Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus

Abstract

The degree of behavioral control that an organism has over a stressor is a potent modulator of the stressor’s impact; uncontrollable stressors produce numerous outcomes that do not occur if the stressor is controllable. Research on controllability has focused on brainstem nuclei such as the dorsal raphe nucleus (DRN). Here we find that the infralimbic and prelimbic regions of the ventral medial prefrontal cortex (mPFCv) in rats detect whether a stressor is under the organism’s control. When a stressor is controllable, stress-induced activation of the DRN is inhibited by the mPFCv, and the behavioral sequelae of uncontrollable stress are blocked. This suggests a new function for the mPFCv and implies that the presence of control inhibits stress-induced neural activity in brainstem nuclei, in contrast to the prevalent view that such activity is induced by a lack of control.

It is generally recognized that the emotional and health-related consequences associated with aversive experiences are much less severe when organisms have control over the aversive event. Lack of control has been proposed to contribute to mood and anxiety disorders such as depression¹ and post-traumatic stress disorder (PTSD)². Much of what is known about the effect of control comes from studies of what is called learned helplessness or behavioral depression. The learned helplessness paradigm uses a triadic design which compares animals in two conditions that experience a series of stressors with animals in a third condition that do not experience the stressor. Rats in one of the stress conditions (escape condition) have control over the duration of the stressors: they can learn a response that terminates or escapes from each stressor. Rats in the other stress condition have no control over any aspect of the stress experience (inescapable condition). For these rats, the duration of each stressor is determined by the animal in the escape condition to which the animal in the inescapable condition is yoked.

The importance of control was first noted in the mid 1960s, with reports that animals exposed to a series of inescapable (uncontrollable) shocks later failed to learn to escape in a different environment³ and developed ulcers⁴, whereas identical escapable (controllable) shocks produced neither consequence. Stressor controllability effects have since been demonstrated in a broad range of species, including humans, and have been shown to extend to a wide spectrum of behavioral and neurochemical consequences of exposure to stressors⁵.

Research directed at understanding the neural mechanisms that mediate how stressor controllability influences the sequelae of stressors has focused on aminergic nuclei in the brainstem. Both the serotonergic (5-HT) DRN and the noradrenergic (NE) locus coeruleus (LC) have received intensive study. These nuclei are particularly interesting because the DRN provides the majority of the 5-HT with innervation of cortical and limbic structures, as does the LC for the majority of the NE. Both 5-HT and NE have been implicated in depression and anxiety.

Work in our laboratory has focused on the DRN. Our most salient findings are that (i) uncontrollable stress activates DRN 5-HT neurons far more than equal controllable stress does, as indicated both by c-Fos expression in 5-HT neurons⁶ and by extracellular 5-HT within the DRN⁷ and projection regions of the DRN^8–10; (ii) intense activation of DRN 5-HT neurons by uncontrollable stress sensitizes these neurons for a period of time⁸; and (iii) this activation and subsequent sensitization are both necessary and sufficient¹¹ to produce the behavioral changes characteristic of learned helplessness (behavioral depression). Similar results have been reported for the LC^1,12, and the changes in the DRN and LC may be related¹³.

The DRN is a small brainstem structure (as is the LC) and is unlikely to be capable of performing the complex computations necessary to process or detect whether there exists a contingency between the organism’s behavior and outcomes, and thus whether a stressor is, or is not, controllable. In addition, the DRN does not receive direct inputs from either primary sensory or motor regions, so it may not even receive the information required for such a computation to be made. The prefrontal cortex (PFC) is a likely site to process contingency/controllability and then to regulate DRN function accordingly. The PFC has been implicated in executive function, the aspect of cognition affording the ability to act flexibly in a way not locked to environmental stimuli¹⁴, as well as in affective processing¹⁵. Within the PFC, the ventral medial prefrontal cortex (mPFCv) is of special interest. Anatomically, the DRN receives virtually all of its cortical input from infralimbic (IL) and prelimbic (PL) regions within the mPFCv^16,17. Glutamatergic projections from these areas synapse onto predominantly GABAergic neurons within the DRN¹⁸, which in turn inhibit 5-HT neurons. Thus, activation of neurons within IL and PL regions might be expected to inhibit DRN 5-HT neurons, and indeed, electrical stimulation in the mPFCv inhibits DRN unit activity¹⁹. However, to our knowledge there are no data that indicate what sorts of environmental conditions might activate pathways that descend from the mPFCv to the DRN or other brainstem nuclei. In addition, although it has been noted²⁰ that PFC executive functions might extend to the control of aminergic brainstem nuclei, as yet there has been no evidence for this.

