Alfred Pope

INTRALAMINAR DISTRIBUTION OF ADENOSINETRIPHOSPHATASE ACTIVITY IN RAT CEREBRAL CORTEX

IN recent years, the principles and techniques of quantitative histochemistry have been used for a number of investigations on the biochemical architecture of the cerebral cortex. The principle object has been to provide detailed information on the chemical structure and metabolism of this tissue that could be related to its microscopic anatomy and electrophysiological properties. To this end, studies have been reported on the intralaminar architectonic distributions of enzymes in rat isocortex (POPE, 1952; POPE, HESS, WARE and THOMSON, 1956), rabbit hippocampus (LOWRY,

Intralaminar distribution of cytochrome oxidase activity in human frontal isocortex.

I N THIS laboratory, the principles and techniques of quantitative histochemistry (LINDERSTRBM-LANG, 1939) are being used to study the biochemical architecture of human cerebral cortex. The immediate aim has been to determine relationships between the intracortical distributions of certain enzymes, representative of different metabolic pathways, and the histological structure of the cortex. The long range objectives are to establish a matrix of biochemical knowledge concerning n o d human cortex and to investigate possible quantitative histochemical changes in certain nervous and mental diseases. Previous reports have described the intralaminar distributions of acetylcholinesterase (POPE, CAVENFSS and LIVINGSTON, 1952) and L-alanylglycine dipeptidase (POPE, 1959) activities in cortical biopsy specimens excised during frontal lobotomy. A similar investigation has been carried out upon the intracortical distribution of cytochrome oxidase, the results of which have already been presented ia prehmmary form (POPE and HESS, 1954; POPE, HESS and ALLEN, 1957). In this article, the details of this study are described in full. Cytochrome oxidase was selected for study because of its critical importance in brain oxidative metabolism as mediator for electron transport between cytochrome c and oxygen.: Its activity is a simple and direct index of cortical oxidative capacity. Since it is readily inactivated, its assay in freshly excised tissue is desirable. For its assay, a microspectrophotometric method was developed (HESS and POPE, 1953). applicable to microgram samples of human cortex. This method has been used in conjunction with a frozen section sampling technique which provides samples who% dry weight, anatomical localization, and histological composition can readily be determined, so that the intracortical profile of the enzyme may be charted. These techniques have been used previously (POPE, HESS, WARE and THoMSON, 1956) to study the distribution of cytochrome oxidase in the layers of rat somatosensory isocortex. In this tissue, oxidase activity was high throughout the first five layers,

THE INTRALAMINAR DISTRIBUTION OF DIPEPTIDASE ACTIVITY IN HUMAN FRONTAL ISOCORTEX

QUANTITATIVE information upon the distribution of biochemical components in relation to microscopic anatomy is an important aspect of the biology of the human nervous system and an essential prerequisite for a comparative, quantitative histochemistry of nervous and mental diseases. Unfortunately, it is seldom possible to obtain specimens of relatively normal human brain in a state suitable for biochemical analyses, including those for metabolites and most enzymes. . The use of psychosurgery for the amelioration of mental disease and the relief of intractable pain provided, for a time, an opportunity t o excise biopsy specimens of human cerebral cortex satisfactory for certain types of biochemical analysis. For investigations on this sort of material, the methods of quantitative histochemistry are particularly appropriate. Such techniques enable simultaneous analysis of the biochemical architecture of a tissue such as human cerebral cortex and the acquisition of precise neurochemical information for eventual comparison with clinical data. Investigations utilizing biopsy specimens of human cortex extirpated during frontal lobotomy were carried out in this laboratory for several years. For the most part, they consisted of studies on the quantitative intralaminar distributions of certain enzymes selected as indices of the location and turnover rates of intracellular metabolic systems critical for the general or special metabolism of brain. The methods used for sampling the cortical layers and sublayers, for quantitative microchemical measurements of enzyme activities, and for correlating the results with cortical anatomical fine structure have represented adaptations to brain of the principles and techniques of quantitive histochemistry originally devised and developed by LINDERSTR0M-LANG (1939) and HOLTER (1951). The nature, rationale and results of experiments upon the activity and intralaminar distribution of acetylcholinesterase in the frontal cortex of psychotic and non-psychotic patients have been presented previously (POPE, CAVENESS and LIVINGSTON, 1952). Subsequent, analogous studies on proteolytic and oxidative enzymes have also been the subjects of partial, preliminary reports (POPE and HESS, 1954; POPE, HESS and ALLEN, 1957). In this article, experiments on the architectonic distribution of r-alanylglycine dipeptidase activity are described in detail. Only the quantitative histochemical aspects are considered herewith. Possible correlations between the results and certain clinical descriptive parameters will be presented in a separate communication.

