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A Nitric Oxide Synthase Inhibitor Impairs Memory Storage in Mice
NEUROBIOLOGY OF LEARNING AND MEMORY ARTICLE NO.
65, 197–201 (1996)
0023
A Nitric Oxide Synthase Inhibitor Impairs Memory Storage in Mice CARLOS M. BARATTI
AND
SILVIA R. KOPF1
Laboratorio de NeurofarmacologıB a de Procesos de Memoria, Ca´tedra de FarmacologıB a, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, RA-1113 Buenos Aires, Argentina
produced by the enzyme, NO synthase (NOS) (Bredt, Hwang, Glatt, Lowenstein, Reed, & Snyder, 1991b) which involves oxidizing one of the terminal guanidino nitrogens of the semiessential amino acid Larginine (Moncada et al., 1991). In brain, a constitutively Ca2/-dependent NOS is expressed in specific neuronal populations (Bredt, Glatt, Hwang, Fotuhi, Dawson, & Snyder, 1991a). The possible involvement of NO in long-term potentiation (LTP) (Bohme, Bon, Stutzmann, Doble, & Blanchard, 1991) has prompted several investigations to determine the role of NO in animal learning and memory processes. Although research into NO in learning and memory has only just started, some studies suggest that NO may participate in the acquisition of learned behaviors (Schuman & Madison, 1994). We report here, for the first time, that the posttraining systemic administration of L-NAME, a competitive inhibitor of NOS (Dwyer, Bredt, & Snyder, 1991), impairs retention of a step-through inhibitory avoidance task in mice.
Posttraining administration of the L-enantiomer of the competitive inhibitor of nitric oxide synthase, NG-nitro-Larginine methyl ester (L-NAME, 3–100 mg/kg, ip), impaired 48-h retention of a one-trial step-through inhibitory shock-avoidance task in male Swiss mice. The effects were dose-dependent and were not observed when the D-enantiomer (D-NAME, 3–100 mg/kg, ip) was injected instead of L-NAME. Retention latencies of mice that had not received a footshock during training were not affected by L-NAME. The memory impairment produced by L-NAME was timedependent, suggesting an action on memory storage. The effects of L-NAME on memory were overcome by the injection of L-(but not D-)arginine (300 mg/kg, ip) along with the inhibitor. Considered together, these findings suggest that the L-arginine/nitric oxide pathway may be involved in memory storage of an inhibitory avoidance response in mice. q 1996 Academic Press, Inc.
INTRODUCTION Evidence accumulated over the past 30 years indicates that, if administered immediately or shortly after a learning experience, a variety of treatments can retroactively impair or enhance memory storage (McGaugh, 1991). Such findings have suggested that retention may be modulated by the endogenous activity of physiological systems that do not themselves serve as the locus of the changes for the formation of long-term memory. The free radical gas nitric oxide (NO) is a recently identified neuronal messenger (Garthwaite, 1991) that has been shown to function in a wide variety of central and peripheral processes (Moncada, Palmer, & Higgs, 1991; Nathan, 1992; Schuman & Madison, 1994). Nitric oxide is
MATERIALS AND METHODS Experiments were conducted in male Swiss mice (Roux-Ocefa Labs., Argentina) weighing approximately 30 g. They were housed 20 per cage in stainless steel cages, with food and tap water ad lib., except during the training and testing procedures. Lighting conditions were 12 h on/12 h off with the light phase of the cycle beginning at 7:00 AM. The mice were trained in a one-trial step-through inhibitory shock-avoidance task. On the training trial, the mouse was placed on a lighted platform outside a hole leading to a dark compartment. When the mouse stepped into the dark compartment a footshock (0.8 mA, 50 Hz, for 1 s) was delivered. For the retention test 48 h later, each mouse was again placed on the same platform and the latency to step
1 Work supported by Grant 5100/92 from CONICET to Carlos M. Baratti. Correspondence and reprint requests should be addressed to Dr. C. M. Baratti. Fax: 54-1-962-5341. E-mail: baratti @cafarm.ffyb.uba.ar.
