A HISTOCHEMICAL STUDY OF THE EiSTRIBUTION
OF OXIDATSON AND HYDROLYfiC ENZYMES IN
THE BRAlfi OF THE ADUL? PER-GMYSCES POUQNDTUS

Thesis for the Degree of M. S.
Mil-MEAN STATE UNIVERSITY
”GO-N A. HAY
1987

 

  
 
    

 
 

LIBRARY

Michigan State
University

 

ABSTRACT

A HISTOCHEMICAL STUDY OF THE DISTRIBUTION
OF OXIDATIVE AND HYDROLYTIC ENZYMES IN THE
BRAIN OF THE ADULT PEROMYSCUS POLIONOTUS

by Don A. Hay

Four brain centers of wild mice (Peromyscus polionotus)

were studied in detail by the use of histochemical procedures,

1.9., the cerebral cortex, cerebellar cortex, superior col-
liculus, and the medulla oblongata. Seven NAB-linked dehy-
drogenases (a-glycerophosphate, isocitrate, glucose96-
phosphate, lactate, malate, alcohol, and glutamate dehydro-
genases) and the carboxylic esterases were localized within
these areas. The presence of enzymes (dehydrogenases) in
regions of the brain or in individual cells of the brain
indicated that either the glycolytic, citric acid, or per-
haps the pentose pathway were utilized for respiration.
Upon removal from the cranium, some specimens of brain
tissue were immediately transferred to tissue holders and
frozen by quenching in acetone and solid-002. Others were
pre-fixed for 2h hours in cold formalin prior to freezing.
This was necessary for esterase identification. Sagittal
sections, eight to twelve microns in thickness, were prepared
, with a cryostat, and incubated in solutions containing either

the monotetrazolium salt MTT, or the ditetrazolium salt

. Don A. Hay
Nitro-BT. The former salt was used according to the pro?
cedures of Chason and Pearse (1961) and Thomas and Pearse
-,(196l). The incubating solution containing Nitro-BT was
prepared by the method of Hess, Scarpelli, and PearSe (1958).
Only alcohol and glutamate dehydrogenases required Nitro-BT.
Both the d-naphthyl acetate method (Pearse, 1960) and the
indoxyl acetate method (Holt and Withers, 1952; Holt, 1958).
for esterase identification were used. After incubation in
air at 37°C, the sections were fixed in cold formalin,
mounted in glycerine with or without a counterstain.

Enzyme distributions were studied with the light micro-
scope, and with phase contrast. The level of enzyme inten;
sity was evaluated on a subjective basis, with zero (0)
representing no activity; one (l),weak; two (2), moderate;
three (3), strong; and four (4), intense activity.

Enzyme activity (both oxidative and hydrolytic) was
uniformly distributed in the cerebral cortex. However, the
pyramidal cells in layers III and V exhibited slightly more
activity. . .' T ' '

The greatest oxidative enzyme activity in the cerebellar
cortex was confined to the glomeruli cerebellosi. The
abundant synapses of mossy and climbing fibers located in
the glomeruli may be responsible for the high activity.
Hydrolytic enzymes were most active within Purkinje cells.

Enzyme activity in the superior colliculi varied from
layer to layer, but was most intense in the stratum griseum

superficiale. The inferior colliculi did not exhibit

Don A. Hay
differential staining according to layers. Rather, meta-
bolic activity was concentrated in,a spherical regional
nucleus. The stratified enzyme distribution found in the
superior colliculi is thought to indicate a greater comp
plexity in function.

Regional nuclei within the medulla oblongata exhibited
all ranges of dehydrogenase activity. In some nuclei the
neurOpil was most active, whereas in others the neurons .
were. White fiber tracts remained non-reactive.

Very intense hydrolytic activity along capillaries
was localized within pericytes (perivascular cells). Capil-
lary endothelium contained moderate amounts of esterase
activity. Oxidative enzyme activity could not be accurately
assessed, as the capillaries and pericytes were almost
imperceptible.

The caudate nucleus of the basal ganglia exhibited
tintense esterase activity, but only moderate levels of
dehydrogenase activity identical to that in surrounding
tissues. .

Tissue sections incubated in Nitro-BT solutions lack-
ing substrates were strongly positive. The distribution
and intensity of the formazan deposits produced by alcohol
and glutamate dehydrogenases were similar to those found in
the control sections. Thus, enzyme localizations observed
within brain sections must be considered inaccurate and

possibly false.

Don A- Hay
LITERATURE CITED IN ABSTRACT '

Chason, J.L., and A.G.E. Pearse. 1961. Phenazine methosul-
phate and nicotinamide in the histochemical demonstra-
tion of dehydrogenases in rat brain. J. Neurochem.,

6: 259-266.

Hess, R., D.G. Scarpelli, and A.G.E. Pearse. 1958. The
cytochemical localization of oxidative enzymes. II.
Pyridine nucleotide-linked dehydrogenases. J. Bio-
phys. Biochem. Cytol., h: 753- 760.

Holt, pS. J. 1958. Indigogenic staining methods fix'esterases,
.375-398. In "General Cytochemical Methods", Ed.
J. F. Danieilli, Vol. I.

Holt, S.J., and R.F.J. Withers. 1952. Cytochemical locali-
zation of esterases using indoxyl derivatives. Nature,
170: 1012-1014.

Pearse, A.G.E. 1960. Histochemistry theoretical and
ap lied. 2nd ed. J. and A. Churchill, Ltd., London.

99

Thomas, E., and A. G. E. Pearse. 1961. The fine localization
of dggydgogenases in the nervous system. Histochemie,

A HISTOCHEMICAL STUDY OF THE DISTRIBUTION
OF OXIDATIVE AND HYDROLYTIC ENZYMES IN THE
BRAIN OF THE ADULT PEROMYSCUS POLIONOTUS

BY

Don A. Hay

A THESIS

Submitted to
Nuchigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

.Department of Zoology

' 1967

_ giaOp
bis/951.07

ACKNOWLEDGMENTS

I am grateful to many individuals for their cooperation
during the course of this study and would like to make
specific mention of them.

My sincere appreciation is extended to Dr. John A.
King, both for his advice and for providing the mice used in
this study, which were from a colony operated under his
direction.

’I wiSh to thank Mrs. Bernadette M. Henderson for all of
the assistance and encouragement She gave me time and time
again. .

were it not for the knowledge and efforts of two fellow
students who aided me in preparing the photomicrographs, I .
would still be loading the camera. I am especially in-
debted to Dan Williams and Donald Schultz for the long hours
they spent in helping me.

I am grateful to Dr. W. D. Collings and Dr. Ralph W. Pax
of Michigan State University for their helpful criticisms.

Special gratitude is due Dr. Richard A. Fennell under
whose supervision this study was conducted.. For his many
suggestions, advice, criticism, and, above all, patience,

I offer him my most sincere thanks.

Lastly, my wife Judy deserves special-mention. In
spite of the many days she Spent alone while I was accumu-
lating data and writing this paper, she did not complain.
Rather, she continually assisted me with her sincere-

encouragement and secretarial skills.

11

TABLE OF CONTENTS

Acmva-IEDGP‘ENTS O O O O O O O O O O 0
INTRODUCTION 0 O O O O O O

I.

II.

III 0

A.
B.
c.

Dehydrogenases . . . .
Esterases . . . . . . .

Objectives . . . . . .

.MATERIALS AND METHODS . . . . .

A.
B.
C.

Tissue sectioning techniques

Incubation of tissues .

Controls . . . . . . .

RESULTS 0 O O O O O O O O O

A.

Cerebral cortex . . . .

l. Dehydrogenases . .

2. Esterases . . . . .

Cerebellar cortex . . .
l. Dehydrogenases ._.
2.9 Esterases . . . . .
Superior colliculus . .
1. Dehydrogenases . .
2. Esterases . . . . .
Medulla oblongata . . .
1. Dehydrogenases . .
2. Esterases . . . . .
Special areas of enzyme

1. Pericytes . . . . .

iii

activity

Page

94.
F4 I*

\Oxowxloxmmwwl—I

a: no a: rd P‘ r4 F‘ id P‘ hi F4 l4
\» Ma ta m) so a; \n \n s- 10 A) rd

IV.

V.
VI.

2. Choroid plexuses
3. The basal ganglia
DISCUSSION . . . . . . .
A. Cerebral cortex . . .
l. Dehydrogenases .
2. Esterases . . . .
B. Cerebellar cortex . .
1. Dehydrogenases .
2. Esterases . . ?1‘
C. Superior colliculus .
1. Anatomical review
2. Dehydrogenases .
3. Esterases . . . .
D. Medulla oblongata . .
l. Dehydrogenases .

2. Esterases . . . .

E. Special areas of enzyme

1. Pericytes . . . .
2. Choroid plexuses

activity

3. Basal ganglia . . . . . . . .

F. Nothing dehydrogenase reactions .

SUWARY O O O O C O C O 0
LITERATURE CITED . . . .

iv

23
2h
26
26
26
28
30

.30

32
33
33
31+
36
37
37
38
38
38
39
41
1+2
1+5
1+8

Table
1.

2.

3.

LIST OF TABLES

Page
Enzymatic activity of brain regions
Dehydrogenases of the glycolytic
energy cycle . . . . . . . . . . . . . . . . 53

Enzymatic activity of brain regions
Dehydrogenases of the Krebs' cycle . . . . . 54

Enzymatic activity of brain regions
Carboxylic esterases . . . . . . . . . . . . 55

Figures
1.

LIST OF FIGURES

Cerebral cortex: LDH activity . . . . .
Cerebral cortex: GDH activity . . . . .
Cerebral cortex: Esterase activity . .
Cerebral cortex: Esterase activity . .

Layers of cerebellar cortex: .
LDH actiVity O O O O O O O O O O O O O

Layers of cerebellar cortex:
a-GPDH aCLIVitY o o o o o o o o o o o

Layers of cerebellar cortex:
Nothing dehydrogenase . . . . . . . .

Layers of cerebellar cortex:
a-GPDH actiVitY o o o o o c o o o o o

Purkinje cells: d-GPDH activity . . . .

b.&c. Layers of the cerebellar cortex:

d.

a.

apGPDH activity . . . . . . . . . .
Cerebellum: GDH activity . . . . . . .
Cerebellar cortex:. Esterase activity .
Purkinje cells: Esterase activity . . .

Layers of the cerebellar cortex:
Eaterase aetiVity 5 o o o c 'o o o o 0

Superior colliculus: CDH activity . . .
Superior colliculus: u-GPDH activity .

Superior and inferior colliculi:
Esterase activity . . . . . . . . . .

Superior colliculus: Esterase activity.

Medulla oblongata: a-CPDH activity . .

Ventral aspect of medulla oblongata:
GDH actiVity O O O O O O O O O O O O 0

vi

Page
57
57
59
59

61

61

61

61 -
63

63
63
65
65

65
67
67

69
69
71

71

c. Neurons in the medulla oblongata:
GDH actiVity O O O O O O O O O O O 0

d. Hyperactive neurons in medulla:
mu actj-Vity O O O O O O O O O O O O

9. a. Regional nucleus in medulla oblongata:
Nothing dehydrogenase . . . . . . .

b. Neuron in medulla oblongata:
Esterase activity . . . . . . . . .

c. .Solitary hyperactive neuron in medulla:
Esterase activity . . . . . . . . .

d. Solitary hyperactive neuron in medulla:
Esterase activity . . . . . . . . .

10. a.-d. Pericytes: Esterase activity . . .

11. a. Choroid plexus: GDH activity . . . .
b. Choroid plexus: LDH activity '. . . .
c. Choroid plexus: d-GPDH activity . . .
d. Choroid plexus: Esterase activity . .

l2. Choroid plexus: LDH activity . . . . . .

13. a.-d.' Caudate and lenticular nuclei of

the basal ganglia: Esterase

actiVity O O O O O O O O O O O O

14. a. .Caudate and lenticular nuclei:
Esterase activity . . . . . . . . .

b. Caudate nucleus: Esterase activity .

c. Caudate nucleus: Esterase activity .

vii

INTRODUCTION

Dehydrogenases. The dehydrogenases are oxidative
enzymes that among other things, are involved in cellular
respiration. Each of the enzymes consists of a substrate-
specific protein which Operates in conjunction with a co-
enzyme, NAD, NADP, or FAD.* When the substrate and
coenzyme are bound to the enzyme protein, hydrogen atoms
are removed from the substrate and transferred to the
coenzyme. Neither of the reduced pyridine nucleotides
(NADH or NADPH) can react directly with oxygen. Thus, in
order to be oxidized themselves, they must in turn reduce
another substrate or depend upon specific enzymes which
employ FAD as a prosthetic group. The flavOprotein then
introduces the hydrogen atoms (or electrons) to the
icytochrome system, and ultimately to molecular oxygen,

with the production of water (White~§t‘§l., 1959).

