Automated analysis of behavior: A computer‐controlled system for drug screening and the investigation of learning

Efforts to understand cognition will be greatly facilitated by computerized systems that enable the automated analysis of animal behavior. A number of controversies in the invertebrate learning field have resulted from difficulties inherent in manual experiments. Driven by the necessity to overcome these problems during investigation of neural function in planarian flatworms and frog larvae, we designed and developed a prototype for an inexpensive, flexible system that enables automated control and analysis of behavior and learning. Applicable to a variety of small animals such as flatworms and zebrafish, this system allows automated analysis of innate behavior, as well as of learning and memory in a plethora of conditioning paradigms. We present here the schematics of a basic prototype, which overcomes experimenter effects and operator tedium, enabling a large number of animals to be analyzed with transparent on‐line access to primary data. A scaled‐up version of this technology represents an efficient methodology to screen pharmacological and genetic libraries for novel neuroactive reagents of basic and biomedical relevance. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

The urbilaterian brain revisited: novel insights into old questions from new flatworm clades

Flatworms are classically considered to represent the simplest organizational form of all living bilaterians with a true central nervous system. Based on their simple body plans, all flatworms have been traditionally grouped together in a single phylum at the base of the bilaterians. Current molecular phylogenomic studies now split the flatworms into two widely separated clades, the acoelomorph flatworms and the platyhelminth flatworms, such that the last common ancestor of both clades corresponds to the urbilaterian ancestor of all bilaterian animals. Remarkably, recent comparative neuroanatomical analyses of acoelomorphs and platyhelminths show that both of these flatworm groups have complex anterior brains with surprisingly similar basic neuroarchitectures. Taken together, these findings imply that fundamental neuroanatomical features of the brain in the two separate flatworm groups are likely to be primitive and derived from the urbilaterian brain.     

Free-living flatworms under the knife: past and present

Traditionally, regeneration research has been closely tied to flatworm research, as flatworms (Plathelminthes) were among the first animals where the phenomenon of regeneration was discovered. Since then, the main focus of flatworm regeneration research was on triclads, for which various phenomena were observed and a number of theories developed. However, free-living flatworms encompass a number of other taxa where regeneration was found to be possible. This review aims to display and to compare regeneration in all major free-living flatworm taxa, with special focus on a new player in the field of regeneration, Macrostomum lignano (Macrostomorpha). Findings on the regeneration capacity of this organism provide clues for links between regeneration and (post-)embryonic development, starvation, and asexual reproduction. The role of the nervous system and especially the brain for regeneration is discussed, and similarities as well as particularities in regeneration among free-living flatworms are pointed out.     

Computational modeling of evolutionary learning processes in the brain

The evolutionary selection circuits model of learning has been specified algorithmically. The basic structural components of the selection circuits model are enzymatic neurons, that is, neurons whose firing behavior is controlled by membrane-bound macromolecules called excitases. Learning involves changes in the excitase contents of neurons through a process of variation and selection. In this paper we report on the behavior of a basic version of the learning algorithm which has been developed through extensive interactive experiments with the model. This algorithm is effective in that it enables single neurons or networks of neurons to learn simple pattern classification tasks in a number of time steps which appears experimentally to be a linear function of problem size, as measured by the number of patterns of presynaptic input. The experimental behavior of the algorithm establishes that evolutionary mechanisms of learning are competent to serve as major mechanisms of neuronal adaptation. As an example, we show how the evolutionary learning algorithm can contribute to adaptive motor control processes in which the learning system develops the ability to reach a target in the presence of randomly imposed disturbances.     

A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science.     

Automating insect identification: exploring the limitations of a prototype system

Automated identification systems based on computer image analysis technology provide an attractive, but as yet unexploited potential solution to the growing burden of routine species identifications presently faced by a dwindling community of expert taxonomists. DAISY (the Digital Automated Identification SYstem) is a prototype novel automated identification system, developed to explore this possibility. In its pilot phase, the DAISY algorithms were developed to discriminate five species of parasitic wasp, based on differences in their wing structure. Here, again using wing form, the ability of DAISY to discriminate amongst an order of magnitude more species – 49 species of closely related biting midges is examined. In so doing an attempt is made to establish a set of basic 'benchmark' tests of the efficacy, and weaknesses, of such an automated identification system.     

