Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety

Abstract

Data will be reviewed using the acoustic startle reflex in rats and humans based on our attempts to operationally define fear vs anxiety. Although the symptoms of fear and anxiety are very similar, they also differ. Fear is a generally adaptive state of apprehension that begins rapidly and dissipates quickly once the threat is removed (phasic fear). Anxiety is elicited by less specific and less predictable threats, or by those that are physically or psychologically more distant. Thus, anxiety is a more long-lasting state of apprehension (sustained fear). Rodent studies suggest that phasic fear is mediated by the amygdala, which sends outputs to the hypothalamus and brainstem to produce symptoms of fear. Sustained fear is also mediated by the amygdala, which releases corticotropin-releasing factor, a stress hormone that acts on receptors in the bed nucleus of the stria terminalis (BNST), a part of the so-called 'extended amygdala.' The amygdala and BNST send outputs to the same hypothalamic and brainstem targets to produce phasic and sustained fear, respectively. In rats, sustained fear is more sensitive to anxiolytic drugs. In humans, symptoms of clinical anxiety are better detected in sustained rather than phasic fear paradigms.

Figure 1

Context conditioning after paired CS shock (predictable shocks), unpaired CS shock (unpredictable shocks), and non-aversive conditionings (control) in a between-group design. During non-aversive conditioning, the US was a signal for button press. Subjects underwent conditioning in two experimental sessions separated by 4–5 days. Context conditioning was assessed by delivering startle stimuli at the beginning of sessions 1 and 2, before conditioning occurred. The figure shows that when subjects received unpaired CS–US, startle magnitude was significantly larger when they returned for testing (session 2) compared with before initial conditioning (session 1). In contrast, during the non-aversive condition, startle decreased (because of long-term habituation) between sessions 1 and 2. Startle magnitude in the paired CS–US condition was intermediate between these two conditions, suggesting weak context conditioning. ^*Significant difference in startle magnitude between sessions 1 and 2.

Figure 2

Verbal instruction experiment. Subjects were verbally instructed that they would be safe in the no shock (N) condition, that they would receive aversive stimuli signaled by a threat cue in the predictable (P) condition, and that they would receive unsignaled aversive stimuli in the unpredictable (U) condition. An 8-s duration cue was presented in each context. The cue signaled the aversive stimulus in the P context, but had no signal value in the N and U contexts. Two types of aversive stimuli were used in this between-group design: a shock, or a blast of air directed to the throat at the level of the larynx. In the P condition, startle was larger during the CS compared with ITI (fear-potentiated startle). In addition, startle during ITI (green bars) increased linearly from the control, to the P, to the U condition. However, such a pattern of response was not seen in the airblast group. ^*Significant increase in startle magnitude during the cue compared to ITI.

Figure 3

Context conditioning using virtual reality. Subjects were presented with three virtual environments in which they underwent different types of aversive conditioning counterbalanced across contexts in a within-subjects design. The three contexts were a casino, a bank, and a restaurant. Subjects were safe in the no-shock (N) context. They received paired CS shock in the predictable (P) context and unpaired CS shock in the unpredictable (U) context. An 8-s cue (a light) was presented in each context (data not shown). The cue signaled the shock in the P context, but had no signal value in the N and U contexts.

Figure 4

Magnitude of startle in each virtual context in the presence and absence of the CS (during inter-trial interval or ITI). As expected, startle was significantly larger during the CS compared with ITI (fear-potentiated startle) only in the predictable condition, when the cue signaled the shock (two middle bars). Startle during ITI (green bars) is a measure of context conditioning, reflecting the degree of contextual anxiety associated with each context. Startle increased linearly from the control, to the predictable, to the unpredictable contexts confirming that (1) context conditioning develops to environments associated with an aversive event and (2) context conditioning is affected by the predictability of the aversive event, with unpredictable environments resulting in greater context conditioning compared with predictable environments. ^*Significant increased in startle magnitude during the cue compared with ITI.

Figure 5

Sustained Startle Test and Conditioning Procedure. For conditioning, rats received eight presentations of variable duration (3, 10, 20 s, 1, 2, 4, 6, and 8 min) of 60-Hz clicker stimulus together with co-terminating footshock. Startle amplitude to 50 ms of 95-dB noise bursts (ISI=30 s) was measured before and after conditioning, for 8 min in the absence and then for 8 min in the presence of the clicker. In normal rats, the clicker did not increase startle before conditioning, but did increase startle after conditioning. Blue bars indicate periods when the clicker was present and arrows indicate footshock.

Figure 6

Mean startle amplitude over minutes for 8 min before the CS, the 8 min during the CS, and the 8 min after the CS. In this particular case, startle amplitude increased abruptly with the CS onset and returned abruptly to pre-conditioning baseline with CS offset. However, on several other occasions we have observed that startle amplitude remains elevated for up to several minutes after CS offset.

Figure 7

Photomicrographs prepared and provided by Dr Chungjun Shi of 30-μm horizontal sections through a rat brain, cut at a slight angle to include the amygdala and BNST in the same sectional plane. Infusions of the anterograde tracer biotinylated dextran-amine (BDA) into the posterior BLA (BLA_P) show strong projections both to the medial and lateral CeA (labeled here as CM and CL) and also to the BNST. As those that project to the BNST pass directly through the CeA, electrolytic CeA lesions or intra-CeA infusions of sodium channel blockers such as TTX would interrupt this pathway.

Figure 8

Infusions of NBQX, an AMPA receptor antagonist, into the caudal rather than the rostral BLA blocked light-enhanced startle. It can be noted that the caudal part provides most of the input from the BLA to the BNST_L.

Figure 9

Fear-potentiated startle to a 3.7-s visual CS, dependent on glutamate receptors in the CeA_M, was occluded by fear-potentiated startle to a 3.7-s auditory CS, also dependent on glutamate receptors in the CeA_M (left bars), but not by CRF-enhanced startle, which depends on CRF receptors in the BNST (right bars). Percent potentiation scores to the visual CS are indicated above each set of bars. Dashed lines indicate baseline startle (ie, on noise-alone trials).

Figure 10

The effect on fear-potentiated startle to 8-min auditory CS was evaluated in rats after intra-cranial infusions of the AMPA receptor antagonist NBQX (3 μg per side in 0.5 μl phosphate-buffered saline). Intra-BNST infusions (N=11) decreased the sustained component of fear-potentiated startle, but augmented the early component, relative to vehicle infusions (N=25, pooled across structures). Owing to an extreme outlier in the PBS group (606% potentiation during block 1) that distorted the normal distribution, these data were analyzed non-parametrically by the Mann–Whitney test on block 2–block 1 difference scores, reflecting an interaction effect (p<0.019). After histological verification of cannula placement, rats were divided into BLA (N=6) or CeA (N=8) groups. Intra-BLA infusions disrupted both the early and sustained components of fear-potentiated startle, whereas intra-CeA infusions disrupted neither.

Figure 11

Rats were tested for light-enhanced startle and then fear-potentiated startle. Before each test, the selective CRF-R1 antagonist GSK876008 was administered orally (for each test, each rat received the same dose that it received in the other test). The selective CRF-R1 antagonist GSK876008 non-monotonically disrupted light-enhanced startle (a); significant quadratic trend), but did not disrupt fear-potentiated startle (b).

Figure 12

In two different experiments using slightly different sustained fear conditioning procedures (see Figure 5), the selective CRF-R1 antagonist GSK876008 disrupted potentiated startle to an 8-min CS, but did not disrupt potentiated startle to a 3.7-s presentation of the same stimulus.

Figure 13

The effect of the CRF-R1 antagonist GSK876008 on pre- to post-shock changes in ‘baseline' startle (ie, on test trials without the 3.7-s CS), which may be a conditioned response to the context CS. The shape of the dose–response curve was similar to that seen earlier for light-enhanced startle, in which intermediate doses of GSK876008 disrupted these increases.

Figure 14

Schematic illustrating the hypothetical involvement of the CeA and BNST in short- and long-duration startle increases. Sensory information enters the basolateral amygdala complex (BLA—lateral, basolateral, and basomedial nuclei), which sends prominent projections to the medial division of the central nucleus of the amygdala (CeA_M) as well as projections to the lateral division of the bed nucleus of the stria terminalis (BNST_L). It also sends light projections to the lateral division of the central nucleus of the amygdala (CeA_L), which sends a heavy, CRF-containing projection to the BNST_L. As inactivation of the BLA blocks the increase in startle produced by CRF given intraventricularly, we suggest that CRF may act presynaptically to enhance glutamate release from the BLA terminals in the BNST_L. The CeA_L also receives projections from cortical areas as well as from the highly stress-sensitive PVT. We hypothesize that a fear-eliciting stimulus rapidly activates the BLA and CeA_M to produce a short-acting phasic fear response. At the same time, inputs to the CeA_L result in a release of CRF into the BNST to produce a more slowly acting, but long-lasting sustained fear response akin to anxiety. Inhibitory feedback from the BNST and/or CeA_L to the CeA_M may turn off the phasic fear response in order to produce a seamless transition from phasic to sustained fear.

Figure 15

An intraventricularly infused cocktail of NBQX and muscimol infused into the BLA completely blocked CRF-enhanced startle. On the basis of this observation and some preliminary microdialysis, we suggest that CRF may act presynpatically on BLA terminals in the BNST to facilitate glutamate release.

Figure 16

Chronic administration of the progesterone metabolite, allopregnanolone, blocked sustained fear measured with CRF-enhanced startle (left panel) but had no effect on phasic fear measured with fear-potentiated startle to a 3.7-s CS (right panel).

Figure 17

Testosterone reduced sustained fear measured with light-enhanced startle but had no effect on phasic fear measured with fear-potentiated startle to a 3.7-s CS.

Figure 18

Effect of the benzodiazepine alprazolam on verbally mediated cued fear and contextual anxiety. The paradigm was the same as presented in Figure 4. Subjects were informed that there would be three conditions: (1) no shock (N), (2) predictable (P) shocks, and unpredictable (U) shocks. Each subject received placebo, 0.5 mg of alprazolam, 1 mg of alprazolam, or 50 mg of diphenhydramine (Benadryl). Diphenhydramine was used as an active control to match the sedative properties of alprazolam on startle. (left panel) Startle magnitude during the cue and ITI in the P condition during the P conditions only. The difference scores between cue and ITI is a measure of fear-potentiated startle. Alprazolam did not affect fear-potentiated startle. (right panel) Startle during ITI (contextual anxiety) in the N, P, and U conditions. As in Figures 3 and 4, startle increased linearly from the N to the P to the U condition with placebo. This effect was replicated with the low dose of alprazolam and with diphenhydramine. However, there was a significant reduction in startle with the high dose of alprazolam, indicating a substantial reduction in contextual anxiety. This effect was not caused by sedation because diphenhydramine, which reduced baseline startle to the same extent as 1 mg alprazolam, did not affect contextual anxiety. ^*(left panel) Significant increase in startle magnitude during the cue compared with the ITI. ^*(right panel) Significant condition X drug linear trend between conditions.

Figure 19

Patients with panic disorder have normal phasic fear to predictable airblasts but elevated sustained fear to unpredictable airblasts (left). Patients with PTSD or GAD have greater phasic fear to predictable airblasts but only patients with PTSD have great sustained fear to unpredictable airblasts (right).