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.     

Prepulse inhibition （PPI） of tactile startle response in recombinant congenic strains of mice： QTL mapping and comparison with acoustic PPI

Prepulse inhibition (PPI) of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems.We analyzed the tactile startle response (TSR) and PPI of TSR (tPPD,using light as a prepulse stimulus,in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them.Parental strains were significantly different for TSR,but were comparable for tPPI.Among the congenic strains,variation for TSR was significant in both genetic backgrounds,but that of tPPI was significant only for the C57BL/6J background.Provisional mapping for loci modulating TSR and tPPI was carded out.Using mapping data from our previous study on acoustic startle responses (ASR) and PPI of ASR (aPPI),no common markers for aPPI and tPPI were identified.However,some markers were significantly associated with both ASR and TSIL at least in one genetic background.These results indicate cross-modal genetic regulation for the startle response but not for PPI,in these mouse strains.     

Independent genetic loci for sensorimotor gating and attentional performance in BXD recombinant inbred strains

A startle reflex in response to an intense acoustic stimulus is inhibited when a barely detectable pulse precedes the startle stimulus by 30-500 ms. It has been theorized that this phenomenon, named prepulse inhibition (PPI) of a startle response, is an automatic early-stage gating process contributing to the ability to focus attention. Deficits in PPI may therefore contribute to deficits in attentional processing. Both deficits are observed in schizophrenia spectrum disorders. Here, we investigated whether there is overlap in genetic control of PPI and attentional processing phenotypes in the panel of BXD recombinant inbred strains of mice. Using an individually titrated prepulse intensity to handle differences in perceived prepulse intensities among strains, we identified a significant quantitative trait locus (QTL) for PPI at the mid-distal end of chromosome 17. A measure of attentional processing in the five-choice serial reaction time task, response variability, mapped to a different locus on proximal-mid chromosome 16. In addition, the estimated genetic and environmental correlations between PPI and several attentional phenotypes were low and not significant. Taken together, the observation of separate genetic loci for PPI and attention and the absence of genetic and environmental correlations indicate that differences in sensorimotor gating do not contribute to differences in attentional performance. Therefore, it is worth pursuing the causative genes residing in both attention and PPI QTL, as these may contribute to separate molecular pathways implicated in neuropsychiatric diseases, such as schizophrenia.     

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.     

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.     

Effect of 5HT2 receptor blockade on the startle reflex and its prepulse inhibition in mice and rats of various strains]

Effects of 5-HT2 receptor blockade on the amplitude of startle reflex, induced by an unexpected sound, and on its prepulse inhibition (PPI) were studied on mice of CBA strain and rats of Wistar and the genetically predisposed to catalepsy (GC) strains. The effect was dependent on type and dose of 5-HT2 antagonist used: 5-HT2A antagonist ketanserin increased startle amplitude at the dose of 0.5 mg/kg and decreased it at the dose of 2 mg/kg. Mixed 5-HT2A/2C antagonist ritanserin (0.1 and 0.2 mg/kg) markedly increased startle in mice. Ketanserin and cyproheptadine produced opposite effects on startle reflex in rats with inherited neuropathology and in rats with normal genotype: marked decrease in GC rats and increase in Wistar rats was shown. Ketanserin and cyproheptadine produced a pronounced potentiation of PPI in mice and rats of both strains, ritanserin was ineffective. Results suggest 5-HT2 receptors implication in both startle and PPI regulation with 5-HT2C receptors in startle response and 5-HT2A in PPI predominant involvement.     

Correlation between cataleptic freezing and prestimulation inhibition of the startle reflex in rats

In rats of GC strain bred for predisdposition to cataleptic freezing, a significant negative correlation between the duration of freezing and the level of prepulse inhibition (PPI) of the startle reflex, has been found. Besides, in a group of GC rats specific by their "nervousness" and jumpiness, there was also a negative correlation between the duration of freezing and the habituation to the startle reflex. None of this correlation have been found in Wistar rats. Since impairment of the PPI and habituation of the startle reflex is considered to be characteristic of schizophrenia, it is believed than cataleptic freezing in the GC rats may be used as a model of schizophrenic psychopathology.     

