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Nitric oxide is involved in long-term potentiation in the medial but not lateral amygdala neuron synapses in vitro
BRAIN
RESEARCH Brain Research 688 (1995) 233-236
ELSEVIER
Short communication
Nitric oxide is involved in long-term potentiation in the medial but not lateral amygdala neuron synapses in vitro Yasumasa Watanabe, Hiroshi Saito, Kazuho Abe
*
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan Accepted 9 May 1995
Abstract
Possible involvement of nitric oxide (NO) in the induction of long-term potentiation (LTP) in the amygdala was investigated using rat brain slice preparations in vitro. The induction of LTP in the medial amygdala was blocked by NO synthase inhibitors, N G-nitro-L-arginine and NG-nitro-L-arginine methyl ester, and an NO scavenger hemoglobin. On the other hand, the lateral amygdala LTP was not blocked by inhibition of endogenous NO. These results suggest that endogenous NO is involved in the induction of LTP in the medial amygdala but not in the lateral amygdala. Keywords: Nitric oxide; Long-term potentiation; Medial amygdala; Lateral amygdala; In vitro
Recent evidence suggests that nitric oxide (NO) may function as a neuronal messenger in the brain [3,9]. The NO-synthesizing enzyme NO synthase is constitutively expressed in neuronal cells and activated by Ca 2÷ influx following activation of N-methyl-D-aspartate (NMDA) receptors [3,9]. Long-term potentiation (LTP) of excitatory synaptic transmission is a form of activity-dependent synaptic plasticity which may underlie learning and memory [1]. The mechanism underlying LTP may be different among brain regions, but activation of NMDA receptors and an increase of intracellular Ca 2÷ concentration in postsynaptic cells are essential events for LTP induction at least in the CA1 region and the dentate gyrus of the hippocampus [7,8,13,15]. It is therefore possible that NO participates in the induction of hippocampal LTP. Supporting the hypothesis, several laboratories have reported that NO synthase inhibitors blocked the induction of LTP in the CA1 region of rat hippocampal slices [12,17,19] and in the dentate gyrus of anesthetized rats [16]. However, to our knowledge, there has been no report about the role of NO in LTP in brain regions other than the hippocampus. The amygdala is thought to be involved in certain types of learning and memory besides emotional and motivational aspects of behavior [14,18]. Amygdala LTP has been demonstrated in vitro [4,5,10,20,22], but the cellular mech-
* Corresponding author. Fax: (81) (3) 3815-4603. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0006-8993(95)00563-3
anism of amygdala LTP is not yet well understood. Since the presence of NO synthase in the amygdala is well demonstrated by histochemical studies [2,21], it is likely that NO functions in the amygdala. Furthermore, intense NO synthase immunoreactivity is observed especially in the medial amygdaloid nucleus [2,21], implying that the role of NO is different among subnuclei of the amygdaloid complex. Therefore, in the present study, we investigated possible roles of NO in the induction of LTP in the medial and lateral amygdala by using brain slice preparations in vitro. Preparation of brain slices and recording of evoked potentials were made as described in our previous paper [22]. Briefly, the amygdala slices (400-500 /zm thickness) were prepared from male Wistar rats (7-9 weeks old), and were allowed to recover for more than 1 h in an incubation chamber containing artificial cerebrospinal fluid (ACSF) which was maintained at 34°C and continuously bubbled with 95% 0 2 / 5 % CO 2. The composition of ACSF was as follows: 124.0 mM NaC1, 5.0 mM KCI, 2.4 mM CaCI 2, 1.3 mM M g S O 4 , 1.24 mM K H 2 P O 4 , 26.0 mM NaHCO 3 and 10.0 mM glucose. Each slice was transferred into a submersion chamber (3 ml) where warmed (34°C) and oxygenated (95% 0 2 / 5 % CO 2) ACSF was continuously perfused at a rate of 1 ml/min. A bipolar tungsten electrode with 0.15-mm tip separation was placed on the stria terminalis or the external capsule to stimulate the afferent fibers, and the evoked potential was extracellularly
234
Y. Watanabe et aL /Brain Research 688 (1995) 233-236
recorded from the medial amygdaloid nucleus or the lateral amygdaloid nucleus, respectively (Fig. 1A and B). A glass capillary microelectrode filled with 0.9% NaC1 (tip resistance, 2-3 M/2) was used for the recording. A single test stimulation (0.05 ms duration) was applied at intervals of 20 s. The stimulus intensity was adjusted to produce a population spike of about 50% of the maximum amplitude.
