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SCHWYN, Robert Conrad, 1938- AN AUTORADIOGRAPHIC STUDY OF NEUROGLIA.

The Ohio State University, Ph.D., 1966 Anatomy

University Microfilms, Inc., Ann Arbor, Michigan ( t ) Copyright by

Robert Conrad Schwyn AN AUTORADIOGRAPHIC STUD! OF NEUROGLIA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Robert Conrad Schwyn, B .S ., M.Sc.

* * * * * * *

The Ohio State University 1966

Approved by

Adviser Department of Anatomy ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to Dr.

James L. H all, Department of Anatomy, School of Medicine, The Ohio

State University, for his assistance and encouragement during this p ro je c t.

A special word of thanks is extended to my wife, Jane, whose patience and devotion have made this endeavor possible, and to our daughter, Christine, who did her best.

i i VITA

June 8, 1938 . Born - Toledo, Ohio

1960 ..... B .S ., The Ohio S tate U niversity, Columbus, Ohio

1960-1962 . . United States Aimed Forces, U.S. Array, Medical Service Corps, Landstuhl, Germany.

1962-196^ • • Graduate A ssistan t, Department o f Anatomy, The Ohio S tate U niversity, Columbus, Ohio

1963 ...... M.Sc., The Ohio S tate U niversity, Columbus, Ohio

1965 ...... Teaching A ssistan t, Department o f Anatomy, The Ohio S tate U niversity, Columbus, Ohio

1965-1966 . . Teaching A ssociate, Department o f Anatomy, The Ohio S tate U niversity, Columbus, Ohio

PUBLICATIONS

"Studies of Neuroglial Activity in Autonomic Ganglia during Electrical Stimulation and Drug Administration." Anat. Rec., 151: March 1965. (Abstract) "A Modified Tri-basic Eye Technique for Neuroglia in Autonomic Gang­ lia." Ohio J. Sci., 6j>: 20^208. 1965.

FIELDS OF STUD!

Major F ield: Anatomy

Studies in Neuroanatomy. Professor James L. Hall

Studies in Gross Anatomy. P rofessors Linden F. Edwards and George R. L. Gaughran

Studies in Comparative Anatomy. Professor W. James Leach

Studies in Histology. Professor G. Adolph Ackerman

Studies in Etabryology. Professor John C. Weston

i i i CONTENTS

Page

ACKNOWLEDGMENTS i i

VITA i i i

INTRODUCTION 1

HISTORICAL REVIEW 5 METHODS 18

A. General operative procedures B. Autoradiographic techniques C. Autoradiographic development technique D. Staining procedure E. H istochem istry F. Standard histological technique

OBSERVATIONS...... 27 I A* Observations of control ganglia removed from unstimulated animals B. Observations of stimulated superior cervical ganglia C. Observations of stimulated ganglia removed from drug-injected animals D. Histochemical observations of superior cervical ganglia

DISCUSSION...... 41

CONCLUSIONS ...... 56

SUMMARY ...... 58 APPENDIX ...... 60

BIBLIOGRAPHY...... 67

iv INTRODUCTION

The neuron doctrine, formulated by Waldeyer in 1891 and championed by Ramon y Cajal during the years to follow (Rasmussen,

19^7)» described the neuron as the anatomical, functional, embryo- logical, and trophic unit of the nervous system. Under the guidance of these fundamental aspects of neurology, our knowledge of nerve structure and function surged forward very rapidly, gathering momen­ tum as it progressed. In comparison, information related to the functional significance of the non-nervous cellular components of the nervous system remained relatively stagnant. These cells consist of a small nucleus, while their cytoplasm is drawn out into thin pro­ cesses. The original descriptive term applied to this mass of supporting cells was translated into the English language as "nerve glue" and was appropriate at that time. This trend of thought per­ sisted during the great boom of neurological investigation. Although there were earlier suggestions that neuroglia cells were involved in processes other than their own maintenance and neuron suspension, there was insufficient experimental data to lend much support to the concept. Within the last twenty years, our understanding of neuro­ glia elements has increased; but it has remained in the shadow of investigations conducted on neuronal function. At present, sufficient

1 data has been compiled to implicate these cells in the metabolic support of neurons.

The supporting cellular elements located in peripheral ganglia were also designated as neuroglia because they demonstrated anatomical and physiological relationships similar to oligodendrocytes identified within the central nervous system. The functional capacities of these cells are even more obscure than those in the central nervous system.

Although the term neuroglia, in reference to these cells, appears in most textbooks and periodicals, many prominent neuroanatomists today hesitate to identify them as neuroglia, perhaps avoiding terminology that may not'correctly reflect their functional or anatomical signi­ ficance. For want of better descriptive terminology, the supporting cellular elements located in the superior cervical sympathetic gang­ lion will be referred to collectively as neuroglial cells. These neuroglial cells will also be considered as interstitial or peri- neuronal satellite cells because of their anatomical location in relation to the neurons. Furthermore, perineuronal satellite cells will be referred to as either primary or secondary satellite cells, again determined by their location. The group of satellite cells excludes Schwann cells, endothelial cells, fibroblasts, or other

connective tissue cells that may be present in the superior cervical

ganglion.

The premise of this investigation was the possibility that neuroglia within autonomic ganglia are involved in maintenance of

environmental conditions which are conducive to efficient transmission 3 of an impulse. Many processes are involved during the transmission of a nervous impulse from preganglionic fibers to end organs receiving postganglionic fiber terminations. Synaptic transmission has been the subject of many histological, physiological, and pharmacological investigations. Most of the evidence, however, has been concerned with neuronal function, rarely implicating the presence of neuroglia or their functional activities. With the aid of previous data on the anatomical and biochemical properties of neuroglia cells and of autoradio graphic and histochemical techniques employed in this in­ vestigation, an attempt is made here to implicate neuroglial activity in relation to repetitive neuronal transmission.

Autoradiographic techniques, although relatively new and in early stages of application, have been utilized very successfully during the past decade in determining the dynamics of cellular activity. Almost any biochemical compound or precursor can be labeled with various radioisotopes. Autoradiographic identification of a cell component, then, depends upon the availability of the labeled compound' and the time involved for its cellular absorption.

Histochemical procedures were undertaken in order that the labeling of a cell component could be correlated with enzymatic and chemical activities occurring simultaneously within the ganglion. By apply­ ing electrical stimulation to preganglionic fibers terminating in the superior cervical ganglion, and in some instances introducing certain drugs into the organism, an attempt was made to alter and control the activity of gLial cells under specific known conditions. It is hoped that the results of this investigation will shed additional light upon the possibility of neuroglial involvement in synaptic activity and neuronal metabolism in autonomic ganglia. The following information may be of value to those interested in the functional relationship existing between neurons and neuroglia.

Since facets of various disciplines are encountered during this study, it is presumed that individuals involved in anatomical, physiological, biochemical, and pathological research may find some aspect of this paper applicable to their own work. HISTORICAL REVIEW

The earliest accounts of neuroglia investigation, according to Glees (1955)» date back to 18^6 -when Virchow first described these structures as independent cellular elements of the nervous system. This marked the beginning of an era concerned primarily with the morphological characteristics and identification of these newly identified elements. Descriptions of neuroglia cells and their processes were more numerous after the turn of the century when many new staining techniques were developed. Two of the most widely known staining methods are those of Golgi and Weigert.

Glees (1955) stated that Held (1909) developed a technique by which he was able to show that glia cellular fibers form a syncytial net in which the primary nervous structures are suspended. In addition to advancing the theory that the neuroglia cells form a mechanical

supporting network, Held suggested that they are involved in the metabolic activity of neurons. It was his identification of terminal end feet on glia processes attached to blood vessels which led him to suggest that glia elements may serve as a transport for metabolic products. According to Glees (1955)» Held also observed granular inclusions in glia processes which he thought to be secretion; further substantiating a function other than mechanical support.

Held's observations, however, were highly advanced for his time, and

5 6 very few follow-up investigations were performed in support of his work. Ramon y Cajal (1911) added considerably to our organized knowledge of neuroglia by classifying than into three types. The cell types that he identified are known today as astrocytes, oligo­ dendrocytes, and microglia. Following Ramon y Cajal were two of his students, del Rio Hortega and Achucarro, who contributed much to our present knowledge of neuroglia morphology, and completed the era of investigation devoted primarily to the descriptive morphology of neuroglia with the light microscope.

In regard to the functional significance of neuroglia cells in the central nervous system, we find that these cells have been first identified with myelin formation by del Rio Hortega in 1928

(Glees, 1955)• De Castro (1951) strongly supported the idea that glia cells are in some fashion concerned with synaptic activity; however, this idea was not very well accepted at this time. Glees

(1956), summarizing at the Conference on the Biology of Neuroglia, concluded that the intense arborizations of neuroglia cells may be the morphological expression of a special metabolic activity (Windle,

1958). He further indicated that these cells may also be secretory in nature, but the actual functional role in the central nervous system s till remains to be answered.

Within the past decade many investigations have indicated that neuroglia are metabolically related to neurons in the central nervous system. Although this study is primarily concerned with neuroglia in 7 autonomic ganglia, a few of these reports deserve mention in this

section to identify specific patterns of neuroglia relationships and

to establish a basis for comparison to structures in autonomic

ganglia. Wyckoff and Young (1956) observed with the electron micro­

scope cytoplasmic processes of neuroglia entwined among nerve fibers,

dendrites, and nerve cell bodies of the ventral horn. From these observations they proposed an exchange between neurons and capillaries presumably through glia cytoplasm. A short while later Niessing

(1959) proposed that neuroglia must be metabolically related to the neuron, and this may be a secretory functional relationship. Hyden

(1961) reported nerve cells rich in ATP, while neuroglia were rela­

tiv e ly poor in ATP. Conversely, g lia c e lls are ric h in ATPase,

which releases the energy in ATP, while the neurons lack ATPase on

the surface membrane. He suggests the existence of an active ATPase

capable of releasing energy for the transport of nutrients from

blood capillaries to the surface of the neuron. ATPase may also be

capable of transporting a wide variety of substances across the cell

membrane. Hamberger and Hyden (1963) proposed a regulatory meta­

bolic mechanism existing between neurons and surrounding glia in

response to vestibular stimulation and hypoxia. Studying levels of

cytochrome oxidase, succinic oxidase, HNA, and anaerobic glycolysis

in neurons and glia, they found that "the neuron has priority in its

high energy requirements when function so demands."

Most recent studies involve neuroglia in the blood brain

barrier participating in lieu of or as the extracellular space in 8 the central nervous system. Astrocytes, therefore, may be involved in water and electrolyte metabolism and in their transfer from blood to neuron (De Robertis and Gerschenfeld,' 1961) • This view is further supported by Dan (1964), who has shown by electron microscopy that neuroglia is the single free pathway between the blood and neurons.

He further advocated that neuroglia may be significantly connected with nervous excitability itself.

Galambos (1961) has indicated that neuroglia may play a func­ tio n a l ro le in the phenomenon o f memory. He suggests th a t g lia c e lls may actually organize and program neuron activity. Hyden (1961) also speculates on the possibility that memory may be explained on the basis of arrangement and rearrangement of RNA molecules in supporting neuroglia cells. Some investigators have even suggested that such an intimate relationship’ exists between neurons and neuroglia that together the neuron and its surrounding cells serve as the func­ tional unit of the nervous system (Terio and Martino, 1963).

While a vast amount of effort is expounded upon the functional activities of neuroglia in the central nervous system, relatively few studies have been carried out on functional activities of sup­ porting cells existing in peripheral ganglia, and particularly the superior cervical ganglia. In view of the relative sparseness of scientific-data related to the functional significance of supporting c e lls in autonomic ganglia, i t has become necessary to pursue tangential material and attempt to correlate it with studies of autonomic ganglia. Scattered throughout the literature are various descriptive, quantitative, and experimental studies performed on the superior cervical ganglia of man, cats, mice, and other experimental animals.

The anatomical relationships and physiological activities existing in the superior cervical ganglia presented in the following para­ graphs are gathered from many sources. It is an attempt to provide a composite of our present knowledge of the ganglion and a basic understanding of anatomical and physiological terminology referred to throughout this investigation.

Macroscopically, the superior cervical ganglion is located at the base of the skull adjacent to the sensory inferior ganglion of the vagus nerve. I t receives preganglionic fibers ascending from tpper thoracic levels, and gives off cranially, postganglionic fibers to smooth muscles and glands of the head. It also supplies the first four spinal nerves with their sympathetic components, as well as sending a superior cardiac nerve to the heart and cardiac plexus..

Ihe ganglion is encapsulated with fibrous connective tissue and receives its primary blood supply from the carotid artery. Ebbesson

(1963) found that the ratio of preganglionic neurons to post­ ganglionic neurons in the s^erior cervical ganglion of man ranged from 1:63 to 1:96. Ihe ratio obtained from cats was approximately one-tenth that of human ganglia. Ihe degree of difference between the two animals may have been due to greater differentiation of p erip h eral e ffe c to rs or to the to ta l volume o f e ffe c to r tiss u e supplied. 10

Neuroglia cells within the ganglion were considered to be homologous to oligodendrocytes of the central nervous system, and were classified according to their position as capsular and inter­ stitia l by Kuntz and Sulkin (19^7a and 19^7b). Applying the electron microscope to the study of glia cells, Palay (1958) was able to determine definitely that glia fibers are not extracellular. Berg and Kallen (1959). studying neuroglia in tissue culture, suggested that neuroglia could be identified by distinct movement patterns as well as by physical properties and appearances.

Perhaps the most recent and complete description of the ultra­ structure of the superior cervical ganglion of the cat was presented by ELfvin (1963). All ganglion cells and their processes in the superior cervical ganglion are covered by a sheath or capsule of satellite cells. The space between the cell bodies is, to a large extent, occupied by their processes and by myelinated as well as unmyelinated fibers of apparently, extraganglionic origin. All these fibers are ensheathed in satellite cells. An extracellular space is distrib­ uted relatively uniformly throughout the ganglion. The satellite cells which surround the nervous elements are separated from this space by a basement membrane. Colla­ gen fibers are the predominant structures in the extra­ cellular spaces. Frequently observed also are connective tissue cells of varying shape and with variable organiza­ tion of their cytoplasm. No basement membrane appears around these cells. Blood capillaries are regularly seen in the ganglion.

ELfvin also described contacts between processes and cell bodies

(axosomatic) and contacts from process to process (axodendritic and dendrodendritic). In regard to axodendritic contacts, preganglionic fibers run parallel to or wind around the dendrites and establish 11 contacts with the dendrites at several points. He also states that except for the areas of interneuronal contacts, the ganglion cell processes are almost surrounded by satellite cells. The part of the surface of the satellite cells which does not border the neurons or continuous satellite cells is bound by a basement membrane outside of which there is an appreciable connective tissue space similar to the organization of a peripheral nerve. The large extracellular space is much different from the pattern of the central nervous system.

Cravioto and Merker (1963) have also utilized the electron microscope for studying sympathetic ganglia. They observed a space of approximately 150 to 200A° between cell membranes. Their des­ cription of satellite cells in the ganglion was not morphologically the same as those in the central nervous system. They may, however, be compared to oligodendroglia cells of the GNS in view of their abundance of endoplasmic reticulum. A further suggestion is implied that the connective tissue cells of autonomic ganglia take over the function of water metabolism performed in the CNS by astrocytes.

More than thirty years ago, Bishop and Heinbecker (1932) and

Eccles (1935) suggested, by means of electrophysiological evidence, the existence of functionally distinct neural pathways through the superior cervical ganglia of cats. They described four discrete groups of fibers of the sympathetic cervical trunk, and each of these groins of fibers synapsed with a specific group of ganglion cells. 12

These groups of cells were not able to be correlated morphologically and physiologically. This view was later substantiated by Hertzler

(1961), Levy (1962), and Voile (1962), who found the cell groups to be pharmacologically distinct by the use of various stimulating and blocking agents.

Takeshige and Voile (1962) observed a bimodal response of

sympathetic ganglia to acetylcholine. Using eserin, a drug known to potentiate acetylcholine, and repetitive stimulation of preganglionic nerve fibers to the ganglia, they found that two separate responses occurred in the ganglia following injection of acetylcholine. An early response was blocked by d-tubocurarine but not by atropine. A later response was blocked by atropine but not by d-tubocurarine.

They further reported that administration of large doses of eserin provoked a postganglionic response blocked by atropine and not by d-tubocurarine. With these observations in mind, they considered their findings to be in harmony with the proposal of many authors that the asynchronous discharges induced by antiesterase agents or repetitive preganglionic stimulation were due to the liberation of acetylcholine from presynaptic nerve endings. Acetylcholine may then diffuse across the synaptic area to act on different postganglionic cholinoceptive sites. Koelle (1962) described the release of acetyl­

choline from intra-axonal sites as one of the series of events that occurs vhen an impulse traverses a cholinergic synapse. In addition to this primary release of acetylcholine, he also suggests a secondary 13 release of acetylcholine which actually acts at the post synaptic receptor site. The site of secondary release is not indicated, and the possibility of neuroglia involvement cannot be eliminated. The most recent evidence supporting this theory is offered by Friesen et al. (196*0. Utilizing the newest techniques of paper chromato­ graphy, paper electrophoresis, and Carbon-1 k lab eled compounds, they have submitted strong evidence that acetylcholine is present in paravertebral ganglia, and functions as the chemical mediator. They presented negative evidence on the presence of any other choline esters in the ganglia.

Neostigmine and atropine, drugs known to influence the trans­ mission of nervous impulses across the synaptic area, have been used extensively in the last decade. To include all the experimentation performed with these drugs on autonomic ganglia would be quite repetitive. However, some of the most recent advancements are very pertinent to this study and should be mentioned. Levy and Ahlquist

(1962) observed that neostigmine stimulates ganglion cells and atropine blocks neostigmine. Mason (1962a) also presented evidence that neostigmine is capable of a direct stimulating action of autonomic ganglia. The effect is a rapid and reversible depolar­ ization of ganglion cells, and not related to anticholinesterase activity. After injection of small doses of neostigmine, the ganglion cell response to acetylcholine was greatly increased in magnitude and duration. Shortly thereafter, Takeshige and Voile (1963a) suggested that neostigmine (1) has actions on ganglia other than those attributable to inactivation of cholinesterase, (2) may possess presynaptic and postsynaptic sites of action, and (3) may unmask a cholinoceptive site which can be blocked by atropine. In later papers, Takeshige et al. (1963) and Takeshige and Voile (1963b) actually suggested a third cholinoceptive site, and further supported the possibility of two pharmacologically distinct receptor sites located on postsynaptic structures, giving rise to responses by acetylcholine.

