Mats-Olof Mattsson

Two Glutamate Decarboxylase Forms Corresponding to the Mammalian GAD65 and GAD67 are Expressed During Development of the Chick Telencephalon

The γ‐aminobutyric acid (GABA)‐synthesizing enzyme glutamate decarboxylase (GAD) was studied during development of the chick telencephalon. By means of reverse‐phase HPLC analysis, we showed that GABA indeed accumulates during embryogenesis, whereas the levels of glutamate, the substrate for GAD, are more or less unchanged up to later developmental stages. The enzyme activity increased ∼25‐fold from embryonic day 3 to embryonic day 17. Immunoblotting data revealed that two GAD proteins, of ∼65 and 67 kDa, were present during the period investigated. Furthermore, Northern blot analysis with probes obtained from rat cDNA sequences, as well as a chicken‐specific probe for GAD65 generated by means of reverse transcriptase‐polymerase chain reaction (RT‐PCR), strengthened the interpretation that the chick embryo expresses genes corresponding to GAD65 and GAD67. The rat probes recognized transcript sizes of 3.9 kb (GAD65) and 5.6 kb (GAD67), sizes which are different from those of the rat brain (Erlander et al., Neuron, 7, 91−100, 1991). Sequencing of the RT‐PCR products revealed a high level of homology (82% at the nucleotide level) between the mammalian and chick GAD65 genes. Taken together, these findings suggest that the chick embryo expresses two GAD genes during embryogenesis. The functional properties of each gene product remain to be investigated.

ORNITHINE DECARBOXYLASE ACTIVITY AND POLYAMINE LEVELS ARE DIFFERENT IN JURKAT AND CEM-CM3 CELLS AFTER EXPOSURE TO A 50 HZ MAGNETIC FIELD

Abstract The present study was undertaken to see if ODC activity in cell lines with similar origin (lymphoblastic leukemia) respond in a similar fashion to a 0.10 mT magnetic field, and also if ‘down-stream’ effects, i.e. synthesis of polyamines, are influenced by changes in ODC activity. Furthermore, we wanted to investigate how various parameters such as induced current and the direction of the applied field contributed to the effect.

Morphological and GABA-immunoreactive development of the embryonic chick telencephalon

The development of neurons utilizing γ‐aminobutyric acid (GABAergic neurons) in prosencephalon and telencephalon from chicken embryonic days 4–14 (E4–E14) was studied by means of immunohistochemistry. Furthermore, routine histology and transmission electron microscopy, respectively, were performed in order to study the morphological development in the designated area. The main finding is that development of GABAergic neurons in the chick telencephalon is rapid; the GABA neurons are appearing in bulk at day 8, being “overexpressed” at days 10–11, decreasing in numbers thereafter and achieving mature morphology on day 14, which is considerably faster than in the rodent.

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A, a GABA uptake inhibitor, studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the γ-aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [3H]d-aspartate and [14C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [3H]d-aspartate and [14C] GABA was increased by [K+] and time, and a minimum value was obtained at [Ca2+] 1.05 mM. The release of both [3H]d-aspartate and [14C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.

DOWN-REGULATION OF ORNITHINE DECARBOXYLASE BY AN INCREASED DEGRADATION OF THE ENZYME DURING GASTRULATION OF XENOPUS LAEVIS

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.

Expression pattern of glutamate decarboxylase (GAD) in the developing cortex of the embryonic chick brain