All drugs were delivered by perfusion. Tetanic stimulation (100 pulses at 100 Hz) was applied at the same stimulus intensity through the same electrode as used for test stimulation. As reported in our previous paper [22], the negativegoing field potentials recorded from the medial and lateral amygdaloid nuclei correspond to population spikes. The
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Fig. 1. Recording of evoked potentials in the medial and lateral amygdaloid nuclei of rat brain slices. A and B: Schematic illustrations of coronal amygdala slices showing locations of stimulating and recording electrodes. The stimulating electrode was placed on the stria terminalis (A, ST) or the external capsule (B, EC) and the field potential was recorded from the medial amygdaloid nucleus (A, MA) or the lateral amygdaloid nucleus (B, LA), respectively. C-H: Sample records of evoked potentials in the ST-MA synapses (C, E, G) and in the EC-LA synapses (D, F, H). C and D: Field potentials recorded in normal and Ca2+-free ACSF. Test stimulation was delivered at the time indicated by arrowheads, Calibration bars: vertical 0.5 mV, horizontal 5 ms. The voltage difference between the sharp negative onset and the negative peak (a) and that between the negative peak and succeeding positive peak (b) were measured, and the amplitude of the population spike was calculated as (a + b)/2. E and F: Field potentials evoked at different stimulus currents in normal ACSF. G and H: Field potentials immediately before and 30 rain after tetanic stimulation in the presence of 10 /zM picrotoxin.
235
Y. Watanabe et al. / Brain Research 688 (1995) 233-236
evoked potentials completely disappeared when perfusing medium was changed from normal ACSF to Ca2+-free ACSF (Fig. 1C and D), confirming that they represent synaptic responses. Furthermore, the latency shift of the evoked potentials at increasing stimulus intensity was small and typical for monosynaptic responses (Fig. 1E and F). We have previously found that the amygdala LTP in vitro could be reliably reproduced under blockade of GABAergic inhibitory processes [22]. Consistent with our previous observation [22], 10 /zM picrotoxin, a GABA g chloride channel blocker, did not affect the baseline synaptic potentials. Before or after tetanic stimulation in the presence of picrotoxin, epileptiform response (e.g. multiple population spikes) was not seen in any slices tested (Fig. 1G and H). Therefore, all LTP experiments were conducted in the presence of picrotoxin. Both in the medial amygdala and in the lateral amygdala, application of tetanic stimulation in the presence of 1 0 / z M picrotoxin induced LTP in all slices tested (Fig. 2). In the medial amygdala, addition of 100 /xM N~-nitro-L arginine (NO-Arg), an NO synthase inhibitor, did not affect the baseline synaptic potentials, but significantly blocked the induction of LTP (Fig. 2A). Another NO synthase inhibitor N~-nitro-L-arginine methyl ester (NAME; 100/xM) also blocked the medial amygdala LTP. The population spike amplitude 30 rain after tetanus in the NAME-treated slices was 113.2 _+ 9.7% (n = 5) of baseline. Furthermore, the medial amygdala LTP was blocked by hemoglobin (100 /zM), which can bind NO to its heme moiety and prevent extracellular NO diffusion (Fig. 2C). On the contrary, the lateral amygdala LTP was not blocked by 100 /zM NO-Arg (Fig. 2B) nor 100 /xM hemoglobin (Fig. 2D). The effects of NO-Arg and hemoglobin at a 300~
