Establishment and transfer of classical eyeblink conditioning using electrical microstimulation of the hippocampus as the conditioned stimulus

Abstract

The present experiment was designed to determine whether classical eyeblink conditioning (EBC) can be established by using electrical microstimulation of the hippocampus as a conditioned stimulus (CS) paired with an air-puff unconditioned stimulus (US). We intended to examine whether EBC transfer could occur when a CS was shifted between microstimulation of the hippocampus as a CS (Hip-CS) and tone as a CS (tone-CS) and to compare the difference in transfer effectiveness between delay EBC (dEBC) and trace EBC (tEBC). Eight groups of guinea pigs, including 4 experimental groups and 4 control groups, were included in the study. First, the experimental groups received either a Hip-CS or a tone-CS paired with a US; then, these groups were exposed to a shifted CS (tone-CS or Hip-CS) paired with the US. The control groups received the corresponding Hip-CS or tone-CS, which was, however, pseudo-paired with the US. The control groups were then shifted to the tone-CS (or Hip-CS) paired with the US. The results show that EBC can be successfully established when using microstimulation of the hippocampus as a CS paired with an air-puff US, and that the acquisition rates of EBC are higher in the experimental groups than in the control groups after switching from the Hip-CS to the tone-CS or vice versa, indicating the occurrence of learning transfer between EBC established with the Hip-CS and tone-CS. The present study also demonstrated that the EBC re-acquisition rates were remarkably higher in dEBC than in tEBC with both types of transfer, which suggests that the saving effect was more evident in dEBC than tEBC. These results significantly expand our knowledge of EBC transfer as well as the functional neural circuit underlying EBC transfer.

Fig 1. Experimental design.

(A) Diagram of EBC measurement. The upper left eyelid movements were measured by a high-resolution spring-return potentiometer that was attached via a thread lead hooked with a nylon loop, which was sutured into the left upper eyelid. Bipolar electrodes were implanted in the right hippocampus (Hip). Electrical stimulation of right hippocampus (Hip-CS) or auditory stimulation (pure tone) was used as the conditioned stimulus (CS). Air-puff presented to the left cornea was used as the unconditioned stimulus (US). (B) Diagram of the sagittal section of a guinea pig brain, showing the electrical stimulation sites. (C, D) Schematic diagrams illustrate the temporal relationship between CS and US and analysis periods of CR and UR in delay (C) and trace (D) paradigms. In delay paradigm, US co-terminated with the offset of CS; and in trace paradigm, a stimulus-free trace interval of 250 ms was interposed between the CS offset and the US onset. The durations of CS and US were 350 ms and 100 ms, respectively. In each trial, parameters of the conditioned eyeblink response (CR; 50–250 ms period after the CS onset) and unconditioned eyeblink response (UR; 0–300 ms period after the US onset) were analyzed. These responses were defined based on the average magnitude of the baseline (a 0~800 ms period prior to the onset of the CS).

Fig 2. Locations of the electrode tips in the hippocampus of guinea pigs.

(A) A representative of toluidine blue-stained coronal hippocampus section (30 μm) from a guinea pig that received hippocampal electrical stimulation as the CS. Scale bar represents 1.0 mm; (B, C) Schematic illustration of the locations of all electrode tips. (B) For delay paradigm: (□ group of from Hip-CS (paired) to tone-CS (paired), n = 6; ■ group of from Hip-CS (pseudo-paired) to tone-CS (paired), n = 4; ○ group of from tone-CS (paired) to Hip-CS (paired), n = 7; ● group of from tone-CS (pseudo-paired) to Hip-CS (paired), n = 4); (C) For trace paradigm: (△ group of from Hip-CS (paired) to tone-CS (paired), n = 6; ▲ group of from Hip-CS (pseudo-paired) to tone-CS (paired), n = 4; ◇ group of from tone-CS (paired) to Hip-CS (paired), n = 5; ◆ group of from tone-CS (pseudo-paired) to Hip-CS (paired), n = 4). The coronal brain plates are adapted from the atlas of Rapisarda and Bacchelli [39].

Fig 3. Acquisition curves of eyelid conditioned responses in delay and trace paradigms when CS shifted from central (Hip–CS) to peripheral (tone-CS) or vice versa.

