Consomic mouse strain selection based on effect size measurement, statistical significance testing and integrated behavioral z-scoring: focus on anxiety-related behavior and locomotion.

BACKGROUND: Selecting chromosome substitution strains (CSSs, also called consomic strains/lines) used in the search for quantitative trait loci (QTLs) consistently requires the identification of the respective phenotypic trait of interest and is simply based on a significant difference between a consomic and host strain. However, statistical significance as represented by P values does not necessarily predicate practical importance. We therefore propose a method that pays attention to both the statistical significance and the actual size of the observed effect. The present paper extends on this approach and describes in more detail the use of effect size measures (Cohen's d, partial eta squared - η p (2) ) together with the P value as statistical selection parameters for the chromosomal assignment of QTLs influencing anxiety-related behavior and locomotion in laboratory mice. RESULTS: The effect size measures were based on integrated behavioral z-scoring and were calculated in three experiments: (A) a complete consomic male mouse panel with A/J as the donor strain and C57BL/6J as the host strain. This panel, including host and donor strains, was analyzed in the modified Hole Board (mHB). The consomic line with chromosome 19 from A/J (CSS-19A) was selected since it showed increased anxiety-related behavior, but similar locomotion compared to its host. (B) Following experiment A, female CSS-19A mice were compared with their C57BL/6J counterparts; however no significant differences and effect sizes close to zero were found. (C) A different consomic mouse strain (CSS-19PWD), with chromosome 19 from PWD/PhJ transferred on the genetic background of C57BL/6J, was compared with its host strain. Here, in contrast with CSS-19A, there was a decreased overall anxiety in CSS-19PWD compared to C57BL/6J males, but not locomotion. CONCLUSIONS: This new method shows an improved way to identify CSSs for QTL analysis for anxiety-related behavior using a combination of statistical significance testing and effect sizes. In addition, an intercross between CSS-19A and CSS-19PWD may be of interest for future studies on the genetic background of anxiety-related behavior.

Disrupted cross-laminar cortical processing in ß amyloid pathology precedes cell death.

Disruption of neuronal networks in the Alzheimer-afflicted brain is increasingly recognized as a key correlate of cognitive and memory decline in Alzheimer patients. We hypothesized that functional synaptic disconnections within cortical columnar microcircuits by pathological ß-amyloid accumulation, rather than cell death, initially causes the cognitive impairments. During development of cortical ß-amyloidosis with still few plaques in the transgenic 5xFAD mouse model single cell resolution mapping of neuronal thallium uptake revealed that electrical activity of pyramidal cells breaks down throughout infragranular cortical layer V long before cell death occurs. Treatment of 5xFAD mice with the glutaminyl cyclase inhibitor, PQ 529, partially prevented the decline of pyramidal cell activity, indicating pyroglutamate-modified forms, potentially mixed oligomers of Aß are contributing to neuronal impairment. Laminar investigation of cortical circuit dysfunction with current source density analysis identified an early loss of excitatory synaptic input in infragranular layers, linked to pathological recurrent activations in supragranular layers. This specific disruption of normal cross-laminar cortical processing coincided with a decline of contextual fear learning.

Animal welfare at the group level: more than the sum of individual welfare?

Currently assessment and management of animal welfare are based on the supposition that welfare status is something experienced identically by each individual animal when exposed to the same conditions. However, many authors argue that individual welfare cannot be seen as an 'objective' state, but is based on the animal's own self-perception; such perception might vary significantly between individuals which appear to be exposed to exactly the same challenges. We argue that this has two implications: (1) actual perceived welfare status of individuals in a population may vary over a wide range even under identical environmental conditions; (2) animals that appear to an external observer to be in better or poorer welfare condition may all in fact perceive their own individual status as the same. This would imply that optimum welfare of a social group might be achieved in situations where individual group members differ markedly in apparent welfare status and perceive their own welfare as being optimal under differing circumstances. Welfare phenotypes may also vary along a continuum between self-regarding and other-regarding behaviour; a variety of situations exist where (social) individuals appear to invest in the welfare of other individuals instead of maximising their own welfare; in such a case it is necessary to re-evaluate individual welfare within the context of a social group and recognise that there may be consequences for the welfare of individuals, of decisions made at the group level or by other group members.

Can we measure accurately the prevalence of doping?

Questionnaires are used in the majority of the studies on doping prevalence in sport. Nevertheless, prevalence is not easy to evaluate and previous epidemiologic studies demonstrated a wide variance. This variance has mostly been explained by sample differences. The way to evaluate doping prevalence in the survey is questioned in this paper. A questionnaire was administered to 1810 amateur athletes (993 males, 817 females). Doping use was ascertained in various ways, using different definitions of doping and types of question in the survey. Depending on the definition of doping and the type of question used, the prevalence of doping obtained can differ enormously, between 1.3 and 39.2% of athletes. Marijuana and drugs for asthma were the two banned substances most used. The majority of athletes often ignored the banned list and did not use prohibited substances to dope. Using various ways to question athletes, observing the usage of substances, cross checking the data, taking into account the aim of substances uses and the various definitions of doping are necessary to give more reliable prevalence of doping. Moreover, doping at an amateur level seems to be less of a sport problem than a social problem.

Resonance phenomena in the human auditory cortex: individual resonance frequencies of the cerebral cortex determine electrophysiological responses.

The brain can be considered a dynamical system which is able to oscillate at multiple frequencies. To study the brain's preferred oscillation frequencies, the resonance frequencies in the frequency response of the system can be assessed by stimulating the brain at various stimulation frequencies. Furthermore, the event-related potential (ERP) can be considered as the brain's impulse response. For linear dynamical systems, the frequency response should be equivalent to the frequency transform of the impulse response. The present study test whether this fundamental relation is also true for the frequency transform of the ERP and the frequency response of the brain. Results show that the spectral characteristics of both impulse and frequency response in the gamma frequency range are significantly correlated. Thus, we speculate that the resonance frequencies determine the frequency spectrum of the impulse response. This, in turn, implies that both measures are determined by the same, individually specific, neuronal generator mechanisms.

Consequences of chronic social stress on behaviour and vasopressin gene expression in the PVN of DBA/2OlaHsd mice--influence of treatment with the CRHR1-antagonist R121919/NBI 30775.

Accumulating evidence suggests that corticotropin-releasing hormone (CRH) neurocircuitry modulate the neuroendocrine and behavioural phenotypes in depression and anxiety. Thus, the administration of the selective CRH-receptor 1 (CRHR1)-antagonist R121919/NBI 30775 has proven its ability to act as an anxiolytic in rats. It is still unclear whether vasopressinergic neuronal circuits, which are known to be involved in the regulation of emotionality, are affected by R121919/NBI 30775. Using DBA/2OlaHsd mice, we investigated the effects of chronic social defeat and concomitant treatment with R121919/NBI 30775 on 1) the behavioural profile in the modified hole board test and 2) in-situ hybridization analysis-based expression of arginine vasopressin (AVP) and CRH mRNA in both the hypothalamic paraventricular nucleus and supraoptic nucleus. The results suggest that chronic social defeat leads to increased avoidance behaviour and reduction in directed exploration, general exploration, and locomotion. Chronic treatment with the CRHR1-antagonist was effective in reversing the directed exploration to control level. The dissection of the antagonist-treated group into responders and non-responders using the parameter time spent on board revealed further positive effects of R121919/NBI 30775 on avoidance behaviour and locomotion. Behavioural changes were accompanied by alterations in AVP gene expression in the paraventricular nucleus. Taken together, the anxiolytic action of the CRHR1 antagonist was found in a subgroup of animals only, and further studies have to be done to clarify the inter-individual biological differences in response patterns to this compound to optimise its application under clinical conditions.

Substrain and light regime effects on integrated anxiety-related behavioral z-scores in male C57BL/6 mice-Hypomagnesaemia has only a small effect on avoidance behavior.

Magnesium (Mg) has been described to possess an anxiolytic function, but a number of studies present inconsistent results on this matter. In this study the effect of Mg deficiency on anxiety-related behavior, brain and blood plasma Mg in young adult male C57BL/6JOlaHsd and C57BL/6NCrl mice was studied. The animals were put on a control or Mg deficient diet from day 0 and significant hypomagnesaemia was evident from day 12 onwards in the test animals. Housing and test conditions were under either conventional light regime (white light behavioral test conditions) or reverse light regime (red light behavioral test conditions). The animals were tested in three tests for unconditioned anxiety: the modified Hole Board (day 14), the light-dark test (day 21) and the elevated plus maze (day 28). Overall integrated behavioral z-scores were calculated over these three behavioral tests. Mg showed a structure dependent distribution at the level of the brain, that differed between C57BL/6 substrain and light regime (conventional versus reverse), respectively. Likewise, total brain Mg did differ between substrain and light regime, but was not affected by the diet. Animals on the Mg deficient diet housed under conventional light regime had a higher final (day 28) blood plasma corticosterone level as compared to controls. Animals housed under reverse light regime exhibited no diet effect of plasma corticosterone levels. The significant hypomagnesaemia at blood plasma level resulted in an effect of Mg deficiency on avoidance, but not overall anxiety-related behavior. Significant differences regarding avoidance behavior were found between the two substrains and light regimes, respectively.

Animal models of anxiety.

Animal models for anxiety-related behavior are based on the assumption that anxiety in animals is comparable to anxiety in humans. Being anxious is an adaptive response to an unfamiliar environment, especially when confronted with danger or threat. However, pathological variants of anxiety can strongly impede the daily life of those affected. To unravel neurobiological mechanisms underlying normal anxiety as well as its pathologi- cal variations, animal models are indispensable tools. What are the characteristics of an ideal animal model? First, it should display reduced anxiety when treated with anxiolytics (predictive validity). Second, the behavioral response of an animal model to a threatening stimulus should be comparable to the response known for humans (face validity). And third, the mechanisms underlying anxiety as well as the psychological causes should be identical (construct validity). Meeting these three requirements is difficult for any animal model. Since both the physiological and the behavioral response to aversive (threatening) stimuli are similar in humans and animals, it can be assumed that animal models can serve at least two distinct purposes: as (1) behavioral tests to screen for potential anxiolytic and antidepressant effects of new drugs and (2) tools to investigate specific pathogenetic aspects of cardinal symptoms of anxiety disorders. The examples presented in this chapter have been selected to illustrate the potential as well as the caveats of current models and the emerging possibilities offered by gene technology. The main concepts in generating animal models for anxiety-that is, selective breeding of rat lines, experience-related models, genetically engineered mice, and phenotype-driven approaches-are concisely introduced and discussed. Independent of the animal model used, one major challenge remains, which is to reliably identify animal behavioral characteristics. Therefore, a description of behavioral expressions of anxiety in rodents as well as tests assays to measure anxiety-related behavior in these animals is also included in this chapter.

Memory performance in tree shrews: effects of stressful experiences.

Male tree shrews were exposed to alternating non-stressful and stressful conditions, and their memory performance was tested during three different stress periods and after rest periods of various lengths using a modified holeboard. This paradigm circumvents confounding factors, e.g. food or water deprivation, transport to a special testing arena, and excludes the development of automatic cognitive processes by complex memory tasks. In experimental tree shrews, psychosocial conflict caused elevated cortisol levels during the stress phases. While this resulted in an impairment of the stress-sensitive declarative memory during the second stress phase, no memory deficit was found during the first and third stress phase. Despite normalized cortisol levels, significant memory deficits in experimental animals were observed even 10 weeks after the last stressful experience. The alternating order of stressful events revealed that the negative correlation between the level of adrenal steroid hormones and memory performance does not account for the long-lasting effects of psychosocial stress in tree shrews.

Repetitive transcranial magnetic stimulation increases the release of dopamine in the mesolimbic and mesostriatal system.

Repetitive transcranial magnetic stimulation (rTMS) is suggested to be a potentially useful treatment in major depression. In order to optimize rTMS for therapeutic use, it is necessary to understand the neurobiological mechanisms involved, particularly the nature of the neurochemical changes induced. Using intracerebral microdialysis in urethane-anesthetized and conscious adult male Wistar rats, we monitored the effects of acute rTMS (20 Hz) on the intrahippocampal, intraaccumbal and intrastriatal release patterns of dopamine and its metabolites (homovanillic acid, 3,4-dihydroxyphenylacetic acid). The stimulation parameters were adjusted according to the results of accurate MRI-based computer-assisted reconstructions of the current density distributions induced by rTMS in the rat brain, ensuring stimulation of frontal brain regions. In the dorsal hippocampus, the shell of the nucleus accumbens and the dorsal striatum the extracellular concentration of dopamine was significantly elevated in response to rTMS. Taken together, these data provide the first in vivo evidence that acute rTMS of frontal brain regions has a modulatory effect on both the mesolimbic and the mesostriatal dopaminergic systems. This increase in dopaminergic neurotransmission may contribute to the beneficial effects of rTMS in the treatment of affective disorders and Parkinson's disease.

Regulation of the developing hypothalamic-pituitary-adrenal axis in corticotropin releasing hormone receptor 1-deficient mice.

During postnatal development, mice undergo a so-called stress hyporesponsive period, which is characterized by low basal corticosterone levels and the inability of mild stressors to induce a corticosterone response. The stress hyporesponsiveness is in part regulated by maternal factors. Twenty-four hours of deprivation results in an activation of basal and stress-induced corticosterone and a down-regulation of corticotropin releasing hormone (CRH), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression in the brain. It has been hypothesized that the CRH receptor 1 (CRHr1) may play an important regulatory role during development by mediating the effects of maternal deprivation. Using CRHr1-deficient mice we examined the role of this receptor on the maternal deprivation effects and in regulating the expression of hypothalamic-pituitary-adrenal axis-related genes. We could demonstrate that the CRHr1 is essential for the activation of the corticosterone response following maternal deprivation, most likely due to the lack of the receptor in the pituitary. Furthermore, we could show that the CRHr1 is regulating the expression of CRH and MRs. In contrast, effects of maternal deprivation during postnatal development on GRs are not mediated by this receptor.

Effect of chronic psychosocial stress and long-term cortisol treatment on hippocampus-mediated memory and hippocampal volume: a pilot-study in tree shrews.

We investigated the impact of chronic psychosocial stress and long-term cortisol treatment on hippocampus-mediated memory processes and hippocampal volume in male tree shrews. By combining cognitive tests on a hole board, magnetic resonance imaging (MRI), and saliva cortisol analysis, we were able to follow in individual animals the stress- and cortisol-induced temporal effects on HPA axis activity and the hippocampus and its executive functions during 15 weeks. Four weeks of either cortisol treatment or psychosocial stress affected hippocampus-mediated memory. Cortisol-induced impairments were observed only at the end of the treatment phase while in stressed animals a negative effect on hippocampus-mediated memory was monitored after 7 weeks of recovery. A trend towards a reduction of the hippocampal volume was found in both experimental groups. The present preclinical study demonstrated that major life events, such as psychosocial stress, or chronic cortisol treatment leave traces in hippocampus-dependent memory. This finding requires systematic analysis to understand how brain areas critical for information processing such as the hippocampal formation are affected by stress and stress hormones.

Dimensions of emotionality in a rat model of innate anxiety.

Emotionality is thought to be multidimensional, with "anxiety" representing one dimension. Dissecting emotional dimensions in animal models is an essential prerequisite for investigating the neurobiological mechanisms that underlie anxiety. The authors used factor analysis to investigate emotional dimensions in normal rats and rats bred for either high or low anxiety-related behavior. Hyperanxious rats were reduced in emotional dimensions in the elevated plus-maze by selection pressure, and a modified hole board test revealed a dissection of their emotionality with precisely defined dimensions. This enabled clear differentiation of "anxiety" from other emotional dimensions including risk assessment behavior and exploration. Factors extracted by analyzing data from a multiple-test battery corresponded to particular test characteristics rather than to emotional dimensions. The approach used might help to develop specific treatment strategies for anxiety disorders.

Differential effects of chronic stress on memory processes in the tree shrew.

Unpredictable and uncontrollable stressful events have been shown to affect cognitive processes. Interestingly, only hippocampus-mediated memory processes are thought to be sensitive to the effects of chronic stress. In contrast, the hippocampus-independent memory processes have been shown to be resistant to chronic stressful experiences. A central feature of the stress response is the activation of the hypothalamus-pituitary-adrenocortical (HPA)-axis, resulting in increased plasma levels of glucocorticoids, and several studies suggested that the performance of hippocampus-mediated memory processes might be directly modulated by these adrenal steroids. We investigated the impact of chronic psychosocial stress on hippocampus-mediated and hippocampus-independent memory processes in male tree shrews. By using a modified holeboard we followed memory performance during 23 weeks of alternating stress-free and stressful conditions. This schedule was designed to mimic a more realistic situation with stress-free conditions being sequentially interrupted by challenging events. The results indicate that chronic stress differentially affects hippocampus-mediated and hippocampus-independent memory processes in tree shrews. While hippocampus-independent memory processes remained unimpaired throughout the study, hippocampus-mediated memory was persistently impaired, not only during stress periods but also during recovery periods. This persistent impairment seems not to be exclusively triggered by glucocorticoids because urinary free cortisol concentration returned to normal during recovery periods. The present study is the first to evaluate the consequences of sequential stress exposure on memory performance in animals. Apparently, the mechanisms modulating cognitive processes are far from being understood and need a very systematic analysis in animal models with a high face and predictive validity to human stress-related memory disorders.

Spatial representation of frequency-modulated tones in gerbil auditory cortex revealed by epidural electrocorticography.

The present study investigated the topography of epidurally recorded middle latency components P1 and N1 evoked by spectrally dynamic stimuli (linearly frequency-modulated (FM) tones) with respect to the tonotopic structure of the right primary auditory cortex, field AI. Whereas the gross topography corresponded to the spectral content of the FM tones, specific tonotopic offsets were found between the potential distributions evoked by FM tones of different modulation direction (i.e. 'rising' vs. 'falling'). Potentials evoked by rising FM tones were located at tonotopic positions corresponding to higher frequencies compared with potentials evoked by falling FM tones. Data indicated that the magnitude of these offsets can be attributed to the local tonotopic resolution in AI and are not dependent on the modulation rate.

Differential analysis of behavior and diazepam-induced alterations in C57BL/6N and BALB/c mice using the modified hole board test.

A variety of test procedures are used in preclinical research on behavioral pharmacology and to dissociate behavioral differences or pharmacologically induced behavioral alterations several independent tests are usually performed. In the present study we introduce a modified hole board procedure for mice which allows us to investigate a variety of behavioral parameters such as anxiety, risk assessment, exploration, locomotion, food-intake inhibition, novelty seeking, and arousal by using only one test. The modified hole board was established by investigating the behavior of two inbred mouse strains, C57BL/6 and BALB. Significant differences in terms of locomotor activity, general exploration, and other parameters were found. Moreover, strain-specific exploration strategies could be detected in the modified hole board. Further, the test was validated by investigating the effects of diazepam as standard anxiolytic on the behavior in both mouse strains. Acute administration of diazepam (1 and 3 mg/kg) induced strong sedative effects in a dose-dependent manner in C57BL/6 mice. In BALB mice, the lower dosage of diazepam showed an activating and anxiolytic action while the 3 mg dosage revealed a slight sedative but still anxiolytic effect in these animals. Taken together, the results demonstrate that the modified hole board enables to differentially investigate behavioral phenotypes and also pharmacologically-induced behavioral alterations in mice. Therefore, this new strategy allows to reduce the number of experimental animals and the time needed, thus, representing an effective screening-tool for behavioral investigations.

Gender-specific alterations of cerebral metabolites with aging and cortisol treatment.

Excess availability of the adrenocortical glucocorticoid hormone cortisol has been correlated with structural brain changes and a decline of cognitive functions during aging. Pertinent studies need to consider gender as a potential confound because of sexual dimorphism in the regulation of hypothalamus-pituitary-adrenal axis activity. In vivo localized proton magnetic resonance spectroscopy of male and female tree shrews revealed similar concentrations of cerebral metabolites in young adult animals but gender-specific alterations with aging as well as in response to cortisol treatment. In comparison with adult tree shrews, aged males had reduced concentrations of N-acetylaspartate (-33%; P<0.01) and total creatine (-34%; P< 0.01). These findings are in line with the occurrence of neuronal loss. In contrast, aged females exhibited increased concentrations of choline-containing compounds (+27%; P<0.05) which--together with a tendency for increased creatine (+24%) and myo-inositol (+14%)--is indicative of glial proliferation. After chronic administration of cortisol (4 mg/day for 28 days), male but not female tree shrews showed a specific reduction of the choline-containing compounds (-29%; P< 0.05). The observed sex differences with age are likely to result from differences in the regulation of stress-related hormones which is further supported by the gender-specific responses to cortisol.