higher concentration (1 mM) on the lateral amygdala LTP were tested, but neither showed significant effects (n = 5, data not shown). The effects of NO synthase inhibitors on hippocampal LTP are controversial. Earlier reports described the blockade of CA1 LTP by NO synthase inhibitors [12,17,19], but many laboratories have failed to replicate the earlier observations [1]. The discrepancies seem to be, at least in part, due to differences in experimental conditions such as temperature, ages of animals and conditions of tetanic stimulations [6,11,23]. Since we compared the medial and lateral amygdala LTPs under the identical experimental conditions, the differential effects of NO synthase inhibitors on the medial and lateral amygdala LTPs are suggestive of real regional differences in mechanisms of LTP. Furthermore, the effects of NO synthase inhibitors were similar to that of hemoglobin. Therefore, the present data suggest that endogenous NO production and its extracellular diffusion are involved in the induction of the medial amygdala LTP, while the lateral amygdala LTP does not require endogenous NO. Histochemical studies have demonstrated the abundant presence of NO synthase in the medial amygdala neurons but little immunoreactive NO synthase in the lateral amygdala [2,21]. Furthermore, it has been reported that the induction of LTP in the medial amygdala requires activation of NMDA receptors [5,20,22], while the lateral amygdala LTP is independent of NMDA receptors [4,22]. The correlation between the dependence on NMDA receptors and endogenous NO production seems to support the linkage of NMDA receptor activation and NO production in the LTP induction cascade. The mechanism underlying NMDA receptor-indepen300"
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Fig. 2. Effects of 100 /xM NO-Arg (A, B) and 100 /LM hemoglobin(C, D) on the induction of LTP in the medial (A, C) and lateral amygdala(B, D). During the time indicated by bold bars, 10 /xM picrotoxinalone (open circles) or picrotoxinplus NO-Argor hemoglobin(filled circles) was peffused,and a tetanic stimulation (100 pulses at 100 Hz) was applied at time 0. Ordinates indicate population spike amplitude expressed as a percentage of baseline values immediately before tetanic stimulation. All data are represented as the means± S.E.M. of the data obtained from six slices. *P < 0.01: Mann-Whitney U-test.
236
E Watanabe et al. /Brain Research 688 (1995) 233-236
dent and NO-independent form of LTP in the lateral amygdala is not clear so far, but we have recently found that the lateral amygdala LTP is blocked by scopolamine, a muscarinic cholinergic receptor antagonist [22]. The lateral amygdala LTP may be induced by activation of non-NMDA glutamate receptors or stimulation of other neurotransmitter receptors including muscarinic receptors. The lateral amygdala LTP may be a useful model for investigating the NO-independent form of synaptic plasticity in the brain. In conclusion, we have shown for the first time that NO is involved in the induction of LTP in the stria terminalismedial amygdala synapses but does not contribute to LTP in the external capsule-lateral amygdala synapses. The present report is of great significance, showing the involvement of NO in LTP in brain regions other than the hippocampus and the existence of NO-independent form of synaptic plasticity in the brain.
References [1] Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31-39. [2] Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615-624. [3] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [4] Chapman, P.F. and Bellavance, L.L., Induction of long-term potentiation in the basolateral amygdala does not depend on NMDA receptor activation, Synapse, 11 (1992) 310-318. [5] Chapman, P.F., Kairiss, E.W., Keenan, C,L. and Brown, T.H., Long-term synaptic potentiation in the amygdala, Synapse, 6 (1990) 271-278. [6] Chetkovich, D.M., Klann, E. and Sweatt, J.D., Nitric oxide synthase-independent long-term potentiation in area CA1 of hippocampus, NeuroReport, 4 (1993) 919-922. [7] Collingridge, G.L., Kehl, S.J. and McLennan, H,, Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. [8] Errington, M.L., Lynch, M.A. and Bliss, T.V.P., Long-term potentiation in the dentate gyms: induction and glutamate release are blocked by D( - )aminophosphonovalerate, Neuroscience, 23 (1987) 279-284.
