Effects of repeated maternal separation on prepulse inhibition of startle across inbred mouse strains

A growing body of research implicates genetic factors and childhood trauma in the etiology of neuropsychiatric diseases such as schizophrenia. However, there remains little understanding of how genetic variation influences early life stress to affect later disease susceptibility. Studies in rats have shown that postnatal maternal separation (MS) results in later deficits in prepulse inhibition of the acoustic startle response (PPI), an impairment in sensorimotor gating found in schizophrenic patients. In the present study, genetic differences in the effects of repeated MS on PPI were examined in eight inbred strains of mice (129S1/SvImJ, 129P3/J, A/J, BALB/cJ, BALB/cByJ C57BL/6J, DBA/2J and FVB/NJ). Mice were assigned to either MS (180 min/day on postnatal days P0-P13), 'handling' (15 min/day, P0-P13) or facility-reared conditions and tested for PPI at 12 weeks of age. Results demonstrated major strain differences in the production of viable offspring irrespective of MS, leading to the exclusion of 129P3/J, A/J and BALB/cJ from the study. Pups from the five remaining strains exhibited marked differences in the acoustic startle response and PPI, confirming previous strain comparisons. However, MS produced no significant effects on PPI in any of the strains tested. A second form of postnatal stress (repeated footshock) also failed to alter PPI in the one strain studied, C57BL/6J. Present results demonstrate that the form of MS studied herein does not provide a robust model of early life stress effects on PPI in the mouse strains tested. The development and validation of a reliable mouse model of early life stress remains an important research goal.     

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.     

Analysis of sensory, motor and cognitive functions of the coloboma (C3Sn.Cg-Cm/J) mutant mouse

The coloboma mutant mouse (C3Sn.Cg-Cm/J) has been proposed as an animal model of attention-deficit hyperactivity disorder (ADHD) because of excessive locomotion in the open field, yet few studies have looked at other behavioral measures in these mice. We analyzed activity levels of male and female Cm mice and their littermate controls (C3H) in two different types of open field, as well as their hearing (acoustic startle) and sensorimotor gating (prepulse inhibition), pain responsiveness (tail flick and hot plate), motor control (balance beam), motor learning (Rotarod), hippocampal working memory (spontaneous alternation in a Y-maze) and olfactory learning and memory (conditioned odor preference). We found hyperactivity and a lack of habituation in the small and large open fields and a deficit in prepulse inhibition in these mice, as well as a learning deficit in male Cm mice in conditioned odor preference but no deficits in pain perception or spontaneous alternation. Results from the rotarod and balance beam tasks indicate that Cm mice have severe motor co-ordination and balance problems compared to their C3H littermates, suggesting that Cm mice may be a more suitable model of ataxia than ADHD.     

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.     

Behavior and mutagenesis screens: the importance of baseline analysis of inbred strains

Random mutagenesis as a means of identifying the function of genes has been used extensively in a variety of model organisms. Until recently it has been used primarily in the identification of single-gene traits that cause visible and developmental mutations. However, this genetic approach also has the power to identify genes that control complex biological systems such as behavior. Mutagenesis screens for behavioral mutations require careful consideration of many factors, including choice of both assays and background strains for use in mutagenesis and subsequent mapping of the affected gene or genes. This paper describes behavioral assays for monitoring motor coordination on the accelerating rotarod, anxiety-related behaviors in the elevated zero maze and sensorimotor reactivity, gating, and habituation of acoustic startle. These five physiological or neurological behaviors can represent potential endophenotypes for a variety of neurological and psychiatric disorders. The significant degree of strain- and sex-specific differences in the performance of four inbred strains of mice (C57BL/6J, C3HeB/FeJ, DBA/2J, and 129/SvlmJ) in these behavioral assays illustrates the importance of performing baseline analysis prior to behavioral mutagenesis screens and genetic mapping of selected mutations. Received: 16 December 1999 / Accepted: 17 December 1999     

