Separate effects of a classical conditioning procedure on respiratory pumping, swimming, and inking in Aplysia fasciata

Abstract

We examined whether swimming and inking, two defensive responses in Aplysia fasciata, are facilitated by a classical conditioning procedure that has been shown to facilitate a third defensive response, respiratory pumping. Training consisted of pairing a head shock (UCS) with a modified seawater (85%, 120%, or pH 7.0 seawater--CSs). Animals were tested by re-exposing them to the same altered seawater 1 hr after the training. For all three altered seawaters, only respiratory pumping is specifically increased by conditioning. Swimming is sensitized by shock, and inking is unaffected by training, indicating that the conditioning procedure is likely to affect a neural site that differentially controls respiratory pumping. Additional observations also indicate that the three defensive responses are differentially regulated. First, different noxious stimuli preferentially elicit different defensive responses. Second, the three defensive responses are differentially affected by shock. Inking is elicited only immediately following shock, whereas swimming and respiratory pumping are facilitated for a period of time following the shock. Third, swimming and respiratory pumping are differentially affected by noxious stimuli that are delivered in open versus closed environments. These data confirm that neural pathways exist that allow Aplysia to modulate separately each of the three defensive behaviors that were examined.

Figure 1

Training procedures. Before all training procedures, the rate of respiratory pumping, as well as the presence of swimming and inking, were measured during 10 min in 100% (pH 7.8) seawater (Before Training). Animals were then transferred to a second chamber, where they received one of four treatments: UCS and CS paired, UCS and CS unpaired, UCS alone, or CS alone. Exposure during a treatment to the CS, an altered seawater (85%, 120%, or pH 7.0 seawater), is depicted by a shaded bar, whereas the presence of normal seawater is depicted by an open bar. The UCS, shock, is depicted by an arrowhead. The animals remained in the training chamber for 5 min. Inking and swimming (but not respiratory pumping) in response to the training procedures were noted. The animals were then transferred for 1 hr to a new chamber of 100% seawater. Respiratory pumping, swimming, and inking were measured during the first and last 10 min of this hour. At the end of the hour, animals were transferred to a test chamber with an altered seawater concentration. Respiratory pumping, swimming, and inking were measured during 10 min in this solution. Responses during this period were compared with those in naive animals, which had not been trained.

Figure 2

Respiratory pumping and swimming in altered seawaters after conditioning. The effects of four treatments are shown on respiratory pumping and swimming in response to exposure to 85%, 120%, or pH 7.0 seawaters. Treatments occurred an hour before the tests shown in the figure. (Paired) Shock delivered in an altered seawater; (Unpaired) exposure to an altered seawater followed by shock; (Shock) exposure to a shock delivered in 100% seawater; (85%, 120%, or pH 7.0 seawater) exposure to one of these altered seawaters. Respiratory pumping and swimming in naive animals exposed to the three altered seawaters is also shown (shaded bars). For respiratory pumping, s.e.s are shown. The data show pairing-specific increases in respiratory pumping in response to all three altered seawaters, as well as sensitization of swimming in response to 85% and 120% seawaters.

Figure 3

Defensive behaviors elicited by the graded application of three stimuli. The thresholds required to elicit swimming, respiratory pumping, and inking were measured by stimulating animals with different levels of seawater of increased and decreased concentration and with different levels of seawater with a decreased pH.

Figure 4

Defensive behaviors elicited by shock during and subsequent to the training. Animals were examined for 10 min before receiving one of seven treatments. (A) Animals were shocked in 100% seawater. (B–D) Animals were pre-exposed for 5 min to one three altered seawaters (B, dark-shaded bar; pH 7.0; C, medium-shaded bar, 85%; D, light-shaded bar, 120%), then transferred to 100% seawater where they were shocked. (E–G) Animals were shocked in one of the three altered seawaters (E, dark-shaded bar, pH 7.0; F, medium-shaded bar, 85%; G, light-shaded bar, 120%). After this treatment, animals were transferred to a new chamber of 100% seawater. Data are shown separately for respiratory pumping (means and s.e.s of the number of pumps in each 10-min period) and swimming (percent of the animals tested that responded with this behavior) during the 10 min before the training, during the training in which they were shocked, and during the first and last 10 min of the hour in 100% seawater after the training. For each of the seven experimental treatments shown, respiratory pumping during the first and last 10 min in 100% seawater was compared with respiratory pumping before the training procedure. All seven comparisons were significant for the first 10 min in altered seawater [for shock alone, t(32) = 8.88; for pH 7.0 unpaired, t(24) = 5.14; for pH 7.0 paired, t(28) = 6.09; for 85% unpaired, t(14) = 3.29; for 85% paired, t(14) = 6.53; for 120% unpaired, t(32) = 6.55; for 120% paired, t(32) = 6.04; for all seven tests, P < 0.001; two-tailed t-tests], whereas none of the seven comparisons was significant during the last 10 min in altered seawater (P ⩾ 0.10, t ≤ 1.7). For each of the seven treatments, a χ² test was used to determine whether swimming was increased over baseline values during either the first or the last 10 min in 100% seawater. All seven treatments led to a significant increase in swimming during the first 10 min (for shock alone, χ² = 28.22; for pH 7.0 unpaired, χ² = 14.44; for pH 70 paired, χ² = 4.05; for 85% unpaired, χ² = 8.90; for 85% paired, χ² = 8.76; for 120% unpaired, χ² = 37.81; for 120% paired, χ² = 58.22; for all seven tests, P < 0.05; χ² tests with 1 df) but no significant increase during the last 10 min (P ⩾ 0.31, χ² ≤ 1.02).

Figure 5

Respiratory pumping and swimming are differentially affected in open (shaded bars) and closed (open bars) spaces. Animals were stimulated with one of four noxious stimuli: shock, pH 6.0 seawater, 140% seawater, or ink collected from another animal. The number of respiratory pumps in response to the stimulus, as well as the time spent swimming, were then measured over a 10-min period. These measurements were made in a large, open space, as well as in a small cage that was immersed within the open space. There was no significant difference in respiratory pumping between animals in open and closed spaces when the animals were shocked [P = 0.19, t(10) = 0.92] or were exposed to pH 6.0 seawater [P = 0.2, t(12) = 0.86]. However, there was significantly more respiratory pumping in the closed space in animals that were stimulated with 140% seawater [P = 0.001, t(14) = 3.77] or with ink [P = 0.04, t(10) = 1.97]. For swimming, there was no significant difference between open and closed spaces in animals that had been shocked [P = 0.40, t(10) = 0.87], but swimming was significantly increased in open spaces in response to pH 6.0 seawater [P = 0.04, t(12) = 1.92], 140% seawater [P = 0.001, t(14) = 3.77] and ink [P = 0.03, t(10) = 2.24; all comparisons are one-tailed t-tests]. These data show that environmental conditions, such as whether animals are in closed or open areas, can differentially affect the likelihood of animals to respond to a stimulus by swimming or by respiratory pumping.