* The following abbreviations will be used throughout this
paper: Nitro-BT, 2:2 di-p-nitrOphenyl-S:5-diphenyl-(3:3-
dimethoxy-h:h bi-phenyline) ditetrazolium chloride;.MTT,
3-(h:5 dimethylthiazoly1-2)-2:5-diphenyl tetrazolium bromide;
ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; MDH,
malate dehydrogenase; a-GPDH, alpha-glycerOphosphate
dehydrogenase; Iso-CDH, isocitrate dehydrogenase (NAD linked);
GDH, glutamate dehydrogenase; C-6-PDH, glucose-6-ph05phate
dehydrogenase; NAD, nicotinamide adenine dinucleotide;

NADP, nicotinamide adenine dinucleotide phosphate; FAD,
flavine adenine dinucleotide.

-1-

-2-

Within recent years, the methods of identifying and
localizing enzymes in the brain centers of experimental
animals have increased both in kind and in specificity.
With the introduction of such highly specific tetrazolium
salts as Nitro-BT (Tsou §t_§l., 1956) and MTT (Pearse,
1957), it became possible to examine oxidative enzymes in'
ultra-thin sections of nervous tissue. The number of
dehydrogenases that could be localized by employing MTT
was increased by the modifications of Thomas and Pearse
(1961).

As these new techniques came into use, investigators
approached the analysis of nervous tissue in different ways.
wolfgram and Rose (1959) used Nitro-BT to show the intra-
cellular distribution of NAB-linked dehydrogenases in
neuroglia. Robins (1960) compared the distribution and
intensity of enzymes in gray matter with that in white
matter. Other investigators (Sidman, 1960; Romanul and
Cohen, 1960; Chason, Gonzales, and Landers, 1963) contrasted
the deposition of dehydrogenases in neurons and neuroglial
cells. Brain regions have been mapped (qualitatively) for
their enzyme content (POpe g§_§1., 1956; Friede, 1959a,
1959b, 1959c, 1960, 1961; Thomas and Pearse, 1961; Tewari
and Bourne, 1962a, 1962c; Lazarus 25 a1., 1962; Friede,
Fleming, and Knoller, 1963). ,

The significance of the enzymic distribution is that
their presence is an indication of the prevailing type of
energy cycle. Metabolic activity varies intracellularly

-3-
and from region to region, both in degree and derivation of
energy (Pearse, 1957: Hess and Pearse, 1961; Friede and
i Fax, 1961; Friede and Fleming, 1962; Tewari and Bourne,
1962d; Nandy and Bourne, 196ha, 196th: Takemori, 1965:
Bartonicek and Lona, 1966). ’

Esterases. Esterase identification was introduced by
Nachlas and Seligman (19t9). The substrate beta-naphthyl
acetate was hydrolyzed, liberating naphthol which combined
with a diazonium salt in the culture medium to form an
insoluble dye at the site of enzyme activity. Gomori (1952)
modified this by substituting a-naphthol, which was hydro-
lyzed by the same enzyme. Barrnett and Seligman (1951) and
Holt (1952) found that esterases hydrolyzed indoxyl acetate
to form indoxyl. The latter was oxidized to produce an
insoluble dark blue indigo dye at the site of the enzyme
activity. Utilizing these methods, as well as the modifi-
cations of Holt (1954, 1958), Holt and Withers (1952), and
Delellis and Fishman (1965), the ester-splitting enzymes
have been demonstrated in various regions of the nervous
system (Pearse, 1956; Pearse, 1960; Fishman and Hayashi,
1962; David 23 a1., 1962; Tewari and Bourne, 1962a, 1962b;
Adams 33 11,, 1963; Bartonicek and Lojda, 1961.; Nandy and
Bourne, 1966).

Objectives. The aforementioned investigations concern-
ing the histochemical analysis of nervous tissue centered

in such domesticated animals as albino rats, albino mice,

-4-

rabbits, cats and dogs. Occasional work was performed
with monkeys and through post-mortem examination of humans.

It is the intent of the present study to show the
existence and distribution of seven'oxidative enzymes
(dehydrogenases), as well as the esterases, within the
brain centers of wild mice (Peromyscus polionotus). Both
regional, intercellular, and intracellular localizations
were examined in the cerebral and cerebellar cortices, the
anterior colliculus and the medulla oblongata. Further-
more, any areas of the brain other than the preceding, where
intense reaction occurred, were identified. The intensity

of such reactions were evaluated on a subjective basis.

MATERIALS AND METHODS

Fully-grown, mature mice, Peromyscus polionotus of
both sexes, were used in this investigation. All animals
used for exPerimentation were raised by Dr. John A. King;
they ranged in weight from 10.8 to 15.3 grams. The mice
were sacrificed by transection of the upper cervical spinal
cord while under a light ether anesthetic.- The skin, under-
lying tissue, and the dorsal surface of the cranium were
quickly removed, exposing the brain. After cutting each of
the cranial nerves close to their point of exit from the
skull, the brain was lifted out and put into cold physiologi-
cal saline solution. Then each brain was bisected sagit-
tally using a razor blade. Each half was placed on mouse
liver attached to a microtome chuck (tissue holder). The
lateral surface of the brain was in contact with the liver,
thus leaving the inner brain surface exposed.

I The tissue was simultaneously frozen and attached to
tissue holders at -70°C using acetone and solid-C02. It
was then transferred to a freezer and stored at ~20°C. The
length of the storage interval was approximately two hours.

The tissues were sectioned in the laboratory of Dr.

R. A. Fennell, utilizing a Harris-International cryostat
(refrigerated microtome). Fresh-frozen, unfixed brain was
found to section best when the cryostat temperature was
between -9°C and -12°C. Sagittal sections of the entire

brain were cut at eight microns and occasionally at twelve

microns in thickness.

-5-

-6-

When esterases were localized, the brain was fixed in
formol-calcium for 2h hours prior to freezing it in the
n'acetone-dry ice mixture. Under these conditions, it was
necessary to keep the temperature of the cryostat at -25°C
to -30°C.

Tissue sections were removed from the microtome blade
by touching them with warm glass coverslips held with a
rubber bulb suction pickup. All sections were then kept in
the cryostat cabinet until cutting was completed.

The tissues were incubated in one of two ways, depending
upon whether an incubating media for dehydrogenases or for
esterases was to be used. For the identification of the
former, the sections were covered with 0.1 to 0.2 ml of
incubating media. Incubation continued for 30 minutes in
air at 37°C. For the localization of the esterases, the
tissues were placed in Columbia staining jars containing
-the incubating solution. The length of incubation varied
from 30 minutes to several hours.

The incubating media used for the demonstration of
a-glycerOphosphate, glucose-6-phosphate, isocitrate (NAD),
lactate, and malate dehydrogenases were prepared according
to the methods of Chason and Pearse (1961), and Thomas and
Pearse (1961). These workers utilized the cobalt-chelating
system employing MTT introduced by Pearse (1957). Alcohol
and glutamate dehydrogenases were identified using the method
of Hess, Scarpelli, and Pearse (1958) in which Nitro-BT was

used as an electron acceptor. Non-specific esterases were

-7-
studied using the a-naphthyl acetate method of Pearse
(1960), and the indoxyl acetate method of Holt and
Withers (1952), and Holt (1958).

Incubation (for dehydrogenases) was halted by rinsing
the sections in distilled water and fixing them in formal-
calcium for 15 minutes. After fixation, the tissues were
either counterstained in.Mayer's carmalum and then mounted
upon slides and covered with glycerine jelly, or mounted in
a similar manner unstained.

'Considerable time and effort were spent in an attempt
to use cresyl violet (Kluver and Barrera, 1953) as a counter-
stain. It is highly specific for neurons and provides
accurate identification of brain structures. However, only
limited success was obtained due to the required differen-
tiation of cresyl violet through several changes of 95%
ethanol.

Control sections for each enzyme were incubated in
media in which the substrate was omitted. Additional controls
consisted of kidney and liver sections placed in the normal
incubating media.

Enzyme reactions were studied in four areas of the
brain; namely, the cerebral cortex, superior colliculus,
cerebellar cortex and the medulla oblongata. As a means of
examining the same specific locale of the cerebral cortex
in each brain, the hippocampus was utilized as a landmark.
Particular attention was given to that region of the cerebral

cortex directly dorsal to the hippocampus. Because of its

-8-
relatively small size, the entire length of the anterior
colliculus was analyzed. In the cerebellar cortex, all
layers were subjected to scrutiny. Tissue in the medulla
oblongata was examined at the level of the inferior olivary
nucleus. All sections were taken from the first two milli-
‘meters of brain beginning at the midline and progressing
laterally. This procedure reduced the possibility of A
variable staining reactions caused by regional metabolic

differences.

RESULTS

A total of 1,087 slides was prepared for this inves-
‘tigation. Approximately 350 slides were randomly chosen
for evaluation of enzyme distribution and intensity. Tissue
sections were taken from four to thirteen mice for each
enzymatic test. Frequently, several dehydrogenases were
localized simultaneously in adjacent sections of the same
mouse brain, since the stock solutions of MTT or Nitro-BT
are made specific by the addition of apprOpriate substrates

and coenzymes. Thirty mice were used in this study.

THE CEREBRAL CORTEX

Dehydrogenases

Alpha—glycerOphosphate dehydrogenase (MTT) activity
was relatively evenly distributed in moderate concentrations
throughout the cortical tissue. Only that region (layer VI)
closely associated with the corpus callosum presented a
slightly reduced enzyme reaction (Table I).
_ Intracellular deposition was more distinct in the
pyramidal cells of layers III and IV. Formazan granules
were arranged in a perinuclear position (+2), with the
nuclei showing no enzyme (d-glycerophosphate dehydrogenase)
activity. The peripheral cyt0plasm had considerably less
activity. Neuroglial cells were weakly positive, but the
exact intracellular localization of granules was not obtain-

ed, due to the masking effect of the formazan in the neur0pil

surrounding the cells.

.9...

I10-

Lactate and malate dehydrogenases present a distri-
bution pattern very similar to that of a-glycerophosphate
dehydrogenase (Fig. la). Formazan granules produced by
lactate dehydrogenase were less abundant (+1) in layers I
and VI, but were found in about the same concentration in
all other layers (Tables I and II). .Malate dehydrogenase
activity was less intense only in layer VI. Intracellular.
distribution was identical, though slightly reduced in
degree. NeurOpil contained a moderate amount (+2) of
formazan, but not in any particular orientation.

Glucose-6-phosphate and isocitrate (NAD) dehydrogenases
did not exhibit any activity (Table II). Sections of kidney
incubated in the same solutions presented weak-to-moderate
(+15) amounts of formazan in the cortical tissue, but little
in the glomeruli. Sections of tissue incubated in solutions
in which the substrate was omitted did not show enzyme
activity.

Glutamate and alcohol dehydrogenases were identified
by utilizing NitroéBT as the tetrazolium salt. The distri-
bution and intensity of reaction Were quite similar for both
enzymes (Tables I and II). A careful and thorough examination
of tissue sections showed weak reactions (layer I) for
glutamate dehydrogenase, whereas alcohol dehydrogenase
activity was less intense. Glutamate dehydrogenase was
usually localized along unmyelinated nerve fibers (Fig. 1b).
The surrounding neurOpil contained moderate and diffuse

formazan deposits. The cytoplasm of pyramidal cells exhibited

-11..
a dark, diffuse reaction (+2£) closely associated with the
nuclear membrane, along with distinct, medium-sized granules
in the peripheral cytOplasm. Few granules were observed in
the intermediate cytoplasm. A 7

Alcohol dehydrogenase reactions were weak or absent in
the outer layer of the cortex, increased to a maximum in
layers IV and V, and then decreased to lower levels in
layer VI (Table I). Unmyelinated nerve fibers were pre-
cisely outlined by small formazan granules. The basal
region of layer I and much of layer VI showed reactive fibers
in abundance. .