[9] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [10] Gean, P., Chan, F., Huang, C,, Lin, J. and Way, L., Long-term enhancement of EPSP and NMDA receptor-mediated synaptie transmission in the amygdala, Brain Res. Bull., 31 (1993) 7-11. Ill] Haley, J.E., Malen, P. and Chapman, P.F., Nitric oxide synthase inhibitors block long-term potentiation induced by weak but not strong tetanic stimulation at physiological brain temperature in rat hippocampal slices, Neurosci. Lett., 160 (1993) 85-88. [12] Haley, J.E., Wilcox, G.L. and Chapman, P.F., The role of nitric oxide in hippocampal long-term potentiation, Neuron, 8 (1992) 2ll-216. [13] Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of N-methyl-t>-aspartate receptors, Brain Res., 323 (1984) 132-137. [14] McGaugh, J.L., Introini-Collison, I.C., Nagahara, A.H., Cahill, L., Brioni, J.D., Castellano, C., Involvement of the amygdaloid complex in neuromodulatory influences on memory storage, Neurosci. Re~., 14 (1990) 425-431. [15] Mizutani, A., Saito, H. and Abe, K., Evidence for involvement of endogenous glycine in the induction of long-terrn potentiation in the dentate gyrus of anesthetized rats, Eur. Z Pharmacol., 205 (199l) 303-305. [16] Mizutani, A., Saito, H. and Abe, K., Involvement of nitric oxide in long-term potentiation in the dentate gyrus in vivo, Brain Res., 605 (1993) 309-311. [17] O'Dell, T.J., Hawkins, R.D., Kandel, E.R. and Arancio, O., Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger, Proc. Natl, Acad. Sci. USA, 88 (1991) 11285-11289. [18] Sarter, M. and Markowitsch, H.J., Involvement of the amygdala in learning and memory: a critical review with emphasis on anatomical relations, Beha~'. Neurosci., 99 (1985) 342-380. [19] Schuman, E.M, and Madison, D.V., A requirement for the intercellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. [20] Shindou, T., Watanabe, S., Yamamoto, K. and Nakanishi, H., NMDA receptor-dependent formation of long-term potentiation in the rat medial amygdala neuron in an in vitro slice preparation, Brain Res. Bull., 31 (1993) 667-672. [21] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784. [22] Watanabe, Y., Saito, H. and Abe, K., Roles of GABA A, NMDA and muscarinie receptors in induction of long-term potentiation in the medial and lateral amygdala in vitro, Neurosci. Res., 21 (1995) 317-322. [23] Williams, J.H., Li, Y.-G., Nayak, A., Errington, M.L., Murphy, K.P.S.J. and Bliss, T.V.P., The suppression of long-term potentiation in rat hippocampus by inhibition of nitric oxide synthase is temperature and age dependent, Neuron, 11 (1993) 877-884.
RESEARCH Brain Research 688 (1995) 233-236
ELSEVIER
Short communication
Nitric oxide is involved in long-term potentiation in the medial but not lateral amygdala neuron synapses in vitro Yasumasa Watanabe, Hiroshi Saito, Kazuho Abe
*
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan Accepted 9 May 1995
Abstract
Possible involvement of nitric oxide (NO) in the induction of long-term potentiation (LTP) in the amygdala was investigated using rat brain slice preparations in vitro. The induction of LTP in the medial amygdala was blocked by NO synthase inhibitors, N G-nitro-L-arginine and NG-nitro-L-arginine methyl ester, and an NO scavenger hemoglobin. On the other hand, the lateral amygdala LTP was not blocked by inhibition of endogenous NO. These results suggest that endogenous NO is involved in the induction of LTP in the medial amygdala but not in the lateral amygdala. Keywords: Nitric oxide; Long-term potentiation; Medial amygdala; Lateral amygdala; In vitro
Recent evidence suggests that nitric oxide (NO) may function as a neuronal messenger in the brain [3,9]. The NO-synthesizing enzyme NO synthase is constitutively expressed in neuronal cells and activated by Ca 2÷ influx following activation of N-methyl-D-aspartate (NMDA) receptors [3,9]. Long-term potentiation (LTP) of excitatory synaptic transmission is a form of activity-dependent synaptic plasticity which may underlie learning and memory [1]. The mechanism underlying LTP may be different among brain regions, but activation of NMDA receptors and an increase of intracellular Ca 2÷ concentration in postsynaptic cells are essential events for LTP induction at least in the CA1 region and the dentate gyrus of the hippocampus [7,8,13,15]. It is therefore possible that NO participates in the induction of hippocampal LTP. Supporting the hypothesis, several laboratories have reported that NO synthase inhibitors blocked the induction of LTP in the CA1 region of rat hippocampal slices [12,17,19] and in the dentate gyrus of anesthetized rats [16]. However, to our knowledge, there has been no report about the role of NO in LTP in brain regions other than the hippocampus. The amygdala is thought to be involved in certain types of learning and memory besides emotional and motivational aspects of behavior [14,18]. Amygdala LTP has been demonstrated in vitro [4,5,10,20,22], but the cellular mech-
