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Scientific Activities (past 5 years)
Highlights of Previous Work

1) Neuronal Correlates of Space Perception in Monkey Neocortex
The ventral intraparietal area (VIP) of macaque monkeys is located in the fundus of the intraparietal sulcus It bridges the gap between the dorsal visual processing stream (LIP) and the somatosensory and premotor system (MIP). The VIP area in macaques contains neurons responding in a directional selective manner to visual stimuli. Furthermore, VIP neurons also respond to somatosensory stimuli. Receptive field locations for visual and tactile stimuli correspond, and directional preferences for both kinds of stimuli are identical. Area VIP is thus considered to play an important role in the analysis of self-motion and multimodal representation of the three-dimensional movement space. Our recordings revealed directionally selective responses for vestibular stimulation, and to visual as well as tactile stimulation. There was, however, one peculiar characteristic of the visual vestibular interactive processing in these neurons that requires further study. Visual and vestibular on-directions were always in the same direction (not opposite) which, in fact, would generate a visual-vestibular conflict situation. Despite such an a priori mutually inhibitory interaction, the neuronal responses were actually amplified, and not diminished. The same neurons often responded also to optic flow stimuli (expansion and contraction) that simulate self-motion. Many neurons in area VIP also revealed an influence of eye position during active fixation in darkness similar to findings in areas LIP and 7a, and also play a role in eye- versus head-centered spatial coding. This was shown by mapping visual receptive field structures that could remain retinocentric, or remain stationary in space during eye movements.

2) Transneuronal Labeling of Eye Movement Circuits with Rabies Virus in Guinea Pigs and Primates
Retrograde transneuronal tracing with rabies virus (CVS strain) was used to visualize the neuronal circuitry involved in horizontal eye movement control. After unilateral injection (1 痞) into the medial rectus muscle (MR), viral transfer was studied immunohistochemically at 6 hour intervals from 1.5 to 5 days post-inoculation. Specificity of uptake was demonstrated by double immuno-fluorescence for rabies and choline acetyl transferase (CAT). The kinetics of retrograde transneuronal transfer of the tracer show an exponential increase in categories of labeled neurons over time. The results signal an ever increasing complexity of afferent pathways and control circuits converging onto the different layers of the investigated networks and onto the final motoneuronal pathway in order to produce a desired motor output, i.e., a behavior, in our case horizontal eye movements.

Transneuronal transfer was time-dependent. Initially (2 days), only the motoneuronal pathway appears (first-order neurons). At a later stage (2.5-3 days), the transneuronally labeled second-order neurons largely comprise the vestibulo-ocular reflex (VOR) control circuits. However, at this level, additional cell groups are interconnected with this circuitry whose involvement cannot be interpreted in a straight-forward fashion. For example, labeling of the Y group, a nucleus generally associated with vertical eye movements, has to be seen in a recently described context of adaptive plasticity

At longer times (3.5 days), third-order transneuronal labeling already seems to involve the entire machinery underlying the eye movement repertoire such as saccade generation, optokinetic reflex and motor learning mechanisms, up to and including precursors of cortical perceptive functions (if a guinea-pig possesses such a capacity).

With regard to the olivo-cerebellar system, labeled Purkinje cells (PCs) appeared at 3.5 days in the ipsilateral flocculus (FL) in a single band that ran diagonally from caudomedial to rostrolateral in an intermediate position. This band corresponds to the so-called "horizontal zone". In some cases, labeling continued laterally across the posterolateral fissure into the ventral paraflocculus. At longer survival times (between 3.5 and 4 days), the initial band became slightly broader and additional separate bands of labeled PCs appeared in the rostromedial and caudolateral FL. At these times, an intermediately positioned band also appeared in the contralateral FL that mirrored the one initially labeled in the ipsilateral FL. The time difference in the appearance between these two bands reflects a similar time difference in labeling in ipsi- versus contralateral magnocellular medial vestibular nucleus neurons. After 4 days, the empty areas adjacent to the "horizontal zone" became filled with labeled PCs that were at first scattered, but at 5 days covered basically all areas. This increase suggests involvement of PCs belonging to the "vertical zones" in horizontal eye movement circuits via neurons in the vestibular nuclei or the deep cerebellar nuclei.