Control sections containing Nitro-BT but no exogenous
substrate consistently yielded the same distribution and
intensity as that found for alcohol dehydrogenase. These
sections exhibited both diffuse and distinct granular
formazan depositions.

‘Esterases

Ester-splitting enzymes were found to be quite abundant
in the cerebral cortex, but not homogeneous in distribution.
Since there are no exact boundaries of the various layers,
assessment of the intensities of enzyme reaction within
particular strata is at best an approximation, i.e., an
estimate of the overall activity. The molecular layer
(layer 1) was very weakly positive when indoxyl acetate was
utilized, and negative with a-naphthyl acetate (Table III).
The pericapillary cells (pericytes) found within the pie

mater were intensely reactive (Fig. 2a).

-12-

The processes of neuroglial cells located in the
layers exhibited randomly-distributed indigo crystals.
In addition, much of the cellular material localized
between neuronal and neuroglial cells showed less intense
(+1-lé) reactions. The cytoplasm of medium-sized pyramidal
cells of layer III exhibited moderate activity with indoxyl
acetate (Fig. 2b), slightly less (+1) when a-naphthyl
acetate was used as a substrate (Table III). No particular
perinuclear distribution was apparent. A

'The most intense (+3) activity observed in the cortex,
aside from that within the pericytes, was exhibited by the
large pyramidal cells of layer V (Table III). Crystals of
indigo were distributed throughout the cytOplasm and into
the axon hillocks. Tissue near and adjacent to the corpus
\callosum exhibited a horizontal distribution of enzyme
activity (+1). Neuroglial cells containing indigo crystals
were dispersed throughout the corpus callosum, separated

from one another by non-reactive tissue. All control sections

were negative.

_ THE CEREBELLAR-CORTEX
Dehydrogenases
The outer (molecular) layer of the cerebellum is com-
prised of a few small, stellate-shaped nerve cells and many
unmyelinated nerve fibers. Dehydrogenase activity was
moderate in intensity (+2) and uniformly distributed through-
out this layer. Only the nuclei of the neurons were com-

pletely negative. Isocitrate, malate, and lactate (Fig. 3a)

-13-
dehydrogenase (MTT) reactions were weak, whereas alcohol
(Nitro-BT) and¢1-glycer0phosphate (MTT) dehydrogenases
exhibited moderate (+2) activity (Fig. 3b). Glutamate
dehydrogenase (Nitro-BT) reactions were more intense
(+25) than they were in either of the two preceding enzymes
(Tables I and III). The presence of glucose-6-phosphate
dehydrogenase was never shown when using MTT as the
hydrogen acceptor, but Nitro-BT produced a diffuse (+1)
formazan deposition.

All sections incubated in an MTT solution without the
apprOpriate substrate were devoid of activity. Those con-
trols immersed in a similar solution of Nitro-BT exhibited
moderate degrees of activity (Fig. 3c).

Adjacent to the molecular layer is a single row of
Purkinje cells. The cells are flask-shaped, with several
dendrites extending into the molecular layer. Formazan
‘deposition in the cytOplasm of cells was distinct, although
the intensity of enzyme activity varied considerably, e.g.,
some dehydrogenase reactions were weak, whereas others were
stronger (Tables I and II). Glucose-éephosphate activity
was not identified, and lactate and isocitrate dehydrogenases
reacted weakly (+1). Most enzyme activity was concentrated
in that portion of the cytOplasm near or adjacent to the
molecular layer (Fig. 3d). The explanation for this dis-
tributiOn was quite apparent when the position of the nucleus
was considered. It was found to be eccentrically located,
leaving only a thin rim of cytoplasm adjacent to the granu-

lar layer. Weak to moderate amounts of formazan granules

-14-
(+l§) were produced by malate dehydrogenase.) Alpha-
glycerOphosphate, alcohol, and glutamate dehydrogenase
reactions were moderate in intensity. It is evident in
Figure 3d that a-glycerOphosphate reactions were intense
in the cytoplasm and eccentrically localized due to the
position of the nucleus. Likewise, an intense concentra-
tion of granules was observed at the cell periphery, in
close association with the cell membrane (Fig. ha).

With the exception of glucosee6-ph05phate, which was
negative, all dehydrogenase activity in the granular layer
was limited to the glomeruli cerebellosi. The reactions
were more intense than those identified in the molecular
layer and in the Purkinje cells. The granule cells were
devoid of enzyme activity. Thus, the overall appearance
of formazan deposition is spotty, patchy, and therefore in
direct contrast to the uniformity of distribution of enzyme
reactions in the molecular layer (Figs. hb, c, and d).
(Malate, isocitrate, and lactate dehydrogenases reacted
weakly (Fig. 3a), whereas a-glycerophosphate, glutamate,
and alcohol dehydrogenases produced moderate amounts of
formazan (Figs. 3b and Ad). Dehydrogenase activity in the
branches of myelinated fibers (arbor vitae) coursing through
the cerebellum was not observed, although positive dehydro-
genase reactions were seen in the cytoplasm of some neuro-
glial cells.

Esterases
The regional and cellular distribution of esterases as

shown by the d-naphthyl acetate and indoxyl acetate methods

.15-
were essentially the same, although differences were found
in the degree of enzyme activity (Table III). The molecular
layer consistently presented more intense esterase reactions
than those observed in the granular layer. The dye was
quite evenly distributed in the former in both the scattered
cellular elements and unmyelinated fibers (Fig. 5a). Enzyme
activity in the granular layer was localized in the glial
cells, the glomeruli, and the unmyelinated axons which run
through this layer.

Esterase activity in the Purkinje cells was frequently
-strong (+3), although enzyme reactions in individual cells
ranged from weak to intense. That portion of the cytoplasm
closest to the molecular layer (including the axon hillock)
exhibited the most intense stain (Figs. 5a, b, and c).
Dendrites arise from this end of the Purkinje cell and then
ascend into the molecular layer.

The rays of white matter (arbor vitae) passing through
the cerebellum appeared to exhibit weak to moderate enzyme
activity in all regions of the tissue. Closer examination
revealed that the moderate staining should be attributed to
interspersed glial cytOplasm and processes, and probably to

synaptic endings of myelinated nerve fibers.

THE SUPERIOR COLLICULUS

Dehydrogenases

The zonal, or outer layer of the superior colliculus
was devoid of all dehydrogenase activity, with the exception

of weak glutamate dehydrogenase reactions. Immediately

-16-
adjacent to the above area is the thick stratum griseum
superficiale, which exhibited a few myelinated fibers and
nerve cell bodies. Tissues of this layer exhibited moderate
amounts of formazan, following incubation in either u-gly-
cerophosphate or malate substrate solution (Fig. 6a),
whereas lactate dehydrogenase (MTT) activity was less intense
(Tables I and II). A weakly positive enzyme reaction for
glutamate and alcohol dehydrogenases (Nitro-BT) was visible
in unmyelinated fibers. Occasional neurons exhibited weak
perinuclear concentrations of formazan,whereas the rest of
the cytoplasm exhibited less activity. All formazan granules
in the stratum griseum were small, spherical, and distinct.
The dehydrogenases specific for isocitrate and glucose-6-
phosphate, respectively, were not identified here or in any
other region of the superior colliculus.

Longitudinally-oriented fiber tracts within the deeper
stratum opticum were responsible for moderate depOsitions of
formazan following incubation incl-glycerophosphate, lactate,
and glutamate substrate solutions (Fig. 6a). Alcohol and
malate dehydrogenases were weakly active (Tables I and II).
The granules within this region were half again as large as
those identified in the stratum griseum, and were less
abundant. Fiber tracts in this area exhibited moderate
malate and glutamate dehydrogenase reactions along their
periphery with less activity in the central portions.

The basal region of the layer referred to in the pre-

ceding paragraph exhibited multipolar neurons with weak

-17-
enzyme reactions. Examination of the cell bodies showed
that lactate dehydrogenase was more concentrated in the
perinuclear cytOplasm. Other dehydrogenases, e.g.,
a-glycerOphosphate and alcohol dehydrogenases, were not
identified in the cytoplasm adjacent to and surrounding the
nuclei. Formazan deposits in low concentrations were
localized in the peripheral cytOplasm. Enzymatic activity
appeared to follow the outlines of the cell membrane.

The deepest layers, namely, strata lemnisci and album
profundum, exhibited moderate enzyme reactions in the
neuropil following incubation in a-glycerOphosphate, lactate,
malate, and glutamate dehydrogenase substrate solutions
(Fig. 6b). Alcohol dehydrogenase yielded strong depositionS'
of formazan granules. _Commissural tracts consistently
presented moderate activity along their periphery, and
considerably less activity in the central region (Fig. 6b).

During the course of this study, it was noted that as
one passed from superficial layers to the deeper layers
of the superior colliculus, the size of the formazan granules
increased. The small, fine granules were localized in
layers I and II, whereas in the deeper layers the granules
became progressively larger.

7 A Control sections incubated in MTT without substrate

did not exhibit formazan deposits, in contrast to tissue
sections incubated in Nitro-BT, which were strongly positive
(+25). _The distribution and intensity of enzyme activity
in the controls closely paralleled that found in alcohol

and glutamate dehydrogenases.

-18-
Kidney sections placed in the complete media (MTT)
, were weakly positive (+lé), whereas those in the control
solution showed no enzyme reaction.
Esterases ,

The esterases presented a rather unique distribution
in the superior colliculus. Intensity of enzyme reactions
was greater than that observed in the inferior colliculus
(Fig. 7a). Only the pericytes within the colliculi reacted
with equal intensity (+A). It is evident in Figure 7a that
the peripheral layer (zonal) exhibited the greatest degree
of activity (+3), and that there was a gradual decrease in
enzyme activity in the deeper layers (Table III). There
was, however, one exception to the step-wise reduction in
the intensity of enzyme reactions. The stratum album
profundum showed strong, irregularly-distributed, enzyme-
active areas (Fig. 7a).

CytOplasm of neurons varied in the degree of staining
(+1-3), and nuclei did not stain. The cytOplasm in contact
with the nucleus showed slightly more activity than the
peripheral cytoplasm. '

The stratum griseum superficiale showed an abundance
of large, circular areas in which enzyme reactions were not
observed. Tissues adjacent to the circular areas exhibited
moderate enzyme activity. Tissues which did not react con-
sisted largely of myelinated nerve fibers, whereas reactive
peripheral materials appeared to be sheathes of Schwann cells.

The intensely active pericapillary cells were frequently

-19-

observed throughout the superior colliculi (Figs. 7a and b).
Additional details are presented in a separate section.

Enzyme distribution was found to be identical with
either of the methods cited in the preceding pages. The
only variation in intensity was observed in the zonal
layer, where arnaphthyl acetate gave a weak (41%) reaction
and indoxyl acetate gave a strong (+3) reaction. Esterase

activity was weak to moderate in neuroglial cells and their

processes.

THE MEDULLA OBLONGATA

Dehydrogenases I

Considerable variation in the intensity of enzyme re-
actions, apparently correlated with the diversity of its
components, was observed in tissues in the medulla.
Regional nuclei, including neurons and surrounding neuropil,
exhibited considerably different distribution patterns from
the abundant fiber tracts which course throughout the area
(Tables I and II, Fig. 8a).

_ Myelinated fiber tracts showed large formazan granules
when examined for the presence of<i~glycerophosphate, lactate,
and malate dehydrogenases (MTT).. It was found in this study
that most of the tissue between the large granules was
.devoid'of enzymatic activity. Glutamate, alcohol, and
a-glycerophosphate dehydrogenases (Nitro-BT) presented a
fine, diffuse formazan deposition within the myelinated

tracts, varying from weak to intense in reactivity (Fig. 8b).

-20-

Isocitrate and glucose-6-phosphate dehydrogenases
were not demonstrated in any area of the medulla. Large,
branched, crystalline artifacts were observed occasionally
when cresyl Violet was used as a counterstain. The tissue
adjacent to the branched particles completely lacked
formazan granules. Sections that were stained in.Mayer's
carmalum did not exhibit artifacts or other types of granu-
les. Kidney sections placed in the complete incubating
medium exhibited a weak to moderate formazan deposition.

NeurOpil in some regional nuclei showed more intense
enzyme reactions than those found in general gray matter
(Fig. 8b). This, however, was reversed in other nuclei..
The reactivity of glutamate and a-glycerophosphate dehydro-
genases produced moderate amounts of formazan granules in
the neurOpil, whereas malate, alcohol, and lactate dehydro-
genases were only weakly active (Tables I and II).