Genome-tagged mice (GTM): two sets of genome-wide congenic strains

An important approach for understanding complex disease risk using the mouse is to map and ultimately identify the genes conferring risk. Genes contributing to complex traits can be mapped to chromosomal regions using genome scans of large mouse crosses. Congenic strains can then be developed to fine-map a trait and to ascertain the magnitude of the genotype effect in a chromosomal region. Congenic strains are constructed by repeated backcrossing to the background strain with selection at each generation for the presence of a donor chromosomal region, a time-consuming process. One approach to accelerate this process is to construct a library of congenic strains encompassing the entire genome of one strain on the background of the other. We have employed marker-assisted breeding to construct two sets of overlapping congenic strains, called genome-tagged mice (GTMs), that span the entire mouse genome. Both congenic GTM sets contain more than 60 mouse strains, each with on average a 23-cM introgressed segment (range 8 to 58 cM). C57BL/6J was utilized as a background strain for both GTM sets with either DBA/2J or CAST/Ei as the donor strain. The background and donor strains are genetically and phenotypically divergent. The genetic basis for the phenotypic strain differences can be rapidly mapped by simply screening the GTM strains. Furthermore, the phenotype differences can be fine-mapped by crossing appropriate congenic mice to the background strain, and complex gene interactions can be investigated using combinations of these congenics.     

The effects of neonatal saline injections on behaviours in DBA/2 male mice

DBA/2 male mice were exposed to the injections of the saline (0.01 ml/g i.p.) on 1-th, 3-th, 5-th, 7-th, 9-th days after birth. Intact males were used as a control group. Adult saline-treated males displayed the increased number of crossed squares, entries in the centre and time spent in the centre during the open "field" test in comparison with intact animals. The time spent in the light compartment of the light-dark box was decreased in saline treated mice compared with intact animals. During the test of acoustic startle response the magnitude of startle reflex and prepulse inhibition didn't change the startle reflex. Saline administration in males did not affect corticosterone basal level. Sexual motivation was revealed to decrease in saline treated males. These data suggest that neonatal administration of saline induced a stable behavioral syndrome in adult DBA/2 male mice: hyperactivity, a decrease of open space fear and simultaneously an increase of some indices of anxiety.     

Auditory and visual prepulse inhibition in mice: parametric analysis and strain comparisons

Prepulse inhibition (PPI) is a multimodal phenomenon where the prepulse and the startling stimulus can be presented in either the same or the different sensory modalities. The aim of the present study was to characterize intramodal and cross-modal PPI in mice. We first examined the effects of varying prepulse intensity and prepulse duration on auditory and visual PPI in three inbred mouse strains C57BL/6J, 129S2 and BALB/cByJ mice. Increasing the intensity (5-15 dB above the background) and the duration (1-25 milliseconds) of the acoustic prepulse increased auditory PPI, and maximum level of inhibition was reached with each prepulse intensity at specific prepulse duration (between 5 and 15 milliseconds). Varying the intensity (30-300 lux) and the duration (1-25 milliseconds) of the light flashes had similar impact on visual PPI level (optimal durations between 1 and 10 milliseconds). There were also marked strain differences in PPI performances, with 129S2 and BALB/cByJ mice displaying the highest and the lowest scores of auditory PPI, respectively. In contrast, opposite strain ranking was obtained for visual PPI. The temporal expression of PPI was then studied in the same mouse strains using a wide range of interstimulus intervals (2-2000 milliseconds between the prepulse offset and the pulse onset). The time-course of the auditory and the visual PPI were relatively comparable (bell-shaped curve) with optimal lead-times between 10 and 100 milliseconds, but the shape of the temporal function varied between the mouse strains depending on the prepulse modality. These findings demonstrate that PPI has many physiological and genetic determinants that vary greatly across temporal and intensity domain, as well as stimulus modality.     

Startle responding and context conditioning. Naloxone pretreatment and stimulus intensity

The possibility that acoustic startle stimuli could support a conditional response (freezing) to contextual stimuli was investigated. Rats were exposed to three acoustic startle stimuli on the first day, and one on the second day. On day 1, 20 rats received naloxone pretreatment and another 20 received saline (placebo) pretreatment. Half of each group received a high-intensity acoustic stimulus, the other half a low-intensity acoustic stimulus. Both the higher stimulus intensity and the naloxone pretreatment led to greater freezing behavior during the 3-minute test period before the single startle stimulus on day 2. These findings support the notion that increased actual or perceived intensity of the acoustic startle stimulus increases conditioning to contextual stimuli as indexed by freezing behavior.     