The results show the expected and the unexpected. Basically, all neuronal relay stations involved in horizontal eye movement control have been labeled. Quite unexpectedly, labeling involves also a number of nuclei which are thought to be only involved in vertical eye movement control (Y group, interstitial nucleus of Cajal). In general, horizontal and vertical systems comprise different functional entities, although cross-activation has been demonstrated and interpreted as a necessity for spatial coordination of three-dimensional eye movements and for vertical/ horizontal adaptive plasticity. Other labeled regions have not as yet been associated with an eye movement control function, such as the dorsal tegmental nucleus or certain limbic structures. Their role clearly remains to be determined. For example, labeling of the hypothalamus may be explained by its known projections to the horizontal saccade related nucleus reticularis cuneiformis (RCf). Another unexpected finding is the absence of labeling at 3 days in areas that could be expected to contain it, such as the visual relay nuclei mediating optokinetic reflexes (nucleus of the optic tract, NOT; accessory optic system, AOS). For some of these nuclei, direct projections to oculomotor motor neurons have been postulated. However, the labeling of these nuclei at 3.5 days would suggest the existence of more polysynaptic relays. Noticeably, the only structure of the AOS labeled at 3.5 days is the contralateral dorsal terminal nucleus (DTN), that only contains horizontal visually direction-selective neurons. The lateral terminal nucleus (LTN) and medial terminal nucleus (MTN) are related to vertical directions and are not labeled.

All in all, the transneuronal labeling technique with rabies virus is a powerful tool to determine entire functional neuronal networks involved in a specific behavior.

3) Flatfish Neurobiology
Flatfish offer a natural paradigm for studying adaptive changes in the vestibulo-ocular reflex (VOR). During metamorphosis, flatfish tilt 90 deg to one side or the other to become bottom-adapted adult animals. In this position, the labyrinths are rotated 90 deg relative to their premetamorphic orientation in space. Structurally, this arrangement requires a neuronal pathway from the horizontal semicircular canals to muscles that move the eye vertically. The morphological substrate subserving adaptation of the vestibulo-ocular reflex in post-metamorphic (adult) flatfish was obtained with a number of morphological and electrophysiological methods. Single cell morphology of second-order vestibular neurons linked to the right or left horizontal canal, visualized with the intracellular HRP injection method, revealed a qualitatively bilaterally symmetric distribution of neurons with terminals in vertical extraocular muscle motoneuron pools, in either both the superior rectus and inferior oblique subpopulations or with the antagonists to these muscles, the inferior recti and superior obliques (trochlear nuclei). Surprisingly, all second-order neurons had contralaterally ascending main axons. Vertical canal related second-order neurons were not different from that described in other vertebrates. Visualized by extra- and intracellular HRP methods, primary afferents from the semicircular canals terminated in the five subnuclei of the vestibular complex (anterior, magnocellular, descending, tangential, posterior), and also in the eminentia granularis and the medial reticular formation, as described in other teleost fishes.

In summary, our data indicate a thoroughly bilateral symmetric distribution of post-metamorphic second-order vestibulo-ocular reflex neurons for both excitation and inhibition linking the horizontal canals to vertical eye muscles. In light of normal primary vestibular afferent, second-order vertical vestibular neuron, and oculomotor organization, the species-specific horizontal vestibular neurons are both necessary and sufficient for adaptation of the vestibulo-ocular reflex in the adult flatfish.

A peculiar reaction to hemilabyrinthectomy was found in the adult flatfish. While lesions of the down-side labyrinth make the animal perform the classical hemilabyrinthectomy symptoms with circling towards the side of the lesion, extirpation of the up-side labyrinth made the animal circle away from the lesion. The neuronal mechanisms and possible consequences for the ontogeny and evolution of the flatfish are presently being investigated. We interpret the peculiar bilaterally asymmetric labyrinthine organization of the adult flatfish to be of fundamental importance for maintaining the flatfish as a flatfish.