These observations suggested to us a set of experiments to determine the effects of temporarily inactivating the mPFCv during exposure to equated (yoked) controllable and uncontrollable stressors on (i) activation of DRN 5-HT neurons and 5-HT release in the DRN during the stressor and (ii) later behaviors typical of learned helplessness (behavioral depression). If the mPFCv is critical to the detection of whether a stressor is controllable and modulates the brainstem DRN accordingly, controllable and uncontrollable stress should not differ with regard to either immediate neurochemical or delayed behavioral consequences. Here, we used microinjections of the GABA_A receptor agonist muscimol in the mPFCv to temporarily inhibit mPFCv activity. We then exposed rats to either controllable or yoked uncontrollable stress. In separate experiments, we measured expression of c-Fos, the protein product of the immediate early gene c-fos, a marker of neuronal activity²¹, in 5-HT–labeled neurons within the DRN after the stressor. We assessed extracellular levels of 5-HT in the DRN before, during and after the stressor. 5-HT efflux in the DRN is a measure of DRN activation²². We also measured fear conditioning and escape learning 24 h after the stressor. These are behaviors that are altered differentially by stressors differing in controllability²³.

We observed that the ability to control the stressor prevented the increase in DRN activation as well as subsequent behavioral changes produced by uncontrollable stress. However, mPFC inhibition with muscimol during controllable stress, while not affecting the ability to learn the wheel-turn response during stressor exposure, completely blocked the impact of stressor controllability on these outcomes.

RESULTS

Wheel-turn escape learning

The critical experiments are only possible if animals with mPFCv inactivation can learn to escape (control) the initial stressors. Thus, rats received a series of 100 escapable tailshocks while restrained in boxes with a wheel mounted in the front that could be turned to terminate each shock. Either muscimol or vehicle was microinjected through indwelling cannulae aimed at the juncture between IL and PL regions of the mPFCv (see Methods) 60 min before the start of the session. As in all of our previous research (for example, ref. 23), the number of wheel turns required to terminate each shock (in quarter turns of the wheel) was increased as the rats became more proficient at escape (see below). Thus, to measure the quality of escape performance, both the response requirement attained across trials (that is, the number of quarter turns to terminate the shock; Fig. 1a) and the time taken to terminate each tailshock (Fig. 1b) must be assessed. As is evident, animals injected with muscimol learned to escape quite well. Repeated-measures ANOVA did not indicate a difference between muscimol- and vehicle-treated groups on either time to terminate (F_1,12 = 0.76) or response requirement (F_1,12 = 1.49). In addition, there was no interaction between Groups and Trials on either measure (F_19,228 = 1.11 and 1.08, respectively). The locations of the cannula placements within the mPFCv for this and the subsequent experiments are shown in Figure 2.

Figure 1.

Efficiency of wheel-turn escape behavior during exposure to controllable tailshock in rats that had received mPFCv muscimol or vehicle 60 min before the shock session. (a) The mean (± s.e.m) number of quarter turns of the wheel attained as the escape requirement on each trial. (b) The mean (± s.e.m) time to reach escape criterion per trial. Filled symbols represent the group given muscimol and open symbols the group given vehicle.

Figure 2.

