Fear-potentiated Startle In Rats Research Articles

Acute, but not repeated, administration of the neurotensin NTS1 receptor agonist PD149163 decreases conditioned footshock-induced ultrasonic vocalizations in rats

Neurotensin is an endogenous neuropeptide that has significant interactions with monoamine neurotransmitter systems. To date, neurotensin NTS1 receptor agonists, such as PD149163, have been primarily evaluated for the treatment for schizophrenia, drug addiction, and pain. Recently, PD149163 was found to attenuate fear-potentiated startle in rats, an experimental procedure used for screening anxiolytic drugs. The present study sought to assess these findings through testing PD149163 in a conditioned footshock-induced ultrasonic vocalization (USV) model. Conditioning was conducted in male Wistar rats using chambers equipped with shock grid floors and an ultrasonic vocalization detector. PD149163 and the 5-HT1A receptor partial agonist buspirone produced a statistically significant reduction of 22kHz USV counts. The typical antipsychotic haloperidol also reduced 22kHz USV counts, but did so at cataleptic doses. Ten days of repeated administration of PD149163 abolished the inhibitory effects of PD149163 on 22kHz USVs. These findings further support an anxiolytic profile for PD149163. However, tolerance to these effects may limit the utility of these drugs for the treatment of anxiety.

Retrograde amnesia for fear-potentiated startle in rats after complete, but not partial, hippocampal damage

We assessed the involvement of the hippocampus in recall of learned fear of a discrete visual stimulus using a fear-potentiated startle (FPS) procedure. Recall was measured by an increase in acoustic startle in the presence of a light that was paired with footshock. In Experiment 1, rats either received sham, dorsal, ventral, or complete (dorsal and ventral) NMDA-induced damage of the hippocampus following FPS acquisition. During the post-surgery retention test, only the rats with complete hippocampal damage showed a significant FPS deficit. In Experiment 2, we examined whether recent and remote memory for FPS would be differentially affected by complete hippocampal damage. Rats received sham or complete hippocampal damage 1- or 4-wk after FPS acquisition. During the retention test, sham rats exhibited significant FPS, whereas rats with hippocampal damage showed a large FPS deficit that was equivalent for recent and remote memories. In Experiment 3, we found that rats with complete hippocampal damage induced before conditioning showed levels of FPS that did not significantly differ from sham rats. Combined, these findings suggest that extensive damage to the hippocampus causes retrograde amnesia for a memory involving a light–shock association that is not temporally graded. The same damage does not cause anterograde amnesia in the same memory task. Partial damage of the hippocampus, whether of the dorsal or ventral region, was insufficient to cause retrograde amnesia. Thus, the hippocampus normally has a critical and long-lasting role enabling recall of fear conditioning to a discrete visual stimulus. In the absence of the hippocampus other memory systems support new learning.

Role of corticosterone in trace and delay conditioned fear-potentiated startle in rats.

Emotional events often lead to particularly strong memory formation. Corticosterone, the final product of hypothalamic-pituitary-adrenal (HPA)-axis activation, has been suggested to play a critical role in this effect. Although a great deal of work has implicated the amygdala as a necessary structure for the effects of corticosterone, other studies have suggested a critical role for the hippocampus in determining the involvement of corticosterone. The current experiments examined this question by disrupting corticosterone synthesis with administration of metyrapone (25 or 100 mg/kg) prior to training in either dorsal hippocampus-independent delay fear conditioning or dorsal hippocampus-dependent trace fear conditioning. Metyrapone administration 2 hrs prior to training significantly attenuated corticosterone secretion during training, but these effects were transient as corticosterone levels were similar to control subjects following the test session. As hypothesized, only trace fear conditioning was impaired. This suggests that only fear conditioning tasks that are dependent on the dorsal hippocampus require HPA-axis activation in order to be learned.

Intra-amygdaloid infusion of Ginkgo biloba leaf extract (EGb761) facilitates fear-potentiated startle in rats

