interface chamber where the
slices were continuously perfused at 1 mL/min with
saturated ACSF at 32 ºC.
Potentials were recorded extracellularly by use of glass microelectrodes (1-2
M
resistance, filled with NaCl
2 mol/L) placed in the pyramidal cell layer of the CA1 region, and stimuli were
applied to the Schaffer collateral-commissural pathway through an insulated
tungsten wire electrode. The test stimuli (0.017 Hz, with duration of 0.1 ms)
adjusted to produce 80 % of the maximal population spike (PS) amplitude were
applied. In some experiments, tetanic stimulation (100 Hz, 2 trains, 60 s of
interval) was delivered. Potentials were fed through a microelectrode amplifier
(MEZ-7101; NIHON KOHDEN, Japan) to a dual-beam memory oscilloscope (VC-10; NIHON
KOHDEN, Japan) and recorded by a X-Y electronic recorder (NIHON KOHDEN, Japan).
Statistical analysis Data were expressed as mean±SD, and analyzed by either paired t test in the same group or standard t test between two groups (P<0.05).
RESULTS
Reciprocal facilitation of LTP by nicotine and tetanic stimulation The effect of nicotine on LTP preestablished by tetanic stimulation was investigated. With test stimuli of giving 80 % of the maximal PS amplitude, tetanic stimulation caused the schaffer collateral LTP (150 %±8 % at the time point of 15 min) compared with baseline (P<0.01, Fig 1A). Twenty minutes after tetanic stimulation was delivered, nicotine 10 µmol/L was perfused for 20 min. Nicotine caused further increase of the PS size (163 %±9 % at the time point of 60 min). Nicotine facilitated LTP elicited by tetanic stimulation compared with the values after nicotine exposure and the values at the time point of 15 min (P<0.01, paired t test, n=5).
Similarly, the effect of tetanic stimulation on LTP induced by nicotine was observed. Nicotine 10 µmol/L was firstly given to elicit stable expression of LTP in the CA1 region (149 %±8 % at the time point of 35 min) compared with baseline (P<0.01, Fig 1B). Forty-five minutes after nicotine exposure, tetanic stimulation was delivered. Tetanic stimulation also further increased the PS size (178 %±10 % at the time point of 60 min) compared with the values after tetanic stimulation delivering and the values at the time point of 35 min (P<0.05, paired t test, n=5).
Fig 1. Nicotine and tetanic stimulation (TS) reciprocally facilitated LTP by each other. Calibration: 5 ms, 1 mv. A) Nicotine 10 µmol/L further facilitated LTP induced by tetanic stimulation. n=5. Mean±SD. cP<0.01 vs the values at the time point of 15 min. Insets, representative original traces at baseline, 15 min and 50 min after TS delivering are shown, respectively. B) Tetanic stimulation further facilitated LTP induced by nicotine 10 µmol/L. n=5. Mean±SD. bP<0.05, cP<0.01 vs the values at the time point of 35 min. Insets, representative original traces at baseline, 35 min and 60 min after nicotine application are shown, respectively.
Effects of MK-801 on LTP induced by nicotine MK-801 10 µmol/L incubated with the hippocampal slice for 2 h inhibited completely the induction of LTP by tetanic stimulation (113 %±8 % at 30 min after tetanic stimulation delivering) compared with the control (160 %±14 % at 30 min after tetanic stimulation delivering) (P<0.01, standard t test, n=5) (Fig 2A).
The slice was incubated with MK-801 10 µmol/L for 2 h, then transferred to the interface chamber. Nicotine 10 µmol/L together with MK-801 10 µmol/L was perfused for 20 min. At the presence of MK-801, nicotine still induced a pronounced potentiation of the PS amplitude (154 %±8 % at 30 min after nicotine exposure) (Fig 2B). The difference was not statistically significant compared with the group only treated with nicotine (150 %±6 % at 30 min after nicotine exposure) (P>0.05, standard t test, n=5).
Fig 2. The different effect of MK-801 on LTP induced by tetanic stimulation
(TS) and nicotine. Calibration: 5 ms, 1 mv. A) MK-801 10 µmol/L markedly
inhibited LTP evoked by tetanic stimulation. (
)
control, (
) MK-801 10 µmol/L.
n=5. Mean±SD. cP<0.01 vs control group.
Insets, representative original traces at baseline and 30 min after TS delivering
are shown, respectively. B) MK-801 10 µmol/L had no effect on LTP induced
by nicotine 10 µmol/L. ()
nicotine 10 µmol/L, ()
nicotine 10 µmol/L+MK-801 10 µmol/L. n=5. Mean±SD. aP>0.05
vs nicotine group. Insets, representative original traces at baseline
and 30 min after nicotine application are shown, respectively.