Placements of microinjection cannula and microdialysis probe. Numerals indicate distance from bregma (mm). (a) Summary of microinjection cannula placements in the mPFCv for all experiments. Not all cannulae are shown owing to overlapping placements. (b) Summary of microdialysis probe placements in the DRN.

c-Fos expression in 5-HT–labeled neurons in the DRN

We have previously reported⁶ that inescapable shock (IS) produces greater activation of DRN 5-HT neurons (as assessed by c-Fos protein expression in 5-HT–labeled neurons) than is produced by exactly equal escapable shock (ES). Moreover, this effect of controllability was especially prominent in middle and caudal regions of the DRN. In the present experiment, rats received ES, received yoked IS or remained as home cage controls (HCC). Half of each group received microinjections of muscimol as in the above experiment before treatment, whereas the remaining half received vehicle. Thus, the design was 3 (stress condition) × 2 (muscimol versus vehicle) factorial. Rats were sacrificed at 2 h after the session, the time of maximal c-Fos expression after IS⁶.

As in our prior studies, neither IS nor ES treatment altered the number of 5-HT–labeled neurons (data not shown). We determined the percentage of 5-HT–labeled neurons expressing c-Fos for the caudal (Fig. 3a) and rostral (Fig. 3b) DRN (coordinates in Methods) by immunohistochemistry. Again, as in our earlier studies, no 5-HT–labeled cells expressed c-Fos in HCC rats. This was true for both vehicle- and muscimol-injected HCC rats. Thus, HCC rats are not shown, as they are at zero with zero standard error, nor are they included in the statistical analysis. IS led to much greater c-Fos expression in caudal 5-HT neurons than did ES: a difference of approximately 300%. Muscimol abolished the difference between IS and ES, largely by increasing c-Fos expression in 5-HT neurons in the ES rats. ANOVA showed significant effects of stress condition (F_{1, 22} = 4.27, P = 0.05) and an interaction between stress condition and muscimol (F_1,22 = 10.41, P < 0.004). Fisher’s PLSD post-hoc comparisons showed that the ES-vehicle group differed from the other groups, which did not differ from each other. Thus, muscimol significantly increased c-Fos expression in caudal 5-HT neurons in ES rats, whereas the small reduction in IS rats was not statistically reliable. There was no effect of stressor controllability or muscimol in rostral DRN.

Figure 3.

Percentage of neurons double-labeled for 5-HT and c-Fos (mean ± s.e.m). (a) Caudal DRN. (b) Rostral DRN. Gray bars represent rats that had received escapable stress; white bars represent rats that had received inescapable stress.

Extracellular 5-HT in the caudal DRN

We have previously found⁷ that IS produces much greater 5-HT efflux than does equal ES within the caudal DRN, as measured by in vivo microdialysis. This 5-HT is released by axon collaterals and perhaps from dendrites themselves and is likely to reflect the activity of the DRN 5-HT neurons²². Therefore, we used the same experimental design as in the above c-Fos experiment and measured extracellular levels of 5-HT in the DRN before, during and after the stress session. Muscimol injected into the mPFCv had no detectable effect on 5-HT efflux within the DRN (Fig. 4, insert), and so the HCC groups with and without muscimol were pooled for simplicity. Measuring extracellular 5-HT levels for the controls and for the groups given IS showed that IS produced a sustained increase in 5-HT that persisted during the IS session and for the period measured after the session (Fig. 4a). As in prior studies, ES (Fig. 4b) produced only a transient increase in 5-HT, with 5-HT returning to baseline levels by 40 min after the onset of the stress session. This rapid reduction in 5-HT if the stressor is controllable is notable, as the stressor exposure continued for another 80 min, but 5-HT now remained at baseline values. Muscimol, which had no effect in the IS rats, produced a marked increase in 5-HT efflux in the ES rats and elevated 5-HT to levels comparable to those observed in the IS rats. ANOVA on the baseline samples taken before the start of the stressor did not show any differences (all F values <1.0). During the stressor there were significant effects of stress condition (F_{2, 27} = 190.0, P < 0.00001), the interaction between stress condition and muscimol (F_{2, 27} = 4.31, P < 0.03), Time (F_4,108 = 10.17, P < 0.0001), and the interaction between time, stress condition and muscimol (F_8,108 = 2.34, P < 0.05). Fisher’s PLSD indicated that the IS-vehicle, IS, muscimol, and ES-muscimol groups differed from the other groups but did not differ from each other. ANOVA conducted on post-stress samples indicated significant effects of shock condition (F_2,25 = 4.65, P < 0.02). Fisher’s PLSD indicated the same group differences as occurred during the stress treatment. Probe placements within the DRN are shown in Figure 2b.

