AbstractHPPD‐Inhibitoren verhindern die Bildung der pflanzlichen Carotinoidpigmente, was wiederum zum Chlorophyllabbau führt. Dieser “Bleaching”‐Wirkmechanismus kommt bei Produkten zur Kontrolle von Schadpflanzen in unterschiedlichen Kulturen wie Reis, Mais und Getreide zum Einsatz. In Kombination mit einem geeigneten Safener lässt sich das Potential dieser Verbindungsklasse bei der selektiven Bekämpfung der wichtigsten Schadpflanzen, einschließlich der rasch zunehmenden Fälle von Resistenzen gegen andere wichtige Herbizidklassen, voll ausschöpfen.

The neurotransmitter GABA (gamma-aminobutyric acid) functions as the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates. In vertebrates GABA signals both through ionotropic receptors (GABA(A), GABA(C)), which induce fast synaptic inhibitory responses, and through metabotropic receptors (GABA(B)), which play a fundamental role in the reduction of presynaptic transmitter release and postsynaptic inhibitory potentials. Whilst GABA(A) and GABA(C) receptors have been cloned from vertebrates as well as invertebrates, GABA(B) receptors have only been identified in vertebrate species to date, although indirect evidence suggests their existence in arthropods, too. Here we report the cloning of three putative invertebrate GABA(B) receptor subtypes (D-GABA(B)R1, R2 and R3) isolated from Drosophila melanogaster. Whilst D-GABA(B)R1 and R2 show high sequence identity to mammalian GABA(B)R1 and R2, respectively, the receptor D-GABA(B)R3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three D-GABA(B)R subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila melanogaster embryos shows that two of the D-GABA(B)Rs (D-GABA(B)R1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, whilst the third D-GABA(B)R (D-GABA(B)R3) displays a unique expression pattern. In agreement with these results we have only been able to functionally characterize D-GABA(B)R1 and R2 when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, whilst D-GABA(B)R3 was inactive in any combination. The pharmacology of the coexpressed D-GABA(B)R1/2 receptor was different from the mammalian GABA(B)Rs: e.g. baclofen, an agonist of mammalian GABA(B)Rs, showed no effect.

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene-carrying retroviral vectors is described. Use of a vector encoding the oncogene v-myc results in lines of nestin-positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate, In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin-positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.

GABAA receptor activation in cerebellar granule cells induced a complex physiological response, namely the activation of a Cl− conductance in concert with a blockade of the resting K+ outward conductance (by 71% as compared to controls). Both responses were mediated by the activation of GABAA receptors, since they were both mimicked by the GABAA receptor agonist muscimol and antagonized by picrotoxin and bicuculline. A substantial decrease of the mean open time of single, outwardly rectifying K+ channels was triggered by GABA as revealed from cell-attached recordings; this finding implies that an intracellular pathway links GABAA receptors and K+ channels. Furthermore, this action of GABA is mediated through the cytoplasm, as experiments with the cell-attached patch-clamp technique show. GABA induced a prominent membrane depolarization ranging from 10 to 25mV as revealed by current-clamp recordings of gramicidin (or nystatin) permeabilized patches, thus selecting conditions not to perturb the physiological Cl− gradient across the cell.Our findings imply that the GABA-activated Cl− current depolarized the membrane as described for immature neurons. The blockade of the resting K+ channel conductance acts in concert and both mechanisms lead to this substantial depolarizing event.

Interactions among receptor neurons, glial cells and neurons intrinsic to the antennal lobe of the moth underlie the formation of olfactory glomeruli. To isolate these interactions, as well as to understand the effect of a variety of humoral agents on differentiation of the neurons and glia, we generate primary cultures of neurons or glia. These methods are described. In addition. we describe a protocol for producing slice preparations of the developing moth brain that we are using to study the biophysical and morphological development of glial cells. This technique allows us to examine a class of glial cells associated with the glomeruli that otherwise are nearly inaccessible using standard intracellular recording techniques. It also preserves the 3-dimensional arrangement of glia that may strongly influence the development of glomeruli.

While Bergmann glial cells play an important role in the development of the cerebellum they were thought to serve as passive insulators of the Purkinje cell dendritic tree and its synaptic connections. New results challenge this view and demonstrate that Bergmann glial cells are equipped with a large repertoire of receptors allowing them to sense the activity of synapses. These receptors have distinct biophysical and pharmacological features activating second-messenger pathways in the Bergmann glial cells. It is evident that the synapse has to be viewed as consisting of three elements, the presynaptic and postsynaptic region and the glial ensheathment. All three elements of this synaptic complex may undergo plastic changes as a prerequisite for central nervous system plasticity. Glial cells could interfere with synaptic transmission by communicating with neurons via the extracellular space, e.g., by modulating ion concentrations or transmitter levels in the cleft (Fig. 6).

The electrophysiological properties of glial precursor cells and oligodendrocytes were studied in an intact tissue preparation, the corpus callosum slice, with the patch-clamp technique. The pattern of voltage-gated currents exhibited by different cell types in this white matter tract was compared with that from cultured cells of the O-2A lineage. Precursor cells from the in vitro and the in situ preparation were strikingly similar in most aspects. In contrast, oligodendrocytes in the intact tissue differed from their culture correlates and displayed the ability to redistribute excess K+. To study glial transmitter receptors in neuron-glial interactions during development, Bergmann glial cells were investigated in a slice preparation of the cerebellum.

Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells, although they yield a prominent staining of both the Purkinje cells and the granule cells. These changes in the Bergmann glial cell membrane properties and GABAA receptor expression suggest a transition between functional states during development of the Bergmann glial cells.

NMDA receptors play a crucial role in synaptic plasticity of the central nervous system and were thought to be exclusive to neurones. In this study we provide evidence that Bergmann glial cells from mouse cerebellar slices show intrinsic responses to NMDA. As in neurones, NMDA increased membrane conductance and the responses were blocked by the NMDA antagonist ketamine, but not by the non-NMDA glutamate receptor antagonist CNQX. In contrast to responses in neurones, the current voltage relation of the glial NMDA-induced current was linear, reversed at -40 mV, currents were not blocked by Mg2+ or enhanced by glycine and NMDA did not induce an increase in cytosolic Ca2+ as recorded with a fura-2 imaging system. These data imply the presence of distinct NMDA receptors on Bergmann glial cells; these glial receptors could be the substitute for complex neurone-glia interactions in the cerebellum.

Glutamate receptors, the most abundant excitatory transmitter receptors in the brain, are not restricted to neurons; they have also been detected on glial cells. Bergmann glial cells in mouse cerebellar slices revealed a kainate-type glutamate receptor with a sigmoid current-to-voltage relation, as demonstrated with the patch-clamp technique. Calcium was imaged with fura-2, and a kainate-induced increase in intracellular calcium concentration was observed, which was blocked by the non- N -methyl-D-aspartate (NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by low concentrations of external calcium, indicating that there was an influx of calcium through the kainate receptor itself. The entry of calcium led to a marked reduction in the resting (passive) potassium conductance of the cell. Purkinje cells, which have glutamatergic synapses, are closely associated with Bergmann glial cells and therefore may provide a functionally important stimulus.

The basic physiology courses for students in biology and medicine include experiments on live animals or animal tissue. This article describes alternatives by replacing such experiments with simulation programs on personal computers. Programs for a neurophysiological experiment were developed at the Department of Neurobiology, University of Heidelberg, within the last years and have served to replace animal experiments in the basic training of biologists.