(A, B) Learning curves of dEBC (A) and tEBC (B) for groups of experiment (square, n = 6, for both dEBC and tEBC) and control (roundness, n = 4, for both dEBC and tEBC) when CS shifted from central to peripheral. Central CS (black in A, B) was presented during first 6 (dEBC) or 12 (tEBC) sessions in stage I and paired (black square in A and B) or pseudo-paired (black roundness in A, B) with US, then CS was switched to peripheral and paired with US (space square and space roundness, A and B) in sessions 7–12 (dEBC) or 13–18 (tEBC) of stage II. (C, D) Learning curves of dEBC (C) and tEBC (D) for groups of experiment (square, n = 7, for dEBC; n = 5, for tEBC) and control (roundness, n = 4, for both dEBC and tEBC) when CS shifted from peripheral to central (C, D). Central CS (space in C, D) was presented during first 6 (dEBC) or 12 (tEBC) sessions in stage I and paired (space square in C and D) or pseudo-paired (space roundness in C, D) with US, then CS was switched to central and paired with US (black square and black roundness, C, D) in sessions 7–12 (dEBC) or 13–18 (tEBC) of stage II. Data represent mean ± SEM. A two-way repeated measures ANOVA followed by the LSD post hoc test showed that there were significant differences in the percentages of the conditioned responses (CR) between groups of experiment and control in stage II in both delay and trace paradigms, either shifting CS from central to peripheral or vice versa. [Fig 3A and 3F (1, 8) = 40.028, *p < 0.05; Fig 3B and 3F (1, 8) = 8.905, *p < 0.05; Fig 3C and 3F (1, 9) = 154.691, *p < 0.05; Fig 3D and 3F (1, 7) = 16.299, *p < 0.05]. In recognition tests of stage III in the above 4 conditions, animals were all able to recall the original CR% to CS1.

Fig 4. Comparisons of pre-shift CR acquisition rates between dEBC and tEBC, and between with Hip-CS and with tone-CS.

Four curves depicting pre-shift CR acquisition rates in Fig 3 were rearranged and illustrated. Data represent mean ± SEM. (A, B), comparison of CR acquisition between paradigms of dEBC and tEBC. dEBC establishment (black square) showed higher acquisition rate than tEBC (space square), for both learning with Hip-CS (A, n = 6, for both dEBC and tEBC) and tone-CS (B, n = 7, for dEBC; n = 5, for tEBC), confirmed by statistically significant main effects of group [Fig 4A and 4F(1, 10) = 439.401, *p < 0.05; Fig 4B and 4F(1, 10) = 57.1, *p < 0.05], a two-way repeated measures ANOVA, followed by the LSD post hoc test; Only 6 sessions of data from trace paradigm are displayed (Fig 4A and 4B) to equal the time course with delay paradigm. (Fig 4C and 4D), comparison of CR acquisition between with Hip-CS and with tone-CS, across 6 or 12 training sessions (n = 6, for Hip-CS/dEBC; n = 7, for tone-CS/dEBC; n = 6, for Hip-CS/tEBC; n = 5, for tone-CS/tEBC). dEBC establishment showed higher acquisition rates when cued with Hip-CS (black square) than with tone-CS (space square), but tEBC establishment showed lower acquisition rates when cued with Hip-CS (black square) than with tone-CS (space square), confirmed by statistically significant main effects of group [Fig 4C and 4F(1, 11) = 5.635, *p < 0.05; Fig 4D and 4F(1, 9) = 70.117, *p < 0.05], a two-way repeated measures ANOVA, followed by the LSD post hoc test.

Fig 5. Comparisons of post-shift CR acquisition rates between dEBC and tEBC, and between with Hip-CS and with tone-CS.

Four curves depicting post-shift CR acquisition rates in Fig 3 were rearranged and illustrated (for comparison, pre-shift CR acquisition rate in the last day of stage I was also demonstrated). Data represent mean ± SEM. (A, B) comparison of CR acquisition between paradigms of dEBC and tEBC (n = 6, for tone-CS/dEBC; n = 6, for tone-CS/tEBC; n = 7, for Hip-CS/dEBC; n = 5, for Hip-CS/tEBC). For post-shift learning with both tone-CS (A) and Hip-CS (B), establishment of dEBC (square, space or black) showed higher acquisition rates than of tEBC (roundness, space or black), confirmed by statistically significant main effects of group [Fig 5A and 5F(1, 10) = 53.918, *p < 0.05; Fig 5B and 5F(1, 10) = 92.772, *p < 0.05], a two-way repeated measures ANOVA, followed by the LSD post hoc test. (C, D) comparison of CR acquisition between with Hip-CS and with tone-CS (n = 6, for tone-CS/dEBC; n = 7, for hip-CS/dEBC; n = 6, for tone-CS/tEBC; n = 5, for Hip-CS/tEBC). Post-shift learning with Hip-CS (black square) showed significant difference relative to with tone-CS (space roundness) for establishment of dEBC (Fig 5C and 5F(1, 11) = 26.796, *p < 0.05), but not of tEBC (Fig 5D and 5F(1, 9) = 0.113, p = 0.745), confirmed by a two-way repeated measures ANOVA, followed by the LSD post hoc test.