Ginkgo biloba extract, EGb761, is one of the most commonly used herbal supplements throughout Western society. It has been used in the treatment of various common geriatric complaints including short-term memory loss. We showed that acute systemic administration of EGb761 enhanced fear-potentiated startle (FPS) in rats. Little is known about the behavioral effects of centrally administered EGb761 on FPS. The current study was performed to evaluate the involvement of basolateral nucleus of amygdala (BLA) in the EGb761 facilitation effect on FPS. Male adult SD rats were used. EGb761 was infused into cerebroventricle or basolateral nucleus of amygdala 10 min prior to fear conditioning. Animals were then tested for FPS 24 h later. Results showed that (1) intra-cerebroventricular infusion of EGb761 (0.1, 1.0, or 3.0 microg/3.0 microl per side, bilaterally) and intra-amygdaloid infusion of EGb761 (1.0, 14.0, or 28.0 ng/microl per side, bilaterally) 30 and 10 min prior to fear conditioning, respectively, facilitated FPS in a dose-dependent manner. (2) Administration of EGb761 did not impair an animal's basal startle response or pain perception. (3) Subsequent control experiment's results indicated that the facilitation effect of EGb761 on the acquisition was not due to anxiogenic effect or non-specific effect. These results suggested that a single dose of EGb761 also has memory-enhancing effects in young animals. In addition, BLA is the central locus for EGb761 facilitation effect on FPS.

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.

Neurotoxic lesions of the dorsal and ventral hippocampus impair acquisition and expression of trace-conditioned fear-potentiated startle in rats

Pavlovian delay conditioning, in which a conditioned stimulus (CS) and unconditioned stimulus (US) co-terminate, is thought to reflect non-declarative memory. In contrast, trace conditioning, in which the CS and US are temporally separate, is thought to reflect declarative memory. Hippocampal lesions impair acquisition and expression of trace conditioning measured by the conditioned freezing and eyeblink responses, while having little effect on the acquisition of delay conditioning. Recent evidence suggests that lesions of the ventral hippocampus (VH) impair conditioned fear under conditions in which dorsal hippocampal (DH) lesions have little effect. In the present study, we examined the time-course of fear expression after delay and trace conditioning using the fear-potentiated startle (FPS) reflex, and the effects of pre- and post-training lesions to the VH and DH on trace-conditioned FPS. We found that both delay- and trace-conditioned rats displayed significant FPS near the end of the CS relative to the unpaired control group. In contrast, trace-conditioned rats displayed significant FPS throughout the duration of the trace interval, whereas FPS decayed rapidly to baseline after CS offset in delay-conditioned rats. In experiment 2, both DH and VH lesions were found to significantly reduce the overall magnitude of FPS compared to the control group, however, no differences were found between the DH and VH groups. These findings support a role for both the DH and VH in trace fear conditioning, and suggest that the greater effect of VH lesions on conditioned fear might be specific to certain measures of fear.

Activation of group II metabotropic glutamate receptors induces depotentiation in amygdala slices and reduces fear-potentiated startle in rats

There is a close correlation between long-term potentiation (LTP) in the synapses of lateral amygdala (LA) and fear conditioning in animals. We predict that reversal of LTP (depotentiation) in this area of the brain may ameliorate conditioned fear. Activation of group II metabotropic glutamate receptors (mGluR II) with DCG-IV induces depotentiation in the LA. The induction of depotentiation is independent of NMDA receptors, L-type Ca++ channels, and calcineurin activity, but requires presynaptic activity and extracellular Ca++. (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) depotentiation is accompanied by a decrease in the frequency but not the amplitude of miniature excitatory post-synaptic currents (mEPSCs) and could be mimicked by endogenously released glutamate. DCG-IV inhibited the release of glutamate evoked by 4-AP but not that evoked by ionomycin, suggesting that the effect of DCG-IV is not mediated by an action downstream of Ca++ entry. Intra-amygdala infusion of mGluR II agonist blocks the consolidation of fear memory measured with fear-potentiated startle. Taken together, the present results characterize the properties of DCG-IV depotentiation and reveal a close parallel between depotentiation in the amygdala slice and the reduction of conditioned fear in animals.

Fear-potentiated startle in rats is mediated by neurons in the deep layers of the superior colliculus/deep mesencephalic nucleus of the rostral midbrain through the glutamate non-NMDA receptors.

The amygdala sends heavy and broad projections to the rostral midbrain including the periaqueductal gray (PAG), the deep layers of the superior colliculus/deep mesencephalic nucleus (deep SC/DpMe), and the lateral mesencephalic reticular formation (MRF) that in turn project to the nucleus reticularis pontis caudalis (PnC), an obligatory relay in the primary acoustic startle circuit. Chemical lesions or inactivation of these areas blocked fear-potentiated startle, suggesting that these areas serve as a relay between the amygdala and the PnC. In the present study, we tried to determine more precisely which of these sites were critical for fear-potentiated startle and the role of glutamate receptors in this site in mediating fear-potentiated startle. Local infusion of the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline (NBQX) dose-dependently blocked fear-potentiated startle when infused into the deep SC/DpMe before testing but had no effect on baseline startle amplitude. NBQX did not block fear-potentiated startle when infused before training. The same dose of NBQX infused into the dorsal/lateral PAG, the lateral MRF, or the superficial layers of the SC did not affect fear-potentiated startle. However, NBQX tended to reduce contextual freezing when infused into the dorsal/lateral PAG. These findings suggest that the deep SC/DpMe is the site that serves as a critical output relay between the amygdala and the PnC in mediating fear-potentiated startle and that glutamatergic transmission is required for this action.

Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats

Recent research suggests that in addition to its prominent role in appetitive learning, the nucleus accumbens (NAC) may also be involved in fear conditioning. In the present study, we investigated whether temporary inactivation of the NAC, by injection of tetrodotoxin (TTX), affects acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). TTX injection into the NAC totally blocked acquisition and markedly decreased expression of conditioned fear to a discrete visual conditioned stimulus (CS). Interestingly, temporary inactivation of the NAC did not affect shock sensitization of startle, indicating that both the perception of the shock and short-term contextual conditioning was not affected by intra-accumbal TTX injection. Taken together, these results show that the NAC is crucial for acquisition and expression of long-term conditioned fear, as measured by fear-potentiated startle, to discrete CSs, but not short-term conditioned fear to a context.

Inhibition of fear potentiated startle in rats following peripheral administration of secretin.

Previous results indicate that peripheral administration of secretin leads to robust Fos protein expression in the central nucleus of the rat amygdala. The implications of this observation on rat brain function, if any, remain unclear. We examined the effect of systemic secretin administration on the expression of fear-potentiated startle in rats, a behavioral response known to require an intact, functional central nucleus of the amygdala. Rats were trained to associate a neutral light conditioned stimulus (CS) with footshock, a fear-inducing unconditioned stimulus (US). Twenty-four hours later, rats were administered secretin or vehicle and were tested immediately for their startle response to a loud noise in the presence or absence of the light. Within a dose range relevant to its clinical use in autistic children, secretin dose-dependently decreased the magnitude of fear-potentiated startle in rats. This investigation provides additional evidence that systemically administered secretin can influence a neural network implicated in the acquisition and expression of emotional behaviors, including fear and anxiety.

LY354740, an mGlu2/3 Receptor Agonist as a Novel Approach to Treat Anxiety/Stress

Metabotropic glutamate (mGlu) receptors, which include mGlu1-8 receptors, are a heterogeneous family of G-protein coupled receptors (GPCRs) that function to modulate neuronal excitation and plasticity via pre-synaptic, post-synaptic and glial mechanisms. Agonists for group II mGlu receptors (mGlu2 and mGlu3), such as LY354740, have been shown to suppress enhanced glutamatergic excitations in brain synapses known to be involved in the expression of fear/anxiety in animals and humans. Systemic administration of LY354740 increases open-arm time in the elevated plus maze in mice under conditions of moderate to severe stress, blocks the expression but not development of fear-potentiated startle in rats, prevents lactate-induced panic-like responses in panic-prone rats, and attenuates certain physiological, behavioral, and neurochemical consequences of acute stress in rodents. In these preclinical models, LY354740 does not produce the side-effects (e.g. sedation) that are associated with other anxiolytic agents such as benzodiazepines. Early clinical results with LY354740 have demonstrated safety and efficacy in a human anxiety model (panic provocation induced by CO 2 challenge). Collectively, these data indicate mGlu2/3 receptor agonists such as LY354740 represent a promising new approach for treatment of anxiety and stress-related disorders in humans.

Facilitation of Conditioned Fear Extinction by Systemic Administration or Intra-Amygdala Infusions of d-Cycloserine as Assessed with Fear-Potentiated Startle in Rats

NMDA receptor antagonists block conditioned fear extinction when injected systemically and also when infused directly into the amygdala. Here we evaluate the ability of D-cycloserine (DCS), a partial agonist at the strychnine-insensitive glycine-recognition site on the NMDA receptor complex, to facilitate conditioned fear extinction after systemic administration or intra-amygdala infusions. Rats received 10 pairings of a 3.7 sec light and a 0.4 mA footshock (fear conditioning). Fear-potentiated startle (increased startle in the presence vs the absence of the light) was subsequently measured before and after 30, 60, or 90 presentations of the light without shock (extinction training). Thirty non-reinforced light presentations produced modest extinction, and 60 or 90 presentations produced nearly complete extinction (experiment 1). DCS injections (3.25, 15, or 30 mg/kg) before 30 non-reinforced light exposures dose-dependently enhanced extinction (experiment 2) but did not influence fear-potentiated startle in rats that did not receive extinction training (experiment 3). These effects were blocked by HA-966, an antagonist at the glycine-recognition site (experiment 4). Neither DCS nor HA-966 altered fear-potentiated startle when injected before testing (experiment 5). The effect of systemic administration was mimicked by intra-amygdala DCS (10 microg/side) infusions (experiment 6). These results indicate that treatments that promote NMDA receptor activity after either systemic or intra-amygdala administration promote the extinction of conditioned fear.

