Nitric oxide is necessary for multiple memory processes after learning that a food is inedible in aplysia

Abstract

Nitric oxide (NO) signaling was inhibited via N(omega)-nitro-L-arginine methyl ester (L-NAME) during and after training Aplysia that a food is inedible. Treating animals with L-NAME 10 min before the start of training blocked the formation of three separable memory processes: (1) short-term, (2) intermediate-term, and (3) long-term memory. The treatment also attenuated, but did not block, a fourth memory process, very short-term memory. L-NAME had little or no effect on feeding behavior per se or on most aspects of the animals' behavior while they were being trained, indicating that the substance did not cause a pervasive modulation or poisoning of many aspects of feeding and other behaviors. Application of L-NAME within 1 min after the training had no effect on short- or long-term memory, indicating that NO signaling was not needed during memory consolidation. Treating animals with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazdine-1-oxy-3-oxide before training also blocked long-term memory. Memory was not blocked by D-NAME, or by the simultaneous treatment with L-NAME and the NO donor S-nitroso-N-acetyl-penicillamine, confirming that the effect of L-NAME is attributable to its effect as a competitive inhibitor of L-arginine for NO synthase in the production of NO rather than to possible effects at other sites. These data indicate that NO signaling during training plays a critical role in the formation of multiple memory processes.

Fig. 1.

Lack of effects of l-NAME on feeding behavior. Shaded bars, Treated with l-NAME;open bars, treated with seawater. A, Percentage of time spent feeding in 1 hr of ad libitumexposure to food (n = 7 seawater controls;n = 6 animals treated with l-NAME).B, Latency to respond to food touching the lips in previously unaroused animal (n = 7 seawater-treated animals; n = 11 l-NAME-treated animals). C, Number of bites in a 5 min period in which the lips were stimulated continuously with food (n= 8). D, Swallow amplitude. E, Swallow period (n = 10 seawater-treated animals;n = 5 l-NAME-treated animals). In this and in subsequent figures, means and SE are shown.

Fig. 2.

Lack of effects of l-NAME on responses to seaweed extract in n = 8 seawater-treated andn = 8 l-NAME-treated animals.Shaded bars, Treated with l-NAME;open bars, treated with seawater. A, Latency to lift of the head. There was no significant difference between l-NAME- and seawater-treated animals (p = 0.39;t₍₁₄₎ = 0.88). B, Latency to the first bite. There was no significant difference betweenl-NAME- and seawater-treated animals (p = 0.67;t₍₁₄₎ = 0.43). C, Number of bites in 5 min of exposure. There was no significant difference between l-NAME- and seawater-treated animals (p = 0.59;t₍₁₄₎ = 0.55). D, Time to stop responding to the seaweed extract. There was no significant difference between l-NAME- and seawater-treated animals (p = 0.39;t₍₁₄₎ = 0.90). E, Amplitude of each bite elicited by seaweed extract using a four-point scale. There was no significant difference in the relative distribution of the four levels of bite amplitude (p = 0.09; χ²₍₃₎ = 6.42).

Fig. 3.

Lack of effects of l-NAME on training. Three parameters of the training were measured. Shaded bars, Treated with l-NAME; open bars, treated with seawater. A, Time that food is in the mouth during the first 5 min of training. B, Number of attempted swallowing responses performed during the first 5 min of training. C, Length of training needed for the animals to stop responding to the inedible food.

Fig. 4.

l-NAME treatment before training blocks short-term memory. Animals were treated before training withl-NAME (n = 7) or seawater (n = 6). Open bars, Data from the initial training session; shaded bars, data from a second session that measured memory after 30 min. Memory is indicated by a decrease in a value from the first to the second session. Experiments showing a significant decrease in this and in subsequent figures are marked with asterisks. An additional group of previously untreated naive animals (Naive;n = 6) was examined in a blind procedure along with the previously trained animals. A, There was a significant decrease in the time for animals to stop responding to inedible food between the training and testing session for the seawater-treated controls (p < 0.001;t₍₅₎ = 14.16) but not for thel-NAME-treated animals (p = 0.340; t₍₆₎ = 1.035; 2-tailed pairedt tests). There was also a significant difference in the percentage of change in response between the training and testing session between l-NAME- and seawater-treated animals (p < 0.001;t₍₁₁₎ > 100; 2-tailed ttest). B, There was a significant decrease in the time that food was in the mouth during the first 5 min between the two session for the control animals (p < 0.001;t₍₅₎ = 7.57) but not for animals treated with l-NAME (p = 0.227;t₍₆₎ = 1.35; 2-tailed pairedt tests). In addition, there was a significant difference in the percentage of change in response between training and testing between l-NAME- and seawater-treated animals (p < 0.001;t₍₁₁₎ = 9.48; 2-tailed ttest).