The actions of catecholamines and particularly adrenalin have not been found to produce any significant effects directly on autonomic ganglia. Studies by Weir and McLennan (1963) found that injections of catecholamines into autonomic ganglia always produced a depressant effect upon synaptic transmission. They also indicated that even though an adrenalin-like substance is released upon preganglionic stimulation, it is not believed to have any physio­ logical significance.

Koelle and Eriedenwald (19*19) were histochemically able to isolate sites of cholinesterase activity in autonomic -ganglia. A year later, Koelle (1950) demonstrated types of cholinesterases and their locations by histochemical studies of cat tissue. In auto­ nomic ganglia he reported specific cholinesterase located in cytoplasm and nuclei of most ganglion cells. Many capsular glia cells showed high concentrations in th e ir nuclei and somewhat le s s in th e ir 15 cytoplasm. Interstitial glia cells also shoved moderate specific ch o lin esterase concentrations. Heavy concentrations o f the enzyme were demonstrated in the nuclei of Schwann cells and neuroglia cells along preganglionic tracts. Non-specific cholinesterase, -which does not hydrolyze acetylcholine, was localized primarily in the nuclei of neuroglia and neurons and in neuron cytoplasm. Based upon previous evidence by Heyman’s et al. (19*^8), that inactivation of non-specific cholinesterase sensitizes the organism to the effect of small doses of acetylcholine, and upon his own observations, Koelle further suggested that non-specific cholinesterase plays a significant role in the hydrolysis of acetylcholine. Fredricsson and Sjoqvist (1962) reported that most of the acetylcholinesterase in the superior cervical ganglion of cats is located in presynaptic elements; how­ ever, in other autonomic ganglia there was evidence that postsynaptic ganglion cells also release cholinesterase.

Acetylcholine may be capable of depolarizing both presynaptic and postsynaptic elements of the superior cervical ganglion (Pappano and Voile, 1962). They stated that "as a result of depolarization of presynaptic nerve terminals there would be a decrease in the amount of transmitter liberated from a nerve volley and consequently a de­ crease in the amplitude of the postganglionic action potential."

"Thus, depolarization by acetylcholine may produce a refractoriness of some of the ganglion cells and reduce the number of cells capable of responding to-a preganglionic volley." The actions of acetylcholine 16 in their study were apparently sensitive to blockage by small doses of atropine.

Autoradiographic studies of neuroglia in autonomic ganglia pertinent to this investigation have not been reported in the literature. Therefore, it becomes necessary to pursue autoradio­ graphic investigations concerned with neuroglia in the central nervous system and apply some of the principles to studies of neuroglia in autonomic ganglia. By injecting thymidine-H^ into mice, Smart and

Leblond (1961) were able to tag some of the neuroglia. In addition to the labeling of neuroglia, the nuclear configuration of these cells led to the suggestion of amitosis as a mode of cell division. Altman

(1962), again using thymidine-^, reported degenerative and regen­ erative proliferation of neuroglia cells in response to lesions of the lateral geniculate body. Altman (1963) also demonstrated uptake of thymidine-H-j by glia cells in a normal cat brain. Most of the labeled cells, however, were ependymal cells or those cells located bilaterally along the midline. After crushing the hypoglossal nerve,

Sjostrand (1965) observed a metabolic synthesis of DMA in astrocytes in the hypoglossal nucleus during regeneration of the nerve._ The labeling substance was also tritiated thymidine.

From the foregoing discussion, it is apparent that neuroglia cells play a considerably more dynamic functional role in the nervous system than was originally perceived by early investigators.

Although the physical supporting theory of neuroglia function has 17 not been discarded, i t is looked upon presently by many as a passive or secondary activity of glia cells. The present trend of scientific investigations represents neuroglia as individual cellular elements related directly or indirectly to the metabolic activity of neurons.

A certain group of these cells may even be involved in synaptic transmission of an impulse. Ihe author hopes that this investigation will present a greater insight into the functional significance of neuroglia, specifically in autonomic ganglia. This information in turn may be valuable to neurologists, neurophysiologists, pathologists and other investigators interested in the study of the nervous system. METHODS

A. General operative procedures

The superior cervical sympathetic ganglia of 12 healthy adult cats (Felis domesticus) were utilized in the experimental procedures.

Each animal was anesthetized intraperitoneally with Diabutal, 30 milligrams per kilogram of body weight. The cervical sympathetic trunk, lying within the carotid sheath, was then surgically exposed and isolated from the vagus nerve and carotid artery. Ihe contra­ lateral trunk was maintained as a control. Paired electrodes were applied to the very lightly myelinated preganglionic fibers of the exposed sympathetic trunk. A sine wave electric current of 0.01 volts at a frequency of 10 pulses per second for a duration of 10 m illi­ seconds was administered continuously for a period not exceeding

3 hours. Adequate stimulation was indicated by complete retraction of the nictitating membrane and dilation of the pupil on the stimulated side of the animal.

In addition to preganglionic stimulation of the cervical sympathetic trunk, some of the animals were subjected to injection of certain drugs known to affect synaptic transmission in autonomic ganglia. Neostigmine was chosen for its activity at the synaptic junction. This drug was administered intramuscularly to three of the experimental animals at the onset of electrical stimulation and every

18 19

^5 minutes thereafter. Ihe dosage per injection was 1 ml of a 1 per

cent aqueous solution. Atropine sulfate was also selected for its

specific activity at sites of synaptic transmission. Atropine is

antagonistic to neostigmine at synaptic sites. At the onset of

stimulation, and at every ^5 minute interval thereafter, 1 ml of a

1:1000 solution was injected intramuscularly into four of the experi­ mental animals. Four of the remaining five animals received only

electrical stimulation. The fifth animal was maintained for control purposes. It was exposed to all the traumatic effects of the opera­

tion, with the exception of drug administration and the applied

electric current.

Ihe main objective of this study is centered around IMA metabolism by neuroglia elements in the superior cervical ganglion.

It is now accepted that thymidine is a specific building block for

ENA, and that the DNA molecule is stable after formation under

normal circumstances and environmental conditions. For these reasons,

thymidine, which i s composed o f the pyrim idine base thymine and a pentose sugar, was used as a precursor of nuclear DNA. Tritium (H3)

coupled to the No. 6 position of the thymidine molecule was employed

as the labeling substance. Tritium, one of the most commonly used

isotopes in autoradiography, emits a low energy Beta particle (18 KSV maximum energy), lowest of any known isotope. The specific activity

of tritiated thymidine is greater than 10,000 millicuries per milli­ mole. Considering that the microscopic resolution of autoradiograms

is inversely proportional to the energy of the Beta particle emission, 20 tritium is, therefore, capable of producing excellent resolution and sensitivity. Tritiated thymidine^ was obtained in two millicurie quantities, one millicurie being equal to 1 ml of solution. Each animal was injected intravenously with 0.5 millicuries of tritiated thymidine stock solution diluted to 2 ml with double distilled water.

The stimulation period varied for each individual operation so that metabolic changes might be observed in sequence. Stimulation of the sympathetic trunk was terminated for the four non-injected animals at the end of 30» 120, 150 and 180 minutes respectively.

The period of stimulation for those animals administered neostigmine was 105, 120 and 180 minutes, while each atropine injected animal received the maximal 3-hour stimulation.

The superior cervical ganglia were removed bilaterally from each animal immediately after the stimulation period, and placed in

10 per cent formalin fixative for 24 hours. Fixatives containing metal ions were avoided due to their reactive capability with silver halide grains of the nuclear tract emulsion to be applied sub­ sequently. Tissues were then dehydrated in ascending concentrations of ethyl alcohols, cleared in xylol, and imbedded in paraffin.

Sections were cut at 7 raicra and mounted on glass sides from a d is tille d water g e latin media, and placed on a warming ta b le over­ night. The procedures followed hereafter are outlined below.

^ Thymidine (nominally 6-T) sterile aqueous solution TRK-61 was obtained from Nuclear-Chicago Corporation, 333 East Howard Avenue, Des Plaines, Illinois. 21

B. Autoradiographic techniques

1. Solutions required

a. Kodak nuclear track emulsion NTB2

b . Kodak D19 developer

c. Developer stop bath (glacial acetic acid)

d. Acid hardening fixing bath

2. Procedure

a. Nuclear track emulsion NTB2 is removed from the refrigerator approximately one hour before beginning the dipping technique suggested by Joftes (1963), and referred to throughout this study for processing of autoradiograms.

b. Sections are deparaffinized, rehydrated through decreasing grades of alcohols to distilled water, and then trans­ ferred to tap water.

c. With the exception of a safelight, all procedures from this point were performed in darkness.

d. Nuclear track emulsion is removed from its storage boxes; placed in a water bath previously heated to ifO°C.; and warmed to 38°G. The liquid emulsion is stirred periodically, but very gently to prevent air bubble formation in the emulsion.

e. When emulsion reaches 38°C., the slides are removed from water (five slides at a time is recommended), dipped individually in the emulsion, and removed rapidly. Excess emulsion

%odak nuclear track emulsion NTB2 was obtained from Eastman Kodak Company, 3^3 State Street, Rochester, New York. 22 is not allowed to drain back into the emulsion container, and the

slides are never returned to the emulsion.

