NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium-3 (HC-3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [³²P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [^l4C]HC-3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC-3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINOACIDS. GLUTAMATE RECEPTOR OF THE SHRIMP MUSCLE

Abstract— –Muscle of shrimps ( Artemisia longinaris ) were extracted with chloroform-methanol (2:1, v/v) and the proteolipids were separated by column chromatography on Sephadex LH-20. Three peaks of protein were eluted with chloroform and one with chloroform-methanol (4:1, v/v). Only the first peak eluted between 16 and 26 ml of chloroform showed binding for l -(¹⁴C]-glutamate. The type of saturation curve obtained suggests the existence of single type of binding site. The saturation is reached at one mole of l -glutamate per 320,000 g protein and the purification achieved about 3200-fold. The protein binding-glutamate does not bind GABA, aspartate or glutamine. The binding of l -[¹⁴C]-glutamate was inhibited by dl -α -methyl glutamic acid and l -glutamic acid diethyl ester. The binding properties of this hydrophobic protein fraction suggest that it may represent the isolated glutamate receptor of the shrimp muscle.     

Subcellular distribution and chemical nature of the receptor for 5-hydroxytryptamine in the central nervous system

Abstract— In vitro binding experiments with 5-hydroxy[¹⁴C]tryptamine (3.3 × 10^?6 M) were carried out on subcellular fractions of the cat brain. The highest specific activity was observed in some fractions of nerve-ending membranes isolated from the hypothalamus, basal ganglia, and gray areas of the mesencephalon. The specificity of this high affinity binding was demonstrated by competition with reserpine, butanolamide of lysergic acid, and desmethylimipramine. With butanol-water extraction the [¹⁴C]5-HT was found in the butanol while the gangliosides were separated in the water phase. Several experiments with thin layer and column chromatography suggest that in the organic phase the [¹⁴C]5-HT is not bound to the lipids but to a special proteolipid. This proteolipid is different from that found in myelin and has similar chromatographic properties to that previously observed in the proteolipid which binds d-[¹⁴C]tubocurarine in nerve-ending membranes of the cerebral cortex.     

The effect of castration upon the ultrastructure of the rat hypothalamus

Summary The neurosecretory hypothalamic nuclei and the inner zone of the median eminence of castrated rats were studied under the electron microscope. After one month of castration all the neurosecretory neurons of both nuclei show signs of hyperactivity characterized by dilated cisternae of the endoplasmic reticulum containing a macromolecular filamentous material and an increase in the number of ribosomes. After six months of castration, some neurosecretory neurons show an increased number of neurotubules and larger lysosomes than in the controls. Other neurons show a very significant hypertrophy of the endoplasmic reticulum, with large amounts of intracisternal filamentous material. These cells have few neurosecretory granules and in the adjacent synapses the number of granulated vesicles is increased. In the supraoptic nucleus there are two kinds of neurosecretory axons: the clear ones, which are similar to those that appear in control animals and the dark ones, which have smaller elementary granules. In the inner zone of the median eminence the axons show an increase in the number of neurosecretory granules with respect to the controls. After supplementary administration of sexual hormones, all the modifications produced by castration disappear. The ultrastructural changes observed in the neurosecretory nuclei after castration are discussed in relation to those previously described in the neurohypophysis under the same experimental conditions. A feedback regulatory action of sex hormones on hypothalamic neurosecretory neurons is postulated.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.     

Inhibition of Escherichia coli growth by cyclic adenosine 3', 5'-monophosphate

Cyclic AMP inhibits growth rate of E. coli Hfr 3000. Doubling times in glucose minimal medium increased from 60 to about 90 minutes with the addition of 5 mM cAMP. This effect is specific since it was not observed when the cyclic nucleotide was replaced by 5′ AMP, ADP, ATP or adenosine. Half maximal inhibition was obtained with 1 to 3 mM cyclic AMP. This inhibition occurs only with those carbon sources which are known to decrease intracellular cyclic AMP levels, i.e. glucose and pyruvate. No inhibition was observed with succinate, malate or glycerol.     

Electron Microscopy of Retinal Photoreceptors : The Use of Chromation Following Formaldehyde Fixation as a Complementary Technique to Osmium Tetroxide Fixation

The fine structure of the cone and rod outer segments of the toad was studied under the electron microscope after fixation in osmium tetroxide and fixation in formaldehyde followed by chromation. In the OsO₄-fixed specimens, the rod outer segment appears to be built of a stack of lobulated flattened sacs, each of which is made of two membranes of about 40 A separated by an innerspace of about 30 A. The distance between the rod sacs is about 50 A. The sacs in the cone outer segment are originated by the folding of a continuous membrane. The thickness of the membranes and width of the spaces between the cone sacs is the same as in rod, but the sac innerspace is slightly narrower in the cone (~ 20 A). After fixation in formaldehyde and chromation, two different dense lines (l₁ and l₂) separated by spaces of less density appear. One of the lines, l₁, has a thickness of 70 A and is less dense than the other, l₂, which is 30 A thick. The correlation of the patterns obtained with both fixatives is considered and two possible interpretations are given. The possibility that l₂ is related to a soluble phospholipid component is discussed. It is suggested that the outer segments have a paracrystallin organization similar to that found in myelin.     

The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons

XIHbox 6 is an early spatially restricted marker for molecular studies of neural induction. The sequence of the full-length XIHbox 6 protein is reported. An antibody raised against a beta-galactosidase/XIHbox 6 fusion protein was used to analyze the expression of XIHbox 6 proteins during frog embryogenesis. The anterior border of XIHbox 6 expression lies just posterior of the hindbrain/spinal cord junction. Immunostaining extends the entire length of the spinal cord. A much weaker transient expression with a similar anterior border is observed in mesoderm. Almost all nuclei in the newly closed spinal cord contain XIHbox 6. The number of positive nuclei decreases over the next stages of development, until in later embryos XIHbox 6 is restricted to nuclei of the dividing neuroepithelium, and not the mantle or marginal zones of the spinal cord. When the limb buds begin to grow, there is a second burst of XIHbox 6 expression in proliferating neurons of the cervical and lumbar enlargements, where nerves arise that supply the limbs. The data suggest that XIHbox 6 expression is spatially and temporally restricted to immature neurons of the spinal cord, before their differentiation into mature neurons.