Fig. 5.

l-NAME treatment immediately after training does not affect short-term memory. The experiment was identical to that in Figure 4, except that animals were treated withl-NAME or seawater immediately after the training. For animals treated with l-NAME, n = 8; for animals treated with seawater, n = 6; for naive animals, n = 7. A, There was a significant decrease in the time to stop responding between the training and testing session for the control animals (p < 0.001;t₍₅₎ = 8.28) and for the animals treated with l-NAME (p < 0.001;t₍₇₎ = 11.77; 2-tailed pairedt tests). In addition, there was no significant difference in the percentage of change in response between the training and testing session between animals treated with l-NAME and with seawater (p = 0.94;t₍₁₂₎ = 0.07; 2-tailed ttest). B, There was a significant decrease in the time that food was within the mouth during the first 5 min between the training and testing session for animals treated with seawater (p = 0.002;t₍₅₎ = 5.74) and for animals treated with l-NAME (p < 0.001;t₍₇₎ = 7.19; 2-tailed pairedt tests). In addition, there was no significant difference in the percentage of change in response between the training and testing session (p = 0.56;t₍₁₂₎ = 0.60; 2-tailed ttest).

Fig. 6.

l-NAME treatment before training blocks long-term memory. The experiment was identical to that in Figure4, except that the test of memory was performed after 24 hr. For animals treated with l-NAME, n = 7; for animals treated with seawater, n = 4; for naive animals, n = 6. A, There was a significant decrease in the time to stop responding to the inedible food between the training and testing session for the control animals (p < 0.001;t₍₃₎ = 25.37) but not for thel-NAME-treated animals (p = 0.32; t₍₆₎ = 1.08; 2-tailed pairedt tests). In addition, there was a significant difference in the percentage of change in response between the training and testing session between animals treated with l-NAME and seawater (p < 0.001;t₍₉₎ = 10.13; 2-tailed ttest). B, There was a significant decrease in the time that food was in the mouth during the first 5 min from the training and testing session for the control animals (p = 0.005; t₍₃₎ = 7.19) but not for thel-NAME-treated animals (p = 0.33; t₍₆₎ = 1.06; 2-tailed pairedt tests). In addition, there was a significant difference between l-NAME- and seawater-treated animals in the percentage of change in response from the training to the testing session (p = 0.002;t₍₉₎ = 4.14; 2-tailed ttest).

Fig. 7.

l-NAME treatment immediately after training does not affect long-term memory. The experiment was identical to that in Figure 6, except that animals were treated withl-NAME or seawater immediately after the training rather than preceding it. For animals treated with l-NAME,n = 7; for animals treated with seawater,n = 5; for naive animals, n = 5. A, There was a significant decrease in the time to stop responding to the inedible food between the training and testing session for the control animals (p = 0.002;t₍₄₎ = 12.13) and for thel-NAME-treated animals (p = 0.001; t₍₆₎ = 5.80; 2-tailed pairedt tests). In addition, there was no significant difference between these groups in the percentage of change in response from the training and testing session (p = 0.11; t₍₁₀₎ = 1.77; 2-tailedt test). B, There was a significant decrease in the time that food was within the mouth during the first 5 min between the training and testing session for the control animals (p = 0.013;t₍₄₎ = 4.22) as well as for thel-NAME-treated animals (p = 0.019; t₍₆₎ = 3.16; 2-tailed pairedt tests). In addition, there was no significant difference in the percentage of change in response between the training and testing session (p = 0.52;t₍₁₀₎ = 0.67; 2-tailed ttest).

Fig. 8.

Lip stimulation alone is insufficient for memory formation. l-NAME-treated (n = 8) and seawater-treated (n = 7) animals were trained with inedible food. Each trained animal was yoked to an animal whose lips were stimulated (for l-NAME-treated, n = 8; for seawater-treated, n = 7) for a period equivalent to that of the training. Twenty-four hours later, both the trained and the yoked animals were tested with inedible netted food that entered the mouth and produced failed swallows. For animals treated with seawater, significant differences were observed between the animals that had been trained with food entering the mouth and those in which the lips were stimulated for both the time to stop responding to food (p < 0.001;t₍₁₃₎ = 5.68) and the time that food was in the mouth during the first 5 min of the test (p < 0.01;t₍₁₃₎ = 3.10; 2-tailed ttests). However, for animals treated with l-NAME, no significant differences were observed between the trained animals and the yoked controls (for the time to stop, p = 0.22,t₍₁₅₎ = 1.28; for the time that food was in the mouth, p = 0.73,t₍₁₅₎ = 0.36; 2-tailed ttests).