Assessing cognitive functions in tree shrews: visuo-spatial and spatial learning in the home cage.

We developed a holeboard paradigm to investigate the cognitive abilities of tree shrews (Tupaia belangeri). The design allows animals to be tested in their homecages, which reduces possible confounding and stress factors, such as transfer to a special testing arena, food deprivation or other restraints. Nine male tree shrews performed four visuo-spatial and one spatial trial per day in two blocks of 5 consecutive days. Tree shrews needed only 1 day, that is five trials, to learn the complex holeboard paradigm. From the second day onwards the learning scores remained almost stable. To demonstrate the applicability of this paradigm in studies on the influence of stress on cognitive functions, three animals were subjected to and tested under psychosocial stress conditions on 2 consecutive days. Since the experimental animals also performed the task under stressful conditions the home cage holeboard paradigm is a valid tool to study the development of stress induced cognitive impairments.

Volumetric MRI measurements of the tree shrew hippocampus.

Protocols suitable for repeated magnetic resonance imaging (MRI) studies of the tree shrew's brain were established. This included the development of (i) a technique for prolonged inhalation anesthesia by endotracheal intubation; (ii) a reproducible fixation of the animal's head in a stereotaxic frame and finally (iii) the set-up of the hardware (rf coil) and software (MRI sequences) of the MRI system. The endotracheal intubation as well as the repeated and prolonged anesthesia showed no complications. The in vivo measurements of the tree shrew's hippocampal formation revealed a high reproducibility. Right and left hippocampal volume was determined as 85.2 mm3 +/- 8% and 87.4 mm3 +/- 10%, respectively. The utility of MRI in delineating alterations in brain anatomy was demonstrated in three animals receiving cortisol via the drinking water (5 mg/animal/day). After a 4-week treatment, in two of the three tree shrews a reduction in hippocampal volume was observed. Thus, the MRI protocols used here allow for repeated and non-invasive measurements of changes in hippocampal anatomy within the same animal and to monitor the temporal dynamics of structural alterations within this brain structure.

Categorical discrimination of direction in frequency-modulated tones by Mongolian gerbils.

Discrimination of the direction of linearly frequency-modulated tones (FMs) was investigated in adult Mongolian gerbils (Meriones unguiculatus) using a footshock motivated shuttle box avoidance go/no go procedure. Symmetric pairs of FMs with frequency linearly increasing with time (ascending FMs) and with frequency linearly decreasing with time (descending FMs) were used as conditioned stimuli, CS+ and CS-, respectively. Stimuli were presented in randomized order in daily sessions over a period of several months. After a number of sessions, the set of conditioned stimuli was changed with respect to frequency range, steepness of modulation and duration. In experiment 1, we observed that gerbils could discriminate between the ascending 2-4 kHz CS+ and the descending 4-2 kHz CS- after a training period of 10-15 days. In experiment 2, we used FM pairs of six other frequency ranges in successive sessions (6-13; 1-2; 13-25; 0.5-1; 3 6; 0.25 0.5 kHz). We found that in the final session the last FM pair (0.25-0.5 kHz) was discriminated already after 3-4 days. Experiment 3 showed that the animals were able to discriminate five of the FM pairs learned in the separate sessions of experiment 2 (i.e. 10 different stimuli) when they were given in randomized order during one training session. In experiment 4, novel FM pairs (not heard before) and familiar FM pairs (trained in experiments 1-3) were presented within one session. It was found that, except for FMs of very short duration and small frequency range, novel FMs were discriminated according to their modulation direction. These results show that Mongolian gerbils are able to discriminate FM tones by modulation direction and, after familiarization with a number of different FM pairs, transfer the ascending-descending concept to stimuli not heard before.