Comparative Brain Morphology of Cleaning and Sponge-Dwelling Elacatinus Gobies.

INTRODUCTION: Comparative studies of brain anatomy between closely related species have been very useful in demonstrating selective changes in brain structure. Within-species comparisons can be particularly useful for identifying changes in brain structure caused by contrasting environmental selection pressures. Here, we aimed to understand whether differences within and between species in habitat use and foraging behaviour influence brain morphology, on both ecological and evolutionary time scales. METHODS: We used as a study model three species of the Elacatinus genus that differ in their habitat-foraging mode. The obligatory cleaning goby Elacatinus evelynae inhabits mainly corals and feeds mostly on ectoparasites removed from larger fish during cleaning interactions. In contrast, the obligatory sponge-dwelling goby Elacatinus chancei inhabits tubular sponges and feeds on microinvertebrates buried in the sponges' tissues. Finally, in the facultatively cleaning goby Elacatinus prochilos, individuals can adopt either phenotype, the cleaning or the sponge-dwelling habitat-foraging mode. By comparing the brains of the facultative goby phenotypes to the brains of the obligatory species we can test whether brain morphology is better predicted by phylogenetic relatedness or the habitat-foraging modes (cleaning × sponge dwelling). RESULTS: We found that E. prochilos brains from both types (cleaning and sponge dwelling) were highly similar to each other. Their brains were in general more similar to the brains of the most closely related species, E. evelynae (obligatory cleaning species), than to the brains of E. chancei (sponge-dwelling species). In contrast, we found significant brain structure differences between the cleaning species (E. evelynae and E. prochilos) and the sponge-dwelling species (E. chancei). These differences revealed independent changes in functionally correlated brain areas that might be ecologically adaptive. E. evelynae and E. prochilos had a relatively larger visual input processing brain axis and a relatively smaller lateral line input processing brain axis than E. chancei. CONCLUSION: The similar brain morphology of the two types of E. prochilos corroborates other studies showing that individuals of both types can be highly plastic in their social and foraging behaviours. Our results in the Elacatinus species suggest that morphological adaptations of the brain are likely to be found in specialists whereas species that are more flexible in their habitat may only show behavioural plasticity without showing anatomical differences.

Evolution of the visual system in ray-finned fishes.

The vertebrate eye allows to capture an enormous amount of detail about the surrounding world which can only be exploited with sophisticated central information processing. Furthermore, vision is an active process due to head and eye movements that enables the animal to change the gaze and actively select objects to investigate in detail. The entire system requires a coordinated coevolution of its parts to work properly. Ray-finned fishes offer a unique opportunity to study the evolution of the visual system due to the high diversity in all of its parts. Here, we are bringing together information on retinal specializations (fovea), central visual centers (brain morphology studies), and eye movements in a large number of ray-finned fishes in a cladistic framework. The nucleus glomerulosus-inferior lobe system is well developed only in Acanthopterygii. A fovea, independent eye movements, and an enlargement of the nucleus glomerulosus-inferior lobe system coevolved at least five times independently within Acanthopterygii. This suggests that the nucleus glomerulosus-inferior lobe system is involved in advanced object recognition which is especially well developed in association with a fovea and independent eye movements. None of the non-Acanthopterygii have a fovea (except for some deep sea fish) or independent eye movements and they also lack important parts of the glomerulosus-inferior lobe system. This suggests that structures for advanced visual object recognition evolved within ray-finned fishes independent of the ones in tetrapods and non-ray-finned fishes as a result of a coevolution of retinal, central, and oculomotor structures.

The Diversity of the Brains of Ray-Finned Fishes.

Brains are very plastic, both in response to phenotypic diversity and to larger evolutionary trends. Differences between taxa cannot be easily attributed to either factors. Comparative morphological data on higher taxonomic levels are scarce, especially in ray-finned fishes. Here we show the great diversity of brain areas of more than 150 species of ray-finned fishes by volumetric measurements using block-face imaging. We found that differences among families or orders are more likely due to environmental needs than to systematic position. Most notable changes are present in the brain areas processing sensory input (chemosenses and lateral line vs. visual system) between salt- and freshwater species due to fundamental differences in habitat properties. Further, some patterns of brain volumetry are linked to characteristics of body morphology. There is a positive correlation between cerebellum size and body depth, as well as the presence of a swim bladder. Since body morphology is linked to ecotypes and habitat selection, a complex character space of brain and body morphology and ecological factors together could explain better the differentiation of species into their ecological niches and may lead to a better understanding of how animals adapt to their environment.

