Startle responses to electric shocks: measurement of shock sensitivity and effects of morphine, buspirone and brain lesions

The present study developed a new protocol to assess shock sensitivity in rats. Male Wistar rats were subjected to footshock stimuli ranging from 0 to 1.6 mA (0.1 s) in a startle apparatus and startle responses elicited by shocks were measured. Acoustic stimuli (95, 105, or 115 dB) were dispersed within the shock series serving as a control measurement of motor performance. Results indicated that the magnitude of shock startle responses significantly increased with the shock intensity in a linear trend. Morphine (8.0 mg/kg) and buspirone (1.0, 2.5, or 5.0 mg/kg), both of which possessing analgesic effects, depressed shock startle but had no such effect on acoustic startle. The effect of morphine was readily reversed by pretreatment of naloxone (1.0 mg/kg). To investigate the neural basis underlying this response, radio-frequency lesions of various structures implicated in processing of nociceptive or aversive information were undertaken. Lesions of the ventroposterior thalamic nucleus, insular cortex, or amygdala decreased startle reactivity to electric shocks but not to acoustic stimuli. Lesions of the anterior cingulate gyrus or medial prefrontal cortex, while altered the reactivity to acoustic stimuli, had no effect on the shock-elicited startle. These results suggested that the amplitude of startle in response to electric shocks provide a quantitative measurement of shock sensitivity within an extended range of stimulus intensities. Performing this response may engage the the central nociceptive pathway.     

Latent contextual inhibition of the cardiac component in the startle reaction to a sound stimulus in rats

Heart rate (HR), freezing score, and motor component were estimated during acoustic startle (ASR) habituation (two sessions with 24-hour interval, 10 trials in a session). It was shown that rats previously exposed to the experimental context (once for 5 min 24 h before training) demonstrated HR decrease in response to the first stimulus and tachycardia in response to the 4th-10th stimuli during the first session. The interstimulus HR declined from the 4th to the 6th trials. The same profile of cardiovascular response was observed in this group at the beginning of the second session with the following (to the 7th trial) habituation of tachycardia. These rats didn't demonstrate the intertrial HR decrease during the second session. Nonadapted rats responded by bradycardia to the 1st and 2nd trials of the first session. The response didn't change for tachycardia with continuation of stimuli presentation. Tachycardia appeared only in response to the 7th-10th trials of the second session and didn't habituate. The intertrial HR level decreased in this group only during the second session. The results are discussed in terms of contextual latent inhibition of the cardiac acoustic startle response.     

Expression of freezing and fear‐potentiated startle during sustained fear in mice

Fear‐potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear‐potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo‐randomized conditioned–unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano‐electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.     

Neural cell adhesion molecule (NCAM-/-) null mice show impaired sensitization of the startle response

The neural cell adhesion molecule (NCAM) plays important roles in development of the nervous system and in synaptic plasticity and memory formation in the adult. The present study sought to further investigate the role of NCAM in learning by testing habituation and footshock sensitization learning of the startle response (SR) in NCAM null mutant (NCAM-/-) and wildtype littermate (NCAM+/+) mice. Whereas habituation is a form of non-associative learning, footshock sensitization is induced by rapid contextual fear conditioning. Habituation was tested by repetitive presentation of acoustic and tactile startle stimuli. Although NCAM-/- mice showed differences in sensitivity in both stimulus modalities, habituation learning was intact in NCAM-/- mice, suggesting that NCAM does not play a role in the mechanisms underlying synaptic plasticity in the startle pathway. Footshock sensitization was elicited by presentation of electric footshocks between two series of acoustic stimuli. In contrast to habituation, footshock sensitization learning was attenuated in NCAM-/- mice: the acoustic SR increase after the footshocks was lower in the mutant than in wildtype mice, indicating that NCAM plays an important role in the relevant brain areas, such as amygdala and/or the hippocampus.     