Figure 4.

5-HT as a percentage of baseline in the DRN. The insert shows nonshocked home cage controls that received either muscimol or vehicle in the mPFCv (mean ± s.e.m). (a) Groups that received inescapable stress (IS) and controls. The open circles represent rats that had received vehicle before IS, the filled circles represent rats that had received muscimol before IS, and the dotted line represents the controls. (b) Groups that received escapable stress (ES) and controls. The open circles represent rats that had received vehicle before ES, the closed circles represent rats that had received muscimol before ES, and the dotted line represents the controls. The gray bar represents the time of stressor exposure in the wheel-turn boxes.

Fear conditioning and escape learning

IS potentiates subsequent fear conditioning and interferes with escape learning, whereas ES does not²⁴. Thus, we used the same 3 × 2 factorial design as above, with behavioral testing conducted 24 h after the ES/IS session using our typical procedures. However, we added two additional site-specificity control groups. One group was injected 2.0 mm rostral (ventral orbital cortex (VO), n = 4) and the other 2.0 mm caudal (cingulate cortex area 2 (Cg2), n = 4) relative to the usual mPFCv injection site. Both groups received muscimol and were subjected to ES, as pilot data indicated that muscimol altered functioning only in the ES rats. All rats received two shocks (0.6 mA) in a shuttlebox, and then fear conditioning to the environmental context was examined by measuring freezing in that environment for 20 min. It has been previously shown that this freezing is not a reaction to the shock per se, but rather to fear that has been conditioned to the context²⁵. After this fear assessment, all rats received escape training in which two crossings of the shuttlebox (FR-2) were required to terminate each footshock. Freezing was not observed before the two footshocks in the shuttlebox but was observed after footshocks (Fig. 5). The ES muscimol control rats injected rostral and caudal to the critical site were pooled, as they did not differ (F < 1). As is typical, IS potentiated fear conditioning, whereas ES did not. Muscimol had no effect on IS rats but increased fear conditioning in the ES rats to the level in the IS rats. Notably, muscimol injected at the control sites had no effect on the behavior of the ES rats. ANOVA showed significant main effects of group (F_6,48 = 3.49, P < 0.007) and trial block (F_9,432 = 171.20, P < 0.00001), and a significant trial block × group interaction (F_{54, 432} = 3.03, P < 0.0001). Post-hoc Fisher’s PLSD tests (P < 0.05) indicated that the HCC-muscimol, HCC-vehicle, ES–muscimol at control sites and ES-vehicle groups did not differ from each other, but did differ from the IS-muscimol, IS-vehicle and ES-muscimol groups.

Figure 5.

The mean (± s.e.m) number of 8-s intervals spent freezing, in 2-min blocks, after two footshocks 24 h after stressor exposure. Blue symbols represent rats that had received inescapable stress on day 1, red symbols escapable stress, and green symbols are home cage controls. Squares represent rats that received vehicle before day 1 treatment, whereas circles represent rats that had received muscimol. Site-specificity controls are represented by the dashed lines and orange stars.

Shuttlebox escape followed a similar pattern (Fig. 6). IS potently interfered with escape learning, whereas ES had no effect relative to controls. Again, muscimol had little effect on IS-treated rats, but led to poor escape in ES rats, although not when administered at the control sites. ANOVA indicated significant effects of groups (F_6,48 = 7.62, P < 0.0001), trial blocks (F_4,192 = 4.36, P < 0.003) and the interaction between groups and trial blocks (F_24,192 = 2.51, P < 0.003). Post-hoc Fisher’s PLSD comparisons (P < 0.05) indicated that the HCC-muscimol, HCC-vehicle, ES–muscimol at the control sites and ES-vehicle groups did not differ from each other but did differ from the IS-muscimol, IS-vehicle, and ES-muscimol groups.