* Corresponding author. Fax: (81) (3) 3815-4603. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0006-8993(95)00563-3
anism of amygdala LTP is not yet well understood. Since the presence of NO synthase in the amygdala is well demonstrated by histochemical studies [2,21], it is likely that NO functions in the amygdala. Furthermore, intense NO synthase immunoreactivity is observed especially in the medial amygdaloid nucleus [2,21], implying that the role of NO is different among subnuclei of the amygdaloid complex. Therefore, in the present study, we investigated possible roles of NO in the induction of LTP in the medial and lateral amygdala by using brain slice preparations in vitro. Preparation of brain slices and recording of evoked potentials were made as described in our previous paper [22]. Briefly, the amygdala slices (400-500 /zm thickness) were prepared from male Wistar rats (7-9 weeks old), and were allowed to recover for more than 1 h in an incubation chamber containing artificial cerebrospinal fluid (ACSF) which was maintained at 34°C and continuously bubbled with 95% 0 2 / 5 % CO 2. The composition of ACSF was as follows: 124.0 mM NaC1, 5.0 mM KCI, 2.4 mM CaCI 2, 1.3 mM M g S O 4 , 1.24 mM K H 2 P O 4 , 26.0 mM NaHCO 3 and 10.0 mM glucose. Each slice was transferred into a submersion chamber (3 ml) where warmed (34°C) and oxygenated (95% 0 2 / 5 % CO 2) ACSF was continuously perfused at a rate of 1 ml/min. A bipolar tungsten electrode with 0.15-mm tip separation was placed on the stria terminalis or the external capsule to stimulate the afferent fibers, and the evoked potential was extracellularly
234
Y. Watanabe et aL /Brain Research 688 (1995) 233-236
recorded from the medial amygdaloid nucleus or the lateral amygdaloid nucleus, respectively (Fig. 1A and B). A glass capillary microelectrode filled with 0.9% NaC1 (tip resistance, 2-3 M/2) was used for the recording. A single test stimulation (0.05 ms duration) was applied at intervals of 20 s. The stimulus intensity was adjusted to produce a population spike of about 50% of the maximum amplitude.
All drugs were delivered by perfusion. Tetanic stimulation (100 pulses at 100 Hz) was applied at the same stimulus intensity through the same electrode as used for test stimulation. As reported in our previous paper [22], the negativegoing field potentials recorded from the medial and lateral amygdaloid nuclei correspond to population spikes. The
Bregma-3.3mm
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A
B
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stim.
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rec.
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+ picrotoxin
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H
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Fig. 1. Recording of evoked potentials in the medial and lateral amygdaloid nuclei of rat brain slices. A and B: Schematic illustrations of coronal amygdala slices showing locations of stimulating and recording electrodes. The stimulating electrode was placed on the stria terminalis (A, ST) or the external capsule (B, EC) and the field potential was recorded from the medial amygdaloid nucleus (A, MA) or the lateral amygdaloid nucleus (B, LA), respectively. C-H: Sample records of evoked potentials in the ST-MA synapses (C, E, G) and in the EC-LA synapses (D, F, H). C and D: Field potentials recorded in normal and Ca2+-free ACSF. Test stimulation was delivered at the time indicated by arrowheads, Calibration bars: vertical 0.5 mV, horizontal 5 ms. The voltage difference between the sharp negative onset and the negative peak (a) and that between the negative peak and succeeding positive peak (b) were measured, and the amplitude of the population spike was calculated as (a + b)/2. E and F: Field potentials evoked at different stimulus currents in normal ACSF. G and H: Field potentials immediately before and 30 rain after tetanic stimulation in the presence of 10 /zM picrotoxin.