Sex Differences in the Swordtail Xiphophorus hellerii Revealed by a New Method to Investigate Volumetric Data.

Comparing the relative volumes of body parts is a useful tool in morphology, but it is not trivial to do this in animals that differ in overall size. To account for scaling differences, a "reference size" has to be determined and the original absolute volumes have to be "corrected for" by this scaling reference. However, the outcome of a statistical analysis is greatly affected by this "reference size," and it is practically impossible to determine the "overall size" of a structure independent of the changes in the relative size of the parts of it. Here, a new method is introduced to compare the relative volumes of parts that does not need a scaling reference. The method transforms the absolute part volumes into a ratio matrix (volume ratio transformation, VRT). The VRT is free of any scaling factors and can be used to compare groups of animals. This paper also reviews various other errors made frequently when comparing brain morphology between animals. Finally, the VRT is applied to investigate sex differences in the swordtail fish (Xiphophorus hellerii), which show profound differences in the size of the valvula cerebelli.

Two modes of information processing in the electrosensory system of the paddlefish (Polyodon spathula).

Paddlefish are uniquely adapted for the detection of their prey, small water fleas, by primarily using their passive electrosensory system. In a recent anatomical study, we found two populations of secondary neurons in the electrosensory hind brain area (dorsal octavolateral nucleus, DON). Cells in the anterior DON project to the contralateral tectum, whereas cells in the posterior DON project bilaterally to the torus semicircularis and lateral mesencephalic nucleus. In this study, we investigated the properties of both populations and found that they form two physiologically different populations. Cells in the posterior DON are about one order of magnitude more sensitive and respond better to stimuli with lower frequency content than anterior cells. The posterior cells are, therefore, better suited to detect distant prey represented by low-amplitude signals at the receptors, along with a lower frequency spectrum, whereas cells in the anterior DON may only be able to sense nearby prey. This suggests the existence of two distinct channels for electrosensory information processing: one for proximal signals via the anterior DON and one for distant stimuli via the posterior DON with the sensory input fed into the appropriate ascending channels based on the relative sensitivity of both cell populations.

Forebrain organization in elasmobranchs.

It has long been known that many elasmobranch fishes have relatively large brains. The telencephalon, in particular, has increased in size in several groups, and as a percent of total brain weight, it is as large as in some mammals. Little is known, however, about the organization, connections, and functions of the telencephalon in elasmobranchs. Early experimental studies indicated that olfaction does not dominate the telencephalon and that other sensory modalities are represented, particularly in the pallium. We have investigated the intrinsic and extrinsic connections of the telencephalon in two elasmobranch species: the thornback guitarfish, Platyrhinoidis triseriata, and the spiny dogfish, Squalus acanthias. Tracers were injected into various parts of the forebrain and olfactory pathways were found to be extensive and were seen to involve the pallium. Injections into various parts of the pallium revealed a major input from the area basalis, which receives secondary and tertiary olfactory fibers. Nonolfactory input from the diencephalon appeared relatively minor and seemed to converge with olfactory information in the dorsal pallium and area superficialis basalis. Major descending projections were seen to originate in the dorsal pallium and terminate in the hypothalamus and - in the case of Platyrhinoidis - massively in the lateral mesencephalic nucleus. Descending pathways appeared mainly crossed in Platyrhinoidis, but not in Squalus. Our data indicate that the concept of the dorsal pallium as a nonolfactory area in elasmobranchs must be reconsidered, and we suggest that many telencephalic centers, including the dorsal pallium, are involved in olfactory orientation.

Nonlinear dynamics of skin potentials in the electrosensory paddlefish.

It is known that steady skin potentials are present in fishes due to chloride pumps in the gills and in the skin. We have found previously that these skin potentials can fluctuate and oscillate in the electrosensory paddlefish. Here we show that larger, discharge like potentials can be triggered by applying external electric fields in the water surrounding the fish. These resemble action potentials in nerve cells, but have a longer time scale. Like action potentials, these discharges travel laterally in the skin. They start at the tip of the rostrum and propagate caudally to the tip of the gill covers. They follow the all-or-nothing rule and need some refractory period before they can be evoked again. This is the first time that such discharges, so strikingly similar to action potentials, have been described at the level of a whole organism.

