Brain areas activated during visual learning in the cichlid fish Pseudotropheus zebra.

The neural correlates of most cognitive functions in fish are unknown. This project aimed to identify brain regions involved in visual learning in the cichlid fish Pseudotropheus zebra. The expression of the protein pS6 was measured in 19 brain areas and compared between groups of individuals subjected to four different behavioral contexts (control, avoidance, trained, and novelty groups). Control group individuals were sacrificed with minimal interactions. Fish in the avoidance group were chased with a net for an hour, after which they were sacrificed. Individuals in the trained group received daily training sessions to associate a visual object with a food reward. They were sacrificed the day they reached learning criterion. Fish in the novelty group were habituated to one set of visual stimuli, then faced a change in stimulus type (novelty stimulus) before they were sacrificed. Fish in the three treatment groups showed the largest activation of pS6 in the inferior lobes and the tectum opticum compared to the control group. The avoidance group showed additional activation in the preoptic area, several telencephalic regions, the torus semicircularis, and the reticular formation. The trained group that received a food reward, showed additional activation of the torus lateralis, a tertiary gustatory center. The only area that showed strong activation in all three treatment groups was the nucleus diffusus situated within the inferior lobe. The inferior lobe receives prominent visual input from the tectum via the nucleus glomerulosus but so far, nothing is known about the functional details of this pathway. Our study showed for the first time that the inferior lobes play an important role in visual learning and object recognition.

Receptive field organization of electrosensory neurons in the paddlefish (Polyodon spathula).

Paddlefish use their electrosense to locate small water fleas (daphnia), their primary prey, in three-dimensional space. High sensitivity and a representation of object location are essential for this task. High sensitivity can be achieved by convergence of information from a large number of receptors and object location is usually represented in the nervous system by topographic maps. However the first electrosensory center in the brain, the dorsal octavolateral nucleus in the hindbrain, is neither topographically organized nor does it show a higher sensitivity than primary afferent fibers. Here, we investigated the response properties of electrosensory neurons in the dorsal octavolateral nucleus (DON), the lateral mesencephalic nucleus (LMN) and the tectum mesencephali (TM). LMN units are characterized by large receptive fields, which suggest a high degree of convergence. TM units have small receptive fields and are topographically arranged, at least in the rostro-caudal axis, the only dimension we could test. Well-defined receptive fields, however, could only be detected in the TM with a moving DC stimulus. The receptive fields of TM units, as determined by slowly scanning the rostrum and head with a 5 Hz stimulus, were very large and frequently two or more receptive fields were present. The receptive fields for LMN units were located in the anterior half of the rostrum whereas TM units had receptive fields predominantly on the head and at the base of the rostrum. A detailed analysis of the prey catching behavior revealed that it consists of two phases that coincide with the location of the receptive fields in LMN and TM, respectively. This suggests that LMN units are responsible for the initial orienting response that occurs when the prey is alongside the anterior first half of the rostrum. TM units, in contrast, had receptive fields at locations where the prey is located when the fish opens its mouth and attempts the final strike.

Response properties of electrosensory units in the midbrain tectum of the paddlefish (Polyodon spathula Walbaum).

Paddlefish use their peculiar rostrum to detect minute electric fields from their main prey, small water fleas. Electroreceptors over the rostrum and head sense these fields and send the information into a single hindbrain area, the dorsal octavolateral nucleus (DON). From there, information is sent to various midbrain structures, including the tectum. The response properties of primary afferent fibers and DON units has been well investigated, but nothing is known about electrosensory units in the midbrain. Here we recorded the responses of single units in the midbrain tectum and DON to uniform electric fields. Tectal units exhibited little spontaneous activity and responded to sine waves with a few, well phase-locked spikes. Phase locking was still significant at amplitudes one order of magnitude lower than in the DON. If stimulated with sinusoidal electric fields of different frequencies, phase locking in DON units decreased proportionally with frequency whereas the response of tectal units depended little on frequency. This is in agreement with behavioral studies showing that relevant frequencies range from DC to ca 20 Hz.

Effect of temperature and calcium on transneuronal diffusion of DiI in fixed brain preparations.

The lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label neuronal pathways in fixed goldfish brains. The normal procedure involving 4% paraformaldehyde as a fixative, applying DiI and storing the brain in the fixative at 40 degrees C resulted in many cases in a rather diffuse labeling of fiber pathways and the occurrence of transneuronally labeled cells and fibers. We found that calcium and heat both facilitate the diffusion of DiI out of membranes in vibratome sections. We modified the protocol by adding the calcium binding substance ethylenediamine tetraacetate to all solutions and incubated at room temperature. This improved the sharpness of labeled structures and eliminated the transneuronal labeling in our material. Although transneuronal transport of the tracer may still occur under certain circumstances, the present modification of the DiI staining procedure substantially increased the staining quality and reproducibility and decreased the occurrence of transneuronal labeling.

Subdivisions of the olfactory system in the sterlet Acipenser ruthenus.

Subdivisions of the olfactory system of the sterlet Acipenser ruthenus were investigated by means of horseradish peroxidase (HRP) injections into the nose, and by soybean agglutinin binding studies. With both methods primary olfactory fibers were labeled which projected to the ventral part of the glomerular layer of the olfactory bulb. The dorsal part of the olfactory bulb did not bind soybean agglutinin, however, even though HRP tracing showed primary olfactory fibers in that area. This confirms earlier morphological studies which claim the existence of distinct subdivisions of the olfactory system in the sturgeon. The lack of soybean agglutinin binding in the dorsal part of the olfactory bulb suggests, however, that this part is not homologous with the accessory olfactory system of tetrapods.

The extrabulbar olfactory pathway: primary olfactory fibers bypassing the olfactory bulb in bony fishes?

Recent evidence has revealed that some primary olfactory fibers bypass the olfactory bulb and terminate in tel- and/or diencephalic areas (extrabulbar olfactory pathway, EBOP). We investigated the projections of this system in different fishes by means of soybean agglutinin binding studies. In all species in which primary olfactory fibers were labelled, fiber bundles can be traced beyond the olfactory bulb. These run with the medial forebrain bundle and terminate at different targets, depending on the species. In the teleosts Macrognathus, Mogurnda, and Hemichromis, EBOP fibers can be traced into the ventral telencephalon, pars ventralis, pars supracommissuralis and/or into the preoptic nucleus. In most nonteleosts studied (Polypterus, Chalamoichthys, Amia), the EBOP also innervates diencephalic targets. An exception is Acipenser, which displays an innervation pattern similar to that in teleosts. Comparison with results obtained by other techniques suggests that the EBOP consists of primary olfactory fibers, which project not only to the olfactory bulb but also to various other targets in the prosencephalon of anamniotic vertebrates.

Retinopetal projections from diencephalic neurons in a primitive actinopterygian fish, the sterlet Acipenser ruthenus.

Horseradish peroxidase (HRP) injections into the sterlet (Acipenser ruthenus) retina retrogradely label neurons in the dorso-medial thalamus, bilaterally. On the contralateral side, 5-7 cells were labelled, whereas ipsilaterally, only 2-3 cells were backfilled. Such diencephalic retinopetal cells have, so far, only been found in teleosts and in tetrapods. It has, therefore, been suggested that they evolved independently in these two vertebrate groups. Our findings on a primitive actinopterygian fish, suggest a more ancient origin of diencephalic projections to the retina.

Visual-evoked responses in the salmon telencephalon change during smolt transformation.

During smolt transformation, salmon behaviour changes dramatically and the fish are imprinted to their natal stream. This brief episode is accompanied by changes in neurochemistry and connectivity of the brain. Comparing visual-evoked potentials, recorded from optic tectum and telencephalon by glass microcapillaries, we found pronounced differences before and after smolt transformation in the telencephalon. In the forebrain of presmolts, only small short-latency responses are present whereas postsmolts display large long-latency waves which characteristic dynamic properties in addition. These findings suggest changes in the functional role of the telencephalon during smolt transformation.

Interval-specific event related potentials to omitted stimuli in the electrosensory pathway in elasmobranchs: an elementary form of expectation.

