An antisense oligonucleotide reverses the footshock-induced expression of fos in the rat medial prefrontal cortex and the subsequent expression of conditioned fear-induced immobility

Abstract

The immediate-early genes, including c-fos, have been proposed to be involved in learning and memory. In this report, we examine stress-induced Fos-like immunoreactivity (Fos-li) in subregions of the prefrontal cortex during a conditioned fear paradigm. During the acquisition phase, the rats were conditioned to fear a formerly neutral tone by pairing the tone with a mild footshock. The rats were then tested for fearful behavior by reexposure to the tone without additional footshock. During acquisition, Fos-li was increased in the medial prefrontal cortex (infralimbic and prelimbic) but not the anterior cingulate and M1 motor cortex. However, during the extinction phase, no significant increase in Fos-li was observed in any region. These findings indicate that acquisition, but not extinction, of conditioned fear is associated with an increase in Fos-li in subregions of the medial prefrontal cortex. In other animals, an antisense oligonucleotide directed against the c-fos mRNA was injected into the infralimbic/prelimbic cortex 12 or 72 hr before the acquisition session. Antisense treatment given 12, but not 72, hr earlier suppressed Fos production without altering behavior during the acquisition session. Three days after the acquisition session, rats were tested for fearful behavior as before. The antisense oligonucleotide blockade of Fos production during acquisition was associated with a significantly less fearful response during the extinction session. These results support a role for Fos in the medial prefrontal cortex during the acquisition of aversive learning.

Fig. 1.

Representation of subregions of the medial prefrontal cortex and the approximate site of the photomicrograph taken. The drawing is from Paxinos and Watson (1997) and represents 2.7 mm anterior to bregma.

Fig. 2.

Immobility in rats during the acquisition and expression phases of the conditioned fear paradigm. After 3 d of habituation to the testing chamber, rats were subjected to a 2 d paradigm. On day 1 (top), rats were placed into a footshock apparatus and randomly received 10 5 sec tones paired with 0.5 sec footshocks over a 30 min period. Control rats received no footshocks. Some controls and footshocked rats were killed after the day 1 session for biochemical analysis. On day 2 (bottom), the remaining rats were returned to the chamber and exposed to 10 5 sec tones without footshock over a 30 min period. Seconds spent immobile during 1 min intervals starting with each of the 10 tones was measured. *p < 0.05 versus control.

Fig. 3.

Photomicrographs of the prelimbic region from representative control, acquisition (footshock), and expression animals. Rats previously adapted to the testing chamber underwent a 2 d test session. On day 1, rats were placed into the chamber for 30 min and received randomized 10 tones alone (Control) or paired with footshock (Acquisition). After this first session, all rats were returned to the home cages, and, after 90 min, rats in the acquisition group and some control rats were killed, and the brain tissues were prepared to visualize Fos-li. On the second day, the remaining rats were returned to the test chamber and exposed to 10 tones without footshock. Rats previously exposed to footshock on day 1 made up the conditioned fear group (Extinction). Ninety minutes after the end of the session, these rats were killed, and Fos-li was visualized in brain tissues. Scale bar, 100 μm.

Fig. 4.

The density of Fos-li-positive nuclei in the subregions of the mPFC of rats undergoing the acquisition (day 1) or expression (day 2) of conditioned fear. Fos-li immunocytochemistry was performed on the brains of rats subjected to the paradigm described in the legend of Figure 2. *p < 0.05 versus the control group; ❖p < 0.05 versus the extinction group of the same region.

Fig. 5.

The location of the tip of the injection cannula in rats treated with sense or antisense oligonucleotides in the prelimbic/infralimbic subregion of the medial prefrontal cortex. Three groups of animals are represented here. A, Rats treated 12 hr before the acquisition session and killed for immunocytochemistry. B, Rats treated 72 hr before the acquisition session and killed for immunocytochemistry.C, Rats treated 12 hr before acquisition and tested for expression 72 hr later. The brain drawings are from Paxinos and Watson (1997), and the numbers represent the location of the section in millimeters anterior to bregma.

Fig. 6.

A photomicrograph of a representative injection site from rats injected with either sense oligonucleotide (SO) and ASO. Rats were treated with the oligonucleotides (5 nmol in 1 μl) in PBS 12 hr before undergoing the acquisition session (Fig. 2) and killed 90 min after the end of the 30 min session. Brains were prepared for visualization of Fos-li. Scale bar, 100 μm.

Fig. 7.

The density of Fos-li positive nuclei in the square millimeter area surrounding (Middle), above, or below the injection site of PBS, sense oligonucleotide (SO), or ASO. Brain sections from treat rats that underwent the acquisition session of fear conditioning were visualized for Fos-li and drawn on a camera lucida system. The number of Fos-li nuclei were counted in each square millimeter area. *p < 0.05 versus the PBS control group within the same area.

Fig. 8.

The lack of an effect of antisense oligonucleotide treatment, given either 12 or 72 hr before, on behavior during the acquisition session. Rats were treated with sense oligonucleotides (SO), ASO, or vehicle and, after a 12 or 72 hr delay, subjected to the acquisition session of the conditioned fear protocol (Fig. 2). The behavior of the sense oligonucleotide-treated and vehicle-treated controls were not different and were combined into a single control group for graphic purposes only. Immobility was measured for each tone plus footshock pairing over a 1 min interval. There was no difference between the sense oligonucleotide- or ASO-treated groups at either 12 or 72 hr.

Fig. 9.

The effect of antisense oligonucleotide blockade of acquisition-induced Fos-li on the later expression of conditioned fear. Rats were treated with either sense (SO) or antisense (ASO) oligonucleotides and, after 12 hr, subjected to the acquisition session of conditioned fear (Fig. 2). Three days later, ∼84 hr after oligonucleotide treatment, rats underwent the expression session of the conditioned fear protocol. ASO-treated rats showed significantly less fear-induced immobility than the sense oligonucleotide-treated controls at every time point tested.