Figure 6.

Mean (± s.e.m) shuttlebox escape latencies across blocks of five shuttlebox FR-2 escape trails 24 h after stressor exposure. Blue symbols represent rats that had received inescapable stress on day 1, red symbols escapable stress, and green symbols are home cage controls. Squares represent rats that received vehicle before day 1 treatment, whereas circles represent rats that had received muscimol. Site specificity controls are represented by the dashed lines and orange stars.

DISCUSSION

The results of this series of experiments are clear. Inactivation of regions within the mPFCv during exposure to stress eliminated the impact of behavioral control over the stressor on DRN 5-HT activation and subsequent behavior. Thus, the mPFCv is indeed involved in the processing of contingency information and regulation of DRN 5-HT activity. Notably, inactivation of the mPFCv eliminated the difference between controllable stress and uncontrollable stress by substantially altering the neural and behavioral impact of only one of these conditions: namely, controllable stress. Thus, muscimol injected into the mPFCv increased the controllable shock–induced c-Fos expression in 5-HT neurons in the DRN and extracellular 5-HT in the DRN to the levels normally produced by uncontrollable shock, and had no significant effect on either c-Fos expression or 5-HT efflux produced by the uncontrollable condition. Furthermore, inactivation of the mPFCv altered the behavioral consequences of stressor exposure measured 24 h later. Again, this manipulation had no effect in uncontrollably shocked rats, as they later showed potentiated fear conditioning and escape deficits. However, controllably stressed rats now behaved as if the stressor had been uncontrollable 24 h earlier: they showed potentiated fear conditioning and poor escape behavior. It would seem reasonable to conclude that aversive events per se drive the DRN²⁶, and that if the events are controllable, the mPFCv inhibits that aversive stimulus–driven DRN 5-HT activity and prevents the cascade of events that follow prolonged activation of DRN 5-HT neurons. In addition, the DRN sends 5-HT projections to the mPFCv, and uncontrollable stress produces greater 5-HT efflux within the mPFCv than does controllable stress⁹. Because 5-HT tends to inhibit pyramidal neurons in the mPFC²⁷, this large 5-HT efflux produced by uncontrollable stress may further contribute to differential activation of the mPFCv by stressors of differing controllability. Thus, processes within the DRN alter mPFCv function, and these two structures, at very different levels of the brain, are likely to interact closely to regulate stressor controllability–related phenomena. The present results also strengthen the tie between DRN 5-HT activation and learned helplessness and behavioral depression. mPFCv inactivation during controllable stress increased DRN 5-HT activation to the level observed with uncontrollable stress, and behaviors characteristic of learned helplessness and behavioral depression followed.

A potential difficulty with part of these conclusions is that our data here do not preclude the possibility that the mPFCv regulates the DRN not directly, but through projections to other structures that then in turn regulate the DRN. For example, the mPFCv projects to the central nucleus of the amygdala²⁸, which in turn projects to the DRN¹⁶. Although the particular possibility that the central nucleus of the amygdala is a necessary relay is unlikely, as large lesions of this structure have no effect on the escape deficits produced by uncontrollable stress²⁹, the general point still holds. However, even if this type of indirect regulation proves to exist, the critical role of the mPFCv in controllability effects and in brainstem regulation remains.