Effects of L-NAME on LTP induced by nicotine L-NAME 100 µmol/L inhibited the baseline spike (data not shown here), while L-NAME 20 µmol/L did not. L-NAME 20 µmol/L completely abolished LTP induced by tetanic stimulation (97 %±9 % at 30 min after tetanic stimulation delivering) compared with control (161 %±14 % at 30 min after tetanic stimulation delivering) (P<0.01, standard t test, n=3) (Fig 3A).
L-NAME 20 µmol/L was perfused for 40 min, and at 20 min after L-NAME was delivered, nicotine 10 µmol/L was delivered for 20 min. The PS size increased gradually and reached to the maximal response at 30 min after nicotine exposure (146 %±16 % at 30 min after nicotine exposure) (Fig 3B). LTP induced by nicotine was not inhibited significantly by the existence of L-NAME, compared with the group only treated with nicotine (149 %±12 % at 30 min after nicotine exposure) (P>0.05, standard t test, n=5).
Fig 3. The different effect of NG-nitro-L-arginine
methyl ester (L-NAME) on LTP induced by tetanic stimulation (TS) and
nicotine. Calibration: 5 ms, 1 mv. A) L-NAME 20 µmol/L completely
abolished LTP induced by tetanic stimulation. ()
control, () L-NAME
20 µmol/L. n=3. Mean±SD.
cP<0.01 vs control group. Insets, representative
original traces at baseline and 30 min after TS delivering are shown, respectively.
B) L-NAME 20 µmol/L did not inhibit LTP induced by nicotine 10 µmol/L.
() nicotine 10 µmol/L,
() nicotine 10 µmol/L+L-NAME
20 µmol/L. n=5. Mean±SD. aP>0.05 vs
nicotine group. Insets, representative original traces at baseline and 30 min
after nicotine application are shown, respectively.
DISCUSSION
After LTP induced by tetanic stimulation became stead, nicotine further increased the PS size. Similarly, tetanic stimulation also further potentiated LTP induced by nicotine in the CA1 region. These suggested that LTP induced by nicotine and tetanic stimulation could share different mechanisms.
Electrophysiological experiments disclosed some roles of the neuronal nicotinic acetylcholine receptors (nAChR) on the glutamergic fibres. Application of nanomolar concentrations of nicotine to the synapse between the neurons of the medial habenula nucleus and the interpeduncular nucleus enhanced the fast excitatory transmission of the glutamatergic synapses by activation of presynaptic nAChR containing the alpha 7 subunit[7]. Submicromolar concentrations of nicotine prompted the release of glutamate by activating the presynaptic nAChR containing the alpha 7 subunit on the terminal of the mossy fibres in the hippocampal CA3 region[8]. Those results intrigued us great interests whether glutamate released from the glutamergic fibres by activation of presynaptic neuronal nAChR participated in LTP induced by nicotine. LTP induced by nicotine was not inhibited by MK-801, showing that it did not require the activation of NMDA receptor. Earlier reports also showed that NMDA receptor-independent LTP in hippoacmpal CA1 region was induced in the presence of 2-amino-5-phosphonovaleric acid by high frequency (200 Hz) tetanic stimulation and suppressed by nifidipine, an antagonist of L-type Ca2+ channel[9].
LTP caused by tetanic stimulation in the hippocamal CA1 region was originally postsynaptic, but it was related to the enhancement of the release of transmitters from the presynaptic fibres[10]. Nitric oxide (NO) may act as a candidate retrograde messenger during the induction of LTP, which was produced in the postsynaptic neurons and acted on the presynaptic terminals to increase the release of transmitters[11]. LTP induced by nicotine was not obviously affected by the existence of L-NAME, which suggested that NO was not involved in the induction of LTP by nicotine. Some experiments also suggested that nicotine protected cultured cortical neurons against glutamate-induced cytotoxicity by reducing formation of NO[12]. Considering that the activation of the presynaptic nAChRs resulted in the entry of Ca2+ into the presynaptic terminals and the increase of the release of transmitters[7], the potentiation of synaptic transmission prompted by nicotine was probably independent of postsynaptically derived retrograde messenger.
Since LTP induced by nicotine did not require the activation of NMDA receptor and NO synthase, it was confirmed that LTP induced by nicotine shared different mechanisms at least in the synaptic level with LTP by tetanic stimulation. Further studies about LTP induced by nicotine would be beneficial to understand intensively the roles of the neuronal nAChR in learning and memory.
REFERENCES