Research report
Role of corticotropin-releasing factor and urocortin within the basolateral amygdala of rats in anxiety and panic responses

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Abstract

The amygdala is a critical temporal lobe structure involved in the expression of anxiety and stress responses. The basolateral nucleus (BLA) of the amygdala in particular, may play a key role in anxiety. Furthermore, corticotropin-releasing factor (CRF), a 41 amino acid peptide, has been strongly implicated in the regulation of stress and anxiety responses. Centrally administered CRF has been shown to increase the anxiety-like behaviors of rodents in several animal models. A recently cloned related peptide, Urocortin (Ucn), appears to have similar affinity for the CRF1 receptor, but higher affinity at the CRF2 receptor. When microinjected into the BLA, we found Ucn was substantially more potent than CRF in producing anxiogenic-like behavior as assessed in the social interaction test. Furthermore, repetitive administration of subthreshold doses of Ucn and CRF resulted in `priming'. Once primed, these animals exhibited behavioral and cardiovascular responses to intravenous sodium lactate, a panicogenic agent in susceptible human patients. These results suggest central CRF and Ucn play a role in generating anxiety which may be similar to that seen in pathological conditions such as panic disorder.

Introduction

Corticotropin-releasing factor (CRF) plays a critical role in initiating the cascade of biological events during the stress response 24, 44. Although initially CRF was recognized for its role in regulating the hormonal response to stress, CRF has been found to have a part in mediating the behavioral aspect of stress as well. CRF administered into the central nervous system will induce anxiety-like behaviors in several animal models [11]. Transgenic mice which overproduce CRF show increased anxiety-like behaviors which are reversed by central administration of the CRF antagonist α-helical CRF 9-41 [29a]. Thus, there is strong evidence that CRF may act as a neurotransmitter/neuromodulator regulating stress and anxiety.

Currently, there have been at least two different CRF receptors identified 5, 6, 19, 21, 27, 28, 47. The anxiogenic behavior produced by CRF in rats appears to be mediated via the CRF-1 receptor [14]. Recently, a peptide which shares sequence identity with both urotensin I and CRF has been discovered and named urocortin (Ucn) [46]. In addition, it appears that Ucn is capable of eliciting anxiety-like behaviors after i.c.v. administration [23].

The amygdala has been implicated as a major anatomical site for regulation of the fight-or-flight response. In previous studies, we have shown that disruption of inhibitory neurotransmission within the BLA using the GABAA antagonist bicuculline methiodode (BMI) can lead to behavioral and physiological responses similar to that seen in anxiety disorders [37]. Further, daily injections of a subthreshold dose of BMI can alter the threshold for BMI effects within the BLA by day 5 [38]. Thus, a 6 pmol (subthreshold dose) injection of BMI on experimental day 5 can induce anxiety-like behaviors similar to a 20 pmol (threshold dose) in an untreated animal. These changes in synaptic activity were termed `priming' and were maintained for at least 6 weeks [38].

The development of anxiety-like behaviors in rats seen during `priming' was similar to panic disorder, a subtype of anxiety. Patients with this type of anxiety exhibit a unique sensitivity to sodium lactate 20, 29, 30, 31. Infusion of sodium lactate into primed rats can induce the development of both anxiety-like behavior and cardiovascular responses in these animals [32]. Thus, the long-term synaptic changes occurring during priming could be similar to subjects with panic disorder.

The first purpose of the study was to compare the effects of Ucn and CRF administered directly into the BLA on anxiety-like behaviors. Second was to characterize the effects of repeated microinjections of subthreshold doses of Ucn and CRF into the BLA on changes in anxiety threshold and lastly we tested the primed animals with lactate infusions to elicit panic responses.

Section snippets

Animals

All experiments were conducted on male Wistar rats (300–350 g) obtained from Harlan Laboratories (Indianapolis, IN). They were individually housed and given food and water ad libitum. The room temperature was controlled at 72°F (22°C) and maintained on a 12-h light, 12-h dark schedule.

Arterial/venous catheterization

Arterial and venous catheters were made up of 8 cm of 0.01 in Teflon tubing (Small Parts, Miami Lakes, FL). A 3-cm section of a 30-cm piece of 0.02 in Tygon tubing (Fisher Scientific, Pittsburgh, PA) was soaked in

Dose response of CRF and Ucn

In the initial experiments, animals received bilateral infusions of vehicle or various doses of CRF and Ucn. Both peptides produced dose-dependent reductions in SI time. Analysis of the resulting data with a one-way ANOVA revealed a significant difference among the doses for both CRF [Fig. 1A: F(3,24)=5.287; P=0.0061] and Ucn [Fig. 1B: F(3,24)=4.880; P=0.0087]. Post hoc analysis between the doses of CRF revealed a significant difference between the 200 fmol dose of CRF and vehicle [P=0.0074] as

Discussion

Central administration of CRF has been shown to have an important role in initiating the biological events known to occur during the stress response 24, 44. It has also been shown to produce anxiogenic-like behavior in several behavioral tests such as open field 17, 41, 43, elevated plus maze [1], conflict test [2], social interaction [12], acoustic startle 18, 42and conditioned fear paradigm [7]. The anxiogenic-like response to CRF in rats appears to be mediated via the CRF-1 receptor [14].

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