Cholinergic septo-hippocampal innervation is required for trace eyeblink classical conditioning

Abstract

We studied the effects of a selective lesion in rats, with 192-IgG-saporin, of the cholinergic neurons located in the medial septum/diagonal band (MSDB) complex on the acquisition of classical and instrumental conditioning paradigms. The MSDB lesion induced a marked deficit in the acquisition, but not in the retrieval, of eyeblink classical conditioning using a trace paradigm. Such a deficit was task-selective, as lesioned rats were able to acquire a fixed-interval operant conditioning as controls, and was not due to nonspecific motor alterations, because spontaneous locomotion and blink reflexes were not disturbed by the MSDB lesion. The deficit in the acquisition of a trace eyeblink classical conditioning was reverted by the systemic administration of carbachol, a nonselective cholinergic muscarinic agonist, but not by lobeline, a nicotinic agonist. These results suggest a key role of muscarinic denervation on the acquisition of new motor abilities using trace classical conditioning procedures. It might also be suggested that muscarinic agents would be useful for the amelioration of some associative learning deficits observed at early stages in patients with Alzheimer's disease.

Figure 1.

Bilateral 192-IgG-saporin microinjection depleted the MSDB-complex cholinergic neuron population. Microphotographs of representative coronal sections through the MSDB complex of control and lesioned animals, immunohistochemically stained for cholineacetyltransferase (ChAT; A,B) or parvalbumin (PARV; C,D). MS, medial septum; DB, diagonal band; n = 10 rats per group.

Figure 2.

Acquisition of eyelid CRs in control and MSDB-lesioned rats. (A) A trace-conditioning paradigm was used to evaluate putative learning deficits in animals with a specific lesion of cholinergic neurons located in the MSDB complex. Learning curves of saline-injected rats (white circles, n = 10), 192-IgG-saporin-injected rats (black circles, n = 10), and pseudoconditioned animals (gray circles, n = 4). Because results obtained with 192IgG-saporin-injected rats during both pseudoconditioning and conditioning programs were similar, the pseudoconditioning curve has been omitted for the sake of clarity. (B) Selective cholinergic MSDB lesions did not affect the retrieval of previously acquired eyelid CRs. Retention index: percentage of CRs during the test session/percentage of CRs during the last conditioning session. White bar, saline-injected rats; black bar, 192-IgG-saporin-injected rats (n = 8 per group). Pseudocond, pseudoconditioning saline-injected rats; ^*, significant differences between saline- or 192IgG-saporin-injected conditioned groups with respect to pseudoconditioned saline-injected groups; +, significant difference between saline- and 192IgG-saporin-injected groups. ^*, +, P ≤ 0.05; ^**, ++, P ≤ 0.01; ^***, +++, P ≤ 0.001.

Figure 3.

Locomotor activity and blink reflex basic properties in saline- and 192-IgG-saporin-injected rats treated with different cholinergic agonists. (A) Locomotor activity, as indicated by the number of broken beams in a 10-min period, of saline- (left panel) and 192-IgG-saporin- (right panel) injected rats measured 1 h after cholinergic drug administration in selected conditioning sessions. Open circles, saline; gray circles, carbachol; black circles, lobeline. (B,C) Bar diagrams representing the mean ±SEM of the latency (B, in msec) and peak amplitude (C, in mV) of the R1 (upper row) and R2 (lower row) components of blink reflex in saline- (left column) and 192-IgG-saporin- (right column) injected animals 1 h after cholinergic drug administration during habituation and selected conditioning sessions (n = 6 per group). Systemic treatments: saline, white bars; 0.1 mg/kg carbachol, gray bars; 1 mg/kg lobeline, black bars. ^*, P ≤ 0.05; ^**, P ≤ 0.01; ^***, P ≤ 0.001. Abbreviations on the x-axis refer to (H) habituation or (C) conditioning sessions.

Figure 4.

Acquisition of operant conditioning in control and MSDB-lesioned rats. (A) Effect of selective cholinergic MSDB lesion on the last three sessions of the training to associate reinforcements with lever pressures. (B) Learning curves of saline- and 192-IgG-saporin-injected rats (n = 10 per group) during the 10 sessions of a 30-sec fixed-interval program. Continuous line, total number of pellets per session; discontinuous line, number of pellets obtained by conditioned lever pressures per session. Saline-injected rats (○), 192-IgG-saporin-injected rats (•).

Figure 5.

Learning curves of classically conditioned eyelid responses in saline- and 192-IgG-saporin-injected rats treated with different cholinergic agonists. (A) Learning curves of saline-injected rats treated with saline (○), 0.1 mg/kg carbachol (▵), or 1 mg/kg lobeline (□). (B) Learning curves of 192-IgG-saporin-injected rats treated with saline (•), 0.1 mg/kg carbachol (▴), or 1 mg/kg lobeline (▪). ^*, P ≤ 0.05; ^**, P ≤ 0.01; ^***, P ≤ 0.001 (n = 6 rats per group). Abbreviations on the x-axis refer to (H) habituation or (C) conditioning sessions.