Paw preferences in mice and rats: Meta-analysis.

Mice and rats are among the most common animal model species in both basic and clinical neuroscience. Despite their ubiquity as model species, many clinically relevant brain-behaviour relationships in rodents are not well understood. In particular, data on hemispheric asymmetries, an important organizational principle in the vertebrate brain, are conflicting as existing studies are often statistically underpowered due to small sample sizes. Paw preference is one of the most frequently investigated forms of hemispheric asymmetries on the behavioural level. Here, we used meta-analysis to statistically integrate findings on paw preferences in rats and mice. For both species, results indicate significant hemispheric asymmetries on the individual level. In mice, 81 % of animals showed a preference for either the left or the right paw, while 84 % of rats showed this preference. However, contrary to what has been reported in humans, population level asymmetries were not observed. These results are particularly significant as they point out that paying attention to potential individual hemispheric differences is important in both basic and clinical neuroscience.

Laterality for the next decade: Costs and benefits of neuronal asymmetries - putting lateralization in an evolutionary context.

In my comment on the target article of Ocklenburg et al. [Laterality 2020: Entering the next decade. Laterality, 1-33. doi:10.1080/1357650X.2020.1804396], I point out the relevance of studies in non-human species within natural settings for understanding the ecological pressures, which shape the direction and degree of brain asymmetries. I therefore outline some major research projects, which are not included in the paper of Ocklenburg et al. and which require comparative animal research.

Pigeons show how meta-control enables decision-making in an ambiguous world.

In situations where the left and right brain sides receive conflicting information that leads to incompatible response options, the brain requires efficient problem-solving mechanisms. This problem is particularly significant in lateralized brains, in which the hemispheres differ in encoding strategies or attention focus and hence, consider different information for decision-making. Meta-control, in which one hemisphere dominates ambiguous decisions, can be a mechanism that ensures fast behavioral reactions. We therefore confronted pigeons with a task in which two stimulus classes were brought into conflict. To this end, we trained pigeons simultaneously on two categories (cats or dogs) whereby each hemisphere learnt only one of the categories respectively. After learning, the birds were confronted with stimulus pairs that combined a picture with a cat (positive for one hemisphere) and a picture with a dog (positive for the other hemisphere). Pecking responses indicated the hemisphere dominating response selection. Pigeons displayed individual meta-control despite equal categorization performances of both brain hemispheres. This means that hemispheric dominance only emerged in interhemispheric conflict situations. The analysis of response latencies indicate that conflict decisions relied on intrahemispheric processes. Interhemispheric components played a role for more complex decisions. This flexibility could be a crucial building block for the evolutionary success of a lateralized brain.

Light-dependent development of the tectorotundal projection in pigeons.

Left-right differences in the structural and functional organization of the brain are widespread in the animal kingdom and develop in close gene-environment interactions. The visual system of birds like chicks and pigeons exemplifies how sensory experience shapes lateralized visual processing. Owing to an asymmetrical posture of the embryo in the egg, the right eye/ left brain side is more strongly light-stimulated what triggers asymmetrical differentiation processes leading to a left-hemispheric dominance for visuomotor control. In pigeons (Columba livia), a critical neuroanatomical element is the asymmetrically organized tectofugal pathway. Here, more fibres cross from the right tectum to the left rotundus than vice versa. In the current study, we tested whether the emergence of this projection asymmetry depends on embryonic light stimulation by tracing tectorotundal neurons in pigeons with and without lateralized embryonic light experience. The quantitative tracing pattern confirmed higher bilateral innervation of the left rotundus in light-exposed and thus, asymmetrically light-stimulated pigeons. This was the same in light-deprived pigeons. Here, however, also the right rotundus received an equally strong bilateral input. This suggests that embryonic light stimulation does not increase bilateral tectal innervation of the stronger stimulated left but rather decreases such an input pattern to the right brain side. Combined with a morphometric analysis, our data indicate that embryonic photic stimulation specifically affects differentiation of the contralateral cell population. Differential modification of ipsi- and contralateral tectorotundal connections could have important impact on the regulation of intra- and interhemispheric information transfer and ultimately on hemispheric dominance pattern during visual processing.

Paw preferences in the Asian small-clawed otter - using an inexpensive, video-based protocol to study laterality of rare species in the zoo.