197 1074-7427/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Effect of posttraining administration of L-NAME on retention. Treatments were given immediately after training. N Å 20 mice per group (shocked mice) and N Å 10 mice per group (unshocked mice). *põ.05, and **p õ .01, compared with saline control group (Newman–Keuls test, põ.05). F.S., foot shock; N.S., no shock.
through was recorded (maximum 300 s). The experiments were performed in such a way that the person doing the testing did not know the makeup of the groups. The drugs used in these experiments were NG-nitro-L-arginine methyl ester hydrochloride, NGnitro-D-arginine methyl ester hydrochloride, L-arginine ethyl ester dihydrochloride, and D-arginine hydrochloride (obtained through Sigma Chemical Co., St. Louis, MO). All drugs were dissolved in saline immediately before being used. All injections were given intraperitoneally (10 ml/kg). Controls received the same volume of saline. All doses were calculated as the free base. Since there was not a significant departure from homogeneity of variance of the data across the experiments (Fmax test) (Sokal & Rohlf, 1969), data are expressed as means { SEM of the latencies to step through during the retention test. They were evaluated using a one- or a two-way ANOVA, depending upon the experimental design (Sokal & Rohlf, 1969). Post hoc analysis with the Newman–Keuls test was made. In all cases p values less than .05 were considered significant.
of mice that received a footshock during training (F(4, 95) Å 6.74; p õ .01, one-way ANOVA). The step-through latencies of mice given 10 or 30 mg/kg (p õ .05) and 100 mg/kg (p õ .01) of L-NAME were significantly lower than those of saline controls. In mice that had not received the footshock on the training trial, the retention test latencies were unaffected by L-NAME (F(4, 45) Å 0.97; p ú .05, one-way ANOVA). Since the effects of NOS inhibitors appear to be enantiomerically specific, we decided to determine whether the post-training administration of DNAME would modify retention. Thus, five different groups of 20 mice each were injected with saline or D-NAME (3–100 mg/kg) immediately after training. There were no significant differences in the retention latencies among these groups (saline, 140 { 18 s; DNAME (3 mg/kg), 138 { 17 s; D-NAME (10 mg/kg), 139 { 19 s; D-NAME (30 mg/kg), 140 { 20 s, and DNAME (100 mg/kg), 140 { 21 s; F(4, 95) Å 0.08; p ú .05, one-way ANOVA). In the following experiment we studied the possible interaction between L-NAME with L-arginine or D-arginine. Thus, six different groups of 20 mice each were submitted to the training procedure. Immediately after it, the mice received saline, L-NAME (100 mg/kg), L-arginine (300 mg/kg), D-arginine (300 mg/ kg), or L-NAME (100 mg/kg) plus L-arginine (300 mg/kg) or D-arginine (300 mg/kg), as a single injection. The results are shown in Fig. 2. Neither Larginine nor D-arginine affected retention on its own (p ú .05, in both cases). The effect of L-NAME on retention was prevented by L-arginine (F(1, 76) Å 7.02; p õ .01), but not by D-arginine (F(1, 76) Å 0.01; p ú .05) (two-way ANOVA, in both cases). The
RESULTS In the first experiment, different groups of 20 mice each were trained as described under Materials and Methods. Immediately after training, the mice received an ip injection of saline or L-NAME (3–100 mg/kg). Other groups of 10 mice each were trained without footshock and received saline or L-NAME according to the same experimental design used for shocked mice. The results are shown in Fig. 1. The posttraining administration of L-NAME induced a dose-dependent decrease in retention performance
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FIG. 2. Effects of L-NAME, L-arginine or D-arginine, and their combination on retention. Treatments were given immediately after training as a single injection. N Å 20 mice per group. **põ.01, compared with saline control group (Newman–Keuls test, põ.05).
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FIG. 3. Effects of delayed posttraining administration of LNAME on retention. Treatments were given immediately or 180 min after training. N Å 20 mice per group. **põ.01, compared with saline control group (Newman–Keuls test, põ.05).
