Gert H. Hansen

Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA.

Neurotransmitter release and changes in the concentration of intracellular free calcium ([Ca++]i) were studied in cultured GABAergic cerebral cortical neurons, from mice, upon depolarization with either an unphysiologically high potassium concentration (55 mM) or the physiological excitatory neurotransmitter glutamate (100 microM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [3H]GABA as well as in [Ca++]i. However, the basic properties of transmitter release and the increase in [Ca++]i under a variety of conditions were different during stimulation with K+ or glutamate. Potassium-evoked release of [3H]GABA consisted of two phases, a rapid, large and transient phase followed by a smaller, more persistent second phase. The rapid phase was inhibited (60%) by nocodazole which reduced the number of vesicles in the neurites by 80%. This rapid phase of the GABA release was also reduced by organic (verapamil) and inorganic (Co++) Ca++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca++ channel antagonists but could be inhibited by SKF 89976A. The glutamate-induced [3H]GABA release, which was mainly mediated by N-methyl-D-aspartate receptors, consisted of a single, sustained phase. This was insensitive to nocodazole, partly inhibited by verapamil and could be blocked by Co++ as well as SKF 89976A. The action of Co++ could be attributed to a block of N-methyl-D-aspartate-associated ion channels. These findings strongly suggest that the majority of the K(+)-stimulated GABA release is dependent upon vesicles whereas the glutamate induced release is non-vesicular and mediated by a depolarization-dependent reversal of the direction of high-affinity GABA transport. The basic differences in the mode of action of the two depolarizing stimuli were reflected in the properties of the increase in [Ca++]i elicited by 55 mM K+ and 100 microM glutamate, respectively. The K(+)-induced increase in [Ca++]i was reduced by both verapamil and Ca(++)-free media whereas the corresponding glutamate response was only sensitive to Ca(++)-free conditions. Exposure of the cells to nocodazole or SKF 89976A had no effect on the ability of K+ or glutamate to increase [Ca++]i. Altogether, the results clearly demonstrate that K(+)-induced transmitter release from these GABAergic neurons is vesicular in nature whereas that induced by the neurotransmitter glutamate is not.

Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum, comb. nov. (=Gyrodinium aureolum) based on morphology, pigment composition, and molecular data

Light and electron microscopy, nuclear‐encoded LSU rDNA sequences, and pigment analyses were performed on five geographically separate isolates of Gymnodinium mikimotoi. The morphological variation between the isolates equals that found within the isolates. The nuclear‐encoded LSU rDNA sequences were nearly identical in all isolates, and molecular analyses using maximum likelihood, parsimony, and neighbor joining showed the geographical isolates as an unresolved clade. Based on the available data it is concluded that the European isolates, formerly identified as Gyrodinium aureolum, Gyrodinium cf. aureolum, or Gymnodinium cf. nagasakiense, are conspecific with the Japanese Gymnodinium mikimotoi. An isolate from the Pettaquamscutt River, USA, is suggested to represent what Hulburt (1957) described as Gyrodinium aureolum. The LSU rDNA sequence data and ultrastructural characters in this isolate closely resemble those of Gymnodinium fuscum, the type species of Gymnodinium, and Gyrodinium aureolum Hulburt is therefore renamed Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov.

GABA influences the ultrastructure composition of cerebellar granule cells during development in culture

The influence of GABA on cerebellar granule cells in culture was followed morphometrically during in vitro development by growing the cells in the absence or presence of 50 μM GABA. The presence of GABA in the culture media increased the number of neurite‐extending cells by 50% after 7 days in culture. At the ultrastructural level GABA treatment led to an increased density of neurotubules, rough endoplasmic reticulum, Golgi apparatus, coated vesicles and other vesicles, whereas structures such as mitochondria and smooth endoplasmic reticulum were not affected by GABA. The density of free ribosomes showed a more pronounced tendency to decrease as a function of the culture period (1–7 days) when the cells were grown in the presence of GABA as compared to control cultures. The results strongly indicate that GABA in addition to being an important neurotransmitter serves as a trophic factor in the development of at least certain types of neurons.

Cultured Neurons as Model Systems for Biochemical and Pharmacological Studies on Receptors for Neurotransmitter Amino Acids

By the use of primary cultures of neurons consisting of cerebral cortex interneurons or cerebellar granule cells it is possible to study biochemical and pharmacological aspects of receptors for GABA and glutamate. Cerebellar granule cells have been shown to express both high- and low-affinity GABA receptors. The latter ones develop, however, only when the neurons are treated with GABA or GABA receptor agonists. It is suggested that the high-affinity receptors play a role in the neurotrophic activity of GABA, whereas the low-affinity GABA receptors are involved in the mediation of the inhibitory action of GABA on evoked release of glutamate, which is the neurotransmitter in cerebellar granule cells. Also glutamate receptors have been studied with regard to the 2 types of neurons. Both cerebral cortex neurons (GABAergic) and cerebellar granule cells (glutamatergic) possess glutamate receptors, which mediate an L-glutamate-induced transmitter release. The pharmacological properties of these glutamate receptors are, however, distinctly different for the 2 types of neurons. While cerebral cortex neurons express both quisqualate-, N-methyl-D-aspartate- and kainate-receptors, the cerebellar granule cells have a receptor which is activated only by L-glutamate and L-aspartate.