Acoustic startle and fear-potentiated startle in rats selectively bred for fast and slow kindling rates: relation to monoamine activity.

The acoustic startle response, prepulse inhibition, fear-potentiated startle and monoamine activity induced by either, a novel stimulus or a cue previously paired with foot-shock (fear-conditioning), were assessed in rats selectively bred for differences in amygdala excitability (Fast vs. Slow kindling epileptogenesis). Comorbid differences of anxiety, which were dependent both on the rats' behavioural style and the kind of stressor, also characterized these strains. In the present investigation, Slow rats exhibited a greater startle reflex to noise relative to Fast rats, suggesting differences in generalized anxiety, but similar rates of startle habituation and prepulse inhibition. The fear-potentiated startle, however, was greater in Fast rats. When movement of the rat was restricted in a new environment, presentation of a novel stimulus (light) increased norepinephrine, dopamine and/or serotonin activity in brain regions typically associated with stressors (e.g. locus coeruleus, paraventricular hypothalamic nucleus). Generally, these effects were more pronounced in Fast rats, and norepinephrine utilization in the central amygdala was particularly highlighted in response to a conditioned fear stimulus. Thus, while generalized anxiety appeared greater in Slow rats, behavioural and central neurochemical reactivity in response to novel stimuli and to fear-eliciting stimuli, was greater in Fast rats. Similarly, basal dopamine activity in the prefrontal cortex was greater in Fast rats, but dopamine utilization elicited by a novel stimulus was more pronounced in Slow rats. This suggested that relative to Slow rats, dopamine neurons in prefrontal cortex of Fast rats do not react normally to environmental stimuli, and this phenomenon could lead to disturbances of attention or impulsivity.

Involvement of NMDA receptors within the amygdala in short- versus long-term memory for fear conditioning as assessed with fear-potentiated startle.

Pretraining intra-amygdala infusions of the NMDA receptor antagonist. D,L-AP5, block fear-potentiated startle in rats tested 24+ hr after training. This may reflect a failure of either acquisition or retention. To evaluate these alternatives, rats were tested for fear-potentiated startle during fear conditioning (30 light-shock pairings [0.6 mA shock]), as well as 1-30 min and 48 hr after fear conditioning. Amygdala lesions abolished fear-potentiated startle at all train-test intervals. Intra-amygdala AP5 infusions (25 nmol/side) abolished fear-potentiated startle during the long-term test and had partial effects at shorter train-test intervals. When the level of fear-potentiated startle during the short-term test was lowered to that of the 48-hr test (i.e., by training rats with a lower, 0.3 mA footshock), AP5 abolished fear-potentiated startle at each timepoint. Thus, amygdala NMDA receptors appear to participate in the initial acquisition of fear memories.

Lasting effect of repeated cocaine administration on acoustic and fear-potentiated startle in rats.

Following cocaine withdrawal, humans may experience an abstinence syndrome with high levels of anxiety. Studying anxious behavior in animals following repeated cocaine administration may help elucidate important variables that contribute to a withdrawal syndrome. This study investigated whether repeated cocaine pre-exposure produced lasting increases in conditioned fear as measured by fear-potentiated startle responses in rats. Startle was measured in response to 50 ms acoustic stimuli of 95, 105 and 115 dB. Cocaine (20 mg/kg, IP) or saline was administered for 7 days and after each injection rats were either placed in startle chambers for 30 min or returned to the home cage. After a 1-week cocaine-free period, most rats were given ten light-footshock pairings in the startle chamber. Fear-potentiated startle was tested by presenting acoustic startle-eliciting stimuli of 95, 105 and 115 dB in the presence or absence of the light. Rats that were not fear conditioned received acoustic stimuli one week after 7 days of 20 mg/kg cocaine. Startle responses, both in the presence and absence of the light CS, were greater in fear-conditioned rats that received cocaine pre-exposure in the startle chamber than in saline pre-exposed rats. Startle responses in the presence of the light CS were further augmented at 115 dB. In contrast, home-cage exposure to cocaine did not enhance startle responses. In rats that were not fear conditioned, cocaine pre-exposure reduced acoustic startle. Repeated cocaine pre-exposure can increase, decrease or not change acoustic startle depending on whether fear conditioning occurred and whether cocaine was given in the testing chamber. The data suggest that cocaine pre-exposure may act as a contextually conditioned occasion setting stimulus that can facilitate anxious behavior similar to the postulated human cocaine abstinence syndrome.

