Günter Rager

The response of cat visual cortex to flicker stimuli of variable frequency

We examined the possibility that neurons or groups of neurons along the retino‐cortical transmission chain have properties of tuned oscillators. To this end, we studied the resonance properties of the retino‐thalamo‐cortical system of anaesthetized cats by entraining responses with flicker stimuli of variable frequency (2–50 Hz). Responses were assessed from multi‐unit activity (MUA) and local field potentials (LFPs) with up to four spatially segregated electrodes placed in areas 17 and 18. MUA and LFP responses were closely related, units discharging with high preference during LFP negativity. About 300 ms after flicker onset, responses stabilized and exhibited a highly regular oscillatory patterning that was surprisingly similar at different recording sites due to precise stimulus locking. Fourier transforms of these steady state oscillations showed maximal power at the inducing frequency and consistently revealed additional peaks at harmonic frequencies. The frequency‐dependent amplitude changes of the fundamental and harmonic response components suggest that the retino‐cortical system is entrainable into steady state oscillations over a broad frequency range and exhibits preferences for distinct frequencies in the θ‐ or slow α‐range, and in the β‐ and γ‐band. Concomitant activation of the mesencephalic reticular formation increased the ability of cortical cells to follow high frequency stimulation, and enhanced dramatically the amplitude of first‐ and second‐order harmonics in the γ‐frequency range between 30 and 50 Hz. Cross‐correlations computed between responses recorded simultaneously from different sites revealed pronounced synchronicity due to precise stimulus locking. These results suggest that the retino‐cortical system contains broadly tuned, strongly damped oscillators which altogether exhibit at least three ranges of preferred frequencies, the relative expression of the preferences depending on the central state. These properties agree with the characteristics of oscillatory responses evoked by non‐temporally modulated stimuli, and they indicate that neuronal responses along the retino‐cortical transmission chain can become synchronized with precision in the millisecond range not only by intrinsic interactions, but also by temporally structured stimuli.

ON and OFF regions in layer IV of striate cortex

In vertical penetrations through the striate cortex in the tree shrew (Tupaia belangeri), we found regions where neural activity was evoked predominantly by light ON. These were followed by regions where responses were evoked predominantly by light OFF. Histological reconstructions indicated that the ON regions were correlated with layer IVa and the OFF regions were correlated with layer IVb. Local application of cobalt chloride produced a transient cessation of visually evoked activity, suggesting that the electrodes sampled cortical activity rather than lateral geniculate nucleus afferents. These data demonstrate that separate ON and OFF regions are present in the tree shrew striate cortex and suggest that spatially separate, parallel ON and OFF afferent channels extend, in this species, at least through the first synapse in the striate cortex.

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum. J. Comp. Neurol. 418:361–372, 2000.

Developmental Expression of Dynamin in the Chick Retinotectal System

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.

Expression of neuroserpin in the visual cortex of the mouse during the developmental critical period

The neuronal serine protease inhibitor neuroserpin is widely expressed in the developing and adult brain. In the neocortex, neuroserpin is displayed particularly during the period of synaptic specification and refinement, indicating a role as modulator of extracellular proteolytic processes. The synaptic connections of the visual system of the mouse are shaped during early postnatal life by an activity‐dependent process. We have studied the expression of the neuronal serine protease inhibitor neuroserpin in the primary visual cortex of mice from birth until the end of the critical period by means of reverse transcription polymerase chain reaction and in situ hybridization. The localization and the level of expression were constant throughout this period. Monocular deprivation with an eyelid sutured induced a decrease in neuroserpin expression in neurons of area 17 after 1 week of deprivation, the decrease being more pronounced on the side contralateral to the closed eye. The expression of neuroserpin in the visual cortex during the critical period and its decrease in parallel to the refinement of synaptic contacts after visual deprivation suggests a regulative role of neuroserpin on these processes.

Transformations of the retinal topography along the visual pathway of the chicken

SummaryIt is still unclear how the retinotectal map of the chick is formed during development. In particular, it is not yet known whether or not the organization of fibres plays a role in the formation of this map. In order to contribute to the solution of this problem, we analysed the representation of the retinal topography at closely spaced intervals along the fibre pathway. We injected HRP into various sites of the tectal surface and traced the labelled fibre bundles back to the retina. The retinal topography was reconstructed at ten different levels, i.e. in the retina, the optic nerve head, the middle of the optic nerve, the chiasm (three levels), the optic tract (three levels), and the optic tectum. We obtained the following results: (1) The labelled fibre bundles as well as the fields of labelled retinal ganglion cells were always well delimited and coherent. (2) The reconstructions show that transformations of the retinal topography occur in the fibre pathway. The first and most important transformation is found in the optic nerve head where the retinal image is mirrored across an axis extending from dorsotemporal to ventronasal retina. In addition, the retinal representation is split in its temporal periphery. Thus, central and centrotemporal fibres are no longer in the centre of the image but close to the dorsal border of the nerve. Peripheral fibres are found along the medial, ventral and lateral circumference of the nerve. In the optic tract a second transformation occurs. The retinal topography is rotated clockwise by about 90 degrees and flattened to a band. The flattening is accompanied by a segregation of fibre bundles so that eventually central and centrotemporal retinal fibres are located centrally, ventral fibres dorsally and dorsal retinal fibres ventrally in the tract. By these two transformations an organization of fibres is produced in the optic tract which can be projected onto the tectal surface without major changes given that dorsal and ventral fibres remain in their relative positions, and that deep lying fibres project to the rostral and central tectum, superficial fibres to the caudal tectum.The transformations which we have observed follow specific rules and thus maintain order in the pathway although retinotopy is lost. In conjunction with our earlier studies on the development of the retinotectal system we conclude that fibres are laid down in a chronotopic order. The transformations take place under particular structural constraints. Thus, an organization of fibres is provided in the optic tract which results in a retinotopic map when projected onto the tectal surface. This is stated for the order of magnitude of fibre bundles as investigated in this study. At the level of individual fibres additional factors may play an important role.