Hypnotizability,Hypnosis and Prepulse Inhibition of the Startle Reflex in Healthy Women: An ERP Analysis

A working model of the neurophysiology of hypnosis suggests that highly hypnotizable individuals (HHs) have more effective frontal attentional systems implementing control, monitoring performance, and inhibiting unwanted stimuli from conscious awareness, than low hypnotizable individuals (LHs). Recent studies, using prepulse inhibition (PPI) of the auditory startle reflex (ASR), suggest that HHs, in the waking condition, may show reduced sensory gating although they may selectively attend and disattend different stimuli. Using a within subject design and a strict subject selection procedure, in waking and hypnosis conditions we tested whether HHs compared to LHs showed a significantly lower inhibition of the ASR and startle-related brain activity in both time and intracerebral source localization domains. HHs, as compared to LH participants, exhibited (a) longer latency of the eyeblink startle reflex, (b) reduced N100 responses to startle stimuli, and (c) higher PPI of eyeblink startle and of the P200 and P300 waves. Hypnosis yielded smaller N100 waves to startle stimuli and greater PPI of this component than in the waking condition. sLORETA analysis revealed that, for the N100 (107 msec) elicited during startle trials, HHs had a smaller activation in the left parietal lobe (BA2/40) than LHs. Auditory pulses of pulse-with prepulse trials in HHs yielded less activity of the P300 (280 msec) wave than LHs, in the cingulate and posterior cingulate gyrus (BA23/31). The present results, on the whole, are in the opposite direction to PPI findings on hypnotizability previously reported in the literature. These results provide support to the neuropsychophysiological model that HHs have more effective sensory integration and gating (or filtering) of irrelevant stimuli than LHs.     

Whole-Body Prepulse Inhibition Protocol to Test Sensorymotor Gating Mechanisms in Monkeys

Prepulse inhibition (PPI) is the decrease of startle reflex amplitude when a slight stimulus is previously generated. This paradigm may provide valuable information about sensorimotor gating functionality. Here we aimed at determining the inhibited and uninhibited startle response of capuchin monkeys (Sapajus spp.), and to evaluate the role of the superior colliculus in PPI. Capuchin monkeys were tested in a whole-body protocol, to determine the best startle amplitude and interstimuli interval. Additionally we tested two subjects with bilateral superior colliculus damage in this protocol. Results show that 115 dB auditory pulse has induced the best startle response. In contrast to reports in other species, no habituation to the auditory stimuli was observed here in capuchins. Also, startle reflex inhibition was optimal after 120 msec interstimuli interval. Finally, there was a downward tendency of percentage inhibition in superior colliculus-lesioned monkeys. Our data provides the possibility of further studies with whole-body protocol in capuchin monkeys and reinforces the importance of the superior colliculus in PPI.     

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.     

Prepulse Inhibition of Acoustic Startle Reflex as a Function of the Frequency Difference between Prepulse and Background Sounds in Mice

Background

Prepulse inhibition (PPI) depicts the effects of a weak sound preceding strong acoustic stimulus on acoustic startle response (ASR). Previous studies suggest that PPI is influenced by physical parameters of prepulse sound such as intensity and preceding time. The present study characterizes the impact of prepulse tone frequency on PPI.

Methods

Seven female C57BL mice were used in the present study. ASR was induced by a 100 dB SPL white noise burst. After assessing the effect of background sounds (white noise and pure tones) on ASR, PPI was tested by using prepulse pure tones with the background tone of either 10 or 18 kHz. The inhibitory effect was assessed by measuring and analyzing the changes in the first peak-to-peak magnitude, root mean square value, duration and latency of the ASR as the function of frequency difference between prepulse and background tones.

Results

Our data showed that ASR magnitude with pure tone background varied with tone frequency and was smaller than that with white noise background. Prepulse tone systematically reduced ASR as the function of the difference in frequency between prepulse and background tone. The 0.5 kHz difference appeared to be a prerequisite for inducing substantial ASR inhibition. The frequency dependence of PPI was similar under either a 10 or 18 kHz background tone.

Conclusion

PPI is sensitive to frequency information of the prepulse sound. However, the critical factor is not tone frequency itself, but the frequency difference between the prepulse and background tones.     

Effects of copper metabolism on neurological functions in Wistar and Wilson's disease model rats

Behavioral functions of Wistar and Long-Evans Cinnamon (LEC) rats, Wilson's disease animal model, were compared by measuring the open-field, acoustic startle reflex and prepulse inhibition (PPI), and shuttle-box avoidance learning tests with or without oral supplementation with copper or D-penicillamine, copper chelator. All of the LEC rats, irrespective of the treatment, exhibited higher locomotor activity, a decreased habituation to startle response or a lower PPI, compared with Wistar rats. The copper content of all brain regions examined, except for the medulla oblongata of LEC rats, was significantly lower than those in Wistar rats. Besides, in the region of the striatum and the nucleus accumbens of the LEC rats, lower content of norepinephrine, and higher content of dopamine and serotonin were observed compared with Wistar rats. Although copper supplementation did not affect the brain copper content, it reduced the PPI in both Wistar and LEC rats. In contrast, D-penicillamine supplementation decreased both the brain copper content and locomotor activity, and enhanced the startle amplitude only in Wistar rats. These findings suggest that an imbalance in copper homeostasis affects monoamine metabolism and behavioral functions.     

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.     