It is still debated whether limb-use preferences represent a common trait in vertebrates, which is based on a shared phylogenetic history. Unravelling the evolutionary origin and pattern of paw preferences in vertebrates requires the analysis of a larger number of species within an ecologically relevant setting. We therefore investigated whether observations in a zoo enable the collection of reliable data sets by quantifying paw use in two independent groups of Asian small-clawed otters (Amblonyx cinerea). Employing a continuous focal animal sampling method, each day one of the ten individuals was video recorded from outside of the enclosure during usual activity. We selected four types of unimanual behaviour (reaching for food, reaching for non-food, reaching into a hole, carrying an object) and quantified paw use for each animal. Our study provides first evidences for individual paw preferences in otters, which were in line with previously reported forelimb use pattern in carnivoran species. Preferences differed between motor acts but for "reaching into a hole" a population-level right paw bias was detected. These data support that observations in a zoological setting are useful to explore task-dependent paw preferences and may facilitate future studies investigating paw preferences under experimentally controlled conditions.

Visuospatial attention in the lateralised brain of pigeons - a matter of ontogenetic light experiences.

The ontogenetic mechanisms leading to complementary hemispheric specialisations of the two brain halves are poorly understood. In pigeons, asymmetrical light stimulation during development triggers the left-hemispheric dominance for visuomotor control but light effects on right-hemispheric specialisations are largely unknown. We therefore tested adult pigeons with and without embryonic light experience in a visual search task in which the birds pecked peas regularly scattered on an area in front of them. Comparing the pecking pattern of both groups indicates that the embryonic light conditions differentially influence biased visuospatial attention under mono- and binocular seeing conditions. When one eye was occluded, dark-incubated pigeons peck only within the limits of the visual hemifield of the seeing eye. Light-exposed pigeons also peck into the contralateral field indicating enlarged monocular visual fields of both hemispheres. While dark-incubated birds evinced an attentional bias to the right halfspace when seeing with both eyes, embryonic light exposure shifted this to the left. Thus, embryonic light experience modifies processes regulating biased visuospatial attention of the adult birds depending on the seeing conditions during testing. These data support the impact of light onto the emergence of functional dominances in both hemispheres and point to the critical role of interhemispheric processes.

Investigating heritability of laterality and cognitive control in speech perception.

Several studies analyzing the ontogenetic origin of cerebral lateralization provide evidences for a genetic foundation of handedness in humans that is modulated by environmental influences. Since other forms of behavioral lateralization are less investigated, it is unclear as to how far different functions display similar heritability. But deeper knowledge is necessary to understand if and how developmental coupling of different functions is based on a shared genetic background or on the impact of environmental influences. Here, we investigated the heritability of language lateralization assessed with the dichotic listening task, as well as the heritability of cognitive control processes modulating performance in this task. Overall, 103 families consisting of both parents and offspring were tested with the non-forced, as well as the forced-right and forced-left condition of the forced attention dichotic listening task, implemented in the iDichotic smartphone app, developed at the University of Bergen, Norway. The results indicate that the typical right ear advantage in the dichotic listening task shows weak and non-significant heritability (h2=0.003; p=0.98). In contrast, cognitive factors, like attention focus (forced right condition: h2=0.36; p<0.01; forced left condition: h2=0.28; p<0.05) and cognitive control (Gain forced right: h2=0.39; p<0.01; Gain forced left: h2=0.49; p<0.01) showed stronger and significant heritability. These findings indicate a variable dependence of different aspects of a cognitive function on heritability and implicate a major contribution of non-genetic influences to individual language lateralization.

A GABAergic tecto-tegmento-tectal pathway in pigeons.

Previous studies have demonstrated that the optic tecta of the left and right brain halves reciprocally inhibit each other in birds. In mammals, the superior colliculus receives inhibitory γ-aminobutyric acid (GABA)ergic input from the basal ganglia via both the ipsilateral and the contralateral substantia nigra pars reticulata (SNr). This contralateral SNr projection is important in intertectal inhibition. Because the basal ganglia are evolutionarily conserved, the tectal projections of the SNr may show a similar pattern in birds. Therefore, the SNr could be a relay station in an indirect tecto-tectal pathway constituting the neuronal substrate for the tecto-tectal inhibition. To test this hypothesis, we performed bilateral anterograde and retrograde tectal tracing combined with GABA immunohistochemistry in pigeons. Suprisingly, the SNr has only ipsilateral projections to the optic tectum, and these are non-GABAergic. Inhibitory GABAergic input to the contralateral optic tectum arises instead from a nearby tegmental region that receives input from the ipsilateral optic tectum. Thus, a disynaptic pathway exists that possibly constitutes the anatomical substrate for the inhibitory tecto-tectal interaction. This pathway likely plays an important role in attentional switches between the laterally placed eyes of birds. J. Comp. Neurol. 524:2886-2913, 2016. © 2016 Wiley Periodicals, Inc.