Dynamics of the level of extracellular dopamine in the rat brain during formation of conditioned fear and acoustic startle habituation

The content of extracellular dopamine (DA) was estimated in dorsal striatum (DS), nucleus accumbens (Nac), and prefrontal cortex (Pfc) of rat brain during two sessions of acoustic startle habituation consisting of 10 trials each with 24-hour intersession and 20-s intertrial intervals. Startle amplitude and freezing behavior were recorded. A decrease in the content of extracellular DA in the DS and an increase in the DA content in Nac were observed during both sessions of habituation with return to a baseline level immediately after termination of the sessions. During the second session, the startle amplitude and change in the DA content in both structures were much lower than during the first session. During the first session, the DA level in DS remained unchanged but dropped in Nac. In the Pfc the DA level increased during both habituation sessions and in the period between the session. Time of freezing prior to acoustic stimulation in the second sessions (that is known to be an indication of conditioned fear) correlated with the DA level in DS on the day of training and with the DA level in Nac just before the beginning of the second session. The role of the dopaminergic system in formation and retrieval of different components of defensive behavior is discussed.     

Increasing selectivity of defense behavior during development of pied flycatcher nestlings

The development of defense reaction was studied in the wildlife and experimentally in 7 broods of altricial pied flycatcher (Ficedula hypoleuca) nestlings. Field studies demonstrated that passive-defense response first appeared on the 4th day of the nest life. It developed from the cessation of begging in young relatively satiated nestlings to characteristic freezing response independent of the level of feeding motivation in older nestlings. Older nestlings also acquire the defense reaction in response to novel visual stimuli. The efficiency of the natural stimulus for defense behavior (species-specific alarm call) nongradually changes during the nest life attaining the 100% level only on the 11th posthatching day. During the initial phase of defense behavior development, the reaction can be induced by different rhythmically organized stimuli. Later it becomes considerably more selective and other rhythmic and acoustic signals become much less effective than the alarm call.     

Effect of Dorsal and Ventral Hippocampal Lesions on Contextual Fear Conditioning and Unconditioned Defensive Behavior Induced by Electrical Stimulation of the Dorsal Periaqueductal Gray

The dorsal (DH) and ventral (VH) subregions of the hippocampus are involved in contextual fear conditioning. However, it is still unknown whether these two brain areas also play a role in defensive behavior induced by electrical stimulation of the dorsal periaqueductal gray (dPAG). In the present study, rats were implanted with electrodes into the dPAG to determine freezing and escape response thresholds after sham or bilateral electrolytic lesions of the DH or VH. The duration of freezing behavior that outlasted electrical stimulation of the dPAG was also measured. The next day, these animals were subjected to contextual fear conditioning using footshock as an unconditioned stimulus. Electrolytic lesions of the DH and VH impaired contextual fear conditioning. Only VH lesions disrupted conditioned freezing immediately after footshock and increased the thresholds of aversive freezing and escape responses to dPAG electrical stimulation. Neither DH nor VH lesions disrupted post-dPAG stimulation freezing. These results indicate that the VH but not DH plays an important role in aversively defensive behavior induced by dPAG electrical stimulation. Interpretations of these findings should be made with caution because of the fact that a non-fiber-sparing lesion method was employed.     

Difference in anxiety and sensitization of the acoustic startle response between the two inbred mouse strains BALB/cAN and DBA/2N

