Jürgen Bolz

Morphology of identified projection neurons in layer 5 of rat visual cortex

We studied the morphology of neurons in layer 5 of rat visual cortex (area 17) projecting to the contralateral hemisphere and the superior colliculus. Double labelling with fluorescent tracers indicated that these projections arise from different populations of cells. To reveal the morphology of the cells we stained retrogradely labelled neurons intracellularly in living brain slices. Callosal projecting pyramidal cells have 3-6 basal dendrites and an apical dendrite which never reaches higher than layer 3. Corticotectal cells have 6-8 basal dendrites and a prominent apical dendrite which always forms a large tuft in layer 1. Thus, neurons in the same cortical layer that give rise to different projections also differ in their morphology. However, each population of neurons has a rather stereotyped dendritic branching pattern, despite a large variation in soma size.

Response latency of brisk-sustained (X) and brisk-transient (Y) cells in the cat retina

1. Several methods for evaluating light‐evoked response latency and its variability in brisk‐sustained (X) and brisk‐transient (Y) retinal ganglion cells were tested. The most accurate procedure proved to be that described by Levick (1973), in which the time of the occurrence of the fourth impulse after stimulus onset is taken as an estimate of the latency.

Tenascin-C Synthesis and Influence on Axonal Growth During Rat Cortical Development

Several putative guidance molecules are restricted to the marginal and subplate zones, the major fibre tracts in the developing cortex. It is presently unknown how their distribution is achieved and how these molecules affect neurite extension. Tenascin‐C is of particular interest in this context, because it may either promote or deflect growing axons depending on its mode of presentation. Therefore, the cellular origin of tenascin‐C in the developing rat cortex and its effects on the extension of cortical afferents and efferents were examined. Tenascin‐C protein is first restricted to the marginal and subplate zones and spreads later into the developing grey matter, in close correlation with afferent innervation. In situ hybridization showed that tenascin‐C mRNA is first confined to the ventricular zone, at some distance from the location of the protein, while at later stages tenascin‐C‐synthesizing cells become scattered throughout the cortical thickness, concomitant with the spread of the protein. In order to assess its function, monoclonal antibodies directed against different domains of tenascin‐C were used in quantitative axonal outgrowth assay. These perturbation experiments suggested that distinct tenascin‐C fibronectin type 111 repeats sustain the growth of thalamic and cortical axons on cortical membrane carpets, whereas the EGF‐type repeats are not involved. The combination of different antibodies revealed that separate fibronectin‐type III repeats exert cooperative effects. These results suggest that ventricular zone cells regulate the establishment of thalamic and cortical axonal projections through locally restricted deposition of tenascin‐C.

Cellular organization and development of slice cultures from rat visual cortex

SummarySlice cultures from the visual cortex of young rats were prepared using the roller culture technique (Gähwiler 1984). After 10 days in vitro the cortical cultures flattened to 1–3 cell layers, surviving for up to 12 weeks. The cultures were organotypically organized, the typical layered structure of the cortex was preserved. The neuronal composition of slice cultures was studied using intracellular staining, Golgi impregnation and GABA immunohistochemistry. Both pyramidal cells and several types of nonpyramidal cells were identified in the slice cultures. Electrophysiological recordings showed that the electrical properties of cells in culture were similar to those measured in acute slice preparations; for some cells, however, the spontaneous activity was higher. The maintained activity was strongly increased by application of the GABA antagonist bicuculline and decreased by GABA, suggesting that GABAergic inhibition is present in these preparations. We could observe the postnatal maturation of some characteristic morphological features in culture. For example, pyramidal cells in 6 day-old rats in situ have very short basal dendrites with growth-cones, and the dendrites are free of spines. After 2–3 weeks in culture growth-cones were no longer observed. Instead, the cells had developed a large basal dendritic field and the dendrites were covered with spines. Slice cultures therefore may provide a useful tool for physiological, anatomical, pharmacological and developmental studies of cortical neurons in an organotypical environment.

The Role of Horizontal Connections in Generating Long Receptive Fields in the Cat Visual Cortex

The cells in the primary visual cortex possess numerous functional properties that are more complex and varied than those seen in the cortical input. These properties result from the network of intrinsic cortical connections running across the cortical layers and between cortical columns. In the current study we relate the long receptive fields that are characteristic of layer 6 cells to the input that these cells receive from layer 5. The axons of layer 5 pyramidal cells project over long distances within layer 6, enabling layer 6 cells to collect input from regions of cortex representing large parts of the visual field. When layer 5 was locally inactivated by injection of the inhibitory transmitter GABA, layer 6 cells lost sensitivity over the portion of their receptive fields corresponding to the inactivated region of layer 5. This suggests that the extensive convergence in the projection from layer 5 to layer 6 is responsible for generating the long receptive fields characteristic of the layer 6 cells.