Multiunit activity and slow local field potentials show Omitted Stimulus Potentials (OSP) in the electrosensory system in rays (Platyrhinoidis triseriata, Urolophus halleri) after a missing stimulus in a 3 to > 20 Hz train of microV pulses in the bath, at levels from the primary medullary nucleus to the telencephalon. A precursor can be seen in the afferent nerve. The OSP follows the due-time of the first omitted stimulus with a, usually, constant main peak latency, 30-50 ms in medullary dorsal nucleus, 60-100 ms in midbrain, 120-190 ms in telencephalon-as though the brain has an expectation specific to the interstimulus interval (ISI). The latency, form and components vary between nerve, medulla, midbrain and forebrain. They include early fast waves, later slow waves and labile induced rhythms. Responsive loci are quite local. Besides ISI, which exerts a strong influence, many factors affect the OSP slightly, including train parameters and intensity, duration and polarity of the single stimulus pulses. Jitter of ISI does not reduce the OSP substantially, if the last interval equals the mean; the mean and the last interval have the main effect on both amplitude and latency. Taken together with our recent findings on visually evoked OSPs, we conclude that OSPs do not require higher brain levels or even the complexities of the retina. They appear in primary sensory nuclei and are then modified at midbrain and telencephalic levels. We propose that the initial processes are partly in the receptors and partly in the first central relay including a rapid increase of some depressing influence contributed by each stimulus. This influence comes to an ISI-specific equilibrium with the excitatory influence; withholding a stimulus and hence its depressing influence causes a rebound excitation with a specific latency.

Peripheral origin of olfactory nerve fibers by-passing the olfactory bulb in Xenopus laevis.

After DiI injections into the diencephalon of Xenopus, two types of retrogradely labelled cells were found in the nasal area: (i) receptor cells in the olfactory epithelium and (ii) a small cell group located between the main olfactory epithelium and the vomeronasal system. These results reveal an extensive extrabulbar olfactory projection of olfactory receptor cells. Fibers of these cells do not terminate in the olfactory bulb but innervate targets in the diencephalon directly. The other type of retrogradely labelled cells, apparently, are not part of any epithelium. They resemble similar cell groups which have previously been regarded as part of the nervus terminalis system in other vertebrates.

Changes in ligand binding to GABAA receptor sites in pacific salmon (Oncorhynchus) brain during spawning migration and "aging".

When several years old, pacific salmon return to the site of birth, to spawn. At this time, a rapid aging process begins and the fish die within a few weeks after reproducing. Age-related changes of high and low affinity GABA binding sites were studied in salmon brains at three different phases of the spawning migration, i.e. shortly after returning to the natal stream, at the time of spawning, and thereafter. High affinity GABA binding slightly increased while the fish deteriorated. The low affinity component showed a remarkable decrease in density and a concomitant increase in affinity during this final episode of salmon life.

Functional subdivisions of the olfactory system correlate with lectin-binding properties in Xenopus.

Soybean agglutinin (SBA) is known to selectively label a portion of neurons in amphibian and mammalian primary olfactory systems. Hitherto, no other distinctive features have been found to correlate with the two neuronal populations. Investigating SBA-HRP binding in olfactory mucosa and CNS of Xenopus, we noted that labelled and unlabelled structures can readily be assigned to different olfactory subsystems. The SBA negative one is utilized to detect air-borne odors, whereas major SBA-positive structures serve a role in the perception of water dissolved molecules. Some labelled fibers by-pass the olfactory bulb, traverse the telencephalon and innervate prosencephalic structures. They are considered to be aberrant olfactory nerve fibers, rather than being part of the terminal nerve.

Subdivisions of the terminal nerve in Xenopus laevis.

The majority of recent studies on the terminal nerve (nt) in various vertebrates either involved tracer injections into the nasal cavity or made use of the LHRH-/FMRFamide-like immunoreactivity (ir) of a portion of its fibers. The present investigation was designed to determine the extent of overlap between data rendered by the two methods in Xenopus. The findings reveal no overlap of nt projections visualized by the two experimental techniques. This result sheds doubt on the validity of current definitions of the nt system.

Histochemical, connectional and cytoarchitectonic evidence for a secondary reduction of the pretectum in the European eel, Anguilla anguilla: a case of parallel evolution.