Measuring flow velocity and flow direction by spatial and temporal analysis of flow fluctuations.

If exposed to bulk water flow, fish lateral line afferents respond only to flow fluctuations (AC) and not to the steady (DC) component of the flow. Consequently, a single lateral line afferent can encode neither bulk flow direction nor velocity. It is possible, however, for a fish to obtain bulk flow information using multiple afferents that respond only to flow fluctuations. We show by means of particle image velocimetry that, if a flow contains fluctuations, these fluctuations propagate with the flow. A cross-correlation of water motion measured at an upstream point with that at a downstream point can then provide information about flow velocity and flow direction. In this study, we recorded from pairs of primary lateral line afferents while a fish was exposed to either bulk water flow, or to the water motion caused by a moving object. We confirm that lateral line afferents responded to the flow fluctuations and not to the DC component of the flow, and that responses of many fiber pairs were highly correlated, if they were time-shifted to correct for gross flow velocity and gross flow direction. To prove that a cross-correlation mechanism can be used to retrieve the information about gross flow velocity and direction, we measured the flow-induced bending motions of two flexible micropillars separated in a downstream direction. A cross-correlation of the bending motions of these micropillars did indeed produce an accurate estimate of the velocity vector along the direction of the micropillars.

Organization of major telencephalic pathways in an elasmobranch, the thornback ray Platyrhinoidis triseriata.

The forebrain of elasmobranchs is well developed, and in some species the relative brain/body weight is comparable to that in mammals. However, little is known about the organization of major telencephalic pathways. We injected biotinylated dextran amines into the olfactory bulb, lateral pallium, dorsomedial pallium, and the forebrain bundles of the thornback ray, Platyrhinoidis triseriata. Secondary olfactory fibers from the bulb innervate the lateral pallium, the ventral division of the rostral telencephalon and area superficialis basalis. Retrogradely labeled cells were seen exclusively in the lateral periventricular area. The projections of the lateral pallium appeared basically similar to those of the olfactory bulb, but labeling was much denser in the superficial part of area basalis. Some fibers were also seen to innervate the posterior tuberal nucleus. Injections into the dorsomedial pallium revealed a major input from area basalis. Only a few cells were retrogradely labeled in the dorsal thalamus and posterior lateral thalamic nucleus. Major efferents of the dorsomedial pallium appear to reach the contralateral inferior lobe of the hypothalamus and the lateral mesencephalic nucleus. Tracer injections into the forebrain bundles retrogradely labeled many cells in the diencephalon and the mesencephalon and also revealed terminal fields in area superficialis basalis. In addition, a large number of cells were labeled in the dorsomedial pallium. Descending telencephalic fibers innervate heavily the inferior lobes and the lateral mesencephalic nucleus. Our results show that higher order olfactory pathway courses from the lateral pallium through area basalis to the dorsomedial pallium and that ascending non-olfactory input is integrated in area superficialis basalis and the dorsal pallium along with olfactory information, rather than being processed in separate, non-olfactory centers.

Lateral line nerve fibers do not code bulk water flow direction in turbulent flow.

The discharges of anterior and posterior lateral line nerve afferents were recorded while stimulating goldfish, Carassius auratus, with bulk water flow. With increasing flow velocity lateral line afferents increased their discharge rates. However, an increased response to flow rates occurred even if flow direction was reversed. Thus, individual lateral line afferents did not encode the direction of running water. Frequency spectra of the water motions quantified with particle image velocimetry revealed flow fluctuations that increased with increasing flow velocity. Maximal spectral amplitudes of the flow fluctuations were below 5 Hz (bulk flow velocity 4-15 cms(-1)). The frequency spectra of the firing rates of lateral line afferents also showed an increase in amplitude when fish were exposed to running water. The maximal spectral amplitudes of the recorded data were in the frequency range 3-8 Hz. This suggests that the lateral line afferents mainly responded to the higher frequency fluctuations that developed under flow conditions, but not to the direct current flow or the lower frequency fluctuations. Although individual lateral line afferents encoded neither flow velocity nor flow direction we suggest that higher order lateral line neurons can do so by monitoring flow fluctuations as they move across the surface of the fish.

Local differences in calretinin immunoreactivity in the optic tectum of the ocellated dragonet.

The optic tectum of the ocellated dragonet (Synchiropus ocellatus) was studied with immunohistochemistry. Antibodies raised against the calcium binding protein calretinin (CR) revealed a lamination similar to that already reported for other ray finned fish. Most immunoreactive fibers could be observed in those layers receiving retinal afferents and most immunoreactive cells occur in the stratum periventriculare. However, there are marked differences in the presence of other calretinin-positive cell types and immunoreactive lamina between the dorsomedial and ventrolateral parts of the tectum. Synchiropus is a bottom dwelling fish with strong functional subdivisions of the visual system into dorsal and lateral visual fields. The differences in calretinin-positive cell bodies and fibers may be a sensitive indicator of functional differences of tectal circuitry.

Temporal analysis of moving DC electric fields in aquatic media.

Many aquatic vertebrates can sense the weak electric fields generated by other animals and may also sense geoelectric or electromagnetic phenomena for use in orientation. All these sources generate stationary (dc) fields. In addition, fields from animals are modulated by respiration and other body movements. Since electroreceptors are insensitive to a pure dc field, it has been suggested that the ac modulation carries most of the relevant information for electrosensory animals. However, in a natural situation pure dc fields are rare since any relative movement between source and receiver will transform a dc field into a time varying signal. In this paper, we will describe the properties of such signals and how they are filtered at the first stage of electrosensory information processing in the brain. We will show that the signal perceived by an animal traversing a dc electric field contains all the information necessary to reconstruct the distance to the source and that the signal conditioning algorithms are perfectly adapted to preserve such information.

Central organization of the electrosensory system in the paddlefish (Polyodon spathula).

The central connections of the electrosensory system were studied in the paddlefish Polyodon spathula by injecting biotinylated dextran amines into the dorsal octavolateral nucleus (DON), the cerebellum, and the mesencephalic tectum. The sole target of primary electrosensory fibers is the ipsilateral dorsal octavolateral nucleus. The principal neurons ascending from this nucleus project to the torus semicircularis, the lateral mesencephalic nucleus, and the mesencephalic tectum. The mesencephalic tectum projects back to the nucleus preeminentialis, which, in turn, projects to the cerebellar auricles and to the DON. The auricles are the main source of parallel fibers in the cerebellar crest ventral to the DON. The DON also receives input from the contralateral DON. These descending feedback loops are very similar to those of other electrosensory fishes. However, the paddlefish is unique in having three mesencephalic targets of electrosensory information. It is the only bony fish known to have extensive projections directly to the mesencephalic tectum and to a lateral mesencephalic nucleus in addition to the torus semicircularis.

The electric sense of the paddlefish: a passive system for the detection and capture of zooplankton prey.

Behavioral and electrophysiological experiments have shown that the elongated paddlefish rostrum, with its extensive population of ampullae of Lorenzini, constitutes a passive electrosensory antenna of great sensitivity and spatial resolution. As demonstrated in juvenile paddlefish, the passive electrosense serves a novel function in feeding serving as the primary, if not exclusive sensory modality for the detection and capture of zooplanktonic prey. Ampullary receptors are sensitive to the weak electrical fields of plankton from distances up to 9 cm, and juvenile paddlefish capture plankton individually with great swimming dexterity in the absence of vision or other stimulus signals. Paddlefish also detect and avoid metal obstacles, the electrical signatures of which are a potential hindrance to their feeding and reproductive migrations. The ampullary receptors, their peripheral innervation and central targets in the dorsal octavolateral nucleus, are described. We also describe the ascending and descending neuronal circuitry of the electrosensory system in the brain based on tracer studies using dextran amines.

A direct projection from the cerebellum to the telencephalon in the goldfish, Carassius auratus.

Recent studies showed that the cerebellum is involved in cognitive functions previously thought to be located in the telencephalon only. Various indirect connections between telencephalon and cerebellum are long known and may be the basis for the interaction of these two brain areas. By means of biotinylated dextran amine injections into the telencephalon and cerebellum in the goldfish, Carassius auratus, we found a direct projection from the valvula cerebelli to the telencephalon. Together with other studies in fish, birds, and mammals, these data suggest that direct connections between cerebellum and telencephalon are more common than previously thought and may contribute to the integration of telencephalic and cerebellar functions.

