Electrophysiological characteristics of feeding-related neurons after taste avoidance Pavlovian conditioning in Lymnaea stagnalis

Abstract

Taste avoidance conditioning (TAC) was carried out on the pond snail, Lymnaea stagnalis. The conditional stimulus (CS) was sucrose which elicits feeding behavior; while the unconditional stimulus (US) was a tactile stimulus to the head which causes feeding to be suppressed. The neuronal circuit that drives feeding behavior in Lymnaea is well worked out. We therefore compared the physiological characteristics on 3 classes of neurons involved with feeding behavior especially in response to the CS in conditioned vs. control snails. The cerebral giant cell (CGC) modulates feeding behavior, N1 medial neuron (N1M) is one of the central pattern generator neurons that organizes feeding behavior, while B3 is a motor neuron active during the rasp phase of feeding. We found the resting membrane potential in CGC was hyperpolarized significantly in conditioned snails but impulse activity remained the same between conditioned vs. control snails. There was, however, a significant increase in spontaneous activity and a significant depolarization of N1M's resting membrane potential in conditioned snails. These changes in N1M activity as a result of training are thought to be due to withdrawal interneuron RPeD11 altering the activity of the CGCs. Finally, in B3 there was: 1) a significant decrease in the amplitude and the frequency of the post-synaptic potentials; 2) a significant hyperpolarization of resting membrane potential in conditioned snails; and 3) a disappearance of bursting activity typically initiated by the CS. These neuronal modifications are consistent with the behavioral phenotype elicited by the CS following conditioning.

Keywords: Lymnaea; central pattern generator; modulatory neuron; motor neuron; taste avoidance conditioning.

Figure 1

Time schedule of Taste Aversion Conditioning. Snails were acclimatized for 10 min then the feeding response to CS was recorded as the pre-conditioning test. Ten minutes later, they received 20 CS-US pairings. Each snail was exposed to the CS followed 5 seconds later by the US. A one minute inter-trial interval was imposed between pairings of the CS-US. A “ten min post-conditioning test” was carried out following the 20 paired CS-US presentations. The CS was again presented to the snails. A “24 h post-conditioning test” was again performed at 24 h later. Electro-physiological recordings were made from semi-intact preparations approximately 1h following the memory test.

Figure 2

Semi-intact preparation used in this study A). The semi-intact preparation consisting of the central ring ganglia along with the buccal ganglia was removed from the snail. In addition the mouth and lips as well as tentacles were left intact. Vaseline dam was constructed around the CNS in order the superfusion of the CS (Sucrose) over the mouth area without coming into contact with neurons in the CNS. Schematic location of N1Ms and B3 motor neurons on the buccal ganglia (right panel). Neuronal circuit involving the feeding behavior, Sensory neurons (SNs), Modulatory neurons, Central Pattern Generator neurons, and Motor neurons B). CGC, N1M and B3 are one of Modulatory neurons, CPG neurons and Motor neurons, respectively. CGC and N1M and B7 are involved in protraction phase (P); SO, N2 and B3 are involved in rasp phase (R); CV1, N3 and B4 are involved in swallow phase (S). Note that in addition to the original scheme by Benjamin et al. RPeD11 has direct inhibitory connection with CGC as shown in Figure 8. Open circles are represented excitatory synapses and closed circles are represented inhibitory synapses. The circuit was modified from Figure 1 of the Benjamin’s review paper (Benjamin et al., 2000).

Figure 3

A scatter plot of feeding scores of TAC conditioned (n=26) and control snails (n=18) are shown. Scores were obtained from pre-test and post-test at 10 min or 24 h after conditioning procedure. Snails were conditioned with 20 paired presentations of sucrose (CS) first then a tactile stimulus to the head (US) or vice versa as forward or backward conditioning, respectively. Naïve animals (n=12) were placed in the experimental chamber for the same period of conditioning. In the CS-US conditioning group (left panel) open circles represented scores obtained from good-performers (n=8), while open squares, open diamond represented poor-performer (n=9) and animals without acquisition of learning (no memory) (n=9), respectively. Control (right panel) was included snails of 12 naïve (+) and 6 backward conditioning (•) with US-CS pairs. Horizontal lines denoted each average scores (Conditioned group: line- good performer; dash- poor performer; dash- no memory| Control group: line-backward; dot-naïve). Note that the behavioral scores of animal without memory, backward conditioner and naïve were statistically identical. Statistical test was performed between the score of pre-test and that of post-test (10 min and 24 h). Even poor-performers showed significant *p<0.05, ⁺p<0.0001, NS: not significant

Figure 4

Representative spontaneous activities of CGC, and B3 were displayed in naïve A) and good performer preparations B). CGC and B3 were recorded simultaneously. Notice that B3 had spontaneous EPSP activities in naïve preparations while they were not relatively less active in the good performer preparation. The CGC activity was not different in good performer vs. naïve preparations though after-hyperpolarization in naïve became smaller. Calibration bars represented 20 mV and 20 second.

Figure 5

Representative spontaneous activities of N1M, and B3 were displayed in naïve A) and good performer preparations B). Each response was recorded simultaneously. Note that spontaneous impulse generation in N1M after conditioning was obviously more frequent (naïve vs. good performers; 3.3±1.1 Hz vs. 16.3±3.5 Hz) while spontaneous EPSPs in the good performer B3 became less frequent and the amplitude became significantly smaller. Calibration bars represented 20 mV and 20 s.

Figure 6

The amplitude of spontaneous EPSPs in naive preparations was significantly larger than that of the good performer preparations in B3 motoneuron. In addition to the larger amplitude, the EPSP frequency was also higher in naïve preparations.

Figure 7

Simultaneous recordings from N1M and B3 in response to CS presentation from a naïve A) and the good performer preparation B). The dotted line in each record represented the timing to start perfusion of 1 ml of 100 mM sucrose. In the naïve preparation, the sucrose application induced rhythmic fictive feeding activity in N1M and B3 in push-pull manner. However, such rhythmic fictive feeding activities did not induce by the presentation of CS in the good performers preparation.

Figure 8

Simultaneous recordings from RPeD11 and CGC. Depolarizing current injection into RPeD11 caused an inhibitory post-synaptic potential in CGC of approximately 5 mV in amplitude. This inhibitory input was sufficient to inhibit the spontaneous firing of the CGC.