Pharmacological analysis of cortical circuitry

The cortical circuitry of the visual cortex has been worked out in great detail. Anatomical investigations reveal stereotyped connections within cortical columns and specific long-range connections between distant columns. Pharmacological techniques for blocking the activity in individual cortical layers or columns allow the microdissection of the cortical circuit. These studies could relate specific functional roles to particular cortical connections.

Action and localization of gamma‐aminobutyric acid in the cat retina.

The effects of iontophoretically applied GABA (gamma‐aminobutyric acid) and bicuculline on retinal ganglion cells were studied in the optically intact eye of the anaesthetized cat. GABA suppressed both the spontaneous activity and light‐evoked discharge of all retinal ganglion cells, regardless of their type and regardless of the visual stimulus used. Bicuculline antagonized the action of iontophoretically applied GABA. Bicuculline enhanced the spontaneous activity of on‐centre cells, but suppressed the spontaneous activity of most off‐centre cells. The light‐evoked response of on‐centre cells was increased by bicuculline. A more complicated picture emerged for off‐centre cells. Weak light responses were suppressed by bicuculline, but during strong light responses the initial transient phase of the response was dramatically enhanced. Amacrine cells of the inner nuclear layer and displaced amacrine cells of the ganglion cell layer were labelled, using glutamic acid decarboxylase (GAD) immunohistochemistry and [3H]muscimol uptake. GAD‐positive dendrites were found throughout the inner plexiform layer and no sign of dendritic stratification was detected.

Reconstructing cortical connections in a dish

During development of the cortex, efferent projection neurons located in distinct cortical layers send their axons to different targets, and afferent fibers establish connections with cortical target cells of a particular layer. Recent studies have shown that layer- and cell-specific afferent and efferent cortical connections established in culture are similar to those observed in vivo. The results of these experiments provide evidence for the existence of diffusible and membrane-bound guidance factors for specific sets of axons. Furthermore, they suggest the use of different molecules to navigate axons towards their target, regulate target innervation and mediate target cell recognition.

THE SPECIFICATION OF NEURONAL FATE : A COMMON PRECURSOR FOR NEUROTRANSMITTER SUBTYPES IN THE RAT CEREBRAL CORTEX IN VITRO

Neurotransmitter choice is a crucial step in neural development. In the cerebral cortex, pyramidal neurons use the excitatory neurotransmitter glutamate, whereas non‐pyramidal cells use the inhibitory neurotransmitter GABA. We are interested in how these two neuronal types are generated. We labelled precursor cells from embryonic rat cerebral cortex with a retroviral vector in dissociated cell cultures, and examined the neurotransmitter phenotype of their progeny immunohistochemically after 2 weeks in vitro. We discovered, first, that precursor cells in culture generate glutamatergic and GABAergic neurons in proportions similar to those in vivo. Second, we found that neuronal precursor cells gave rise to both GABAergic and glutamatergic neurons. These results suggest that neuronal precursor cells in the cerebral cortex have the potential to generate both neuronal subtypes. Moreover, these data are consistent with a stochastic model of neurotransmitter specification.

Differentiation of Transmitter Phenotypes in Rat Cerebral Cortex

Cortical neurons differ in their neurochemical properties. Projection neurons use excitatory amino acids as transmitters, most local interneurons contain the inhibitory transmitter GABA, and specific subtypes of local circuit neurons express distinct neuropeptides. How this cellular diversity is generated during development is not known. We have been studying the transmitter differentiation of cortical neurons in different in vitro systems using immunohistochemical techniques. Transmitter phenotypes of cortical neurons were examined in slice cultures, i.e. in the absence of extrinsic cortical connections, and in dissociated cortical cell cultures, i.e. in the absence of extrinsic and intrinsic cortical connections. The expression of vasoactive intestinal polypeptide in cortical interneurons occurred normally in slice cultures prepared from neonatal rats between birth and 2 days of age, but was strongly impaired in dissociated cell cultures prepared at the same time. These results suggest that the intact cortical environment present in the slice cultures exerts crucial influences for neuropeptide differentiation. In contrast, the transmitters glutamate and GABA were expressed normally in the appropriate cell types and similar in proportions in dissociated cell cultures prepared from cortices at embryonic day 19. Only cells dissociated during S‐phase failed to express glutamate and GABA in vitro. When cells were kept for 24 h after mitosis in a cortical slice preparation in vitro, however, they later expressed their appropriate transmitter phenotypes. Thus, signals from the local cortical environment that act early in the cell cycle are required for the specification of transmitter phenotypes of cortical neurons.