Renewal and reinstatement of the conditioned but not the unconditioned response following habituation of the unconditioned stimulus

Research on the inhibition of learned fear currently relies almost exclusively on one specific procedure, namely extinction of the conditioned stimulus (CS). Importantly, however, learned fear responses can be reduced by a number of other procedures, including habituation of the unconditioned stimulus (US). We recently demonstrated that reductions in learned fear following US habituation, like CS extinction, were subject to both renewal and reinstatement (Storsve et al., 2010). The present study further investigates the associative and non-associative processes shared between habituation and extinction. Given that habituation is typically context-independent (Mackintosh, 1987), in the present study we directly compared renewal and reinstatement of both a conditioned response (CR; freezing) and an unconditioned response (UR; startle) following habituation. It was found that the reduction in conditioned freezing resulting from habituation was context specific (i.e., a change in context led to a renewal of the conditioned fear response; Experiment 1) and was attenuated when a pre-test shock was given (i.e., reinstatement of conditioned fear was observed; Experiment 2). In contrast, habituation of an unconditioned response elicited by the US (i.e., a startle response) was unaffected by either a change in test context or administration of a pre-test shock. This dissociation in the effects of habituation on learned and unlearned responses is discussed in relation to theories of fear extinction.     

Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic β-Blocker

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a β-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.     

A cellular mechanism for prepulse inhibition

In prepulse inhibition (PPI), startle responses to sudden, unexpected stimuli are markedly attenuated if immediately preceded by a weak stimulus of almost any modality. This experimental paradigm exposes a potent inhibitory process, present in nervous systems from invertebrates to humans, that is widely considered to play an important role in reducing distraction during the processing of sensory input. The neural mechanisms mediating PPI are of considerable interest given evidence linking PPI deficits with some of the cognitive disorders of schizophrenia. Here, in the marine mollusk Tritonia diomedea, we describe a detailed cellular mechanism for PPI--a combination of presynaptic inhibition of startle afferent neurons together with distributed postsynaptic inhibition of several downstream interneuronal sites in the startle circuit.     

The characteristics of the startle reflex to an acoustic stimulus in rats with hereditary stress-sensitive arterial hypertension: the effect of chronic stress in the prepuberty period]

Acoustic startle reflex was studied in young and adult rats with hereditary stress-induced arterial hypertension (HSIAH) in comparison with original normotensive Wistar strain. Immediate and long-lasting effects of chronic handling or chronic unpredictable stress at the age of 4 and 5 weeks on manifestations of the startle reflex were studied. The amplitude of the sensorimotor reaction was lower in both young (38-day-old) and adult (4-month-old) HSIAH rats than in age-matched normotensive Wistar rats. Young and adult hypertensives demonstrated significant inhibition of startle reflex when the startling stimulus was preceded by a weak prepulse. Such prepulse inhibition was not expressed in young or adult Wistar rats. Chronic handling as well as chronic unpredictable stress during the 4th and 5th weeks of life potentiated the startle amplitude in young HSIAH rats. The prepulse inhibition level did not depend on the chronic stress. The long-lasting effect of the prepubertal chronic stress depended on the type of the stress factors.     

Genetic correlational analysis of glycogen synthase kinase-3 beta and prepulse inhibition in inbred mice

In humans, GSK-3β activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK-3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzyme's activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK-3β regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.     

Dependency of prepulse inhibition deficits on baseline startle reactivity in a mouse model of the human 22q11.2 microdeletion syndrome

Hemizygous microdeletion at the chromosomal locus 22q11.2 is a copy number variation with strong genetic linkage to schizophrenia and related disorders. This association, along with its phenotypic overlap with the 22q11.2 microdeletion syndrome, has motivated the establishment of Df[h22q11]/+ mice, in which the human 22q11.2 orthologous region is deleted. Previous investigations using this model showed the presence of reduced prepulse inhibition (PPI) of the acoustic startle reflex, a form of sensorimotor gating known to be impaired in a number of psychiatric disorders. Concomitantly to reduced PPI, however, Df[h22q11]/+ mice are also characterized by a robust increase in baseline startle reactivity, which may complicate or confound the interpretation of PPI. Therefore, the present study re‐examined the relationship between acoustic startle reactivity and PPI in this mouse model. We found that while PPI is reduced in Df[h22q11]/+ mice when using its relative indexation (ie, % PPI), this deficit is no longer apparent when using the absolute quantification, that is, the direct comparison between pulse‐alone and prepulse‐plus‐pulse conditions with successively increasing prepulse intensities. We further identified marked negative correlations between % PPI and startle reactivity in Df[h22q11]/+ mice. Moreover, when stratifying Df[h22q11]/+ mice into subgroups displaying low‐ and high‐startle reactivity, only the latter subgroup displayed a significant reduction in % PPI. Collectively, our data suggest that alterations in baseline startle reactivity can confound the outcomes and interpretation of PPI in this mouse model of the human 22q11.2 microdeletion syndrome.