Critical time-window for NO-cGMP-dependent long-term memory formation after one-trial appetitive conditioning

Abstract

The nitric oxide (NO)-cGMP signaling pathway is implicated in an increasing number of experimental models of plasticity. Here, in a behavioral analysis using one-trial appetitive associative conditioning, we show that there is an obligatory requirement for this pathway in the formation of long-term memory (LTM). Moreover, we demonstrate that this requirement lasts for a critical period of approximately 5 hr after training. Specifically, we trained intact specimens of the snail Lymnaea stagnalis in a single conditioning trial using a conditioned stimulus, amyl-acetate, paired with a salient unconditioned stimulus, sucrose, for feeding. Long-term associative memory induced by a single associative trial was demonstrated at 24 hr and shown to last at least 14 d after training. Tests for LTM and its dependence on NO were performed routinely 24 hr after training. The critical period when NO was needed for memory formation was established by transiently depleting it from the animals at a series of time points after training by the injection of the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO). By blocking the activity of NO synthase and soluble guanylyl cyclase enzymes after training, we provided further evidence that LTM formation depends on an intact NO-cGMP pathway. An electrophysiological correlate of LTM was also blocked by PTIO, showing that the dependence of LTM on NO is amenable to analysis at the cellular level in vitro. This represents the first demonstration that associative memory formation after single-trial appetitive classical conditioning is dependent on an intact NO-cGMP signaling pathway.

Fig. 1.

Components of the NO-cGMP signaling pathway and the drugs that were used to target them in the present experiments. PTIO, a nitric oxide (NO) scavenger, was used to temporarily deplete NO from intact Lymnaea.l-NAME was used as an inhibitor of the nitric oxide synthase (NOS) enzyme, and ODQ was used as an inhibitor of the soluble guanylyl cyclase enzyme (for details, see Materials and Methods).

Fig. 2.

Depletion of NO by PTIO (1 mm final concentration) temporarily impairs the feeding response ofLymnaea to sucrose. A, Snails were tested for preinjection feeding responses to sucrose (US test, −60 min) and retested at three different time points (30 min, 60 min, and 120 min) after injection (at 0 min) with PTIO or saline (vehicle control). B, PTIO-injected snails (PTIO, black bars), matched for preinjection response levels (Before injection) with saline-injected snails (Saline, white bars), show a significant impairment of the feeding response to sucrose at 30 min after injection. There is a full recovery of the feeding response by 60 min after injection with PTIO. Data are shown as means ± SE. Asterisk indicates a significant difference (p < 0.003) between postinjection and preinjection response levels.

Fig. 3.

One-trial chemical conditioning of the feeding response results in LTM formation in Lymnaea.A, All snails were tested for feeding responses in the presence of water (a disturbance factor) and amyl-acetate (the CS) 24 hr before training. For each animal, an amyl-acetate minus water response score was generated, and this was used to quantify the feeding response to the CS. The pretested animals were divided into four groups matched for their mean feeding responses to the CS (Bi). Conditioned snails (black bar) were subjected to a single paired CS + US trial (amyl-acetate paired with sucrose), whereas control snails (white bars) were subjected to an unpaired CS and US trial (Unpaired control) or CS or US alone control trials. Water (W) was used to control for volume changes associated with the addition of the CS or US (a disturbance factor). After each trial, the animals were rinsed and transferred back to their home tank. All four groups were retested for the CS 24 hr and 14 d after training.B, Conditioned animals (black bars) show significantly stronger feeding responses to the CS than controls (white bars), at both 24 hr (Bii) and 14 d (Biii) after training. Only the trained group shows a significant increase in the response to the CS (Bii, Biii) compared with pretraining levels (Bi). The feeding responses to the CS data are shown as means ± SE. The groups showed no differences in their pretraining feeding rates in water and amyl-acetate or in their post-training feeding rates in water (see Results). For details of the statistical analyses, also see Results.

Fig. 4.

Blocking of the NO-cGMP pathway impairs LTM after single-trial appetitive classical conditioning. A, For each experiment shown in this figure, a group of snails were pretested for the CS 24 hr before training and divided into four matched groups (pretraining response levels are shown in Bi,Ci, and Di, respectively). Conditioned snails (black bar) were subjected to one-trial CS + US training. Unpaired control snails (white bar) were subjected to a CS + water (W) trial followed after 1 hr by a water + US trial. Water was used to control for volume changes associated with the addition of the CS or US (control groups), or both (conditioned groups). At the end of each trial, the animals were rinsed and transferred back to their home tank. Ten minutes after the training or control trial, animals were injected with NO-cGMP pathway blocking drugs or control substances. Both conditioned and control snails were tested for feeding response to the CS 24 hr after the training trial. B, The effect of NO depletion after training. There is no difference in PTIO-injected conditioned snails (Bii, PTIO,black bar) compared with the PTIO-injected unpaired controls (Bii, PTIO, white bar). In contrast, vehicle-injected conditioned snails (Bii, Saline, black bar) show significantly higher feeding responses to the CS than vehicle-injected unpaired controls (Bii, Saline,white bar). C, The effect of inhibiting NOS after training. Conditioned l-NAME injected snails (Cii, l-NAME,black bar) show no increase in their post-training feeding responses to the CS, which are statistically similar tol-NAME-injected unpaired controls (Cii, l-NAME, white bar). The feeding response to the CS in thed-NAME-injected conditioned snails (Cii, d-NAME, black bar) is significantly higher than the response in bothd-NAME-injected unpaired controls (Cii, d-NAME, white bar) and l-NAME-injected conditioned animals. D, The effect of inhibiting sGC after training. Conditioned ODQ-injected snails (Dii, ODQ,black bar) show no increase in their post-training feeding responses to the CS, which are statistically similar to ODQ-injected unpaired controls (Dii, ODQ, white bar). The feeding response to the CS in the saline + DMSO-injected conditioned snails (Dii, Saline+DMSO, black bar) is significantly higher than the response in both saline + DMSO-injected unpaired controls (Dii,Saline+DMSO, white bar) and ODQ-injected conditioned animals. In all three experiments, only conditioned snails injected with control substances show post-training responses to the CS that are significantly stronger than pretraining response levels (Bi, Ci, Di, black bars). All data in this figure are shown as means ± SE. In each experiment, the groups showed no differences in their pretraining feeding rates in water and amyl-acetate or in their post-training feeding rates in water (see Results). For details of the statistical analyses, also see Results.

Fig. 5.

Critical time window for the impairment of LTM by PTIO-induced depletion of NO. Ai, A single large group of animals were initially tested for feeding response to the CS (white bar, −24 hr) before they were conditioned 24 hr later (0h, CS+US). The conditioned animals were divided into seven groups (matched for pretraining response to the CS), each injected with PTIO at one of seven different time points after training (Group 1,1h; Group 2, 2h;. . .Group7, 21h). All snails were retested for feeding response to the CS at 24 hr after training. Aii, Depletion of NO by injection of PTIO up to 4 hr after training completely prevents the conditioned feeding responses (black bars), which are the same as the feeding responses to the CS before training (white bars). After >4 hr, the effect of NO depletion starts to decline, and after >5 hr after training, NO depletion no longer has an effect on the conditioned feeding responses to the CS, which are significantly greater than before training and greater than the post-training responses in the groups injected between 1 and 4 hr after training. p values indicate significant within-group differences in the response to the CS before and after training. Bi, For comparisons at the most critical time points revealed by the results in Aii, both conditioned and unpaired control groups were used that were injected with PTIO either 4 hr (Group 1) or 6 hr (Group 2) after the training or control trial. Bii, Conditioned animals (black bars) injected with PTIO 4 hr after training show feeding responses to the CS that are not different from either pretraining levels or post-training control levels.Biii, In contrast, conditioned animals (black bars) injected with PTIO 6 hr after training show feeding responses to the CS that are significantly stronger than both pretraining levels and post-training control levels. Data are shown as means ± SE. In each experiment shown in this figure, the groups showed no differences in their pretraining feeding rates in water and amyl-acetate or in their post-training feeding rates in water (see Results). For details of the statistical analyses, also see Results.

Fig. 6.

The behavioral effect of PTIO-induced NO depletion on LTM is amenable to a cellular analysis. A, The same group of conditioned and saline-injected snails (black bars) shows both a behavioral (Ai, feeding) and an electrophysiological (Aii, fictive feeding) response to the CS, and these are both significantly stronger than corresponding response levels in conditioned snails that were subsequently injected with PTIO. Bi, The semi-intact preparation used in this experiment. The positions of the two types of neurons that were recorded are shown in the cerebral ganglia (CV1a, a modulatory neuron) and the buccal ganglia (B3, a motoneuron). The cerebral ganglia are connected to the lips by the lip and tentacle nerves. Bii, A typical preparation from one of the five saline-injected conditioned animals that were used in the quantitative analysis shown in Aii. Both CV1a and B3 show a series of fictive feeding cycles in response to amyl-acetate applied to the lips (start of application indicated byarrowhead). The characteristic N1/protraction, N2/rasp, and N3/swallow phases of fictive feeding (Benjamin and Elliott, 1989) are marked on an expanded time-base trace of a section with one of the cycles (boxed). Biii, A typical preparation from one of the five PTIO-injected conditioned animals that were used in the quantitative analysis shown in Aii. Neither CV1a nor B3 shows fictive feeding cycles in response to amyl-acetate (start of application indicated byarrowhead). However, activation of CV1a by injection of a steady depolarizing current can still drive activity in the feeding CPG, which can be monitored as N1, N2, and N3 phase synaptic inputs on both CV1a and B3 (expanded time-base trace of boxed section). Ci, The depolarization produced in CV1a in response to amyl-acetate (Bii,Biii, arrows) is significantly stronger in preparations derived from saline-injected animals (Saline, black bar) than in those obtained from PTIO-injected conditioned animals (PTIO,white bar). Cii, The resting potential levels of CV1a are virtually identical in the two groups, indicating that the difference in the depolarizing response is not caused by a difference in CV1a membrane potential between the two types of preparation. All data in this figure are shown as means ± SE. For details of the statistical analyses, see Results.