The development of the GABAergic system in the chick embryo telencephalon has been studied. Special emphasis was placed on the development of glutamate decarboxylase (GAD) between embryonic day 8 (E8) and E17. The GABA immunoreactivity and neuron‐specific enolase expression was detected simultaneously in glutardialdehyde fixed sections, which confirmed that GABAergic cells exhibit neuronal phenotype. The GAD expression was studied by means of immunohistochemistry on cryo‐sectioned material both at the light and electron microscopic levels. Furthermore, the presence and localization of GAD65 and GAD67 mRNAs were studied with an in situ hybridization technique with digoxigenin‐labeled RNA probes. Protein expression as well as mRNA appearance mostly coincided both temporally and spatially.In the parahippocampal area, as well as in other regions of the developing cortex, GAD staining was seen from E8 onwards. The number of positive cells increased as did the intensity of staining up to E14. As observed in the electron microscope, the GAD protein was co‐localized with GABA in most cases, although some GAD‐positive cells devoid of GABA‐staining also were observed. The pattern of GAD mRNA expression was in general similar to that of GAD immunostaining. Both GAD65 and GAD67 mRNA were detected during the entire period. Furthermore, GAD67 mRNA localization spatially was more correlated with GAD protein expression. The study provides evidence for the notion that development of the GABAergic system occurs rapidly during embryogenesis and, as suggested from mRNA data, that two forms of GAD with a slight difference in distribution can contribute to this.

Uncharacterized Physical Parameters Can Contribute More Than Magnetic Field Exposure to ODC Activity in Vitro

Exposure of eukaryotic cells to extremely low frequency (ELF) magnetic fields (MF) has among other things been shown to induce changes in ornithine decarboxylase (ODC) enzymatic activity1–4. This enzyme is ubiquitously expressed in living cells, and the first and rate-limiting enzyme in the synthesis pathway for polyamines. The polyamines are very important for cellular growth, and also take part in various cell differentiation processes. However, over-expression of the enzyme, and thus higher activity levels, seem to be related to an increased risk for tumor formation5. That is why studies where magnetic field (MF) exposure cause elevated ODC activity have received such interest.

Factors involved in the formation and stabilization of cell aggregates obtained from amphibian embryonic explants

The effect of factors influencing the formation and stability of animal and vegetal aggregates from Xenopus laevis and Ambystoma mexicanum was examined in the light and scanning electron microscopes. At extreme values of pH the surface coat covering the vegetal aggregates is dissolved and dissociation may take place. Animal aggregates are more resistant. At high tonicities vegetal aggregates may be dissociated, and in the animal aggregates the epidermal differentiation is suppressed. In the absence of Ca2+ the vegetal aggregates are dissociated, but the animal aggregates are not affected. The results obtained with the inhibitor selenate and from incorporation experiments indicate that sulfated glycosaminoglycans are involved in the formation of aggregates in both species. Corresponding observations with tunicamycin suggest that even glycoproteins may play a role in aggregate formation, particularly in the vegetal aggregates.

Differences in the Release of L-Glutamate and D-Aspartate from Primary Neuronal Chick Cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[3H]aspartate andl-[3H]glutamate. Thed-[3H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[3H]glutamate release was calcium dependent, and furthermorel-[3H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[3H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.

115 Gaba transporters during the development of the embryonic chick telencephalon

We have previously reported that the survival of mouse cerebellar granule cells is independent of chronic depolarization. In this regard they differ from their rat counterparts. In the present study we measured mRNA concentration for the GluR2 subunit and the relative concentrations of the GluRl-4 mRNA in parallel cultures of mouse and rat cerebellar granule cells. The cultures were grown under depolarizing and nondepolarizing conditions. The concentration of GluR2 mRNA was measured by competitive RT-PCR and was normalized to the concentration of mRNA for the constitutively expressed histone gene H3.3, measured within the same culture. The relative concentrations of mRNA for GluRl4 were measured by restriction enzyme analysis of a PCR product containing cDNA corresponding to all four subtypes. At 4 days, the concentrations of GluR2 and total GluR mRNA were higher in mouse than in rat cultures, independent of cell depolarization. However, whereas at 7 days the concentrations were unchanged in mouse and non-depolarized rat cultures, the concentrations increased in depolarized rat cultures. In conclusion, we suggest that a fairly high AMPA receptor mRNA concentration is correlated to cerebellar granule cell survival in culture. (Supported by a grant from the Danish Medical Research Council).