Newman–Keuls test showed significant differences for the groups that received L-NAME alone or LNAME plus D-arginine compared with saline control group (p õ .01, in both cases). Finally different groups of 20 mice each were submitted to the training and received saline or LNAME (100 mg/kg) immediately or 180 min after training. Figure 3 shows the results of these experiments. A 2 (saline– L-NAME) 1 2 (training–treatment interval) ANOVA shows significant interaction of treatment 1 time (F(1, 76) Å 9.51; p õ .01). LNAME administered immediately after training significantly impaired retention (p õ .01). However, when the NOS inhibitor was given 180 min after training, retention latencies did not differ significantly from its control group (p ú .05). DISCUSSION Nitric oxide synthase inhibitors (NOSI), such as NG-nitro-L-arginine methyl ester (L-NAME), block LTP-induction both in vitro (Bohme et al., 1991) and in vivo (Iga, Yoshioka, Togashi, & Saito, 1993). The effects may be reversed by L- (but not D-) arginine (Schuman & Madison, 1994). Since LTP remains a candidate mnemonic device and it appears that NO production is necessary for LTP-induction (O’Dell, Hawkins, Kandel, & Arancio, 1991), recent research has clearly emphasized the possible involvement of L-arginine/NO pathway in the acquisition of learned behaviors. Thus, the results of Holscher and Rose (1992) demonstrated for the first time that NO synthesis is necessary for an early phase of memory formation for an inhibitory avoidance response in the chick. The anterograde amnesia induced by the inhibition of NOS was overcome by coadministration of L-arginine. In contrast, in a one-trial shock-inhibi-
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tory avoidance task using rats as experimental subjects, the same NOSI left shock-avoidance learning unaffected (Bohme, Bon, Lemaire, Reibaud, Piot, Stutzmann, Doble, & Blanchard, 1993). Deficits in the acquisition of spatial reference memory in rats, both in the water maze (Chapman, Atkins, Allen, Haley, & Steinmetz, 1992) and in the eight-arm radial maze (Bohme et al., 1992), as well as an impaired acquisition of the classical conditioned eyeblink responses in rabbits (Chapman et al., 1992), following administration of NOSI have also been reported. In addition, Bohme et al. (1993) have shown that the inhibition of NOS impairs learning in a social recognition task (social memory). Although it appears that NO functions as an early signal during the acquisition of new information rather than in its long-term storage (Schuman & Madison, 1994), recently Rickard, Ng and Gibbs (1994) reported that sodium nitropruside, a NO donor (Feelish & Novack, 1987), when given intracerebrally to 1-day-old chicks immediately after a 1-week training trial, facilitates the formation of long-term memory, suggesting that NO may be also important for memory storage. In this sense, the posttraining administration of N-nitroarginine, a NOSI (Nathan, 1992), into the dorsal hippocampus of rats impairs retention of a step-down inhibitory avoidance response (Fin, Da Cunha, Bromberg, Schmitz, Bianchin, Medina, & Izquierdo, 1995). On the contrary, under the same experimental conditions, the administration of the NO donor, S-nitroso-N-acetylpenicillamine (Schuman & Madison, 1994) enhanced retention (Fin et al., 1995). Since both effects were time-dependent, these results suggest an action on memory storage processes. Similar results were reported by Huang and Lee (1995) using N-monometilarginine as a NOSI and sodium nitroprusside as NO donor, injected into dentate gyrus. In both cases it was suggested that hippocampal NO plays a facilitatory role in the memory process of an inhibitory avoidance task in rats (Fin et al., 1995; Huang & Lee, 1995). The present results agree, in general, with these suggestions and extend the participation of the L-arginine/NO pathways to the memory processes that are carried out during memory storage of an inhibitory avoidance response in mice. Indeed, when L-NAME was systemically administered to mice immediately after training, it significantly impaired retention performance. The effects varied in a monotonic fashion as a function of the dosage of the NOSI. Our findings show that L-NAME does not affect retention latencies of unshocked mice, indicating that the effects on retention are not due to a nonspecific action on response latencies. The effects of L-NAME on retention were enantio-
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merically specific, since they were not observed when, in the same range of dose, D-NAME was administered immediately after training instead of LNAME. The conversion of L-arginine to NO is specific because a number of analogs of L-arginine, including its D-enantiomer, are not substrates of the NOS (Moncada et al., 1991). In addition, the substrate analog inhibitors of NOS are competitive for L-arginine, but not for D-arginine (Nathan, 1992); therefore, the effects of L-NAME on retention might be attenuated by the former, but not by the latter amino acid. This view was corroborated by the findings of the present study, which are in agreement with those previously reported by several authors. Considered together, all these evidences suggest a causal relationship between an inhibition of NOS activity and the impairment of retention here reported. Since the effects of L-NAME on retention were time-dependent, that is, the impairment of retention decreased as the training–treatment interval was increased, it suggests the existence of an action on posttraining neural or neurohumoral processes underlying the storage of acquired information (McGaugh, 1991). The present results do not rule out a possible participation of peripheral influences on the effects of L-NAME on memory storage in mice, taking into account that L-NAME also inhibits NOS localized peripherally (Moncada et al., 1991; Nathan, 1992). A comparison of effective doses of L-NAME following icv and ip administration would be desirable and also the use of novel inhibitors of NOS which exhibit more selectivity for the central enzyme (Moore, Babbedge, Wallace, Gaffen, & Hart, 1993). While the present experiments on their own do little to clarify the final mechanism(s) by which posttraining LNAME impairs retention, future studies must take into account the potential role of NO in the regulation of neurotratransmitter release (Schuman & Madison, 1994). In this sense, recent evidence from our laboratory suggests that the memory-impairing effects of posttraining L-NAME may be mediated, at least in part, by a reduction of the central cholinergic tone (Baratti & Kopf, 1995). Finally, many other putative retrograde synaptic messengers, aside from NO, such as carbon monoxide, araquidonic acid, and the platelet-activating factor, may play a coordinate role in memory (Izquierdo, 1994), pointing out the need for further enquiry into the functional role of them in the brain. In summary, we suggest that the L-arginine/NO pathway may be critically involved in memory storage of a one-trial inhibitory avoidance response in mice. Further studies are necessary to elucidate the
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possible participation of NO in memory storage of other learned tasks. REFERENCES Baratti, C. M., & Kopf, S. R. (1995). Effects of physostigmine and oxotremorine on memory impairment induced by a nitric oxide synthase inhibitor in mice. Ninth International Symposium on Cholinergic Mechanisms, Abstract 70, Mainz, Germany. Bohme, G. A., Bon, C., Lemaire, M., Reibaud, M., Piot, O., Stutzmann, J. M., Doble, A., & Blanchard, J. C. (1993). Altered synaptic plasticity and memory formation in nitric oxide inhibitor treated rats. Proceedings of National Academy of Sciences USA, 90, 9191–9194. Bohme, G. A., Bon, C., Stutzmann, J. M., Doble, A., & Blanchard, J. C. (1991). Possible involvement of nitric oxide in long-term potentiation. European Journal of Pharmacology, 199, 379– 381. Bredt, D. S., Glatt, C., Hwang, P. M., Fotuhi, M., Dawson, T. M., & Snyder, S. H. (1991a). Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of mammalian nervous system together with NADPH diaphorase. Neuron, 7, 615–624. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., & Snyder, S. H. (1991b). Cloned and expressed nitric oxide synthase protein structurally resembles cytochrome P450 reductase. Nature, 351, 714–718. Chapman, P. F., Atkins, C. M., Allen, M. T., Haley, J. E., & Steinmetz, J. E. (1992). Inhibition of nitric oxide synthesis impairs two different forms of learning. Neuroreport, 3, 567– 570. Dwyer, M. A., Bredt, D. S., & Snyder, S. H. (1991). Nitric oxide synthase: Irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. Biochemical and Biophysical Research Communications, 176, 1136. Feelish, M., & Noack, E. A. (1987). Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. European Journal of Pharmacology, 139, 19–25. Fin, C., Da Cunha, C., Bromberg, E., Schmitz, P. K., Bianchin, M., Medina, J. E., & Izquierdo, I. (1995). Experiments suggesting a role for nitric oxide in the hippocampus in memory processes. Neurobiology of Learning and Memory, 63, 113– 115. Garthwaite, J. (1991). Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends in Neurosciences, 14, 60– 68. Holscher, C., & Rose, S. P. R. (1992). An inhibitor of nitric oxide synthesis prevents memory formation in the chick. Neuroscience Letters, 145, 165–167. Huang, A. M., & Lee, E. H. Y. (1995). Role of hippocampal nitric oxide in memory retention in rats. Pharmacology Biochemistry and Behaviour, 50, 327–332. Iga, Y., Yoshioka, M., Togashi, H., & Saito, H. (1993). Inhibitory action of N-omega-nitro-L-arginine methyl ester on in vivo long-term potentiation in the rat dentate gyrus. European Journal of Pharmacology, 238, 395–398. Izquierdo, I. (1994). Pharmacological evidence for a role of longterm potentiation in memory. FASEB J. 8, 1139–1145. McGaugh, J. L. (1991). Interaction of hormones and neurotransmitter systems in the modulation of memory storage. In
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NITRIC OXIDE AND MEMORY STORAGE R. C. A. Frederickson, J. L., McGaugh, & D. L. Felten (Eds.), Peripheral signalling of the brain (pp. 391–419). Toronto: Hagrefe & Huber. Moncada, S., Palmer, R. M. J., & Higgs, E. A. (1991). Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacological Reviews, 43, 109–142. Nathan, C. (1992). Nitric oxide as a secretory product of mammalian cells. FASEB J. 6, 3051–3064. O’Dell, T. J., Hawkins, R. D., Kandel, E. R., & Arancio, O. (1991).
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Test of the role of two diffusible substances in long-term potentiation: Evidence for nitric oxide as a possible early retrograde messenger. Proceedings National Academy of Sciences USA, 88, 11285–11289. Rickard, N. S., Ng, N. T., & Gibbs, M. E. (1994). Nitric oxide agonist stimulates consolidation of long-term memory in the 1-day old chick. Behavioral Neurosciences, 108, 3–6. Schuman, E. R., & Madison, D. U. (1994). Nitric oxide and synaptic function. Annual Review of Neuroscience, 17, 153–183. Sokal, R. R., & Rohlf, F. J. (1969). Biometry. New York: Freeman.
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0023
A Nitric Oxide Synthase Inhibitor Impairs Memory Storage in Mice CARLOS M. BARATTI
AND
SILVIA R. KOPF1
Laboratorio de NeurofarmacologıB a de Procesos de Memoria, Ca´tedra de FarmacologıB a, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, RA-1113 Buenos Aires, Argentina
produced by the enzyme, NO synthase (NOS) (Bredt, Hwang, Glatt, Lowenstein, Reed, & Snyder, 1991b) which involves oxidizing one of the terminal guanidino nitrogens of the semiessential amino acid Larginine (Moncada et al., 1991). In brain, a constitutively Ca2/-dependent NOS is expressed in specific neuronal populations (Bredt, Glatt, Hwang, Fotuhi, Dawson, & Snyder, 1991a). The possible involvement of NO in long-term potentiation (LTP) (Bohme, Bon, Stutzmann, Doble, & Blanchard, 1991) has prompted several investigations to determine the role of NO in animal learning and memory processes. Although research into NO in learning and memory has only just started, some studies suggest that NO may participate in the acquisition of learned behaviors (Schuman & Madison, 1994). We report here, for the first time, that the posttraining systemic administration of L-NAME, a competitive inhibitor of NOS (Dwyer, Bredt, & Snyder, 1991), impairs retention of a step-through inhibitory avoidance task in mice.
Posttraining administration of the L-enantiomer of the competitive inhibitor of nitric oxide synthase, NG-nitro-Larginine methyl ester (L-NAME, 3–100 mg/kg, ip), impaired 48-h retention of a one-trial step-through inhibitory shock-avoidance task in male Swiss mice. The effects were dose-dependent and were not observed when the D-enantiomer (D-NAME, 3–100 mg/kg, ip) was injected instead of L-NAME. Retention latencies of mice that had not received a footshock during training were not affected by L-NAME. The memory impairment produced by L-NAME was timedependent, suggesting an action on memory storage. The effects of L-NAME on memory were overcome by the injection of L-(but not D-)arginine (300 mg/kg, ip) along with the inhibitor. Considered together, these findings suggest that the L-arginine/nitric oxide pathway may be involved in memory storage of an inhibitory avoidance response in mice. q 1996 Academic Press, Inc.