— «Enzyme activity was usually associated with neurons.
Neuroglial cells were difficult to identify and showed
little activity. Neurons exhibited a perinuclear distri-
bution that was moderately intense for both glutamate and

alcohol dehydrogenases (Fig. 8c). Large concentrations of
formazan granules were frequently noted in nerve cells whose
nuclei were eccentrically positioned. Alpha-glycerOphosphate
dehydrogenase activity was usually uniform in distribution
and weak in intensity when MTT was used, and in some in-

_ stances perinuclear in position. When Nitro-BT_was used as

the hydrogen acceptor, the intracellular distribution was

similar, but much more intense.

-21-

The perikarya of regional nuclei showed a few very
fine formazan granules following incubation in lactate and
‘=malate substrate solutions. However, occasional solitary
neurons widely scattered in the medulla exhibited consider-
able amounts (+2%) of malate dehydrogenase (Fig. 8d).

Tissue sections incubated without substrate were
negative with MTT. Other controls were weakly to moder-
ately positive when Nitro-BT was utilized (Fig. 9a). It
was noted that in contrast to the light yellow color of the
complete incubating media for lactate, malate, isocitrate,
and glucose-6-phosphate dehydrogenases, an opaque, dark
green colored incubation medium was seen subsequent to the
addition of arglycerophosphate. No precipitant occurred,
and a differential distribution of reaction products was
still observed.
Esterases

The demonstration of esterases by the denaphthyl
acetate and indoxyl acetate methods was similar in the
distribution of the enzymes throughout the area. However,
the intensity of the reaction was always less When using
the former procedure, with the exception of the pericytes,
which showed high activity with both methods (Table III).

Moderately-strong reactions were observed in the ’
regional nuclei but the neurons were consistently strongly
positive, while the surrounding neurOpil was less intense
'(Fig. 9b). Neurons of the inferior olivary nucleus were

much weaker than those in deeper nuclei. The esterase

-22-
distribution was observed to be perinuclear in solitary
neurons when using the arnaphthyl acetate method (Figs. 9c
and 9d). The peripheral areas of the cytOplasm normally
exhibited less intense reactions. However, when indoxyl
acetate was used as the substrate, the esterase activity
was evenly distributed throughout the cytOplasm. Figures
9c and 9d show that axon hillocks and the proximal portion
of the axons exhibited a moderate degree of activity which
exceeded reactions in the peripheral cytoplasm, but was
less intense than reactions in the perinuclear areas.
Cellular nuclei were negative, but were somewhat obscured
by the overlying darkly-staining cytoplasm (Fig. 9b).

Fiber tracts were very weakly active, with small
branched granules appearing throughout. Neuroglial cells
showed a weak-to-moderate reaction with indoxyl acetate,
considerably less with a-naphthyl acetate. The pericapillary
cells (pericytes) were always intensely positive, with only
their nuclei remaining negative.

All controls were negative.

SPECIAL AREAS OF ENZTME ACTIVITY
Perivascular Cells (Pericytes)

While examining the brain centers in this study, it was
not uncommon to observe large, irregularly-shaped cells in
close proximity to small blood vessels (capillaries). These
perivascular cells exhibited very intense esterase activity
throughout their cytoplasm (Fig. 7b). The nuclei remained
enzymically inactive, as was the case in all other types of
cells examined. Pericytes follow the course of the capil-
laries and appear to be wrapped around the vessels, probably
in contact with the endothelial membrane (Figs. 10a and 10b).

It was evident that the intense reactions were localized
in pericytes and not in the endothelial cells (Figs. 10c
and 10d). A thin rim of positively-staining cytoplasm was
seen in the endothelial cells, but intensity of reactions
was less pronounced than that in pericytes. Identification
of pericytes as such was extremely difficult without the use
of esterase incubating medium.

Choroid Plexuses

During the course of observations on the various brain
centers, particular attention was given to the ventricles of
the brain and their accompanying choroid plexuses. Compari-
son of the enzymatic activity within the epithelium of the
choroid plexus with that in any other region, revealed that
enzymes (both dehydrogenases and esterases) were equally as

intense as the most active area, if not greater. Formazan

, -23-

-24-
deposition was localized in the cytoplasm of short, cuboidal
epithelial cells. Figures 11a and 11b illustrate the con-
tinuity between these cells and ependymal cells. It was
also observed that although the ependyma did exhibit a
positive enzyme reaction, the intensity of this reaction
was found to be greater in the cuboidal cells which project-
ed into the lumen of the ventricle and covered the choroid
plexuses (Figs. 11c and 11d). Rarely were the neighboring
ependymal cells seen to be equal in enzyme activity with
the specialized covering of the choroid plexuses.

Furthermore, intensity of enzyme reaction was irregu-
larly distributed throughout the epithelium. Figures 11c
and 12 illustrate the variability in activity between close-
ly associated portions of choroid plexuses. Esterases, as
well as glutamate and alcohol dehydrogenases, showed the
most intense activity, while lactate and aeglycerOphosphate
dehydrogenases were moderately active. Similar evaluations
of the remaining enzymes were not made due to insufficient
numbers of suitable sections.

The Basal Ganglia

In the ventral portion of the forebrain, laterad to the
midline, and just anterior to the Optic chiasma, an intensely
positive reaction for esterases was observed. Reactions
were usually localized in the nuclear components of the
basal ganglia, i.e., in the caudate and lenticular nuclei.
The intensity of reactions decreased laterally from the

midline and disappeared about three millimeters from the

-25-
center (Figs. 13a, b, c, and d). The overall shape of the
enzymatically-active tissue was that of an inverted comma
when viewed in sagittal sections (Fig. 13b).

The esterase activity, as shown by the a-naphthyl
acetate and the indoxyl acetate esterase methods, differed
only in the intensity of reaction, not in the distribution
(Figs. 13b and 14a). The former method always produced an
intense (+4) reaction, whereas the latter consistently
showed moderate activity.

The tail of the caudate nucleus extends directly
ventrally to the genu of the corpus callosum, and is just
anterior to the anterior cOmmissure (Fig. 13c). This highly
reactive tissue extends from the choroid plexuses of the
lateral ventricles (Fig. 14a) to the ventral surface of the
brain, just anterior to the root of the Optic chiasma
(Fig. 13d).

‘*It was apparent, when viewed under greater magnifi-
cation, that not only the neurons but the surrounding
neurOpil stained intensely (Figs. 14b and 14c). The nuclei
Of the nerve cells were again negative of all enzymatic
activity. None of the dehydrogenases exhibited any similari-

ty in distribution or in intensity of reaction within the

basal ganglia.

DISCUSSION

THE CEREBRAL CORTEX

Dehydrogenases

The significance of the distribution and intensity of
oxidative enzymes may best be understood if one realizes
that the level of activity of these enzymes is an approx-
imate indication of the intensity of oxidative metabolism.

The similarity in enzyme distribution and intensity
among the WAD-dependent dehydrogenases indicates that both
the glycolytic and the citric acid energy cycles exist
within cortical tissue of the mice used in this study. The
predominance of axOnal and dendritic fibers in the molec~
ular layer (layer 1) was responsible for the reduced enzyme
activity in that area. This investigator found some en-
zymatic reactions to be greater in perikarya than in neuro-
pil. Although this observation appears to be directly con-
tradictory to the findings of Robins (1960), it is in
agreement with the observations of Thomas and Pearse (1961),
and Chason gt 3;. (i963). Friede 23 3;. (1963) found that
both distributions existed not necessarily for the same
dehydrogenase but for different Ones. Their work indicated
that some enzymes Of the citric acid cycle (SDH and MDH)
predominatewithin neurOpil, whereas the glycolytic enzyme
lactate dehydrogenase prevailed in perikarya (of pyramidal

cells).

-26..

-27-

Indeed, when cortical tissue was incubated for the
demonstration of alcohol and glutamate dehydrogenases,
‘unmyelinated fibers in layers I and VI were conspicuous
(Fig. lb). Neither of the aforementiOned enzymes can be
identified with MTT as the electron acceptor, only with
Nitro-BT. It should be noted also that such enzymically-
active fibers could not be demonstrated for any dehydrogenase
utilizing MTT.

Few nerve cell bodies were Observed until deeper strata
were reached. The intensity of enzymatic activity remained
consistent throughout the layers, even when considering
enzymes of different energy cycles. The slight variation
between cortical layers and between enzymes made it difficult
to arrive at any significant conclusions with respect to
differential metabolic activity (Tables I and II). Thomas
and Pearse (1961) obtained similar results using rat cere-
bral cortex.

The pyramidal cells of layers III and V were only
slightly more active than the neighboring neuropil, as is
evident with lactate dehydrogenase in Figure la. This in-
vestigator was unable to demonstrate the existence of the
pentose-pathway enzyme glucose-6-phosphate dehydrogenase,
nor of WAD-dependent isocitrate dehydrogenase, though other
workers have (Friede 22 al., 1963). Kidney sections incubated
in the complete isocitrate medium exhibited typical formazan
granules, which indicates that this enzyme is either non-

functional within the cerebral cortex or in concentrations

-23-
insufficient for detection. Kidney sections immersed in
glucose-6-phosphate medium were barely reactive, making
this data inconclusive.

It was shown in the preceding pages that five dehy-
drogenases were identified by the cobalt-chelation method
using MTT, i.e., a-glycerophosphate, glucose-6-phosphate,
isocitrate (NAD-dependent), lactate, and malate dehydro-
genases. All control sections placed in media lacking the
substrate were negative. Alcohol and glutamate dehydro-
genaSes were demonstrated with the use Of NitroéBT. However,
control sections immersed in such media exhibited consider-
able formazan deposition of bOth varieties (diffuse and
granular). Rinsing tissue sections in dilute (15%) alcohol '
or in acetone did not decrease the activity. Since this is
of considerable significance, a thorough discussion of this
phenomenon is merited. However, due to the fact that similar
~ results were obtained in the other brain centers covered in
this study but not yet mentioned, discourse on the subject
will be deferred until a later section.

Esterases

If one observes Figure 2a, it-becomes apparent that
the esterases were stratified in their distribution. Glial.
metabolic activity is responsible for the slight reaction
seen in the molecular layer of the cerebral cortex. ‘The
small triangular nerve cells within the second layer and
the pyramidal cells in layers III and V contain moderate

amounts of esterases, as is evident in Figure 2b. It should

-29-
be understood, however, that much of this activity in the
neurOpil is actually at the point of synapse of nerve fibers
c(Pearse, 1956; Tewari and Bourne, 1962b and 1962e).

However, analysis of the reactivity within nerve cell
bodies of the mouse cortices in this study definitely show
more activity than in the surrounding neuropil. This finding
can still be compatible with the results reported above if
one considers the method of esterase identification, i.e.,
the formation of indigo from indoxyl acetate. Pearse (1956)
pointed out that "this_(method) shows not only non-specific
esterases, but also specific cholinesterase and pseudo-
cholinesterase". Thus, it is entirely possible and indeed
quite probable, that different enzymes are responsible for
the variation in results.

Unless the chemical situation (such as optimum pH) is
considered, and unless Specific inhibitors are employed at
certain concentrations, comparison of esterases in different
brain regions and in different species will be futile.

Although the pH was kept constant (7.3) in this study,
inhibitors like E 600 were not used extensively. Therefore,
esterase activity between different cell types, e.g., glial
cells versus pyramidal cells, may be due to different
esterases.

The intensely reactive, elongated structures in figure
2a are pericytes. This fibroblastic-type cell is always
associated with capillary networks throughout the various
brain centers. They will be considered in detail in a later

section.

THE CEREBELLAR CORTEX
‘Qghydrogenases

The molecular layer was found to present a relatively
uniform enzyme distribution for all of the dehydrogenases.
As this layer contains few nerve cells but many unmyelinated
nerve fibers, it was not surprising to find metabolic acti-
vity relatively low (moderate). Other authors (Friede 32
‘31., 1963) have reported a similar distribution of oxidative
enzyme activity within this layer, with Lazarus 33 a1. (1962)
finding metabolic activity to be equal in both neuropil and
perikarya. Friede and Fax (1961), noting a direct corre-
lation between the location of mitochondria and citric acid
cycle enzymes, found that both mitochondria and enzyme ac-
tivity were more abundant in the deeper half of the molec-
ular layer.