The inbred mouse strain BALB has been proposed to be an animal model for pathological anxiety. BALB exhibits a stronger acoustic startle response (ASR) than the 'less emotional' inbred strain DBA. Four experiments were conducted to determine whether this strong ASR is due to a higher anxiety level and/or to greater sensitization in BALB than in DBA, with the following results: (1) The ASR to the very first startle stimulus was found to be much stronger in BALB than in DBA, and freezing behavior evoked by startle stimuli was more pronounced in BALB than in DBA. These findings indicate a higher level of anxiety in this strain. (2) ASR amplitudes of BALB initially rose much higher during consecutive startle stimuli and remained at a high level much longer than in DBA. Thereafter, ASR amplitude dropped more slowly and to a lesser degree than in DBA. Startle amplitudes decreased similarly in both strains (strong exponential decrease) only when a low sound pressure level (SPL) was used which elicited approximately the same low ASR in both strains. These results can only be explained by increased sensitization in BALB. (3) The slope of the i/o-function, which represents the relation between sensory input and motor output, was steeper in BALB than in DBA. As it has been shown recently, sensitization increases the slope of the startle i/o-function indicating increased sensitization in BALB. It is discussed, however, whether anxiety also contributes to this effect. (4) Footshocks increased the ASR much less in BALB than in DBA, again showing increased sensitization in BALB. Both a higher level of anxiety and greater sensitization therefore determined the greater strength of the ASR in BALB than in DBA.     

Foraging strategy switching in an antlion larva

Prepulse inhibition (PPI) refers to the process wherein startle responses to salient stimuli (e.g., startling sound pulses) are attenuated by the presentation of another stimulus (e.g., a brief pre-pulse) immediately before the startling stimulus. Accordingly, deficits in PPI reflect atypical sensorimotor gating that is linked to neurobehavioral systems underlying responsivity to emotionally evocative cues. Little is known about the effects of changes in visual contextual information in PPI among humans. In this study, the effects of introducing unexpected changes in the visual scenes presented on a computer monitor on the human auditory startle response and PPI were assessed in young adults. Based on our animal data showing that unexpected transitions from a dark to a light environment reduce the startle response and PPI in rats after the illumination transition, it was hypothesized that novel changes in visual scenes would produce similar effects in humans. Results show that PPI decreased when elements were added to or removed from visual scenes, and that this effect declined after repeated presentations of the modified scene, supporting the interpretation that the PPI reduction was due to novel information being processed. These findings are the first to demonstrate that novel visual stimuli can impair sensorimotor gating of auditory stimuli in humans.     

Modification of acoustic startle by microwave pulses in the rat: a preliminary report.

Single, 1.25-GHz microwave pulses of 0.8- to 1.0-microseconds duration were presented to each of four rats 100 ms before presentation of a startle-inducing acoustic stimulus. This sequential pairing of microwave pulse and acoustic stimulus was found to modify the startle response. At an energy dose to the head of 22-43 mJ/kg per pulse (peak SAR, 23-48 kW/kg), the mean latency to the startle response was longer and the mean amplitude of the response was smaller with respect to control responses that occurred to acoustic stimuli alone. However, at a higher energy dose per microwave pulse in the range of 59-107 mJ/kg (peak SAR, 63-111 kW/kg), the mean latency and amplitude of the startle response were not statistically different from the respective means of control responses.     

High predisposition to catalepsy decreases intermale aggression and increases acoustic startle reflex amplitude

Reaction of freezing (a pronounced motor inhibition, catalepsy) is suggested to be associated with fear in response to predator appearance or attack of aggressive congener. In order to evaluate association between a kind of behavior such as freezing, aggressiveness and fear, the effects of high predisposition to catalepsy on intermale aggression, acoustic startle response and anxiety-related behavior in the light/dark test were studied. Mice of 14th and 15th generations of selective breeding for high predisposition to catalepsy were characterized by a significant decrease in aggressive behavior. The marked decrease in the percentage of aggressive mice in the catalepsy-prone strain is consistent with the notion that aggression and catalepsy represent two alternative kinds of behavior in intermale conflicts. A positive correlation was found between high predisposition to catalepsy and startle reflex amplitude (but not anxiety-related behavior).     

Habituation and prepulse inhibition of acoustic startle in rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents(1). Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see( 2, 3). The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level(3). We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)(4). Habituation is stimulus specific(5). Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus(6). The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used. Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating(7). PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment(8-10). Both habituation and PPI are disrupted in patients suffering from schizophrenia(11), and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics(12, 13). However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)(11, 14, 15) Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs(16), but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.     

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.     