Effects of dopaminergic drugs and telencephalic ablation on eye movements in the goldfish, Carassius auratus.

The effect of the dopamine agonist apomorphine and the antagonist haloperidol on eye movements was tested in normal and telencephalon ablated goldfish. Reflex eye movements evoked by a rotating striped cylinder were not affected, which suggests that basic sensory and motor functions were not influenced by neither dopaminergic drugs nor the telencephalon. However, profound changes were observed in spontaneous eye movements. Particularly, the effect of apomorphine was similar to changes in eye movements observed in mammals after suppression of dopaminergic functions either by means of drugs or in patients suffering from Parkinson's disease or schizophrenia.

Responses of diencephalic neurons to sensory stimulation in the goldfish, Carassius auratus.

We studied the responses to sensory stimulation in two diencephalic areas, the central posterior nucleus of the dorsal thalamus (CP) and the anterior tuberal nucleus of the hypothalamus (TA). In both the CP and the TA, units sensitive to acoustic (500-Hz sound), hydrodynamic (25-Hz dipole stimulus), and visual (640-nm light flash) stimuli were found. In the CP, most units were unimodal and responded exclusively to visual stimulation. In contrast, in the TA, most units responded to more than one modality. The data suggest that the CP is primarily involved in the unimodal processing of sensory information, whereas the TA may be involved in multisensory integration.

Response properties of diencephalic neurons to visual, acoustic and hydrodynamic stimulation in the goldfish, Carassius auratus.

We studied the responses to sensory stimulation of three diencephalic areas, the central posterior nucleus of the dorsal thalamus, the anterior tuberal nucleus of the hypothalamus, and the preglomerular complex. Units sensitive to acoustic (500 Hz tone burst), hydrodynamic (25 Hz dipole stimulus) and visual (640 nm light flash) stimuli were found in both the central posterior and anterior tuberal nucleus. In contrast, unit responses or large robust evoked potentials confined to the preglomerular complex were not found. In the central posterior nucleus, most units were unimodal. Many units responded exclusively to visual stimulation and exhibited a variety of temporal response patterns to light stimuli. In the anterior tuberal nucleus of the hypothalamus, most units responded to more than one modality and showed a stronger response decrement to stimulus repetitions than units in the central posterior nucleus. Our data suggest that units in the central posterior nucleus are primarily involved in the unimodal processing of sensory information whereas units in the anterior tuberal nucleus of the hypothalamus may be involved in multisensory integration.

VIEWER: a program for visualising, recording, and analysing animal behaviour.

In order to determine the influence of environmental interventions, medical substances, drugs, or diseases on an animal, it is a common to observe the behaviour of the animal in the open field or in other environments. Changes in animal behaviour can be the first indicators for an influence of a substance or an intervention on the organism and certain kinds of behavioural changes can permit speculations about the underlying physiological mechanisms. Behavioural properties which are of interest in this respect are movement parameters such as velocity or direction, activity patterns, spatial distribution and occurrence of several types of standard behaviours of an animal. Observation of simple behaviours such as determining the average activity level can help to detect general changes of the internal state of an animal, while changes in complex behaviours may point to specific influences of a substance or an intervention. Our system 'VIEWER' uses a microcomputer (IBM-compatible PC running WINDOWS 95, WINDOWS 98, WINDOWS NT, or WINDOWS 2000), a low budget framegrabber card (e.g. WinTV, Hauppauge) and a standard black-and-white video camera (if necessary with infrared sensitivity). The software records the position of the animal online with a sample rate of up to 25 frames/s. After identification of the animal in the arena, animal location and orientation with respect to time is determined. In addition, movement velocity and direction, general activity level, and several other behavioural parameters which can include complex behavioural patterns are processed online. Data are presented as graphics and/or tables. Results may also be exported into those programs that are capable of importing graphics (wmf or bmp format) or ASCII-files. VIEWER offers an inexpensive, fast and easy way for analysing simple and complex behaviours of many species of animals in a variety of behavioural situations.