f. Slides are then held on end; tapped lightly on a

gauze pad; placed on end in a wooden slide holder; and allowed to drain fo r JO seconds. The slide surface opposite to which the tissue

is affixed is cleaned thoroughly with a gauze pad.

g. An evenly distributed emulsion coat may be seen when slides have drained sufficiently. They are then removed from

the rack and placed on a smooth, level, dry surface and allowed to harden for 15 minutes. At this time, the emulsion remaining in its

container should be covered; returned to its storage boxes; and re frig e ra te d a t 2°G.

h. Coated slides are then placed in a small plastic

slide box (maximum 25 slides per box) and covered. The box with

slides is subsequently placed in a desiccator containing Brierite

(anhydrous CaSOij.).

i. The desiccator is sealed with clear grease and black masking tape around its edges.

j. The lights now may be turned on. The desiccator is placed in a refrigerator at approximately 5°G. Exposure of radioactive material to the nuclear track emulsion at room tempera­

tures is perfectly acceptable; however, better results are obtained at lower temperatures in this particular procedure. 23

k. The ideal exposure time was found to be 10 days; thus, the coated slides remained in the refrigerator for this period of time before further processing,

C. Autoradiographic development technique

The slides are removed from the desiccator in the darkroom with the safelight on, and immersed in the following solutions pre­ viously cooled to 18°C,

1. Kodak D19 developer, b to 5 minutes: This solution reduces

.the exposed silver halide grains to silver granules which appear black in the emulsion over the tissue section. Slides allowed to remain in developer too long will exhibit artifactual background by developing unstable silver halide grains not actually altered by the radio­ isotope, Underdevelopment also provides undesirable results,

2. Glacial acetic acid stop bath, 10 seconds: This solution inhibits further activity of the D19 developer solution. The concen­ tration of glacial acetic acid was a 28 per cent stock solution diluted further to 44 cc per liter of water,

3. Acid hardening fixing bath for at least 4 minutes: Joftes* original method calls for a time equal to twice the clearing time; however, a time slightly in excess of this will not be detrimental to the sections, and will assure removal of all undeveloped silver grains from the emulsion.

Running tap water, 1 hour: At this time the emulsion on the slides consists of developed silver grains in a hardened gelatin medium. 24

D. Staining procedure

1. This process is necessary to localize the exact sites of radioactive tracer in the tissue underlying the emulsion. A dilute aqueous toluidine blue staining solution was selected since it readily penetrates the emulsion coat with minimal absorption by the gelatin component of the emulsion. This stain is also excellent for nuclear detail and nervous tissue in general.

2. The slides are removed from water and immersed in the following solutions.

a. Freshly filtered 0.5 per cent aqueous toluidine blue staining solution, 1 to 2 minutes: Slides may be dipped in distilled water occasionally in order to observe staining density.

b. Transfer to 70 per cent ethanol for destaining and differentiation, 2 minutes.

c. Dehydrate in two changes of n-butanol, 3 minutes for each change.

d. Clear sections in xylol, 2 changes, 10 minutes each.

e. Mount with p icco ly te.

3. Completed autoradiograms are now ready for histological examination and preparation of photomicrographs.

E. Histochemistry

The Feulgen-nucleal reaction was utilized in this investiga­ tion for the histochemical demonstration of nuclear DNA. Paraffin- imbedded tissue sections were obtained from stimulated and unstimulated 25 ganglia as previously described in Section A. After sections were deparaffinized and rehydrated, they were processed according to the method recommended by Barka and Anderson (19^3).

Histochemical techniques were also employed for the identi­ fication of certain enzyme activities present in various concen­ trations and locations in the superior cervical ganglion. Material was obtained from a healthy live adult cat and frozen immediately after removal at temperatures approaching -100°C. Cryostat sections of the unfixed ganglia were cut at 7 micra and mounted on thin cover slips. After sections had dried for at least 5 minutes, they were processed according to the following techniques for the identifica­ tion of cholinesterase, succinic dehydrogenase, and lactic dehydro­ genase activities. The acetylthiocholine iodide method, described by Barka and Anderson (1963) and originally suggested by Gerebtzoff

(1953)» was utilized for the localization of cholinesterase activity.

Incubation times and solutions prepared for the histochemical demon­ stration of succinic dehydrogenase activity are also described by

Barka and Anderson (1963) as modifications of the original method presented by Nachlas et al. (1957). Procedures and solutions for localization of lactic dehydrogenase activity were employed according to the method suggested by Pearse (i960).

F. Standard histological technique

A few ganglion tissue sections from the unstimulated control animal were handled according to the procedures outlined by Lapham 26

et al. (196*0. This method is performed with paraffin sections and

utilizes phyloxine B for myelin, fast green FCF for cell processes,

and gallocyanin for nuclear details. The sections were prepared to

illustrate the basic cellular components within the superior cervical

ganglion and to identify their shape and anatomical relationships

-prior to electrical stimulation and drug administration. OBSERVATIONS

Autoradiograms from this investigation m il be examined and described in three separate groups. Superior cervical ganglia of non-stimulated non-injected animals will be presented initially to establish the general pattern of radioisotope activity in a normal ganglion. Observations of autoradiograms from control sections will serve as a basis for comparison with the stimulated and unstimulated ganglia of the other two groups. The second group to be examined will be those ganglia removed from animals receiving only unilateral preganglionic electrical stimulation. The third group consists of ganglia removed from animals that were administered drugs, viz. neostigmine and atropine, in addition to receiving preganglionic stimulation. Autoradiograms and tissue sections were examined under the light microscope at magnifications of 500x and under o il immer­ sion at a magnification of 1300x .

A. Observations of control ganglia removed from unstimulated animals

Autoradiograms prepared from bilateral ganglia removed from the unstimulated control animal (Fig. 2), revealed no evidence of any labeled cell nuclei. The multipolar neurons within the superior cervical ganglion displayed an abundance of Nissl substance, which

27 28 was homogeneously dispersed throughout the cytoplasm. Nuclei of neurons were located near the center of the cell body. Supporting neuroglia cells exhibited round or slightly oval nuclei, and were identified by their location as either perineuronal or interstitial satellite cells. Some of the perineuronal satellite cells could be observed as primary capsular cells forming an incomplete layer of cells adjacent to the neurons. Other secondary satellite cells were positioned slightly outside the layer of primary cells and were related to the preganglionic or postganglionic nerve process directly associated with the neuron which they partially surrounded. Inter­ stitial satellite cells were arranged in a relatively even and randomly distributed pattern. Ganglion sections represented by

Figure 1 were prepared specifically to illustrate the above descrip­ tion of cellular morphology and anatomical relationships in the superior cervical ganglion. The nuclei of Schwann cells, forming a sheath for preganglionic sympathetic nerve processes joining the ganglion were confined to neuron processes. In contrast to neuro­ glia, the nuclei of Schwann cells appeared more dense, more oblong, and were arranged in uniform parallel patterns along the neuron processes. Nuclei of Schwann cells do not contain the dense centrally located chromatin granule that is usually prominent in the nuclei of neuroglia cells. If fibroblasts are present within the ganglia, they could not be recognized by the procedures followed in this investigation. Mast cells were present throughout the ganglia and 29 along the nerves joining the ganglia, and displayed evidence of cyto­ plasmic disintegration.

B. Observations of stimulated superior cervical ganglia

The examination of autoradiograms from the superior cervical ganglion stimulated for 30 minutes revealed a few labeled nuclei of neuroglia cells. The labeled cells contained at least three, but rarely more than three, black granules over their nuclei. Most of the radioactivity was localized within blood vessels and interstitial areas. The shape and arrangement of nuclei of neuroglia cells was similar to that of control ganglia previously described. Most neurons revealed no morphological indications of increased metabolic activity.

The nuclear position of the neurons was normal, and Nissl substance was homogeneously dispersed throughout the neuron cytoplasm. The unstimulated ganglion revealed no evidence of any labeled nuclei.

There was a definite sparsity of radioactivity in blood vessels and interstitial areas of the unstimulated ganglion.

A small increase in the quantity of labeled nuclei of neuroglia cells was noticed in the ganglion stimulated for 120 minutes. More contrast was observed in the degree of radioactivity in blood vessels and interstitial areas of the ganglion. Blood vessels in this ganglion were not as highly concentrated with radioactive precursor as vessels in the ganglion stimulated for 30 minutes;, in contrast, high concen­ trations of radioisotope was present in tissue fluid and cytoplasm of 30

the cellular elements of the ganglion stimulated for 120 minutes.

Some of the neurons showed the initial characteristics of chromato­

lysis, the peripheral migration of Nissl substance and peripherally

located nuclei. Structures within the unstimulated control ganglion

demonstrated characteristics similar to the ganglia of the control

animal described in Section A.

The greatest degree of neuroglial activity was observed in

autoradiograms prepared from the ganglion stimulated for 150 minutes.

Many nuclei of neuroglia cells were heavily labeled (Fig. 3)• Some

of the labeled cells were so densely covered with black granules that

it was difficult to determine the specific details of the nucleus or

its outline. Densely labeled nuclei appeared singly or in clusters

of two or three. The entire ganglion demonstrated a high degree of

labeling selectivity. Many heavily labeled nuclei or groups of

labeled nuclei were identified adjacent to or surrounded by neuro­

glia cells completely void of radioisotope.