235
Y. Watanabe et al. / Brain Research 688 (1995) 233-236
evoked potentials completely disappeared when perfusing medium was changed from normal ACSF to Ca2+-free ACSF (Fig. 1C and D), confirming that they represent synaptic responses. Furthermore, the latency shift of the evoked potentials at increasing stimulus intensity was small and typical for monosynaptic responses (Fig. 1E and F). We have previously found that the amygdala LTP in vitro could be reliably reproduced under blockade of GABAergic inhibitory processes [22]. Consistent with our previous observation [22], 10 /zM picrotoxin, a GABA g chloride channel blocker, did not affect the baseline synaptic potentials. Before or after tetanic stimulation in the presence of picrotoxin, epileptiform response (e.g. multiple population spikes) was not seen in any slices tested (Fig. 1G and H). Therefore, all LTP experiments were conducted in the presence of picrotoxin. Both in the medial amygdala and in the lateral amygdala, application of tetanic stimulation in the presence of 1 0 / z M picrotoxin induced LTP in all slices tested (Fig. 2). In the medial amygdala, addition of 100 /xM N~-nitro-L arginine (NO-Arg), an NO synthase inhibitor, did not affect the baseline synaptic potentials, but significantly blocked the induction of LTP (Fig. 2A). Another NO synthase inhibitor N~-nitro-L-arginine methyl ester (NAME; 100/xM) also blocked the medial amygdala LTP. The population spike amplitude 30 rain after tetanus in the NAME-treated slices was 113.2 _+ 9.7% (n = 5) of baseline. Furthermore, the medial amygdala LTP was blocked by hemoglobin (100 /zM), which can bind NO to its heme moiety and prevent extracellular NO diffusion (Fig. 2C). On the contrary, the lateral amygdala LTP was not blocked by 100 /zM NO-Arg (Fig. 2B) nor 100 /xM hemoglobin (Fig. 2D). The effects of NO-Arg and hemoglobin at a 300~
higher concentration (1 mM) on the lateral amygdala LTP were tested, but neither showed significant effects (n = 5, data not shown). The effects of NO synthase inhibitors on hippocampal LTP are controversial. Earlier reports described the blockade of CA1 LTP by NO synthase inhibitors [12,17,19], but many laboratories have failed to replicate the earlier observations [1]. The discrepancies seem to be, at least in part, due to differences in experimental conditions such as temperature, ages of animals and conditions of tetanic stimulations [6,11,23]. Since we compared the medial and lateral amygdala LTPs under the identical experimental conditions, the differential effects of NO synthase inhibitors on the medial and lateral amygdala LTPs are suggestive of real regional differences in mechanisms of LTP. Furthermore, the effects of NO synthase inhibitors were similar to that of hemoglobin. Therefore, the present data suggest that endogenous NO production and its extracellular diffusion are involved in the induction of the medial amygdala LTP, while the lateral amygdala LTP does not require endogenous NO. Histochemical studies have demonstrated the abundant presence of NO synthase in the medial amygdala neurons but little immunoreactive NO synthase in the lateral amygdala [2,21]. Furthermore, it has been reported that the induction of LTP in the medial amygdala requires activation of NMDA receptors [5,20,22], while the lateral amygdala LTP is independent of NMDA receptors [4,22]. The correlation between the dependence on NMDA receptors and endogenous NO production seems to support the linkage of NMDA receptor activation and NO production in the LTP induction cascade. The mechanism underlying NMDA receptor-indepen300"
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B
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Fig. 2. Effects of 100 /xM NO-Arg (A, B) and 100 /LM hemoglobin(C, D) on the induction of LTP in the medial (A, C) and lateral amygdala(B, D). During the time indicated by bold bars, 10 /xM picrotoxinalone (open circles) or picrotoxinplus NO-Argor hemoglobin(filled circles) was peffused,and a tetanic stimulation (100 pulses at 100 Hz) was applied at time 0. Ordinates indicate population spike amplitude expressed as a percentage of baseline values immediately before tetanic stimulation. All data are represented as the means± S.E.M. of the data obtained from six slices. *P < 0.01: Mann-Whitney U-test.
236
E Watanabe et al. /Brain Research 688 (1995) 233-236
dent and NO-independent form of LTP in the lateral amygdala is not clear so far, but we have recently found that the lateral amygdala LTP is blocked by scopolamine, a muscarinic cholinergic receptor antagonist [22]. The lateral amygdala LTP may be induced by activation of non-NMDA glutamate receptors or stimulation of other neurotransmitter receptors including muscarinic receptors. The lateral amygdala LTP may be a useful model for investigating the NO-independent form of synaptic plasticity in the brain. In conclusion, we have shown for the first time that NO is involved in the induction of LTP in the stria terminalismedial amygdala synapses but does not contribute to LTP in the external capsule-lateral amygdala synapses. The present report is of great significance, showing the involvement of NO in LTP in brain regions other than the hippocampus and the existence of NO-independent form of synaptic plasticity in the brain.
