Russell J. Barrnett

Hypothalamic Lesions in Goldthioglucose Injected Mice.

Summary Goldthioglucose injections, in the dose which produces obesity in mice, cause in these animals extensive hypothalamic damage involving the ventromedial nuclei. Such lesions are permanent in animals which in fact become obese, but are not apparent in animals unsuccessfully injected. Goldthiomalate, though as toxic as goldthioglucose, does not cause obesity; its injection does not lead to hypothalamic lesions. Implications of these findings as regards the mechanism of the regulation of food intake are discussed.

HISTOCHEMICAL DEMONSTRATION OF NOREPINEPHRINE AT A FINE STRUCTURAL LEVEL

The present work describes a method for the histochemical demonstration of, and the differentiation between, norepinephrine and epinephrine at a fine structural level in cells of the adrenal medulla. The two catecholamines are definitely contained in granules which occur in two different cell types; one for norepinephrine, the other for epinephrine. No evidence could be adduced from the present material that norepinephrine material served as a precursor for epinephrine granules. Preliminary observations are included on the occurence of catecholamine-containing granules in neuronal processes of the hypothalamus.

Hormone-Sensitive Adenyl Cyclase: Cytochemical Localization in Rat Liver

An electron microscopic procedure has been developed, using rat liver, for the localization of hormone-sensitive adenyl cyclase. Isoproterenol-sensitive adenyl cyclase is located almost exclusively in the parenchymal cells. In contrast, glucagon-sensitive adenyl cyclase is located primarily in the reticulo-endothelial cells but is also present in parenchymal cells. Sodium fluoride-sensitive adenyl cyclase is found in both cell types.

ALDEHYDE FIXATION FOR MORPHOLOGICAL AND ENZYME HISTOCHEMICAL STUDIES WITH THE ELECTRON MICROSCOPE.

Instead of discussing in detail the conditions under which fixation techniques for electron nucroscopv can be best adapted to maintain the levels of enzymatic activity necessary for their histochemical demonstration, we will take this opportunity at the 14th Histochemical Society Symposium to illustrate some of the work that has been done at the Department of Anatomy of Yale University and at the Department. of Cytology of the Rockefeller Institute utilizing aldehydes as fixatives. These reagents have been found of value for studies which require the combination of histochemist.rv and electron microscopy and have also been useful in pure morphological studies with the electron microseOl)e (19). It is almost needless to say that none of the aldehycles (glyoxal, glutaraldehyde, hydroxyadipaldehyde, crotonaldehyde, methacrolein, pyruvic aldehyde, and acetalaldehyde’) (19) used in these works, to which malonaldehyde, malialdehyde, and succinaldehyde (2) were recently added, meet all the requirements of an ideal universal fixative. Some of these substances, such as methacrolein and (rotonaldehyde, which are closely related to acrolein (12), are very active cross linking agents which combine with olyamino and polyhydroxy (-oml)ounds and readily block sulfhvdrvl groups and, in fact, almost all active hydrogens. When used for morphological studies, either alone or with heavy metal stains, they give what can be considered as a rather good fine structural preservation, l)ut naturally, the more active the aldehydes, the more stringent the limitations are for the histochemical applications resulting from the denaturation and inactivation

THE FINE STRUCTURAL LOCALIZATION OF GLUCOSE-6-PHOSPHATASE IN RAT LIVER

Glucose-6-phosphatase activity was demonstrated histochemically in rat liver using either the Wachstein-Meisel medium or a modified Chiquoine medium, and the characteristic properties of the enzyme activity were confirmed. The distribution of activity in both unfixed and hydroxyadipaldehyde-fixed material was demonstrated with the electron microscope. Activity was found in both smooth- and rough-surfaced elements of the endoplasmic reticulum of hepatic cells, including the nuclear envelope, but was absent from the plasma membrane. These findings further implicate the endoplasmic reticulum as an organelle of transport, and in addition suggest that the nuclear envelope has functional as well as morphological continuity with the endoplasmic reticulum.

Cytochemical studies on chromosome ultrastructure.

This paper deals with the staining of nuclear proteins, presumably histones, in the meitoic chromosomes of the prophase nuclei of lily microsporocytes. In the pachytene chromosones the lateral element of the synaptinemal complex was particularly reactive with alcoholic PTA. The reactive material was extractable with dilute acid, and staining was inhibited by prior acetylation. Various in vitro quantative studies were performed on the PTA reactivity of isolated histones and deoxyribonucleohistones. These studies suggest that the lateral element of the synaptinemal complex may well contain a histone which may be synthesized in the early part of prophase.

A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.

The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at -100 degrees C, began to react at -70 degrees C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Ss(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, rpesulting in a final mixture which was 50-60% Os(III). The results support a scheme for the reaction os osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.

NUCLEOSIDE PHOSPHATASE ACTIVITY IN MEMBRANOUS FINE STRUCTURES OF NEURONS AND GLIA

The reaction product resulting from the hydrolysis of various nucleoside phosphate esters in the Wachstein-Meisel medium at a neutral pH has been localized by means of electron microscopy to various glial and neuronal membranous fine structures. Enzyme activity following the use of adenosine triphosphate as a substrate was localized to the interspace between the plasma membranes of neurons and glial dendrites adjacent to the neuronal cell body, the proximal axon and synaptic terminals. The relationship of this enzymatic activity to the transfer of cations, especially sodium and potassium, as well as to the synthesis of acetylcholine at synapses is discussed. A reaction product was also found in the Golgi apparatus of neurons and although it was most prominent following the use of inosine diphosphate as a substrate, it was also noted with other nucleoside phosphates. In addition, enzymatic activity was noted on the plasma membrane of oligodendroglia, particularly when adenosine triphosphate and cytidine triphosphate were used as substrates.

THE LOCALIZATION OF NUCLEOSIDEPHOSPHATASE ACTIVITY IN DIFFERENT TYPES OF SMALL BLOOD VESSELS.

In aldehydefixed rat tissues, the distribution of nucleoside phosphatase activity in three different types of capillaries is presented. In some unfenestrated vessels (lung) activity was localized to pinocytic vesicles of endothelial cells while in others (retina), the endothelium was unreactive. In the latter case activity was present in the basement membrane of the capillary and associated with the surface of glial cells. Fenestrated capillaries contain no enzymatic activity associated with their walls. These findings are discussed in relation to current concepts of transport across capillary walls.