Selective retrograde labelling of the rat olivocerebellar climbing fiber system with D-[3H]aspartate.

Selective retrograde labelling of the olivocerebellar climbing fiber system with D-[3H]aspartate has been observed in the rat, and the results have implications for the identification of a transmitter candidate as well as the neuroanatomical understanding of these cerebellar afferents. Microinjections of D-[3H]aspartate (50 nl, ca 10-2 M) were made into various parts of cerebellar cortex. Survival times were 6, 12 or 24 h. Pronounced diffusion of the tracer resulted in large injection sites. Within the zone of injection, glial elements were labelled over background. Most granule cells exposed to the tracer were unlabelled; the small numbers demonstrating labelling were believed to have been injured by the micropipette penetration. Beneath injection sites, large numbers of well-labelled nerve fibers appeared in the white matter and could be followed through the brainstem to the contralateral inferior olive, where labelled perikarya were found. After the inferior olivary neurons had been effectively destroyed with 3-acetylpyridine, evidence of cerebellar afferent labelling with D-[3H]aspartate was missing. Retrograde labelling of the olivocerebellar system was also observed after superfusion of the vermis with D-[3H]aspartate at concentrations in the range of Km for high affinity uptake (10(-5) or 10(-4) M, for 2 h). Mossy fiber or monoaminergic afferents to the cerebellum were never labelled with D-[3H]aspartate. The distribution of labelled cells in the olivary subnuclei after injections in different cerebellar areas was in line with the olivocerebellar organization previously described in the cat. Moreover, it was demonstrated that fibers from the different subnuclei follow different routes through the brainstem towards the cerebellum. Labelling of climbing fiber collaterals in uninjected parts of cerebellum indicated that some of the retrogradely migrating D-[3H]aspartate was directed in anterograde direction at axonal branching points. Collaterals were demonstrated in all deep cerebellar and Deiters' nuclei, and the results of intranuclear injections suggested that virtually every olivary neuron sends collaterals to these nuclei. Intracortical collaterals were organized in sagittal zones. Midline injections into the anterior lobe and VI lobule labelled collaterals in several zones of the posterior lobe spinal area and uninjected parts of the anterior lobe vermis. Hemispheral injection into copula pyramidis labelled collaterals in two prominent bundles in the anterior lobe.(ABSTRACT TRUNCATED AT 400 WORDS)

Release of endogenous and accumulated exogenous amino acids from slices of normal and climbing fibre-deprived rat cerebellar slices.

Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for gamma-aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low-Ca2+ (0.1 mM), high-Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+-induced efflux was similar under normal and low-Ca2+ concentrations. The K+-induced efflux of exogenously accumulated L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low-Ca2+, high-Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3-acetylpyridine, release of endogenous aspartate and exogenous L-[3H]glutamate and D-[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, L-[3H]glutamate and L-[3H]aspartate differ in several aspects: (1) L-[3H]glutamate uptake was 4 times higher than that of L-[3H]aspartate; (2) fractional rate constant of K+- evoked release of L-[3H]aspartate was 7% X 2.5 min-1, and of L-[3H]glutamate 36% X 2.5 min-1; and (3) specific activity of L-[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for L-[3H]aspartate, specific activities in the eluate and tissue were similar.

In vitro release of endogenous beta-alanine, GABA, and glutamate, and electrophysiological effect of beta-alanine in pigeon optic tectum.

The efflux of 20 amino acids, induced by either high K+ concentration or veratrine, was determined in pigeon tectal slices. Ca2+-dependent, K+-induced release of beta-alanine, gamma-aminobutyric acid (GABA), and glutamate was observed. Veratrine caused release of the same amino acids plus glycine in a tetrodotoxin-sensitive manner. beta-Alanine had a strong inhibitory effect on the activity of tectal neurons which was blocked by strychnine but not by bicuculline. The results indicated a transmitter function for beta-alanine in the optic tectum, and were consistent with the previously proposed transmitter role of GABA and glutamate in this structure.

Phlorizin as a probe of the small-intestinal Na+,D-glucose cotransporter. A model.

(1)'Uptake' of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of D-glucose, by the simultaneous presence of Naout+ and delta psi much less than 0. However, phlorizin contrary to D-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na+,D-glucose cotransporter. (2) The stoicheiometry of Na+/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Naout+-dependence of phlorizin binding. (3) The preferred order of binding at delta psi much less than 0 is Na+ first, phlorizin second. (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to delta psi. These observations, while agreeing with the effect of delta psi much less than 0 on the Kd of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na+, delta psi-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na+, D-glucose cotransport. (6) The kon, koff and Kd constants of phlorizin interaction with the Na+,D-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.

Aspartate: possible neurotransmitter in cerebellar climbing fibers.

Autoradiography demonstrated prominent retrograde labeling of olivocerebellar climbing fiber neurons after injection of tritiated D-aspartate into the rat cerebellar cortex or deep nuclei. Mossy fiber systems originating in the brainstem and spinal cord remained unlabeled. Potassium ion-induced depolarization of cerebellar slices resulted in calcium ion-dependent release of endogenous L-aspartate, L-glutamate, gamma-aminobutyric acid, and glycine. A 26 percent decrease in aspartate release was observed after 3-acetylpyridine-induced destruction of the inferior olive, supporting the hypothesis that aspartate is a neurotransmitter in climbing fibers.

Asparagine as precursor for transmitter aspartate in corticostriatal fibres.

The role of asparagine as precursor for the neurotransmitter aspartate was investigated in rat striatum in vitro. 14C-asparagine incubated with striatal slices is converted to a great extent to 14C-aspartate which is released in a calcium-dependent manner by high KCl. Furthermore, a frontoparietal cortex ablation of two weeks produces a decrease of more than 70% in the striatal release of newly synthetized 14C-aspartate, whereas the striatal GABA release is unaffected. This suggests that asparagine is a possible precursor in vitro for transmitter aspartate in the striatum. This reaction is dependent on intact corticostriatal fibres.

Similarity in effects of Na+ gradients and membrane potentials on D-glucose transport by, and phlorizin binding to, vesicles derived from brush borders of rattit intestinal mucosal cells.

Both the presence of sodium and of an electrical potential difference across the membrane have been found to be necessary in order to achieve optimal D-glucose-protectable phlorizin binding to brush border membranes from rabbit small intestine. The effect of delta approximately muNa on phlorizin binding shows a close similarity to that on D-glucose transport, confirming that phlorizin is indeed bound to the D-glucose transporting protein. Possible modulations of binding by a transmembrane potential are discussed on the basis of some models.

High-affinity phlorizin binding to brush border membranes from small intestine: identity with (a part of) the glucose transport system, dependence on Na +-gradient, partial purification.

In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd similar or equal to 4.7 micron) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min.). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2-3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein.