Fear-potentiated Startle Test Research Articles

Role of the extended amygdala in short-duration versus sustained fear: a tribute to Dr. Lennart Heimer

The concept of the "extended amygdala", developed and explored by Lennart Heimer, Jose de Olmos, George Alheid, and their collaborators, has had an enormous impact on the field of neuroscience and on our own work. Measuring fear-potentiated startle test using conditioned stimuli that vary in length we suggest that the central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST(L)) are involved in short-term versus long-term fear responses we call phasic versus sustained fear, respectively. Outputs from the basolateral amygdala (BLA) activate the medial division of the CeA (CeA(M)) to very rapidly elicit phasic fear responses via CeA(M) projections to the hypothalamus and brainstem. The BLA also projects to the BNST(L), which together with other BNST(L) inputs from the lateral CeA (CeA(L)) initiate a slower developing, but sustained fear response, akin to anxiety. We hypothesize this occurs because the CeA(L) releases the peptide corticotropin releasing hormone (CRF) into the BNST(L) which facilitates the release of glutamate from BLA terminals. This activates the BNST(L) which projects to hypothalamic and brainstem areas similar to those innervated by the CeA(M) that mediate the specific signs of fear and anxiety. The generality of this idea is illustrated by selective studies looking at context conditioning, social defeat, drug withdrawal and stress induced reinstatement.

Fear‐Potentiated Startle and Light‐Enhanced Startle Models in Drug Discovery

Described in this unit are the fear-potentiated startle (FPS) and light-enhanced startle (LES) tests. These protocols have proven reliable in detecting the anxiolytic properties of test compounds. The principle of these tests is that the magnitude of the acoustic startle reflex is an index of anxiety. The FPS test includes two training sessions in which an intrinsically aversive foot shock is paired with a neutral cue light. In the test session presentation of this cue light is subsequently used to elicit startle potentiation. In the LES test startle reactivity is increased by presentation of bright light. Because LES is based on the innate aversion of rodents for bright light it does not require training sessions. Although LES has been used less frequently than FPS for screening compounds, it has an advantage in that drug effects on startle potentiation are independent of memory retrieval. Further, the contextual anxiety measured in the LES test could be more relevant for pathological anxiety than the conditioned fear associated with the FPS test.

Amygdala infusions of an NR2B-selective or an NR2A-preferring NMDA receptor antagonist differentially influence fear conditioning and expression in the fear-potentiated startle test.

Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the NR1/NR2B receptor antagonist CP101,606 (0.5, 1.5, or 4.5 microg/amygdala) or the NR1/NR2A-preferring antagonist NVP-AAM077 (0.075, 0.25, 0.75, or 2.5 microg/amygdala) into the amygdala prior to either fear conditioning (i.e., light-shock pairings) or fear-potentiated startle testing. CP101,606 nonmonotonically disrupted fear conditioning but did not disrupt fear expression. NVP-AAM077 dose-dependently disrupted fear conditioning as well as fear expression. The results suggest that amygdala NR1/NR2B receptors play a special role in fear memory formation, whereas NR1/NR2A receptors participate more generally in synaptic transmission.

Fear-Potentiated Startle to Threat, and Prepulse Inhibition Among Young Adult Nonsmokers, Abstinent Smokers, and Nonabstinent Smokers

Evidence suggests that the transition from experimental to regular smoking is facilitated by the influence of tobacco on affective and attentional mechanisms. The objective of this study was to examine affective and attentional responses in young adult smokers using fear-potentiated startle and prepulse inhibition. Participants were 56 college nonsmokers, nonabstinent smokers, and overnight-abstinent smokers. The fear-potentiated startle test examined phasic responses to imminent threat cues and more sustained responses to unpredictable aversive events. Prepulse inhibition investigated responses to attended and ignored prepulse stimuli. Abstinent and nonabstinent smokers showed increased sustained potentiation of startle to contextual cues, compared to controls. Abstinent smokers showed increased fear-potentiated startle to threat cues, compared to nonsmokers. PPI did not discriminate between abstinent or nonabstinent smokers and controls. These findings suggest that negative affectivity or anxiety is associated with smoking, particularly during short withdrawal. Potentiated startle may provide a valuable tool in understanding the biologic mechanisms underlying nicotine withdrawal and inform cessation and prevention efforts.

In vivo pharmacological characterization of the structurally novel, potent, selective mGlu2/3 receptor agonist LY404039 in animal models of psychiatric disorders

Data from both preclinical and clinical studies have provided proof of concept that modulation of limbic and forebrain glutamate, via mGlu2/3 receptor agonists, might provide therapeutic benefits in many psychiatric disorders including schizophrenia and anxiety. The aim of this study was to assess the efficacy of a structurally novel, potent, selective mGlu2/3 receptor agonist with improved bioavailability (LY404039) in animal models predictive of antipsychotic and anxiolytic efficacy. LY404039 was assessed in amphetamine- and phencyclidine-induced hyperlocomotion, conditioned avoidance responding, fear-potentiated startle, marble burying, and rotarod behavioral tests. Monoamine release and turnover were assessed using microdialysis and ex vivo tissue levels. LY404039 attenuated amphetamine- and phencyclidine-induced hyperlocomotion (3-30 and 10 mg/kg, respectively). LY404039 (3-10 mg/kg) inhibited conditioned avoidance responding. LY404039 also reduced fear-potentiated startle in rats (3-30 microg/kg) and marble burying in mice (3-10 mg/kg), indicating anxiolytic-like effects. Importantly, LY404039 did not produce sedative effects or motor impairment as measured by rotarod performance and lack of escape failures in the conditioned avoidance task (at doses up to 30 and 10 mg/kg, respectively). LY404039 (10 mg/kg) also increased dopamine and serotonin release/turnover in the prefrontal cortex. These results demonstrate the broad preclinical efficacy of LY404039 across multiple animal models of antipsychotic and anxiolytic efficacy. Additionally, this compound modulates mesocortical neurotransmission and provides a novel mechanism for the treatment of psychiatric disorders that may be associated with improved efficacy and reduced incidence of undesirable side effects. As glutamatergic dysfunction has been linked to the etiology of schizophrenia, clinical studies with more potent mGlu2/3 agonists, such as LY404039, may be useful to explore the validity of this hypothesis.

Early postnatal protein malnutrition causes resistance to the anxiolytic effects of diazepam as assessed by the fear-potentiated startle test

Given that protein malnutrition induces structural, neurochemical and functional changes in the CNS, the present study aimed to investigate the effects of different early periods of protein malnutrition on the behavior and reactivity to diazepam (DZ) in a model of anxiety: the fear-potentiated startle (FPS). Male Wistar rats (n = 110) from well-nourished (16%-protein) or malnourished (6%-protein) litters were distributed in five different groups: W (well-nourished), M7 (malnourished for 7-days, since day 0), M14 (14-days), M21 (21-days) and M28 (28-days). The results obtained in FPS revealed that malnourished-animals acquired the startle response, irrespective of the time they were exposed to the diet. Besides, DZ reduced the startle amplitude in the noise-alone and light-noise trials. The data concerning the total freezing time showed that the expression of this response was affected by malnutrition and varied in accordance with the findings of previous studies in which malnutrition procedures was imposed for long periods (more than 50 days). Therefore, we suggest that early protein malnutrition: (a) did not produce deficits in the associative learning process of these animals in the FPS, and (b) decreased freezing time in the FPS and produce hyporeactivity to the effects of DZ in rats malnourished for 21 days or more, indicating alterations in the GABAergic neurotransmitter system.

An Overview of SSR149415, a Selective Nonpeptide Vasopressin V1b Receptor Antagonist for the Treatment of Stress-Related Disorders

Vasopressin (AVP) and corticotropin-releasing factor (CRF) are key mediators in the organism's neuro-adaptive response to stress. Through pituitary and central vasopressin V(1b) receptors, AVP participates in the control of the hypothalamic-pituitary-adrenal axis (HPA) and is involved in various emotional processes. SSR149415 is the first selective, orally active vasopressin V(1b) receptor antagonist yet described. It is a competitive antagonist with nanomolar affinity for animal and human V(1b) receptors and displays a highly selective profile with regard to a large number of receptors or enzymes. In vitro, SSR149415 potently antagonizes functional cellular events associated with V(1b) receptor activation by AVP, such as intracellular Ca(2+) increase or proliferation in various cell systems. Pharmacological studies, performed by measuring ACTH secretion induced by various stimulants such as hormones (AVP or AVP + CRF) or physical stress (restraint or forced swimming stress and dehydration) in conscious rats or mice, confirm the antagonist profile of SSR149415 and its efficacy in normalizing ACTH secretion in vivo. SSR149415 is active by the oral route, at doses from 3 mg/kg, it potentiates CRF effect and displays a long-lasting oral effect in the different models. At 10 mg/kg p.o. its duration of action is longer than 4 h. This molecule also decreases anxiety and exerts marked antidepressant-like activity in several predictive animal models. The anxiolytic effects of SSR149415 have been demonstrated in various Generalized Anxiety Disorders (GAD) models (four-plate, punished drinking, elevated plus-maze, light dark, mouse defense test battery, fear-potentiated startle and social interaction tests). It is as effective as the benzodiazepine diazepam in the acute stress exposure test. SSR149415 has similar efficacy to the reference antidepressant drug, fluoxetine, in acute (forced-swimming) and chronic (chronic mild stress and subordination stress) situations in rodents. SSR149415 also reduces offensive aggression in the resident-intruder model in mice and hamsters. Depending on the model, the minimal effective doses are in the range of 1-10 mg/kg i.p. or 3-10 mg/kg p.o. SSR149415 is devoid of adverse effects on motor activity, sedation, memory or cognitive functions and produces no tachyphylaxis when administered repeatedly. It is well-tolerated in animals and humans and exhibits an adequate ADME profile. Thus, SSR149415 is a new dual anxiolytic/antidepressant compound, which appears to be free of the known side effects of classical anxiolytic/antidepressant drugs. Clinical trials are in progress, they will hopefully demonstrate its therapeutical potential for treating stress-related disorders.

The expression of fear-potentiated startle during development: Integration of learning and response systems.

Relative to freezing, fear-potentiated startle (FPS) is developmentally delayed. Rats trained on Postnatal Day (PD) 18 expressed conditioned stimulus learning on PD 19 in freezing but not in FPS, whereas rats trained on PD 24 and tested on PD 25 expressed both freezing and FPS (Experiment 1). According to a neural maturation hypothesis, this delay results from functional immaturity of pathways mediating FPS. When rats were trained on PD 18, neither delaying the FPS test, allowing FPS pathways to develop, nor administrating the "reminder" treatment, the expression of FPS was promoted (Experiments 1, 2, and 2A). PD 18 learning was expressed in FPS on PD 25 when nontarget conditioned stimulus-unconditioned stimulus training occurred prior to the test, and this effect was modality dependent (Experiments 3 and 4). The authors conclude that engaging mechanisms of associative encoding when FPS pathways are functional is a critical condition for integrating learning and FPS response systems in development.

Isolation-Induced Changes in Ultrasonic Vocalization, Fear-Potentiated Startle and Prepulse Inhibition in Rats

