L. G. Abood

HISTOCHEMISTRY AND CHARACTERIZATION OF HYALURONIC ACID IN AXONS OF PERIPHERAL NERVE

CATIONIC adsorption, or accumulation, within tissues has been attributed to many substances, including proteins (see LING (1955) for discussion), phospholipids (FOLCH, 1952), soluble constituents, such as polyphosphates (STEINBACH, 1950), chondroitin sulphate (MEYER and RAPPORT, 1951), and to ground substance colloids (JOSEPH, ENGEL, and CATCHPOLE, 1952). In view of certain di!liculties concerning the hypothesis that the internal potassium concentration exists in a free form and is maintained by a “sodium pump mechanism,” LING (1955) has postulated that “fixed charges” within the nerve are responsible for the selective accumulation of potassium. Studies of the energy requirements of nervous tissue during excitation have suggested that “high-energy phosphates” are primarily concerned with the maintenance of structural relationships essential for activity and, presumably, are not speci6dy involved in a sodium pumping mechanism (AWOD and GOLDMAN, 1956). In an effort to determine whether nucleic acids, or the microsomes of which they are an integral part, are involved in ionic accumulation (ABOOD and ROMANCHEK, 1955), the suggestion offered itself that mucopolysaccharides may play a role, since they are implicated in ionic transfer elsewhere in the organism. ’The present communication reports the histochemical identification of hyaluronic acid (HA): in nerve, its isolation and chemical characterization, as well as certain cationic-exchange studies pointing to its possible role in nerve conduction.

ISOLATION FROM RAT BRAIN OF MITOCHONDRIA DEVOID OF GLYCOLYTIC ACTIVITY.

BRAIN tissue is composed of a variety of cell types, ranging in diameter size from a few, to over 100, micra. Even inore significant is the fact that the protoplasmic bulk of brain is largely due to dendritic, axonal, and glial processes which intertwine about the diverse cellular elements. Although homogenization will readily rupture the cell bodies of larger neurons and glia, numerous processes and smaller cellular elements may be only partly disrupted, even after extensive homogenization or even ultrasonic disintegration. When conventional techniques of differential centrifugation are used in the isolation of mitochondria and other cellular elements from brain, it is extremely difficult to obtain reasonably homogeneous fractions, since many of the partly disrupted processes and cellular elements have similar sedimentation constants. Although brain mitochondrial preparations obtained by conventional methods are adequate for many purposes, they may often yield results which are misleading. The many reports that such preparations exhibited glycolytic activity (HESSELBACH and D u s w , 1953), have led some observers (BALAZS and RICHTER, 1958 ; GALLAGHER et al., 1956), to conclude that brain mitochondria contain glycolytic enzymes not present in liver or heart mitochondria. In one such study ABOOD et al. (1959) indicated that with hypertonic sucrose it was possible to obtain a fraction rich in mitochondria but almost devoid of glycolytic activity. The present study is devoted to a closer re-examination of the problems of the source of glycolysis in the mitochondrial fractions of rat brain. A more refined procedure for preparing relatively pure mitochondria as well as other cellular entities from rat brain is described. M E T H O D S

HISTOLOGICAL DEMONSTRATION OF HYALURONIC ACID IN THE CENTRAL NERVOUS SYSTEM

MUCOPOLYSACCHARIDES (MP)f have been known to exist in tissue for several decades, but it is only within the past ten years that interest in MP has extended to the nervous system (ABCOD and ABUL-HAJ, 1956; BRANTE, 1957). The presence of MP in the central nervous system has been demonstrated chemically by BRANTE (1957, 1958), but an extensive characterization has not been accomplished. The preponderance of evidence for MP in the central nervous system, however, is histologic. One of the earliest studies of MP in the CNS is that of FRUBERC and RINCERTS (1956), who demonstrated that upon the injection of 35s into rat embryos, the uptake of sulphur was very low in both peripheral nerve and CNS up to 10 days, but at 17 to 18 days there was a fairly high uptake in the corpus striaturn and certain cortical layers, as judged by PAS-staining properties. Neuroglia, particularly astrocytes, were found to contain PAS-positive granules which were not cerebroside or lecithin (LUMSDEN, 1958). WOLMAN (1956, 1957) reported that during degeneration of the myelin sheath, MP is released, presumably from the myelin. H ~ s s (1955) suggested that MP may be related to the blood-brain barrier, since the disappearance and reappearance of the barrier correlated well with the reappearance of the PAS-staining reaction. Insofar as H-ase did not alter PAS staining, the material was probably not HA. OZELLO et al. (1958), on the other hand, did not detect PAS staining comparable to the ground substance of other tissues, except in the walls of blood vessels. The use of H-ase in demonstrating the presence of MP in the central nervous system has been surprisingly limited. H-ase produced a greater diffusion of trypan blue through either normal or traumatized brain tissue (ARTETA, 1956), and facilitated the penetration of Indian ink around the nerve cells (BAIRATI, 1953). FREEDMAN (1953) also was able to demonstrate a difference in the metachromatic staining of brain sections with thionine after treatment with H-ase, particularly in the ‘intercellular matrix of the grey matter.’ Since a previous study had demonstrated the presence of HA in the axoplasm and neurilemma sheath of amphibian and mammalian nerve, there was reason to believe that the neurons of the central nervous system should likewise contain the material. The present study is devoted to an extensive histological exploration of MP in the mammalian brain and definitely establishes the existence of an MP within the neurons, which is readily attacked by testicular H-ase. Furthermore, it will be