Differential expression of neuronal calcium sensor-1 in the developing chick retina

Neuronal calcium sensor‐1 (NCS‐1) is a Ca2+ binding protein that has been implicated in the regulation of neurotransmission and synaptogenesis. In this study we investigated the developmental expression and localization of NCS‐1 in the chick retina. Single‐ and double‐labeling experiments with three‐dimensional reconstruction as well as ultrastructural data of the distribution of NCS‐1 suggest that this protein is also involved in axonal process outgrowth. We found an early expression of NCS‐1 in ganglion cells and their axons, in amacrine, and in horizontal cells, whereas photoreceptors were immunonegative at embryonic stages. In the early posthatching days we found strong immunostaining for NCS‐1 in horizontal cells and their processes in the outer plexiform layer. In contrast, synaptic vesicle protein 2 (SV2) was prominent only in photoreceptor synaptic terminals. Ultrastructural analysis confirmed that NCS‐1 was localized postsynaptically in horizontal cell processes, whereas presynaptic terminals were immunonegative. However, at late posthatching days we observed that photoreceptor ribbon synapses (from rods and/or cones) also expressed NCS‐1. Thus the results support the notion that NCS‐1 is involved in neuronal process outgrowth and is localized in pre‐ and postsynaptic compartments including mature photoreceptor synapses. J. Comp. Neurol. 449:231–240, 2002.

Classes of axons and their distribution in the optic nerve of the tree shrew (Tupaia belangeri)

It has been suggested that retinal ganglion cells (RGCs) of Tupaia can be subdivided into three classes that correspond to the X, Y, and W classes in the cat. Estimates of these classes as determined by electrophysiological experiments and by histological studies of the retina are at variance. Because the RGC classes differ in axon diameter, this parameter could serve as a reliable criterion for the evaluation of RGC classes and their proportions.

Developmental expression of amphiphysin in the retinotectal system of the chick: from mRNA to protein

The role of amphiphysin in clathrin‐mediated endocytosis of synaptic vesicles is well established. However, it is still uncertain if the protein is also involved in developmental mechanisms, e.g. axon outgrowth and synapse formation. To investigate the developmental changes in the expression of amphiphysin we used the retinotectal system of the chick, a highly ordered and easily accessible primary neuronal pathway. Reverse transcription polymerase chain reaction (RT‐PCR) of total RNA from chick retina and tectum revealed first transcripts for amphiphysin, dynamin and synaptotagmin at embryonic day 5 (E5) for both regions. Surprisingly, Western blots of the retina revealed an increase of protein expression for amphiphysin only after E11 in the retina and the tectum. Immunofluorescence for amphiphysin was not detectable before E10 in the developing chick retina, while other presynaptic proteins like synaptotagmin showed already intense signals in the inner and outer plexiform layers. Subsequently, amphiphysin immunoreactivity follows the expression of synaptotagmin and synaptic vesicle protein 2 (SV2) as seen in the retina and the tectum, and exhibits the same staining as the other proteins in the mature chick brain. Ultrastructural data revealed for the first time that amphiphysin is not only limited to conventional synapses but is also abundant in retinal ribbon terminals. Taken together our data reveal that: (i) there is a developmental delay between mRNA transcription and protein expression for key proteins involved in endocytosis; (ii) amphiphysin gets upregulated after synapse formation; and (iii) amphiphysin is present in the synaptic vesicle cycle in retinal ribbon synapses.

Differential expression of the mRNAs of the axonal glycoproteins axonin-1 and NgCAM in the developing chick retina.

Cell adhesion molecules expressed on the axonal membrane have been thought to be involved in the guidance of axons to their target area. In the chick, axonin-1 and NgCAM have been shown to promote, through reciprocal interactions, neurite outgrowth in vitro. We have recently shown that chick retinal ganglion cells (RGC) express both proteins as early as the axonal elongation begins. Their expression continues throughout the development of the retinotectal system synchronously with the chronotopic spread of axons. To further investigate the spatiotemporal distribution of axonin-1 and NgCAM in the retina, we have analysed the expression of their mRNAs in the present study. From stage 36 (E10) until hatching photoreceptors express axonin-1 but not NgCAM. In the inner nuclear layer groups of amacrine cells were strongly labelled with both probes but they seemed to belong to different subgroups. These patterns of expression might indicate a differential influence of the two proteins on the development of the local neural circuits of the retina.