Asymmetric top-down modulation of ascending visual pathways in pigeons.

Cerebral asymmetries are a ubiquitous phenomenon evident in many species, incl. humans, and they display some similarities in their organization across vertebrates. In many species the left hemisphere is associated with the ability to categorize objects based on abstract or experience-based behaviors. Using the asymmetrically organized visual system of pigeons as an animal model, we show that descending forebrain pathways asymmetrically modulate visually evoked responses of single thalamic units. Activity patterns of neurons within the nucleus rotundus, the largest thalamic visual relay structure in birds, were differently modulated by left and right hemispheric descending systems. Thus, visual information ascending towards the left hemisphere was modulated by forebrain top-down systems at thalamic level, while right thalamic units were strikingly less modulated. This asymmetry of top-down control could promote experience-based processes within the left hemisphere, while biasing the right side towards stimulus-bound response patterns. In a subsequent behavioral task we tested the possible functional impact of this asymmetry. Under monocular conditions, pigeons learned to discriminate color pairs, so that each hemisphere was trained on one specific discrimination. Afterwards the animals were presented with stimuli that put the hemispheres in conflict. Response patterns on the conflicting stimuli revealed a clear dominance of the left hemisphere. Transient inactivation of left hemispheric top-down control reduced this dominance while inactivation of right hemispheric top-down control had no effect on response patterns. Functional asymmetries of descending systems that modify visual ascending pathways seem to play an important role in the superiority of the left hemisphere in experience-based visual tasks.

Functional and structural comparison of visual lateralization in birds - similar but still different.

Vertebrate brains display physiological and anatomical left-right differences, which are related to hemispheric dominances for specific functions. Functional lateralizations likely rely on structural left-right differences in intra- and interhemispheric connectivity patterns that develop in tight gene-environment interactions. The visual systems of chickens and pigeons show that asymmetrical light stimulation during ontogeny induces a dominance of the left hemisphere for visuomotor control that is paralleled by projection asymmetries within the ascending visual pathways. But structural asymmetries vary essentially between both species concerning the affected pathway (thalamo- vs. tectofugal system), constancy of effects (transient vs. permanent), and the hemisphere receiving stronger bilateral input (right vs. left). These discrepancies suggest that at least two aspects of visual processes are influenced by asymmetric light stimulation: (1) visuomotor dominance develops within the ontogenetically stronger stimulated hemisphere but not necessarily in the one receiving stronger bottom-up input. As a secondary consequence of asymmetrical light experience, lateralized top-down mechanisms play a critical role in the emergence of hemispheric dominance. (2) Ontogenetic light experiences may affect the dominant use of left- and right-hemispheric strategies. Evidences from social and spatial cognition tasks indicate that chickens rely more on a right-hemispheric global strategy whereas pigeons display a dominance of the left hemisphere. Thus, behavioral asymmetries are linked to a stronger bilateral input to the right hemisphere in chickens but to the left one in pigeons. The degree of bilateral visual input may determine the dominant visual processing strategy when redundant encoding is possible. This analysis supports that environmental stimulation affects the balance between hemispheric-specific processing by lateralized interactions of bottom-up and top-down systems.

Shaping a lateralized brain: asymmetrical light experience modulates access to visual interhemispheric information in pigeons.

Cerebral asymmetries result from hemispheric specialization and interhemispheric communication pattern that develop in close gene-environment interactions. To gain a deeper understanding of developmental and functional interrelations, we investigated interhemispheric information exchange in pigeons, which possess a lateralized visual system that develops in response to asymmetrical ontogenetic light stimulation. We monocularly trained pigeons with or without embryonic light experience in color discriminations whereby they learned another pair of colors with each eye. Thereby, information from the ipsilateral eye had to be transferred. Monocular tests confronting the animals with trained and transferred color pairs demonstrated that embryonic light stimulation modulates the balance of asymmetrical handling of transfer information. Stronger embryonic stimulation of the left hemisphere significantly enhanced access to interhemispheric visual information, thereby reversing the right-hemispheric advantage that develops in the absence of embryonic light experience. These data support the critical role of environmental factors in molding a functionally lateralized brain.