A further investigation of the developmental emergence of fear-potentiated startle in rats.

This series of experiments was designed to reexamine the ontogenetic emergence of the fear-potentiated startle response in rats. Previous results (Hunt, Richardson, & Campbell, 1994) indicated that potentiated startle to a light conditioned stimulus (CS) paired with an acoustic unconditioned stimulus (US) was not observed until 30 days of age. In the present experiments, subjects were given pairings of a light CS with a brief footshock unconditioned stimulus (US) and were tested for fear-potentiated startle 24 hr later. Subjects 23 and 30 days of age exhibited significant potentiated responding in the presence of the light, while 17-and 20-day-olds did not. Subjects 17 days of age did reliably express conditioned decreases in heart rate to the light at the 24-hr test. The failure to observe fear-potentiated startle at the youngest age was shown not to be due to a general disruption of conditioned fear responding by either (a) pretest startle stimulus presentations or (b) contextual characteristics of the startle testing apparatus. The capacity to express fear through a potentiated startle response develops later than the capacity for other defensive responses in the rat.

Muscimol in the deep layers of the superior colliculus/mesencephalic reticular formation blocks expression but not acquisition of fear-potentiated startle in rats.

Deposits of the retrograde tracer Fluoro-Gold into the ventrolateral nucleus reticularis pontis caudalis labeled neurons in the deep layers of the superior colliculus/mesencephalic reticular formation (deep SC/Me). To test the involvement of this area in the fear-potentiated startle effect, rats were implanted with cannulas into the deep SC/Me and trained for fear-potentiated startle after infusion of the GABA(A) agonist muscimol (0.1 microg/0.5 microl). Two days later, they were tested for fear-potentiated startle. Rats then received a 2nd training session without any infusions, and 2 days later they were reinfused with muscimol (0.1 microg/0.5 microl) and tested for fear-potentiated startle. Local infusion of muscimol into the deep SC/Me completely blocked the expression but not the acquisition of fear-potentiated startle. These results indicate that a synapse in the midbrain is critical for the expression of fear-potentiated startle.

Muscimol in the deep layers of the superior colliculus/mesencephalic reticular formation blocks expression but not acquisition of fear-potentiated startle in rats.

Muscimol in the deep layers of the superior colliculus/mesencephalic reticular formation blocks expression but not acquisition of fear-potentiated startle in rats.

Different regions of the periaqueductal grey are involved differently in the expression and conditioned inhibition of fear-potentiated startle.

The fear-potentiated startle paradigm is a useful model for investigating the neural systems underlying the mediation and modulation of fear and anxiety. A series of previous studies showed that the periaqueductal grey is involved in the expression and inhibition of fear-potentiated startle, but it was uncertain whether the different columns of the periaqueductal grey play different roles. In the present study, picrotoxin, a GABAA (gamma-aminobutyric acid) chloride-channel blocker, was injected into the dorsal, lateral and ventrolateral columns of the periaqueductal grey in order to examine the effects of neural disinhibition on expression and conditioned inhibition of fear-potentiated startle. Picrotoxin injections into the lateral periaqueductal grey increase potentiation of the startle response by conditioned fear, injections into the ventrolateral periaqueductal grey block fear-potentiated startle. Picrotoxin injections into the dorsal periaqueductal grey decreased conditioned inhibition but did not affect expression of fear-potentiated startle. The baseline startle amplitude was not influenced by picrotoxin injections into the different columns of the periaqueductal grey. Picrotoxin injections into neighbouring brain areas did not have any effect on baseline startle amplitude nor did they have an effect on expression or conditioned inhibition of fear-potentiated startle. Based on the present results, the possible pathways underlying the expression and conditioned inhibition of fear-potentiated startle in rats are discussed.

Lack of a temporal gradient of retrograde amnesia following NMDA-induced lesions of the basolateral anygdala assessed with the fear-potentiated startle paradigm.

M. Kim and M. Davis (1993b) previously reported that electrolytic lesions of the central nucleus of the amygdala, made 6 or 30 days after training, completely blocked the expression of fear-potentiated startle in rats. The present study shows that excitotoxic lesions of the basolateral amygdala also block fear-potentiated startle and do so whether the lesions are made soon (i.e., 6 days) or long (i.e., 30 days) after training. The relevance of these findings to various theories of amygdala function is discussed.