The chemical constitution and biochemical effects of psychotherapeutic and structurally related agents.

Although the number and diversity of pharmacological agents having rather well-defined effects on metabolism continues to grow, very little effort has been made to correlate the metabolic disturbance with either the pharmacological activity or chemical structure of these agents. One area in which such investigations have been carried on to excellent advantage is in the case of the cholinergic and adrenergic’ drugs. Attempts to correlate metabolic disturbances with narcosis have been numerous, although the evidence in favor of such a view is not conclusive. In recent years electron microscopy2’ and physicochemical studies4’ have revealed that the mitochondria, which are the cellular sites of energy production, consist of an elaborate structure surrounded by and intertwined with a lipid (or lipoprotein) membrane. The maintenance of the intramitochondrial geometric pattern, or of some smaller structure within it,6 is apparently essential for carrying on oxidative phosphorylation. A wide diversity of agents has been found to inhibit oxidative pho~phorylation,~ but no attempt has been made to classify such agents according to their chemical constitution. In studying the relation of structure to the inhibition of cytochrome oxidase (a mitochondria1 enzyme) in a series of organic compounds, the conclusion was reached that the basic structure essential to inhibition was a “dipheny1”t moiety.8 With the introduction of chlorpromazine (10-[y-dimethylaminopropyl] 2-chlorphenothiazineJ or CPZ) into the field of mental health, interest was renewed in this problem, for the structural configurations of this compound suggested that it was related to the series of diphenyl compounds studied previously. This relationship was confirmed and, furthermore, it was found that CPZ was an effective inhibitor of oxidative phosph~rylation.~ Certain peculiarities regarding the inhibitory effect of CPZ, such as its complete reversibility, indicated that its reactivity with the phosphorylating complex of mitochondria from the brain resembled the relationship of the diphenyl compounds to cytochrome oxidase. In the present report, the problem of structure-metabolic activity relationships is re-examined, and a diverse number of diphenyl and related compounds, including reserpine and other tranquilizing drugs, are studied. An attempt also has been made to correlate the metabolic disturbances produced by some of the neurotropic drugs in ziivo, as well as in vitro, to their neuropharmacological properties.

CHOLINERGIC BLOCKADE AS AN APPROACH TO THE DEVELOPMENT OF NEW PSYCHOTROPIC AGENTS

The emergence of the psychotropic drugs as effective therapeutic agents in the treatment of mental disorders and the subsequent attempts to elucidate their mechanism of action, brought to the forefront a number of endogenous substances that had previously been thought to be concerned only in the control and transmission of autonomic nerve impulses (FIGURE 1). The effects of the psychotropic agents were described in terms of mimicking, potentiating, or inhibiting the actions of these endogenous substances, and this led to the conclusion that the subcortical centers of the brain might be subject to similar autonomic controls as the remainder of the body physiology. Thus Brodie’ expanded the hypothesis of Hessz (FIGURE 2), which postulated an ergotropic (excitatory) center and a trophotropic (depressant) center in the hypothalamus. Norepinephrine was thought to be the neurohormone involved in ergotropic stimulation while serotonin or acetylcholine were concerned in trophotropic stimulation. Substances that mimicked or potentiated the actions of norepinephrine such as the sympathomimetic amines and its “masked” analogues (pipradol, methylphenidate) or prevented its metabolic destruction (MA0 inhibitors) produced mood elevation, antifatigue effects, and an antidepressant action in depressed mental patients. Conversely, an inhibition of the ergotropic stimulator, norepinephrine, by the administration of blocking agents such as the chlorpromazine-type drugs or ethoxyb~tamoxan~ resulted in a calming and tranquilizing action. This theory was further substantiated by the development of substances that prevented the synthesis of norepinephrine from its carboxylic acid precursor dihydroxyphenylalanine (DOPA) , the socalled DOPA decarboxylase inhibitors (FIGURE 3 ) . These compounds likewise exerted a sedative and depressant effect on the CNS.4 By the same token, blockade of trophotrophic stimulators should lead to central stimulation and excitation. While serotonin antagonists have exhibited no such properties, a number of drugs with potent anticholinergic properties have demonstrated antidepressant effects (FIGURE 4). For instance, Benadryl is a fairly potent sedative, while its o-methyl congener, Disipal, is not a t all sedative and may even cause e ~ p h o r i a . ~ Its anticholinergic action is twice that of Benadryl. Benactyzine is a potent anticholinergic that has been applied successfully in the treatment of depression when used in conjunction with meprobamate.6 Tofranil, one of the newer antidepressant drugs, is a moderately potent anti~holinergic~ and Pacatal whose action is less sedative than that of chlorpromazine is also a much more potent inhibitor of acetylcholine.8 The work of Finkg with several experimental anticholinergic compounds