References [1] Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31-39. [2] Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615-624. [3] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [4] Chapman, P.F. and Bellavance, L.L., Induction of long-term potentiation in the basolateral amygdala does not depend on NMDA receptor activation, Synapse, 11 (1992) 310-318. [5] Chapman, P.F., Kairiss, E.W., Keenan, C,L. and Brown, T.H., Long-term synaptic potentiation in the amygdala, Synapse, 6 (1990) 271-278. [6] Chetkovich, D.M., Klann, E. and Sweatt, J.D., Nitric oxide synthase-independent long-term potentiation in area CA1 of hippocampus, NeuroReport, 4 (1993) 919-922. [7] Collingridge, G.L., Kehl, S.J. and McLennan, H,, Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. [8] Errington, M.L., Lynch, M.A. and Bliss, T.V.P., Long-term potentiation in the dentate gyms: induction and glutamate release are blocked by D( - )aminophosphonovalerate, Neuroscience, 23 (1987) 279-284.
[9] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [10] Gean, P., Chan, F., Huang, C,, Lin, J. and Way, L., Long-term enhancement of EPSP and NMDA receptor-mediated synaptie transmission in the amygdala, Brain Res. Bull., 31 (1993) 7-11. Ill] Haley, J.E., Malen, P. and Chapman, P.F., Nitric oxide synthase inhibitors block long-term potentiation induced by weak but not strong tetanic stimulation at physiological brain temperature in rat hippocampal slices, Neurosci. Lett., 160 (1993) 85-88. [12] Haley, J.E., Wilcox, G.L. and Chapman, P.F., The role of nitric oxide in hippocampal long-term potentiation, Neuron, 8 (1992) 2ll-216. [13] Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of N-methyl-t>-aspartate receptors, Brain Res., 323 (1984) 132-137. [14] McGaugh, J.L., Introini-Collison, I.C., Nagahara, A.H., Cahill, L., Brioni, J.D., Castellano, C., Involvement of the amygdaloid complex in neuromodulatory influences on memory storage, Neurosci. Re~., 14 (1990) 425-431. [15] Mizutani, A., Saito, H. and Abe, K., Evidence for involvement of endogenous glycine in the induction of long-terrn potentiation in the dentate gyrus of anesthetized rats, Eur. Z Pharmacol., 205 (199l) 303-305. [16] Mizutani, A., Saito, H. and Abe, K., Involvement of nitric oxide in long-term potentiation in the dentate gyrus in vivo, Brain Res., 605 (1993) 309-311. [17] O'Dell, T.J., Hawkins, R.D., Kandel, E.R. and Arancio, O., Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger, Proc. Natl, Acad. Sci. USA, 88 (1991) 11285-11289. [18] Sarter, M. and Markowitsch, H.J., Involvement of the amygdala in learning and memory: a critical review with emphasis on anatomical relations, Beha~'. Neurosci., 99 (1985) 342-380. [19] Schuman, E.M, and Madison, D.V., A requirement for the intercellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. [20] Shindou, T., Watanabe, S., Yamamoto, K. and Nakanishi, H., NMDA receptor-dependent formation of long-term potentiation in the rat medial amygdala neuron in an in vitro slice preparation, Brain Res. Bull., 31 (1993) 667-672. [21] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784. [22] Watanabe, Y., Saito, H. and Abe, K., Roles of GABA A, NMDA and muscarinie receptors in induction of long-term potentiation in the medial and lateral amygdala in vitro, Neurosci. Res., 21 (1995) 317-322. [23] Williams, J.H., Li, Y.-G., Nayak, A., Errington, M.L., Murphy, K.P.S.J. and Bliss, T.V.P., The suppression of long-term potentiation in rat hippocampus by inhibition of nitric oxide synthase is temperature and age dependent, Neuron, 11 (1993) 877-884.