Induction of Enhanced Acoustic Startle Response by Noise Exposure: Dependence on Exposure Conditions and Testing Parameters and Possible Relevance to Hyperacusis

There has been a recent surge of interest in the development of animal models of hyperacusis, a condition in which tolerance to sounds of moderate and high intensities is diminished. The reasons for this decreased tolerance are likely multifactorial, but some major factors that contribute to hyperacusis are increased loudness perception and heightened sensitivity and/or responsiveness to sound. Increased sound sensitivity is a symptom that sometimes develops in human subjects after acoustic insult and has recently been demonstrated in animals as evidenced by enhancement of the acoustic startle reflex following acoustic over-exposure. However, different laboratories have obtained conflicting results in this regard, with some studies reporting enhanced startle, others reporting weakened startle, and still others reporting little, if any, change in the amplitude of the acoustic startle reflex following noise exposure. In an effort to gain insight into these discrepancies, we conducted measures of acoustic startle responses (ASR) in animals exposed to different levels of sound, and repeated such measures on consecutive days using a range of different startle stimuli. Since many studies combine measures of acoustic startle with measures of gap detection, we also tested ASR in two different acoustic contexts, one in which the startle amplitudes were tested in isolation, the other in which startle amplitudes were measured in the context of the gap detection test. The results reveal that the emergence of chronic hyperacusis-like enhancements of startle following noise exposure is highly reproducible but is dependent on the post-exposure thresholds, the time when the measures are performed and the context in which the ASR measures are obtained. These findings could explain many of the discrepancies that exist across studies and suggest guidelines for inducing in animals enhancements of the startle reflex that may be related to hyperacusis.     

Sleep,activity, temperature and arousal responses of mice deficient for muscarinic receptor M2 or M4

AimsThe type 2 muscarinic receptor (M2R) differs from the other G-protein-coupled muscarinic receptor (type 4, or M4R) in tissue distribution and physiologic effects. We studied the impact of these receptors on sleep and arousal by using M2R and M4R knock-out (KO) mice.Main methodsM2R and M4R KO and genetically intact mice were compared in terms of normal patterns of sleep, responses to sleep loss, infectious challenge and acoustic startle, and acoustic prepulse inhibition of startle (PPI).Key findingsUnder basal conditions, M2R and M4R KO mice do not differ from the background strain or each other in the amount or diurnal pattern of sleep, locomotor activity, and body temperature. After enforced sleep loss, M2R KO mice, in contrast to the other two strains, show no rebound in slow-wave sleep (SWS) time, although their SWS is consolidated, and they show a greater rebound in time spent in REMS (rapid-eye-movement sleep) and REMS consolidation. During influenza infection, M2R KO mice, as compared with the other strains, show marked hypothermia and a less robust increase in SWS. During Candida albicans infection, M2R KO mice show a greater increase in SWS and a greater inflammatory response than do the other strains. M2R KO mice also show greater acoustic startle amplitude than does the background strain, although PPI was not different across the 3 strains over a range of stimulus intensities.SignificanceTaken together, these findings support different roles for M2R and M4R in the modulation of sleep and arousal during homeostatic challenge.     

Behavioral effects of ivermectin in mice.

BACKGROUND AND PURPOSE: Ivermectin is a common anthelmintic drug, widely used in laboratory rodents for treatment of pinworm and mite infestations. We evaluated the action of ivermectin on sensitive behavioral tasks in mice during treatment for mites within a barrier facility. METHODS: A total of 21 (5 males, 16 females) mice (129/SvEv) were used for measuring body weight, open field locomotor activity, and rotarod motor coordination. For acoustic startle and prepulse inhibition, 20 C57BL/6J and 29 AKR/J mice were studied. For the Morris water task, the same 20 C57BL/6J mice were studied. Ivermectin (0.08% sheep drench) was administered in the drinking water of the home cage for 8 weeks. Control groups received normal tap water in identical bottles. RESULTS: Ivermectin did not affect general health, body weight, motor coordination, swimming behavior, or spatial learning in several inbred strains of mice. However, it induced a small but significant effect on some sensitive behaviors. CONCLUSIONS: A cautious approach to initiating ivermectin treatment in mice should be used for sensitive behavioral experiments.     