Visual asymmetries and the ascending thalamofugal pathway in pigeons.

The lateralized visual systems of pigeons and chickens are excellent models to study neural asymmetries at the functional and anatomical level. The aim of the current study was to reveal why these two species closely resemble each other with respect to left-right differences in behavior but not with respect to the pathways involved: While pigeons show an asymmetrically organized tectofugal system, only transient lateralizations of the thalamofugal system have been observed in chickens. Four possible explanations are conceivable. (1) Adult pigeons might also show a hitherto undiscovered thalamofugal asymmetry like chickens. (2) The thalamofugal asymmetry might be transient in both species. (3) Prehatch light stimulation could differentially affect the two visual pathways of chickens and pigeons that mature with different speeds. (4) Tecto- and thalamofugal asymmetries represent species differences, independent of developmental factors. To test these explanations, we injected retrograde tracers into the Wulst of adult pigeons, of hatchlings, and of dark reared pigeons which were monocularly deprived on their left or right eye for one week after hatch. Subsequently we counted labeled cells within the ipsi- and contralateral n. geniculatus lateralis pars dorsalis in search for possible lateralizations of ascending pathways. None of the experimental groups displayed significant differences in the thalamofugal projection pattern. This indicates that visual lateralization in pigeons and chickens depends on tectofugal and thalamofugal asymmetries, respectively. Thus, in different species a highly similar pattern of behavioral asymmetries can be subserved by diverse neural systems.

Protein biomarkers for in vitro testing of toxicology.

The vision of the toxicology in the 21st century movement is to overcome the currently used animal tests and identify molecular pathways of toxicity, using human in vitro systems with the aim to provide the most relevant mechanistic information for human risk assessment. It is expected to translate key surrogate biomarkers to novel types of toxicity-related high throughput screening of the many thousands of compounds which need to be tested during development phases of the pharmaceutical industry and with regard to the REACH legislation in Europe. Systems biology, an emerging and increasingly popular field of research, appears to be the discipline of choice to integrate results from transcriptomics, proteomics, epigenomics and metabonomics technologies used to analyze samples from toxicological models. The challenges, however, with respect to data generation, statistical treatment, bioinformatic integration and interpretation or in silico modeling remain formidable. One of the main difficulties is the fact that the sheer number of molecular species is inflated enormously in the course of translation from genes to proteins due to post-translational modifications. Moreover, at the level of proteins, time scales of cellular reactions to toxic insults can be very fast, ranging from milliseconds to seconds. Linear dynamic ranges of concentration differences between conditions can also differ by several orders of magnitude. So, the search for protein biomarkers of toxicity requires sophisticated strategies for time-resolved quantitative differential approaches. The statistical principles, normalization of primary data and principal component and cluster analysis have been well developed for genomics/transcriptomics and partly for proteomics, but have not been widely adapted to technologies like metabonomics. Also, the integration of functional data, in particular data from mass spectrometry, with the aim of modeling pathways of toxicity for human risk assessment, is still at an infant stage.

The impact of asymmetrical light input on cerebral hemispheric specialization and interhemispheric cooperation.

Hemispheric specialization potentially provides evolutionary advantages by enhancing cognitive capacities. However, separation of function might be advantageous only with the presence of commissural systems allowing for efficient information exchange and cooperation between the hemispheres. Here we investigate hemispheric cooperation in pigeons as they possess an asymmetrically organized visual system that develops in response to biased ontogenetic light stimulation. This allows comparison of the integration capacities of lateralized (light-incubated) and non-lateralized (dark-incubated) animals. We show that pigeons integrate information learnt separately with each hemisphere when confronted with a transitive reasoning task that they cannot solve with the knowledge of one hemisphere alone. Impairments in dark-incubated birds demonstrate that this ability depends on asymmetrical embryonic light stimulation. Our study provides for the first time direct evidence that lateralized environmental experience not only induces hemispheric specialization, but also affects the efficiency of interhemispheric crosstalk. Environmental factors can influence the tight interplay between the hemispheres, which in turn determines cognitive abilities.