Most labeled cells were either primary or secondary peri­

neuronal satellite cells (Fig. 3» and 5)* The remaining labeled

cells were interstitial satellite cells (Fig. 6). Most neurons

appeared to be in a high degree of metabolic activity as determined

by chromatolysis, a peripheral ring of Nissl substance, and peripheral

nucleus. The nuclei of some neurons appeared to touch the plasma membrane. Labeled perineuronal satellite cells appeared to be

associated normally with neurons having the characteristics of 31 increased metabolic activity (Fig. **• and 5). Labeled secondary cap­ sular cells were related to the processes directly adjacent to neurons exhibiting increased activity. The interstitial satellite cells that were labeled appeared randomly, and no pattern of cellular distribution could be identified. Many satellite cells did not display the round or slightly oval nuclear configuration presented by the nuclei of ganglia processed from the control animal. The atypical nuclei of neuroglia cells were distorted in such a fashion as to appear oblong, fusiform, and occasionally dumbell-shaped. A thin strand of nuclear material, varying in length and width, joined the two nodular ends of the dumbell-shaped nuclei. Atypical nuclei were not considered to be technique artifacts, since many normal nuclei were intermingled with the atypical nuclear configurations.

An occasional labeled cell or group of cells appeared in the un­ stimulated ganglion of this operation. Frequently the labeled cells were directly associated with a neuron, or the fibers immediately adjacent to the neuron. Mast cells were prominent, although quanti­ tatively less and displaying less evidence of cytoplasmic disinte­ gration than those in the stimulated ganglion. Morphological characteristics of neuronal activity was similar to the character­ istics displayed by neurons observed in unstimulated ganglia taken from the control animal. Ihe shape and distribution of neuroglia was similar to the neuroglia cells observed in sections of control ganglia. 32

Microscopic observations of autoradiograms from the ganglion

stimulated for 180 minutes were essentially identical to the ganglion

stimulated for 150 minutes. The only measurable difference was an

increase in the number of cell clusters and individually labeled

cells. Most of the labeled neuroglia cells displayed a greater

density of label over the nucleus. The appearance of respective

unstimulated ganglia from both animals was identical.

An observed increase in quantity of neuroglia cells in ganglia

stimulated for at least 150 minutes is consistent with previous

investigations performed in this laboratory (Schwyn and Hall, 1965).

The numerical difference in neuroglia cells was more evident when

the ganglion stimulated for 180 minutes was compared to the contra­ lateral control ganglion of the same animal. The only observable

indications of cellular proliferation was the appearance of un­ labeled dumbell-shaped nuclear configurations normally identifiable

in interstitial areas.

On the stimulated side of each animal in this section, the p u p il was maximally d ila te d , and the n ic tita tin g membrane was com­ pletely retracted from the external surface of the eye. The opposite pupil was almost completely constricted and the nictitating membrane was drawn incompletely over the surface of the eye. The dilator pupillae and nictitating membrane on the stimulated side remained

contracted, with very minute deviation, until the stimulation was

terminated. 33

C. Observations of stimulated ganglia removed from drug- injected animals

1. Ganglia exposed to neostigmine

Each neostigmine injected animal will be examined in

sequence relative to the stimulation period. The stimulated ganglion will be compared to the contralateral unstimulated ganglion. In

addition, both stimulated and unstimulated ganglia will be compared

to the other ganglia in this sequence and to the ganglia removed

from non-injected animals described in Sections A and B.

In the neostigmine exposed ganglion, stimulated for 105 minutes, many labeled nuclei were observed singly and in clusters ranging from

three to ten nuclei per cluster. Most ganglionic areas containing

cell clusters also displayed a more active environment surrounding

the clusters. Environmental activity was determined primarily by the density of radioisotope present. Other areas of the ganglion, free of cell clusters, were relatively void of radioactivity; however,

-singly labeled cells were quite prominent. Although the number of labeled cells in this ganglion was approximately 2 to ^ times greater

than the number observed in the stimulated ganglia not exposed to drugs, the density of radioactivity over the labeled nuclei was not. as heavy.

The neurons appeared to be very active — exhibiting chromato­ lysis, peripheral migration of Nissl substance, and eccentrically located nuclei. Neurons from this ganglion did not appear to be in the state of metabolic stress observed in the neurons of stimulated ganglia not influenced by drugs. Most of the nuclei of neuroglia cells within the ganglia were not the typical round or oval shape.

On the contrary, they displayed many different shapes and sizes in d ic a tiv e of c e ll movement, as reported by Berg and Kallen (1959).

A few labeled cells also were observed randomly distributed throughout the unstimulated ganglion. The neurons showed no evidence of in­ creased metabolic activity. Hie shape and distribution of neuroglia within the unstimulated ganglion were similar to the control ganglia described in Section A.

The ganglion stimulated for 120minutes showed a s lig h t in ­ crease in the number and density of labeled cells in comparison to the ganglion stimulated for 105minutes. Morphological and physio­ logical characteristics of neurons and neuroglia were relatively similar to the characteristics of cells observed in the ganglion stimulated for 105 minutes. An increase in the number of labeled cells and density of labeling over each nucleus was observed upon comparison of the two unstimulated ganglia. Neurons and neuroglia in the unstimulated ganglia have the appearance of cells in a typical control ganglion.

Labeled neuroglia cells observed from the ganglion stimulated for 180 minutes varied extensively in the density of labeling granules over the nucleus (Fig. 7)». Many cells were heavily labeled, some were moderately labeled, and others displayed only 35 minimum labeling. Cytoplasmic areas were highly concentrated with radioactivity. Many cell clusters were observed and seemed to be invested in a highly active environment. T he labeled cells in this ganglion outnumbered the labeled cells in the ganglion stimulated for 120 minutes by a ratio of 2:1. Ihe proportion of increase over labeled cells in the stimulated ganglia of non-injected animals is approximately 10-1 (compare Fig. 6 and 7). On the average, the concentration of radioactivity per labeled cell was not as dense.

Ihe unstimulated ganglion contained many lightly labeled cells, and was similar in appearance to the neostigmine exposed ganglion stim­ ulated for 120 minutes.

Ihe neurons in the three operations of this section did not display the high degree of metabolic stress that was prevalent in ganglia not exposed to drugs and stimulated for an identical period of time. Nuclei were moderately migrated toward the periphery of the neuron; chromatolysis was not as evident; and the peripheral ring of

NisaL substance was not as prominent. Many nuclei were observed in clusters and displayed various deviations from normal appearing n u clei.

Pupillary dilation was not as prominent with neostigmine on the stimulated side of the animal as was observed in the non-injected anim als. R etraction o f the n ic tita tin g membrane was comparatively the same in both groups of animals. It should be noted that the n ic tita tin g membrane receiv es only sympathetic adrenergic nerve 36 terminations (Ambache et a l.. 1956). The muscular components of the pupil are antagonistically innervated by cholinergic and adrenergic nerve endings. Differences in pupillary reactions may be explained on this basis and will be discussed more completely in the following chapter. The ptpil on the unstimulated side was slightly dilated and the n ic tita tin g membrane was m oderately re tra c te d .

2. Observations of stimulated gangLia exposed to atropine sulfate

The stimulated and contralateral unstimulated ganglia of those animals injected with atropine sulfate were very similar in appearance to each other, and to the control gangLia described in

Section A. Snail quantities of radioactive tracer were evident within the ganglia. Very rarely were any labeled nuclei observed.

When a labeled cell did appear, it was usually a perineuronal satel­ lite cell within a stimulated ganglion. The neurons in all ganglia appeared to be in a normal functional state, demonstrating nearly centrally positioned nuclei and evenly distributed Nissl substance.

Neuroglia were randomly and evenly distributed, and most nuclei were round or slightly oval shaped (Fig. 8).

During the three-hour stimulation period, abnormal dilation o f the p u p ils and re tra c tio n o f the n ic tita tin g membranes o f both eyes was observed. These reactions were considered to be the normal results of atropine influence at nerve terminations rather than a result of induced electrical stimulation. This effect could not 37 be altered regardless of the strength or duration of electrical

stimulation.

D. Histochemical observations of superior cervical ganglia

1. Feulgen-nucleal reaction for deoxyribose nucleic acid (DNA) (Figure 9)

Neurons in both stimulated and unstimulated ganglia have a very light pink colored nucleus, and are identical in appear­ ance in both ganglia. The nuclei of Schwann cells demonstrated dark reddish purple staining characteristics. The density and degree of

staining was relatively uniform in Schwann cells throughout both ganglia. The greatest degree of differentiation was observed in the nuclei of neuroglia cells. The staining characteristic of nuclei of neuroglia cells in the unstimulated ganglion was predominantly dark reddish purple. These nuclei could be distinguished by the typical round or oval shape, the same pattern observed from sections stained with to lu id in e b lu e. There were approximately the same number of dark reddish purple staining nuclei of neuroglia cells in the stim­ ulated ganglion. In addition, many lightly or moderately colored nuclei were observed in the stimulated ganglion (Fig. 9). The stimulated ganglion appeared to contain twice as many neuroglia nuclei as the unstimulated ganglion. Many nuclear clusters were also observed in the stimulated ganglion while the nuclear components of the clusters varied from light pink to dark purple in staining charac teristic s. 2. Acetylthiocholine iodide method for cholinester­ ase activity (Figure 10)

Sites of cholinesterase activity appear as yellowish brown deposits, and are observed in various concentrations throughout the ganglion. Nuclei of neurons present a very light yellow coloring indicating minimal cholinesterase activity. While the neuron nuclei do not differ from each other in staining density, the cytoplasm of each cell body varies from a very light coloring to dark yellow brown.