Of the dehydrogenases considered in this study, only
isocitrate (NAD) exhibited weak enzyme reactions, while
glucose-6ephosphate dehydrogenase was entirely non-reactive.
Since positive controls using kidney sections could not be
obtained for the latter enzyme, no conclusions can be made
regarding its distribution.

Metabolic activity in Purkinje cells frequently equaled
or exceeded that found in the molecular layer. In the case
Of lactate dehydrogenase, Purkinje cells were difficult to
distinguish from the adjacent molecular layer, as their
activities were identical. In the main, however, enzyme
reactions in Purkinje cells agreed with the findings of

-30-

-31-

- other investigators (Tewari and Bourne, l962e; Chason,
Gonzalez, and Landers, 1963; Friede and Fax, 1961).
Considerable dehydrogenase activity was apparent, but
varied from cell to cell in intensity.

Thomas and Pearse (1961) were unable to demonstrate
a-glycerophosphate dehydrogenase activity within Purkinje
cells, whereas this worker found moderate formazan deposi-
tions with the same enzyme, when using either MTT or Nitro-
BT (Figs. 3b, d, and 4a-c). In addition, there was, if
anything, a drOp in enzyme activity in the immediate
vicinity of the Purkinje cells. In contrast, Thomas and
Pearse (1961) reported considerable activity. It should be
noted that Friede and Pax (1961) found decreased numbers of
mitochondria and reduced oxidative enzyme reactions in this
same area.

Occasional sections showed an increased metabolic
activity at the junction of axons and their respective Pur-
kinje cells. This was especially the case with the large,
unmyelinated fibers which enter the molecular layer.

In the granular layer, utilization of either the gly-
cOlytic or the citric acid cycle enzymes was confined to
the glomeruli cerebellosi, which, according to Friede and
Pax (1961), "represent the synaptic areas of the mossy and
climbing fibers and the dendrites of the granular cells".
The actual level of metabolic activity within these areas
was found to be higher than in any other tissue of the

cerebellar cortex. These results are in agreement with

-32-
those of many other authors (Friede, 1960; Friede and Fax,
1961; Thomas and Pearse, 1961; Lazarus 33 31., 1962;
Tswari and Bourne, 1962d and 1962c).

White matter coursing through the cerebellum (arbor
vitae) contained little oxidative enzyme activity, although
the perikarya of glial cells was faintly positive. It
should be noted, however, that a diffuse, positive reaction
was observed when Nitro-BT was used, but none witthTT.
Esterases

The distribution of esterases in the four layers of
the cerebellar cortex presents a considerably different
picture than that described for the dehydrogenases. The
molecular layer remained homogeneous in appearance, but it
exhibited a more intense enzyme reaction, with only the
nuclei Of scattered cells being negative.

Purkinje cells contained varying amounts of stain.
Even adjacent cells were observed at Opposite extremes of
metabolic activity. Tewari and Bourne (1962b) reported
similar findings, and were of the Opinion that the cells
were exhibiting cyclic activity. In spite of the variations,
the perikarya as a whole was more reactive than in any
other layers of the cerebellum.

The granular layer was much less conspicuous. Granu-'
lar cells still were without enzyme activity, and the.
glomeruli were considerably weaker in their esterase content.
Myelinated fibers were Observed throughout the granular

layer, showing continuity both with the Purkinje cell layer

-33-
and the deeper white matter tracts. Figure 5a shows that
the esterase activity in the fibers manifests itself in
the form of tiny beads of naphthol. This is in agreement
with the results of Tewari and Bourne (l962b). They explain
the beaded appearance as representing the localization of
the enzyme at synaptic endings. These same workers (19626)
felt that much of the esterase activity observed in the
molecular layer and in the glomeruli cerebellosi is actually
at points of synapse.

.The arbor vitae of the cerebellar cortex exhibited es-
terase activity only along a few fibers, and then just at
synaptic endings and in glial cytOplasm.

THE SUPERIOR COLLICULUS
The following brief review of anatomical and functional
-aspects of the superior and inferior colliculi is presented
so that the reader might understand more easily the sig-
nificance of enzymatic distribution differences.

It has been determined that the colliculi (both superior
and inferior) receive the terminations of most of the optic
nerve fibers, and at the same time have become massive au-
ditory reflex centers (Zeman and Innes, 1963). In the
mouse the inferior colliculi are considerably smaller than
the superior, whereas in man both are nearly alike in size.
This is due primarily to a decrease in the functional im-
portance of the superior colliculus in man, and therefore

results in a decrease in its size.

-34-

Since the colliculi are important reflex centers,
they receive many different kinds of fibers coming from the
spinal cord, medulla oblongata, and the forebrain. Some of
these fibers terminate in the inferior colliculi, while
others divide, sending a branch into the superior colliculus.
Still other fibers send a branch into the colliculi, but
continue up further in the brain. Those fibers ending in
the colliculi are thought to form the chief reflex auditory
pathway, while those traveling up into higher centers of the
brain form the cortical pathway. By means of this latter
tract, auditory impulses pass into consciousness and into
the auditory cortex (Zeman and Innes, 1963).

The superior colliculi differ basically from the in-
ferior colliculi because of divergence during their develOp-
ment. That is, the inferior colliculus increased in size
but not in complexity as the auditory paths increased. The
superior colliculus exhibits a secondary differentiation'
due to the migration of neurons during embryonic develOp-
ment to form peripheral layers. According to Huber and
Crosby (1943), this "layering....favors localization and
permits specificity Of reception and projection of impulses".
Dehydrogenases

The inferior colliculi exhibit no differential Staining
but rather a moderate degree of oxidative enzyme activity
concentrated in a sphere identical to regional nuclei found
elsewhere in the brain. Synaptic neurons are probably

responsible for the activity.

. 4 -35-

The stratification described above is evident when the
distribution of the oxidative enzymes is considered. Little
activity is Observed in the peripheral (zonal) layer, which
is comprised of fibers that turn and course downward into
the lower layers. Directly below the zonal layer, the
stratum griseum superficiale exhibits a moderate degree of
metabolic activity. Much of this layer is comprised of un-
myelinated fibers, their synapses, and an occasional neuron.
Since the superior colliculus is a reflex center with
fibers from various parts Of the brain synapsing here, it
is entirely possible that synaptic endings might be respon-
sible for much of the oxidative enzyme activity observed
(here. When studying synaptic areas in the olfactory bulb
of the rat, Nandy and Bourne (1966) found that synapses
were much more metabolically active than the nerve cell
bodies.

. Before accurate conclusions can be made, further analy-
ses are necessary. A possible avenue of investigation would
involve comparison of enzymes and their intensities between
layers of the superior colliculi and areas of known synap-
tic endings, e.g., glomeruli cerebellosi and portions of
the Olfactory bulb. Although a detailed examination was not
conducted in this study, the enzyme activities listed in
Tables I and II for the colliculi and the glomeruli indi-
cate the plausibility of such an undertaking. ' .

Enzyme reactions were gradually reduced in intensity in

the deeper layers, as entering Optic fibers are encountered

' ~36-

and as ascending systems are received and correlated with
optic and cortical terminals. It seemed to this worker
that as the number of myelinated fiber tracts increased,
the intensity of oxidative enzyme activity decreased. It
was also noted that small, spherical formazan granules in
the outer layers were replaced by larger, irregularly-
shaped granules that were fewer in number. The latter
were very similar in size and shape to those found in the
arbor vitae of the cerebellum, and in other concentrations
Of white matter. Therefore, perhaps the increased amounts
of lipids found within myelin cause the formation Of large
aggregates of formazan, drawing smaller granules from their
point of origin.
Esterases

The stratification observed in the superior colliculus
with dehydrogenases was not quite so apparent with ester-
ases. The two peripheral layers were strongly reactive,
with a reduction in activity occurring in the stratum
-Opticum. Koelle (1954) reported strong cholinesterase
activity in this layer, attributing it to neurons which
synapsed directly with afferent motor neurons. Hydrolytic
activity in the adjacent stratum lemnisci was intense, due
perhaps, to a concentration of synapses from large commis-
sural fiber tracts.

The superior colliculi possessed considerably more
esterase activity than the inferior colliculi(Fig. 7a).

Although the former structures were known to be more complex

, ~37- .
due to the inward migration of neurons during embryonic
develOpment, there have been few reports of a distinct pre-
dominance in their (superior colliculi) esterase activity.
Extensive enzymatic study is necessary before the functional

significance of such a difference is completely understood.

THE MEDULLA OBLONGATA
Dehydrogenases D A A

Regional nuclei differed considerably in the location
of dehydrogenase activity, and consequently, of metabolic
activity. In the inferior olivary nucleus, both a-glycero-
phosphate and glutamate dehydrogenases (Nitro-BT) were
active primarily in the neuropil, whereas formazan deposits
were not identified in many neurons. In contrast, other
nuclei showed the same two enzymes along with malate dehy-
drogenase concentrated in the perikarya of neurons with only
_diffuse deposits in neurOpil. This variety in localization
Of both glycolytic and citric acid cycle enzymes is in agree-
ment with the findings of Friede and Fax (1961), Thomas and
Pearse (1961), and of Friede £2.31- (1963).

Myelinated fibers usually did not Show dehydrogenase
activity. When MTT was used, the fibers occasionally
exhibited large, angulan,irregularly distributed formazan
granules. In contrast, when using Nitro-BT the formazan
deposition was indistinct and not clearly granular. The
fibers exhibited a diffuse, gray hue. ‘With either tetra-

zolium salt, granules were not found consistently in glial

-38-
cells, or in a distinctively beaded fashion, nor were they
just on the periphery of the tracts. Therefore, due to
the variability in distribution from one tissue sectiOn to
the next, both localizations are thought to be false.
Esterases "

Esterase activity varied greatly among regional nuclei,
but the inferior olivary nucleus consistently exhibited the
weakest hydrolytic activity. In most nuclei, however, the
neuropil remained non-reactive. The perikarya of many
neurons showed moderate concentrations of esterases in a
perinuclear position. The occasional solitary hyperactive
cells seen within the medulla alSo exhibited this perinu-
clear distribution. In addition, the hydrolytic activity

appeared to be continuous with that surrounding the axis

cylinder (Fig. 9c).

SPECIAL AREAS OF ENZYME ACTIVITY
Pericytes . _ A - '7 1'

Throughout the brain centers considered in this study,
large cells identified as pericytes were observed. They
were extremely reactive to esterase incubating medium, but
when using dehydrogenase solutions they were difficult to
identify and therefore to evaluate. Pearse (1956) discussed
the enzymatic activity of pericytes and concluded that al-
though these cells were intensely reactive to indoxyl acetate,
an actual esterase was not being shown. Rather, he stated
that "the esterase of the pericytes is not a true esterase

but an intracellular peptidase or cathepsin".

-39-

Though found to be distinct from the endothelial cells,
pericytes appear to be wrapped about the vessel. Farquhar
and Hartman (1956), and Donahue and Pappas (1961) have re-
ported the Specific location of these cells, noting that they
are external to the endothelium but are surrounded by a
basement membrane which splits to enclose them.

Landers, Chason, Gonzalez, and Palutke (1962) considered
the possibility that pericapillary cells were of reticulo-
endothelial origin, but could detect none of the prOperties
related to such cells, i.e., phagocytosis or acid phos-
phatase activity. Therefore, they cOncluded that on the
basis of "morphologic relationships, enzymatic activities,
and lack of phagocytic ability....that these cells are a
modified form of smooth muscle". It was not implied,
however, that pericytes were necessarily contractile in
function. Thus, at the present time, the natural activity
of these cells remains unknown.

Choroid.§lgxg§

Ependyma and choroid plexuses consistently exhibited
intense dehydrogenase and esterase activity; Hess and
Pearse (1961) found mitochondrial asglycerOphOSphate dehy-
drogenase activity to be high in the ependyma and choroid
plexuses. Lazarus 22.21! (1962) reported similar findings
for both lactate and glucose-6-phOSphate dehydrogenases.
Alpha-glycerOphOSphate, lactate, glutamate, and alcohol
dehydrogenase activity of the choroid plexus epithelium
consistently exceeded that of the adjacent ependymal cells,
irrespective of the definite continuity of the two.‘

-40-

When working with white laboratory mice, rats, guinea
pigs, and golden hamsters, Bartonicek and Lojda (1966)
Obtained essentially the same results. Furthermore, they
supported the work of Thomas and Pearse (1961), who found
that the luminal half of the ependyma was more reactive
than the basal portion. This investigator was neither able
to confirm the luminal enzyme concentration nor refute its
existence. The majority of the plexuses examined contained
a relatively uniform distribution of formazan granules.
Because of the orientation of these oxidative enzymes,
Thomas and Pearse (1961) contended that the ependymal cells
probably secrete substances into the ventricular lumen.