Animal learning and intelligence

The ability to learn is common to most animal species: the need to exploit past experience being obviously extremely important for survival, many animals have evolved ways of coping with it. Although the complexity of learning needed for optimal survival may be different in different species, the basic mechanisms appear to be fairly constant even in phylogenetically distant ones. This homogeneity across species in learning mechanisms is in some ways surprising in view of the large phylogenetic differences and of the considerable variability not only in the general plan of their bodily structures, but also, more specifically, in their neural organization and in their behavioral adaptations. One possible explanation is that animals have acquired learning very precociously, and that the original and basic mechanisms have proved so efficient and faultproof as to be preserved from then on without any significant modification. Most researchers of the subject seem to accept the equation «intelligence=learning capability», operationally very useful because it leads to a variety of formal tests. Some researchers, stressing that behavior is subject to the same evolutionary principles as any other character of the organism and acknowledging some problems in the accepted laws of learning, have tried to find a satisfactory answer to the question of animal intelligence by attempting a synthesis between the concepts of animal learning psychology and those of ethology. To some extent, dissatisfaction with established learning theories originated within the theories themselves: the study of phenomena such as autoshaping, selective attention, preferential learning of some responses amongst the many possible, conditioned learning of taste aversions, etc. Further difficulties for conditioning theories arose from the discovery of ethological phenomena. Other researchers have attempted to check the hypothesis that animals possess cognition. A number of complex experimental situtations have been devised to this purpose, but the results still are far from conclusive.     

Hox genes and the parasitic flatworms: new opportunities, challenges and lessons from the free-living

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with >100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.     

Rapid, automated screening method for enzymatic transformations using a robotic system and supercritical fluid chromatography

An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.     

Analysis of classical neurotransmitter markers in tapeworms: Evidence for extensive loss of neurotransmitter pathways

Parasitic flatworms have complex neuromuscular systems that serve important functions in their life cycles. However, our understanding of neurotransmission in parasitic flatworms is limited. Pioneering studies have suggested the presence of several classical neurotransmitter systems, but their molecular components have not been characterized in most cases. Because these components are conserved in bilaterian animals, we searched the genomes of parasitic flatworms for orthologs of genes required for neurotransmitter synthesis, vesicular transport, reuptake, and reception. Our results indicate that tapeworms have lost the genes that are specifically required in other animals for synaptic signaling using the classical neurotransmitters dopamine, tyramine, octopamine, histamine and gamma-aminobutyric acid (GABA). These results imply that these signaling pathways are either absent in these parasites, or that they require completely different molecular components in comparison with other animals. The orthologs of genes related to histaminergic and GABA signaling are also missing in trematodes (although Schistosoma-specific histaminergic receptors have been previously described). In contrast, conserved genes required for glutamatergic, serotonergic and cholinergic signaling could be found in all analyzed flatworms. We analyzed the expression of selected markers of each pathway in the tapeworm Hymenolepis microstoma by whole-mount in situ hybridization. Each marker was specifically expressed in the nervous system, although with different patterns. In addition, we analyzed the expression of proprotein convertase 2 as a marker of peptidergic cells. This gene showed the widest expression in the nervous system, but was also expressed in other tissues, suggesting additional roles of peptidergic signaling in tapeworm development and reproduction.     

Tracking and predation on earthworms by the invasive terrestrial planarian Bipalium adventitium (Tricladida, Platyhelminthes)

The potential ecological impact of exotic terrestrial planarians will be determined in part by their sensory abilities and predatory behavior. It has been suggested that these flatworms may only encounter their earthworm prey by chance, hence restricting the breadth of species they will feed upon and the number of microhabitats in which predator-prey interactions occur. We hypothesized that those flatworms that have already successfully invaded North America (genus Bipalium) actually detect and follow chemical trails of earthworms and possess the behavioral repertoire needed to feed on the prey in a range of microhabitats. We examined: (1) the tendency of Bipalium adventitium to follow chemical trails left by injured and un-injured earthworms; (2) the behavioral repertoire and predatory success of B. adventitium feeding on three earthworm species in subterranean tunnels; and (3) the response of flatworms to the reportedly defensive secretions of the earthworm Eisenia fetida in tunnels. B. adventitium detected and followed trails of earthworm mucus and secretions left by injured and un-injured earthworms. Flatworms followed trails on a range of substrates and pursued and captured three species of earthworms in subterranean tunnels, including individuals many times their mass. Although most behavior exhibited during underground attacks was similar to that reported for surface encounters, the flatworms also behaved in ways that blocked earthworm escape from tunnels. The flatworms were less successful at preying on E. fetida than on Lumbricus rubellus and Lumbricus terrestris in underground tunnels and showed some aversion to the secretions from E. fetida.     

Effects of estrogens on learning of rats with chronic brain cholinergic deficit in Morris water maze

A chronic deprivation of brain cholinergic functions in rats caused by intracerebroventricular injection of neurotoxin AF64A increases the escape latency in Morris water maze test as compared to control sham-operated animals. Measurements and analysis of rat movement tracks using an original computerized "Behavioral Vision" system revealed the ability of 17 beta-Estradiol and its synthetic isomer J-861 (both administered daily in per os dose 0.2 mg/kg during 7 days before and 10 days after a single intracerebroventricular injection of AF64A) to improve learning of the animals. Directivity of search trajectories was estimated by a novel index of track straightness. The introduction of an index of "passive swimming" made it possible to reveal episodes of immobility in water-maze behavior of AF64A-injected animals. Unlike J-861, 17 beta-Estradiol almost completely eliminated these episodes. The newly developed indices (especially straightness) seem to be very useful in differentiating learning ability of rats from a decrease in their mobility in the Morris water-maze test, in particular, in case of the estrogens under study.     