Ihis may be interpreted as an indication of various degrees of cholinesterase activity when the ganglion was removed. Many inter­ stitial areas, in which a nerve process can be identified adjacent to a neuron, are highly active. Higher magnification reveals that the cellular components responsible for these dark areas are nerve pro­ cesses, neuroglia cytoplasm, and the nuclei of neuroglia. Most all heavily colored areas, surrounding one neuron or a group of neurons, contain dark brown colored nuclei of neuroglia cells. Ihe prevailing components of lighter areas in the ganglion are nerve tracts. Ihe - nuclei of Schwann cells are relatively clear except in a few widely dispersed areas. The nuclei of neuroglia cells, when they appear in light areas, are normally lighter colored than when they appear in areas of high cholinesterase activity. The overall picture gives the appearance of high cholinesterase activity in areas that most likely would be involved in synaptic activity. Other areas of the ganglion display lesser degrees of enzyme activity. The nuclei of neuroglia cells normally exhibited higher enzyme activity than other components of the ganglion, and were darker colored when they were located in areas demonstrating high cytoplasmic cholinesterase a c tiv ity .

3. Nitro BT method for succinic dehydrogenase activity (Figure 11)

Sites of succinic dehydrogenase activity appear blue to purple, depending upon the degree of activity of the enzyme. Areas of highest activity are neuron cytoplasm. Many dense granules appeared along the periphery of nerve processes, while the central areas of the processes were relatively clear. Dense granules also appeared localized within the nerve cell body. Cytoplasm of neuro­ glia cells contained purple coloring indicative of moderate succinic dehydrogenase activity. Some neuroglia cells contain a few heavily concentrated granules in their cytoplasm, which are identical with those in the nerve cells. Hie nuclei of neuroglia cells were clear.

The nucleolus of each neuron was also clear; however, a weblike pattern of fine purple granules could be distinguished in the nuclei of neurons.

Nitro BT method for lactic dehydrogenase activity ( Figure 12)

Sites of lactic dehydrogenase activity appear bluish purple. The nuclei of neuroglia and neurons were absolutely void of any coloring. Nerve cell bodies and the periphery of nerve processes 1*0 show high concentrations of enzyme activity. Although all neuroglia cytoplasm contains some traces of lactic dehydrogenase activity, the contrast of coloring in many cells indicated varying degrees of enzyme activity. Ihe nuclei of perineuronal satellite cells give the appearance of vacuoles in the nerve cell body and their bluish purple cytoplasm seems to blend with the cytoplasm of the neuron. Two or three neuroglia cells cam be seen bordering pro­ cesses adjacent to a neuron. Ihe cytoplasmic coloring of neuroglia cells also seems to blend with the cytoplasm of nerve processes. DISCUSSION

Repetitive faradic stimulation of preganglionic nerve fibers terminating in the superior cervical ganglion has indirectly altered the activity of neuroglial elements within the stimulated ganglion.

This phenomenon was further modified and controlled by the injection of certain drugs known to affect transmission of a nervous impulse from presynaptic to postsynaptic structures. The degree of neuro­ glial activity also could be controlled by varying the duration of preganglionic stimulation.

The orientation of the nuclei of neurons and the concentration of Nissl substance in the stimulated ganglia were identical to the characteristics of metabolically disturbed neurons described by

Richins and Hall (1958). Since postganglionic neurons exhibiting characteristics of metabolic stress were not directly stimulated, it must be assumed that their altered metabolic activity is due to preganglionic stimulation. Even though neurons were observed in varying degrees of metabolic stress, gross observations of ocular structures innervated by superior cervical ganglion neurons indicated no deviation from normal functional efficiency under the stimulatory conditions.

In view of the stress conditions exhibited by ganglionic neurons from preganglionic electrical stimulation, it can be assumed that the metabolic activity and nutritional requirements of neurons

receiving impulses from preganglionic fibers are greatly increased.

Synaptic activity within the ganglion also is highly accelerated.

More specifically, when a nerve action potential of a preganglionic

neuron arrives at the termination of the axon, it stimulates release

of the chemical transmitter acetylcholine from synaptic vesicles

■within the axon. Acetylcholine diffuses across the narrow synaptic

cleft between cell membranes and reaches postganglionic receptor

sites located either on the dendrites or the cell body of the post­

ganglionic neurons. Ihe arrival of acetylcholine creates a localized

postsynaptic potential which initiates electrogenically a nerve

action potential. The impulse is then propagated along the post­

ganglionic fiber in the same manner as it passes along the pre­

ganglionic fib e r. R estoration of the postsynaptic membrane is

brought about by rapid destruction of acetylcholine by another enzyme,

acetylcholinesterase. It has been well established physiologically

and biochemically that ionic and chemical changes occurring during

the transmission, integration, and recovery thereafter of a single

impulse requires vast amounts of nutrients, oxygen, and energy, as

well as metabolic pathways for removal of waste products (Winton and

Bayliss, 1955). An attempt to calculate the metabolic turnover in

the ganglion during a continuous three-hour stimulation period is beyond the scope of this study. However, the greatly accelerated metabolic and synaptic activity created during stimulation procedures are considered to be the most significant factors initially responsible for altered satellite cell activity within the stimulated ganglia.

The neuroglial reaction to the environmental conditions within the stimulated ganglia was thymidine uptake from the blood and incorp­ oration of this compound into nuclear substance.

Thymidine is a specific precursor of DNA, and it is generally considered that synthesis of thymidine reflects some process of DNA turnover. When thymidine is readily available in vascular channels, a condition artificially produced by injection in this investigation, its uptake and incorporation into nuclear structure would equally represent a process of IMA turnover. Except during chromosomal replication, nuclear DNA is considered to be metabolically inert.

Therefore, autoradiographic demonstration of thymidine-H-j uptake by cell nuclei indicates that labeled cells either were preparing for cell division while the thymidine was available, or they are products o f previously lab eled c e lls (Taylor e t a l . . -1957; Hughes e t a l . . 1958;

Leblond et a l., 1959; and Altman, 1963).

Upon interpretation of autoradiograms prepared from ganglia, removed from stimulated non-injected animals, it was evident that the quantity of labeled satellite cells increased in direct proportion to the stimulation period. Since the neurons also displayed meta­ bolic disturbances in direct proportion to increased stimulation time, one could deduce that neuroglia are reacting to these conditions by synthesizing nuclear IMA. Comparison of results from non-drugged ganglia with results obtained from ganglia removed from animals ad­ ministered neostigmine, in addition to electrical stimulation, reveals an interesting question. Why does such a noticeable difference exist in the quantity of labeled satellite cells in the neostigmine exposed ganglion as opposed to the drug-free ganglion? An attempt to explain th is phenomenon must be considered with resp ect to the a ffe c t o f neostigmine upon impulse transmission in the superior cervical ganglion.

Acetylcholine is the only known chemical transmitter in autonomic gangLia and it is very rapidly deactivated by cholin­ esterase immediately after it has performed its function of depolar­ izing the postsynaptic membrane. It is the speed of this inactivation process which allows for rapid restoration of the postsynaptic membrane and enables the neuron to respond to the next stimulus in approximately one millisecond. Neostigmine antagonistically acts to break down cholinesterase. Consequently, acetylcholine is allowed to accumulate at synaptic areas, creating constant depolarization of postsynaptic membranes and increased metabolic activity of neurons involved. Neostigmine does not inhibit the synthesis of cholin­ esterase; therefore, the presence of large volumes of acetylcholine and constant d epolarization o f postsynaptic membranes would con­ tinuously stimulate production and release of cholinesterase from anatomical sites not yet specifically identified. Pappano and Voile

( 1962) have suggested that acetylcholine may cause depolarization of k5 both presynaptic and postsynaptic elements. According to their pro­ posal, the depolarization by acetylcholine of nerve terminals would re s u lt in a decrease in the amount o f tra n sm itter lib e ra te d by induced electrical stimulation, and consequently a decrease in the amplitude of the postganglionic action potential. Incomplete pupillary dilatation and less obvious neuronal chromatolysis ob­ served from neostigmine administered animals in this investigation lends support to this hypothesis. Conversely, the hypothesis may be utilized in an attempt to explain the observed effects of neo­ stigmine. Mason (1962a and 1962b) has presented evidence that neostigmine is capable of exerting a direct stimulating action on the ganglion cells as well as acting in an anti-cholinesterase capacity. This may partially account for the presence of labeled cell nuclei in unstimulated neostigmine exposed ganglia. Koelle

(1962) advanced the possibility that acetylcholine is continuously liberated in small quantities from nerve terminals in a resting state. If cholinesterase were inhibited from immediately hydrolyzing acetylcholine liberated in synaptic areas, it seems probable that sufficient quantities would build up over a period of time that would be capable of depolarizing postsynaptic membranes. Thus, the same conditions would e x is t in an unstim ulated ganglion with admin­ istration of neostigmine, varying only in the degree of intensity.

Under these conditions neuroglia would be expected to respond in the same capacity as they would in a stimulated ganglion, but delayed in 4 6

their response. The existence of many labeled neuroglia cells in the unstimulated neostigmine exposed ganglia is consistent with the apparent physiological conditions. It appears that the presence of neostigmine is either directly or indirectly responsible for the neuroglia reactions in unstimulated neostigmine exposed ganglia.

Attempts to explain the normal appearance of neurons and neuroglia in atropine-exposed ganglia are somewhat le s s complicated.

Atropine sulfate in autonomic ganglia is thought to compete with acetylcholine for postganglionic receptor sites (Lewis, 1960). With atropine blocking sites of impulse transmission mediated by acetyl­ choline there will be no postsynaptic depolarization, no impulse transmission, no increased metabolic activity of ganglion cells, and no effect of preganglionic stimulation relayed to pupillary structures of the eye. The activity of neuroglia would be expected to remain unchanged after preganglionic stimulation, if their response was to either increase synaptic transmission or increase neuronal activity, or both. Except for a few labeled neuroglia cells in the stimulated ganglia, the morphology and activity of neuroglia in atropine-exposed ganglia appeared to exhibit characteristics identical to normal neuroglia in the control ganglia. Perhaps the existence of a few labeled satellite cells in stimulated ganglia can be explained on the basis of the most recent evidence submitted by Roszkowski (1961).

He reported the existence of pharmacologically distinct cholino- ceptive sites on postsynaptic sites. He also observed that one of these sites could be blocked by atropine; the other site was not *7 affected. Takeshige et al. (1963) further supported the existence of two cholinoceptive sites and suggested the possibility of a third distinct site. It may be possible that there is sufficient synaptic activity induced at specific loci to stimulate a neuroglial response.

The quantity of atropine immune receptor sites is apparently in­ sufficient to create abnormal metabolic stress to ganglion cells, or to perpetuate an impulse capable of producing effective movement of structures supplied by postganglionic nerve fibers.

In order to create a working hypothesis related to the func­ tional significance of labeled neuroglia cells, it is necessary to consider the biochemical properties and anatomical relationships of these cells. Evidence has been established in support of neuroglia increasing their Hi-IA component in response to either highly increased neuronal activity, or greatly increased synaptic activity within the ganglion, or to both. Results of previous investigations performed in th is lab o rato ry (Schwyn and H all, 1965) and observations o f neuroglia cells in both stimulated and unstimulated ganglia indicate that neuroglia have increased in number from 50 to 100 per cent over a three-hour preganglionic stimulation period. It seems reasonable to presume that labeled cells observed in autoradiograms are the same c e lls responsible for the increased number when q u a n tita tiv e calculations are performed.

Nuclear configurations suggesting mitotic division have not been observed under the conditions of this or similar previous k8 investigations in our laboratory. If cellular proliferation is by mitotic mechanisms, one would expect to see mitotic figures in various phases of division. Prophases and telophases should at least be fairly prominent since this type of cell division requires a considerable amount of time for completion. This pattern was not observed in any histological sections, which coincides with results observed of neuroglia in tissue culture by Wolfgram and Rose (1957).

The probability of inconspicuous or unidentifiable mitotic figures is very unlikely; therefore, it became necessary to explore the likelihood of more feasible proliferative mechanisms.

Amitosis or endom itosis seemed to be the most lo g ic a l mech­ anisms to consider, since dumbell-shaped nuclear configurations could be identified in many of the stimulated ganglia. Kuntz and Sulkin

(19^7b) reported amitotic proliferation of interstitial and capsular glia cells in autonomic ganglia after 72 hours of preganglionic electrical stimulation. Many of the newly formed cells, that were products of amitotic division, were devoid of processes and encroached upon ganglion c e lls . The reactio n of g lia c e lls , however, was con­ sidered to be in response to neurons exhibiting pathological char­ acteristics. In contrast, neurons and their innervated structures did not appear to be in a pathological state of degeneration during the short three-hour maximal stimulation administered in this study.

If neuroglia are actively participating in the metabolic support of neurons or in the release of certain substances involved in the impulse transmission cycle, the demand placed upon these cells would be immense during three hours o f continuous stim ulation. The dan and

■ay be so great that neuroglia supporting cells would be stimulated to increase their number in order to meet the increased requirements.

Although very little is known about amitosis, it apparently does not involve the cytoplasm to any large extent. With this type of prolif­ eration there would be minimal disturbance of the physiological activity of the cell and relatively no interruption of its function.

Lapham (1961) suggests the following information after observing amitotic division of astrocytes in the central nervous system.

If the reaction is chiefly a functional response of the cell rather than a response to form new tissue, it is not surprising that a mechanism of nuclear division should be evolved which would minimally disrupt the activity of a cell. Direct nuclear division would thus provide for in­ creased nuclear surface and enhanced transfer of chemical substances from nucleus to cytoplasm in a hypertrophied cell, rather than being related to cell replication.

Whether the newly formed cells observed in this study were devoid of processes, as reported by Kuntz and Sulkin (19^7b), could not be deter­ mined from the staining techniques employed in this study. Exact identification of these cells coordinated with processing of auto­ radiograms awaits further development and refinement of histological techniques.

Experimental data, compiled from this investigation and from the works of previous investigators, tends to support the hypothesis that labeled neuroglia cells are directly related to accelerated synaptic activity. Electron microscopic studies of Elfvin (1963), 50

Cravioto and Merker (19&3), and others have provided us with specific

information regarding the exact anatomical relationship of neuroglia to neurons and their processes. From these studies, it has been

established that every neuron and its processes are covered by a

sheath or capsule of satellite cells except at the synaptic point

between cell membranes. The extracellular space, ranging from 150 to

200 A0, is bound laterally by satellite cells which have a very ir­

regular boundary. A few synaptic contacts have been found to exist

between preganglionic fibers and ganglion cell bodies. The most

abundant type of contact in autonomic ganglia, however, is between

preganglionic fibers and ganglion cell dendrites, which extend only

a short distance from the cell body. This type of contact is char­

acterized by preganglionic fibers running parallel to or winding

around the dendrite to establish contacts at several different points.

Both types (axosomatic and axodendritic) are primarily ensheathed by primary or secondary perineuronal satellite cells. Most labeled

neuroglia cells, observed from autoradiograms of stimulated ganglia,

were either primary or secondary perineuronal satellite cells, and

could be identified normally in relation to highly active neurons.

The few labeled cells observed in stimulated atropine-exposed ganglia

and in unstimulated ganglia also could be identified as perineuronal

satellite cells. Furthermore, it was noted that labeled cells were located adjacent to processes that were directly related to meta- bolically active neurons. Thus, the prevailing direct relationships 51 existing within stimulated ganglia consisted of a stimulated neuron, the process that would most likely make contact with its cell body or dendrites, and a single or cluster of labeled perineuronal satel­ lite cells.

Histochemical observations of cholinesterase activity in the superior cervical ganglion are consistent with data presented by

Koelle (1950) and Fredricsson and Sjoqvist (1962), and lend further support to a direct relationship of neuroglia to ganglionic synaptic activity. According to Koelle (1950), high concentrations of specific cholinesterase were demonstrated in nuclei of many capsular glia cells and to a lesser extent their cytoplasm. Moderate concen­ trations were found in the nuclei of many interstitial neuroglia cells. Most neurons also showed degrees of specific cholinesterase in the cytoplasm and nuclei. Non-specific cholinesterase was local­ ized in the nucleus of glia cells and ganglion cells. In protoplasmic tracts, the concentration of non-specific cholinesterase was confined largely to the nuclei. Low magnification observations of histo­ chemical tissue sections prepared in this investigation revealed many areas of high cholinesterase activity immediately surrounding neurons and areas of low concentration where the likelihood of synaptic contacts was greatly diminished. The nuclei of neuroglia and their cytoplasm appear more saturated in the highly concentrated areas of the ganglion. The evidence is meager that non-specific cholin­ esterase is able to hydrolyze acetylcholine; however, it is a 52 relatively simple biochemical process to convert this compound to specific cholinesterase. It may be functionally significant in its own state since Heyman's et al. (19^) found that inactivation of non-specific cholinesterase sensitizes an organism to the effect of small doses of acetylcholine. Koelle (1950) also suggested that at certain sites the two enzymes, viz. specific and non-specific cholinesterase, may complement each other, inasmuch as specific cholinesterase functions at relatively low substrate concentrations and non-specific cholinesterase functions optimally at higher sub­ strate concentrations. It could be that the selectivity of thymidine uptake by perineuronal satellite cells is indicative of neuroglia cell proliferation directly or indirectly involved in the elaboration of acetylcholinesterase at synaptic sites within autonomic ganglia.

Perhaps labeled interstitial satellite cells represent cellular recruitment in an attempt to meet increased demands of perineuronal cells directly involved in the process. This seems to be true especially if the ganglion is exposed to the actions of neostigmine.

Intracellular synthesis of specific and non-specific cholinesterase ‘ and their precursors must also be considered as an integral part of this process even though the final product emitted from the cell is specific cholinesterase. Initiation of the entire process could be attributed to a combination of increased acetylcholine concentrations and depolarization of postganglionic membranes.

The other possibility to explore is that neuroglia are reacting to increased nutritional requirements of neurons. Most labeled 53 neuroglia cells were related to neurons exhibiting characteristics of increased metabolic stress. Most all neurons within ganglia stimulated for the maximal three-hour period shoxred signs of metabolic dis­ turbances. Metabolites entering and leaving the ganglion cells are thought to pass through glia cytoplasm. The activity of neuroglia would be expected to increase if they -were directly involved in relaying metabolites from capillaries to neurons. When the metabolic requirements of a neuron reach a certain critical point, neuroglia mijght increase in number in an attempt to fu lfill the demands. Mast cells observed in moderate numbers in the superior cervical ganglion, and appearing in slightly increased quantities in stimulated ganglia, may express their functional significance by releasing histamine.