Choroid plexus epithelium within the same ventricle
varied considerably in the degree of enzyme activity, but'
each epithelial cell contained granules that were evenly
distributed throughout the cytoplasm. This agrees with the
observations of Bartonicek and Lojda (1966), who felt that
this variation in cell activity actually indicated different
phases of metabolic activity. The capillaries surrounded
by the choroid plexus epithelium Were not enzymatically
reactive which is Similar to the results of Bartonicek and
(Lojda (1966), and Felgenhauer and Stammler (1962).

When utilizing arnaphthyl acetate or indoxyl acetate
as substrates for esterase activity, a strong positive
reaction was Observed. This is in agreement with the find-
ings of Fishman and Hayashi (1962) and Bartonicek and Lojda
(1964). The distribution did not differ significantly from

~41-
that exhibited by the dehydrogenases, as the present study
utilized the same animal species throughout. However, ‘
Bartonicek and Lojda (1964) found that the type of hydro-
1ytic enzyme detected varied considerably from species to
Species, but not among members of the same species. The
presence of the several oxidative and hydrolytic enzymes
is a strong indication that the choroid plexuses play an
important role in the formation of cerebrospinal fluid.
{Basal Ganglia (Qgrpgs Striatum)

(The basal ganglia were found to be exceedingly rich in
esterase activity, whereas the surrounding tissue exhibited
only weak hydrolytic activity. Only the choroid plexuses
rivaled this area in the intensity of esterase reaction.
Examination of NAD-linked dehydrogenases in the same area
showed moderate enzyme activity which was no different than
that in adjacent tissues. That is, the caudate and lenti-
cular nuclei were not unique in the intensity of their oxi-
dative enzyme activity. This is in contrast to the findings
Of Thomas and Pearse (1961), which indicated a very high
dehydrogenase activity in two components of the basal ganglia
(putamen and pallidum). In the present study, glycolytic
activity within this region was considered to be moderate,
as both a-glycerophosphate and lactate dehydrogenases (MTT)
produced moderate amounts of formazan. The same situation
existed for enzymes of the citric acid energy cycle, in

that malate (MTT), alcohol, and glutamate (Nitro-BT)

-42-
dehydrogenase activities were Observed, but no greater than
that found in other tissues of the telencephalon. The
possible utilization of the pentose pathway in this area
could not be ascertained, since glucose-6-phosphate dehy-
drogenase could not be demonstrated.

Examination of the esterase-active tissue in the basal
ganglia indicated that both perikarya and neuropil stained
with equal intensity. The circular, non-reactive areas
were apparently the individual nuclei. The basal ganglia
contain fiber connections, making this region a relay
center, as it appears to be in rats (Zeman and Innes, 1963).
At the present time, the exact significance of a concentra-

tion of highly-active esterases in this region is not clear.'

NOTHING DEHYDROGENASE REACTIONS

During this investigation, seven NAD-linked dehydro-
genases were Studied for their distribution and their inten-
sity in the various brain centers. Five of these enzymes
were analyzed by using MTT at pH 7.2 as the electron acceptor.
Three enzymes, upglycerOphOSphate, alcohol, and glutamate
dehydrogenases, were studied by means of Nitro-BT (also at
pH 7.2). The activity of aeglycerophosphate dehydrogenase
was examined by using both of the tetrazolium salts (in
separate tissue sections). Just NitrO-BT was utilized with
the other two enzymes.

Distinct formazan deposits were produced with the

addition of appropriate substrates. In addition to Speci-

-43-
fically-localized granules, a diffuse positive reaction
was observed. However, sections immersed in incubating
medium (Nitro-BT) without exogenous substrates exhibited
considerable amOunts of formazan granules. These deposits
were equal in intensity and similar in distribution to both
alcohol and glutamate dehydrogenase activities. Therefore,
the accuracy of certain enzyme localizations reported
herein is subject to question, and the possibility of false
localization must be considered.

Zimmermann and Pearse (1959) reported similar findings
with other NAD-linked dehydrogenases. By using particular
inhibitors, they fOund that sulfhydryl groups (SH) appar-
ently reduced NAD or NADP to NADH and NADPH, respectively.
If the appropriate diaphorases are present in the tissue,
these substances can pick up and transfer electrons directly
to the tetrazolium salt, with the production of formazan.
Adding support to this concept are the findings of Racker
(1955) and Nachlas, Walker, and Seligman (1958), Which
indicated that the final localization of formazan appeared
to resemble the pattern formed by the histochemical reactions
for the diaphorases.

Thomas and Pearse (1961) reported Similar distribution
patterns in nerve tissue for glutamate dehydrogenase and
control solutions containing Nitro-BT. Pearse and Hess
(1961) concluded that the great affinity (substantivity)

of NitrO-BT for lip0protein structures was reaponSible for

this "nothing dehydrogenase" reaction.

-44-

The recent efforts of Shaw and Koen (1965) have shown
that alcohol dehydrogenase may be the major agent of "nothing
dehydrogenase" activity. They determined that pure alcohol
dehydrogenase reduced the tetrazolium salt in the absence
of added substrate. Furthermore, liver extracts with no
substrate were electrOphoretically compared to pure alcohol
‘dehydrogenase. The incubating medium contained ethyl
alcohOl and no substrate. The resulting protein bands were
identical. The sulfhydryl reagent parahydroxy-mercuri-
benante completely inhibited the appearance of the bands
both with-and-without substrate. However, Shaw and Koen
(1965) were unable to explain the basis for the hydrogen
(electron)-transferring behavior of alcohol dehydrogenase
in the absence of added substrate.

Thus it appears that both experimental and control
sections incubated in Nitro-BT may actually be presenting
the localization of endogenous alcohol dehydrogenase.
Results obtained during this investigation support this

conclusion.

SUMMARY

Oxidative and hydrolytic enzyme localizations and
intensities were studied within the central nervous
system using slide histochemical methods. Fresh-frozen
sagittal sections of brain were prepared from mice of the
species Peromyscus polionotus. The NAD-dependent dehy-
drogenases, i.e., a-glycerOphosphate, glucose-6—phosphate,
isocitrate, lactate, and malate were identified by using
the monotetrazolium salt MTT. Two other NAD-linked enzymes,
alcohol and glutamate dehydrogenases, were analyzed with
the use of Nitro-BT. Esterases were localized by both the
d-naphthyl acetate and the indoxyl acetate methods.

Four brain centers were examined in detail; namely,
the cerebral cortex, cerebellar cortex, superior colliculus,
and the medulla oblongata. Pericapillary cells, choroid
plexuses, and the basal ganglia were also Observed in
detail.

In the cerebral cortex, amidst relatively uniform
dehydrogenase distribution, slightly greater enzyme activity
was noted in the neurons of layers III and V. Enzymes of
both the glycolytic and the citric acid energy cycles were
present. Very little enzyme activity was obtained for
isocitrate dehydrogenase (NAD-dependent), none for glucose-
6-phosphate dehydrogenase. Hydrolytic enzyme activity was
centered in the perikarya of pyramidal cells in layers III

and V.
-45-

(all-[I‘ll Il‘bln'lw : [(llll'l A 'lllfll‘ll) II A

, ~46.

In the cerebellar cortex, glomeruli cerebellosi
showed the strongest dehydrogenase activity, but Purkinje
cells were also prominent, exhibiting moderate activity.
The high reaction within the glomeruli was correlated
with a concentration of synaptic endings in the same
areas. Purkinje cells contained the greatest esterase
activity, whereas glomeruli were considerably reduced.

The stratum griseum superficiale, an intermediate
layer in the superior colliculus and known to contain
abundant synapses, exhibited high metabolic activity. The
formazan distribution was stratified, indicating consider-
able complexity, in contrast to the inferiOr colliculus,
where layering was absent. The superior colliculus showed
strong esterase activity, whereas little was Observed in
the inferior colIiculus. Commissural tracts deep within
the colliculus (in the stratum lemnisci) exhibited strong
hydrolytic enzyme activity. A

Metabolic activity in the medulla oblongata was pri-
marily localized in the regional nuclei, but varied greatly
in intensity. In some nuclei the perikarya was highly
active; in others, the neuropil was predominant. Scattered
throughout the general gray matter of the medulla were
solitary cells that were intensely active for esterases.

Esterase activity within pericytes was extremely high,
but distinct enough to Show moderate levels in the endothel-
ium. Oxidative enzyme activity was difficult to ascertain
within these cells.

-47-

Choroid plexus epithelium exhibited wide variations
of dehydrogenase activity in the same ventricle with the
same enzyme. It was concluded that this indicated a
fluctuation in the metabolic activity. The distribution
and intensity of oxidative and hydrolytic enzymes remained
consistent between different ventricles of the same mouse
and between ventricles of different mice of the same
species.

. The basal ganglia showed very strong esterase activity
especially in the caudate and lenticular nuclei. The dehy-
drogenases were moderately active in these nuclei and
also in the surrounding tissues, which made differentiation
of the separate areas almost impossible.

Results obtained when using Nitro-BT must be regarded
as inaccurate and possibly false. Control sections were
quite positive, and similar in all respects to the locali-
-zation given by alcohol dehydrogenase. Thus the highly-
1ipoprOtein character of brain tissue appears to cause a

"nothing dehydrogenase" reaction when Nitro-BT is employed.

alt." II In! {I III 'III(

1 {I [I‘ll .I’l [I 1)

LITERATURE CITED

.Adams, C.W.M., A.N. Davison, and N.A. Gregson. 1963.
“ Enzyme inactivity of myelin; histochemical and bio-
chemical evidence. J. Neurochem., 10: 383.

Barrnett, R.J., and A.M. Seligman. 1951. Histochemical
demonstration of esterases by the production of indigo.
Science, 114: 579-582.

Bartonicek, V., and Z. Lojda. 1964. TOpochemistry of
enzymes of choroid plexus and ependyma of four animal
species. I. Hydrolytic enzymes. Acta Histochem.,‘
19: 357-368.

Bartonicek, V., and Z. Lojda. 1966. TOpochemistry Of
enzymes of choroid plexus and ependyma of four animal
species. II. Diaghorases and dehydrogenases. Acta
Histochem., 23: ll ~126.

Chason, J.L., and A.G.E. Pearse. 1961. Phenazine methosul-
phate and nicotinamide in the histochemical demonstra-

tion of dehydrogenases in rat brain. J. Neurochem.,

Chason, J.L., J.E. Gonzales, and J;W. Landers. 1963.
Respiratory enzyme activity and distribution in the
postmortem central nervous system. J. Neuropath.
Exp. Neurol., 22: 248-254.

David, G.B., D.F. deAlmeida, G. deO. Castro, and A.N. Brown.
1962. The fine localization of certain hydrolytic,
oxidative and hydrogen-transfer enzymes within living
nerve cells of vertebrates. International symposium
on enzymic activity of the central nervous system.
Abstracts. Acta Neurol. Scand., 38 (Suppl. 1).

Delellis, R., and R. Fishman. 1965. The variable of pH in
the bromo-indoxyl acetate method for the demonstration
of esterase. Letters to Editor. J. Histochem. Cyto-
chem., 13: 297. -

Donahue, S., and G.D. Pappas. 1961. The fine structure Of
capillaries in the cerebral cortex of the rat at various
stages of develOpment. Amer. J. Anat., 108: 331.

Farquhar, M.G., and J.F. Hartman. 1956. Electron micro-
scOpy of cerebral capillaries. Anat. Rec., 124: 288.

-48-

-49-

Felgenhauer, K., and A. Stammler. 1962. Das Verteilungs-
muster der Dehydrogenasen and Diaphorasen im Zentral-
nervensystem deS Meerschweinchens. Z. Zellforsch.,

58: 219-233.

Fishman, J.S., and M. Hayashi. 1962. Enzymorphology of
rat brain; beta-glucuronidase, alkaline phosphatase,
esterase. J. Histochem. Cytochem., 10: 515-519.

Friede, R.L. 1959a. Histochemical investigations on
succinic dehydrogenase in the central nervous system.
I. The postnatal develOpment of the rat brain.
J. Neurochem., 4: 101-110.