INTRODUCTION Evidence accumulated over the past 30 years indicates that, if administered immediately or shortly after a learning experience, a variety of treatments can retroactively impair or enhance memory storage (McGaugh, 1991). Such findings have suggested that retention may be modulated by the endogenous activity of physiological systems that do not themselves serve as the locus of the changes for the formation of long-term memory. The free radical gas nitric oxide (NO) is a recently identified neuronal messenger (Garthwaite, 1991) that has been shown to function in a wide variety of central and peripheral processes (Moncada, Palmer, & Higgs, 1991; Nathan, 1992; Schuman & Madison, 1994). Nitric oxide is
MATERIALS AND METHODS Experiments were conducted in male Swiss mice (Roux-Ocefa Labs., Argentina) weighing approximately 30 g. They were housed 20 per cage in stainless steel cages, with food and tap water ad lib., except during the training and testing procedures. Lighting conditions were 12 h on/12 h off with the light phase of the cycle beginning at 7:00 AM. The mice were trained in a one-trial step-through inhibitory shock-avoidance task. On the training trial, the mouse was placed on a lighted platform outside a hole leading to a dark compartment. When the mouse stepped into the dark compartment a footshock (0.8 mA, 50 Hz, for 1 s) was delivered. For the retention test 48 h later, each mouse was again placed on the same platform and the latency to step
1 Work supported by Grant 5100/92 from CONICET to Carlos M. Baratti. Correspondence and reprint requests should be addressed to Dr. C. M. Baratti. Fax: 54-1-962-5341. E-mail: baratti @cafarm.ffyb.uba.ar.
197 1074-7427/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Effect of posttraining administration of L-NAME on retention. Treatments were given immediately after training. N Å 20 mice per group (shocked mice) and N Å 10 mice per group (unshocked mice). *põ.05, and **p õ .01, compared with saline control group (Newman–Keuls test, põ.05). F.S., foot shock; N.S., no shock.
through was recorded (maximum 300 s). The experiments were performed in such a way that the person doing the testing did not know the makeup of the groups. The drugs used in these experiments were NG-nitro-L-arginine methyl ester hydrochloride, NGnitro-D-arginine methyl ester hydrochloride, L-arginine ethyl ester dihydrochloride, and D-arginine hydrochloride (obtained through Sigma Chemical Co., St. Louis, MO). All drugs were dissolved in saline immediately before being used. All injections were given intraperitoneally (10 ml/kg). Controls received the same volume of saline. All doses were calculated as the free base. Since there was not a significant departure from homogeneity of variance of the data across the experiments (Fmax test) (Sokal & Rohlf, 1969), data are expressed as means { SEM of the latencies to step through during the retention test. They were evaluated using a one- or a two-way ANOVA, depending upon the experimental design (Sokal & Rohlf, 1969). Post hoc analysis with the Newman–Keuls test was made. In all cases p values less than .05 were considered significant.