Manifestation of active and passive defensive behavior in juvenile rats

The dynamics of freezing and flight reactions in juvenile rats was investigated. The rats were tested on the 20th, 25th, 35th, and 40th postnatal days. A sound of 6-sec duration (bell) was used as a threatening stimulus. The following parameters were recorded: number of rearing reactions and defecations within 5 min prior to stimulation, reactions to the bell, latent periods and durations of freezing reactions, freezing posture rigidity, and time of recovery of movements after freezing. It was shown that the intensity of freezing reduced in the period from the 20th to 35th postnatal day. The flight reactions were highest on the 25th and 40th days. Correlations between freezing indices and numbers of rearing and defecation reactions were different in rats of all age groups. The results suggest that the structure of defensive behavior changes with maturation of principal defensive reactions in rats within the first 40 postnatal days.     

Intensity Characteristics of the Noctuid Acoustic Receptor

Spiking activity of the more sensitive acoustic receptor is described as a function of stimulus intensity. The form of the intensity characteristic depends strongly on stimulus duration. For very brief stimuli, the integral of stimulus power over stimulus duration determines the effectiveness. No response saturation is observed. With longer stimuli (50 msec), a steady firing rate is elicited. The response extends from the spontaneous rate of 20–40 spikes/sec to a saturated firing rate of nearly 700 spikes/sec. The characteristic is monotonic over more than 50 db in stimulus intensity. With very long stimuli (10 sec), the characteristics are nonmonotonic. Firing rates late in the stimulus decrease in response to an increase in stimulus intensity. The non-monotonic characteristics are attributed to intensity-related changes in response adaptation.     

Influence of pre- and postnatal exposure of rats to 2.45-GHz microwave radiation on neurobehavioral function

Rats exposed to microwaves prenatally (2,450 MHz, 10 mW/cm2, 3 h/day, days 5-20 of gestation) or perinatally (same as above plus days 2-20 postnatally) were examined by a neurobehavioral test battery on postnatal days 30 and 100. Body mass, locomotor activity, startle to acoustic and air-puff stimuli, fore- and hindlimb grip strength, negative geotaxis, reaction to thermal stimulation, and swimming endurance were assessed. The prenatally and the perinatally exposed rats (male and female) weighted more than sham-exposed rats at 30, but not at 100, days of age. In addition, the perinatally exposed animals had less swimming endurance at 30, but not at 100, days of age relative to sham-exposed rats. For the other measures, only the air-puff startle response was altered and was limited to the prenatally exposed female pups; ie, at postnatal day 30, the startle response was increased in magnitude, and at postnatal day 100, the response was decreased. No other reliable effects were observed. In a second experiment, rats treated as described above were examined for alterations in body mass, locomotor activity, reaction to air-puff stimuli, reaction to thermal stimulation, and swimming endurance at postnatal days 30-36. Again, perinatally exposed rats were larger in body mass and had less swimming endurance compared with sham-exposed rats. The latency to the air-puff startle response was longer in female pups exposed prenatally. These data indicate that altered endurance and gross motor activity result from perinatal exposure to microwave irradiation.     

Signaling without the risk of illegitimate receivers: do predators respond to the acoustic signals of <Emphasis Type="Italic">Cyprinella</Emphasis> (Cyprinidae)?

Acoustic and visual signals are commonly used by fishes for communication. A significant drawback to both types of signals is that both sounds and visual stimuli are easily detected by illegitimate receivers, such as predators. Although predator attraction to visual stimuli has been well-studied in other animals, predator response to acoustic stimuli has received virtually no research attention among fishes and snakes. This study assessed whether the calls of male tricolor shiner (Cyprinella trichroistia) made during the breeding season would attract potential predators. We also examined the effect of visual stimulus of tricolor shiners on predators. Predators used were red eye bass (Micropterus coosae) and midland water snakes (Nerodia sipedon pleuralis). Neither predator was attracted to tricolor sounds when presented alone. Micropterus coosae responded significantly more to a visual stimulus, and to a combination of visual and acoustic stimuli, but with no greater intensity in the latter. Nerodia sipedon pleuralis did not responded to visual stimulus presented alone, but did respond to visual and acoustic stimuli presented simultaneously, and with greater intensity to the latter, indicating that acoustic signals may play a role in prey detection by N. sipedon pleuralis.     