Automated proximity sensing in small vertebrates: design of miniaturized sensor nodes and first field tests in bats

Social evolution has led to a stunning diversity of complex social behavior, in particular in vertebrate taxa. Thorough documentation of social interactions is crucial to study the causes and consequences of sociality in gregarious animals. Wireless digital transceivers represent a promising tool to revolutionize data collection for the study of social interactions in terms of the degree of automation, data quantity, and quality. Unfortunately, devices for automated proximity sensing via direct communication among animal‐borne sensors are usually heavy and do not allow for the investigation of small animal species, which represent the majority of avian and mammalian taxa. We present a lightweight animal‐borne sensor node that is built from commercially available components and uses a sophisticated scheme for energy‐efficient communication, with high sampling rates at relatively low power consumption. We demonstrate the basic functionality of the sensor node under laboratory conditions and its applicability for the study of social interactions among free‐ranging animals. The first field tests were performed on two species of bats in temperate and tropical ecosystems. At <2 g, this sensor node is light enough to observe a broad spectrum of taxa including small vertebrates. Given our specifications, the system was especially sensitive to changes in distance within the short range (up to a distance of 4 m between tags). High spatial resolution at short distances enables the evaluation of interactions among individuals at a fine scale and the investigation of close contacts. This technology opens new avenues of research, allowing detailed investigation of events associated with social contact, such as mating behavior, pathogen transmission, social learning, and resource sharing. Social behavior that is not easily observed becomes observable, for example, in animals living in burrows or in nocturnal animals. A switch from traditional methods to the application of digital transceiver chips in proximity sensing offers numerous advantages in addition to an enormous increase in data quality and quantity. For future applications, the platform allows for the integration of additional sensors that may collect physiological or environmental data. Such information complements social network studies and may allow for a deeper understanding of animal ecology and social behavior.     

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze^1,2 that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze^1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal''s behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.     

Evolution of Canine Information Processing under Conditions of Natural and Artificial Selection

The paper first argues that under conditions of domestication animals are necessarily selected (incidentally or otherwise) for (1) responsitivity to a broad band of stimuli and (2) behavioral plasticity. The consequent tractability of domestic animals is contrasted with the stimulus-boundness and response stereotypy of the fixed action patterns observed in wild animal behavior. It is suggested that this difference accounts for the differential trainability of wolves and dogs. The second section of the paper presents observational evidence that although the wolf is not very amenable to instrumental conditioning, it possesses a highly developed capacity for observational learning. It is then noted that since observational learning requires recognition of means-ends relationships this conclusion is inconsistent with the claim that wolf behavior is largely instinct-bound. Finally, these conclusions are reconciled by hypothesizing that the wolf possesses a “duplex” information-processing system, a primitive “instinctual” system that mediates basic survival responses and a more recently acquired “cognitive” system that evolved as the wolt became a group hunter. Neurobehavioral and developmental comparisons of wolf and dog suggest that these two systems have become integrated into a single scheme in the course of the dog's domestication.     

The Hox gene complement of acoel flatworms, a basal bilaterian clade

Several molecular data sets suggest that acoelomorph flatworms are not members of the phylum Platyhelminthes but form a separate branch of the Metazoa that diverged from all other bilaterian animals before the separation of protostomes and deuterostomes. Here we examine the Hox gene complement of the acoel flatworms. In two distantly related acoel taxa, we identify only three distinct classes of Hox gene: an anterior gene, a posterior gene, and a central class gene most similar to genes of Hox classes 4 and 5 in other Bilateria. Phylogenetic analysis of these genes, together with the acoel caudal homologue, supports the basal position of the acoels. The similar gene sets found in two distantly related acoels suggest that this reduced gene complement may be ancestral in the acoels and that the acoels may have diverged from other bilaterians before elaboration of the 8- to 10-gene Hox cluster that characterizes most bilaterians.     

Latency to first naloxone-induced jump as a measure of precipitated abstinence intensity in morphine-dependent mice and the interaction of set on this test

A method developed in this laboratory uses latency to time of first jump after injection of naloxone rather than the number of jumps in a specified period or the number of animals jumping as a measure of the degree of morphine physical dependence. For the test, mice are placed in a glass cylinder used as a test chamber, after being injected with the antagonist. During the development of this method it was observed that repeated exposures of dependent mice to both naloxone and the chamber yielded shorter latencies to first jump than did repeated exposures to naloxone alone in animals with the same degree of physical dependence. It appears that learning develops when naloxone injections are given repeatedly and followed by exposure to the test chamber and that this learned behavior is manifested by a reduced latency to first jump which may be confused with increased intensity of the opiate-withdrawal syndrome.