of mice that received a footshock during training (F(4, 95) Å 6.74; p õ .01, one-way ANOVA). The step-through latencies of mice given 10 or 30 mg/kg (p õ .05) and 100 mg/kg (p õ .01) of L-NAME were significantly lower than those of saline controls. In mice that had not received the footshock on the training trial, the retention test latencies were unaffected by L-NAME (F(4, 45) Å 0.97; p ú .05, one-way ANOVA). Since the effects of NOS inhibitors appear to be enantiomerically specific, we decided to determine whether the post-training administration of DNAME would modify retention. Thus, five different groups of 20 mice each were injected with saline or D-NAME (3–100 mg/kg) immediately after training. There were no significant differences in the retention latencies among these groups (saline, 140 { 18 s; DNAME (3 mg/kg), 138 { 17 s; D-NAME (10 mg/kg), 139 { 19 s; D-NAME (30 mg/kg), 140 { 20 s, and DNAME (100 mg/kg), 140 { 21 s; F(4, 95) Å 0.08; p ú .05, one-way ANOVA). In the following experiment we studied the possible interaction between L-NAME with L-arginine or D-arginine. Thus, six different groups of 20 mice each were submitted to the training procedure. Immediately after it, the mice received saline, L-NAME (100 mg/kg), L-arginine (300 mg/kg), D-arginine (300 mg/ kg), or L-NAME (100 mg/kg) plus L-arginine (300 mg/kg) or D-arginine (300 mg/kg), as a single injection. The results are shown in Fig. 2. Neither Larginine nor D-arginine affected retention on its own (p ú .05, in both cases). The effect of L-NAME on retention was prevented by L-arginine (F(1, 76) Å 7.02; p õ .01), but not by D-arginine (F(1, 76) Å 0.01; p ú .05) (two-way ANOVA, in both cases). The
RESULTS In the first experiment, different groups of 20 mice each were trained as described under Materials and Methods. Immediately after training, the mice received an ip injection of saline or L-NAME (3–100 mg/kg). Other groups of 10 mice each were trained without footshock and received saline or L-NAME according to the same experimental design used for shocked mice. The results are shown in Fig. 1. The posttraining administration of L-NAME induced a dose-dependent decrease in retention performance
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FIG. 2. Effects of L-NAME, L-arginine or D-arginine, and their combination on retention. Treatments were given immediately after training as a single injection. N Å 20 mice per group. **põ.01, compared with saline control group (Newman–Keuls test, põ.05).
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FIG. 3. Effects of delayed posttraining administration of LNAME on retention. Treatments were given immediately or 180 min after training. N Å 20 mice per group. **põ.01, compared with saline control group (Newman–Keuls test, põ.05).
Newman–Keuls test showed significant differences for the groups that received L-NAME alone or LNAME plus D-arginine compared with saline control group (p õ .01, in both cases). Finally different groups of 20 mice each were submitted to the training and received saline or LNAME (100 mg/kg) immediately or 180 min after training. Figure 3 shows the results of these experiments. A 2 (saline– L-NAME) 1 2 (training–treatment interval) ANOVA shows significant interaction of treatment 1 time (F(1, 76) Å 9.51; p õ .01). LNAME administered immediately after training significantly impaired retention (p õ .01). However, when the NOS inhibitor was given 180 min after training, retention latencies did not differ significantly from its control group (p ú .05). DISCUSSION Nitric oxide synthase inhibitors (NOSI), such as NG-nitro-L-arginine methyl ester (L-NAME), block LTP-induction both in vitro (Bohme et al., 1991) and in vivo (Iga, Yoshioka, Togashi, & Saito, 1993). The effects may be reversed by L- (but not D-) arginine (Schuman & Madison, 1994). Since LTP remains a candidate mnemonic device and it appears that NO production is necessary for LTP-induction (O’Dell, Hawkins, Kandel, & Arancio, 1991), recent research has clearly emphasized the possible involvement of L-arginine/NO pathway in the acquisition of learned behaviors. Thus, the results of Holscher and Rose (1992) demonstrated for the first time that NO synthesis is necessary for an early phase of memory formation for an inhibitory avoidance response in the chick. The anterograde amnesia induced by the inhibition of NOS was overcome by coadministration of L-arginine. In contrast, in a one-trial shock-inhibi-
