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.

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.

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.

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.

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.

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.

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.

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.

Dual coding of visual asymmetries in the pigeon brain: the interaction of bottom-up and top-down systems.

The pigeon's visual system is an excellent model to investigate the ontogenetic and the neuronal foundations of cerebral asymmetries. Before hatching, lateralized visual stimulation induces structural asymmetries within the tectofugal pathway during a critical time window. Interhemispheric control mechanisms emerge presumably after hatching and stabilize these induced asymmetries. Once established, visual asymmetry in pigeons displays a left hemispheric dominance for complex learning and discrimination tasks and unravels how the interplay between bottom-up and top-down mechanisms generate a lateralized, hemispheric-specific visual analysis. The ascending visual (tectofugal) pathway displays cell size asymmetries and directs more bilateral visual information towards the left hemisphere. This bottom- up system is controlled by telencephalic top-down projections, which affect intra- and/or interhemispheric inhibitory systems in a presumably lateralized manner. Such a flexible organization allows the control of information transfer depending on the visual input and hence adapt the dominant processing mode to environmental requirements.

Development of the diencephalic relay structures of the visual thalamofugal system in pigeons.

To compare the developmental pattern of the visual tecto- and thalamofugal pathways in the altricial pigeon, we examined the posthatch differentiation of the retinothalamic system. Choleratoxin was injected into the left and right eye to visualize the retinal innervation pattern of the lateral geniculate nucleus of the thalamus (GLd). The calcium-binding proteins parvalbumin and calbindin and GABA(Abeta) receptors were used as indicators for the functional development of the GLd. Although all retinorecipient thalamic target structures were invaded by retinal fibers directly after hatching, density of the projection increased during the first week. While the adult GLd was characterized by a substantial number of cells displaying calbindin-immunoreactivity and by a sparse innervation by parvalbumin-immunoreactive fibers, after hatching no labelling for calcium-binding proteins could be detected. Calbindin-immunoreactivity appeared not before posthatching day 7, while parvalbumin-immunoreactive fibers were detected only after the third week. In contrast, a dense but diffuse GABA(Abeta) receptor-labelling was present from hatching onwards that decreased during development. The delayed expression of calbindin as well as changes in the density of GABA(Abeta) receptors indicate that maturation of GLd neurons extends long into the posthatch period. It is likely that the GABAergic interneurons mainly develop within this posthatch timeframe. Combined with the delayed development of the parvalbumin-positive innervation, the developmental pattern of GLd neurons suggests that the thalamofugal networks are immature after hatching and therefore still sensitive to modulations of posthatch visual experience.

A morphological study of the nucleus subpretectalis of the pigeon.

In pigeons, the tectofugal system is functionally as well as structurally lateralized. So for example the right nucleus rotundus is less modulated by right forebrain influences than the left nucleus rotundus by the left ones. This functional lateralization pattern may depend on a dynamic balance between left and right tectal processing. Apart from inhibitory interactions at tectal level, suppressive influences might directly affect rotundal neurons by GABAergic input from a cluster of nuclei, the bed nuclei of the tecto-thalamic tract. A major afferent of these nuclei is the side branch of the tectorotundal projection which is of bilateral origin and which is involved in the regulation of ipsilateral as well as bilateral visual processing. Hence, an important role of the bed nuclei could be the interhemispheric communication and in turn the mediation of functional asymmetries. In a first step to unravel asymmetric influences of these nuclei, the present study investigated if the largest of the bed nuclei, the nucleus subpretectalis displays morphological asymmetries in the pigeon. We found that the nucleus subpretectalis in fact exhibits asymmetric cell sizes with larger cell bodies on the left side. This asymmetrical pattern was not present in dark-incubated animals indicating that cell size asymmetries within nucleus subpretectalis are induced by asymmetric photic stimulation during embryonic development.

Calcium-binding proteins label functional streams of the visual system in a songbird.