Lipid raft organization and function in brush borders of epithelial cells (Review)

Polarized epithelial cells of multicellular organisms confront the environment with a highly specialized apical cell membrane that differs in composition and function from that facing the internal milieu. In the case of absorptive cells, such as the small intestinal enterocyte and the kidney proximal tubule cell, the apical cell membrane is formed as a brush border, composed of regular, dense arrays of microvilli. Hydrolytic ectoenzymes make up the bulk of the microvillar membrane proteins, endowing the brush border with a huge digestive capacity. Several of the major enzymes are localized in lipid rafts, which, for the enterocyte in particular, are organized in a unique fashion. Glycolipids, rather than cholesterol, together with the divalent lectin galectin-4, define these rafts, which are stable and probably quite large. The architecture of these rafts supports a digestive/absorptive strategy for nutrient assimilation, but also serves as a portal for a large number of pathogens. Caveolae are well-known vehicles for internalization of lipid rafts, but in the enterocyte brush border, binding of cholera toxin is followed by uptake via a clathrin-dependent mechanism. Recently, ‘anti-glycosyl’ antibodies were shown to be deposited in the enterocyte brush border. When the antibodies were removed from the membrane, other carbohydrate-binding proteins, including cholera toxin, increased their binding to the brush border. Thus, anti-glycosyl antibodies may serve as guardians of glycolipid-based rafts, protecting them from lumenal pathogens and in this way be part of an ongoing ‘cross-talk’ between indigenous bacteria and the host.

The trophic effect of gaba on cerebellar granule cells is mediated by gaba-receptors.

The effect of GABA, its agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol), and its antagonist bicuculline methobromide on the ultrastructure of cerebellar granule cells was studied. It was found that while the density of mitochondria, the ribosomes and smooth endoplasmic reticulum was unaffected by this treatment the density of neurotubules, rough endoplasmic reticulum, Golgi apparatus and coated as well as other vesicles was greatly enhanced by treatment of the cultured neurons with GABA (50 μM) or THIP (150 μM). The effects of GABA could be completely blocked by the simultaneous presence of bicuculline methobromide (150 μM) in the culture media. The findings strongly suggest that the trophic action of GABA is mediated by GABA receptors and not by an intracellular action of GABA.

Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTBs raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation

The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti‐sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization. The aminopeptidase N gene is expressed along the whole length of the villus with a maximum at its base. Expression was not detected in the crypt cells. The distribution of aminopeptidase N mRNA correlates with the presence of active enzyme as monitored by histochemical staining. The results are compatible with onset of transcription of the aminopeptidase N gene at the crypt/villus transition zone during the enterocyte differentiation.

Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (= Gymnodinium chlorophorum )

The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara was studied in detail. The nuclear envelope possessed numerous chambers each furnished with a nuclear pore, a similar arrangement to that found in other gymnodinioids. The flagellar apparatus was essentially identical to Gymnodinium chlorophorum Elbrächter et Schnepf, a species also containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber and a microtubular extension of the R3 flagellar root. This feature has not been found in other dinoflagellates and suggests a close relationship between these two species. This was confirmed by phylogenetic analysis based on partial sequences of the large subunit (LSU) rDNA gene of L. viride, G. chlorophorum and 16 other unarmoured dinoflagellates, including both the ‘type’ culture and a new Tasmanian isolate of G. chlorophorum. These two isolates had identical sequences and differed from L. viride by only 3.75% of their partial LSU sequences, considerably less than the difference between other Gymnodinium species. Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was emended to encompass L. chlorophorum comb. nov.

Light and electron microscopical observations on the type species of Gymnodinium, G. fuscum (Dinophyceae)

Abstract Gymnodinium fuscum (Ehrenberg) Stein is the type species of Gymnodinium, one of the largest genera of dinoflagellates. It has the typical fine structure of dinoflagellates, but also possesses several unusual features. The nuclear envelope is specialized by having vesicular chambers in which the nuclear pores are located; the pusular complex includes an internal collectjon chamber; and a nuclear fibrous connective links the nucleus with the longitudinal microtubular root of the flagellar apparatus. A delicate horseshoe-shaped apical groove is visible by scanning electron microscopy. The same morphological features have been observed in a few other gymnodinioids, indicating a close phylogenetic relationship, and have recently been used, together with large subunit (LSU) rDNA data, in redefining the genus Gymnodinium [Daugbjerg et al. (2000) Phycologia 39: 302–317]. However, one of the most unusual features of G. fuscum is the absence of a transverse striated flagellar root, a feature presently known only in the zoospores of the otherwise very different genus Noctiluca. This feature, together with the lack of striated collars around the flagellar canals, suggests a phylogenetically more isolated position for G. fuscum. Future investigations may reveal the need for further ‘splitting’ of the genus Gymnodinium.