Notably, the mPFCv has not been implicated in escape learning per se, and the periaqueductal gray has generally been regarded as the key mediator of this type of learning³⁰. Indeed, acquisition of the wheel-turn escape response occurred normally during mPFCv inactivation. Thus, the mPFCv is likely not to be critical to the acquisition of the escape (controlling) response, but rather is the location of circuitry that detects that a contingency is present and then regulates other structures (here, the DRN) accordingly. Motor responses can be learned (that is, habits formed) in a manner separated from higher cognitive processing and ‘expectational-like’ phenomena³¹. Other circuitry then uses this information to regulate other neural and behavioral processes. It would seem that the mPFCv is the structure that performs this ‘utilizing’ function for responses that escape aversive events. The escape response can be acquired without the involvement of the mPFCv, but the mPFCv is necessary to use the existence of successful escape to regulate other circuitries that respond to aversive events and that influence later emotion and behavior. Thus, this interpretation of the data implies that when stressor termination is made contingent upon a particular response, at least two types of learning occur: (i) low-level processes are engaged that result in strengthening of the escape response (that is, a habit may be learned) and (ii) higher processes are engaged enabling the organism to detect, and/or use the knowledge, that there is a contingency between behavior and outcome and that its responses control the stressor. It is the latter process that mitigates the consequences associated with prolonged uncontrollable stress.

A related argument has been made with regard to the role of the PL in the learning of instrumental responses that obtain appetitive rewards^32,33. Rats with PL lesions learned to make instrumental responses such as pressing a lever for food, but were sensitive only to the pairing or the temporal contiguity between the response and food, and not to the contingency between response and food. Thus, when the probability of food in the absence of the instrumental response was increased to be the same as the probability of food after responses (that is, no contingency, but still pairing between response and food), control rats responded less, but PL-lesioned rats continued to respond.

The present data and conclusions fit well with recent studies exploring the role of the mPFCv in fear-related processes. mPFCv lesions have proven to have no effect on the acquisition of conditioned fear³⁴ or on the extinction of fear under some circumstances³⁴. However, mPFCv lesions, in the same regions as inactivated here, interfere with the memory³⁵ or the use of extinction to guide behavior³⁶. Similarly, in the present experiments, the effects of mPFCv inactivation can be described as a failure to use the presence of an escape response to modulate the impact of the stressor on neural activation within the brainstem and on later behavior. At a neural level, it has been argued³⁷ that the mPFCv mediates the memory of extinction, and perhaps the more general use of extinction information, by inhibiting amygdala function. Glutamatergic neurons from the IL region of the mPFCv project to GABAergic inhibitory interneurons within the capsular division of the central nucleus of the amygdala, which inhibit output from this latter region³⁸. Consistent with this sort of arrangement, combat veterans with PTSD show enhanced amygdala and reduced mPFCv activation when shown combat scenes, relative to combat veterans that do not suffer from PTSD³⁹. The suggestion has been that amygdala function is exaggerated because mPFCv inhibition of the structure is reduced. Notably, controllable stress produces less conditioned fear and anxiety than does uncontrollable stress⁵, findings that would follow from the activation of the mPFCv by the presence of control. It may be that the absence of a perception of control contributes to the occurrence of intense anxiety, as occurs in PTSD. Highly aversive events are likely to drive limbic and brainstem structures that induce negative affective and motivational experiences; control, perceived control or, more generally, the ability to cope may activate mPFCv inhibition of these limbic and brainstem processes. From an evolutionary perspective, it may be sensible that activation of ‘lower’ centers by strongly aversive events came first, and that as species developed the ability to cope with such events by behavioral means, inhibition from ‘higher’ centers under conditions of behavioral coping then developed.

The present findings also fit with more general conceptions of the functions of the mPFC. The mPFC is generally believed to mediate ‘executive control’, a set of processes that involves the scheduling and optimizing of subsidiary processes⁴⁰. These subsidiary processes are typically viewed as residing in other cortical and perhaps limbic regions, although, as noted above, the possibility of brainstem locations has been suggested²⁰. Here we extend to brainstem nuclei the general concept of mPFC control, with controllability as the regulator and the DRN as the target.

METHODS

Rats.

In all experiments, rats were male Sprague-Dawley rats (Harlan Labs) weighing 275–325 g, housed four per cage on a 12-h light/12-h dark cycle (on at 0700 and off at 1900). Experiments were conducted between 0800 and 1200 h. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado at Boulder.

Surgery and cannulation.