Isolation causes important changes in the behavioral reactivity of rats to environmental stimuli. These changes include deficit in sensorimotor gating and altered fear-like responses to aversive stimuli. Measures of ultrasound vocalizations at 20–22 kHz when rats are exposed to threatening conditions, such as novelty, have been taken as a good measure of fear. The fear-potentiated startle to loud sounds and the prepulse inhibition tests have been considered reliable indicators of anxiety and attention impairments, respectively. Rats reared under conditions of isolation from weaning display clear deficits in prepulse inhibition. Taking into account that housing condition changes the emotional state of the animals, we evaluated in this work the performance of rats in the fear-potentiated startle test, prepulse inhibition and emission of ultrasound vocalizations to novelty when isolated for 10 days and after resocialization for 1 week in comparison to grouped rats. Isolated rats showed greater reactivity to loud sounds in the fear-potentiated startle test than grouped animals. They also emitted less ultrasound vocalizations at 20–22 kHz than grouped animals when exposed to a novel environment. In contrast to the well-known deficit in prepulse inhibition displayed by isolation-reared animals, in the present study isolation for 10 days caused a significant increase in prepulse inhibition. Resocialization was not able to counteract the effects of isolation in all three tests. The results suggest that the emotional state of the animals is altered by 10 days of isolation; they do not vocalize characteristically as grouped rats when submitted to novelty; unconditioned responses to loud sounds are enhanced and increased prepulse inhibition is shown rather than a deficit as largely documented in studies with isolation-reared animals. It is suggested that the assessment of the emotional state of the animals is a prerequisite in the evaluation of prepulse inhibition. The level of defensive reactivity displayed by isolated animals is crucial for the functioning of sensory gating and, by extension, to the expression of prepulse inhibition.

Families at high and low risk for depression: A three-generation startle study

Anxiety symptoms might be a vulnerability factor for the development of major depressive disorder (MDD). Because elevated startle magnitude in threatening contexts is a marker for anxiety disorder, the present study investigated the hypothesis that enhanced startle reactivity would also be found in children and grandchildren of individuals with MDD. The magnitude of startle was investigated in two tests (anticipation of an unpleasant blast of air and during darkness) in children (second generation) and grandchildren (third generation) of probands with (high risk) or without (low risk) MDD (first generation). Startle discriminated between the low- and high-risk groups. In the probands' children, the high-risk group showed increased startle magnitude throughout the fear-potentiated startle test. In the probands' grandchildren, a gender-specific abnormality was found in the high-risk group with high-risk girls, but not boys, exhibiting elevated startle magnitude throughout the procedure. Increased startle reactivity in threatening contexts, previously found in patients with anxiety disorder and in children of parents with an anxiety disorder, might also constitute a vulnerability marker for MDD. These findings suggest that there might be common biologic diatheses underlying depression and anxiety.

Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression

The basolateral amygdala's involvement in fear acquisition and expression to visual and auditory stimuli is well known. The involvement of the basolateral and other amygdala areas in fear acquisition and expression to stimuli of other modalities is less certain. We evaluated the contribution of the basolateral and medial amygdala to olfactory and to context fear and fear conditioning by infusing into these areas the NMDA receptor antagonist AP5, the AMPA/kainate receptor antagonist NBQX, or vehicle prior to either odor-shock pairings or fear-potentiated startle testing. Pre-training AP5 infusions into the basolateral amygdala disrupted fear conditioning to the odor but not the context conditioned stimulus (CS). Pre-test NBQX infusions disrupted fear-potentiated startle to the odor but not context CS. Neither compound blocked fear conditioning when infused into the medial amygdala prior to training, but pre-test NBQX infusions did block fear-potentiated startle. The results confirm and extend recent findings suggesting a role for the basolateral amygdala in olfactory fear and fear conditioning, reveal an unexpected dissociation of the basolateral amygdala's involvement in discrete cue versus context fear and fear conditioning, and implicate for the first time the medial amygdala in fear-potentiated startle.

Fear‐Potentiated Startle in Mice

Pavlovian fear conditioning is frequently used to assess the behavioral, physiological, genetic and molecular correlates of learning and memory. In the typical Pavlovian conditioned fear procedure a neutral stimulus, such as a tone, is paired with a mildly aversive stimulus such as a foot shock. The tone conditioned stimulus (CS) comes to elicit a variety of behaviors that are indicative of learned fear. One of the more prominent of these behaviors is a potentiated acoustic startle response. While fear-potentiated startle in mice is qualitatively similar to that in rats, the stimulus parameters and procedures for producing optimum fear-potentiated startle in mice differ considerably from those used in rats. Procedures outlined in this unit include initial assessment of startle, fear conditioning and fear-potentiated startle testing. Special attention is paid to the parameters that affect the magnitude of fear-potentiated startle and procedures designed to systematically examine these parameters are included.