There are at least three different patterns of pretectal organization in teleost fishes: a simple pattern observed in cyprinids, an elaborate pattern present in percomorphs, and an intermediately complex pattern seen in many other teleost groups. The taxonomic distribution of the pretectal patterns indicates that the simple and the elaborate patterns are both evolutionarily derived (apomorphic) from the primitive (plesiomorphic) intermediately complex one. In anguillids, the pretectal pattern observed cytoarchitectonically has an anatomical configuration similar to that of the simple pattern in cyprinids. The distribution of acetylcholinesterase positivity in the pretectum (namely acetylcholinesterase positivity in the parvo- and magnocellular superficial and posterior pretectal nuclei, and acetylcholinesterase negativity in the pretectal cell plate and the ovoid preglomerular cell aggregate), as well as the retinal projections (namely retinal terminals in the parvocellular superficial and central pretectal nuclei, and absence of such terminals in the magnocellular superficial and posterior pretectal nuclei and the pretectal cell plate), strongly supports the interpretation suggested by the cytoarchitectonic analysis. As anguillids (elopomorpha) and cyprinids (ostariophysi) are related only distantly, this secondary simplification in the pretectum likely occurred independently, i.e. this simplification represents a case of parallel reduction.

Event-related potentials in the retina and optic tectum of fish.

1. Compound field potentials were recorded with up to 18 microelectrodes in comb, brush, or spear arrays on and in the optic tectum and with suction electrodes from the distal stump of the cut optic nerve and from the optic nerve head in the opened eye in elasmobranchs and teleosts. Diffuse light flashes of different durations and submaximal intensities were delivered in trains with regular or irregular interstimulus intervals (ISI). 2. Event-related potentials (ERPs) are visible in single trials and begin at 50-200 ms after an "oddball" flash, especially one that is slightly weaker, briefer, or delayed by as little as 6% of ISI, compared with the more frequent stimulus. ERPs to the opposite condition are not of the same form or size. 3. One or more stimuli were omitted from a train or the train terminated after various conditioning times. Deflections occur beyond the expected visual-evoked potentials (VEPs) to the last flash and are called omitted-stimulus potentials (OSPs). They occur on schedule--approximately 100 ms after the next flash would be due--almost independent of intensity, duration, or conditioning time. They are considered to be ERPs without any necessary implication or denial of a temporally specific expectation. 4. Three components of OSP occur alone or in combination: an initial fast peak, a slow wave, and an oscillatory spindle up to ls or more in duration. This resembles the OFF response to steady light. 5. All these components are already present in the retina with optic nerve cut. 6. The same mean ISI with a high proportion of jitter gives OSPs with only slightly longer latencies and smaller amplitudes; the OSP acts as though the retina makes an integrated prediction of ISI, intensity, and duration. 7. During a conditioning train the equilibrium between excitation and inhibition after each flash changes according to frequency, intensity, duration, and conditioning time; the VEP reflects this in a shape unique to the ISI; inhibition increases rapidly after each flash and then decays slowly according to the recent mean ISI. This allows rebound disinhibition after missing, weak, or delayed flashes (OSP or ERP) or causes an altered VEP after a longer or stronger oddball. 8. It seems unlikely that the OSP or oddball ERP in fish tectum is equivalent to mammalian ERPs under the same regime or signals higher cognitive events, because they are already present in the retina, require flash frequencies greater than 1 Hz, and grow with frequency up to and beyond flicker fusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Tectal afferents in Rana pipiens. A reassessment questioning the comparability of HRP-results.

Studying afferents to the optic tectum in Rana pipiens by means of retrograde HRP transport, results were obtained, that cast doubt on the reliability of this neuronal tract tracing technique. The tectum was found to receive afferents from e.g. lateral mesencephalic tegmentum, torus semicircularis, contralateral large-celled pretectal nucleus, and ipsilateral thalamic nuclei, which have not previously been demonstrated. In addition, the data obtained lack evidence of projections reported earlier. We suggest, that neuronal projections may be subject to hormone and/or activity dependent changes, that alter the probability of accumulating sufficient amounts of the tracer to become labeled.

Dynamic properties of visual evoked potentials in the tectum of cartilaginous and bony fishes, with neuroethological implications.