Friede, R.L. 1959b. Histochemical investigations on
succinic dehydrogenase in the central nervous system.
II. Atlas of the medulla oblongata of the guinea pig.
J. Neurochem., 4: 111-123.

Friede, R.L. 1959c. Histochemical investigations on
succinic dehydrogenase'in the central nervous system.
III. Atlas of the midbrain of the guinea pig, includ-
ing pons and cerebellum. J. Neurochem., 4: 290-303.

Friede, R.L. 1960. Histochemical investigations on succinic
dehydrogenase in the central nervous system. IV. A
histochemical mapping of the cerebral cortex of the
guinea pig. J. NeuroChem., 5:156-171.

Friede, R.L. 1961. Histochemical investigations on succinic
dehydrogenase in the central nervous system. V. The
diencephalon and basal telencephalic centers in the
guinea pig. J. Neurochem., 6: 190-199.

Friede, R. L., and L. M. Fleming. 1962. A mapping of oxi-
dative enzymes in the human brain. J. Neurochem.,
9: 179-198.

Friede, R.L., L.M. Fleming, and M. Knoller. 1963. A com-
parative mapping of enzymes involved in HMP shunt and
citric acid cycle in the brain. J. Neurochem., 10:

2 3 277

Friede, R.L., and R.A. Pax. 1961. Mitochondria and mito-
chondrial enzymes. A comparative study of localization
in the cat's brain stem. Histochemie, 2: 186-191.

Gomori, G. 1952. Histochemistry of esterases. Int. Rev.
CYL. , 1: 323-3350

Hess, R., and A. G. E. Pearse. 1961. Histochemical and homo-
genization studies of mitochondrial alpha-glycerOphos-
phate dehydrogenase in the nervous system. Nature,

191. 718-719

11.41)") (llllulclllll‘lalllllll All.) 'llllu'l‘ll‘lli)lll|ll,lll(lll" .

-50-

Hess, R., D.G. Scarpelli, and A.G.E. Pearse. 1958. The
cytochemical localization Of oxidative enzymes. 11.
Pyridine nucleotide-linked dehydrogenases. J. Bio-
phys. Biochem. Cytol., 4: 753- 760.

Holt, 8. J. 1952. A new principle for the histochemical
localization of hydrolytic enzymes. Nature, 169:

271-273-

Holt, 8. J. 1954. A new approach to the cytochemical
iggalization of enzymes. Proc. Roy. Soc., B 142:
-1 9 .

Holt, S.J. 1958. Indigogenic staining methods for
esterases, p. 375-39 . In "General Cytochemical
[Methods", Ed. J.F. Danieilli, Vol. I.

Holt, S.J., and R.F.J.'Withers. 1952. Cytochemical
localization of esterases using indoxyl derivatives.
Nature, 170: 1012-1014.

Huber, G. C., and E. C. Crosby. 1943 A comparison of the
mammalian and reptilian tecta. J. Comp. Neurol.,

78: 133- 190.

Kluver ., and E. Barrera. 1953. A method for the com-
bined staining of cells and fibers in the nervous
system. J. Neuropath. Exp. Neurol., 12: 400-403.

Koelle, G. B. 1954 The histochemical localization of chol-
inesterases in the central nervous system of the rat.
J. Comp. Neurol., 100: 211-235.

Lenders J. W., J. L. Chason, J. E. Gonzalez, and'W.Pa1utke.
1962 Morphology and enzymatic activity of rat cere-
bral capillaries. Lab. Invest., 11: 1253-1259.

Lazarus s. s., B. J. Wallace, G. w. F. Edgar, and B. w. Volk.
1962. Enzyme localization in rabbit cerebellum and
effect of postmortem autolysis._ J. Neurochem., 9:
227-232.

Nachlas, M.M., and A.M. Seligman. 1949. The comparative
distribution of esterase in the tissues of five mam-
mals by a histochemical technique. Anat. Rec.,

105: 677-695

Nachlas, M .M., D. G. ‘Walker, and A. M. Seligman. 1958.
A histochemical method for the demonstration of DPN
nucleotide diaphorase. J. BiOphys. Biochem. Cytol.,

4: 29-35.

-51-

Nandy, K., and G.H. Bourne. 1964a. A histochemical study
of the localization of the oxidative enzymes in the
neurons of the spinal cord in rats. J. Anat., 98:

647-653-

Nandy, K., and G.H. Bourne. 1964b. A histochemical study
of the localization of SDH, CYO, and DPN-diaphorase
in the synaptic regions of the spinal cord in the rat.
J. Histochem. Cytochem., 12:188-193.

Nandy, K., and G. H. Bourne. 1966. Histochemical study
of the oxidative enzymes in the olfactory bulb of
the rat. Acta Histochem., 23:86-93.

Pearse, A. G. E. 1956. Esterases of the hypothalamus and
neurohypOphysis and their functional significance.
Pathophysiologica Diencephalica, International
Symposium. May, Milan.

Pearse, A.G.E. 1957. Intracellular localization of
dehydrogenase systems using monotetrazolium salts
and metal chelation of their formazans. J. Histo-
chem. Cytochem., 5: 515-527.

Pearse, A.G.E. 1960. Histochemistry theoretical and
ap lied. 2nd ed. J. and A. Churchill, Ltd., London.

99

Pearse, A. G. E., and R. Hess. 1961. Substantivity and
other factors responsible for formazan patterns in
dehydrogenase histochemistry. Experientia (Basel),
17: 136-141.

POpe, A., H.H. Hess, J.R. Ware, and H.H. Thomson. 1956.
Intralaminar distribution of cytochrome oxidase and
DPN in rat cerebral cortex. J. NeurOphysiol., 19:

259-270.

Racker, E. 1955. The mechanism of action and the prOper-
ties of pyridine-linked enzymes. Physiol. Rev.,

35: 1-56.

Robins, E. 1960. The chemical composition of central
tracts and of the nerve cell bodies. J. Histochem.
Cytochem., 8: 431-436.

Romanul, F.C.A., and R.B. Cohen. 1960. A histochemical
study of dehydrogenases in the central and peripheral
nervous systems. J. NeurOpath. exp. Neurol., 19:

135-136.

Shaw, C.R., and A.L. Koen. 1965. On the identity of
"nothing dehydrogenase". J. Histochem. Cytochem.,

13: 431-433.

II. I all [ I I all] «IIIAII. '1» (III I) A

-52-

Sidman, R.L. 1960. Localization of chemicals in histo-
logical sections of mammalian nervous system. J.
Histochem. Cytochem., 8: 412-418.

Takemori, A.E. 1965. Effect of central depressant agents
on cerebral glucose-6-phosphate dehydrogenase activity
of rats. J. Neurochem., 12: 407-415.

Tewari, R.B., and G.H. Bourne.' l962a. Histochemical
evidence of metabolic cycles in spinal ganglion cells
of rat. J. Histochem. Cytochem., 10: 42-64.

Tewari, R.B., and G.H. Bourne. l962b. Histochemical
studies on the distribution of Specific and non-
specific cholinesterases and simple esterase in the
cerebellum Of the rat. Acta Anat., 51: 349-368.

Tewari, R.B., and G.H. Bourne. 1962c. Some new aspects
of the nucleo-cytoplasmic relationship in neurons of
rat. J. Histochem. Cytochem., Letter to Editor.,
10: 767-768.

Tewari, R.B., and G.H. Bourne. 1962d. Histochemical
studies on the distribution of oxidative enzymes in
the cerebellum of the rat. J. Histochem. Cytochem.,
10: 619-627.

Tewari, R.B., and G.H. Bourne. l962e. A comparative study
on the distribution of certain enzymes and their meta-
bolic significance in the cerebellum of the rat.
Abstracts. Acta Neurol. Scand., 38 (Suppl. 1).

“Thomas, E., and A.G.E. Pearse. 1961. The fine localization
of dggydgogenases in the nervous System. Histochemie,
2: 2 2 2.

Tsou, K-C., C.S. Cheng, MQM. Nachlas, and A.M. Seligman.
, 1956. J. Amer. Chem. Soc., 78: 6139.

White A., P. Handler, E.L. Smith, and D. Stetten. 1959.

Principles of biochemistry. 2nd ed. McGraw-Hill Book

Company, Inc., New York. 1149 p.

Wolfgram, 1., and A.S. Rose. 1959. The histochemical
demonstration of dehydrogenases in neuroglia. Exp.
Cell Res., 17: 526-530.

Zeman, WT, and J.R.M. Innes. 1963. Craigie's neuroanatomy
of the rat. Academic Press, New York. 230 p.

Zimmermann, H., and A.G.E. Pearse. 1959. Limitations in
the histochemical demonstration of pyridine nucleotide-
linked dehydrogenases ("nothing dehydrogenase").

J. Histochem. Cytochem., 7: 271-275.

(III 0" All]! ill-III! II (I. Alt-Ill ‘- llil I. I [I'll '1'- l.i ll! 1"). A I 4| r.

-53-

Table 1. Enzymatic Activity of Brain Regions

Dehydrogenases of the Glycolytic Energy Cyc1e*

Region Alpha-GPDH ADH LDH
A B C A B C A B C

Cerebellar Cortex

Purkinje cells 2 0-4 (13) 2 1-3 (5) 1 0-2 (6)
Molecular layer 2 0-3 (13) 2 0-3 (5) 1 0-2 (6)
Granular layer 2 0-4 (13) 2 0-3 (5) 1% 0-2 (6)
White matter 0 0-1 (13) 0 0-2 (5) 0 0-1 (6)
Cerebral Cortex .
Layer No. I 2 0-3 (9) 0 0-1 (4) 1 0-2 (8)
Layer No. II 2 0-3 (9) 1 0-2 (4) 2 0-2 (8)
Layer No. III 2 0-3 (9) 1%; 0-2 (4) 2 0-2 (8)
Layer No. IV 2 0-3 (9) 2 0-2 (4) 2 0-2 (8)
Layer No. V 2 0-3 (9) 2 0-2 (4) 2 0-2 (8)
Layer No. VI ‘2 0-3 (9) 1% 0-3 (4) 1 0-2 (8)
Corpus Callosum 1 0-2 (9) 0 0-1 (4) 1 0-2 (8)
Medulla Oblongata
Inf. Olivary Nucl. % 0-2 (9) 1 0-1 (5) 0 0-2 (4)
Neurons 1 0-4 (9) 1 0-3 (5) 0 0-3 (4)
Tracts 1 0-1 (9) 2 1-2 (5) 1 0-2 (4)
Neurophil 2 0-2 (9) 1 0-2 (5) 1 0-2 (4)
Superior Colliculus A
Zonal layer 0 0-2 (11) 0 0-1 (5) 0 0 (6)
Stratum Griseum 2% 0-3 (11) 1 0-1 (5) 1% 0-2 (6)
Stratum Opticum 2 0-3 (11) 1 0-3 (5) 2 0-2 (6)

Stratum Lemniscus .
and A1. Profundum 2 0-3 (11) 3 0-3 (5) 2 0-2 (6)

* The number in Column A represents the average intensity of enzymatic
activity for the designated structure, based on a subjective evaluation
by this investigator: 0, no activity; 1, weak activity; 2, moderate
activity; 3, strong activity; 4, intense activity.

The value in Column B is the range of enzymatic activity.

The number within the parentheses (in Column C) indicates the number of
mice analyzed for each enzyme.

-54-

Table 2. Enzymatic Activity of Brain Regions

Dehydrogenases of the Krebs Cycle*

Region IDH GDH MDH

Cerebellar Cortex

Purkinje cells 1 0-2 (8) 3 0-4 (3) 1% 0-2 (6)
Molecular layer 1 0-2 (8) 2% 0-3 (3) 1 0-1 (6)
Granular layer 1 0-2 (8) 3 0-4 (3) 1% 0-2 (6)
White matter 0 O (8) 0 0-1 (3) 0 0-1 (6)
Cerebral Cortex
Layer No. I 0 0-1 (8) 1 0-1 (4) 2 0-3 (6)
Layer No. II 0 0-1 (8) 1 0-1 (4) 2 0-2 (6)
Layer No. III 0 0-1 (8) 2 0-3 (4) 2 0-2 (6)
Layer No. IV 0 0-1 (8) 2 0-3 (4) 2 0-2 (6)
Layer No. V 0 0-1 (8) 2 0-3 (4) 2 0-2 (6)
Layer No. VI -0 0-1 (8) 28 0-3 (4) 1 0-2 (6)
Corpus Callosum 0 0-1 (8) 1 0-1 (4) 1 0-1 (6)
Medulla Oblongata
Inf. Olivary Nucl. 0 0 (6) 1 0-3 (4) 0 0-2 (4)
Neurons 0 O (6) 1% 0-3 (4) 0 0-2 (4)
Tracts 0 O (6) 0 0-1 (4) 3 0-1 (4)
Neuropil 0 0-1 (6) 2 0-3 (4) 1 0-2 (4)
Superior Colliculus
Zonal layer 0 0-1 (8) 1 0-3 (5) 0 0-1 (5)
Stratum Griseum 0 0-1 (8) 18 1-3 (5) 2 0-3 (5)
Stratum Opticum 0 0-1 (8) 2 0-2 (5) 1 0-2 (5)
Stratum Lemniscus .
and A1. Profhndum 0 0-1 (8) 2 0-2 (5) 2 0-2 (5)

* For explanation of symbols, see Table l.