Some behavioral effects of short-term exposure of rats to 2.45 GHz microwave radiation

Rats were tested for neurobehavioral alterations immediately after exposure to 2.45-GHz (CW) microwave radiation at 10 mW/cm2 for 7 h. Behavioral tests used were locomotor activity, startle to an acoustic stimulus and acquisition and retention of a shock-motivated passive avoidance task. Both horizontal and vertical components of locomotor activity were assessed in 5-min epochs for a period of 30 min using photoelectric detectors. Microwave-exposed animals exhibited less activity than sham-exposed animals. This was most evident during the last 10-15 min of the 30-min test session. Twenty identical acoustical stimuli (8 KHz, 110 dB) were delivered to each rat at 40-s intervals. The microwave-exposed animals were less responsive to the stimuli than sham-exposed animals. Microwave exposure had no effect on the retention of a passive avoidance procedure when tested at 1 week after training. Both the locomotor activity and acoustic startle data demonstrate that, under the conditions of this experiment, microwave exposure may alter responsiveness of rats to novel environmental conditions or stimuli.     

Low tryptophan diet increases stress-sensitivity, but does not affect habituation in rats

Cerebral dysfunction of 5-HT (serotonin) has been associated with stress response and with affective disorders. Stress alone is insufficient to induce depression, since only a minor proportion of subjects that have experienced stressful life events develop depressive episodes. We investigated whether long-term brain 5-HT depletion induced in rats by a diet with low content of its precursor tryptophan affects stress-responsiveness in rats. Stress-sensitivity was measured through various physiological parameters and by measuring the rats' response to acoustic stimuli. One group of rats was subjected to daily acoustic stimulus sessions for 5 days. Other groups received both immobilization stress and acoustic stimulus sessions daily for either 9 days (chronic experiment) or 1 day (acute experiment). A low tryptophan diet led to decreases in plasma tryptophan levels, low ratio of tryptophan/large neutral amino acid, whole blood 5-HT, and neuronal 5-HT content in the Dorsal and Median Raphe Nuclei, as well as altered c-fos expression in the brain. Without concomitant immobilization, the diet alone did not affect reactivity and habituation to acoustic stimuli, although plasma corticosterone levels, but not the adrenal weights, were increased on day 5. Low tryptophan and chronic immobilization stress together with the acoustic testing procedure increased adrenal weight, plasma corticosterone levels and reactivity to the acoustic stimuli, but not the rate of habituation to acoustic stimuli. These results show that cerebral dysfunction of serotonin achieved through a low tryptophan diet, increases the sensitivity of rats to external and stressful stimuli, but does not impair the capacity to adapt to these stimuli. Accordingly, brain-serotonin modulates reactivity to stress, but not stress coping.     

Startle Responses in Motor Vehicle Accident Survivors: A Pilot Study

The aim of the present study was to investigate startle responses in motor vehicle accident (MVA) survivors to trauma-related, startle, and neutral sounds. Participants were 17 MVA survivors, 11 of whom participated in a controlled treatment study comparing cognitive-behavioral treatment (CBT) and supportive therapy (ST) versus a waitlist condition. Though participants differed significantly in their pretreatment clinical status and symptom severity, these differences were not reflected by group differences in EMG (at orbicularis oculi) to the stimuli at the initial assessment. Some cue-specificity was found, as all participants showed larger startle responses to trauma-related sounds, compared to startle and neutral sounds. At posttreatment, a significant reduction in EMG reactivity to all stimuli was observed in participants who received active treatment (either CBT or ST), compared to waitlist controls. The use of startle responses as a PTSD treatment outcome index is discussed.