Anxiogenic-like effects of opiate withdrawal seen in the fear-potentiated startle test, an interdisciplinary probe for drug-related motivational states.

Anxiety-like effects may be universal to withdrawal from drugs of abuse. The study of withdrawal would benefit from the acoustic startle response (ASR), a discrete, cross-species reflex which is increased by fear-related states. However, existing reports of opiate-related effects on baseline ASR have not validated ASR as a measure of drug-related motivation. The effects of opiate treatment and withdrawal were examined using fear-potentiated startle, a startle test more sensitive to fear than baseline changes. Fear-conditioned rats were treated with Alzet osmotic pumps delivering 0.25 mg/kg per day fentanyl or placebo pumps. Experiment I examined changes before and during opiate treatment on locomotor activity and baseline, prepulse inhibition, and fear-potentiated startle. Experiment 2 examined the same responses during withdrawal precipitated after 4-7 days of treatment using IV naloxone. Experiment 1 revealed an attenuated fear-potentiated startle on the first test after the start of fentanyl treatment (4 h); this was not seen on subsequent tests and suggested tolerance to this acute effect. Experiment 2 found an enhancement of fear-potentiated startle precipitated in fentanyl-treated rats after injection of 0.025 and 0.16 mg/kg naloxone; this was not seen at 1 mg/kg naloxone, even though more physical withdrawal signs were most prevalent at this dose. In neither experiment did locomotor activity, baseline ASR, or prepulse inhibition of the ASR show any treatment effect. Fear-potentiated startle may provide a specific and valid measure of anxiety-like effects of drug withdrawal. Discussed were conditions needed to see this effect and the relevance of the findings for different mechanisms of withdrawal discomfort.

A nonextinction procedure for long-term studies of classically conditioned enhancement of acoustic startle in the rat

In the Fear-Potentiated Startle (FPS) paradigm, the reflexive response to a noise burst is enhanced when it is presented with a stimulus (typically a light) that previously had been paired with the presentation of an aversive stimulus, usually an electric shock. In the FPS paradigm, the conditioned effect is demonstrated under conditions of extinction, i.e., the light is not paired with the shock during FPS testing. Because of this, the FPS paradigm is of somewhat limited value as a longitudinal measure for studying classically conditioned enhancement of acoustic startle. The present studies report a simple and reliable nonshock procedure for studying classically conditioned potentiation of acoustic startle in the rat that does not utilize testing under conditions of extinction. Naive rats were exposed to 5-or 3-days/week startle test sessions for up to 20 weeks. Twenty (20) startle stimuli (115 dB noise bursts; 40 ms in duration) were presented during each session. Half of these startle stimuli were presented in darkness and half were immediately preceded by a 3500-ms presentation of a 15-W light. With this paradigm, the effects of pairing the light with the startle noise burst could be studied across many test sessions in the absence of extinction training. The light did not increase startle amplitude on the first few startle trials of the first test session. By the end of the first session, however, and continuing for many weeks of testing, startle responses in the presence of the light were significantly greater (by 30–40%) than in the absence of the light. The finding that the startle stimulus itself can serve as an unconditioned stimulus (UCS) to enhance subsequent startle responses replicates an earlier finding. The persistence of this Startle-Potentiated Startle (SPS) effect across multiple weeks of testing is in contrast to that earlier report.

