Reevaluating the role of the hippocampus in delay eyeblink conditioning

Abstract

The role of the hippocampus in delay eyeblink conditioning (DEC) remains controversial. Here, we investigated the involvement of the hippocampus in DEC with a soft tone as the conditioned stimulus (CS) by using electrolytic lesions or muscimol inactivation of guinea pig dorsal hippocampus. Interestingly, when a soft tone was used as a CS, electrolytic lesions of the hippocampus significantly retarded acquisition of the conditioned response (CR), and muscimol infusions into hippocampus distinctly inhibited the acquisition and expression of CR, but had no significant effect on consolidation of well-learned CR. In contrast, both electrolytic lesions and muscimol inactivation of hippocampus produced no significant deficits in the CR when a loud tone was used as the CS. These results demonstrate that the hippocampus is essential for the DEC when the delay task was rendered more difficult.

Figure 1. Experimental design.

(A) The upper left eyelid movements were measured by a high-resolution spring-return potentiometer that was attached via a thread lead that was hooked through a nylon loop, which was sutured into the left upper eyelid, and the left lower eyelid was taped open. Two loudness levels of tone (i.e., 60 dB or 85 dB) were presented binaurally, as a soft or loud tone conditioned stimulus (CS), and airpuffs were presented to the ipsilateral cornea as an unconditioned stimulus (US). Moreover, electrolytic lesions or muscimol inactivation were performed in the guinea pigs’ bilateral dorsal hippocampus. Four black circles represent four lesion sites. (B) Diagram of the sagittal section of guinea pig brain, showing the lesion electrode or infusion sites. (C, D) The temporal relationship of the CS, US and analysis periods during delay eyeblink conditioning with a soft (C) and loud (D) tone CS. In each trial, we analyzed the parameters of the startle eyeblink response (SR; 0–120 ms period after the CS onset), conditioned eyeblink response (CR; 120–400 ms period after the CS onset) and unconditioned eyeblink response (UR; 0–300 ms period after the US onset). These responses were based on the average magnitude of the baseline (a 0–800 ms period prior to the onset of the CS). Examples of two typical SR, CR and UR from two records were exhibited during delay eyeblink conditioning with the soft (C) and loud (D) tone CS.

Figure 2. Histological reconstructions of the extents of electrolytic lesions and the placements of infusion cannula tips.

(A, B) Two photomicrographs of two coronal sections showing a representative extent of electrolytic lesion of the dorsal hippocampus and a representative placement of infusion cannula tip in the dorsal hippocampus, respectively. Scale bars represent 2 mm. Red arrows indicate the locations of the infusion cannula tips. (C, D) The largest (gray shaded areas) and smallest (black shaded areas) lesions of the dorsal hippocampus in the Soft-Lesion (n = 7, C) and Loud-Lesion (n = 7, D) groups. (E, F) Schematic illustration of all infusion cannula tips placements in the Soft-MAMA (n = 8), Soft-AAAA (n = 8) (E), Soft-Muscimol (n = 8), and Soft-ACSF (n = 8) groups (F). A red circle, blue circle, red square, or blue square represents a placement of infusion cannula tip in the Soft-MAMA, Soft-AAAA, Soft-Muscimol, or Soft-ACSF group, respectively. Numbers to the left represent distance (mm) from the frontal zero plane. The coronal brain plates are adapted from the atlas of Rapisarda and Bacchelli (1977).

Figure 3. Effects of electrolytic lesions of the hippocampus before conditioning training on delay eyeblink conditioning with the soft tone CS.

(A–D) The mean value ± standard error (SEM) for the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of CR with the soft tone CS in the Soft-lesion (n = 7, red) and Soft-sham (n = 8, blue) groups. (E, F) Averaged eyelid responses of all trials in the Soft-lesion (E) and Soft-sham (F) animals across twenty consecutive training sessions. Note that there were only significant differences in the percentage and peak magnitude of the CR between the Soft-lesion and Loud-Lesion groups. *p<0.05 versus control. The error bars represent the SEM.

Figure 4. Effects of the infusion of muscimol into the hippocampus 30 min before daily conditioning training during the inactivation phases (phases I and III) on delay eyeblink conditioning with the soft tone CS.

MAMA, muscimol-ACSF-muscimol-ACSF infusion; AAAA, ACSF-ACSF-ACSF-ACSF infusion. A solid red line and dash blue line represents muscimol or PBS infusion, respectively. (A–C) The mean value ± standard error (SEM) for the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of the CR with the soft tone CS in the Soft-MAMA (n = 8, green triangle) and Soft-AAAA (n = 10, green circle) groups. (E, F) Averaged eyelid responses of all trials in the Soft-MAMA (E) and Soft-AAAA (F) animals across thirty consecutive training sessions. Note that there were only significant differences in the percentage and peak magnitude of the CR between the Soft-MAMA and Soft-AAAA groups in phases I, II and III. *p<0.05 versus control. The error bars represent the SEM.

Figure 5. Effects of electrolytic lesions of the hippocampus before conditioning training on delay eyeblink conditioning with the loud tone CS.

(A–D) The mean value ± standard error (SEM) for the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of the CR with the loud tone CS in the Loud-Lesion (n = 7, red) and Loud-Sham (n = 7, blue) groups. (E, F) Averaged eyelid responses of all trials in the Loud-Lesion (E) and Loud-Sham (F) animals across six consecutive training sessions. Note that there were no significant lesion effects on the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of the CR between the Loud-Lesion and Loud-Sham groups. The error bars represent the SEM.

Figure 6. Effects of the infusion of muscimol into the hippocampus 30 min before daily conditioning training on delay eyeblink conditioning with the loud tone CS.

(A–D) The mean value ± standard error (SEM) for the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of the CR with the loud tone CS in the Soft-Muscimol (n = 8, red) and Soft-ACSF (n = 8, blue) groups. (E, F) Averaged eyelid responses of all trials in the Soft-Muscimol (E) and Soft-ACSF (F) animals across six consecutive training sessions. Note that there were no significant muscimol infusion effects on the percentage (A), peak magnitude (B), onset latency (C), and peak latency (D) of the CR between the Loud-Lesion and Loud-Sham groups. The error bars represent the SEM.