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.     

Behavioural profiles of inbred mouse strains used as transgenic backgrounds. II: cognitive tests

One of the characteristic manifestations of several neurodegenerative diseases is the progressive decline in cognitive ability. In order to determine the suitability of six mouse strains (129S2/Sv, BALB/c, C3H/He, C57BL/6j, CBA/Ca and DBA/2) as transgenic background strains, we investigated the performance on a variety of tasks designed to identify subtle changes in cognition. In addition, a test of exploratory behaviour was used to probe the level of underlying anxiety in these mouse strains, as anxiety can be a confounding factor on behavioural performance generally. The C3H/He mice exhibited the least anxiogenic behavioural profile spending most time on the open arms of the maze, in contrast to the 129S2/Sv mice which spent the least amount of time in this location and were the quickest to move into a closed arm. The C3H/He mouse strain failed to acquire a visual discrimination task and failed to demonstrate learning on a water maze spatial learning task, in contrast to the CBA/Ca, DBA/2 and C57BL/6j strains which demonstrated a degree of learning in both tasks. No significant strain differences were identified on the object recognition task. These data, taken together, suggest that care must be taken when choosing cognitive tasks to be used with particular mouse strains and that task sensitivity must be considered as a critical element to research protocols with regard to these mouse strains.     

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 of acoustic startle in aromatase knock-out mice: effects of age and gender

Estrogen has been suggested to play a neuromodulatory and neuroprotective role on the brain dopamine system. We used aromatase knockout (ArKO) mice that lack a functional aromatase enzyme and are unable to convert testosterone into estrogen, and assessed prepulse inhibition of acoustic startle, locomotor hyperactivity to amphetamine treatment and rotarod performance. Mice were tested at either 1 month, 4–5 months or 12–18 months of age. In male, but not female ArKO mice, there was an age-related reduction of prepulse inhibition. The 12–18 months old male ArKO mice also showed significantly greater amphetamine-induced hyperactivity. Mice heterozygous for the mutation showed no deficits or were in-between wildtype mice and ArKO mice. We postulate that these data indicate a neuroprotective role of estrogen, particularly in male mice, on ageing of brain mechanisms involved in prepulse inhibition and locomotor activity regulation. It is likely that these brain mechanisms are or include dopaminergic activity.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

Analysis of possibility of genotypic correlation between fear and anxiety

Special features of anxious behavior in the elevated plus maze test and acoustic startle response were analyzed in 11 inbred mouse strains. A significant influence of the genotype both on the startle amplitude and behavior in the elevated plus maze was found. However, analysis of covariance did not reveal a genotype-related association between anxiety and startle amplitude. The data indicates that the fear-induced acoustic startle response and anxious behavior in the elevated plus maze (agoraphobia) are not genetically related.     

Gene-environment interactions differentially affect mouse strain behavioral parameters

Systematic phenotyping of mouse strains and mutants generated through genome-wide mutagenesis programs promises to deliver a wealth of functional genetic information. To this end, the appropriation of a standard series of phenotyping protocols is desirable to produce data sets that are consistent within and across laboratories and across time. Standard phenotyping protocols such as EMPReSS (European Mouse Phenotyping Resource for Standardised Screens) provide a series of protocols aimed at phenotyping multiple body systems that could realistically be adopted and/or reproduced in any laboratory. This includes a series of neurologic and behavioral screens, bearing in mind that this class of phenotype is well represented in targeted mutants and mutagenesis screens. Having cross-validated screening batteries in a number of laboratories and in a number of commonly used inbred strains, our group was interested in establishing whether subtle changes in cage environment could affect behavioral test outcome. Aside from unavoidable quantitative differences in test outcome, we identified significant and distinct genotype-environment-test interactions. For example, specific strain order in open-field center entries and total distance traveled can be reversed depending on the form of enrichment used, while prepulse inhibition of the acoustic startle response is, even quantitatively, unaffected by the enrichment condition. Our findings argue that unless systematically recorded, behavioral studies conducted under subtle variations in cage environment may lead to data misinterpretation, although this could be limited to particular behaviors. Further investigations into the extent and limits of genetic and environmental variables are critical for the realization of both behavioral and functional genomics endpoints.