Surgery was carried out under anesthesia with either 60 mg kg⁻¹ sodium pentobarbital i.p. or halothane. All rats were implanted with dual cannula guides for microinjections (26 gauge) 1 mm center-to-center distance (Plastics One). The tips of the cannulae were at the PL/IL junction region of the medial prefrontal cortex (mPFC): 2.2 mm rostral to bregma, 3.5 mm ventral from the dura matter and ±0.5 mm relative to midline. For the microdialysis experiments, rats were implanted with a second cannula guide for microdialysis probes (CMA 12), with the tip terminating within the DRN: 8.3 mm caudal to bregma and 5 mm ventral from the dura matter at the midline⁴¹. A screw cap of a 15-ml conical centrifuge tube, whose central lid portion was removed, was also affixed to the skull so that its threads were exposed and it encircled the cannulae guide. This was done so that the skull assembly could be protected during microdialysis. Rats were allowed to recover for 1–2 weeks after surgery before experimentation.

Muscimol microinjection.

Rats were injected bilaterally with 0.5 μl of either 50 ng of muscimol (Sigma) or vehicle in the mPFC. Dual 33-gauge microinjectors (Plastics One) attached to PE 50 tubing were inserted through the guides, from which they protruded 1 mm. The other end of the tubing was connected to a 25-μl Hamilton syringe that was attached to a Kopf microinjection unit (Model 5000). The volumes were injected over a period of about 30 s, and the injector was left in place for 2 min to allow diffusion.

Wheel-turn escape/yoked inescapable shock procedure.

Each rat was placed in a Plexiglas box (14 × 11 × 17 cm) with a wheel mounted in the front and a Plexiglas rod extending from the back. The rats’ tails were taped to the Plexiglas rod and affixed with copper electrodes. Rats received shocks in yoked pairs (ES and IS). The treatment consisted of 100 trials with an average intertrial interval of 60 s. Shocks began simultaneously for both rats in a pair and terminated for both whenever the ES rat met a response criterion. Initially, the shock was terminated by a quarter turn of the wheel. The response requirement was increased by one quarter turn when each of three consecutive trials was completed in less than 5 s. Subsequent latencies under 5 s increased the requirement by 50% up to a maximum of four full turns. If the requirement was not reached in less than 30 s, the shock was terminated and the requirement reduced to a single quarter turn. This procedure was used to insure that the ES rats learned an operant response. Shock intensity was 1.0 mA for the first 30 trials, 1.3 mA for the second 30 trials and 1.6 mA for the last 40 trials. Nonshocked home cage control (HCC) rats remained undisturbed in the colony, except during the microdialysis experiments, where they remained undisturbed in the dialysis room.

c-Fos immunohistochemistry.

Tissue preparation. Two hours after the last tailshock, rats were deeply anesthetized with sodium pentobarbital (Nembutal) and transcardially perfused with 100 ml of 0.9% saline followed by 250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and postfixed in the same fixative overnight. After postfixation, brains were transferred to 30% sucrose and stored at 4 °C until sectioning. Brains were rapidly frozen in −40 °C isopentane and 35-μm sections were obtained in a −20 °C cryostat. Free-floating sections were stored at 4 °C in cryoprotectant until staining. Immunohistochemistry (IHC). Immunohistochemical staining for c-Fos and 5-HT was conducted sequentially as previously described⁶. Briefly, staining for c-Fos was conducted first using rabbit polyclonal Fos primary antibody (1:15,000; Santa Cruz) and biotinylated goat anti-rabbit secondary antibody (Jackson Laboratories) with 3,3′-diaminobenzidine (DAB) and nickel as chromogens. Staining for 5-HT used rabbit 5-HT primary antibody (1:10,000; ImmunoStar) and non-biotinylated goat anti–rabbit IgG (Jackson Laboratories) secondary antibody with peroxidase anti-peroxidase (PAP) and DAB as chromogens. Image analysis. Sections were assessed for the number of 5-HT–stained cells and the number of cells double-labeled for both 5-HT and c-Fos. c-Fos-stained nuclei were identified by dark brown or black ovoid particles. Larger reddish-tan particles, with or without Fos-stained nuclei, were counted as 5-HT positive cells. Rostral DRN sections were comparable to an anterior-posterior coordinate of −1.36 mm from interaural zero. Caudal DRN sections were comparable to the −0.7 mm anterior-posterior coordinate. Histology. Verification of cannulae placement in the mPFC was conducted on frontal sections stained with cresyl violet and visualized under a light microscope.