INTRALAMINAR DISTRIBUTION OF ADENOSINETRI‐PHOSPHATASE ACTIVITY IN HUMAN FRONTAL ISOCORTEX*

PREVIOUS publications from this laboratory have described the progress of experimental work designed to outline the biochemical architecture of mammalian cerebral cortex. To this end, the intracortical distributions of certain enzymes important in brain metabolism are being studied in human anterior frontal cortex and in rat somatosensory isocortex by the methods of quantitative histochemistry. A primary objective is to correlate the biochemical architecture with the cytoand myeloarchitecture and with the electrophysiological properties of the cortex. The work serves a wider purpose of building up a body of information helpful in assessing possible metabolic or structural alterations of human cortex in certain nervous and mental diseases. Adenosinetriphosphatases, which release inorganic phosphate from ATin the absence of other added substrates, were among the enzymes chosen for study because early work with myosin suggested involvement of ATPases in the specialized functions of highly differentiated tissues. A survey of ATPase activity in rat somaesthetic cortex was conducted using two assay systems, the first having Ca++, the second Mg++ as activating ion (HESS and POPE, 1959). It was found that Ca-ATPase showed maximal activity in layer I, lesser elevations of activity in layers III(a), III(b), V(c) and VI(a), and an overall decline of activity as a function of subpial depth. This pattern is consistent with occurrence of Ca-ATPase activity in finely subdivided cortical plexuses of axons and dendrites. Mg-ATPase activity, on the other hand, was greatest in layers 11, 111, and IV, subsidiary peaks being seen in layers V(b) or V(c) and in VI(a), a pattern consistent with localization chiefly in neuronal perikarya and dendrites, and at axon terminals. This paper reports the details of an analogous study of Ca-ATPase and MgATPase activities in human cortical biopsy specimens excised during frontal lobotomy. The distributions of the two enzyme activities in human cortex bear a striking resemblance to their respective distributions in rat cortex, and presumably may be interpreted similarly as indicating that in human brain Ca++ and Mg++ predominantly activate separate enzymes having different cytological locations and functional roles.

Quantitative Neurochemical Histology

The uniquely complex histological structure of the vertebrate CNS poses serious problems for interpretation of data on the biochemical composition and metabolism of normal and pathological tissue. Although nervous tissue is comprised of only three principal types of cells, its complexity differs from that of other tissues by several orders of magnitude. First, the neuron population shows enormous variety in morphology. Neuronal processes have unique elaborative tendencies and may extend for long distances from the cell body. In particular, the wealth of neuronal interrelationships through synaptic contacts, which are the basis of the highly organized function of the nervous system, represents a structural and metabolic variant of a complexity without parallel in nonnervous tissues. Second, the glial population, both oligodendroglial and astroglial, also shows some variety in morphology and function. Finally, the absolute numbers and proportions of these three cell types and their varieties differ greatly in regional distribution in the CNS.

Biochemistry of Middle and Late Life Dementias

The disease entities to be considered in this review are characterized clinically by a slow but relentless deterioration of intellectual faculties resulting in profound dementia and eventually death during middle and late life. Alzheimer’s disease, Pick’s disease, Huntington’s chorea, Creutzfeldt-Jakob disease, and senile dementia are included in this group of “primary dementias” where the unifying pathologic feature is a degeneration of the cerebral cortex with varying degrees of involvement of related structures. These conditions, which as yet have no established cause (the special status of Creutzfeldt-Jakob disease is discussed below) and as far as can be determined at present are not related to any known disorder, stand in contrast to the “secondary dementias” that occur in association with syphilitic infection, cerebrovascular disease, brain tumor, myxedema, alcoholism, nutritional deficiencies, encephalitis, trauma, and chronic neurological diseases such as disseminated sclerosis, subacute combined degeneration, Wilson’s disease, and the cerebral lipidoses. These secondary dementias are considered elsewhere in this Handbook.

PROBLEMS OF INTERPRETATION IN THE CHEMICAL PATHOLOGY OF SCHIZOPHRENIA

Publisher Summary This chapter discusses problems related to interpretation in the chemical pathology of schizophrenia. Researchers believe that all of the changes noted in schizophrenic brains must be considered nonspecific or artifactual either on the basis of inadequate criteria for normative ranges of histological appearances, or as the result of age changes or other somatic influences of the many kinds prevalent in a chronic schizophrenic population, or to inadequate standardization of fixation and histological preparative procedures. The first and the most important problem is the nosology of mental disease, the assignments of subjects to class and subclass representing publicly verifiable and operationally or ostensivly definable ensembles. This is absolutely essential for establishment of meaningful correlations between psychopathology and biological phenomena. The second problem (already alluded to) is that of biological variability that, in the case of the nervous system, is a two-dimensional problem. Intraspecies ranges of normative data values, particularly for metabolic indices, are very great and especially so in an outbred species.

From Intelligence to the Microchemistry of the Human Cerebral Cortex

The chasm in current knowledge between phenomena that emerge from the human brain in action for which the languages of psychology and psychopathology are required, and events at the level of brain molecules and electrolytes that are correlates in the physical domain, is vast both in conceptual and practical terms. In the biological sphere alone, the interactive hypercomplexities of anatomical substructure and biophysical function virtually defy definitive analysis, although seemingly providing commensurate degrees of freedom for the manifestations called “mind”. Given the still rudimentary state of knowledge in human neurosciences, it is clear that understanding of the exact nature of cerebral events that translate into cognition, affect and behavior (rational or otherwise) remains a distant goal, even without invocation of more recondite issues in logic and epistemology that, in the author’s view, are inescapable for any comprehensive consideration of these perplexing but transcendingly challenging matters.1

INTRODUCTION: QUANTITATIVE HISTOCHEMISTRY

A PANEL on histochemical technologies, applicable to neurochemical pathology, is especially germane at this Conference because of the complex microscopic anatomy of the central nervous system and the consequent compelling need for analytical methods capable of establishing correlations between chemical composition and histological structure. Three major principles and congruent technical measures have been employed for this purpose :l (1) Qualitative histo- and cytochemistry enabling in situ identification of biochemical constituents in light and electron microscopic preparations. This classical form of histochemistry is elaborated and exemplified by other panelists. (2) Use of differential gradient density techniques for preparation (with subsequent macro analysis) of subcellular cytological constituents (nuclei, mitochrondia, synaptosomes etc.) and of enriched fractions of neurons, astrocytes, oligodendrocytes, myelin sheaths and axons. With respect to the nervous system, important contributions in these spheres have been those of V. Whitaker (U.K.), E. de Robertis (Argentina), S. Rose (U.K.) and W. Norton (U.S.A.) (3) Quantitative histochemistry based on the preparation of microgram or sub-microgram sized tissue samples of known histological composition on which direct analyses for chemical structural components or enzyme activities can be carried out using miniaturized forms of standard biochemistry. Procedures of this sort were developed originally by LinderstromLang and Holter in the Carlsberg Laboratories, Copenhagen.2 Remarkable extensions and refinements of such techniques to levels permitting single cell analysis we owe especialiy to Lowry3 and HydCn4. Our laboratory has been concerned for many years with applying microgram level quantitative histochemical techniques, applicable to samples of histological dimensions, to the normal and diseased nervous system. Within this context a central program has been the one dealing with the microchemical anatomy and pathology of mammalian cerebral cortex referred to in my earlier presentation at this Conference. Its objectives have been to analyze cortical biochemical fine structure in terms of its microscopic anatomy, and to search for chemoarchitectonic alterations in disease. The results also permit inferential correlations between biochemical composition and histological structure, normal and otherwise. The experimental design serving these objectives has consisted in preparing from fresh,