Lateralized reward-related visual discrimination in the avian entopallium.

In humans and many other animals, the two cerebral hemispheres are partly specialized for different functions. However, knowledge about the neuronal basis of lateralization is mostly lacking. The visual system of birds is an excellent model in which to investigate hemispheric asymmetries as birds show a pronounced left hemispheric advantage in the discrimination of various visual objects. In addition, visual input crosses at the optic chiasm and thus testing of each hemisphere is easily accomplished. We aimed to find a neuronal correlate for three hallmarks of visual lateralization in pigeons: first, the animals learn faster with the right eye-left hemisphere; second, they reach higher performance levels under this condition; third, visually guided behavior is mostly under left hemisphere control. To this end, we recorded from the left and right forebrain entopallium while the animals performed a colour discrimination task. We found that, even before learning, left entopallial neurons were more responsive to visual stimulation. Subsequent discrimination acquisition recruited more neuronal responses in the left entopallium and these cells showed a higher degree of differentiation between the rewarded and the unrewarded stimulus. Thus, differential left-right responses are already present, albeit to a modest degree, before learning. As soon as some cues are associated with reward, however, this asymmetry increases substantially and the higher discrimination ratio of the left hemispheric tectofugal pathway would not only contribute to a higher performance of this hemisphere but could thereby also result in a left hemispheric dominance over downstream motor structures via reward-associated feedback systems.

Role of neuronal ras activity in adult hippocampal neurogenesis and cognition.

Hippocampal neurogenesis in the adult mammalian brain is modulated by various signals like growth factors, hormones, neuropeptides, and neurotransmitters. All of these factors can (but not necessarily do) converge on the activation of the G protein Ras. We used a transgenic mouse model (synRas mice) expressing constitutively activated G12V-Harvey Ras selectively in differentiated neurons to investigate the possible effects onto neurogenesis. H-Ras activation in neurons attenuates hippocampal precursor cell generation at an early stage of the proliferative cascade before neuronal lineage determination occurs. Therefore it is unlikely that the transgenically activated H-Ras in neurons mediates this effect by a direct, intracellular signaling mechanism. Voluntary exercise restores neurogenesis up to wild type level presumably mediated by brain-derived neurotrophic factor. Reduced neurogenesis is linked to impairments in spatial short-term memory and object recognition, the latter can be rescued by voluntary exercise, as well. These data support the view that new cells significantly increase complexity that can be processed by the hippocampal network when experience requires high demands to associate stimuli over time and/or space.

Protein biomarkers for in vitro testing of embryotoxicity.

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs ß-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein ß-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.

Navigation-induced ZENK expression in the olfactory system of pigeons (Columba livia).

A large body of evidence indicates that pigeons use olfactory cues to navigate over unfamiliar areas with a differential contribution of the left and right hemispheres. In particular, the right nostril/olfactory bulb (OB) and left piriform cortex (Cpi) have been demonstrated to be crucially involved in navigation. In this study we analysed behaviour-induced activation of the olfactory system, indicated by the expression of the immediate early gene ZENK, under different homing conditions. One experimental group was released from an unfamiliar site, the second group was transported to the unfamiliar site and back to the loft, and the third group was released in front of the loft. To evaluate the differential contribution of the left and/or right olfactory input, the nostrils of the pigeons were either occluded unilaterally or not. Released pigeons revealed the highest ZENK cell density in the OB and Cpi, indicating that the olfactory system is activated during navigation from an unfamiliar site. The groups with no plug showed the highest ZENK cell density, supporting the activation of the olfactory system probably being due to sensory input. Moreover, both Cpis seem to contribute differently to the navigation process. Only occlusion of the right OB resulted in a decreased ZENK cell expression in the Cpi, whereas occlusion of the left nostril had no effect. This is the first study to reveal neuronal activation patterns in the olfactory system during homing. Our data show that lateralized processing of olfactory cues is indeed involved in navigation over unfamiliar areas.

Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin.

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.

Age-dependent posttranslational modifications of voltage-dependent anion channel 1.

The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems. In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, we previously found a set of conserved biomarkers across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, we found age-related redundant isoforms of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms. Building on these results, we examined the expression of VDAC-1 in an in vitro model of excitotoxicity. We show that glutamate-induced calcium toxicity in neurons induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation.