Classical eyeblink conditioning using electrical stimulation of caudal mPFC as conditioned stimulus is dependent on cerebellar interpositus nucleus in guinea pigs

Abstract

Aim: To determine whether electrical stimulation of caudal medial prefrontal cortex (mPFC) as conditioned stimulus (CS) paired with airpuff unconditioned stimulus (US) was sufficient for establishing eyeblink conditioning in guinea pigs, and whether it was dependent on cerebellar interpositus nucleus.

Methods: Thirty adult guinea pigs were divided into 3 conditioned groups, and trained on the delay eyeblink conditioning, short-trace eyeblink conditioning, and long-trace eyeblink conditioning paradigms, respectively, in which electrical stimulation of the right caudal mPFC was used as CS and paired with corneal airpuff US. A pseudo conditioned group of another 10 adult guinea pigs was given unpaired caudal mPFC electrical stimulation and the US. Muscimol (1 μg in 1 μL saline) and saline (1 μL) were infused into the cerebellar interpositus nucleus of the animals through the infusion cannula on d 11 and 12, respectively.

Results: The 3 eyeblink conditioning paradigms have been successfully established in guinea pigs. The animals acquired the delay and short-trace conditioned responses more rapidly than long-trace conditioned responses. Muscimol infusion into the cerebellar interpositus nucleus markedly impaired the expression of the 3 eyeblink conditioned responses.

Conclusion: Electrical stimulation of caudal mPFC is effective CS for establishing eyeblink conditioning in guinea pigs, and it is dependent on the cerebellar interpositus nucleus.

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. One electrode was implanted in the right caudal medial prefrontal cortex (mPFC) and one infusion guiding cannula was implanted in left cerebellar interpositus nucleus. Electrical stimulation of right caudal mPFC was used as a conditioned stimulus (CS), and airpuff was presented to the left cornea as an unconditioned stimulus (US). (B) Diagram of the sagittal section of guinea pig brain, showing the stimulating and infusion sites. (C) Schematic diagram showing the delay, short-trace, and long-trace eyeblink conditioned response (CR) paradigms used in the present study. Note that the CS, US, and total trial lengths were equal in each conditioning. Short and long-trace CR were different in trace interval (TI) length.

Figure 2

Histological reconstructions of the electrode and infusion cannula tips. Schematic illustration of the locations of all electrode tips (A) and all infusion cannula tips (B) for the delay (▪ n=9), short-trace (• n=10), long-trace (▴ n=8), and pseudo conditioned (▾ n=10) groups, respectively. Note that one infusion cannula tip of the delay conditioned group and one infusion cannula tip of the short-trace conditioned group were not in or near the left cerebellar interpositus nucleus. 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

Acquisition of the eyeblink conditioned responses (CR) for delay (n=8), short-trace (n=9), long-trace (n=8), and pseudo (n=10) conditioned groups given training with a right caudal mPFC conditioned stimulus (CS) across 10 acquisition training sessions. (A) CR percentage, (B) CR peak amplitude, and (C) CR relative peak latency are given as mean±standard error (SEM). Error bars represent the SEM.

Figure 4

Delay conditioned group data for the effects of muscimol (▴) and saline (•) infused into the left cerebellar interpositus nucleus. (A) Infusion of muscimol abolished the responses almost completely as illustrated by its effects on the percentage of trials in which the delay CRs were seen, whereas infusion of saline had no significant effect on the delay CRs. (B) Muscimol infusion significantly decreased the peak amplitude of the delay CRs. (C) Eyelid position of an animal after muscimol and saline infusion in the sixth trial. Upper panel: the conditioning paradigm illustrating the timing of the CS and the US. Middle panel: eyelid position after muscimol infusion. Lower panel: eyelid postion after saline infusion. All data are from the same animals. Mean±SEM. n=8. ^bP<0.05 vs control.

Figure 5

Short-trace conditioned group data for the effects of muscimol (▴), and saline (•) infused into the left cerebellar interpositus nucleus. (A) Infusion of muscimol abolished the responses almost completely as illustrated by its effects on the percentage of trials in which the short-trace CRs are seen, whereas infusion of saline had no significant effect on the short-trace CRs. (B) Muscimol infusion significantly decreased the peak amplitude of the short-trace CRs. (C) Eyelid position of an animal after muscimol and saline infusion in the sixth trial. Upper panel: the conditioning paradigm illustrating the timing of the CS and the US. Middle panel: eyelid position after muscimol infusion. Lower panel: eyelid postion after saline infusion. All data are from the same animals. Mean±SEM. n=9. ^bP<0.05 vs control.

Figure 6

Long-trace conditioned group data for the effects of muscimol (▴) and saline (•) infused into the left cerebellar interpositus nucleus. (A) Infusion of muscimol abolished the responses almost completely as illustrated by its effects on the percentage of trials in which the long-trace CRs are seen, whereas infusion of saline had no significant effect on the long-trace CRs. (B) Muscimol infusion had significant effect on peak amplitude of the long-trace CRs. (C) Eyelid position of an animal after muscimol and saline infusion in the sixth trial. Upper panel: the conditioning paradigm illustrating the timing of the CS and the US. Middle panel: eyelid position after muscimol infusion. Lower panel: eyelid postion after saline infusion. All data are from the same animals. Mean±SEM. n=8. ^bP<0.05 vs control.