ITI-Signals and Prelimbic Cortex Facilitate Avoidance Acquisition and Reduce Avoidance Latencies, Respectively, in Male WKY Rats

Abstract

As a model of anxiety disorder vulnerability, male Wistar-Kyoto (WKY) rats acquire lever-press avoidance behavior more readily than outbred Sprague-Dawley rats, and their acquisition is enhanced by the presence of a discrete signal presented during the inter-trial intervals (ITIs), suggesting that it is perceived as a safety signal. A series of experiments were conducted to determine if this is the case. Additional experiments investigated if the avoidance facilitation relies upon processing through medial prefrontal cortex (mPFC). The results suggest that the ITI-signal facilitates acquisition during the early stages of the avoidance acquisition process, when the rats are initially acquiring escape behavior and then transitioning to avoidance behavior. Post-avoidance introduction of the visual ITI-signal into other associative learning tasks failed to confirm that the visual stimulus had acquired the properties of a conditioned inhibitor. Shortening the signal from the entirety of the 3 min ITI to only the first 5 s of the 3 min ITI slowed acquisition during the first four sessions, suggesting the flashing light (FL) is not functioning as a feedback signal. The prelimbic (PL) cortex showed greater activation during the period of training when the transition from escape responding to avoidance responding occurs. Only combined PL + infralimbic cortex lesions modestly slowed avoidance acquisition, but PL-cortex lesions slowed avoidance response latencies. Thus, the FL ITI-signal is not likely perceived as a safety signal nor is it serving as a feedback signal. The functional role of the PL-cortex appears to be to increase the drive toward responding to the threat of the warning signal. Hence, avoidance susceptibility displayed by male WKY rats may be driven, in part, both by external stimuli (ITI signal) as well as by enhanced threat recognition to the warning signal via the PL cortex.

Keywords: anxiety vulnerability; conditioned inhibitor; infralimbic cortex; lever-press avoidance; prelimbic cortex; safety signals.

Figure 1

Lever-press avoidance behavior training occurred over 12 sessions. At the mid-point of training, following session 6, half of the subjects had their flashing light (FL) ITI-signal status switched. Shown in (A) are the mean avoidance responses per condition per session. The facilitation of lever-press avoidance learning by the presence of a FL ITI-signal is evident through session 6, with significant differences between the two initial conditions denoted by an asterisk (*). In the latter half of training, there were no significant differences between the groups, regardless of the presence or absence of the FL during the 3 min ITIs. Shown in (B,C) are the percentage of subjects avoiding on each trial through the first (sessions 1–3) and second (sessions 4–6) session blocks, respectively. (D) (Sessions 7–9) and (E) (sessions 10–12) show the percentage of subjects avoiding after half of each group had an ITI-signal switch. This within-session analysis demonstrates that the presence/absence of the FL during the ITIs does not affect the between-session retention of the learning, as much as the within-session acquisition process.

Figure 2

Male WKY rats, previously trained in lever-press avoidance, were conditioned to reflexively blink to a white-noise conditional stimulus (CS), which was paired with unconditional eyelid muscle stimulation (US). Prior to the CS on trials where the US was to be presented, either a 5 Hz flashing light (FL) or a 1 kHz tone was presented as an occasion setter for the US. Those rats trained with the FL during avoidance, then subsequently trained to emit conditioned eyeblink responses with a FL occasion setter during eyeblink conditioning (denoted in red), acquired conditional eyeblink responses slower than those with an occasion-setter FL that previously were trained in avoidance but did not have a FL during the avoidance ITIs (i.e., novel FL). Rats trained with an occasion setter that approximated the warning tone from the previous avoidance learning were also slower to acquire the response compared to the novel FL occasion-setter group. Significant differences between groups were found across trial block (collapsed over day), see insert. An asterisk (*) represents a significant difference between the two conditions with a FL occasion setter. A cross (†) represents a significant difference between the no-occasion-setter control group and the avoidance ITI-signal/FL occasion-setter group. A double cross (‡) represents a significant difference between the no-occasion-setter control group and the tone occasion-setter group (p < 0.05 Fishers LSD).

Figure 3

Male WKY rats were pretested for startle reactivity prior to avoidance training. Of the 24 startle trials, 8 were preceded by a 5 s 1 kHz tone, 8 were preceded by a 5 Hz FL, 8 were preceded by the combination of both the tone and FL, and 8 were not preceded by any stimuli. The same startle test occurred within 2 days following the end of lever-press avoidance training. The elicited startle responses were lower during the post-avoidance test, regardless of begin trained with or without a FL ITI signal. However, exposure to the 1 kHz tone reduced startle magnitudes approximately 50–60%. The FL did not appear to influence the magnitude of the elicited startle response.

Figure 4

Male WKY rats were randomly assigned to be trained in lever-press avoidance with no ITI-signal or a 5 s flashing light (FL) ITI-signal (in both cases, the total ITI was 3 min). Those assigned to the 5 s FL condition expressed significantly less avoidance responses over the first four sessions of acquisition. An asterisk (*) represents a significant difference between groups for a particular session (p < 0.05, Fishers LSD).

Figure 5

Brains were extracted from subjects, trained in the lever-press avoidance protocol with either the FL ITI-signal or no explicit ITI-signal (nFL), following a randomly assigned number of training sessions. Shown in (A) is the percentage of avoidance responses emitted from the rats on the day they were sacrificed. (B–D) provide the density of c-Fos labeling in the anterior cingulate (AC) cortex, prelimbic (PL) cortex, and infralimbic (IL) cortex, respectively, for those subjects depicted in (A). Behaviorally, the groups with a flashing light (FL) ITI-signal emitted more lever-press avoidance responses during sessions 1(†) and 4 (*) than the groups without an ITI-signal. Neurochemically, only the PL cortex exhibited significant differences between groups. Overall, the density of c-Fos labeling was significantly different between sessions 2 and 4, regardless of the ITI signal (‡).

Figure 6

Male WKY rats were randomly assigned to have bilateral excitotoxic lesions to the PL cortex, IL cortex, or combined PL + IL cortex [see (A)], followed by lever-press avoidance training, beginning at least 10 days later. As shown in (B), the lesion groups did not significantly differ from the sham-lesion group when assessed across sessions (until the PL + IL group was specifically compared to the Sham group). As shown in (C), within-session acquisition of the response did not suggest the lesion altered the characteristic phenotype of WKY rat lever-press avoidance, that is the absence of a warm-up effect (the seemingly reacquisition of the response beginning at a performance level lower than what the rats had attained in the previous session). Even the PL + IL lesion group, which exhibited the slowest avoidance rates, did not exhibit a warm-up effect. Thus, these lesions did not reinstate a typical warm-up pattern of responding.

Figure 7

The change in avoidance response latency was affected by the lesion in male WKY rats. As denoted by the asterisk (*), the PL-cortex lesion group has slower avoidance latencies beginning with session 4, the session where equal or more avoidance responses are typically emitted (Fisher LSD, p < 0.05). Thus, the shorter latencies prior to session 4 are due to much fewer avoidance responses being represented in the calculation, then the more representative mean values for the latencies are longer through session 8. The cross (†) represents the combined PL + IL lesion group being significantly different from the Sham-lesion condition during session 4 only.