Double Dissociation between the Involvement of the Bed Nucleus of the Stria Terminalis and the Central Nucleus of the Amygdala in Startle Increases Produced by Conditioned versus Unconditioned Fear

The amplitude of the acoustic startle response is reliably enhanced when elicited in the presence of bright light (light-enhanced startle) or in the presence of cues previously paired with shock (fear-potentiated startle). Light-enhanced startle appears to reflect an unconditioned response to an anxiogenic stimulus, whereas fear-potentiated startle reflects a conditioned response to a fear-eliciting stimulus. We examine the involvement of the basolateral nucleus of the amygdala, the central nucleus of the amygdala, and the bed nucleus of the stria terminalis in both phenomena. Immediately before light-enhanced or fear-potentiated startle testing, rats received intracranial infusions of the AMPA receptor antagonist 2, 3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)-quinoxaline (3 microg) or PBS. Infusions into the central nucleus of the amygdala blocked fear-potentiated but not light-enhanced startle, and infusions into the bed nucleus of the stria terminalis blocked light-enhanced but not fear-potentiated startle. Infusions into the basolateral amygdala disrupted both phenomena. These findings indicate that the neuroanatomical substrates of fear-potentiated and light-enhanced startle, and perhaps more generally of conditioned and unconditioned fear, may be anatomically dissociated.

Monoamines in the dorsomedial hypothalamus of rats following exposure to different tests of “anxiety”

1. 1. Adult male Sprague-Dawley rats were subjected to the fear-potentiated startle, elevated plus-maze and social interaction tests and the levels of norepinephrine, dopamine and serotonin in the dorsomedial hypothalamus were determined by high-performance liquid chromatography. 2. 2. Only rats subjected to the full fear potentiated startle test and not the other tests of anxiety or components (foot shocks or acoustic startle) of fear-potentiated startle showed significant increases in norepinephrine and dopamine levels after 24 hours. 3. 3. A time course experiment specific for the norepinephrine changes in the dorsomedial hypothalamus of fear-potentiated startle rats revealed a significant increase in tissue content as compared to controls at both the 12 and 24 hour post-test time points. 4. 4. Tyrosine hydroxylase activity in the dorsomedial hypothalamus of the fear-potentiated startle rats did not show a significant change from controls. 5. 5. An in vitro release study found a significant decrease in potassium-stimulated release of norepinephrine in the dorsomedial hypothalamus as compared to controls at 24 hours posttest. 6. 6. These results suggest that animals exposed to fear-potentiated startle and not other tests of “anxiety” have a change in tissue catecholamine levels and that the norepinephrine change may be the result of a decrease in release and not an increase in synthesis.

Mineralocorticoid and glucocorticoid receptor antagonists in animal models of anxiety

The behavioral effects of intracerebroventricular (ICV) administration of a specific mineralocorticoid receptor (MR) antagonist [RU28318 (10–50 ng/2 μl)], a glucocorticoid receptor (GR) antagonist [RU38486 (1–50 ng/2 μl)], or both antagonists (50 ng/2 μl), were studied in two different animal models of fear and anxiety in rats. In the defensive burying paradigm simultaneous blockade of MR and GR increased immobility behavior, whereas a small decrease in defensive burying was seen. In the fear-potentiated startle test concurrent MR and GR blockade led to an increase in fear-potentiated startle at the highest loudness level (105 dB). In both tests the antagonists were not effective when given separately. The findings are discussed in terms of the involvement of GR and MR in neural mechanisms of fear and anxiety.

Fear-potentiated startle elevates catecholamine levels in the dorsomedial hypothalamus of rats

The norepinephrine (NE), dopamine (DA), and serotonin (5-HT) systems are thought to be important in the development of anxiety and stress. The dorsomedial hypothalamus (DMH) of rats has been implicated in the regulation of physiological and behavioral responses associated with fear and anxiety. In order to elucidate the interactions between the monoamine systems and the DMH, we studied the effects of subjecting rats to the fear-potentiated startle test, a commonly used test of anxiety in rats, on the NE, DA, and 5-HT levels in the DMH. Rats in the potentiated startle test, but not those exposed to just foot shocks or acoustic startle, showed significantly higher levels of NE and DA in the DMH compared to cage controls. In contrast, foot shocks significantly elevated the 5-HT levels in the DMH.