Cholinergic modulation of Pavlovian fear conditioning in rats: differential effects of intrahippocampal infusion of mecamylamine and methyllycaconitine

Abstract

The cholinergic system has consistently been implicated in Pavlovian fear conditioning. Considerable work has been done to localize specific nicotinic receptor subtypes in the hippocampus and determine their functional importance; however, the specific function of many of these subtypes has yet to be determined. An alpha7 nicotinic antagonist methyllycaconitine (MLA) (35 microg), and a broad spectrum non-alpha7 nicotinic antagonist mecamylamine (35 microg) was injected directly into the dorsal hippocampus or overlying cortex either 15 min pre-, 1 min post-, or 6h post-fear conditioning. One week after conditioning, retention of contextual and cue (tone) conditioning were assessed. A significant impairment in retention of contextual fear was observed when mecamylamine was injected 15 min pre- and 1 min post-conditioning. No significant impairment was observed when mecamylamine was injected 6h post-conditioning. Likewise, a significant impairment in retention of contextual fear was observed when MLA was injected 1 min post-conditioning; however, in contrast, MLA did not show any significant impairments when injected 15 min pre-conditioning, but did show a significant impairment when injected 6h post-conditioning. There were no significant impairments observed when either drug was injected into overlying cortex. No significant impairments were observed in cue conditioning for either drug. In general, specific temporal dynamics involved in nicotinic receptor function were found relative to time of receptor dysfunction. The results indicate that the greatest deficits in long-term retention (1 week) of contextual fear are produced by central infusion of MLA minutes to hours post-conditioning or mecamylamine within minutes of conditioning.

Figure 1

Histological verification of cannulae placement (n=76). a. A transverse brain section through the dorsal hippocampus stained with cresyl violet. The section represents the approximate site for cannulae placement for targeted dorsal hippocampus. b. A transverse brain section through the dorsal hippocampus representing approximate site for cannulae placement in targeted overlying cortex. c. Approximate spread of drug assessed by dye (Chicago blue) infusion is indicated by circular shaded area. Each closed dot/open square represents the approximate point in which the cannulae were positioned for each animal. Stereotaxic images adapted from (Paxinos, 1998).

Figure 2

General activity level. Mean number of ambulatory crossovers during a 4-min period preceding first conditioning trial.

Figure 3

Context and Tone test. a. Context test. Average freezing level (+/− SEM) of each drug type (mecamylamine, MLA, or PBS) at each time of drug administration [pre-15 min, post-1 min, and post-6 hr] collapsed across the first 8 minutes of context testing. Animals received either mecamylamine, MLA, or PBS 15 minutes before conditioning, 1 minute or 6 hours post-conditioning; one week post-conditioning, they were placed back in the original context. Freezing was scored for 16 minutes and reported as an average percentage value (see text for details). Animals begin to habituate to the same level of exploration after 8 minutes of testing, hence, only an average of the first 8 minutes are shown. b. Animals were placed in a novel context approximately 24 hours after the context test. b. Tone test. After a 2-minute baseline period, the original tone was played continuously for 16 minutes; freezing was scored and reported as an average percentage value during the first 8 minutes after baseline. No differences were observed between any of the groups during the baseline or during the first 8 minutes of tone. (* = p < 0.05)

Figure 4

Context and Tone test - State-dependent control. Animals administered mecamylamine (n=6) or MLA (n=6) injections 15 minutes before conditioning as well as 15 minutes before testing were compared to the groups of animals given mecamylamine (n=8) and MLA (n=8) 15 minutes before conditioning only. All animals were also compared to the PBS control. a. Context test. Freezing behavior was measured in original context for 16 minutes 1 week post-conditioning and 15 minutes after drug/control injections. Average freezing during the first 8 minutes of testing is shown. There were no significant differences between drug groups injected with drug before conditioning and drug groups injected both before conditioning and testing. Additionally, only mecamylamine significantly differed from control. b. Tone test. Approximately 24 hours after the context test and 15 min after drug/control injections and a 2-minute baseline (BL) period, the original tone was played continuously for 16 minutes; freezing behavior was scored and reported as average percent freezing during the first 8 minutes. Animals administered mecamylamine (n=4) or MLA (n=4) injections 15 minutes before conditioning as well as 15 minutes before testing were compared to the groups of animals given mecamylamine (n=8) and MLA (n=8) 15 minutes before conditioning only. All animals were also compared to the PBS control. There were no significant differences among drug groups or PBS control during the baseline or during the presentation of the tone. (* = p < 0.001)