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tory avoidance task using rats as experimental subjects, the same NOSI left shock-avoidance learning unaffected (Bohme, Bon, Lemaire, Reibaud, Piot, Stutzmann, Doble, & Blanchard, 1993). Deficits in the acquisition of spatial reference memory in rats, both in the water maze (Chapman, Atkins, Allen, Haley, & Steinmetz, 1992) and in the eight-arm radial maze (Bohme et al., 1992), as well as an impaired acquisition of the classical conditioned eyeblink responses in rabbits (Chapman et al., 1992), following administration of NOSI have also been reported. In addition, Bohme et al. (1993) have shown that the inhibition of NOS impairs learning in a social recognition task (social memory). Although it appears that NO functions as an early signal during the acquisition of new information rather than in its long-term storage (Schuman & Madison, 1994), recently Rickard, Ng and Gibbs (1994) reported that sodium nitropruside, a NO donor (Feelish & Novack, 1987), when given intracerebrally to 1-day-old chicks immediately after a 1-week training trial, facilitates the formation of long-term memory, suggesting that NO may be also important for memory storage. In this sense, the posttraining administration of N-nitroarginine, a NOSI (Nathan, 1992), into the dorsal hippocampus of rats impairs retention of a step-down inhibitory avoidance response (Fin, Da Cunha, Bromberg, Schmitz, Bianchin, Medina, & Izquierdo, 1995). On the contrary, under the same experimental conditions, the administration of the NO donor, S-nitroso-N-acetylpenicillamine (Schuman & Madison, 1994) enhanced retention (Fin et al., 1995). Since both effects were time-dependent, these results suggest an action on memory storage processes. Similar results were reported by Huang and Lee (1995) using N-monometilarginine as a NOSI and sodium nitroprusside as NO donor, injected into dentate gyrus. In both cases it was suggested that hippocampal NO plays a facilitatory role in the memory process of an inhibitory avoidance task in rats (Fin et al., 1995; Huang & Lee, 1995). The present results agree, in general, with these suggestions and extend the participation of the L-arginine/NO pathways to the memory processes that are carried out during memory storage of an inhibitory avoidance response in mice. Indeed, when L-NAME was systemically administered to mice immediately after training, it significantly impaired retention performance. The effects varied in a monotonic fashion as a function of the dosage of the NOSI. Our findings show that L-NAME does not affect retention latencies of unshocked mice, indicating that the effects on retention are not due to a nonspecific action on response latencies. The effects of L-NAME on retention were enantio-
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merically specific, since they were not observed when, in the same range of dose, D-NAME was administered immediately after training instead of LNAME. The conversion of L-arginine to NO is specific because a number of analogs of L-arginine, including its D-enantiomer, are not substrates of the NOS (Moncada et al., 1991). In addition, the substrate analog inhibitors of NOS are competitive for L-arginine, but not for D-arginine (Nathan, 1992); therefore, the effects of L-NAME on retention might be attenuated by the former, but not by the latter amino acid. This view was corroborated by the findings of the present study, which are in agreement with those previously reported by several authors. Considered together, all these evidences suggest a causal relationship between an inhibition of NOS activity and the impairment of retention here reported. Since the effects of L-NAME on retention were time-dependent, that is, the impairment of retention decreased as the training–treatment interval was increased, it suggests the existence of an action on posttraining neural or neurohumoral processes underlying the storage of acquired information (McGaugh, 1991). The present results do not rule out a possible participation of peripheral influences on the effects of L-NAME on memory storage in mice, taking into account that L-NAME also inhibits NOS localized peripherally (Moncada et al., 1991; Nathan, 1992). A comparison of effective doses of L-NAME following icv and ip administration would be desirable and also the use of novel inhibitors of NOS which exhibit more selectivity for the central enzyme (Moore, Babbedge, Wallace, Gaffen, & Hart, 1993). While the present experiments on their own do little to clarify the final mechanism(s) by which posttraining LNAME impairs retention, future studies must take into account the potential role of NO in the regulation of neurotratransmitter release (Schuman & Madison, 1994). In this sense, recent evidence from our laboratory suggests that the memory-impairing effects of posttraining L-NAME may be mediated, at least in part, by a reduction of the central cholinergic tone (Baratti & Kopf, 1995). Finally, many other putative retrograde synaptic messengers, aside from NO, such as carbon monoxide, araquidonic acid, and the platelet-activating factor, may play a coordinate role in memory (Izquierdo, 1994), pointing out the need for further enquiry into the functional role of them in the brain. In summary, we suggest that the L-arginine/NO pathway may be critically involved in memory storage of a one-trial inhibitory avoidance response in mice. Further studies are necessary to elucidate the
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