This substance is an intense peripheral vascular dilator and would allow fo r increased movement o f m etabolites throughout the ganglion.

Histochemical procedures in this study-have revealed neuroglia cytoplasm to contain both succinic dehydrogenase and lactic dehydro­ genase activity. The functional significance of succinic dehydro­ genase is related to the tricarboxylic acid cycle where it catalyzes the release of two hydrogen atoms from succinate. The released hydrogen atoms w ill then pass to cellular areas concerned with the production of adenosine tri-phosphate, which in turn is an essential source of energy for a cell. Lactic dehydrogenase acts upon the two end products of the tricarboxylic acid cycle, pyruvic acid and hydrogen atoms. This enzyme catalyzes the reduction of pyruvic acid 54 to lactic acid, which may be reconverted to glucose in the liver. One might expect the neuron cytoplasm to contain large concentrations of both enzymes, since transmission and integration of a nervous impulse requires vast amounts of energy. NeurogLia, in comparison, are moderately rich in both enzymes. Although this may be highly spec­ ulative, the possibility exists that neuroglia may be a storage depot or a source of energy for the neurons. It is well known that neurons have a high energy requirement but are incapable of storing glycogen for utilization in meeting abnormal demands. This function may be delegated to the supporting neuroglia cells. Another explanation for the abundance of both enzymes in glia cytoplasm is that transfer of m etabolites from c a p illa rie s through c e ll membranes and through cellular cytoplasm requires certain amounts of energy. This view is supported further by a recent investigation by Hyden (1961). He reported that nerve cells are rich in ATP while glia are relatively poor in ATP. G lia, however, are ric h in ATPase, an enzyme which releases the energy in ATP, while the neuron lacks ATPase on the surface membrane. He fu rth e r suggests th a t an active ATPase e x ists that is able to release energy for the transport of nutrients from blood capillaries to the surface of the neuron and to transport a wide variety of substances across the membrane.

Although the preceding suggestions are in need of further investigation and may be disproved in the future, they present at this time a working hypothesis compiled from pertinent available 55 knowledge of neuroglia in the superior cervical ganglion of cats.

There is definite evidence that neuroglia in the ganglion respond to increased metabolic and synaptic activity by proliferating. The mode of cellular division is something other than normal mitosis; and in all probability it is either endomitosis or amitosis. The neuro­ glial reaction can be controlled by the use of various drugs and variation of the preganglionic stimulation period. This study has been performed on the cat superior cervical ganglion; however, there is no reason to believe that the principles deducted from this data cannot be applied to other autonomic ganglia in which the environ­ mental conditions, cellular elements, and chemical transmitter are similar. As far as the evolution of biological systems is con­ cerned, the autonomic nervous system is one of the oldest. Perhaps the knowledge compiled on the supporting cells within the autonomic nervous system can be applied with or without modifications to more newly developed and more complex portions of the nervous system, whose biochemical properties are still in its infancy of exploration. CONCLUSIONS

1. Preganglionic electrical stimulation of nerve fibers terminating in the superior cervical ganglion altered the metabolic activity of the ganglion cells.

2. Preganglionic electrical stimulation of nerve fibers terminating in the superior cervical ganglion accelerated synaptic activity within the ganglion.

3. Neuroglia supporting cells within the stimulated ganglion responded to increased neuronal and synaptic activity by incorporating pre-injected thymidine-H-j into their nuclear structure.

k . Neuroglia cells demonstrating thymidine-H^ in their nuclei were considered to be in the process of cell division, or the product of a divided cell.

5* Ihe influence of neostigmine in the stimulated ganglion increased the quantity of neuroglia cells responding to the environ­ mental conditions and reduced the time interval in which the response is first observed.

6. In all probability, proliferation of neuroglia cells under the conditions of this investigation was endomitosis or amitosis and was very rap id .

7. Atropine injected during electrical stimulation blocked the effect of preganglionic stimulation on postganglionic neurons and their terminal organs.

56 57

8* Neuroglia within the stimulated atropine-expo sed ganglia appeared normal, with the exception of a few labeled cells located at presumed atropine immune receptor sites.

9* Neuroglia cytoplasm contained an abundance of succinic and lactic dehydrogenase activity, which was not as abundant as that con­ tained in neuron cytoplasm.

10. Neuroglia nuclei, as well as their cytoplasm, exhibited high cholinesterase activity.

11. The superior cervical ganglion demonstrated areas of high and low cholinesterase activity, with the highest concentrations of cholinesterase activity tending to be localized in presumed synaptic areas.

12. Most neuroglia cells demonstrating thymidine-H^ uptake were perineuronal satellite cells located at logical synaptic areas.

13. Interstitial satellite cells demonstrating thymidine-H-j uptake revealed no indication of strategic positioning.

1*K Neuroglia were apparently capable of reacting to increased concentrations of acetylcholine.

15. Ganglion cells, after a three-hour stimulation period, exhibited normal metabolic disturbances under the experimental con­ ditions; but they were not in a state of pathological degeneration.

16. The conduction capabilities of ganglionic neurons after three hours of electrical stimulation were not noticeably diminished. SUMMARY

This study was undertaken to observe and correlate the re­ sponses of neuroglia in .autonomic ganglia to increased neuronal and synaptic activity. By electrically stimulating preganglionic nerve fibers terminating in the superior cervical sympathetic ganglion in

12 adult cats, and concurrently injecting thymidine-H3 into the stimulated animal, it was possible to alter the neuronal and synap­ tic activity within the ganglion, and subsequently observe the neuroglial response from autoradiograms. The specific reaction encountered was incorporation of thymidine-H^ into neuroglia nuclei.

Absorption of thymidine by neuroglia cells and incorporation of this compound into nuclear structure could be accelerated or inhibited by exposing the organisn to specific drugs during the stimulatory period. By means of histochemical identification of DNA, cholin­ esterase, succinic dehydrogenase, and lactic dehydrogenase activities, it was possible to correlate nuclear changes and enzymatic activities within the experimental .ganglion to the absorption of labeled DNA precu rso r.

The results of this investigation combined with information gathered from studies in many other scientific areas implicate neuroglia in the active support of neurons and in the production or release of acetylcholinesterase at sites of synaptic transmission. 59

Neuroglia apparently proliferate amitotically to cope with the demands of increased ganglionic activity elicited by the experimental pro­

cedures employed in this study. Although specific reactions of neuroglia, other than cellular proliferation, were not identified in

this study, their probability may provide a basis for future study.

The results obtained may serve to broaden the scope of neuroglia

significance in autonomic ganglia and to provide a more transparent perspective of their functional activities. APPENDIX PLATE I

Figure 1. A normal superior cervical ganglion showing typical shape and location of (a) primary perineuronal satellite cells, (b) secondary perineuronal satellite cells, (c) interstitial satellite cells, and (d) normal appearing neurons. Fast green FCF and gallo- cyanin. X500.

Figure 2. A control superior cervical ganglion showing normal morphological appearance of the neuron with slightly eccentric nucleus and homogeneously distributed Nissl substance. Capsular neuroglia cells (n) surround the nerve cell body. Mast cells (m) are also p resen t in th is sectio n . Toluidine blue. X1300.

Figure 3. An autoradiogram counter stained with toluidine blue. This section is a representative of a ganglion stimulated for 150 minutes. Note the relationship of labeled neuroglia cells (n) to neuron processes (p). X1300.

Figure 4. An autoradiogram counterstained with toluidine blue. This section is a representative of a ganglion stimulated for 150 minutes. Note the relationship of labeled neurogLia cells (n) to neuron processes (p) . X1300.

61 62 PLATE I I

Figure 5. Labeled secondary perineuronal satellite cells (s) related to synaptic area (a) and to the stimulated neuron (n). Tissues were counterstained with toluidine blue. X1300.

Figure 6. Labeled interstitial satellite cells (arrow) from a ganglion stim ulated fo r 150 m inutes. X1300.

Figure 7. Labeled neuroglia cells (arrow) from a stimulated neostigmine exposed ganglion counterstained with toluidine blue. Note the quantity of labeled neuroglia cells and the density oflabel. Compare with Figures 5 and 6. X1300.

Figure 8. Section from a stimulated atropine-exposed superior cervical ganglion. Note the normal appearance of neurons and neuro­ g lia c e lls . Compare with Figures 6 and 7» X1300.

63 > PLATE I I I

Figure 9. Ganglion section stained by the Feulgen-nudear reaction for nuclear ENA. Note (1) the location of neuron cell bodies (n), (2) completeness of neuroglial capsule (c) surrounding many of the neuron cell bodies, and (3) differences in staining density of neuroglia cells. Compare nucleus (a) with nucleus (b). X1300.

Figure 10. Sites of cholinesterase activity in the superior cervical ganglion. Satellite cell nucleus (sn), synaptic area (sa), and neuron cell body (cb). X1300.

Figure 11. Sites of succinic dehydrogenase activity in the superior cervical ganglion: (a) neuron, (b) nerve cell process, and (c) s a te llite c e ll. X1300.

Figure 12. Sites of lactic dehydrogenase activity in the superior cervical ganglion: (a) neuron, (b) nucleus of primary perineuronal satellite cell, (c) cytoplasm of primary perineuronal satellite cell, (d) secondary perineuronal satellite cell. X1300.

65

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