We have extended the study of Bullock ('84), who reported on visually evoked potentials (VEP) in the tectum of 10 species of elasmobranchs, by adding further stimulus regimes, multichannel recording, and additional taxa, particularly addressing the question of flicker fusion frequency by electrophysiological signs in central processing centers. Using principally the guitarfish, Platyrhinoidis and Rhinobatos, and the bass, Paralabrax, with some additional data from 32 other species, the findings support the following conclusions: 1. Latency of the first main VEP peak, a sharp surface negativity, to a diffuse white flash of submaximal intensity while the eye is moderately light adapted varies from less than 20 ms in some teleosts to greater than 120 ms in agnathans, holocephalans, and some rays. Among the elasmobranchs tested, the sharks are generally faster than the rays. Among the teleosts tested, some species are at least three times slower than others. There is little overlap between the fastest elasmobranchs and the slowest teleosts. 2. After the first VEP peak, later components are more diverse than the classic descriptions of one late surface-negative hump; they may include also sharp peaks, slow humps, and oscillatory waves extending out to greater than 1 s postflash. These are highly labile, variable and similar to OFF responses after a long light pulse. All these components occur already in the retina, whether the optic nerve is intact or cut. Many records do not show the late components; in the same preparation, some tectal loci may and others may not. 3. Ongoing activity (the micro-EEG, over all frequency bands) is depressed between early and late waves after a flash as well as during a long light pulse. 4. Repeated flashes above a few per second do not so much cause fatigue of the VEPs as reduce or prevent them by a sustained inhibition; large late waves are released as a rebound excitation any time the train of flashes stops or is delayed or sufficiently weakened. 5. Repeated flashes depress first the early waves; later waves follow 1:1 up to an upper following frequency (UFF) of approximately 13 Hz in the guitarfishes at optimal intensity and light adaptation (15-17 degrees C). A transition zone of gradual fusion from 15 to 30 Hz is marked by sputtering or irregular sharp VEPs; above a lower fusion frequency (LFF) of 30-40 Hz, the flash train becomes equivalent to steady light.(ABSTRACT TRUNCATED AT 400 WORDS)

The nervus terminalis in larval and adult Xenopus laevis.

Nervus terminalis (nt) projections were studied by HRP injections into one nostril in adult Xenopus and in Xenopus tadpoles. Central nt targets are: medial septum, preoptic nucleus, nucleus of the anterior commissure, and hypothalamus (mainly ipsilaterally). In Xenopus tadpoles, additional fibers reach the ipsilateral dorsal thalamus and the mesencephalic tegmentum, bilaterally; furthermore, hypothalamic projections are bilateral. Xenopus tadpole nt connections resemble those of adult urodeles more closely than the projections of frogs. However, Xenopus tadpoles lack nt innervation of the medial septum.

Thyroxine influences neuronal connectivity in the adult frog brain.

In contrast to results of earlier investigations the influence of thyroxine on CNS connectivity is not restricted to circum-metamorphic stages in frogs. Neuroanatomical findings in adult Xenopus treated with thyroxine reveal a spread of the ipsilateral retino-tectal projection. Sprouting fibers establish a tectal innervation pattern similar to the one found in primitive fish. The question arises, whether thyroxine also has morphogenetic effects in the mature CNS of other species.

Central projections of the nervus terminalis in four species of amphibians.

The central projections of the nervus terminalis were investigated in two anuran and two urodele species by means of horseradish peroxidase injections into one nasal cavity. In anurans, the nervus terminalis projects to the medial septum, to the preoptic nucleus, to the nucleus of the anterior commissure and to the hypothalamus. In addition to these structures, the dorsal thalamus, the infundibulum and the mesencephalic tegmentum are innervated in urodeles. The structure containing the highest density of terminals in the amphibians investigated is the hypothalamus. In one anuran and one urodele species, the contralateral hypothalamus is primarily innervated, whereas in the other two species the majority of fibers remain ipsilateral. A comparison with other vertebrates shows that the terminalis system in urodeles has the greatest diversity of connections. Anurans, in contrast, lack some connections that are present in urodeles and fishes. These findings have implications for a possible relation of the nervus terminalis to an aquatic habitat.