Table 3.

Region

Cerebellar Cortex
Purkinje cells
Molecular layer
Granular layer
White matter

Cerebral Cortex
Layer No. I
Layer No. II
Layer No. III
Layer No. IV
Layer No. V
Layer No. VI
Corpus Callosum

Medulla Oblongata
Inf. Olivary Nucl.
Neurons
Tracts
Neuropil

Superior Colliculus
Zonal layer
Stratum Griseum
Stratum Opticum
Stratum Lemniscus

and Al. Profhndum

* For explanation of symbols, see Table 1.

Enzymatic Activity of Brain Regions

A.

ONMHNNH MONO!

NONN

HNM

Carboxylic Esterases*

Indoxyl

Acetate Esterase
B C
0-4 (4)
1-3 (4)
0-2 (4)
0-3 (4)
0-2 (4)
0-2 (4)
0-3 (4)
1-2 (4)
1-3 (4)
l—3 (4)
0-1 (4)
1-4 (4)
3-4 (4)
0-1 (4)
1-3 (4)
0-3 (4)
1-3 (4)
1-2 (4)
1-3 (4)

a. -Naphthy1

Acetate Esterase
A B C
2 1-3 (5)
2 0-2 (5)
1 0-2 (5)
0 0-1 (5)
0 0-2 (5)
0 - 0-1 (5)
1 0-3 (5)
1 0-2 (5)
2 0-3 (5)
0 0-2 (5)
0 0-1 (5)
1 0-3 (6)
2 0—3 (6)
0 0-2 (6)
l _ 0-2 (6)
18 0-4 (6)
2 0-3 (6)
1 0-2 (6)
2 0-3 (6)

x ‘ II ‘Il‘t'lllt‘ l I hurl. ‘l.l|‘{‘ ll ‘ ll“ l 1"“ {I'll I I II (If r:

-55-

Fig. la. Cerebral cortex. Lactate dehydrogenase
activity. The symbol P represents layer I; Q, layers
II and III: R, layer IV; S, layer V: T, layer VI; U,
corpus callosum. No counterstain. MTT.; x 130.

Fig. 1b. Cerebral cortex. Glutamate dehydrogenase
activity localized in non-myelinated nerve fibers. No
counterstain. Nitro-BT.; x 520.

 

-53-

Fig. 2a.’ Cerebral cortex. Esterase activity. See
Fig. la for explanation of symbols. The most intense
activity is exhibited by pericytes (black structures).
Mayer's carmalum counterstain. Indoxyl acetate; x LB.

Fig. 2b. Cerebral cortex. Esterase activity in la
I, II, and III (See Fig. la for explanation of symbols)
mayer's carmalum counterstain. Indoxyl acetate: x 130.

ers'

_— .. _

 

Fig. 3a. Layers of the cerebellar cortex. Lactate
‘dehydrogenase activity in the molecular (M) layer and

in the granular (G) layer. Counterstained in Mayer's

carmalum. MTT.; x 130.

Fig. 3b. Layers of the cerebellar cortex. upglycero-
phosphate dehydrogenase. The letter P designates the
Purkinje cell layer. See Fig. 3a for explanation of other
symbols. Mayer's carmalum. MTT.; x 520. -

Fig. 3c. The cerebellar cortex. "Nothing dehydrogenase"
activity; control section, substrate lacking. The letter W
represents fiber tracts of white matter. See Fig. 3a and
3b for explanation of the other symbols. No counterstain.
Nitro-BT.; x 130.

Fig. 3d.. Purkinje cells bounded by the granular and the
molecular layers. Note the eccentric distribution of the
cytoplasm (arrow) in the Purkinje cells. a-glycerophosphate
dehydrogenase. Mayer’s carmalum. MTT.; x 520.

-61..

' sh ,;

9'.

.“

3.:

’x

.

at'é.‘

J

 

-62-

Fig. ha. Purkinje cell containing a-glycerophOSphate
dehydrogenase. Note the intense perinuclear distribution,
as well as the increased peripheral activity (arrows).
Mayer's carmalum. MTT.; x 1330.

Fig. Ab and Ac. Layers of the cerebellar cortex. Note
the patchy appearance of enzyme within the granular layer
(G). cz-glycerOphosphate dehydrogenase. No counterstain.
Nitro-BT.; Lb, x 130; he, x 260.

' Fig. hd. Gerebellum. Glutamate dehydrogenase, with
no counterstain. Nitro-BT.; x 260.

 

 

   

o ‘.".

    

. i
._.»)...

 

‘v

s I‘l _ 1' ."
.-“"1~“" NI ‘ ~

-6a-

Fig. 5a. Esterase activity in the layers of the cere-
bellar cortex. See Fig. 3c for explanation of the symbols.
Counterstained in Mayer's carmalum. a-naphthyl acetate;

x 130.

Fig. 5b. Purkinje cells. The esterase activity is
concentrated in that portion of the cytOplasm nearest the
molecular layer (M). Counterstained in Mayer's carmalum.
Indoxyl acetate; x 520.

Fig. 5c. Layers of the cerebellar cortex. See Fig.
3c for explanation of the symbols. Esterase activity.
Indoxyl acetate; x 130.

 

-66-

Fig. 6a. Superior colliculus. Glutamate dehydrogenase.
Z represents the zonal layer; GS, the stratum griseum
superficiale; O, the stratum Opticum; L the stratum lem-
nisci; AP, the stratum album profundum (containing com-
missural tracts). The tissue above the zonal layer is the
ventral aspect of the cerebral cortex. No counterstain.
Nitro-BT.; x 48.

Fig. 6b. Superior colliculus. a-glycerophOSphate
dehydrogenase activity. The distinct line coursing diago-
nally across the upper-right corner is the surface of the
colliculus in contact with the cerebral cortex. See Fig.
6a for gxplanation of symbols. No counterstain. Nitro-
BT.; x 3.

-67-

 

-68-

Fig. 7a. The superior colliculus (SC) and the anterior
half of the inferior colliculus (10). Note the greater
esterase activity within the superior colliculus. The
stratum album profundum (AP) exhibits a strong enzyme re-
action. No counterstain. a-naphthyl acetate; x 130.

Fig. 7b. The superior colliculus (SC) and part of the
inferior colliculus (IC). Note the intense esterase ac-
tivity exhibited by pericytes (see arrows). Counterstained
in Mayer's.carmalum. Indoxyl acetate; x 83.

.1
r

.4
Ax.

4%.

' vly’".

I

 

Ago

‘ A
‘ . . . “LY,- ‘ .
“fi‘b. M“.- .“o \

\

.

.vo..)d.r,‘“ § Q.
up .fiv'd‘
.J...D».Ifl

f.

'

 

Fig. 8a. Medulla oblongata in sagittal section.
a-glycerOphOSphate dehydrogenase. The arrows point out
the inferior olivary nucleus. The letters A and P indi-
cate the anterior and posterior ends of the tissue in the
photograph, respectively. A portion of the choroid
plexus of the fourth ventricle is visible in the lower-
right corner of the picture. No counterstain. Nitro-BT.;
x 2h.

Fig. 8b. Ventral portion of the medulla oblongata, at
the level of the inferior olivary nucleus (IN). Note
myelinated fiber tracts (MT). Glutamate dehydrogenase.
Nitro-BT.; x 130.

Fig. 8c. Neurons of a small regional nucleus within
the medulla oblongata. Note the perinuclear concentration
of glutamate dehydrogenase (arrows). No counterstain.
Nitro—BT.; x 570.

Fig. 8d. Hyperactive neurons within the medulla,
exhibiting malate dehydrogenase activity. Mayer's carmal-
um. MTT.; x 260.

 

 

I‘ II. 4" lull! I'll .l‘llu III II' [I III

-72-

Fig. 9a. Regional nucleus within the medulla oblongata.
"Nothing" dehydrogenase activity. Control section; sub-
strate absent from the incubating media. No counterstain.
Nitro-BT.; x 570.

Fig. 9b. A neuron in the medulla oblongata exhibiting
intense esterase activity. Mayer's carmalum counterstain.
Indoxyl acetate; x 1330.

Fig. 9c. Solitary hyperactive neuron of the medulla.
Esterase. No counterstain. a-naphthyl acetate; x 1330.

Fig. 9d. The same neuron shown in 9c, but photographed
while under phase contrast. Note the increased esterase
activit on the periphery, probably on the cell membrane
(arrows . a-naphthyl acetate; x 1330.

 

 

-74-

Fig. 10a. Pericytes at a capillary junction. Note
portion of capillary membrane (endothelium) by arrow.
Esterase. Mayer's carmalum. Indoxyl acetate; x 570.

Fig. 10b. Pericyte. Esterase. Indoxyl acetate; x 570.

Fig. 10c. Pericyte. Arrows delineate endothelial
esterase activity. Mayer's carmalum. Indoxyl acetate;
x 1330. .

Fig. 10d. Pericyte. Note endothelial nucleus (arrow)
made visible by the surrounding esterase activity of the
cytOplasm. Mayer's carmalum. Indoxyl acetate; x 1330.

 

-76-

Fig. lla. Glutamate dehydrogenase activity within the
choroid plexus of the fourth ventricle. The arrows point
out the continuity between the ependyma (E) and the cuboidal
epithelium (C) covering the blood vessels. No counterstain.

Fig. 11b. Choroid plexus of the feurth ventricle.
Laitate dehydrogenase activity. Mayer's carmalum. MTT.;
x 30.

Fig. llc. Choroid plexus of the fourth ventricle.
u-glycerOphosphate dehydrogenase. No counterstain. Nitro-

BT.; x 260.

Fig. 11d. Choroid plexus of the lateral ventricle.
Esterase. Mayer's carmalum. Indoxyl acetate; x 130.

  
 
  
      
    
 

r“

\
.. u
\ o

. .\‘-'
~ ‘ ‘0'"
$55-‘
"‘Frot .‘
I‘d . -'
"‘.‘3.A»»:'“.‘

“-
. ”
fi‘.“. Q

.31:

     

..-

 

-73-

Fig. 12. Lactate dehydrogenase activity within the
chor01d plexus of the fOurth ventricle. Note the varia-
tion in intensity between different portions of the same
plexus. Counterstained with Mayer's carmalum. MTT.;

Phase contrast, x 570.

-79-

 

 

-80-

Figs. 13a-d. The caudate and lenticular nuclei (neo-
striatum) of the basal ganglia. In sagittal section.
Figures l3a-d show the distribution of esterase activity
from.the medial limit of the ganglia to its lateral
boundary. The following letters represent their respective
structures: AC, anterior commissure; CN, caudate nucleus;
GP, choroid plexus of the lateral ventricles; G, genu
of the corpus callosum; LN, lenticular nucleus; 00, Optic
,chiasma; T, thalamus. All sections represented were
counterstained in Mayer's carmalum. a-naphthyl acetate;
x 24.

(The right side of each photograph is the anterior end

ofdthe tissue shown, the left side is the posterior
en .

-8l-

 

 

-32-

Fig. 14a. Esterase activity in the caudate and lenti-
cular nuclei. For explanation of symbols, see Figures
13a-d. No counterstain. Indoxyl acetate; x 2h.

Fig. lhb. Esterase activity in the caudate nucleus.
Mayer's carmalum. a-naphthyl acetate; x 130.

Fig. 1hc. Caudate nucleus. Esterase activity. Nerve
cell nuclei are negative. Cells at the periphery of the
nucleus (arrows) exhibit reduced activity. a-naphthyl
acetate; x 260. _

 

"'iliifilflnfifllilfifififlffliflimjmlfim‘5