Fig. 9.

d-NAME does not block memory.A, Effects of l- and d-NAME application before training on short-term memory. l-NAME blocked short-term memory, as shown by no significant differences between training and testing (for the time to stop responding to food,p = 0.53, t₍₉₎ = 0. 69; for the time that the food was in the mouth during the first 5 min,p = 0.21, t₍₉₎ = 1.49; 2-tailed paired t tests). In contrast,d-NAME did not affect short-term memory, as shown by significant savings between training and testing (for time to stop responding, p < 0.001,t₍₇₎ = 9.73; for time in the mouth,p < 0.001, t₍₇₎ = 7.97; 2-tailed paired t test). B, Effects of l- and d-NAME before training on long-term memory. l-NAME blocked long-term memory, as shown by no significant difference in the time to stop responding to food (p = 0.59;t₍₆₎ = 0.56) and in the time that the food was in the mouth during the first 5 min (p = 0.88;t₍₆₎ = 0.15; 2-tailed pairedt tests). In contrast, d-NAME did not affect long-term memory, because there were significant differences in both the time to stop responding to food (p < 0.001; t₍₆₎ = 17.28) and in the time in the mouth (p < 0.001;t₍₆₎ = 10.06; 2-tailed pairedt tests). d-NAME-treated animals were run in a blind procedure with seawater-treated controls. There was no significant difference in the percentage of savings betweend-NAME- and seawater-treated animals for the time to stop responding (p = 0.24;t₍₁₀₎ = 1.18), and for the time spent in the mouth, there was a significantly larger percentage of savings for d-NAME-treated animals than for seawater-treated animals (p = 0.4;t₍₁₀₎ = 2.42; 2-tailed ttests).

Fig. 10.

PTIO blocks long-term memory. Animals were treated with PTIO (n = 9) or seawater (n = 7) before training, and memory was measured after 24 hr. A, There was a significant decrease in the time to stop responding between the training and testing session for the control animals (p = 0.025;t₍₆₎ = 2.95) but not for animals treated with PTIO (p = 0.492;t₍₈₎ = 0.71; 2-tailed pairedt tests). However, there was no significant difference in the percentage of change in response between the training and testing session between animals treated with PTIO and seawater (p = 0.09;t₍₁₃₎ = 1.82; 2-tailed ttest). B, There was a significant decrease in the time that food was within the mouth during the first 5 min of a session between the training and testing session for the control animals (p = 0.016;t₍₆₎ = 3.33) but not for animals treated with PTIO (p = 0.085;t₍₈₎ = 1.97; 2-tailed pairedt tests). However, there was no significant difference in the percentage of change in response between PTIO- and seawater-treated animals between the training and testing session (p = 0.07;t₍₁₄₎ = 1.93; 2-tailed ttest).

Fig. 11.

Effects on long-term memory of treating animals with the NO donor SNAP. All animals were treated withl-NAME before training. One group of animals (n = 8) was then immediately treated with SNAP, and another was treated with seawater (n = 5). Memory was examined after 24 hr. A, There was no significant decrease in the time to stop responding between the training and testing session for the animals treated with seawater (p = 0.85;t₍₄₎ = 0.20). In contrast, animals treated with SNAP displayed a decrease in the time to stop responding to food (p = 0.002;t₍₆₎ = 5.44; 2-tailed pairedt tests). There was also a significant difference between the animals receiving SNAP and seawater in the percentage of change in response (p = 0.005;t₍₁₁₎ = 3.44; 2-tailed ttest). B, There was no significant decrease in the time that food was within the mouth during the first 5 min of a session between the training and testing session for the control animals (p = 0.50;t₍₄₎ = 0.75). However, animals treated with SNAP showed significant savings using this parameter (p < 0.001;t₍₇₎ = 16.69; 2-tailed pairedt tests). There was also a significant difference in the percentage of change in response between the animals receiving SNAP and seawater (p = 0.007;t₍₁₁₎ = 3.24; 2-tailed ttest).

Fig. 12.

Effects of l-NAME using spaced training. A, Animals were trained with three 5 min training sessions after being treated with artificial seawater (n = 7) or l-NAME (n = 8). For each of the three training sessions, the time that food was in the mouth was measured. Note that the data shown preceded the test of intermediate memory shown inB. Data on spaced training preceding long-term memory were similar to those shown (see Results). B, Four hours after spaced training, animals were retrained in a single session until they stopped responding to food. The three groups whose data are shown with shaded bars were tested in a blind procedure (for Naive; n = 7). Animals treated with l-NAME showed no memory, whereas memory was seen in seawater-treated controls. C, In a separate population of animals that received spaced training, memory was examined 24 hr after the training. Data on retention were gathered in a blind procedure, with n = 6 naive animals. Animals treated with l-NAME (n = 6) showed no memory, whereas memory was seen in seawater-treated controls (n = 6).

Fig. 13.

Effects of l-NAME on very short-term memory. Very short-term memory was measured by comparing the percentage of time that food was spent in the mouth over three successive 5 min periods during the initial training. Normalized data were used in this experiment, because some animals (4 of 16 l-NAME-treated and 3 of 14 seawater-treated) reached the criterion to stop responding to the food before the end of the 15 min. To compensate, the time that food was in the mouth during each 5 min period was normalized by dividing it by the total number of minutes that the animals responded to food during the 5 min period (the full 5 min for all animals during the first and second 5 min periods as well as for all animals that also responded throughout the third 5 min period; for animals that stopped during the last 5 min period, the time in the mouth was divided by the time during this period that had already gone by before the experiment was stopped).