In vivo microdialysis.

The afternoon before the experiment, a CMA 12 microdialysis probe (0.5 mm in diameter, 1 mm membrane with a 20-kD molecular weight cut-off; CMA/Microdialysis), was introduced through the cannula guide so that the membranous tip of the probe was within the DRN. A portion of a 15-ml Eppendorf tube was screwed onto the skull-mounted screw cap, through which the dialysis tubing, protected within a metal spring, entered and attached to the probe. Each animal was placed individually in a Plexiglas bowl (Bioanalytical Systems) and infused with isotonic Ringer’s solution (Baxter) at a rate of 0.2 μl min⁻¹ overnight. At about 0900 the next day, the flow rate was increased to1.5 μl min⁻¹ and a 90-min stabilization period was allowed. The infusion flow remained constant throughout the experiment. Samples were collected every 20 min. After stabilization, three baseline samples were collected, then muscimol or vehicle was injected in the mPFC and three additional baseline samples were collected. Next the rats were moved into a room containing the shock equipment and placed in Plexiglas wheel-turn boxes that were designed to accommodate the dialysis tubing. There they received 100 ES and yoked IS tail shocks. Five samples were collected during the session. After this, the rats were transferred back to the Plexiglas bowls where three postshock samples were collected. Two HCC control groups received bilateral mPFC injections of either vehicle or muscimol.

5-H T analysis.

5-HT concentration was measured in dialysates by HPLC with electrochemical detection. The system consisted of an ESA 5600A Coularray detector with an ESA 5014B analytical cell and an ESA 5020 guard cell. The column was an ESA MD-150 (C-18, 3 μm, 150 × 3.2 mm) maintained at 26 °C, and the mobile phase was the ESA buffer MD-TM. The analytical cell potentials were kept at −75 mV and +250 mV and the guard cell at +300mV. Dialysate (23 μl) was injected with an ESA 542 autosampler that kept the dialysates at 6 °C. External standards (Sigma) were run each day to quantify 5-HT.

Cannula and dialysis probe verification.

At the end of the experiment an overdose of pentobarbital was administered and brains were removed and frozen. A cryostat was used to take 40-μm sections, which were then stained with cresyl violet for cannula placement verification. The mPFC injections were considered successful if the injector tip was within the prelimbic or infralimbic regions of the mPFC, at about 2.2 mm rostral to bregma. Only rats with the dialysis probe at least 70% within the intermediate and caudal DRN (−7.8 to −8.5 from bregma) were included.

Fear conditioning and shuttlebox escape learning.

Fear conditioning and escape learning occurred in shuttleboxes using procedures previously described¹¹. Freezing was measured for the first 5 min after placement in the shuttleboxes. Each subject’s behavior was scored every 8 s as being either freezing or not freezing. Freezing was defined as the absence of all movement except that required for respiration. The observer was blind with regard to treatment condition, and inter-rater reliability has been calculated to be greater than 0.92. This observation period was followed by two scrambled footshocks (0.6 mA) that could be terminated by crossing to the other side of the shuttlebox (FR-1 trials). IS does not alter FR-1 shuttlebox escape latencies²⁹, and therefore IS and other rats are here exposed to shocks of equal duration. FR-1 latencies were measured in the present experiment, and, as is typical, there were no group differences. These two footshocks were followed by a 20-min observation period during which freezing was scored. This observation period was followed by three further FR-1 escape trials and then 25 FR-2 escape trails. On FR-2 trials, the rats were required to cross to the other side of the shuttlebox and back to terminate each shock. It was here that IS-induced escape deficits typically occurred. Each shock terminated after 30 s if an escape response had not occurred.