INCORPORATION OF ORTHO (32P)PHOSPHATE INTO THE SUBCELLULAR FRACTIONS OF DEVELOPING RAT BRAIN.

The refinement of techniques for the isolation of the cytoplasmic fractions of rat brain has now made it possible to study more accurately the biochemical development and turnover of the various subcellular elements of the neuron. A previous communication (Abdel‐Latif and Abood, 1964) described enzymic studies with cytoplasmic constituents prepared from embryonic and neonatal rat brains by the technique of density gradient centrifugation. It was the object of the present study to examine, with the aid of ortho [32P]phosphate, the formation and turnover of the phospholipids, phosphoproteins, and other phosphate‐containing compounds of the various subcellular components of the rat brain at various stages of development.

A new group of psychotomimetic agents.

Summary A series of synthetic anticholinergic agents have been shown to possess potent psychotomimetic properties. Chemically, the agents are esters of piperidine and benzilic acid. Among the effects produced are megalomanic and paranoid delusions, visual and auditory hallucinations, and a partial loss of contact with the environment. A number of congeners of the compounds have been examined with regard to structure-activity relationships.

IN VIVO INCORPORATION OF l‐[14C]SERINE INTO PHOSPHOLIPIDS AND PROTEINS OF THE SUBCELLULAR FRACTIONS OF DEVELOPING RAT BRAIN*

A study was conducted on the in vivo incorporation of l‐[14C]‐serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine‐containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid‐soluble fraction in addition to l‐[14C]serine.

Fixation of 5-hydroxytryptamine by brain mitochondria.

Summary Distribution of 5-hydroxytryptamine (HT) in various cytoplasmic fractions of rat brain has been determined, and the neurohumor was confined almost exclusively to the mitochondrial fraction. Numerous inhibitors of oxidative phosphorylation were tested for ability to release HT from mitochondria or to prevent binding. Reserpine completely depleted mitochondria of HT, but did not alter their ability to reabsorb HT, while phenyl ether and LSD partly prevented mitochondrial fixation of HT.

THE ACTION OF SOME PSYCHOTOMIMETIC AGENTS ON THE EXCITATORY AND BIOCHEMICAL PROCESSES OF FROG SARTORIUS MUSCLE

As PART of an extensive programme, concerned with the development and investigation of a group of heterocyclic imino esters of substituted glycolic acid possessing psychotomimetic properties (ABOOD et al., 1959), attention has recently been directed towards the possible mechanism of action of these compounds. Earlier studies have failed to reveal any action on a variety of brain enzyme systems (ABOOD et al., 1959). Although these compounds were potent anticholinergic agents, and there was a good correlation between psychotomimetic and anticholinergic potency, it was believed, nonetheless, that their action upon the central nervous system was not due to cholinergic blockade (ABOOD et al., 1959). Structure’activity studies with diphenyl derivatives have indicated that compounds of this nature possessing an ester-type linkage were, in general, not inhibitory to respiratory enzymes (ABOOD, 1961). In the course of investigating the relationship of calcium and phosphates to the excitatory and contractile processes of frog sartorius muscle {ABOOD et al., 1962), it was found that the glycolic acid esters exerted a striking action on both the metabolic and functional properties. ‘Despite the fact that similar actions could not be demonstrated on nervous tissue, the results obtained were assumed to provide information regarding the mode of action of the agents on excitable membranes in general. The present study suggests that this class of agents may prove of value in exploring some of the electrical and other physicochemical properties of excitable tissues.