The vertebrate nervous system has been shown to contain high concentrations of intracellular calcium-binding proteins, each of them with a restricted expression pattern in specific brain regions and specific neuronal subpopulations. Using immunohistochemical staining techniques, we analyzed the expression pattern of calbindin, calretinin and parvalbumin in visual brain areas of a songbird species, the zebra finch (Taeniopyga guttata). Here we show that the analyzed proteins are expressed in a complementary fashion within different brain substructures generally corresponding to functional subpathways of the avian visual system. In detail, calbindin is expressed in the brain structures that belong to the thalamofugal pathway, whereas parvalbumin-positive neurons are found in the brain structures that are part of the tectofugal visual pathway. Originally, the expression of calcium-binding proteins has been associated with specific morphological or neurochemical criteria of neurons. Our results suggest that their expression pattern also indicates a functional segregation of brain substructures linked to vision in the zebra finch brain. As the selective labeling of functional streams has also been shown for the visual system in mammalian species, function-selective expression of calcium-binding proteins might be a general feature of vertebrates.

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.

Visual responses and afferent connections of the n. ventrolateralis thalami (VLT) in the pigeon (Columba livia).

The nucleus ventrolateralis thalami (VLT) in pigeons receives direct retinal and forebrain projections and has reciprocal connections with the optic tectum. Although VLT is a component of the avian visual system, no study directly examined its connections or its cellular response characteristics. We, therefore, recorded from single units in the pigeon's VLT while visually stimulating the ipsi- and/or contralateral eye. In addition, tracing experiments were conducted to investigate its afferent connections. Electrophysiologically, we discovered three types of neurons, two of which were probably activated via a top-down telencephalotectal system (latencies > 100 ms). Type I neurons responded to uni- and bilateral and type II neurons exclusively to bilateral stimulation. Type III neurons were probably activated by retinal or retinotectal input (latencies < 27 ms) and responded to contra- and bilateral stimulation. Retrograde tracer injections into the VLT revealed an ipsilateral forebrain input from the visual Wulst, from subregions of the arcopallium, and bilateral afferents from the optic tectum. Most intriguing was the direct connection between the VLTs of both hemispheres. We suggest that the avian VLT is part of a system that integrates visuomotor processes which are controlled by both forebrain hemispheres and that VLT contributes to descending tectomotor mechanisms.

"Let There be Light!" pigeon eggs are regularly exposed to light during breeding.

Light stimulation before hatching initiates the emergence of avian visual lateralisation. Since several studies show that birds benefit from being lateralised, we can conjecture that their clutch is being exposed to light during breeding. We tested this assumption in pigeons with a semi-natural setup where the animals were systematically recorded using a movement detection system throughout their breeding period. The results show that pigeon pairs perform their relieves in a regular way by abandoning their clutch for a mean of about 55 s at approximately every 43 min. Thus, the developing visual pathways are repetitively stimulated by light for cumulatively over 3h before the breeding period ends. It becomes apparent that both the duration as well as the repetitions of light stimulation play a crucial role in the onset of visual asymmetry.

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.

Differential effects of ocular BDNF-injections onto the development of tectal cells characterized by calcium-binding proteins in pigeons.

The optic tectum of vertebrates bears a set of visual neurons which can be differentiated by the expression of distinct calcium-binding proteins (CaBPs). Using immunohistochemistry, we mapped the distribution of the CaBPs calbindin (CB) and parvalbumin (PV) in the pigeon's optic tectum and examined if their differentiation is affected by retinal brain-derived neurotrophic factor (BDNF)-injections. CB-immunoreactive (ir) and PV-ir cells displayed a lamination pattern which differed from other birds. While PV-ir cells were present in several retinorecipient tectal laminae, CB-ir cells were confined to layer 3 and 5 and - as a specialization of pigeons - were also detected in a subpopulation of layer 13 neurons. Comparison of saline- and BDNF-injected animals revealed that this general expression pattern was not affected by ocular BDNF-injections. In contrast, the size of tectal cells was differentially modulated. While CB-ir cells in layers 3 and 13 were unaffected by retinal BDNF, cells in layer 5 developed enlarged cell bodies. The PV-ir cells displayed smaller soma sizes within both tectal hemispheres suggesting also an indirect effect of retinal BDNF. These data indicate a differential sensitivity of tectal cell types to retinal BDNF, which might be one mechanism by which retinal input modulates tectal circuitries.