ACRID!NE ORANGE FLUORESCENCE
MICROSPECTROPHOTOMETRY N THE ANALYSIS OF
NUCLElC ACIDS lN D!FFERENT MICROENWRONMENTS AND
IN SUNGLE NEURONS

Thesis for the Degree of M. S.
MECH!GAN STATE UNWERSWY
ROBERT GENE CANADA
1976

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ABSTRACT

ACRIDINE ORANGE FLUORESCENCE MICROSPECTROPHOTOMETRY
IN THE ANALYSIS OF NUCLEIC ACIDS IN DIFFERENT MICROENVIRONMENTS
AND IN SINGLE NEURONS
By

Robert Gene Canada

The application of biophysical cytochemistry to single neurons in
culture represents a model system whereby inferences. anent the structure
and functions of a biopolymer engaged in neuronal interactions, may be
devised by studying the interactions of a fluorescent molecular probe
and the macromolecule under question. A large segment of this investi-
gation was concerned with the binding and structure characterization of
acridine orange--nucleic acid complexes embedded in gelatin microdroplets,
exposed to various microenvironmental conditions. This investigation
affirms that acridine orange (A0) has the same binding mode and a
specific affinity for each nucleic acid (NA) conformation investigated
(rRNA, DNA, Poly U, and denatured rRNA), where each AO-NA complex has
a green fluorescence maximum at 536 nm. and a prominent shoulder towards
the longer wavelengths at 604 nm. The alteration of the NA microenvir-
onment altered the binding of A0 to the NA, whereby increasing the AO-NA
interaction time, NA denaturation, pH, and dye concentration increased
the binding of A0 to the NA. In addition, the binding of A0 to the NA

was enhanced in the presence of NaCl. More importantly, there was a

Robert Gene Canada

linearity between the A0-NA emission and the amount of NA available

for binding an unchanging A0 concentration. Fluorescence coefficients
for specific staining conditions were procured and employed in the
calculation of the NA content inside the soma of single neurons iden-
tified in dissociated cell cultures of the rat brain. The NA content
per neuron was found to depend more upon neuronal type and maturity

than size. The binding of A0 to the NA in the neurons and microdroplets

is predominantly in the monomer form with a small degree of aggregation.

ACRIDINE ORANGE FLUORESCENCE MICROSPECTROPHOTOMETRY
IN THE ANALYSIS OF NUCLEIC ACIDS IN DIFFERENT MICROENVIRONMENTS
AND IN SINGLE NEURONS
By

Robert Gene Canada

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Biophysics

1976

@ Copyright by
ROBERT GENE CANADA
1976

a

DEDICATION

To Donna

I thank whatever God there be,'

For the Love, Peace, and Happiness You have given Me.

ii

ACKNOWLEDGMENTS

I wish to communicate my profound appreciation to: Dr. John I.
Johnson, Jr., for his services, advice and support as chairman of my
Masters Thesis research committee and my primary adviser; Dr. Eloise
Kuntz, who gave me my start in Biophysics and who acquainted me with
quantitative scientific techniques of superior quality and served as
a committee member. I gratefully extend my special thanks to Dr.
Richard N. Wagner who introduced me to his dissociated cell culture
techniques and who provided assistance and encouragement throughout
the course of this work; Mrs. Donna Maria Trotter Canada who supplied
the necessary ingredients to make my research a success.

This research was supported by funds from NIH Research Grants No.
N505982. NIH Training Grant No. GM-Dl422. the College of Osteopathic
Medicine, the College of Natural Science. and the College of Human

Medicine of Michigan State University.

iii

TABLE OF CONTENTS

List of Tables ........................

List of Figures .......................

I. INTRODUCTION .......................

D OW)

Biophysical Cytochemistry ..............
The Metachromasy of Acridine Orange .........

The Relationship Between Nucleic Acids and Neuronal
Activities ......................

Neuron Cultures ..... . . . - ..........

II.- EXPERIMENTAL DETAILS ..................

A.
B.

III.

Materials ......................
Methods . . . ....................

l. Microdroplet procedures .............
2. Neuron Culture procedures ............

Instrumentation ...................

RESULTS AND DISCUSSION .................

Introduction .....................
Microdroplet Analysis ................

l. The fluorescence changes of AO-NA complexes as a
function of acridine orange staining concentration
The fluorescence changes of AO-NA complexes as a
function of acridine orange staining time .
The fluorescence changes of AO-NA complexes as a
function of acridine orange staining solution pH .
The fluorescence intensity changes of A0~NA com-
plexes as a function of NaCl content .......
The fluorescence intensities of A0-rRNA complexes
at various microdroplet forming solution
temperatures ...................
6. The fluorescence intensity changes of AO-NA,
gelatin-protein complexes during continuous
irradiation ........ . . . . . . . . . . .

01 45 on N
o o o 0

iv

Page
vi

viii

—I

one (JO-d

23
24

24
26

29
33
33
37
39
45
50
58

64

65

Page

7. The fluorescence intensity changes of AO-NA
complexes as a function of the nucleic acid con-
centration, and the quantitative determination
of nucleic acids contained in a given micro-

environment .................... 69

C. Neuron Analysis .................... 79

IV. CONCLUSIONS ....................... 125
LIST OF REFERENCES ...................... T34

Table

LIST OF TABLES

Page

The relative fluorescence intensity of AO-NA complexes
at 536 nm. and 604 nm ................. 36

The average relative fluorescence intensities of A0-3%
purified pigskin only and AO-NA complexes in 3% pf.

pgsk. at 536 nm. and 604 nm. for different acridine

orange staining concentrations (expressed in molar-

ities), pH 4.1, staining time 15 minutes ....... 41

The average relative fluorescence intensities of AO-NA
complexes in 3% pf. pgsk. at 536 nm. and 604 nm. for
different staining times (expressed in minutes)
acridine orange staining concentration, 5 x 10-5M,

pH 4.1 ................... . . . 46
The average relative fluorescence intensities of 1.0

pg. DNA-A0 and 1.0 pg. rRNA-AO complexes in 3% pf.

pgsk.. and gelatin protein-A0 complexes of 3% purified
pigskin only, at 536 nm. and 604 nm., for different

acridine orange staining solution pH. A0 staining
concentration 5 x 10-5M. staining time 30 mins. . . . 51

The average relative fluorescence intensities of 0.5

pg. DNA-A0 complexes and 1.0 pg. rRNA-AD complexes in

3% pf. pgsk., at 536 nm. and 604 nm.. for various

NaCl contents expressed in picograms. A0 staining
concentration 5 x 10-5M, staining time 30 mins..

pH 4.1 .......... . . . ........... 60

The average relative fluorescence intensities of

1.0 pg. rRNA-AO complexes in 3% pf. pgsk.. at 536 nm.

and 604 nm.. for various microdroplet forming sglution
temperatures. A0 staining concentration 5 x 0' M. at

pH 4.1, staining time 30 mins ............. 65

The average relative fluorescence intensities of A0

bound to DNA or rRNA, at 536 nm. and 604 nm.. in 3%

pf. pgsk. for various DNA and ERNA concentrations, A0
staining concentration 5 x 10' M, staining time 30

minutes, pH 4.1 ........ . .......... 70

vi

Table

Page

The nucleic acid content of 3% purified pigskin micro-
droplets, derived from the sum of the embedded pg.

rRNA + 4.63 (pg. DNA) on the left side of equation (4),
is compared to its value on the right side of the equa-
tion, obtained fr°m[(log F536 _ log b)/f.c.g§gA]' The
fluorescence spectrum and intensity, at 536 nm., of each
microdroplet was obtained with the microspectrophotometer
while irradiating at 400 nm. The average relative fluo-
rescence intensity of 3% pf. pgsk. microdroplets, con-
taining no NA, at 536 nm. is equal to 54.5. The micro-
droplets were stained for 30 mins., with 5 x 10'5M A0,

at pH 4.1 ....................... 78

The R + yD content in the cell bodies of Canada, II,
and Canada, bipolar neurons, at 67 da. and 99 da. old,
as determined microspectrophotometrical1y using equa-
tion (4), R + 70 = (log F536 - log b)/ f.c.§§g , where

y is equal to 4.63 and f.c.g§2A is equal to 0.272. Cells

#1 through 7 were stained with l x 10'4M A0, and neurons

#8 through 12 were stained with 5 x 10'5M A0 for 30 mins.
at a pH 4.1. All of the cells were taken from the

cerebral cortex of a 3 da. old albino female rat and
maintainedin dissociated cell cultures. Neurons #8
through 12 are from the somatic sensory cortex I and II. 93

vii

Figure

LIST OF FIGURES

Absorption spectra of acridine orange in aqueous
solution. Solvent: citrate-phosphate buffer,
pH 6.0, 20°C (from Zanker, 1952) ...........

Fluorescence spectra of acridine orange in aqueous
solution. Solvent: citrate-phosphate buffer, pH
6.0, 20°C (from Zanker, 1952) ............

Acridine orange molecular configuration and schema-
tic molecular configurations of A0-NA complexes in
the monomer form (A0 to nucleotide phosphorus = 1:6)
and aggregate form (A0 to nucleotide phosphorus =
:l.5 ...... . ................

Diagram of the principle of the LEITZ fluorescence
vertical illuminator . . . . . . . ........ .

Diagram of the microspectrophotometer ........

The normalized fluorescence spectra of A0 molecules

bound to different NA conformations and gelatin pro-
teins, A0 staining concentration 5 x 10- M, staining
time 30 mins., pH 4.1 ................

The average relative fluorescence intensities of
A0-3% purified pigskin only and AO-NA complexes in
3% pf. pgsk. at 536 nm. and 604 nm. for different
acridine orange staining concentrations (expressed
in molarities), pH 4.1, staining time 15 minutes

The degree of aggregation of A0 onto 1.0 pg. DNA

or rRNA in 3% pf. pgsk. for different acridine orange
staining concentrations (expressed in molarities),

pH 4.1, staining time 15 minutes ..........

The average relative fluorescence intensities of
AD-NA complexes in 3% pf. pgsk. at 536 nm. and 604

nm. for different gtaining times, A0 staining con-
centration 5 x l0‘ M, pH 4.1 ............

viii

Page

31
32

35

4O

43

47

Figure

10

ll

12

l3

I4

15

16

T7

Page

The average relative fluorescence intensities of

1.0 pg. DNA-A0 and 1.0 pg. rRNA-A0 complexes in

3% pf. pgsk., and gelatin protein-A0 complexes of

3% pf. pgsk. only, at 536 nm. and 604 nm., for

different acridine orange staining solution pH, A0

staining concentration 5 x 10‘5M, staining time

30 mins ....................... 52

The degree of aggregation of A0 onto 1. 0 pg. DNA or

rRNA in 3% pf. pgsk. for different acridine orange

staining solution pH, A0 staining concentrations

5 x 10’ M, staining time 30 mins. ......... 56

The average relative fluorescence intensities of

0.5 pg. DNA-A0 and 1.0 pg. rRNA-A0 complexes in

3% pf. pgsk. and gelatin protein-A0 complexes of

3% pf. pgsk. only, at 536 nm. and 604 nm., for

various NaCl contents (expressed in picograms),

A0 staining concentration 5 x 10'5M, staining

time 30 mins., pH 4.1 ............... 59

The degree of aggregation of A0 onto 0.5 pg. DNA

or 1.0 pg. rRNA in 3% pf. pgsk. for various NaCl

contents, A0 staining concentration 5 x 10‘5M,

staining time 30 mins., pH 4.1 .......... 62

Fluorescence decay of A0—NA complexes at 536 nm. and
604 nm., as fUnctign of the irradiation time, A0 con-
centration 5 x 10‘ M, pH 4.1, staining time 30 mins.,
excitation wavelength 400 nm ............. 67

The average relative fluorescence intensities of

A0 bound to DNA or rRNA, at 536 nm. and 604 nm., in

3% pf. pgsk. for various DNA or rRNA concentrations,

A0 staining concentration 5 x 10'5M, staining time 30

mins. , pH 4.1 ..... _ ............... 71

The average relative fluorescence intensities of A0

bound to rRNA, at 536 nm. and 604 nm., in 3% pf. pgsk.

for various rRNA concentrations, A0 staining concen-
tration 5 x 10'5M, staining time 30 mins., pH 4.1 . . 74

The degree of aggregation of A0 onto DNA or rRNA in

3% pf. pgsk. for different NA cgncentrations, A0

staining concentrations 5 x 10’ M, staining time 30

mins., pH 4.1 .................... 76

ix

Figure

18

19

20

Page

A photomicrograph of a living brain cell found in a
dissociated monolayer cell culture, taken from the
somatic sensory cortex I and II, of a 3 days old
female albino rat. The cell was maintained 23 days
in culture; at the time of photo, it has a diamond
shape with a length of 13- microns and a width of
96 microns. The nucleus of the cell is centrally
located, having an oval shape 30 microns long and
18 microns wide, with several nucleoli. This cell
has been designated the Canada, Type I neuron. The
scale for the photomicrograph is 14.4 microns per
centimeter ...................... 82

A photomicrograph of a living Canada, bipolar neuron
(right of center) found in a dissociated monolayer
cell culture, taken from the somatic sensory cortex

I and II, of a 3 days old female albino rat. The cell
was maintained 32 days in culture. The cell body is
spherical, with a length of 26 microns and a width of
17 microns. Note the fibrous astrocyte above the
center of the photomicrograph. The Canada, bipolar
neuron is making a contact with a Canada, Type II
neuron (bottom of center). The scale for the photo-
micrograph is 14.4 microns per centimeter ...... 84

A photomicrograph of a Canada, Type II neuron found

in a dissociated monolayer cell culture, taken from

the brain of a 3 days old female albino rat. The

neuron was maintained 96 days in culture. stained

with 1 x 10'4M acridine orange, at 22°C, for 30 min-
utes, at a pH of 4.1, and irradiated with 400 nm. light.
The cytoplasm of the neuron is full of orange gran-

ules and the nucleus has a yellow-orange color, indi—
cating that under these conditions the degree of
aggregation of A0 onto the NA macromolecules in the
neuroplasm is very high and greater than that for the

NA macromolecules in the neucleoplasm. The average

soma area of the Canada, Type II neurons found in this
culture was 179 sq. microns. Note that the processes

of the ce11.in the bottom right corner appears to be
making contacts with the soma and processes of the
Canada, Type II neuron: the cell has an orange cytoplasm
with a yellow—orange nucleus and its ratio of the
nucleus to soma areas is greater than that fer the
Canada, Type II neuron ................. 85

Figure

21

22

23

24

Page

A photomicrograph of two Canada, bipolar neurons,
found in a dissociated monolayer cell culture, taken
from the brain of a 3 days old female albino rat.

The cells were maintained 96 days in culture, stained
with l x 10'4M acridine orange at 22°C, for 30 min-
utes at a pH of 4.1, and irradiated with 400 nm.
light. The average area of their somas is 112 sq.
microns, having two processes at opposite poles, with
a bifurcation in one of the processes. Both of their
nuclei are large and round, taking up most of the soma
while stained a yellow-orange color. Their cytoplasm
is orange in color. One of the Canada, bipolar
neurons, with its bifurcated process, is making con—
tact with a process of the other neuron ........ 86

A photomicrograph of living fibrous astrocytes dur-

ing redifferentiation. The cells were maintained 23

days in a dissociated monolayer cell culture, and

were taken from the somatic sensory cortex, I and II,

of a 3 days old female albino rat. As it happens

with cells in brain sections, these cells in culture

may be confused with neurons . . ........... 88

A photomicrograph of three living neurons rediffer-
entiating, the cells were taken from the somatic
sensory cortex, I and II, of a 3 days old female
albino rat. They were grown 23 days in a dissoci-
ated monolayer cell culture. Note that there is a
soma-somatic contact between two of the neurons. The
nucleus of each cell conforms with the general shape
of the cell body, having one or two nucleoli. The
cytoplasm of each cell is dense with Nissl bodies.
The cells in the surrounding environment are glia or
connective tissue ................... 90

A photomicrograph of a single Canada, bipolar neuron
found in the same culture and subjected to the same
conditions as those in Figure 21. The average area
of the soma is 112 sq. microns. The other large
oval—shaped yellow-green objects are the nuclei of
glia cells. Note the local accumulation of dye

'molecules at the orange thickenings in the bifur-

cated process, before and at the bifurcation, due to
an increased aggregation of A0 ........ . . . . 95

xi

Figure

25

26

27

28

29

30

Page

A photomicrograph of a Canada, bipolar neuron found

in a dissociated monolayer cell culture, taken from

the brain of a 3 days old female albino rat. The cell
was maintained 96 days in culture, stained with

l x 10'4M acridine orange, at 22°C, for 30 minutes

at a pH of 4.1, and irradiated with 400 nm. light.

The average area of the soma is 112 sq. microns, hav-
ing two processes at opposite poles, with a bifurcation
in one of the processes. Note that the neuron at the
bottom of the photomicrograph is sending its process
towards the bifurcated process of the Canada, bipolar
neuron ......................... 96

These two cells are Canada, Type II neurons. Canada.
Type II neurons occur in polymorphic form, with 3 to

4 processes, in dissociated monolayer cell cultures of

3 days old female albino rat brains. Note that there

is one contact between the two neurons and a contact
with a common body. The cells w re maintained 96 days
in culture, stained with 1 x 10’ acridine orange, at
22°C for 30 minutes at pH 4.1. The photomicrograph

was taken while exciting the cell with 400 nm. light.
The average area of their soma is 179 sq. microns. The
cytoplasm is stained orange. The yellow-orange nucleus
is usually round or oval in shape, comprising about half
the area of their somas ................. 97

A Canada, Type II neuron, in upper section, found in

the same culture and subjected to the same conditions

as those in Figure 26. Notice the extensive network

of fibers between this cell and the cell in the lower
section. Two of the processes of the Canada, Type II

neuron are making several contacts with the processes

of other cells ..................... 98

The [rRNA + v(DNA)] content, in picograms, per neu-
ronal type fer different neuronal ages ........ 100

The [rRNA + v(DNA)] content, in picograms, per neu-
ronal age for different soma areas of the neurons . . . 101

Ethidium Bromide ................... 108

xii

I. INTRODUCTION

In recent years neuroscientists have gained valuable information
anent the structure and function of macromolecules involved in neuronal
systems during behavioral responses. However, this information is
inadequate to fully understand the molecular interactions between the
central nervous system and the mind. Our knowledge of molecular mechan-
isms for behavior can be characterized as being in the infant phase.

This suggests that neuroscientists are confronted with many challenging
problems that require a variety of innovative and incisive techniques

for their solution. An application of biophysical cytochemistry to single
neurons in culture is one method that may provide salient answers con-

cerning the molecular events engaged in neuron to neuron interactions.

A. Biophysical Cytochemistry

In 1972, Seymour S. Nest and Andrew E. Lorincz [Nest and Lorincz,
1973] introduced the term "biophysical cytochemistry" to the conference
on "Quantitative Fluorescence Techniques as Applied to Cell Biology."
The term suggested the application of biophysical techniques and theories
to cytology: in particular, the use of fluorescent molecular probes as
cytochemical tools to study the behavior of complex biopolymers. Fluor-
escent molecular probes are small planar dye molecules, with emission
properties that respond to alterations within their near and distant
environment. Fluorescent probes bind to unique locations on biological
macromolecules without appreciably disturbing those features of the
macromolecule desired for investigation [Stryer, 1968]. Inferences

l

about the structure and function of the macromolecule can be formu-
lated by studying behavioral variations of the bound probe. This
reduces the problem of investigating a complex macromolecule to a simple
dye molecule. Consequently, the extent to which we understand the
behavior of biological macromolecules will depend upon our knowledge
of the dye, macromolecule, and dye-macromolecule interations.
Fluorescence microspectrophotometry is an invaluable technique
for applying quantitative cytochemistry to cellular components and
their functions. It allows for the investigation of biopolymers within
individual cells by fluorescent molecular probes. Acridine orange (A0)
is a fluorescent probe used in microspectrophotometry for analysis of
various intracellular macromolecules. Because of its high quantum
efficiency and metachromasy, Rigler [1966] used acridine orange to study
the nucleic acids and nucleoproteins in single cells from fixed micro-
scopic preparations. He found that the orthochromatic green fluorescence
is due to A0 binding to helical molecular configurations in a monomer
nolecular fbrm (having a low A0 content), and the metachromatic red
fluorescence is the result of the dye binding to random coil molecular
configurations in an associated molecular fashion (having a high A0
content). Nest [1969, 1973] utilized acridine orange in television
fluorescence microspectrophotometry to examine the nucleic acids
and mucopolysaccharides in living cells. He related the emission pro-
perties of the dye-biopolymer complex to the intracellular dye content
within the cell. A low intracellular dye content produced a green
fluorescence with an emission maximum between 530-540 nm. A high
intracellular dye content elicited a red fluorescence with a long wave-

length emission peak at 690 nm. He also noted that as the intracellular

dye content increased, the fluorescence of the cells cytoplasm pro-
gressively changed from green to yellow, yellow to orange, and orange to
red, with each color depending upon the present intracellular dye content.
The application, by Rigler [1966] and Nest [1969, 1973], of
acridine orange fluorescence microspectrophotometry to nucleic acids in
single cells successfully demonstrated its utility for fixed and living
microscopic preparations. This allows for a possible direct comparison

of jg_situ results with those obtained from in vitro model systems.

 

Neuroscientists should note that most biophysical cytochemistry is per-
formed on non-neural material, such as leukocytes, lymphocytes, bacteri-
ophages, spermatozoa or fibroblasts. A beginning researcher in this area
should acquaint himself with the techniques for fixed nervous material
befbre attempting living tissue. This will enable him to conquer the
many difficulties in using living nervous tissue. As an example, under
certain experimental conditions acridine orange can be used as a vital
stain for nervous tissue [Zieger and Harders. 1951], and under other
experimental conditions it becomes a neurotoxin [Nest, 1969]. Learning
the techniques for fixed single neurons will provide the novice with
enough knowledge to overcome the basic problems involved in fluorescence

microspectrophotometry in conjunction with dissociated neurons in culture.

8. The Metachromasy of Acridine Orange

Acridine orange has been established as a fluorescent dye for vital
staining of nervous tissue and vital studies on the function of the
kidney and liver [Nest, 1969]. The fluorescence colors of cells stained
with A0 is different from the fluorescence color of the dye in dilute
solution. Further, the various intracellular structures themselves

fluoresce with different colors when stained with acridine orange.

Ehrlich [1879; Nest, 1969] termed this effect “metachromasy” and the
color that was different from the dye in dilute solution "metachromatic."
The wavelength shift (change in color) produced by a metachromatic dye

is due to aggregation of the dye molecules. This association results

in the dye's deviation from Beer's law [Nest, 1969].

Zanker [1952] provided the first explanation of the metachromatic
behavior of acridine orange in aqueous solutions. He related the meta-
chromatic shifts of the absorption and emission spectra towards lower and
higher wavelengths, respectively, to the increased association of the dye
molecules at increasing dye concentrations. At low concentrations, acri-
dine orange is a monomer with an absorption maximum of 490 nm. and a
fluorescence peak at 535 nm.. A0 is a polymer at high concentrations
with an absorption peak at 455 nm. and a fluorescence maximum at 660 nm.
The absorption peak of the polymer shifts with dilution, however the
absorption maximum for monomer A0 does not change. There were only
slight changes in maxima for the corresponding fluorescence spectra.
Zanker [1952b, 1959; Rigler, 1966] proposed that the long wavelength
absorption band of 490 nm. was caused by electronic transitions from
the zero vibrational level of the ground state to the zero vibrational
level of the excited electronic state (0--0 transition band). This
absorption band elicits the short wavelength fluorescence peak at 535
nm.. 0n the other hand, the electronic transitions for the A0 polymer
is slightly different. The short wavelength absorption band of 455 nm.
was interpreted to be due to electronic transitions from the zero vibra-
tional level of the ground state to the first or second vibrational
level of the excited electronic state (0--l and 0-—-2 transition bands),

yielding the long wavelength fluorescence peak at 660 nm.. The short

and long wavelength fluorescence bands are the result of electronic
transitions from the zero vibrational level of the excited electronic
state to the zero, first and second vibrational levels of the ground
state. Zanker [1952; Nest, 1969] suggested a stacked-coin model for
the structural configuration of the acridine orange polymer. The
absorption and fluorescence spectra from Zanker's investigation are
shown in Figures 1 and 2; the wavelengths are expressed in wave numbers.
A number of investigators have studied the metachromatic behavior
of acridine orange in solution with nucleic acids (NA), [Bradley and
Half, 1960; Loeser et a1, 1969; Nest, 1969: Rigler, 1966]. They found
that two distinct molecular complexes are formed when A0 binds to NA
in solution. The first complex, relatively stable, is formed at low
dye-to-nucleotide ratios, absorbing at 502 nm. and emitting at 540 nm..
The absorption and fluorescence spectral characteristics of this com-
plex resemble those of the monomer dye alone at low concentrations in
aqueous solutions. A second, less stable complex is formed at high
dye-to-nucleotide ratios, with spectral characteristics similar to that
of the polymer dye alone in solution, absorbing at 465 nm. and emitting
at 660 nm. More importantly, the absorption and fluorescence charac-
teristics of the two complexes gg.ggt distinguish between RNA and DNA
in solution.' However, Lerman [1961, 1963, 1964] and Rigler [1966] pro-
posed a molecular configuration for the two binding modes of acridine
orange to nucleic acids, see Figure 3. The mode of binding of A0 to a
helical configuration, when the dye-to-nucleotide ratio is low, is by
intercalation. The planar A0 cations are sandwiched between two adjacent
base pairs inside the helix. The dimethylamino groups of the dye form

ionic bonds with the negatively charged P04 groups of the nucleotides.

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a“.m..m 11.. 0.0 s. :0 . _ _ on
. 0 § q —u 0.0.. .
.\ .. ,/ tubim II. _
\ _ :Qoo_ I — OI'I' .
_ knee—InOIOIOJ O-
. 3.0.0. .1 It. .
N \J. .O.-O 0'11 . A . 8
grit Ar 1 3‘“..°.- _ H0610 U — — A._A — I V

 

 

 

 

 

 

 

 

 

 

 

 

 

IIIGIIIIIII llltf’l'llrtnl.

 

Figure 3. Acridine orange molecular configuration, and schematic mole-

cular configurations of AO-NA complexes in the monomer form (A0 to

nucleotide phosphorus ratio I 1:6) and aggregate form (A0 to nucleotide
phosphorus ratio - 1:1.5).

ACRIDINE ORANGE

A0 bound in the,

double stranded, helical
regions of the NA
conformation, via
intercalation.

 

A0 bound along the
single strand of
the NA conformation,
via aggregation.

 

This complex is not only stabilized by the hydrophobic interactions
between the A0 ring structure and the hydrophobic interior of the helix,
and the ionic bonds, but also by the dipole-dipole interactions between
the acridine ring and the purine-pyrimidine rings of the upper and lower
base pairs [Rigler, 1966]. Acridine orange is a monomer in this form,
because the distance between two dye molecules is large enough to pre-
vent any dye-dye interaction. Note that the second NA-A0 complex appears
when the dye-to-nucleotide ratio is high: the dye is now considered bound
by almost every nucleotide unit. The A0 molecules are stacked along the
outside of helical configurations or along the singlestrand of random
coil configurations. The close association of A0 molecules results in a
dye-dye interaction that produces a long wavelength emission (660 nm.)
and less stability. The second complex is considered the aggregated

form of NA-AO complexes. In both binding complexes. the planes of the
acridine rings are parallel to the planes of the purine-pyrimidine rings
[MacInnes and Uretz, 1966; Rigler, 1966].

The metachromatic phenomenon is caused by the aggregation of dye
molecules on a given macromolecule. Bradley and Nolf [1959; Nest, 1969]
reported that the aggregation of A0.on a biopolymer can be described in
terms of its "stacking coefficient." A biopolymer's stacking coefficient
is proportional to the free energy of interaction between a pair cf
neighboring dye molecules and its value is related to the conformation of
the biopolymer. A highly ordered biopolymer has a small stacking coef-
ficient. Native DNA has a stacking coefficient of 1.25 and upon de-
naturation it increases to 6.2. RNA has a slightly higher stacking
coefficient of 3. However, some polysaccharides have a stacking coeffi-

cient greater than 800 [Nest, 1969]. This means that if a biopolymer

has a large stacking coefficient, then the aggregation of the dye will
occur at low dye-to-biopolymer ratios. On this basis, at a low intra-
cellular dye content macromolecules in an A0-stained cell will fluoresce
red (660 nm.-690 nm.) if their stacking coefficients are very high, or
green (500 nm.-540 nm.) if their stacking coefficients are low [Nest,
1969]. ’

Forster [1951] and Rigler [1966] attributed the metachromatic be-
havior of aggregated acridine orange molecules to the existence of an
intermediate metastable state. They theorized that two associated dye
molecules are elevated to an excited state with both of their electronic
oscillators vibrating in phase, :: I, along their long axis. The
excited A0 dimer makes a radiationless transition to an intermediate
metastable state of lower energy, with both electronic oscillators vi-
brating out of phase, :_"::. Since the probability of a transition
from a metastable to a ground state is low, this intermediate state is
preserved for a certain period. The metastable dimer reaches the ground
state by a radiationless transition or by a prolonged long wave fluores-
cence and lifetime. A sustained phosphorescence at room temperature
can be ruled out since the emission wavelengths have lifetimes of less
than 10'3 seconds. Therefore, the dimerization of A0 results in a long
wave fluorescence, involving a radiationless transition from the excited
state over an intermediate metastable state to the ground state, with

an additional loss of vibrational energy.

C. The Relationship Between Nucleic Acids and Neuronal Activities
The nucleus is the principal morphological feature within the soma

of neurons. The entire nucleus is surrounded by a perinuclear envelope

10

containing large pores. The nucleoplasm is primarily composed of chro-
matin and contains at least one nucleolus rich in ribonucleic acids
(RNA). The nucleolus manufactures the ribosomal RNA used in the forma-
tion of ribosomes for protein synthesis [Lehninger, 1971, p. 33].
Deoxyribonucleic acid (DNA) molecules, in association with histones and
other proteins, form the genes, which are arranged into the chromosomal
material of chromatin. Chromosomes are pre-programmed with the genetic
information necessary for the development and maintenance of a neuron.
The genetic message, contained in the sequences of DNA nucleotides, is
transcribed and translated by ribonucleic acids for conversion into
proteins. The mitochrondria of neurons also contain small amounts of
DNA that code for the synthesis of a few specific proteins which are
located in the mitochrondria membrane. The DNA content per neuron is
constant fer a given species and it cannot be altered by internal or
external environmental circumstances [Lehninger, 1971, chap. 28-301..
The rapid accumulation of DNA in the brain, i.e., the replication
of DNA molecules, occurs during the proliferation of neuroblasts and
spongioblasts, which are the embryonic precursors of differentiated
neurons and neuroglia, with the proliferation of the neuroblasts, pre-
ceding that of the spongioblasts [Benjamins and McKhann, 1972]. At
some appropriate time the induction and/or repression of specific
chromosomal genes initiates the differentiation of a neuroblast into
a specific neuronal type (Betz, Deiters, granule, mitral, Purkinje, etc.)
and a spongioblast into a specific neuroglia (fibrous astrocyte. oligo-
dendroglia, protoplasmic astrocyte, etc.).. Before differentiation and
the appearance of Nissl substance, neuroblast division ends. The DNA

in differentiated macroneurons (long-axoned, input-output neurons) and

11

most microneurons (short-axoned, modulating interneurons) lack self-
replication; therefore, in most cases a differentiated nerve cell loses
its capacity to divide. However, the DNA in differentiated neuroglia,
and microneurons in certain brain regions, e.g., the outer granular
layer of the hippocampus, olfactory lobe, cerebellar cortex, and the
ventral cochlear nucleus, can self-replicate, resulting in further
accumulation of brain DNA and multiplication of cells [Mahler, 1972].
The total brain DNA, in most species, accumulates in a linear manner
as a function of time until the adult level is reached. As the accumu-
lation of DNA ends, the ratio of RNA to DNA increases [Benjamins and
McKhann, 1972].

As indicated by the measurements of brain DNA levels, the human
brain has two major periods of cell proliferation [Dobbins, and Sands,
1970; Benjamins and McKhann, 1972]. The first period corresponds to the
proliferation of neuroblasts and begins at 15 to 20 weeks of gestation.
In the second period the multiplication of neuroglia occurs, in addi-
tion to a second wave of neurogenesis restricted to microneurons in the
granular layer of the olfactory lobe, cerebellar cortex, and hippocampus.
This second period begins at 25 weeks of gestation and continues into
the second year of postnatal life [Dobbins and Sands, 1970; Benjamins
and McKhann, 1972].

Nissl bodies are considered a prime characteristic of neurons.
Except for the axon hillock and axoplasm, Nissl bodies are found within
the neuroplasm of the soma and the dendrites. The neuroplasm is the
cytoplasm in neurons and should not be confused with nucleoplasm [Jenkins,
1972, p. 47]. Nissl bodies are basophilic granules consisting primarily

of ribosomal RNA. Nissl bodies are involved in protein synthesis and

12

are the ribosomes that stud the rough endoplasmic reticulum of other
cells. Nissl bodies (ribosomes) may even participate in conformational
changes during protein synthesis, where both the polypeptide chain and
the translated mRNA are translocated along the granules [Lehninger,
1970, p. 698]. The amount of Nissl substance contained in the soma of
neurons can be correlated with the physiological and functional activity
of the neuron. They may increase or decrease, depending upon the rate
of neuronal stimulation and development as well as the behavioral and
metabolic changes of the neuron. Nissl bodies reflect the neuronal RNA
content, thus indicating the level of protein synthesis.

Injury to a mammalian neuron results in a decrease in its neuronal
RNA content. In most vertebrate neurons injury is accompanied by a dila-
tion of the soma, and the shrinkage and deformation of the nucleus, with
its displacement toward the periphery of the cell body [Jenkins, 1972,
p. 121]. There can be an incomplete chromatolysis if the insult (nerve
fiber injury) is distal to the soma; here the Nissl bodies partially
dissolve and the residual neuroplasm becomes vacuolated. Complete
chromatolysis occurs when the insult is proximal to the soma, causing
total disintegration of the Nissl bodies. The interruption of protein
synthesis initiates the dissipation of the perikaryon with all organelles,
and is suggestive of impending neuronal death. However, under suitable
conditions, there can be a restoration of the Nissl bodies and protein
synthesis, and the subsequent regeneration of the fiber.

A distinction of neurons is the characteristic possession of a high
RNA content. The RNA level in a neuron changes during the neuron's life-
cycle. A connection between the RNA content and neuronal activities was

reported in retinal ganglion neurons as a function of light stimulation

13

[John, 1967, p. 95]. The RNA levels of the cells were found to be pro-
portional to the total light stimulation received by the cells. In
addition, light deprivation caused a dramatic decrease in the RNA con-
centration of the retinal ganglion neurons. This suggests that there
is a positive relationship between the RNA content and neural activity,
as reflected in the neuron's RNA level. In another experiment, analysis
on the nuclear and ribosomal fractions of centrifuged homogenates taken
from brains of animals trained to perform a conditioned avoidance response
displayed greater incorporation of radioactively labelled RNA precursors
than the identical fractions taken from untrained animals [John, 1967,
p. 97]. This demonstrated that synthesis of RNA by a neuron increases
during increased neural activity, e.g. learning situations, thereby
establishing more directly a positive relationship between the RNA con-
tent and neuronal activity. This suggests that the high RNA levels and
changes during the life of a neuron are attributed to the functional
activity of that neuron.

The classic works by Hyden [1967, p. 200] demonstrated that the
RNA content per neuron increases and the nuclear RNA base ratios change
during a learning experience. In two different experiments, he deter-
mined the amount and base composition of the RNA manufactured by vesti-
bular and cortical neurons. The nuclear RNA of cortical neurons exhibi-
ted a slight increase during the early and acute stages of a learning
situation (right handed rats were induced to use their left hand in
retrieving food from far down a narrow glass tube). The nuclear RNA
fbrmed had a high adenine-uracil value. As learning increases and per-
fbrmance improves, the nuclear RNA of the cortical neurons is enhanced

significantly, and the adenine-uracil rich RNA base ratio changes to a

14

composition similar to ribosomal RNA. Thus, a differentiated formation
of nuclear RNA occurs in cortical neurons engaged in a learning experi-
ence, reflecting a genic stimulation. After physiological stimulation
and work requiring memorization (young rats learn how to balance on

thin wire strung 45° between the floor and a small platform with food

in order to eat), the RNA content of Deiter neurons from the lateral
vestibular nucleus of rats was analyzed. Hyden [1967, p. 200] found

that the RNA content per neuron increased during learning and physiologi-
cal stimulation. The nuclear RNA formed during physiological stimulation
had the base characteristics of a ribosomal RNA. In contrast, the nuclear
RNA formed during learning was an asymmetric, adenine-rich RNA of the
chromosomal type, signify that the increase in the adenine-to-uracil
ratio was specific for learning. The cytoplasmic RNA of the Deiter
neurons increased during learning but did not change in base composition.
Hyden also reported that there occurs a transfer of RNA between glia and
neurons. The two cells communicate during neuronal functions, whereby

an increase or decrease in the RNA content and enzyme activities of the
neuron causes an opposite response in the surrounding glia.

The priceless contribution of Hyden [1967] and others [Cameron et
a1, 1970; Rosenblatt, 1970; Unger, 1970] coincides with the author's pro-
position that there exists a direct functional relationship between the
ribonucleoprotein (RNA) characteristics of a neuronal complex and the
maintenance of memory and learning, and that this relationship is mani-
fested in the form of a proportional change. This may be evidenced when
a change in the concentration, composition.and/or conformation of ribo-
nucleoproteins results during the facilitation or inhibition of behavioral

processes .

15

At present the Edstrom [1953, 1958, 1964] method is widely used
for the determination and analysis of nucleic acids in isolated sub-
cellular units and cell samples. The Edstrom method has some advantages
and disadvantages in comparison to absorption and fluorescence micro-
spectrophotometry. One of the positive aspects of the Edstrom method
is its ability to determine the base composition for the nucleic acids.
However, the sensitivity of this method is only good for about 25 pico-
grams of material, and the extraction and isolation procedures could
result in leaving portions of the nucleic acids undetected. In addition,
the method [Edstrom, 1953, 1958, 1964] is unable to detect any molecular
activities within the ghglg_neuron, due to internal or external environ-
mental circumstances. At this interim,the aforementioned method
[Edstrom, 1953, 1958, 1964] is not recommended or used by this author
for more than one reason. Not only do its negative aspects outweigh
its positive elements, but it is time-consuming and tedious.

A number of investigators [Haltia, 1970; Sobkowicz et a1, 1973]
have noted that changes in metabolic activities (aging) can result in
changes in the RNA content of neurons. A piece of work presented by
Haltia [1970] was concerned with.the RNA content of spinal anterior
horn neurons during the postnatal development of rats (jg_yjyg). The
neurons were isolated from the other cells, but the Edstrom [1953, 1958,
1964] method was employed for determination of the RNA content as a
function of age. A rapid acceleration in the neuronal RNA content was
observed between the ages of 1 and 15 days. At 15 days, a deceleration
in the RNA content occurred and almost reached adulthood values at 30
days of age. No significant change in the RNA content was observed
after 90 days of age. These results were interpeted by Haltia [1970] to

represent a differential growth rate, a growth-curve relationship between

16

the RNA content and the age of the developing neuron, evidenced by a
sigmoid curve (S-shape curve) for the RNA content (ordinate) plotted
against the age (abscissa) of the neuron. This relationship was further
substantiated by Sobkowicz et a1 [1973]. They displayed a growth-curve
correlation compatible to Haltia's [1970] results. The RNA content of
normal and cultured cells from fetal rat spinal ganglia was determined,
again by the Edstrom [1953, 1958, 1964] method, as a function of age.
Both the jn_yitrg_and in yiyg_neurons demonstrated a differential growth
rate. The RNA content of the cells rapidly increased during the first
stages of normal neuronal development. Sobkowicz et a1 [1973] suggested
that the increase in neuronal RNA content was associated with the forma-
tion and differentiation of Nissl material. Also, it was noted that the
cells in the explant cultures develop at a much slower pace than jg_yjyg,
This may be the result of a change in environment or trauma from explan-
ation. In any case, it is evident that there exists a differential
growth rate during the maturation of neurons. A growth-curve relation-
ship, in the RNA content, as a function of age, is found for neurons in
culture as well as jg_yjyg, This particular relationship is an excellent
test situation, in respect to the author's proposed techniques, which
involves the utilization of acridine orange fluorescence microscopy

in the analysis of nucleic acids in single neurons maintained in dis-

sociated cell cultures.

0. Neuron Cultures

The ability to grow nervous tissues in cultures can greatly enhance
our knowledge in the neurosciences. It has been demonstrated that the
morphological, biochemical, organizational and functional developments

of neurons in culture are similar to neurons in the natural situation.

17

The experimental control over neurons in cultures can be utilized to
study the activities of macromolecules under given conditions. Neurons
grown in culture provide an excellent model system to probe the macro-
molecular mechanisms involved in neuronal interactions. The following
paragraphs exemplify some of the similarities between neurons maintained
in culture and neurons grown jg_yjyg,

Vatter and Seeds [1971] demonstrated that the morphological devel-
opment of cells from an embryonic mouse brain in culture is quite similar
to cells j§_yiyg, They observed that dissociated cells reassociated
into highly organized aggregates in a basal Eagle's medium with 10% fetal
calf serum. Vatter and Seeds [1971] reported that the cells differenti-
ated and formed myelinated axons after five weeks in culture. The cells
arranged themselves in the same cytoarchitectural design that exists lg
3119. In addition, synapses matured and increased in number during this
period. These morphological results were correlated to be identical to
the fin yiyg_cytoarchitectural development of the embryonic mouse brain.

In an earlier paper, Seeds [1971] established that the development
of enzymatic activities related to chemical transmission in the reaggre-
gating cell culture are similar to the jg_yjyg_situation. Seeds dis-
sociated neurons from an embryonic mouse brain and allowed them to re-
associate in a rotation culture to form aggregates. The specific acti-
vities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE),
and glutamate decarboxylase (Gluase) were obtained from the aggregates
as a function of time in culture. The results, displayed by their
activity/time curves in culture, were analogous to the typical S-shape
patterns found in the brain. Further, it was observed that at the

curve's maxima, the ChAT activity has a greater propinquity toward the

18

jg_yiyg_levels than the AChE. One possible explanation for this involves
the difference in the rate of development between axons and dendrites

In the aggregates, axon development proceeds at a faster rate than does
dendrite maturation. Because ChAT is feund in the synaptic bouton of
the axon, whereas the AChE activity is largely in the postsynaptic mem-
brane of the dendrite, it seems plausible that the peak levels reached
by the enzymes would be affected by these circumstances [Seeds, 1971].

Nelson and Peacock [1972] provided evidence that substantiated the
equivalence between the electrophysiological experiences of L cells in
culture and experiences of L cells jg yjyg, Utilizing iontophoretic
techniques, they applied acetylcholine to L cells in culture. The
affected cells evoked a prolonged active membrane hyperpolarization;
and the administration of atropine was feund to block this response.
Plus, a cell to cell functional interaction was demonstrated in culture,
whereby a hyperpolarizing activiation response was recorded in cells
adjacent to cells stimulated with acetylcholine. The sensitivity to
this neurotransmitter suggests that L cells in culture are capable of
generating electrophysiological responses similar to L cells in the
natural environment.

The onset and development of functional interneuronal connections
in cultures were described by Crain and Peterson [1967]. They reported
that the complex bioelectrical activities evoked by nervous tissue in
cultures resembles, to a high degree, the functional organization of
synaptic networks inuylyg, Crain and Peterson performed their record-
ings on rat spinal cord ganglia explants. Their results exhibited that
14-15 days old fetal rat explants in culture for 2-3 days elicited

either a simple spike potential or no bioelectrical transmission

19

whatsoever from one neuron to another. However, 3-4 days in culture
demonstrated complex bioelectric activities. Facilitation was observed
with paired stimuli at long test intervals. Also brief tetanic stimula-
tion produced slow waves and a long lasting spike barrage. These results
correlated closely with ya 11!g_conditions, in that they indicated that
polysynaptic networks are beginning to function in the culture. Further-
more, electron micrographs of these explants illustrated that morpho-
logical parameters associated with sequential development of bioelectric
functions increased during culture maturation. Grain and Peterson [1967]
digested their results and concluded that nervous tissue, grown under
suitable cultural conditions, have smiliar morphological, biochemical
and functional characteristics as the 13 1139 state. I

In reiteration, it has thus far been demonstrated that the develop-
ment of a neuronal complex in a cell culture is analogous in function to
its development jg_yiyg, It has not yet been established that the men-
tal behavior of neurons maintained in culture exhibits the same char-
acteristics as the natural state--or, in fact, that there exists a men-
tal behavior in neurons in culture. Nhether or not a behavior is
localized in neuronal complexes or encompasses the entire brain is,
indeed, a controversial issue. However, regardless of the conclusions
one may draw, it must be acknowledged that neuron-to-neuron molecular
interactions are significant in a behavioral process. The complex
molecular interactions of these neurons are the consequence of neuronal
metabolic activities whose functions are related to behavior. Therefore,
the parameters that reflect metabolic functions can be intimately asso-
ciated to behavior. It is for these reasons that this author submits

that a thorough understanding of the neuronal molecular interactions of

20

nucleic acids and proteins will eventually lead to the elucidation of
the molecular mechanisms involved in the functioning neuron.

A number of investigators [Chignell, 1973; Kohen et al, 1973;
Rigler, 1966; Nest and Lorinez, 1973] have used fluorescent probes in
conjunction with cell cultures to study various cellular macromolecules.
Rigler [1966] and Nest and Lorinez [1973] have used the fluorescent
probe, acridine orange, to study, respectively, nucleic acids and muco-
polysaccharides in cell cultures. Kohen et a1 [1973] have used rapid
microfluorometry to examine enzyme reactions and transport mechanisms
in single living cells maintained in cultures. Neurons maintained in
cell culture have a number of advantages for use with fluorescent probes.
The dissociated cell cultures can provide individual neurons; the
macromolecules can be analyzed in non-mutilated neurons (fixed or liv-
ing). The neurons are maintained in an environment that can be con-
trolled and easily manipulated. In addition, they are readily accessible
to diffusible materials and any unbound excess probe can be washed off
freely. Furthermore, neurons and neuronal contacts in a monolayer cell
culture are simple to observe and their fluorescence easily analyzed
with microspectrophotometry. It is evidenced that cell cultures in
combination with fluorescent probes furnish a valuable model system
to study the molecular interactions of neuronal activities.

The objective of this investigation is to survey and increase the
information about the use of fluorescent molecular probes in the study
of macromolecular interactions involved in the functional activities of .
neurons in culture. Specifically, the author intends to obtain the
fluorescence characteristics of the binding of acridine orange to nucleic

acids in different microenvironments. by examining: l. The fluorescence

 

21

changes of AO-NA complexes,iat 536 nm. and 604 nm., in gelatin mitro-_
droplets as a function of the A0 concentration in the microenvironment
of the NA. This is to determine whether the fluorescence intensities

of A0 molecules bound to 1.0 pg. DNA or rRNA in the monomer and/or
aggregated forms are proportional to the amount of A0 permitted to bind
to the NA macromolecules, 2. The fluorescencegchanges of AO-NA complexes
at 536 nm. in gelatin microdroplets as a function of the A0 staining
31mg;_ This is to find out the optimum interaction time for the binding
of A0 to the monomer binding sites in the DNA or rRNA conformations,

3. The fluorescence variations of AD—NA complexes at 536 nm. and 604 nm.

in gelatin microdroplets as a function of the hydrogen ion concentration

 

in the micro environment of the NA. This is to determine the effects

of ionization on the binding of A0 to 1.0 pg. of DNA or rRNA in the
monomer and aggregated forms, 4. The flgprescence intensity changg§_of
AO-NA complexes at 536 nm. and 604 nm. in gelatin migrgdroglets as a
fhnction of the NaCl concentration in the microenvironment of the NA.
This is to ascertain the effects of the environmental ionic strength

on the binding of A0 molecules to the DNA or rRNA conformations in the
monomer and aggregated forms, 5. The fluorescence intensities of A0-rRNA
complexes at 536 nm. and 604 nm. in gelatin microdroplets at various

temporatures. This is to resolve whether the denatured conformations

 

of rRNA binds A0 in different modes, 6. The fluorescence intensity_

changes at 536 nm. and at 604 nm. of A0:NA complexes in gelatin micro-

 

dropletswduring continuous irradiation. This is to determine if the

 

fluorescence of A0 molecules bound to DNA macromolecules decay differently

than that of A0 molecules bound to rRNA macromolecules, and if the

22

fluorescence of A0 molecules bound to NA in the monomer form decay dif-
ferently compared to those bound in the aggregated form, 7. The fluores-
cence intensity changes of A0-NA complexes as a function of the NA con-
centration in the microenvironment of gglatin microdroplets, for the
Quantitative determination of DNA or rRNA contained in a given micro-
environment. The author intends to answer the question--If the nucleic
acid (RNA) content of neurons varied as a result of neuronal activity,
what, then, is the nucleic acid content of specific neuronal types
grown in culture, at two different ages? In addition, another purpose
of this inquiry is to acquaint the author and novice with the basic
techniques involved in the combined use of fluorescence microscopy and

the methods of dissociated neuron cultures.

II. EXPERIMENTAL DETAILS

A. Materials

The nucleic acids used in studying the binding behavior of acri—
dine orange were obtained from Sigma Chemical Company. The deoxyri-
bonucleic acid (Type 1: sodium salt) was in highly polymerized form
isolated from calf thymus. The rRNA (Type XI) was purified by Sigma
from Baker's Yeast ribosomes after Crestfield et a1 [1955] and the
Polyuridylic acid was Type II: potassium salt. The nucleic acids were
suspended in gelatin solutions of 3% purified pigskin in bidistilled
water. The purified pigskin was obtained from Sargent-Nelch Scientific
Company. The gelatin microdroplets served as inert carriers, the pro-
tein matrix of the microdroplets prevented the elution of the nucleic
acids during the staining procedures.

Punified acridine orange (3,6-bis-dimethylamino-acridinium chloride)
was secured from Sigma Chemical Company. Stock solutions of A0 (1x10'3M)
were made up in bidistilled H20 and stored at 4°C in glass-stoppered
low-actinic glass flasks. Aliquots of the acridine orange stock solu-
tions were diluted to a desired concentration by McIlvanine's citric acid-
phosphate buffer. Other than the experiment involving the fluorescence
intensity changes of A0-NA complexes, as a function of pH, the citrate-
phosphate buffer was always buffered to pH 4.1 by the same procedure,
permitting the ionic.strength of each buffer solution to remain constant.
All reagents were of analytical quality and were used without further
purification.

Dissociated cells from the cerebral cortex of 3 days old female

23

24

albino rats were used. The rats were purchased from Spartan Research
Animals Company. The dissociated neurons were cultivated in Culturstat
MEN Eagle's medium (Earle's base without serum) with 17% Fetal calf
serum, incubated at 37°C, as monolayers on glass slides in sterilized
plastic culture dishes. The medium and serum were obtained from Bio
Quest Company, Cockeyville, Maryland. The incubator was a Napco 322
from the National Appliance Company, with a temperature variation of
t0.5°C. The internal environment of the incubator was COZ-air (5:95)
and humid with bidistilled water. All culture glassware was autoclaved

for 30 minutes. The surgical instruments were sterilized by the flame.

B. Methods

1. Microdroplet procedures.

Specific amounts of DNA, rRNA or Poly U stock solutions were mixed
with enough bidistilled water to make a maximum liquid content of 9.7
ml. (see the example on the following p.26), and added to 0.3 gm. of
purified pigskin in a 25 m1. Erlenmeyer flask. The flasks were sealed
with paraffin papers, and the solutions were stirred (using a stirring
bar and magnetic stirrer) at temperatures around 37°C. A mixing temper-
ature of 37°C would avoid denaturation of the nucleic acids. After
equilibrium was reached, 0.1 microliter droplets of each solution were
delicately placed on a clean glass slide with a microsyringe, in a
micromanipulator, at an angle of 90°. A maximum amount of space be-
tween each microdroplet was allowed to prevent adjacent droplets from
being indirectly irradiated by stray excitation light. The microdrop-
lets were fixed in formaldehyde gas for 12 to 48 hours. The formalde-
hyde gas fixation stops the loss of NA when the gelatin swells in water.

25

The glass slides were attached to a holder, and immersed verti-
cally into the reaction solutions. The reaction vessels were matched,
each containing 100 ml. of reaction solution. The reaction solutions
stood 12 to 48 hours in a 22°C water bath, until equilibrium. The
staining process was carried out in the dark, and obeys Fick's diffusion
law for reproducibility. The fbllowing is the basic staining procedure

employed, unless stated otherwise:

1. Citric acid - NazHPD4 buffer, pH 4.1 5 mins.
2. 5 x 10’5M A0 in buffer solution, pH 4.1 30 mins.
3. Rediffusion of A0 into buffer, pH 4.1 5 mins.
4. Rediffusion of A0 into buffer, pH 4.1 ‘ 5 mins.
5. Rediffusion of A0 into buffer, pH 4.1 5 mins.

The rediffusion process removes the excess, unbound A0 molecules from
the microdroplets. The microdroplets were placed 12 hours to 13 days
under formaldehyde gas in the dark, after rediffusion. The length of
fixation, before or after staining, has no appreciable effect on the
fluorescence of the AO-NA complexes in the microdroplets. After fixa-
tion, the diameter of each microdroplet measured 1.0 :_0.1 millimeters,
and the thickness at the center of the microdroplet was 5 microns.
Focused at its center, the microdroplet was irradiated at 400 nm. by
the Fluorescence Vertical Illuminator.

An aperture, 22 microns in diameter, is situated in front of the
monochromator housing. Because of the aperture, the fluorescence
detected by the photomultiplier was restricted to a finite volume in
the center of the microdroplet. The volume was cylindrical in shape,

9

and equal to 1.9 x 10' cubic centimeters. The fluorescence spectra

of the A0-NA complexes in the micro-cylinder were recorded during

26

continuous irradiation. Depending upon the NA concentration of the
microdroplet forming solution, the NA content in the gelatin micro-

cylinder can be accurately calculated. As an example:

DNA stock solution, 5.3 x 10"4 gm. DNA/m1.

 

 

 

 

 

 

 

Sample ml. of stock ml. of H20 gm. of pf. pgsk.
Number sol'n used added in Erlyms.

l. 9.7 - 0.3

2. - 7.7 2 0.3

3. 5.7 4 0.3

4. 3.7 6 0.3

5. 1.7 8 0.3

6. 0.85 8.85 0.3

7. - 9.7 0.3
Sample DNA conc. in DNA content in
Number microd.- forming -9 3

1.9x10 cm
5°]"t10" micro-cyl.

1 5.3x10jfigm./m1. 1.00 pg.

2 4.2x10-4gm./ml. 0.80 pg.

3. 3.1x10_4gm./ml. 0.59 pg.

4. 2.0x10_sgm./ml. 0.38 pg.

5. 9.3x10_59m./m1. 0.18 pg.

6 4.6x10 gm./m1. 0.09 pg.

7 - 0.00 pg.

12

Note: 1.0 pg. = 1.0 x 10' gm.

2. Neuron Culture procedures.

The nature of this endeavor suggest that there may exist a variety
of factors that, if left uncorrected, may lead to erroneous results
and conclusions. For example, the location and condition of the area
to be utilized in the preparation and growing of the neurons are critical.
The entire working area should be sterilized; if not, germs may invade
the cell culture and disrupt the neuronal development. A mixture of
mineral oil and xylene can serve this purpose. The fetal calf serum is

inactivated in a constant temperature water bath at 56°C for 40 min.

27

before use. After the MEM Eagle's Culturstat Preparation a mixture of
17% fetal calf serum in MEM Eagle's Culturstat is prepared and labelled

neuron medium. The excess serum and culturstat is stored at -4°C. The

 

neuron medium is kept sealed at room temperature. The entire culture
preparation procedures require aseptic techniques.

Befbre brain removal, the animals were anesthetized with ether and
were sterilized in 70% ethanol and iodine - 70% ethanol. The surgical
instruments were rinsed in ethanol from water, before flaming with a blue
flame. The whole brains from 3 day old female albino rats were removed
via a calvarium dislocation. The brains were placed in culture dishes
containing neuron medium. An attempt was made to dissect the somatic
sensory cortex, I and II according to the location and external limiting
boundaries described by Nelker and Sinha [1972]. These sections and the
entire cerebral cortex were mechanically dissociated without the utiliza-
tion of trypsin to reduce unwanted chemical interferences. The disso-
ciated tissue and neuron medium were intimately associated in a test tube
with a vortex. This mixture was agitated for 5 mins. and allowed to
settle (by gravity) for 30 mins. at room temperature. 2.0 ml. of the
supernatant from the test tube were diffused on a glass slide in a cul-
ture dish, and incubated for 24 hours. The next day, 10 m1. of neuron
medium were added to each dish. The cultures were incubated at 37°C
with a humid COZ-air (5:95) environment. Two weeks after the cultures
were set up, 2 ml. of fresh neuron medium were added to each culture.
Three weeks later, the old medium was poured off and 12 ml. of fresh
medium were added to each culture. This neuron feeding procedure was
continued to allow the cultures to develop satisfactorily. The age of

the neurons in culture equaled the age in days at the time of sacrifice

28

plus the number of days spent in culture. The cultures met a morpho-

logical criterion (axonal and dendritic redifferentiation) before

rinsing and fixation. The glass slide was removed from the cultures
and rinsed, at 22°C, by gently placing in a dish of 0.87% physiological
saline. The glass slide (with cells attached) was fixed for 3 hours at
room temperature in 1:1 ethanol and acetone. The entire staining pro-
cess was performed, in the dark, at a constant temperature of 22°C, to
maintain a diffusion equilibrium with the solution:

1. Acetylation of amino groups. The amino groups of the protein frac-
tion in the nucleoproteins were blocked with acetic acid anhydride
in pyridine:

(a) Pyridine, water-free 5 mins.
(b) Acetic acid anhydride:pyridine (2:3) 15 mins.
2. Purification and rehydration. The cells were purified from acetic

acid anhydride, and transferred into an aqueous medium: '

(a) Ethanol 100 percent 5 mins.
(b) Ethanol 95 percent 5 mins.
(c) Ethanol 60 percent 5 mins.
(d) Ethanol 30 percent 5 mins.
(e) bidistilled H20 5 mins.
3. Staining process. 5 x 10-5M A0 in Citric acid - NaZHP04 buffer,

pH 4.1:

(a) Citric acid - NaZHPO4 buffer 5 mins.
(b) A0 in citrate-phosphate buffer 30 mins.

4. Rediffusion process. Nashing away of unbound A0 with citrate-
phosphate buffer, pH 4.1:
(a) Citric acid - NazHP04 buffer 5 mins.

29

(b) Citric acid - Na2HP04 buffer 5 mins.

(c) Citric acid - NazHPD4 buffer 5 mins.
The reaction vessels were matched and each contained 100 ml of reaction
solution. The solutions were allowed to reach equilibrium in the thermo-
static waterbath for 24 hours. After the rediffusion process, the excess
buffer solution was drained off. The cells were sealed in the same citric
acid - NaZHP04 buffer solution, pH 4.1, with a coverslip and paraffin.
The areas of the cell bodies were determined by projecting and tracing
the cell outlines on paper. A planimeter was used to measure the areas

of the cells' profiles.

C. Instrumentation

The observations and emission spectra were registered with a micro-
spectrophotometer constructed from commercially available components.
The microscope was a Leitz 0rtholux, with fluorescence equipment.

The light source was a Xenon arc lamp, type X80 150, housed in a
Universal Lamp housing model #250. A blue filter, #GS 5-56 (Corning
Glass Norks), was placed in front of the light exit aperture on the Lamp
housing. The Xenon lamp was powered by a power supply which produces a
constant current that maintains the light output within :_1% (E. Leitz,
New York).

For fluorescence excitation of samples, in incident light, the
energy source was focused into the entrance slit of a Leitz Fluorescence
Vertical Illuminator (according to Ploem). The fluorescence excitation
was produced by exciting filters, 4 mm 86 38 and 5 mm BG 12, in the lamp
housing. A dichroic beam-splitting mirror, TK495, was also used. The
fluorescence of the samples was filtered through a built-in suppression

filter, K495, and a Sharp-Cut filter, 3-70 (Corning Glass Norks),

30

contained in the suppression filter slide (see Figure 4). The radia-
tion emitted from the irradiated sample was collected and projected
into a 4 in. diameter, 180°, wedge interference filter (linear from
400 nm. to 700 nm.). The monochromator was motorized, and powered

by a Heathkit IP32 regulated power supply (see Figure 5).

The monochromatic radiation was focused onto a R 4468 (200 nm.
to 800 nm.) potted photomultiplier tube (American Instrument Company).
The phototube high voltage electrical power was manufactured by two
Heathkit IP32 regulated power supplies. The electrical responses were
amplified by an instrument modelled after the Aminco Solid-State
Blank-Substract Photomultiplier Microphotometer 10-180 [St. Pierre,
1972]. The emission spectra were recorded on a 135C X-Y Recorder

(Hewlett Packard, Mosely Division) (see Figure 5).

 

 

 

 

 

 

 

 

 

Figure 4. Diagram of the principle of the LEITZ
fluorescence vertical illuminator

l.
2.
3.

O‘DQNO‘U‘b

suppression filter slide

suppression filter

turret with suppression filters and beam-splitting
mirror

beam-splitting mirror

stray light stop

field diaphragm

exciting-filter combination (in the lamp housing)
ultra-high-pressure Xenon arc lamp

objective

object

 

lull! ‘Illll...lle])] I l )i ’9] \IIII . e.

R 4468 Phototubs

Honochromator

Sharp-cut
Light Source filter (3-70)

(Xenon 130 150

@. __._.__-__-

Blue filter
(#05 5-56)

Figure 5.

 
 
 

32

Phototubs
Power Supply
(two Heathkits)

   

 

Photo-ultiplisr
Microphotometer

L

motor

 

T7

 

 

 

. L__fi

sits Fluorescence
Vertical Illuina-

 

x-r Recorder

 

 

Monochromator Power Supply
(Heathkit)

Diagram of the microspectrophotometer.

III. RESULTS AND DISCUSSION

A. Introduction

The extent to which we understand the behavior of biological macro-
molecules will depend upon our knowledge of the dye molecule, and dye-
macromolecule interaction. Acridine orange (A0), a small planar dye
molecule, is a fluorescent molecular probe used in the investigation of
nucleic acids (NA). Zanker [1952; Nest, 1969] has studied the nature of
A0 alone in aqueous solutions, while other investigators have examined
the conduct of A0 and NA together, in aqueous solutions and in gelatin
microdroplets, under various environmental conditions [Bradley and Nolf,
1959, 1960; Loeser et a1, 1960; Rigler, 1966]. Under certain conditions
the spectral characteristics of the acridine orange-nucleic acid com-
plexes can be employed in determining the A0 and/or NA content, as well
as to delineate the nucleic acids within individual fixed or living cells
[Rigler, 1966; Nest, 1969]. Inferences about the structure and function
of macromolecules that are involved in neuronal phenomena, such as plas-
ticity (memory), energy transduction, specificity and aging, may be de-
vised by studying the interactions of the fluorescent probe and macro—
molecule. Biophysical cytochemistry provides the neurobiophysicist with
valuable tools to investigate the salient features of various neuronal
phenomena.

A large segment of my research was concentrated on the binding and
structure characterization of AO-NA complexes embedded in gelatin micro-
droplets exposed to various microenvironmental conditions. The spectral
distribution of the emission of different AD-NA complexes subjected to

33

34

the same staining conditions is displayed in Figure 6. Notice that equi-
valent amounts of different A0-NA complexes (AD-DNA, AO-RNA, A0-Poly U)
have identical spectral patterns, with a green fluorescence maximum at
536 nm. and a prominent shoulder toward the longer wavelengths at 604 nm.
Table 1 displays the relative fluorescence intensities for the various
AD-NA complexes under the same conditions. The differences in the inten-
sities suggest that AD has a specific binding affinity for each of the
NA; this theme is observed in all of the microdroplet experiments.

In summary, my research on other gelatin microdroplet systems re-
vealed a considerable amount of information about the binding of acridine
orange to nucleic acids. It affirms that no significant difference in
the aggregation of A0 on the various NA exists, as evidenced by only
minute variations in the degree of aggregation for each nucleic acid.

The degree of aggregation, a, is a measure of the degree of dye asso-
ciation of A0 on a particular nucleic acid configuration [Rigler, 1966],
and can be calculated from the ratio of the emission intensity at the
longer wavelength (604 nm., associated A0) to the short wavelength

(536 nm., monomer A0) fluorescence maximum intensity. In the presence
of an increasing dye concentration, the degree of aggregation and bind-
ing of the dye molecules onto the nucleic acid in both the monomer and
associated forms intensified linearly within a specific A0 concentration
range. Increasing the interaction time of the acridine orange molecules
with the biopolymers enhanced the binding of the dye molecules to the
nucleic acids. My results also showed that the binding of A0 to the
nucleic acids increased as the pH increased from 4.0 to 8.4. The degree
of aggregation fbr rRNA decreased slightly as the pH increased. No
significant change in the degree of aggregation for DNA was noticed,

NORMALIZEI FLUORESCENCE INTENSITY

1.00

0.75

0. 50

0. 15

0. 10

0.05

35

" — 0 - 1.0 pg. DNA in 31 pf.pgsk.

—A-— 1.1 pg. Poly 0 in 32 pf.pgsk.

-x — 3: put-med Pigskin Only

 

 

 

L- o
/A
b
x
A- /
I t r r I
502 536 570 60‘ 638 672 706 III.

RAVI-21.8116!!! (exp. in nanameters)

Figure 6. The normalised fluorescence spectra of A0 molecules
bound to different NA conformations and gelatin proteins, A0
staining concentration 5 s 10"5 M, staining time 30 mins., pH 4.1.

36

TABLE 1.

The relative fluorescence intensity of AO-NA complexes
at 536 nm. and 604 nm.

Staining Condition: 5 x 10'5M Acridine Orange, 30
minutes staining time, pH 4.l
1_0.l, at 22°C.

536 nm. 604 nm.

l. l.0 pg. DNA in 3% pf. pgsk. 822 l35
2 1.0 pg. rRNA in 3% pf. pgsk. 122 30.1
3. l.l pg. Poly U in 3% pf. pgsk. 98.1 23
4 3% purified pigskin only 64.3 17.9

indicating that the binding of A0 to nucleic acids in the monomer
fashion is preferred at high pH levels or at low hydrogen ion concentra-
tions. More importantly a linearity between the emission of the AO-NA
complexes and the amount of NA available for binding was established,
whereby increasing the amount of NA proportionately increased the amount
of A0 bound by the NA. Under the proper micro-environmental conditions,
the degree of aggregation for DNA and rRNA are tantamount and stable as
the NA content increases. Increasing the ions in the NA micro-environ-
ment increases the binding of A0 and decreases its degree of aggregation
onto the NA, suggesting that a high ion content favors the binding of
A0 to NA in a monomer formation. The denaturation of rRNA does not
appreciably affect the degree of aggregation of A0 or rRNA. however, the
binding of A0 to rRNA increases slightly with increasing temperature.
These results affirm that A0 has the sggg_binding modes with different
affinities for different NA conformations.

Another segment of my research involved the microspectrophotometric

analysis of AO-NA complexes within individual neurons identified in

37

dissociated cell cultures of specific and nonspecific areas of the rat
brain. From the microdroplet inquiry, fluorescence coefficients for
specific staining conditions were procured and used to calculate the NA
content inside the soma of single neurons. The nucleic acid content of
two neuronal types was determined at two ages, and the NA content per
neuron was found to depend more upon neuronal type and maturity than size.
The average value of the NA content of each neuronal type was 28% greater
at the older age than at the younger age. The NA content of one neuronal
type always averaged 8% greater than the NA content of the other type.
However, the NA content per neuron. as a function of neuronal soma area,
presented no definite pattern. Under the same staining conditions, the
spectral patterns of AO-NA complex emissions in single neurons were found
to be analogous to A0-NA complex emissions embedded in gelatin microdrop-
lets. The fluorescence characteristic of the AO-NA complexes in neurons
and in microdroplets are attributed to the binding of A0 to the NA pre-
dominantly in the monomer conformation, with a small degree of aggrega-

tion.

B. Microdroplet Analysis
Inasmuch as the main thrust of this inquiry was to develop and

apply a microspectrophotometric technique to the qualitative and quan-
titative determination of intrasomatic nucleic acids in single neurons,
the binding mechanisms of acridine orange to nucleic acids were examined
in a protein carrier system, in hopes of understanding, in detail. the
nature of AO-NA complexes within various microenvironments. In view of
the fact that neurons have cell and nuclear membranes. organelles, and

other biopolymers, the interactions of acridine orange and nucleic acids

38

within living and even fixed cells are more complicated than that within
simple protein microdroplet systems.

In each of the subsequent results, an average of three microdrop-
lets was analyzed per relative fluorescence intensity value (these being
actual experimental values without mathematical manipulations). The use
of an average of three microdroplets proves to be a sufficient quantity
since, on numerous occasions, different microdroplets with identical con-
tents yield tantamount fluorescence intensities and spectra, when treated
to the same conditions. In calculating the degree of aggregation, defined
earlier, necessary mathematical corrections were made. since the fluores-
cence of A0 fades during excitation and the protein environment inter-
acts with the dye molecules. Because the scanning speed of the X-Y re-
corder is slow in the x direction (0.l4 cm/sec) the fluorescence inten-
sities at the 604 nm. wavelength were the result of a longer irradiation
time than that of the 536 nm. wavelength. The relative fluorescence
intensity values for the 536 nm. wavelength had to be multiplied by a
0.76 correction factor, making the fluorescence intensity equal to the
intensity values if irradiated the same length of time as the 604 nm.
wavelength (see the Fluorescence Decay during Continuous Irradiation
section, p. 65). Before calculating the degree of aggregation for a
given condition, the relative fluorescence intensity values for 3%
purified pigskin (pf. pgsk.) only (containing no nucleic acids) were
used to eliminate any environmental influences of the AO-protein (gel-
atin) complexes from the AO-DNA and AO-rRNA complexes in the microdrop-
lets. This was accomplished by subtracting the values for 3% pf. pgsk.
only from that of AO-DNA and AO-rRNA in 3% pf. pgsk. at each wavelength.
An example of the calculations of the degree of aggregation of A0 onto

DNA fbr a specific staining condition is as follows:

39

 

_ 1 (F604 of DNA in 3% pf. pgsk.)-(F604 of 3% pf. pgsk. only)
a _ 0.76 {F536 of UNA in 3% pf. pgsk.)-(F536 of 3% pf. pgsk. only)

1. The flggrescence changgs of AO-NA complexes as a function of
acridine orange staining concentration. .

The relative fluorescence intensities of the AO-NA complexes at
536 nm. and 604 nm. reflect the amount of A0 bound to the NA in the
monomer formation and in the aggregated structure, respectively. In
this experiment, groups of microdroplets were subjected to a different
A0 staining solution concentration, while the rRNA or DNA contents,
analyzed within each microdroplet, were held constant over the entire
A0 staining concentration range (5xl0'6M to leO'4M). The microdroplets
were stained for 15 minutes each, and all of their reaction solutions
had pH 4.l. With the microspectrophotometer, the fluorescence spectra
of the AO-DNA, AO-rRNA, or A0-gelatin protein complexes were recorded
from each microdroplet while excited at 400 nm. The average relative
fluorescence intensities of the AO-DNA, AO-rRNA, and AO-gelatin protein
complexes. at 536 nm. and 604 nm. for each A0 staining concentration are
tabulated in Table 2 and graphically displayed in a semilogarithmic plot

in Figure 7.

In Figure 7. the relative fluorescence intensities, at 536 nm. and
604 nm., for A0 bound to l.0 pg. rRNA and 1.0 pg. DNA in 3% pf. pgsk.,
and to 3% purified pigskin only were found to increase linearly as the

6M to 5 x 10"

A0 staining concentration increased from 5 x 10' M. This
indicates that the total amount of A0 bound to the NA and gelatin proteins,
in both the monomer and aggregated forms. depends proportionately on

the quantity of free dye initially available for binding a NA or protein

macromolecule. The slope (m) for l.0 pg. DNA in 3% pf. pgsk. at 536 nm..

RELATIVE FLUORESCENCE INTENSITY

10000

5000

1000

500

100

50

10

40

 

 

E.—
-
b
-
L——
' /1 pg. DNA, 536nm.
p.
___ 1 pg. DNA, 604nm.
E:
—
-
_- 1 pg. rRNA, 536nm.
)—
i::- 32 pf.pgsk., 536nm.
E3. 1 pg. rRNA. 604nm.
_
L— , . ’ I ’ " , - 32 pf.pgsk., 604nm.
4" ._"‘ '
I’I‘ ’vx"’
.” “x.'
b b "'
L: w“
-
P
a...
P
b
h
l l l l l l l l I l l I l

 

lo-fu 10-521 10"“ 10'3"
ACRIDINE ORANGE STAININC CONCENTRATION

Figure 7. The average relative fluorescence intensities
of AO—3Z purified pigskin only and AO-NA complexes in

32 pf. pgsk. at 536 nm. and 604 nm. for different acridine
orange staining concentrations (expressed in molarities),
pH 4.1, staining time 15 minutes.

41

TABLE 2

The average relative fluorescence intensities of A0-3% purified pigskin
only and AO—NA complexes in 3% pf. pgsk. at 536 nm. and 604 nm. for
different acridine orange staining concentrations (expressed in molar-
ities), pH 4.1, staining time 15 minutes.

Acridine Orange Concentrations

 

5x10'5M 1x10'5M 5x10'5M 1x10’4M 5x10'4M
3% pf. pgsk. - 535 nm. 32.5 40.3 59.2 59 194
604 nm. 9.5 10.8 14.7 18.5 45.5
1.0 pg. rRNA - 535 nm. 47.3 59.5 122 155 503
604 nm. 11 8 15.3 29 42 127
1.0 pg. DNA - 535 nm. 54 102 480 1540 2825
604 nm. 13 24 95 325 790

where m = 9.5 x 103/M A0, was 1.9 times the value of the slope for 1.0
pg. rRNA in 3% pf. pgsk. at the same wavelength. Further, the slope
for 1.0 pg. rRNA in 3% pf. pgsk. (m = 5.0 x 103/M A0) was 2.2 times
higher than the slope for 3% purified pigskin only at 536 nm., whose m

equals 2.3 x 103/" A0. At A0 staining concentrations between 1 x 10'5

M
and 5 x 10'4M, the relative fluorescence intensities, at 536 nm. and at
604 nm.. of 1.0 pg. DNA in 3% pf. pgsk. are greater than the correspond-
ing intensities for 1.0 pg. rRNA in 3% pf. pgsk. However, at lower dye
concentrations, the amount of A0 bound to 1.0 pg. DNA in the monomer
and aggregated form are equivalent to the amount of A0 bound to 1.0 pg.
rRNA. as indicated by the intersection of the lines for 1.0 pg. DNA and
1.0 pg. rRNA in 3% pf. pgsk. around 5 x 10'5" A0. This suggests that
certain regions within the DNA and rRNA conformations have equal capa-
bilities for binding A0, and these regions are probably located on the

surface or outer portions of the NA macromolecule. For each A0 staining

concentration, the relative fluorescence intensities, at 604 nm.. of

42

each AO-NA complex, were sizeably less than their intensities at 536 nm.,
suggesting that within this AO staining concentration range (5 x lO'GM
- 5 x 10'4M) A0 binds to NA and to gelatin proteins preferentially in
the monomer conformation. Although the results in Figure 7 are not
enough to calculate binding constants for DNA and rRNA, they do suggest
that the constant for each biopolymer may be different, such that the
binding constant for a DNA macromolecule is greater than a rRNA macro-
molecule and, due to a greater number of binding sites and/or regions,
the DNA macromolecule has the capacity to bind more A0 molecules than
has the rRNA macromolecule.

Figure 8 displays the degree of aggregation of A0 on 1.0 pg. DNA
and on 1.0 pg. rRNA as a function of the acridine orange staining con-
centration. This figure demonstrates that the degree of dye aggrega-
tion for DNA and rRNA depends upon the A0 staining concentration,
whereby increasing the amount of dye available for binding the NA
augments the association of A0 on the NA conformations. The degree of

4

aggregation at 5 x 10' M A0, for both DNA and rRNA, is only 1.7 times

'SM. Therefore, to

larger than their degrees of aggregation at 5 x 10
produce a small change (2x) in the degree of aggregation requires a
large change in the staining concentration (loo-fold). This implies
that each DNA and rRNA macromolecule must contain a large amount of A0
molecules to secure at least a degree of aggregation equal to 1, and,
in order to evoke a red fluorescence, the degree of aggregation and the

A0 content per NA biopolymer must be tremendous. Therefore. for all

6

practical purposes at an A0 staining concentration between 5 x 10' N

4

and 5 x 10’ M, the degreelof aggregation for rRNA (from 0.204 to 0.347)

is equivalent to the degree of aggregation for DNA (from 0.214 to 0.372),

DEGREE OP AGGREGATION

.40

.35

.30

.25

.20

.15

.10

.05

 

43

 

hm
"' -1.0 pg. DNA
'7 ‘;D 1.0 pg. rRNA
/
_. .r
3’”.
(5.. o
n. (I ""‘f)
I s
_
O
_. o»
—
—
1 1 1 1 1 1 1 1 1 1 1
10-611 10'514 10"" 10"31
ACRIDINE ORANGE STAINING CONCENTRATION
Figure 8. The degree of aggregation of A0 onto 1.0 pg. DNA or

rRNA in 3% pf. pgsk. for different acridine orange staining
concentrations (expressed in molarities), pH 4.1, staining time
15 minutes.

44

and as the A0 staining concentration increases, they both follow the

same pattern. The author proposes that there are sites or regions within
the DNA and rRNA conformations, specific for binding A0 in the aggreg-
ated form, with similar binding behaviors as a function of the dye
concentration.

Utilizing a fluorometer, West [1969; Zanker, 1952] measured the
fluorescence of monomer acridine orange as a function of dye concentra-
tion. He found that in aqueous solutions (0.154M NaCl, pH 7) the flu-
orescence emission at 538 nm. is linear up to an A0 concentration of
1 x 10'6M. The slope was approximately equal to unity. More signi-
ficantly, the emission at 538 nm. deviates from linearity (i.e.,
decreases) at higher A0 concentrations, due to the formation of A0
polymers. My results indicate that in gelatin microdroplets the
fluorescence emission of monomer AO-NA complexes, as a function of dye
concentration, is linear up to 5 x 10'4" A0. with a slope nearly equal
to 1 x 104/" for 1.0 pg. DNA and 0.5 x 104/n for 1.0 pg. rRNA. Because
of the rediffusion proCess (removal of free dye molecules) the reader
should note that, within gelatin microdroplets. the actual A0 concen-
tration bound per NA macromolecule is much less than the A0 staining
concentration; this confines the interactions to dye molecules primarily
bound by a macromolecule. In comparison, A0 molecules free in aqueous
solutions have greater diffusion rates than A0 molecules bound and
unbound to NA in gelatin microdroplets. Therefore. due to the lack of
mobility and the medium, the collision rates of A0 molecules bound to
the inflexible NA macromolecules are negligible. This lowers the inter-
action between A0 molecules considerably, thus limiting the formation

of unbound A0 polymers, i.e., free aggregates are washed away during

45

rediffusion. In addition, the results from Figures 7 and 8 indicate
that within gelatin microdroplets the aggregation of A0 onto a NA
macromolecule may be proportional to the A0 staining concentration.
This suggests that there are specific binding sites on the DNA and
rRNA macromolecule for independently binding A0 in either the monomer
or aggregated formations.

Rigler [1966] measured the degree of aggregation and the fluores-
cence of monomer A0 at 530 nm. in a fixed tetraploid mouse fibroblast
cell, as a function of A0 staining concentration. He published results
showing that the degree of aggregation increased to 0.53, while the
fluorescence intensity at 530 nm. decreased by 25%. as the A0 staining
concentration increased from 1 x 10’4N to l x 10'3H. As one might
expect, unlike the simple gelatin microdroplet systems. the nuclear
and cell membranes, along with other intracellular structures, served
to concentrate the A0 content within the fixed cell by obstructing the
diffusion of excesses outside the cell. This increases the quantity
of A0 molecules per NA macromolecule which enhances aggregation. The
quenching of the monomer A0 fluoreScence suggests an increased formation
of unbound A0 polymers. In the opinion of this author. the monomer A0
formation is the primary mode of binding onto the NA biopolymers, and
the disposition of each A0 formation depends heavily upon microenviron-

mental conditions, such as the dye concentration.

2. The fluorgscence changes of A0-NA complexes as a function of
acridine oragge staining time.
In this experiment, groups of microdroplets were subjected to

different reaction intervals, while the rRNA or DNA contents analyzed

46

within each microdroplet, and the acridine orange concentration

(5 x lO'SM) were held constant over the entire range of staining times
(5 minutes to 120 minutes). The staining solutions and all other reac-
tion solutions had a pH 4.1. The fluorescence spectra of the AO-DNA
and AO-rRNA complexes were recorded from each microdroplet while irrad-
iating at 400 nm.. The average relative fluorescence intensities of
A0 bound to 1.0 pg. DNA or 1.0 pg. rRNA in 3% pf. pgsk., at 535 nm. and
604 nm. for each staining time are tabulated in Table 3 and graphically
displayed, in a semilogarithmic plot, in Figure 9.

TABLE 3
The average relative fluorescence intensities of AO-NA complexes in
3% pf. pgsk. at 536 nm. and 604 nm. for different staining times
(expressed in minutes), acridine orange staining concentration,
5 x 10-5M, pH 4.1.
Staining Time

5m 10m 15m 30m 45m 60m 90m 120m

 

3% pf. pgsk.

-536 nm. - - 53.3 61.3 - - - -
-604 nm. - - 13.8 16 - - - _
1.0 pg. rRNA .

-536 nm. 55.5 71.3 68.3 81.5 98.5 96.5 131.5 145
-604 nm. 19.9 21.8 17.5 22 22.5 24.5 28.5 30.5
1.0 pg. DNA

-536 nm. 366 400 435 634 715 - 825 990
-604 nm. - 74.5 88 80 124 153 - 190 255

In Figure 9, the relative fluorescence intensities. at 536 nm.
'and 604 nm., for A0 bound to 1.0 pg. DNA and 1.0 pg. rRNA in 3% pf.
pgsk., demonstrated an enhancement along with prolonged reaction inter-
vals. The relative fluorescence intensities of AO-rRNA complexes in

3% pf. pgsk. increased linearly, at 536 nm. and 604 nm., as a function

47

 

 

 

10000 .—
h
h
5000 Z.
1—
10 _
00 : .__v . 1 98s DNA,
— C
500 .—
—.’/'
h
.
*— -----'—‘- 1 p8. DNA,

RELATIVE FLUORESCENCE INTENSITY

;’ / l P8.tm.
100 I’ ' C 0

”fl, ’ ‘ t 1 past“.

10

U!
1 111111”

 

llllllllLLlll

10 30 50 70 90 110 130m.
STAINING TIME (expressed in minutes)

Figure 9. The average relative fluorescence intensities of AD-NA
complexes in 3% pf. pgsk. at 536 u. and 604 nm. for different
staining times, A0 staining concentration 5 x 10'”5 11, p11 4.1.

536nm.

604nm.

536nm.

604nm .

48

of staining time, whereas the amount of A0 bound to rRNA in the monomer
and aggregated forms were proportional to the length of time, in min-
utes, the dye molecules were allowed to bind to the rRNA macromolecules.
The slope of the line for A0 bound to rRNA in the monomer form, at

536 nm., is equal to 2.9 x 10-3/mins. of staining and is a little larger
than the slope for A0 bound to rRNA in the aggregated form at 604 nm.
Contrarily. the relative fluorescence intensities of AD-DNA complexes,
at 536 nm., rapidly doubled between 5 minutes and 35 minutes staining
times with a slope equal to 9.8 x 10’3/mins. of staining. Prolonging
the staining time above 35 mins. changed the slope, such that the slope
significantly decreased 86% of its original value, to 1.4 x 10'3/mins.
where, at the 120 mins. staining interval, the fluorescence intensity
had only increased 1.3 times greater than its value at 35 mins. stain-
ing time. Clearly, two phases are-involved in the binding of A0 mole-
cules to DNA macromolecules: during the first 35 mins. of staining,
there is a rapid uptake of dye molecules by the DNA macromolecules;
later, the rate of uptake of the dye molecules substantially decreases
by more than 80% of the initial value. This suggests that there exist
highly seductive binding sites within unique regions inside the DNA
biopolymer, which promptly bind A0 molecules in the monomer form; these
sites, as a result of such binding, are capable of being completely
filled within 35 mins. There are other regions, with hindered binding
sites, that secure the dye molecules at a much slower rate. The slopes

for DNA as compared to rRNA, infer that initially the binding rate for

A0 molecules onto the DNA macromolecules is approximately 3.4 times
faster than the rate for rRNA--the binding rate is taken to be the

quantity of A0 molecules bound per minute in a specific conformation

49

per NA macromolecule. In addition, for each reaction interval, the
fluorescence intensities of A0 - 1.0 pg. rRNA and A0 - 1.0 pg. DNA
complexes at 536 nm. averaged 3.9 and 4.6 times greater, respectively,
than the intensities at 604 nm.; and the fluorescence intensities of
AO—DNA complexes averaged 6.2 times larger than the intensities of
AO-rRNA complexes. These results conclude that AD molecules selec-
tively bind to NA in the monomer form and the dye molecules prefer-
entially bind to the DNA macromolecule rather than the rRNA macromolecule.
In comparison, the relative fluorescence intensity changes of
AO-DNA complexes in gelatin microdroplets, as a function of staining
time, in this study are similar to the results of AO-NA complexes in
a tetraploid cell. Rigler [1966] published the fluorescence intensity
changes of AO-NA complexes, in a fixed tetraploid mouSe fibroblast cell,
at 530 nm., as a function of staining time. He found that the average
fluorescence intensity increased as the staining time increased from
15 mins. to 30 mins., having a slope equal to 1 x lO'Z/mins. Prolong-
ing the staining time above 30 mins. to 100 mins. substantially decreased
the fluorescence intensity, with a line having a negative slope equal to
-2 x 10'3/nn'ns. During the initial 30 mins. of staining, the A0 mole-
cules are quickly bound by all of the available monomer binding sites
on the NA macromolecules. Above 30 mins. further uptake of the dye
molecules increases the degree of aggregation, thus decreasing the
monomer fluorescence at 530 mn. The graphic pattern for AO—NA complexes.
as a function of staining time in tetraploid cells, is primarily due to
the A0 molecules binding to chromosomal DNA macromolecules in the monomer
form, whereby the dye molecules readily bind to specific regions of

high affinity inside the DNA conformation in less than 35 mins. The

50

binding rates of A0 molecules in the monomer form to a DNA macromolecule
are greater in a fixed teraploid mouse fibroblast cell than in a gela-
tin microdroplet. This elevation, reflected by different corresponding
slopes in each of the environments, is principally due to the nuclear
and cell membranes concentrating the intracellular A0 content within

the cell. This serves to increase the collision rates between the
molecules, whereby the binding of the dye molecules onto the NA macro-
molecules in the monomer and aggregated forms is intensified consider-
ably. Therefore, the amount of dye molecules bound to a DNA macro-
molecule and the binding rates of A0 molecules onto the DNA macromolecule
depend more on the microenvironmental circumstances (specifically, the
A0 content per NA macromolecule in the intracellular environment versus
that of a gelatin microdroplet) than the interaction time between the

dye molecules and the DNA macromolecule.

3. The fluorescence changes of AO-NA complexes as a function of

acridine orange staining_solutionng.

 

Undoubtedly, the pH of the reaction solutions, i.e. the degree
of ionization of the acridine orange and nucleic acid molecules, affect
the binding of acridine orange onto the nucleic acids. In this experi-
ment, groups of microdroplets were exposed to a variety of acridine
orange staining solution pH. The rRNA or DNA contents analyzed within
each microdroplet were held at 1.0 picograms. The A0 staining concentra-
tion was held constant at 5 x 10'5M, and the microdroplets were each
stained for 30 mins. apiece. The pH of the rediffusion solutions were
the same as the staining solution. After rediffusion, the emission
spectrum of each microdroplet was recorded, with the microspectrophoto-

meter, during irradiation at 400 nm. The average relative fluorescence

51

intensities of AO-DNA, AO-rRNA, and AO—gelatin protein complexes, at
536 nm. and 604 nm., for each A0 staining solution pH, are tabulated

in Table 4 and graphically displayed in a semilogarithmic plot in Figure

10.

TABLE 4

The average relative fluorescence intensities of 1.0 pg. DNA-A0 and
1.0 pg. rRNA-A0 complexes in 3% pf. pgsk., and gelatin protein-A0
complexes of 3% purified pigskin only, at 536 nm. and 604 nm., for
different acridine orange staining solution pH, A0 staining concentra—
tion 5 x 10-5M, staining time 30 mins.

Staining Solution pH

 

pH 4.0 pH 5.7 pH 7.2 pH 8.4
3% pf. pgsk. - 536 nm. 80 297 675 790
604 nm. 19 56.5 98 115
1.0 pg. rRNA - 536 nm. 125 943 3050 1990
- 604 nm. 31 133 450 240
1.0 pg. DNA - 536 nm. 715 3075 4170 7050
- 604 nm. 125 615 850 1150

The relative fluorescence intensities of A0 bound to NA and gelatin
protein, at 536 nm. and 604 nm., exhibited an enlargement as a function
of pH; see Figure 10. The fluorescence intensities of A0 - 1.0 pg.

DNA complexes in 3% pf. pgsk., at 536 nm., increased by 5.3 times, from
a pH of 4.0 to 5.7, with a slope equal to 3.8 x 10-1/pH. Above pH 5.7,
the slope decreased by more than 65% (m = 1.2 x 10'1/pH), while the
fluorescence increased another 140% at pH 7.2, and over 200% at pH 8.4.
The graphic pattern of the fluorescence intensities, at 604 nm., of
AO-DNA complexes as a function of pH, is tantamount to the pattern at
536 nm.; however, at each pH unit, the fluorescence intensities at 604
nm. averaged 19% of the intensities at 536 nm. In addition, the rela-

tive fluorescence intensities for A0 bound to 1.0 pg. rRNA in 3% pf.

10000

RELATIVE FLUORESCENCE INTENSITY

5000

1000

500

100

50

10

52

   
  

—
_
t: ' 1 pg. DNA, 536nm.
p.
—
P O
F- \
0’
-- ~~1 pg.rRNA, 536nm.
_ / ’0’ 1 pg. DNA, 604nm.
il-I- / . x, 3
P z pf. gsk.,536nn1.
— / x/ P
1—
i-—-
.. °\
‘\
h— ‘\ ‘~
°\l pg.rRNA, 604nm.

I .— .—X¢' 31 pf.pgsk. ,604nn.
— x.— “T.
1—- / I I
: 4x / ’ I
(- / X I
-— / I

/

- ’l ’

o I
h I ’

I

— "

,3!
:2
—
p
h
1—

l l l l I l .l l 1, I. l l

 

 

4.0 5.0 6.0 7.0 8.0 9.0
pH OF STAINING SOLUTION

Figure 10. The average relative fluorescence intensities of 1.0 pg.
DNA-A0 and 1.0 pg. rRNArAO complexes in 31 pf. pgsk., and gelatin
protein-A0 complexes of 32 pf. pgsk. only, at 536 an. and 604 nm., for
different acridine orange staining solution pH, A0 staining concentration
5 x 10"5 H, staining time 30 mins..

53

pgsk., at 536 nm., itensified by 24.4 times, from pH 4.0 to 7.2, having
a slope equal to 4.2 x lO'I/pH. At pH 8.4 the fluorescence intensity
decreased to 65% of it value at pH 7.2 and the slope changed its value to
-l.8 x 10'1/pH. The fluorescence intensity changes at 604 nm. for
AO-rRNA complexes as a function of pH were the same as the changes at
536 nm., except the slopes were slightly lower and the fluorescence
intensities at each pH were 12 to 25% of the 536 nm. values. It has
been reported that AD exists as a cation completely ionized at pH 7

with a pK of 10.45 [Zanker, 1952; West, 1969]. The lower fluorescence
intensities for AO-DNA and AO-rRNA complexes in 3% pf. pgsk. at acid

pH are due to an augmented positive charge on the A0 molecules and on
the negative phosphoric acid residues of the NA nucleotides. This leads
to an enhanced molecular repulsion, suppression of the NA dye binding
sites, along with a slight denaturation of the NA macromolecule, thereby
decreasing the binding of A0 molecules to a NA macromolecule. Decreas-
ing the positive charge concentration to neutral pH values maintains a
relatively stable NA conformation and permits the phosphoric acid resi-
dues to become ionized [Levene and Simons, 1925]. This favors the mole-
cular attraction and hydrophobic interactions of the NA nucleotides and
A0 molecules. thus increasing the binding of A0 to the NA macromolecule.
The dye molecules can readily f0rm molecular complexes with the monomer
and aggregated binding sites on the NA macromolecule, as hinted by an
intensification of fluorescence for AO-DNA and AO-rRNA complexes in 3%
pf. pgsk., at pH 7.2. The slopes for A0 bound to 1.0 pg. DNA, between
pH 4.0 and 5.7, at 536 nm. and 604 nm., are practically equivalent to
the slopes for A0 bound to l.0_pg. rRNA, m = 4.0 x 10'1/pH. at the same
wavelengths. These circumstances imply that between a pH of 4.0 and 5.7

54

the binding of A0 to DNA and rRNA macromolecules uniformly increases as

a function of pH. This unifbrmity in the binding behavior of A0 to DNA
and to rRNA macromolecules, between pH 4.0 and 5.7, is offset by dis-
similar DNA and rRNA conformations. The dissimilar conformations are
reflected by the slope of the lines deviations from 4 x 10‘1/pH at pH 5.7
for DNA, and pH 7.2 for rRNA as a function of pH, and the quantity of A0
bound to the DNA conformation in the monomer and aggregated forms aver-
aged 1.6 times greater than the rRNA conformation at pH 7.2, compared to
4.9 times greater at pH 4.0. These results suggest that the DNA con-
formation has a greater structural stability and capaCity to bind A0
molecules in both molecular forms. than the rRNA conformation, especially
at low pH values.

Also, in Figure 10, the relative fluorescence intensities of A0-
gelatin protein complexes (3% pf. pgsk. only), at 536 nm. and 604 nm.,
rise as a function of pH. The fluorescence intensity, for 536 nm., at
pH 8.4 is 10 times greater than the intensity at pH 4.0, and 6 times
greater than the intensity at pH 4.0 fer 604 nm. It appears that the
fluorescence intensities begin to plateau at pH 8.4. and the slope be-
tween pH 4.0 and 7.2 is 2.9 x 10'1/pH at 536 nm. The lower fluorescence
intensities at pH 4.0 are due to molecular interferences produced by an
increased positive charge concentration. However. at higher pH values
the A0 molecules can freely bind to unobstructed carboxylic groups in or
near hydrophobic regions of the gelatin protein conformation. The slope
at 536 nm. for A0 bound to gelatin protein, between 4.0 and 5.7, aver-
aged 28% less than the slopes fer A0 bound to NA, and the fluorescence
amounts of A0 bound to the gelatin protein at each pH are considerably

less than the amount of A0 bound to rRNA and DNA. This suggests that

55

specific types and/or conformations of macromolecules have distinctive
behaviors in binding A0 molecules. The binding rates of AD. as a func-
tion of pH to NA, are greater than the rates for gelatin proteins and
the highly ordered DNA conformation, and to a lesser extent, the rRNA
confOrmation have higher potentials for binding A0 molecules than has
the gelatin protein conformation. The fluorescence intensities, at 536
nm., for A0 bound to gelatin protein, are significantly greater, in
comparison, than the intensities at 604 nm., demonstrating that AD pre-
dominantly binds to gelatin proteins in the monomer form.

The degree of dye aggregation on DNA and rRNA macromolecules
changes as a function of pH; see Figure 11. The degree to which A0
aggregates onto rRNA macromolecules decreased by 54%, from 0.35 at pH
4.0 to 0.19 at pH 7.2, and another 14% at pH 8.4. In addition, the
degree of aggregation for rRNA macromolecules at pH 4.0 is 1.6 times
larger than the value for DNA macromolecules. This indicates that
lowering the pH disturbs the less stable rRNA conformation, thus chang-
ing the hydrophobic microenvironment of the helical regions capable
of binding A0 in the monomer form. (These regions may not necessarily
have the same fine structure as the DNA double helix.) 'This lowers
the number of binding sites on the rRNA conformation that ordinarily
bind A0 in the monomer form. Concurrently, the binding sites for A0 in
the aggregated form are undisturbed, thereby increasing the ratio for
the degree of aggregation at low pH. The degree of aggregation for 1.0
pg. DNA increases by 27%, from 0.22 at pH 4.0 to 0.28 at pH 7.2. and is
1.5 times greater than the value for 1.0 pg. rRNA at pH 7.2. It then
decreases to 0.22 at pH 8.4. In contrast. the hydrogen bonds in the
hydrophobic interior of the DNA double helix are intact at pH values

DEGREE OF AGGREGATION

56

 

 

0‘0 -
.35 - <3
.30 -
\\ /.
s25 — \
— O \ O
\ 1 pg. DNA
020 —
\. C)
a”"’ \\
" \ ”,,r \\\\\
.15 - ‘,
(3 1 pg. rRNA
.10 -
.05 -
l l l l l .1 l l. 1 l l l
4.0 5.0 6.0 7.0 8.0 9.0
pH 0? STAINING SOLUTION
Figure 11. The degree of aggregation of A0 onto 1.0 pg. DNA or

rRNA in 32 pf. pgsk. for different acridine orange staining solution
pH, A0 staining concentrations 5 x 10'5M, staining time 30 mins..

57

above 4.0 and are reversibly destroyed at pH values below 4.0. There-
fbre, between pH 4.0 and 8.4, the A0 monomer binding sites on the more
stable DNA conformation are unimpaired, and are located in similar micro-
environments. Once the A0 monomer binding sites in a NA conformation
are filled, further binding of the dye molecules are in the aggregate
mode of binding. Since the A0 monomer binding sites were nearly all
full, any variations in the binding of A0 to the DNA conformation above
pH 4.0 would be in the aggregated mode and would depend upon the ionic
interactions of the dye molecules and DNA nucleotides, and less on
hydrophobic interactions. Enhancing the positive charge concentration
disrupts the binding of A0 molecules to the DNA macromolecules, thus
reducing dye aggregation. Enhancing the negative charge concentration
favors the binding of A0 to DNA, thus increasing the dye aggregation.
The degree of aggregation, at each pH value, for 1.0 pg. DNA and 1.0 pg.
rRNA is less than 0.36, indicating that A0 preferentially binds to DNA
and rRNA in the monomer mode.

-Langridge et a1 [1957; Nest. 1969] reported that. for the living
cell, the intranuclear pH is 7.6-7.8, while the cytoplasmic pH is 6.8.
Digesting the microdroplet results, the author concludes that the intra-
cellular pH values would allow for optimal binding of A0 to NA in the
living neuron. Along with the nuclear and cellular membranes. the bind-
ing of A0 to the intracellular NA would greatly increase, resulting in
a tremendous dye-to-nucleotide ratio. The amount of A0 bound per NA
macromolecule would make the degree of aggregation large enough to pro-
. duce orange to red colors, provided the staining conditions are appro-

priate.

58

4. The fluorescence intensity changes of A0-NA complexes as a
function of NaCl content.

The ionic strength of the molecular microenvironment should have
profbund effects on the binding behavior of acridine orange molecules
to a NA macromolecule, since acridine orange not only binds to NA via
hydrophobic interactions, but also by a strong ionic bond between the
positive charged dimethylamino group of the acridine orange ring and
the negative charged phosphoric acid group of a NA nucleotide. In order
to study these effects, different amount of NaCl were embedded in gelatin
microdroplets and analyzed along with 0.5 pg. DNA or 1.0 pg. rRNA. Each

microdroplet was stained with 5 x 10"5

M acridine orange for 30 mins. at

a pH of 4.1. To avoid complications, the ionic strength and pH of all

of the reaction solutions were held constant throughout the entire experi-
ment. The emission spectrum of each microdroplet was recorded with the
microspectrophotometer, while exciting at 400 nm. The average relative
fluorescence intensities of AO-DNA, AO-rRNA. and AO-gelatin protein com-
plexes, at 536 nm. and 604 nm., for a particular NaCl content, are tab-

ulated in Table 5 and graphically displayed in a semilogarithmic plot

in Figure 12.
Figure 12 demonstrates that the fluorescence intensity of A0 bound

to 0.5 pg. DNA and 1.0 pg. rRNA in 3% pf. pgsk., and to 3% pf. pgsk.
only. at 536 nm. and 604 nm., is dependent upon the NaCl content within
the microdroplet. The average fluorescence intensities of monomer AO-DNA
complexes rapidly increased from 0.00 pg. NaCl to 98% of its maximum
value at 29.5 pg. NaCl, having a slope equal to 4.0 x 10'2/pg. NaCl.
Above 29.5 pg. NaCl. the slope decreased dramatically to a plateau.
Dissimilarly, the plots f0r AO-rRNA complexes never plateau. The

RELATIVE FLUORESCENCE INTENSITY

10000

5000

1000

500

100

10

59

 

r

1—

E

h—

_ ' # 0.5pg. DNA, 536nm.
— /.--_— -—~-_.“/-’-/.§ l psstm. 5361)..
1— ’ O 0.5pg. DNA, 604nm.

I O
1..

 

/ x x- 32 pf.pgsk.,536nm.
I
/ - .— — ’0. l ng’RNA. 60‘“.
x -° - "

x 32 pf.pgsk.,604nn.

llllllll

“physiological saline
(17 pg. NaCl)

 

lLLllllJlllll-

0.0 20 40 60 80 100 120pg.
NaCl CONTENT (expressed in picograms)

Figure 12. The average relative fluorescence intensities of 0.5 pg.
DNA-A0 and 1.0 pg. rRNA-A0 complexes in 32 pf. pgsk. and gelatin protein-
A0 complexes of 31 pf. pgsk. only, at 536 nm. and 604 nm., for various
NaCl contents (expressed in picograms), A0 staining concentration

5 x 10-511, staining time 30 mins., on 4.1.

60

TABLE 5

The average relative fluorescence intensities of 0.5 pg. DNA-A0 com-
plexes and 1.0 pg. rRNA-A0 complexes in 3% pf. pgsk. and gelatin pro-

tein-AO complexes of 3% pf. pgsk., at 536 nm. and 604 nm.. for various
NaCl contents expressed in picograms, A0 staining concentration
5 x 10-5M, staining time 30 mins., pH 4.1.

NaCl Content
0.00 pg. 29.5 pg. 78.3 pg. 111.7 pg.

 

3% pf. pgsk. - 536 nm. 61.2 81.5 105 106

- 604 nm. 20.5 22.5 28.5 31.5
1.0 pg. rRNA - 536 nm. 154 223 298 367

- 604 nm. 42.5 47.5 65 71
0.5 pg. DNA - 536 nm. 166 2580 2640 2220

- 604 nm. 42.5 420 370 340

average fluorescence intensities increased gradually from a minimum
value, at 0.00 pg. NaCl, to a maximum value 2.4 times larger, at about
112 pg. NaCl. having a slope of 3.3 x 10'3/pg. NaCl. These results
suggest that increasing the NaCl content elevates the ionic strength
within the external microenvironment of the NA macromolecules. :flhfig
serves to fortify_and enhance the hydrophobic interactions between the
acridine and nucleotidg_bgse ring_structures within the interior of

the NA macromolecule. thus increasingthe b1nding_of A0 to NA in the
monomer form via hydrophobic bonding, Even though the DNA content was
50% less than the rRNA content. the binding of acridineorange, due to
the differences in the two NA conformations, was still satisfactorily
manifested by different fluorescence intensities and slopes. At equi-
valent amounts of NaCl, the fluorescence intensities for AO-DNA com-
plexes were significantly greater than the intensities for rRNA, at 536
nm. and 604 nm., and the initial slope for monomer AO-DNA complexes was

approximately 12 times larger than the slope for monomer A0-rRNA

61

complexes indicating that acridine orange discriminately binds to the
DNA conformation over the rRNA conformation. The average fluorescence
intensities of AO-gelatin protein complexes at 536 nm. gradually increased,
as a function of the NaCl content, from 0.00 to 78.3 pg. NaCl, having

a slope equal to 3.2 x 10'3/pg. NaCl. This implies that the increased
binding of A0 to gelatin proteins. in the monomer form, may be due pri-
marily to elevated interactions between the hydrophobic regions within
the protein conformation and the ring structures of the acridine orange
molecules. As reflected by similar initial slopes (3 x 10'3/pg. NaCl),
the binding behavior of A0 to gelatin proteins, as a function of the
NaCl concentration, is synonymous to that of rRNA, suggesting that there
nay be similarities in their structures while binding acridine orange

in the presence of ions.

Since the degree of structural order of nucleic acids is decreased
at low ionic strengths [Rigler, 1966], varying the ionic strength of the
external environment of DNA and rRNA macromolecules would change their
conformations and their internal microenvironments. The results dis-
played in Figure 12 show that the binding behavior of acridine orange to
DNA, rRNA, and gelatin proteins relies heavily on the ions in the exter-
nal environment of the macromolecules. Further, the lowest NaCl concen-
tration that allows for the maximum binding of A0 to each macromolecule
is different. Note that the NaCl content of physiological saline is
well below the plateau point for each macromolecule, and the DNA confor-
mation is the lowest, at 29.5 pg. NaCl. suggesting greater stability.
and structural order at lower ionic strengths.

The aggregation of A0 molecules onto DNA and rRNA macromolecules

depends on the ionic strength of the microenvironment (see Figure 13).

DEGREE 0F AGGREGATION

.40

.35

.30

.25

.20

.15

.10

.05

62

1—

#-

_physiological saline
(17 pg NaCl)

 

I I I I I I I I I I I I

0.0 20 40 6O 80 100 120pg.
NaCl CONTENT (expressed in picograns)

Figure 13. The degree of aggregation of A0 onto 0.5 pg. DNA or
1.0 pg. rRNA in 3% pf. pgsk. for various NaCl contents, A0 staining
concentration 5 x 10'5M, staining time 30 mins., pH 4.1.

63

The degree of dye aggregation on 0.5 pg. DNA and 1.0 pg. rRNA decreased
as the NaCl content within the external environment increased. The
degree of A0 aggregation for rRNA decreased from 0.312, at 0.0 pg. NaCl,
to 0.199, at 112 pg. NaCl, a reduction of 36%. The degree of A0 aggre-
gation for DNA decreased from 0.278 at 0.00 pg. NaCl to 0.192 at 112 pg.
NaCl, a reduction of 31%. suggesting that the lower tendency of A0 to
aggregate, at a high ionic strength, on DNA and rRNA macromolecules may
be induced by the increased interaction of ions with equal charge (NA+).
The pattern of the degree of A0 aggregation on rRNA as a function of the
NaCl content is similar to the pattern for DNA, implying that AD aggre-
gates on both macromolecules in similar regions and modes. However,
the degree of dye aggregation on rRNA at each NaCl concentration aver-
aged 17% larger than DNA. suggesting thatAO has a slightly higher ten-
dency to aggregate on the rRNA conformation than the DNA conformation.
The aggregating behavior of A0 onto DNA and rRNA, as a function of the
NaCl content. is in contraposition to the binding behavior of A0 to DNA
and rRNA in the monomer form. This is evidenced when a loo-fold increase
,in the NaCl concentration produces a small decrease in the degree of dye
aggregation on DNA and rRNA, versus a very large increase in the binding
of A0 to DNA and rRNA in the monomer form. The degree of dye aggrega-
tion on rRNA and DNA never exceeded 0.35. accompanied by significantly
greater fluorescence intensities at 536 nm. than at 604 nm., concluding
that the binding of A0 to DNA and rRNA is predominantly in the monomer
farm and is sensitive to ions within the microenvironment.

Rigler [1966] examined the effects of the ionic strength of the
reaction solution upon the degree of A0 aggregation and fluorescence

intensity, at 530 nm., of a mouse fibroblast cell. He found that an

64

increase in the ionic strength caused a rapid increase of the fluores-
cence intensity at 530 nm. and a simultaneous decrease in the degree of
dye aggregation. Rigler's results were analogous to the author's, in
that the aggregation of A0 onto NA in mouse fibroblast cells and in gel-
atin microdrOplets was inversely proportional to the ionic strength,

and the binding of A0 in the monomer from had an opposite behavior.

5. The fluorescence intensities of AO-rRNA complexes at various

 

 

microdroplet forming solution temperatures.

I The binding of A0 molecules to a particular NA macromolecule de-
pends upon the NA native and denatured conformations. Raising the
temperature of the microdroplet forming solution results in the denatur—
ation of the NA macromolecules contained in the solution. In this
experiment, various microdroplet forming solutions containing rRNA were
slowly raised to different denaturation temperatures. The solutions were
held at a specific temperature for 3 mins.; immediately afterwards, the
droplets were formed and cooled rapidly. The rRNA content analyzed in
each microdroplet was 1.0 picograms. Each microdroolet was stained with
5 x 10'5M acridine orange for 30 mins. at a pH of 4.1. The emission
spectrum of each microdroplet was recorded with the microspectrophoto-
meter, while exciting at 400 nm. The average relative fluorescence
intensities of AO-rRNA complexes in 3% purified pigskin, at 536 nm. and
604 nm., for a particular forming solution temperature are tabulated in
Table 6.

Table 6 shows that a relative large increase in the microdroplet
forming solution temperature produced only a small increase in the

fluorescence intensities of A0 bound to 1.0 pg. rRNA in 3% pf. pgsk.,

65

TABLE 6

The average relative fluorescence intensities of
1.0 pg. rRNA-A0 complexes in 3% pf. pgsk., at

536 nm. and 604 nm., for various microdroplet
forming solutiog temperatures, A0 staining concen-
tration 5 x 10' M, at pH 4.1, staining time 30 mins.

Forming Solution Temperatures
55°C 82°C 100°C

 

1.0 pg. rRNA - 536 nm. 85 86 100
604 nm. 20 23 27

536 nm. and 604 nm. As the temperature increased from 55°C to 82°C, the
fluorescence intensity increased only 1% at 536 nm. and 15% at 604 nm.
Increasing the temperature further, to 100°C, increased the fluorescence
at 536 nm. by 16% and at 604 nm. another 17%. In addition, the fluores-
cence intensities of A0-rRNA complexes at 536 nm. averaged 3.9 times
greater than the intensities at 604 nm. These results suggest that a
rRNA conformation of low structural order of a completely denatured rRNA
macromolecule (at 100°C) has a slightly larger capacity to bind A0, pre-
ferentially in the monomer form, than a rRNA conformation of higher
structural order having a less disarranged conformation (55°C). This
was in contrast to the expectation that an ordered rRNA conformation
should bind more A0 molecules in the monomer form than a random rRNA
structure. Unfortunately, there is not sufficient data to give a com-

prehensive explanation.

6. The fluorescence intensity changes of AO-NA, gelatig-protein
complexes duriggcontinuous irradiation. .
Like many other fluorescent molecular probes, i.e. small planar

dye molecules, the fluorescence emission of acridine orange slowly

66

diminishes during electronic perturbation of excitation wavelength.
The concern of this experiment was the establishment of the fluores-
cence decay behavior of acridine orange bound to NA and gelatin pro-
teins in the monomer and aggregated forms, as a function of excitation
time. Since the emission maxima of acridine orange fluorescence spectra
are invariable with respect to the irradiation time, the 536 nm. and 604
nm. wavelengths were chosen as the emission wavelengths at which a
measurement of fluorescence emission amplitude was taken at various
irradiation intervals. The excitation, 400 nm., was continuous through-
out the course of the measurements. The contents analyzed in each micro-
droplet were either 0.5 pg. DNA in 3% pf. pgsk., 1.0 pg. rRNA in 3% pf.
pgsk., or 3% purified pigskin only, and were stained with 5 x lO-SM
acridine orange for 30 mins. at a pH of 4.1. The average relative
fluorescence intensities of AO-DNA, AO-rRNA, and AO-gelatin protein com-
plexes, at 536 nm. and 604 nm., as a function of the irradiation inter—
val, are graphically displayed in Figure 14. The intensities are normal-
ized to the instantaneous fluorescence emission amplitude at time zero.
The curves shown in Figure 14 exhibit that, as a consequence of an
extended irradiation time, there is a decrement in the fluorescence in-
tensities, at 536 nm. and 604 nm., of A0 bound to DNA, rRNA, and gelatin
protein macromolecules in 3% purified pigskin microdroplets. The two
fluorescence molecular species of acridine orange, the monomer and
aggregated forms, have different fluorescence decay behaviors as a
function of the irradiation time: the fluorescence fading of AO-DNA,
rRNA, and gelatin protein complexes at 536 nm. displays somewhat faster
decay rates than the corresponding fluorescence fading at 604 nm.,

depreciating over 50% of the initial fluorescence intensities at time

67

536 nm. 604 nm.
0.5 pg. DNA O—O .---.
1.0 pg.rRNA o—o 0.0.0
37. pf.pgsk. x—K x---x

 

3‘“
IT 417 4r I
/~
/. x
I
I
I
I,’
o
[I
I
II
II I
I
I
x

 

111111111111111111

10 30 50 7O 90 110 130 150 1809.
IRRADIATION TIME (expressed in seconds)

Figure 14. Fluorescence decay of AO-NA complexes, at 536 nm. and 604 nm.,
as function of the irradiation time, A0 concentration 5 x 10‘5 M, pH 4.1,
staining time 30 mins., excitation wavelength 400 nm..

68

zero after 30 sec. of irradiation for 536 nm., versus 60 sec. of
irradiation for 604 nm. The fluorescence decay behavior of AO-DNA
complexes, at 536 nm. and 604 nm., resembles quite closely the fading
behavior of AO-rRNA complexes at the same wavelengths. Where. after

10 sec. of irradiation, the fluorescence intensities of AO-DNA and
A0-rRNA complexes rapidly decline from 1.00 to 0.70 at 536 nm., and

to 0.76 at 604 nm., increasing the irradiation time to 30 sec., moder-
ately decreased the emission, further, to approximately 0.46 at 536 nm.
and to 0.56 at 604 nm. At a slower decay rate, the fluorescence intenj
sities of AO-DNA and AO-rRNA complexes faded roughly to 0.35 at 536 nm.
and to 0.44 at 604 nm. fellowing 60 sec. of irradiation. Finally, after
3 mins. of continuous excitation, the fluorescence emission of A0 bound
to either DNA or rRNA macromolecules gradually decayed to nearly 20%,
at 536 nm. and 28%, at 604 nm. of their original values at time zero.
Similarly, the fluorescence decay behavior of A0 gelatin protein com-
plexes at 536 nm. and 604 nm. parallels that of A0-NA complexes at the
same wavelength. These results suggest that the fluorescence decay
behavior of A0 molecules bound to a biopolymer, as a function of the
irradiation time, is more dependent on the binding mode of acridine
orange, the monomer of aggregated forms. than on the conformation or
type of the biopolymer.

Comparatively, the results presented here are similar to the
results obtained from single cells stained with acridine orange. Nest
and Lorincz [1973] studied the fluorescence intensity changes of living
human leukocytes, containing 1.05 x 10"14 moles AD/cell, at 540 nm.
and 660 nm., as a function of the irradiation time. They found that

the fluorescence fading at 660 nm., during irradiation with exciting

69

light, diminished at a slower rate than the fluorescence at 540 nm.

The fluorescence intensities at 660 nm. and 540 nm. decline to almost
50% and 40%, respectively, of their original values at time zero, after
25 secs. of irradiation. Continuing the irradiation time to 50 secs.
decreased the fluorescence emission roughly 5% at 660 nm. and 10% at
540 nm. The results of West and Lorincz were analogous to the author's,
in that the fluorescent molecular species of acridine orange differed
from each other on the basis of fluorescence fading. The fluorescent
dacay behavior of A0 bound in the monomer form, as a function of time.
fades at a faster rate than that of A0 bound in the aggregated form.
From our results we infer that the two binding modes of AD (the monomer
and aggregated forms) are independent of each other and photochemically
differentiable (i.e., fading), regardless of the microenvironment or

the confbrmation of the biopolymer.

7. The fluorescence intensity changes of Ao-NA complexes asge
fonction of the nucleic acid concentration,_and the quantitative deter-
mination of nucleic acids contained in aggiven microenvironment.

In collating the two molecular species of acridine orange, the
monomer complex was found to be more sufficient as a fluorescent mole-
cular probe for the detection of nucleic acids than was the aggregated
complex. The previous experiments established that A0 predominantly
binds to DNA, rRNA, and Poly U in the monomer farm with a small degree
of aggregation. The A0 molecules bound to a NA macromolecule in the
monomer farm are more stable and less sensitive to variations in the
microenvironment, i.e. changes in the pH, temperature and ionic strength,

than when the dye molecules are bound in the aggregated form. In this

70

experiment, gelatin microdroplets containing different amounts of rRNA
and/or DNA were treated to identical staining conditions. The A0 stain-

ing concentration was 5 x 10'5

M, and each microdroplet was stained for
30 mins. The pH of the staining and rediffusion solutions was 4.1.

The fluorescence spectrum of each microdroplet was recorded with the
microspectrophotometer, during irradiation with 400 nm. exciting light.
The average relative fluorescence intensities of A0 bound to DNA or
rRNA in 3% pf. pgsk., at 536 nm. and 604 nm., for a particular DNA or

rRNA concentration are tabulated in Table 7 and graphically displayed

in a semilogarithmic plot, in Figures 15 and 16.

TABLE 7

The average relative fluorescence intensities of A0 bound to DNA or
rRNA. at 536 nm. and 604 nm., in 3% pf. pgsk. for various DNA and
rRNA concentration, A0 staining concentration 5 x 10'5H, staining
time 30 mins., pH 4.1.

DNA and rRNA Concentration (exp. in pg.)

 

0.00 0.09 0.18 0.38 0.59 0.80 1.00

DNA - 536 nm. 63.5 77.5 84 144 295 485 875
- 604 nm. 17.2 22.0 23.5 31.5 61.0 98.0 210

rRNA - 536 mm. 68.3 58.5 61.5 73.5 96.0 134 115
- 604 nm. 18.3 16.0 16.0 19.5 27.5 32.5 25

rRNA Concentration (exp. in pg.)
0.00 0.51 1.00 1.90 2.95 3.99 5.04

 

rRNA - 536 nm. 53.2 86.0 121 197 373 1160 1460
- 604 nm. 16.2 22.5 30.0 41.0 67.5 210 285

The relative fluorescence intensities. represented in Figure 15,
of AO-DNA complexes in 3% purified pigskin, at 536 nm., were found to

increase linearly as the DNA concentration increased from 0.00 pg. to

71

10000

5000

[1'11"]

1

1000

500

11l1111l
.5
E"

1

604 nm.

100 536 on.

U1
0

11]1111[

 

rRNA, 604 nm.

RELATIVE FLUORESCENCE INTENSITY

1
\
1
1
9
1
1
1
1

10

11?!111[

1

 

IIIIIIJIIIIII

0.00 0.20 0.40 0.60 0.80 1.00 pg.
NUCLEIC ACID CONCENTRATION (exp. in picograms)

Figure 15. The average relative fluorescence intensities of A0 bound
to DNA or rRNA, at 536 nm. and 604 nm., in 31 pf. pgsk. for various

DNA or rRNA concentrations, A0 staining concentration 5 x 10'5 H, stain-
ing time 30 mins., pH 11.1. °

72'
1.00 pg. The equation of the line, eq. 1, is as fellows:

DNA:

109 F536 mDNA(pg. DNA) + 109 b (l)

where log Fggg is the logarithm of the fluorescence intensity, at 536

nm., of a microdroplet containing only DNA; m is the slope 0f the

DNA
line; (pg. DNA) is the quantity of DNA contained in the microdroplet
expressed in picograms; and log b is the logarithm of the fluorescence
intensity, at 536 nm., of the microdroplet containing no DNA. The line
has a slope, mDNA’ equal to 1.26/99. DNA. This means that the total
amount of A0 bound to the DNA macromolecules is proportional to the
quantity of DNA available for binding the dye molecules. implying that

a DNA macromolecule has a specific number of binding sites for A0
molecules in the monomer form. As a function of the acridine orange
concentration, the fluorescence emission of monomer A0 molecules linearly
increases until the polymerization of the dye molecules occurs, and,
whenever bound to NA macromolecules. the fluorescence emission of monomer
A0 molecules are independent and unaffected by the emission of aggrega-
ted A0 molecules [Nest, 1969]. In addition, since the effects of un-
bound excess dye molecules are eliminated from the system. equation (1)
can be used to determine any amount of DNA contained in a 3% pf. pgsk.
microdroplet. where the amount of DNA (in picograms) is equal to

(log Fggg - log b)/mDNA' Displayed in Figure 15 are the fluorescence
intensities of A0-rRNA complexes, at 536 nm. in 3% pf. pgsk. as a func-
tion of the rRNA concentration, where the fluorescence intensity pro-
portionally increased 1.7 times as the rRNA concentration increased

from 0.00 pg. to 1.00 pg. rRNA. In comparison. the fluorescence inten-
sities of A0 bound to 1.00 pg. DNA at 536 nm. and at 604 nm. averaged

73

eight times greater than the corresponding intensities of A0 bound to
rRNA at the same concentration, indicating that a DNA macromolecule

has a significantly greater number of A0 binding sites, in the monomer
and aggregated farms. than does a rRNA macromolecule. These results
suggest that with my methodology, the smallest amounts of nucleic acids,
easily detectable, are about 0.38 pg. DNA and 0.8 pg. rRNA.

In Figure 16, the rRNA concentration was extended, permitting a
more lucid portrayal of the binding behavior of A0 molecules to rRNA,
as a function of the rRNA concentration. The average relative fluores-
cence intensities of AO-rRNA complexes, at 536 nm., demonstrated a
commensurable intensification, while the rRNA concentration enlarged
from 0.00 pg. to 5.04 pg. This indicates that there exist a constant
number of binding sites on rRNA macromolecules that bind A0 in the

monomer form. The equation of the line, eq. (2), is written as fellows:

log FgggA = erNA (pg. rRNA) + log b . (2)

where log Fgng is the logarithm of the fluorescence intensity of a

microdroplet containing only rRNA, at 536 nm.; erNA is the slope of

the line; (pg. rRNA) is the quantity of rRNA contained in the micro-
droplet expressed in picograms; and log b is the logarithm of the fluo-
rescence intensity, at 536 nm., of the microdroplet containing no rRNA.
The slope of the line. erNA’ isequal to 0.272/pg. rRNA. Equation (2)
can be used to calculate an unknown quantity of rRNA in a gelatin micro-
droplet, where the amount of rRNA (in pg.) is equal to (log F??? -

log b)/m Hith respect to the NA concentration. the binding rate of

rRNA'
A0 molecules to DNA macromolecules is greater than the rate for rRNA

macromolecules, since the slope mDNA is 4.63 times larger than the slope

RELATIVE FLUORESCENCE INTENSITY

74

10000

11111111|

0’ rRNA, 536 nm.

1000

500

1 1111111!

/ ’01 rRNA, 604 nut.
I
O

100

50.

11'1111'
‘\
\
\
0

1

10

r1l1111l

1

 

IIIIIIIIIIILJ

0.00 1.00 2.00 3.00 4.00 5.00 pg.
rRNA CONCENTRATION (exp. in picograms)

Figure 16. The average relative fluorescence intensities of A0 bound
to rRNA, at 536 nm. and 604 nm., in 3% pf. pgsk. for various rRNA
concentrations, A0 staining concentration 5 x 10"5 M, staining time
30 mins., pH 4.1.

75

erNA' Therefore, after having identical fluorescence spectra, the
binding of A0 molecules to the DNA and rRNA conformations are only
distinguishable by different binding affinities.

Throughout the course of the increments in concentration, the
degree of dye aggregation on the DNA and rRNA macromolecules was, for
all practical purposes, constant; the degree of A0 aggregation on the
DNA and rRNA conformations averaged 0.242 :_0.016 at diverse NA concen-
trations; see Figure 17. (Because of sensitivity limitations, DNA
contents below 0.38 pg. were not included in the average.) The aggre-
gation mode of A0 on the DNA and rRNA conformations was similar, inas-
much as each DNA and rRNA macromolecule had a specific number of binding
sites for A0 in the aggregate form, and their aggregate-to-monomer
binding sites ratio was the same. At each NA concentration, the fluores-
cence intensities, at 536 nm., of AO-DNA and AO-rRNA complexes in 3%
pf. pgsk., were 4.27 :_0.62 and 4.81 :_0.74 times larger. respectively,
than their affiliated intensities at 604 nm.. with a low degree of dye
aggregation on the DNA and rRNA macromolecules. once more confirming
that AD preferentially binds to NA in the monomer form.

The linearity between the fluorescence intensity of AO-NA com-
plexes and the amount of NA available for binding the dye molecules
suggests the possibility of determining the quantity of DNA and/or rRNA
contained in a given microenvironment. If a gelatin microdroplet con-
tains a mixture of DNA and rRNA macromolecules, a measurement of the
microdroplet's monomer A0 emission may be regarded as the sum of the
fluorescence intensities characteristic for each constituent of the
system. The logarithm of the fluorescence intensity at 536 nm. is then
denoted as:

DEGREE OF AGGREGATION

.40

.35

76

DNA CONCENTRATION (exp. in picograms)
0.00 0.20 0.40 0.60 0.80 1.00 pg.
1 f I I I I l I l

1

e30 (—

s
N
U!

N
O

.15

.104

.05

._ O \ ,.—-—-——-' ,OrRNA
'\ aav""' ~~::7_~" DNA

 

I 1 I 1 I 1 I 1 I 1 I

 

0.00 1.00 2.00 3.00 4.00 5.00 pg.
rRNA CONCENTRATION (exp. in picograms)

Figure 17. The degree of aggregation of A0 onto DNA or rRNA in
32 pf. pgsk. for different NA concentrations, A0 staining concentra-
tions 5 x 10"5 M, staining time 30 mins., pH 4.1.

77

DNA rRNA
log F536 = D(f.c.536) + R(f.c.536 ) + log b (3)
where log F536 = the logarithm of the relative fluorescence inten-

sity at 536 nm. of the gelatin microdroplet.

D,R the quantity of DNA and rRNA contained in the gela-
tin microdroplet system given in picograms of

nucleic acids,

DNA rRNA

f.C.5369f.C.536

= "fluorescence coefficients“; the slopes of the lines
for the fluorescence intensities of AO-DNA and A0-
rRNA complexes at 536 nm., as a function of the DNA
and rRNA contents, respectively (see equations 1 and

2).

the logarithm of the fluorescence intensities of the
gelatin microdroplet containing no nucleic acids,
at 536 nm.

log b

In simplifying equation (3) the total NA content of a system is
given by:

 

R + yD = rm (4)

where y is a theoretical constant equal to the ratio of the DNA to rRNA
fluorescence coefficients for any microenvironment, and is equal to 4.63.

Gelatin microdroplets containing different mixtures of known amounts
of DNA and rRNA macromolecules were employed to test the validity of this
equation. 'The value on the right side of equation (4) was compared to
the value on the left side of the equation. For each microdroplet, the
experimental (R and 70) value obtained from the [(109 F536 - log b)/
f.c.;§2A] averaged 87% of the theoretical value derived from the known
quantities of the embedded rRNA and DNA macromolecules. The results are
tabulated in Table 8.

Since the validity of this methodology was based on only a few

samples, these results should be regarded as a first approximation.

78

 

 

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m m4m<h

79

However, a correlation does exist between the fluorescence intensities
of AO-NA complexes, in the monomer form. and the quantity of NA avail-
able for binding the dye molecules. The application of equation (4)
requires that the amount of A0 bound by the NA in a given system must
be proportional to the fluorescence intensity of the monomer A0 mole-
cules, and must be proportional to the quantity of the NA macromole-

cules in the system.

C. Neuron Analysis

With the recognition that the metabolic functions of nucleic acids
are significant in the neuron to neuron interaction, it is postulated
that the molecular events underlying neuronal activities are manifested
in the cellular nucleic acid content during the development of nervous
tissue. However, the intricate anatomical and cytoarchitectural design
of the nervous system poses certain problems for the use of fluorescent
molecular probes, in the study of a few intracellular components. Study-
ing neurons ig_§1§g is complicated by the dense packing of nervous tis-
sue, sheaths, glia, and blood-brain barriers. These problems may be
obviated by means of dissociated cell cultures. Under various micro-
environmental conditions. the binding behavior of acridine orange to
nucleic acids has afforded investigators with information about the
structure and function of the probed macromolecule [Rigler, 1966; Nest.
1969]. In association with my microdroplet results, these findings
substantiate the concept that the utilization of acridine orange as a
fluorescent molecular probe, in conjunction with dissociated neuron
cultures, facilitates the microspectrophotometric determination of

nucleic acids in single neurons.

80

The gross appearances of neurons grown in dissociated cell cul-
tures are different than those jg situ (i.e., the morphology in a normal
developing animal). Therefore, if any meaningful correlations are to be
realized from this investigation, it is of great importance that the
nucleic acid determinations be made on specific neuronal types. In
order to accomplish this, several neuronal types are identified in the
cell cultures. The classification of these neurons were made with
reference to their morphological descriptions. The neuronal types
characterized are as follows:

1. Canada, Type I neuron. This cell has a large diamond-shaped
cell body. having a length over 30% greater than its width. The cell
body is customarily surrounded by perineuronal satellites [Jenkins,
1972. p. 59] see Figure 18. The nucleus is oval, generally located
toward the center of the cell body, and contains several nucleoli.

The nucleus comprises about 20% of the cell body. Each of the points
on the diamond soma has a process that can measure up to several hun-

dred microns. The cytoplasm is moderately filled with Nissl bodies.
and is a little darker than the nucleus.

 

2. Canadg,_Type II neuron. These are very small cells. their
somas being much smaller1than that of the Canada, Type I neuron. The
cell body exists in a polymorphic form with 3 to 4 processes: see
Figures 20 and 26. The nucleus is round or oval-shaped, involving
about half the area of the cytoplasm, and is usually located close to
one side of the cell body. This cell is found in close association
with other cells, particularly of the same type, and with astrocytes.

3. Canada, bipolar neuron. Distinguishing features of this
neuron are the two primary processes. at opposite poles, of a round or
spherically-shaped cell body. A bifurcation is regularly seen in one
of the processes; see Figures 19 and 21. The nucleus is large, taking
up most of the cell body, and conforms to the general shape of the cell
body. The nucleus customarily contains at least one nucleoli.

A number of glial cells were found jg_situ, particularly the fibrous
astrocytes (see Figures 19 and 22) which are spider-shaped cells

[Jenkins, 1972, p. 59]. The neuronal type of a cell in culture is
almost impossible to determine if the cell is in the process of re-

differentiation (see Figures 22 and 23). The reader should note that

81

Figure 18. A photomicrograph of a living brain cell found in a disso-
ciated monolayer cell culture. taken from the somatic sensory cortex

I and II, of a 3 days old female albino rat. The cell was maintained
23 days in culture; at the time of photo, it has a diamond shape with
a length of 130 microns and a width of 96 microns. The nucleus of

the cell is centrally located. having an oval shape 30 microns long
and 18 microns wide, with several nucleoli. This cell has been desig-
nated the Canada, Type I neuron. The scale for the photomicrograph

is 14.4 microns per centimeter. ‘

_../ -\_..J‘.--W ;.—AJH‘U\W Hm... H \__._‘\__ /'

"—4M gM H‘e wm,.fi A

 

83

Figure 19. A photomicrograph of a living Canada, bipolar neuron (right

of center) found in a dissociated monolayer cell culture. taken from

the somatic sensory cortex I and II, of a 3 days old female albino rat.

The cell was maintained 32 days in culture. The cell body is spherical.
with a length of 26 microns and a width of 17 microns. Note the fibrous
astrocyte above the center of the photomicrograph. The Canada, bipolar
neuron is making a contact with a Canada. Type II neuron (bottom of
center). The scale far the photomicrograph is 14.4 microns per centimeter.

H—Iwwhv—h—‘flavw

’\ m‘H—W g‘MH—Jm WJ-WM ~———d

 

85

 

Figure 20. A photomicrograph of a Canada, Type II neuron found in a
dissociated monolayer cell culture, taken from the brain of a 3 days
old female albion rat. The neuron was maintained 96 days in culture,
stained with 1 x 10' M acridine orange. at 22°C, for 30 minutes, at

a pH of 4.1, and irradiated with 400 nm. light. The cytoplasm of the
neuron is full of orange granules and the nucleus has a yellow-orange
color, indicating that under these conditions the degree of aggrega-
tion of A0 onto the NA macromolecules in the neuroplasm is very high
and greater than that for the NA macromolecules in the neucleoplasm.
The average soma area of the Canada, Type II neurons found in this
culture was 179 sq. microns. Note that the processes of the cell in
the bottom right corner appears to be making contacts with the soma
and processes of the Canada, Type II neuron; the cell has an orange
cytoplasm with a yellow-orange nucleus and its ratio of the nucleus
to soma areas is greater than that for the Canada, Type II neuron.

86

 

Figure 21. A photomicrograph of two Canada. bipolar neurons, found in
a dissociated monolayer cell culture, taken from the brain of a 3 days
old female albino rat. The cells were maintained 96 days in culture,
stained with l x 10' M acridine orange at 22°C, for 30 minutes at a pH
of 4.1, and irradiated with 400 nm. light. The average area of their
somas is 112 sq. microns, having two processes at opposite poles, with
a bifurcation in one of the processes. Both of their nuclei are large
and round, taking up most of the soma. while stained a yellow-orange
color. Their cytoplasm is orange in color. One of the Canada. bipolar
neurons, with its bifurcated process, is making contact with a process
of the other neuron.

87

Figure 22. "A photomicrograph of living fibrous astrocytes during re-
differentiation. The cells were maintained 23 days in a dissociated
monolayer cell culture, and were taken from the somatic sensory cortex,
I and II, of a 3 days old female albino rat. As it happens with cells
in brain sections, these cells in culture may be confused with neurons.

 

 

 

89

Figure 23. A photomicrograph of three living neurons redifferentiating,
the cells were taken from the somatic sensory cortex, I and II, of a 3
days old female albino rat. They were grown 23 days in a dissociated
monolayer cell culture. Note that there is a soma-somatic contact
between two of the neurons. The nucleus of each cell conforms with

the general shape of the cell body, having one or two nucleoli. The
cytoplasm of each cell is dense with Nissl bodies. The cells in the
surrounding environment are glia or connective tissue.

 

91

the names and characteristics of these neuronal types pertain to neurons
taken from the rat cortex and maintained in dissociated cell cultures.
The development of neurons in culture closely resembles the maturation
of neurons in the natural state. A number of investigators have shown
that changes in age can result in changes in the RNA content, establish-
ing a differential growth rate in the development of neurons [Haltia,
1970; Sobkowicz et a1, 1973]. It is for these reasons that the author's
proposed technique was applied in the determination of the nucleic acid
content of single neurons in culture, at two different ages.

Cellular or nucleic acids in the intact cell are usually found in
a nucleoprotein complex; i.e. 1_h!1!g, nucleic acids are usually found
intimately associated with proteins. The amphoteric nature of the pro-
tein fraction of the nucleoprotein complex elicits a slight problem dur—
ing staining with acridine orange. The cationic portion of the protein
competes with A0 for binding sites on the nucleic acids. and the anionic
part of the protein competes with the nucleic acid for binding acridine
orange; both situations result in an emission spectral change for the
various nucleic acid-A0 complexes. A procedure was developed to minimize
the interactions between the protein fraction and the nucleic acid
fraction of nucleoproteins [Rigler, 1966]. The nucleic acids could be
exposed and loosened from their nucleoproteins, by the blocking of the
cationic amino groups of the proteins with acetic acid anhydride acetyl-
ation. A high hydrogen-ion concentration was faund to halt the unspeci-
fic binding of A0 to proteins. The anionic carboxyl groups of the pro-
teins were blocked at a pH of 4.1, the maximum hydrogenaion concentration
which the hydrogen bonds in highly organized nucleic acids are not

irreversibly destroyed. This causes the fluorescence spectra of

92

AO-nucleoprotein complexes to be identical to their free nucleic acid—
A0 complexes emission spectra [Rigler, 1966].

In addition to water comprising approximately 70% of its total
weight, a cell is also predominantly composed of proteins and nucleic
acids which are about 22% of the total weight [Lehninger, 1970, p. 20].
During fixation, a neuron is dehydrated, reducing it essentially to a
gelatin microdroplet. The internal microenvironment of a neuron is
dependent upon its living condition and neuronal type. The neurons
were stained for 30 mins., allowing the A0 molecules to bind to almost
all of the monomer binding sites on the NA conformations. Using an

AO-staining concentration between 5 x 10'6N and 5 x 10"4

N at a pH 4.1
permits equation (4), from the gelatin microdroplet experiments, to be
directly applied to the fluorescence of neurons stained with A0, since
the fluorescence coefficients of DNA and rRNA at 536 nm. are constant
under these conditions (rRNA is the major type of ribonucleic acid
found in the cell, comprising over 80% of total cell RNA) [Lehninger,
1970, p. 254]. The fluorescence spectrum of each neuron was recorded
with the microspectrophotometer, during irradiation with 400 nm. excit-
ing light. The pg. rRNA + y(pg. DNA) content of the Canada. Type II
and bipolar neurons' cell bodies, at 67 da. and 99 da; old are tabulated
in Table 9. Except for neuron #10, the neurons at each age were grown
in the same culture; and were taken from a 3 day old female albino rat.
The age of a neuron, in days, is equal to the sum of the age of the rat
at the time of sacrifice plus the number of days the neuron spent in
culture before fixation.

The logarithm of the background fluorescence, log b. for each cell

was equal to zero, even though the A0 staining concentrations were

93

TABLE 9

The R + yD content in the cell bodies of Canada, Type II, and Canada,
bipolar neurons. at 67 da. and 99 da. old, as determined microspec-

trophotometrically using equation (4), R + 70 = (log F
where y is equal to 4.63 and f.c.

f.c.

rRNA
536 ’

rRNA
536

535 ' ‘°9 b”
is equal to 0.272.

Cells #1 through 7 were stained with l x 10-4M A0, and neurons #8
through 12 were stained with 5 x 10-5M A0 for 30 mins., at a pH 4.1.
All of the cells were taken from the cerebral cortex of a 3 da. old

 

albino female rat and maintained in dissociated cell cultures. Neurons
#8 through 12 are from the somatic sensory cortex I and 11.
Time Spent Soma 2 F536 Aperture 8 + 70

Neuronal Type 1n Culture area (u ) Correction 1n pg.
1 Canada, Type II 96 da. 179 7200 2.1 15.4
2 Canada, Type II 96 da. 179 5700 2.1 15.0
3 Canada, Type II 96 da. 179 5100 2.1 14.8
4 Canada, bipolar 96 da. 112 7650 3.4 16.2
5 Canada, bipolar 96 da. 112 6500 3.4 16.0
6 Canada, bipolar 96 da. 112 8000 3.4 16.3
7 Fibrous astrocyte 96 da. 116 7250 3.3 16.1
8 Canada, Type II 64 da. 89 436 4.3 12.0
9 Canada, Type II 64 da. 114 300 3.3 11.0
10 Canada, Type II 64 da. 108 600 3.5 12.2
11 Canada, bipolar 64 da. 119 1075 3.2 13.0
12 Canada, bipolar 64 da. 319 1900 1.2 12.3

94

different, since the fluorescence of the area surrounding each cell body
analyzed was either nil or negligible for both of the A0 staining con-
centrations. Because the neurons were found living either on the glass
slide alone or on top of an glial cell, and the major subcellular com-
ponents in the cytoplasm of most glial cells are made of proteins [Hyden,
1967, Chap. 4], the background area of the neuron contains no appreci-
able amount of nucleic acids, if any. The negligible background fluores—
cence intensities harmonize with results found by Rigler [1966]: after
enzymatic extraction of the nucleic acids, the fluorescence emission,

at 530 nm., of the protein residual of AO-stained cells at pH 4.1,
amounted to about 2% of the total fluorescence intensity at 530 nm. of
AO-stained cells containing nucleic acids. In this investigation, the
fluorescence spectral patterns of the cells stained at 1 x 10'4H A0 were

5

identical to the spectral patterns of the cells stained at 5 x 10' M A0.

The degree of dye aggregation for the cells stained at l x 10'411 averaged

0.29, and for cells stained at 5 x 10'5

M it averaged 0.25. Visually, the
cells stained at the higher A0 staining concentration had orange cyto-
plasms and yellow-orange nuclei (see Figures 24 through 27). In contrast,
the cells stained at the lower A0 staining concentration were green all
over (nucleus and cytoplasm) similar to the appearance of the gelatin
microdroplets under the same staining conditions. These differences

may be due to the older neurons' (99 da.) greater capacity to retain A0
than the younger neurons (67 da.), resulting in a higher intracellular
dye content for the older neurons. Since the amount of fluorescence
light reaching the phototube is dependent on the slit size. aperture

correction factors were introduced in Table 9 in order to correct for

intensity differences caused by the smaller apertures used for the cell

95

 

Figure 24. A photomicrograph of a single Canada, bipolar neuron
found in the same culture and subjected to the same conditions as
those in Figure 21. The average area of the soma is 112 sq. microns.
The other large oval-shaped yellow-green objects are the nuclei of
glia cells. Note the local accumulation of dye molecules at the
orange thickenings in the bifurcated process, before and at the bi-
furcation, due to an increased aggregation of A0.

96

 

Figure 25. A photomicrograph of a Canada, bipolar neuron found in a
dissociated monolayer cell culture, taken from the brain of a 3 days
old female albino rat. The cell was maintained 96 days in culture.
stained with 1 x 10‘4M acridine orange, at 22°C, for 30 minutes at a
pH of 4.1, and irradiated with 400 nm. light.' The average area of the
soma is 112 sq. microns, having two processes at opposite poles, with
a bifurcation in one of the processes. Note the neuron at the bottom
of the photomicrograph is sending its process towards the bifurcated
process of the Canada, bipolar neuron.

97

 

Figure 26. These two cells are Canada, Type II neurons. Canada, Type
II neurons occur in polymorphic form. with 3 to 4 processes, in dis—
sociated monolayer cell cultures of 3 days old female albino rat
brains. Note that there is one contact between the two neurons and

a contact with a common body. The cells were maintained 96 days in
culture, stained with l x 10‘4M acridine orange, at 22°C, for 30
minutes at pH 4.1. The photomicrograph was taken while exciting the
cell with 400 nm. light. The average area of their soma is 179 sq.
microns. The cytoplasm is stained orange. The yellow-orange nucleus
is usually round or oval in shape, comprising about half the area of
their somas,

98

 

Figure 27. A Canada, Type II neuron, in upper section, found in

the same culture and subjected to the same conditions as those in
Figure 26. Notice the extensive network of fibers between this cell
and the cell in the lower section. Two of the processes of the Canada,
Tyge II neuron are making several contacts with the processes of other
ce 5.

99

bodies. The fluorescence coefficients were determined for an aperture
area of 380 sq. microns.

The average R + YD values for the Canada, Type II and the Canada.
bipolar neurons demonstrated an increase as a function of neuronal age,
where the R + yD value per neuronal type averaged 28% greater at age
99 da. than at age 67 da.; see Figure 28. The slopes of the line for
the R + 70 values of Canada, Type II, and Canada, bipolar neurons, as a
function of time, are equal to 1.1 x 10"1 pg/da.. suggesting that both
neuronal types have identical growth rates in culture; between 64 da.
and 96 da. in culture, the R + yD value per neuron increased 0.1 pg.
per diem. Since the y(pg. DNA) content per neuron is constant at each
age, the slope can reflect the rate at which the RNA content of each
neuronal type increases as a function of age. The low value for the
slopes infer that neurons, grown in culture, grow at a very slow rate.
This agrees with the results published by Sobkowicz et al in 1973, in
which they reported that the RNA content of spinal ganglion neurons in
culture increased at a much slower rate than when grown jg_gjxg, Figure
28 also demonstrates that the amount of RNA contained in a given neuron
in culture is dependent upon its neuronal type, inasmuch as the R + YD
value of the Canada, bipolar neurons averaged 8% greater than the
R + yD content of Canada, Type II neurons at each age.

Table 9 shows that the area of a neuron's cell body is dependent
upon the neuronal age and type. The average cell body area of the Canada,
Type II neurons increased 73%, as the neuronal age increased from 67 da.
old to 99 da. old. In contrast. the average area of the Canada, bipolar
neuron's cell body decreased as their neuronal age increased. However,

in Figure 29, the R.+ 70 content per neuron was not dependent upon the

 

100

, Canada, bipolar neurons

Canada, Type II neurons

1" —

logF +logb 12 '-

 

536
f . c. rRNA '-
536
10 -
8 —
6 —

 

l l I l l l

50 6O 70 80 90 100 da.
AGE OF 111111110115 (exp. in days)

Figure 28. The {rRNA + 1011”} content,

in picograms, per neuronal type for dif-
ferent neuronal ages.

101

—99 da. old
F- "“67 da. old

16 r-

14—

lo F +10 6
8 535 3 12 *"

 

f.c.rRNA -

536
10 '-

-—-----------
-

 

 

 

F-_--___-_-------

1 41 1 1 In _)(_
80 90 100 110 120 179
AREA OF 8011A (exp. in eq. microns)

 

Figure 29. The {rRNA + 1(DNA)} content, in
picograms, per neuronal age for different
soma areas of the neurons.

U
p-s
\D
I
N

102

size of its cell body; the R'+ yD content per neuron presented no defi-
nite pattern as a function of the neuron's soma area. Clearly, these
results suggest that during the normal development of neurons in cul-
ture their RNA content and the size of their cell bodies are highly
correlated with age. The same is true jg.§jtg_[Haltia, 1970. Sobkowicz
et a1, 1973].

No doubt, a better experimental design could be conceived for
determining the NA content in single neurons. The ability of this modus
operandi to yield the absolute value of the NA content in the soma of a
single neuron must be questioned. Logically, the R + yD value of each
neuron is greater than the value of 70, but never less than the value
of 70. However, the experimental R + yD value (315.4 pg.) of each neu—
ron was found to be less than the expected yD value of 30.1 pg. The
theoretical yD value, 30.1 pg., was determined by multiplying the re-
ported DNA content of a mammalian neuron. 6.5 pg. [Mahler, 1972] by
the-yvalue of 4.63. The use of 4.63 as the value of 7 was based on the
assumption that a fixed cell was analogous to a gelatin microdroplet,
whereby under similar staining conditions the binding behavior of A0
molecules to NA macromolecules inside a fixed cell is identical to the
binding behavior of A0 molecules to NA macromolecules in a gelatin micro-
droplet. Obviously, this assumption was wrong, since the experimental
yD value of each neuron appears to be considerably less than 30.1 pg.
Therefore..the y value of a fixed neuron must be less than the y, 4.63,
value of a gelatin microdroplet.

The fluorescence coefficients obtained from the gelatin microdrop-
let experiments, f.c.g§2 = 1.26/pg. DNA and f.c.gggA = 0.272/pg. rRNA,

cannot be used to calculate the absolute NA content within the soma of

103

a single fixed neuron. Evidence suggests that the fluorescence coef-

ficients of the fixed neuron are different from the fluorescence coef-

ficients of the gelatin microdr0plet, in that the f.c.gggA and f.c.ggg
of the fixed neuron may be greater than the corresponding coefficients

of the gelatin microdroplet, or the f.c.rRNA of the fixed neuron is

536
greater than its f.c.ggg. or both may be possible. (Note: 9 = f.c.ggg/

f.c.gggA.) More importantly, Beer's law may not prevail at the staining
concentrations used in the fixed neurons, as it does with regards to

the gelatin microdroplets, resulting in an erroneous application of the
fluorescence coefficients and equation (4) to the fixed neurons. Under
these conditions, the fluorescence intensity of A0 bound to the macro-
molecules in the monomer form, and the amount of the macromolecules in
question are not proportional to the amount of A0 inside the fixed neuron,
as compared to the gelatin microdroplet. Consequently, experiments must
be devised to determine the staining concentrations for which Beer's

law holds true for a fixed neur0n. An example of such an experiment
might be the examination of the fluorescence emission properties of the
Nissl bodies. contained in the cytoplasm of a specific A0-stained neu-
ronal type, as a function of their staining concentrations.

The potential modification of the fluorescence coefficients, de-
rived from the gelatin microdroplet experiments, can be created by the
cellular and nuclear membranes and the dissimilar internal environment
of the fixed neuron. As one might expect. unlike the nonmembranous gela-
tin microdroplet the nuclear and cellular membranes serve to concentrate
the A0 content within the fixed neuron by obstructing the diffusion of

excesses outside the neuron. This enhances the binding of A0 molecules

104

to the NA macromolecules. Also, the binding of A0 molecules to NA
macromolecules increases, as a function of the ion concentration in the
microenvironment and, due to the higher ionic strength in the internal
environment of the fixed neuron, the amount of A0 molecules bound per
NA macromolecule in a fixed neuron is greater than the amount of dye
molecules bound per NA macromolecule in a gelatin microdroplet. This
.would cause the f.c.ggé and f.c.gggA of the fixed neuron to be greater
than the corresponding coefficients of the gelatin microdroplet. Unlike
the gelatin microdroplet, the internal environment of the fixed neuron
contains other A0 binding biopolymers that are intimately associated
with the NA macromolecules, such as the ribosomal proteins, histones,
etc. [Rigler, 1966]. These biopolymers may serve to indirectly increase
the amount of A0 molecules associated with a NA macromolecule. Since
they are not found in the gelatin microdroplets. the quantity of A0
molecules bound per NA macromolecule in a fixed neuron is greater than
the amount of dye molecules bound per NA macromolecule in a gelatin
microdroplet, resulting in higher fluorescence coefficients for the
fixed neuron. The conditions inside a fixed neuron can cause the
f.c.gggA to be larger than the f.c.ggg. Due to a higher concentration
of A0 molecules in the neuroplasm, the amount of dye molecules bound

per rRNA macromolecule in the neuroplasm is greater than the quantity
of A0 molecules bound per DNA macromolecule in the nucleoplasm. This

is brought about by a greater ionic strength and concentration of A0-
binding biopolymers in the neuroplasm of the fixed neuron than in its
nucleoplasm, and the obstruction by the nuclear membrane of the diffusion

of dye molecules into and out of the nucleus. These circumstances are

evidenced by the orange cytoplasm and yellow nucleus of the A0-stained

105

neurons in Figures 24 to 27. The orange cytoplasm indicates a high
degree of dye aggregation, resulting from the high concentration of A0
molecules in the neuroplasm. The cellular and nuclear membranes and
the higher ionic strength, along with the other biopolymers that bind
A0 molecules, would cause the internal A0 concentration of the fixed
neuron to be greater than the gelatin microdr0p1et, under the same
staining conditions. The staining concentration that deviates from
Beer's law for the fixed neuron would be lowered, thus preventing the
direct application of equation (4). Since A0 obeys Beer's law up to

a concentration that would cause its aggregation [Nest, 1969], at the

411 AD, the amount of A0

staining concentration of 5 x 10'5M and l x 10'
inside the fixed neuron may not be proportional to the fluorescence
intensity of the monomer A0 molecules and the amount of the NA macro-
molecules in the neuron.

The aperture (area) correction factors may be a source of error in
calculating the R + v0 value of each neuron. Although the thickness of
the gelatin microdroplets and the neurons were about the same a volume
correction factor would be more accurate to use than the area correction
in correcting for the size differences between the gelatin microdroplet
and the cell bodies. The different fixation processes of the gelatin
microdroplet and the fixed neuron may be another source of error. result-
ing in different binding behaviors of A0 molecules to the NA macromole-
cules. No doubt, there may be a need to introdute other correction
factors; only further experimentation can yield this answer. Nonetheless,
the quantitative aspects of this investigation may be regarded as a first

approximation, since the fluorescence intensity of AO-NA complexes at

536 nm. is proportional to the amount of NA available for binding the

106

dye molecules, reflecting the quantity of NA in AO-stained neurons. The
R + TD values can be used to calculate the growth rate in the RNA content
per neuronal type, as a function of time, since only the RNA content of
the neuron is changing.

The reliability of this technique to yield accurate data should be
tested against other biochemical and biophysical-cytochemical methods
fer the determination of NA in single cells. The biochemical procedures
developed by Edstrom [1953, 1958, 1964] and ethidium bromide fluorescence
microspectrophotometry [LePecq. 1973] are suitable methodologies to
examine the competency of this technique. Hyden [1967, p. 191] modified
the Edstrom methods for the analysis of brain cell material, involving
the extraction of RNA and its determination by a microphotographic-
photometric procedure in which a single neuron is removed from brain
tissue, placed in a special chamber under liquid paraffin,and incubated
with ribonuclease to enzymatically break down the large molecules of RNA.
The depolymerized RNA is dried as specks on a glass slide and redissolved
in a glycerine solution, forming microdr0ps. The microdrops and a den-
sity scale are photographed with 357 nm. ultraviolet radiation. Curves
showing the RNA content of each microdrop and the density scale are
photometrically produced by scanning the images on the photographic plate
with light while recording the responses with a photocell-amplifier-
recorder. The method can determine at least 20 pg. of RNA with a varia-
tion coefficient of the whole procedure being about 5%. Hyden [1967,
p. 197] employed this technique to study the motor neurons in man.
Unlike many other cells whose RNA content increases during the prenatal
period, the RNA content in the nerve cell increases during the postnatal

period of development. After birth the RNA content in the motor neuron

107

increases significantly until the ages around 40. Between the ages of 0
and 20 years, the RNA content in each cell averages 402 :_28 pg., and
between 21 and 40 years it averages 553 i 38 pg. Between 41 and 50 years
of age, the RNA content in each cell is maintained at a high level,
averaging 640 :_55 pg. per neuron. After the age of 60 years, the RNA_
content in each cell decreases significantly. Between the ages of 61
and 80 years the RNA content per neuron averages 554 1,31 pg. Over the
age of 80 years the RNA content per neuron averages 460 1_30 pg. [Hyden,
1967, p. 197]. The integrity of the Edstrom procedure [1953, 1958,
1964] is confirmed by the following investigators--Hyden [1967], Haltia
[1970], and Sobkowicz et a1 [l973]--and used in the demonstration of a
growth-curve relationship (a differential growth rate) between the RNA
content and the age of the developing neuron.

The compound, ethidium bromide (Figure 30) is a fluorescent mole-
cular probe of the NA structure, binding to the NA confbrmation in two
different forms [Le Pecq, 1973]. Similar to the acridine orange molecule,
ethidium bromide (EthBr) preferentially binds to the NA conformation in
the monomer molecular form. In this form, the dye molecule is intercal-
ated between two base pairs, forming a "sandwich complex." The plane of
the dye molecule is parallel to the plane of the base pairs. The binding
of EthBr to the nucleic acids increases the fluorescence quantum effici-
ency, 20 times greater than that of the free dye in aqueous solution. to
a value of 0.14, and shifts the absorption spectrum to longer wavelengths,
having an emission peak at 575 nm. The bound dye molecule has a fluores-
cence lifetime equal to 24 ns. [LePecq, 1973]. The intercalation of
EthBr is specific for the double stranded regions in either the DNA,

RNA, or DNA-RNA hybrid structures. The fluorescence intensity of the

 

Figure 30.

108

R ' CZHS

Ethidium Bromide

109

dye is increased proportional to the amount of double stranded regions
available for binding, and is negligible in the presence of single
stranded structures. The fluorescence enhancement of the bound dye is
due to the immersion of the dye molecule into the hydrophobic medium

of the intercalation site [LePecq, 1973]. Unlike the acridine orange
molecules, ethidium bromide molecules cannot readily bind to the NA
conformation in the aggregated form. The stacking of the dye molecules,
along the single stranded regions and on the outside of the double
stranded regions in the NA conformation, is obstructed by the phenyl
substituent, in the sixth position, the latter being oriented perpen-
dicular to the main ring of the molecule. The fluorescence quantum
efficiency of the dye molecule bound in the second sites is considerably
less than the fluorescence quantum efficiency of the intercalation sites
[LePecq, 1973]. The binding constant for DNA is less than that for RNA
and the binding constant for both structures is dependent upon the salt
concentration in the microenvironment. The binding constant of EthBr

to the monomer binding sites (intercalation sites) of the NA conformation,

6M, and the binding

at low ionic strength, is of the order of l x 10
constant decreases slightly as the ionic strength is increased. In con-
trast, the binding constant of EthBr to the NA conformation, in the
aggregated form, is completely eliminated at high salt concentrations.
The conformation and the conformational changes of a NA macro-
molecule were studied, 13 XIEEQ’ using the fluorescence properties of
EthBr bound to the macromolecule. The binding constant of EthBr for
a naturally super-coiled DNA conformation is larger than that for a

nicked circular DNA conformation [Bauer and Vinograd, 1968; LePecq, 1973].

The fluorescence intensity of EthBr bound to a given concentration of

110

supercoiled DNA is higher than the fluorescence intensity of EthBr
bound to the same concentration of nicked circular DNA under identical
conditions, thus demonstrating the affinity differences of EthBr for
the two different DNA confbrmations. The binding of EthBr to naturally
supercoiled DNA progressively releases (denatures) the superhelix into
a relaxed circle because the intercalation process of EthBr induces a
change in the torsion of the double helix. The conformational changes
of the DNA superhelix is a function of the EthBr concentrations, whereby
increasing the EthBr concentration gradually unwinds the naturally
supercoiled DNA conformation, and the number of turns in the superhelix
is proportional to the amount of EthBr bound by macromolecule [Bauer
and Vinograd, 1968; LePecq, 1973]. The fluorescence intensity of the
EthBr—induced circular DNA (covalently closed circular DNA). the DNA
structure resulting from the increased binding of EthBr, is identical
to the fluorescence intensity of EthBr bound to the nicked circular DNA
conformation, under similar reaction and fluorescence measurement con-
ditions. This means that the affinity of EthBr fer the completely re-
leased superhelix is equivalent to the affinity of EthBr for the nicked
circular DNA conformation. Increasing the EthBr concentration to excess
induces the circular DNA to form a superhelix of the opposite sign,
compared to the sign of the original superhelix. The affinity of EthBr
for the supercoiled DNA conformation of opposite sign is less than that
of the nicked circular DNA conformation under identical conditions,
binding less ethidium bromide molecules, and it is less fluorescent
than the nicked circular DNA conformation. These experiments can yield
the number of dye molecules bound per macromolecule of circular DNA,

and if the change in torsion connected with the intercalation process

111

is ascertained, the sign of the superhelix and the degree of superhelicity
of the original supercoiled DNA conformation can be determined [LePecq,
1973]. The difference in affinity of EthBr for the supercoiled DNA

and the nicked circular DNA conformation, at highggye concentrationg.

is used to investigate the conformational changes induced by an enzymatic
reaction [Paoletti et a1, 1971; LePecq, 1973]. In the presence of excess
EthBr, a single nick is made on the supercoiled DNA conformation; the
break in the nucleotide sequence is made by the enzymatic reaction of
DNase. As the topological constraint limiting the binding of the dye
molecules is eliminated, the nicked superhelix relaxes into the nicked
circular conformation, accompanied by an increase in the fluorescence
intensity. The reverse is true for the transformation of the nicked
circular DNA conformation into the supercoiled DNA conformation. The
enzyme. Ligase, is used to repair the break in the nucleotide sequence

of the nicked circular DNA conformation. The result is a decrease in
fluorescence intensity, associated with the conversion of the nicked
circular conformation into the supercoiled DNA conformation. The
fluorescence intensity changes accompanying the transformation of the

DNA conformations provides the basis for studying the details of these
enzymatic reactions [LePecq, 1973].

A conformational change in a macromolecule, involving a minor
modification of the distance between two chromophores, can be perceived
by measuring the changes in the energy transfer rate of the chromophores.
The rate of energy transfer between a donor and an acceptor chromophore
is inversely proportional to the sixth power of the distance between
them, and proportional to their orientation factor [Forster, 1959:

LePecq, 1973]. Minor variations in the intermolecular distance between

112

the base of phenylalanine t-RNA of yeast and EthBr bound to the strong-
est binding site were uncovered by Tao et a1 [1970; LePecq, 1973] who
measured the variations of the energy transfer rate between the chromo-
phores. They found that the rate of energy transfer, and thus the
intermolecular space between the chromophores. change. as a function of
the change in the salt concentration of the solution. This suggests
that the degree of a conformational change in the t-RNA biopolymer is
dependent on the ionic strength of the microenvironment.

Although the DNA macromolecule has a large molecular weight the
fluorescence lifetime of the rigidly bound, intercalated EthBr molecules
is of a sufficient duration to detect the dynamic structure of DNA
[Kelly and Sinsheimer, 1967; LePecq, 1973]. The rotational relaxation
time of a macromolecule can be determined by measuring the fluorescence
depolarization of the bound probe [Stryer, 1968]. The decay in the
fluorescence anisotropy of the bound EthBr monomer, as a function of
time in the nsec. range, implies that the DNA macromolecule in solution
has an internal oscillatory Brownian motion, possessing a relaxation
time of 28 nsec. and an amplitude of 35° for the oscillation [LePecq,
1973].

The facility and limitations of using EthBr fluorescence micro-
spectrophotometry to obtain detailed information on the structure and
function of nucleic acids, in respects to the intact cell, can be
examined in complicated systems of nucleoproteins like ribosomes.
chromosomes, etc. [Bollen et a1, 1970; Angerer and Moudrianakis. 1972;
LePecq, 1973]. In the cell the nucleic acids are found intimately
associated with proteins. The existence of proteins on the surface

of the nucleic acids hinders the binding of EthBr to the NA macromolecule.

113

In experiments involving the reconstitution of the ribosome out of its
protein and nucleic acid components, Bollen et a1 [1970; LePecq, 1973]
investigated the binding behavior of EthBr molecules to rRNA macromole-
cules in the presence of ribosomal proteins. The fluorescence inten-
sity of EthBr bound to rRNA was measured as the ribosomal proteins were
serially added to the rRNA. When the concentration of the ribosomal
proteins increased, the fluorescence intensity of the EthBr—rRNA com~
plexes exhibited a drastic decline. The EthBr molecules were bound

into the double stranded regions of the rRNA macromolecule, in the
monomer form, and were expelled from the rRNA macromolecules during the
formation of the ribosome. The reconstituted ribosome was contradis-
tinctive to the native ribosome in that it binds some EthBr and the
latter did not [Bollen et a1, 1970]. Since fluorometry can be used to
follow the reconstitution kinetics of ribosomes _i_n_ 1193, the formation
of Nissl bodies, i.e. polyribosomes, and the NA-dye-protein interactions
may be investigated by EthBr fluorescence microspectrophotometry in the
living neuron. The stimulation of a living neuron would result in an
increase in the Nissl body content of the neuron, which would be accom-
panied by a decrease in the fluorescence intensity of EthBr bound to the
free rRNA macromolecules in the neuron.) In the dissociation and recon-
stitution of nucleohistones, and in the trypanosoma (which are parasitic
flagellate protozoans that infest the blood stream of animals) the
binding behavior of EthBr to DNA macromolecules in the presence of
chromosomal proteins is similar to the binding of EthBr to rRNA macro-
molecules in the presence of ribosomal proteins [Angerer and Noudrianakis,

1972; LePecq, 1973]. The EthBr molecules can readily bind to a DNA

114

macromolecule that is free of proteins, in contrast, the binding of
EthBr to DNA macromolecules in deoxyribonucleoproteins is limited by
the close association of the chromosomal proteins with the surface of
the DNA macromolecules. The binding of EthBr molecules to the DNA
macromolecule may be further limited by the foldings in the DNA struc-
ture. The fluorescence characteristics of EthBr bound to the DNA of
intact phages has been measured as a function of the folding constraint
of the DNA macromolecules in the phage [LePecq, 1973]. The variations
of the constraints imposed by the DNA folding which induces conforma-
tional changes in the DNA helix are produced by the smaller amount of DNA,
packed in the same head volume, of different deletion mutants of the
phage. As the amount of DNA is decreased in the mutants, the folding
constraint of the DNA macromolecules is progressively released. The
fluorescence characteristics of EthBr bound to the DNA tightly confined
in the deletion mutants, gradually returned to the fluorescence charac-
teristics of EthBr bound to DNA macromolecules free in solution. The
constraints imposed by the increased DNA folding restricts the inter-
caltion of EthBr, but favors the binding of the dye molecules to the
second binding site [LePecq, 1973]. The limitations in using EthBr
fluorescence microspectrophotometry must be known before any meaningful
interpretations of the results on its application to the neuron can be
expressed, especially the restrictions put upon by the folding con-
straint of the native NA structures and the protein components of nucleo-
proteins. The facility of using EthBr on single living neurons is the
detection of changes in the conformations of the DNA macromolecules in
the chromosomes, and changes in the cellular pool of free RNA macro-

molecules, as the result of alterations in the internal and/or external

115

environment of the neuron. The fluorescence of EthBr can be quenched
through the transfer of energy between EthBr and a non-fluorescent

EthBr derivative, and through proper energy transfer experiments, this
nay facilitate the maping of superhelices, the measuring of inter-helix
spacing, and the study of DNA packing in the chromosomes, provided the
superhelices can be specifically labeled [LePeco. 1973], and may facili-
tate the study of the NA-dye-protein interactions, in the neuron. The
energy transfer and the quenching of fluorescence can be measured. as a
function of changes in the microenvironment of the bound probe. yielding
information on the molecular interactions of the probe before, during,
and after any microenvironmental change.

Acridine orange and ethidium bromide are suitable fluorescent
probes to explore the details of the structures and functions of nucleic
acids in the single neuron. The A0 and EthBr molecules can selectively
bind, in the monomer form, into the double stranded regions of the NA
macromolecules. In a similar manner, both dye molecules are intercalated.
in a "sandwich complex," between two adjacent base pairs causing a slight
distortion of the double strands; the three ring structure of the dye
molecule is immersed into the hydrophobic interior of the stacked bases,
with the electrically charged phosphate groups that are bound to the
hydrophilic sugar residues of the nucleotides being located on the peri-
phery of the complex. The differences between the binding behavior of
A0 and EthBr to the NA, and the nature of the investigation, prescribes
which fluorescent probe is to be used on the NA in the neuron. Dissimilar
to EthBr, A0 molecules can readily bind to the native ribosomes and
chromosomes. The existence of proteins on the surface of the NA macro-

molecules does not prevent the binding of A0 to the NA, in fact, the

116

anionic portions (carboxyl groups) of the proteins in the organized
nucleoprotein compete with the negatively charged phosphate groups of the
NA to bind the cationic A0 molecules. Since the amount of A0 bound by

a NA is proportional to the amount of the NA, the greater sensitivity of
A0 infers that in experiments involving the microspectrophotometric
determination of the quantity of rRNA in the soma of single neurons, as
a function of a neuronal parameter such as age; A0 would be the better
fluorescent probe to use than the employment of EthBr. The amount of
EthBr bound by a NA is a measure of the amount of double stranged regions
in the NA [LePecq, 1973]. Although the binding affinity of A0 for
nucleoproteins is greater than that of EthBr. EthBr would be preferred
over AD, as a fluorescent probe, in investigations of the molecular
interactions of rRNA macromolecules, during the initial phases of the
restoration of the neuron to its resting state following neuronal excit-
ation or inhibition. Since the expulsion of EthBr occurs in the associ-
ation of rRNA macromolecules with ribosomal proteins, the fluorescence
intensity of EthBr bound to free rRNA macromolecules would decrease when
the free rRNA macromoleculesare integrated into ribosomes, and when the
ribosomes are aggregated into polyribosomes. Keeping adult rats in the
dark for 3 days, and then exposing them to laboratory lights and sounds
of 15 mins., has been reported to increase the polyribosome to monomer
(i.e. a monomer ribosome) ratio by 83% in their cerebral cortex while
nothing happens in their liver [Mahler, 1972]. Electroshock has been
reported to decrease the polyribosome to monomer ratio, because it dis-
sociates the polyribosomes [Mahler, 1972]. This suggests that when the
estate of aggregation of the ribosomes in the neurons. the polyribosome

t0 monomer ratio, increases the fluorescence intensity of EthBr bound

117

to the rRNA macromolecules in the ribosomes would decrease, and vice
versa.

If the binding behavior of A0 to the NA, as a function of the
folding constraint of the NA, is similar to that of EthBr. then increas-
ing the folding of the NA conformation would progressively strengthen
the constraint limiting the binding of A0 to the NA in the monomer form.
Then the constraint imposed by the folding of the NA in organized systems
like Nissl bodies, i.e. polyribosomes of different degrees of aggrega-
tion, and chromosomes inside the living or fixed neuron can hinder the
binding of A0 to the NA. The amount of A0 molecules bound, in the monomer
form, by the NA macromolecules in a gene or a ribosome of the neuron,
would be less than the quantity of monomer A0 molecules bound to the
same NA macromolecules free in a gelatin microdroplet. Since the fluores-
cence coefficient of a NA at 536 nm. is a measure of the amount of A0
molecules bound per pg. of the NA macromolecules, in the monomer form.
Possibly the f.c.ggg and f.c.gggA for the fixed neuron may be really
less than the corresponding fluorescence coefficients for the gelatin
microdroplet, advocating that the lower y value for the fixed neuron is
rRNA being greater than the f.c.DNA In addi-

536 536 '
tion, the folding constraint may favor the binding of A0 to the NA in

actually due to the f.c.

the aggregated form, and since the A0 molecule does not have a phenyl
group lieing perpendicular to the plane of the dye molecule like EthBr,
the aggregation of A0 on a NA macromolecule is greater and occurs faster
than that of EthBr. Then the amount of A0 molecules aggregated on a
given NA macromolecule in the neuron would be greater than the amount .
of dye molecules aggregated on the same NA macromolecule in a gelatin

microdroplet. under the same staining conditions. This suggest that

118

the staining concentrations where Beer's law prevails for the fixed
neuron is less than that for the gelatin microdroplet. At the

5 x 10'5N and l x 10-4

M AO staining concentrations, the fluorescence
coefficients and equation (4) of the gelatin microdroplet experiments
can not be directly applied to the fixed neuron, in determining its
rRNA + y(DNA) content.

The RNA metabolism of a neuron is strongly correlated to the
behavioral and physiological functions of the neuron. The existing
functional state of the neuron establishes the RNA level in the neuron,
where, at the time of observation, the RNA content of the activated or
depressed neuron is greater than or less than, respectively. the proper
quantity of RNA in a normal quiescent neuron of the same age and type.
The external and internal environmental circumstances of the function-
ing neuron affects the synthesis of RNA in the neuron. The rate of RNA
synthesis in the giant neurons of the abdominal ganglia in the sea hare ‘
Aplysia, jg_!jxg, is increased significantly as the result of prolonged
neuronal stimulation; the incorporation of radioactively labeled nucleo-
sides into the RNA of excited neurons is greater than that of unstimulated
controls [Mahler, 1972]. This is consistent with the effects of stimul-
ation on the RNA synthesis in the rat Purkinje neurons. and the frog
retinal ganglion and cat sympathetic ganglion neurons. The correlation
between the functional activity of a neuron and the synthesis of RNA
in the neuron, as influenced by the environment, is demonstrated in
experiments investigating the effects of visual stimulation on the
incorporation of orotate (pyrimidine derivative of RNA precursors) into
the RNA of the visual cortex in rats [Mahler, 1972]; and the effects

of strychnine sulfate and of marijuana on the brain RNA and DNA content.

119

and on the maze learning (consolidation of memory) of rats [McGaugh

and Petrinovich, 1959 and 1965; Carlini and Carlini, 1965; John, 1967].
The rate of labeling of RNA in the visual cortex of a totally blind
strain of rats is delayed and decreased, in comparison to the rate of
RNA labeling in the visual cortex of normal sighted rats [Mahler, 1972].
The RNA labeling of the visual cortex in the sighted animals is more
distinctive when using a flashing light rather than continuous illu-
mination for the stimulation. and the labeling rate of RNA in the motor
cortex of the blind rats is similar to that of the sighted rats. This
phenomenon can be seen in animals that are kept in the dark from birth
or blinded, in that, they exhibited a deficit in the RNA content of
various cells in their visual system [Mahler, 1972]. The level of RNA
in a neuron can influence its performance in behavioral functions like
memory and learning. Strychnine can cause hyperactivity and increase
the synthesis of RNA in a neuron by prolonging the action potential and
producing repetitive spike discharge in the neuron [Hashizu et a1.

1961; John, 1967]. The injection of strychnine sulfate into rats before
or after learning trials increased their speed of consolidation (of
memory) and enhanced the brain RNA content of the rats, with no effects
on the brain DNA content. However, marijuana can only facilitate learn-
ing in rats when administered before the training session: the marijuana
increases the concentration of the brain DNA but not RNA. The facilita-
tion of learning by marijuana may not be directly related to the consol-
idation process. but due to general factors [John, 1967]. These results
suggest that the rate of learning for rats can be increased by increas-
ing the level of RNA in their neurons, and possibly by raising the

nuclear DNA level of other brain cells such as the neuroglia.

120

B-azaguanine is known to inhibit the synthesis of RNA in the rat brain,
and rats injected with 8-azaguanine show significantly poorer retention
in learning a maze than compared to controls [Dingman and Sporn, 1961;
John, 1967]. To reiterate, clearly, there is a strong correlation
between the RNA metabolism and the behavioral or physiological functions
of the neurons. The existing functional activity of a neuron can set
the amount of RNA present in the neuron. The continuous activity of
neuronal aging effects the RNA content of a neuron much more slowly
than transient activities like neuronal excitation. Therefore, in
order to accurately calculate the differential accumulation rate of RNA
for a specific neuronal type, as a function of time (postnatal), the
experimental neurons at the time of fixation or observation must be in
the same transient state. i

The incorporation kinetics of radioactively labeled precursors
of RNA into the RNA of an isolated neuron can express the various mole-
cular species of the RNA (newly synthesized) in the neuron [Mahler,
1972]. The nuclear and nucleolar RNA become radioactively labeled
immediately after the administration of the precursors to the neuron.
The ribosomal precursors are first labeled in the nucleolus, having
sedimentation coefficients of 455, 355, and 325; a little later, a
polydisperse species is labeled in the nucleué, this is not the direct
precursor of the equally polydisperse mRNA. but it has sedimentation
coefficients varying from 505 to 55 and is capable of effective hybrid-
ization with DNA. Several minutes after the nucleus is extensively
labeled mRNA makes its first appearance in the cytoplasm associated
with ribonucleoprotein particles. The labeling of the stable rRNAs,

which are integrated into the ribonucleoproteins, follows the

121

cytoplasmic mRNA within 30 mins. to 1 hr., proceeding until an isotopic
steady state is reached [Mahler, 1972]. In addition, in the adult rat.
the rRNAs and the ribosomal proteins of the ribosome turnover as a
unit in about 12 days [Mahler, 1972]. It would be nice if there were
specific fluorescent molecular probes for each of the various molecular
species of RNA and DNA. Then the metabolic changes of each NA species,
in relation to the state of the neuron, could be studied by fluorescence
microspectrophotometry. A thorough understanding of the molecular
events associated with the various NA species during behavioral or
physiological functions of the neuron may eventually lead to the cures
of some neural disorders, i.e. paralysis, mental illness. etc.. and
the enhancement of some normal neural functions like memory and learning.
Although, at present, only the relative values for the NA concen-
tration in the soma of neurons can be determined by the author's meth-
odology, the utilization of the appropriate staining conditions in the
method may yield the absolute amounts of rRNA and DNA inside the soma
of living neurons. The application of equation (4) to the single neuron
requires that the fluorescence intensity of AO-rRNA and A0-DNA complexes
at 536 nm. be proportional to the amount of A0 molecules, DNA, and rRNA
macromolecules available for interaction. Therefore. the ratio of dye
to neuron (intracellular dye content per cell) is very important, since
A0 obeys Beer's law up to a concentration that would cause its aggre-
gation. The cellular framework of the neuron serves to concentrate the
amount of A0 inside the neuron; a high dye-to-neuron ratio would increase
the aggregation of the A0 molecules onto the rRNA and DNA macromolecules,
causing the fluorescence intensity of the AO-complexes at 536 nm. to be

non-proportional to the concentration of dye molecules and NA ,

122

macromolecules in the neuron. The utilization of a low dye-to-neuron
ratio would eliminate the problem caused by the polymerization of the
A0 molecules, and may be accomplished by using low dye concentrations
for the staining of the neuron. The results of the gelatin microdroplet

511

experiments indicate that an A0 staining concentration of l x 10'
should be low enough so that Beer's law is obeyed, and toxicity or
damage to the living neuron is prevented. At this concentration, the
amount of A0 bound per rRNA macromolecule is equivalent to the amount
bound per DNA macromolecule and proportional to the monomer A0 fluores—
cence intensity at 536 nm., and the degree of aggregation of A0 onto
the NA macromolecules is very low (see Figures 7 and 8). (An experi-
ment to determine the staining concentrations for which Beer's law
holds true for the living neuron is the examination of the fluorescence
emission properties of the rRNA in Nissl bodies and the DNA in chromo-
somes of a specific A0-stained neuronal type and age, as a function

of low staining concentrations.) ,The staining time for the neuron
should allow for the maximum amount of monomer binding sites on the NA
macromolecules to be filled, and the equilibrium between the cell and
dye molecules to be reached. A neuronal staining time of 1 hr. should
permit the monomer binding sites on the DNA and rRNA macromolecules

to be roughly 70% full with all of the binding sites in the outer
regions of the macromolecules completely filled. In order to keep the.
neurons healthy and alive during the staining process, physiological
saline can be employed as a solvent for the dye and rediffusion solu-
tions. Unfortunately, the results from the microdroplet experiments
involving the binding of A0 molecules to NA macromolecules in the pre—

sense of ions cannot be directly applied to the neuron because there

123

is no valid way to measure the ion concentration in the microenvironment
of the NA in the neuron. However. the results (Figures 12 and 13) do
show that ions in the microenvironment of the NA can increase the bind-
ing of A0 in the monomer form, and decrease the aggregation of the A0
molecules. The physiological saline can be buffered to pH 7.2 with
McIlvanine's citrate-phosphate buffer, minimizing the changes in the
ionic strength. The pH 7.2 would allow for the optimum binding of A0
to the NA, and the amount of A0 bound per rRNA macromolecule is nearly
equivalent to the amount bound per DNA macromolecule (see Figure 10;
for the living cell the intranuclear and cytoplasmic pH average 7.3).
Bear in mind that not only is the interaction of A0 molecules and the
NA macromolecules affected by the dye concentration, staining time.
staining solution pH and ionic strength, but also by the temperature
at which the reaction is taking place. The neuron could be incubated
in the reaction solutions, of constant volume, in its incubator at
37°C with a humid COZ-air (5:95) environment. A preliminary experiment
must be perfbrmed to ascertain the effects of the incubation and of
the rediffusion time on the interaction of A0 with the NA in the living
neuron.

Ideally, it would be desirable to have the binding behavior of
A0 molecules to the DNA and rRNA macromolecules in the microdroplet
identical to that in the living neuron. This implies that the NA micro-
environment of the microdroplet may be made similar, as much as possible,
to the microenvironment of the NA in the neuron. The cellular components,
void of NA, that make up neurons can be employed as the material for the
microdroplets. Also, the microdroplets may be subjected to the same

circumstances as the neuron. In addition, the above staining condition

124

can be applied to the microdroplets to obtain the proper fluorescence
coefficients. If, under these conditions, the binding behavior of A0
molecules to the NA macromolecules is identical to that in the neuron,
then as a possible check, the F536 of 6.5 pg. DNA in the microdroplet

should be equal to the F536 of the nucleus of neurons, and other mammal-

ian cells treated in the same manner. This suggests that v.(f.c.g§él
rRNA DNA rRNA
f.c.536 ).may be equal to 1 or less, and the f.c.536 and f.c.536

rRNA

are equivalent or the f.c.536 is greater than the f.c.DNA

536'
The RNA content in the soma of single neurons can be obtained as

a function of various parameters such as age, drugs, excitation, etc.

As an example, the neuron may be stimulated ionophoretically for various
periods of time at a specific frequency; after each period the NA content
in the neuron could be determined. Before the staining process, the
neuron should be permitted to rest and given a chance to respond to the
parameter. Staining procedures such as that used in Figures 24 to 27

may be devised to delineate AO-DNA complexes in the nucleus from AD-rRNA
complexes in the cytoplasm. Also. the NA content of isolated nuclei

or ribosomes may be determined chemically and compared to that determined
by this method.

The author strongly advocates that the identification of future
neurons found in dissociated or reassociating cell cultures of the cere-
bral cortex of rats, fitting the morphological descriptions of Canada,
Type»I, Canada,1ype II, and Canada, bipolar neurons be characterized

as these neuronal types.

IV. CONCLUSIONS

 

In this investigation, the author examined the fluorescence char-
acteristics of the binding of acridine orange to nucleic acids in dif-
ferent microenvironments. The fluorescence emission spectra of acridine
orange molecules bound to deoxyribonucleic acids, ribosomal ribonucleic
acids, and polyuridylic acids in gelatin microdroplets and in single
fixed neurons were found to be the same under a number of different cir-
cumstances (polyuridylic acids were not examined in the cells). The
fluorescence spectrum of A0 bound to each NA had a maximum at 536 nm.
and a secondary peak at 604 nm. The relative fluorescence intensity
at 536 nm. and at 604 nm. was interpreted to reflect the amount of A0
molecules bound to the NA macromolecules in the monomer form [Bradley
and Nolf, 1960; Loeser et al, 1960; Rigler, 1966; Nest, 1969, 1973] and
aggregated form, respectively. It was determined that AD has a specific
binding affinity for each NA: the binding constant for a DNA macromole-
cule is greater than a rRNA macromolecule, which is greater than a Poly
U macromolecule. The binding affinity of A0 for gelatin proteins is less
than that for the NA. 1

The binding behaviors of A0 molecules onto DNA and rRNA macro-
molecules were found to be different. as a function of the A0 staining
concentration, dye-NA interaction time, staining solution pH, and the

NaCl concentration. 1. In the presence of an increasing dye concen-

 

tration the binding of the dye moletules onto the nucleic acids'

 

macromolecules demonstrated a linear intensification; indicating that

 

125

I . J I‘ll» I} .
1“ O. ‘1]11‘ (‘1.l‘. 1160811511 .111

126

the total amount of A0 bound to the NA in the monomer form, depends pro-
portionately on the qUantity of free dye molecules initially available

in the microenvironment for binding a NA macromolecule. 2. Increasing

 

the interaction time of the A0 molecules with the NA macromolecules
enhanced the binding of the dye molecules to the NA macromolecule. The
amount of A0 bound to rRNA in the monomer form is proportional to the
length of time, in minutes, the dye molecules were allowed to bind to the
rRNA macromolecules. There were two phases involved in the binding of
A0 molecules to the DNA macromolecules in the monomer form; during the
first 35 mins. of staining, there was a rapid uptake of dye molecules

by the DNA macromolecules--approximate1y 3.4 times faster than that of
the rRNA macromolecules, later, the uptake of dye molecules by the DNA
macromolecules substantially decreases by more than 80% of the initial
value. The majority of the monomer binding sites on the DNA conformation
were completely filled, during the initial 30 mintues of interaction,

and on the rRNA conformation they were only partially filled. 3. The

binding of A0 to the nucleic acidsfiincreased, as the pH increased from

 

4.0 to 8.4. The lower amounts of A0 bound to DNA or rRNA at acid pH
were due to an augmented positive charge on the A0 molecules and on the
negative phosphoric acid residues of the nucleotides; which leads to

an enhanced molecular repulsion, along with the suppression of the bind-
ing sites, thereby decreasing the binding of A0 molecules to the NA
macromolecule. Decreasing the positive charge concentration in the
environment to neutral pH maintains a relatively stable NA conformation
and permits the phosphoric acid residues to become ionized, favoring

. the molecular attractions and hydrophobic interactions of the NA nucleo-

tides and the dye molecules. This increases the binding of A0 to the

127

NA macromolecule. 4. The binding of A0 to the nucleic acids increased
in the presence of ions. Since the degree of structural order ofnucleic
acids is reduced at low ionic strengths, increasing the NaCl concentration
in the microenvironment would serve to fortify and enhance the hydro-
phobic interactions between the acridine and nucleotide base ring
structures, within the interior of the NA macromolecule. This increases
the binding of A0 to the NA in the monomer form via hydrophobic bonding.
At low NaCl concentrations, the DNA conformation has greater sensitivity
in binding A0 in the monomer form than the rRNA conformation.

5. The denaturation of the rRNA conformation slightly increased
the binding of A0 to the magromolecule. The spectral distribution of A0
molecules bound to the different denatured conformations of rRNA macro-
molecules are identical.

6. The two fluorescence molecularspecies of acridine orange, the
monomer and_egggegated forms. were found tofihave different fluorescence

decay behaviors_as 3 function of the irradiation time: the fluorescence

 

fading of AO-DNA, rRNA and gelatin protein complexes at 536 nm. displays
somewhat faster decay rates than the corresponding fluorescence fading
at 604 nm.; the fluorescence fading is more dependent on the binding
mode of acridine orange than on the conformation or type of the biopolymer.
7. It was established that the total amount of A0 bound bygDNA
and rRNA macromolecules isgproportional to theggantity of NA available
for bindigg_the dye molecules, in the monomer form. This implies that

each NA macromolecule has a specific number of binding sites for A0

moleculesin the monomer form. Fluorescence coefficients, f.c.ggg = 1.26
and f.c."RNA = 0.272, were procured and employed in the calculation of

536

128

the NA content in gelatin microdroplets. The equation

 

can be used to calculate the total NA content of a given gelatin micro-
droplet system; y = (f.c.gg2)/(f.c.;§gA), and b = background fluorescence.
With the author's methodology, the smallest amounts of nucleic acids,
easily detectable, were about 0.38 pg. DNA and 0.8 pg. rRNA.

There were three neuronal types identified in dissociated cell
cultures of female albino rat cortices: the Canada Type 1, Canada, Type
II. and the Canada, bipolar neurons. The NA content in the Canada,

Type II, and Canada, bipolar neurons between the ages of 67 do. and 99
da. old demonstrated identical growth rates, 0.1 pg. NA per diem. The
NA content in the Canada, bipolar neurons were always greater than that
in the Canada, Type II neurons by 8%. The monomer form of A0 is the
primary mode of binding onto the NA macromolecules in the neurons and
in the gelatin microdroplets, and the disposition of this AD formation
depends heavily on microenvironmental conditions.

In recent years neuroscientists have gained valuable information
anent the structure and function of macromolecules involved in neural
systems during behavioral responses. This information is inadequate
to fully understand the molecular interactions between the central
nervous system and the mind. However, if the neuroscientists were'
able to determine the molecular events underlying the behavioral or
physiological functions of neural systems, drugs which affect specific
molecular mechanisms can be administered to organisms in order to

facilitate specific functions of their neurons [Hashizu et a1. 1961;

129

McGaugh and Petrinovich, 1959 and 1965; Cameron et al, 1961 and 1966;
John, 1967; Unger, 1970; Gaito, 1971; Mahler, 1972]. The application
of this concept to the human organism would improve our p0tentia1 for
specific behavioral characteristics, such as better work performance,
increased longevity, higher intelligence, better mental makeup (mental
health), and other aspects [Gaito, 1971]. The administration of yeast
RNA to senile and arteriosclerotic individuals is reported to improve
their memories and alleviate their emotional problems; however, these
improvements would diminish when the RNA is discontinued [Cameron and
Solyom, 1961; Gaito, 1971]. This may be due to the yeast RNA being
readily attacked by the highly active RNase of the aged patients, per-
mitting more of their native RNA to be free from attack since the RNase
activity in the blood of humans increases with age [Kral and Sved,
1963; Gaito, 1971]. If neuroscientists could ascertain the molecular
mechanisms of the learning process, the appropriate chemical could be
fiadministered to intellectual retardates or normal individuals to in-
crease their intellecutal potential and performance. A considerable
amount of time in the life of a human being is spent engaged in the
performance of work. Improving the work performance of the individual
would be of great value to man. Specified drugs administered to
individuals could improve their performance and productivity during the
work experience, thereby raising the level of the human society. The
environmental circumstances of an individual affects the development
of his intellectual potential. The author concludes that the concept
of regulating specific endogenous chemicals, which affect the molecular
events of specific behavioral or physiological functions of the neural

systems in the human, may allow the individual to approach or reach

130

his true potential under the existing internal and/or external environ-
ment. The chemical approach could supplement the current principles

in maximizing the intellectual and physical performance of the individual
[Gaito, 1971]. .

The metabolic modifications of the structural and functional
characteristics of the nucleic acids due to neuronal aging affects the
functions of the normal living neuron. The RNA content in the neurons
of humans may tend to decrease when the individual reaches the twilight
years of life, resulting in the decline of behavioral and physiological
functions of the individual's neurons, and reflected by the deterioration
of the individual's physical and mental capabilities [Gaito, 1971]. The
prevailing metabolic circumstances might cause the diminuation of the
RNA content and the functions of the neurons in the aging human. Kral
and Sved [1963; Gaito, 1971] reported that the RNase activity in the
blood of humans increased with age of the individual. They found that
the enzymatic activity of RNase in the blood of individuals at 60 years
of age was about 25% greater than that in individuals at age 20; and at
100 years old, about 50% greater. The enzyme RNase attacks the RNA and
degrades it by hydrolyzing the ester linkage between the hydroxyl and
phosphoric acid groups of the phosphodiester bonds of the RNA backbone
[Lehninger, 1970, pp. 254—255]. Provided that the RNase activity in
the blood is representative of enzymatic activity in the brain, the
increased activity of RNase in aged individuals would reduce the amount
of RNA in the neurons of their brains [Gaito, 1971]. The template
activity of DNA for RNA synthesis is less effective with increasing age
[Gaito, 1971]. This would decrease the quantity of RNA in the aged

neuron; coupled with the increase in the RNase activity, they may cause

131

fonctional changes in the aging human. To consider another metabolic
circumstance of aging, the hydrogen bonding in polyribosomes is signi-
ficantly different in neurons of aged rats (24 months) than in neurons
of young rats (3 months) [Gaito, 1971]. In the aged rats the larger
polyribosomes of the neuron exhibited less hydrogen bonding, and the
smaller polyribosomes, conversely, exhibited greater hydrogen bonding,
than that in corresponding polyribosomes of the younger rats. The I
change in hydrogen bonding in polyribosomes of the aged neuron is greater
when the temperature is raised, than that of younger neurons [Gaito, 1971].
This suggests that the polyribosomes of aged neurons have markedly dif-
ferent internal structures, and are deficient in the capacity to syn-
thesize protein, compared to polyribosomes of young neurons. The age-
altered conformation of the RNA in the polyribosomes of aged neurons
can be restored to the characteristics of the young normal state by the
administration of the drug. diphenylhydantoin or NP-ll3 (developed by
the Newport Pharmaceuticals. Inc., Newport Beach, California) to the
aged animal [Gaito, 1971]. These drugs enhanced the synthesis of brain
RNA and protein and improved the learning and memory functions of the
aged animals. Therefore, if the neuroscientists were able to determine
the affects of aging on the molecular mechanisms of behaviors, the proper
chemicals could be administered to aged individuals to increase their
functional capabilities.

The regulation of the molecular events underlying behavioral or
physiological functions of the neuron would be of great value to man;
it could raise the performance and productivity of individuals in the
total human society. Obviously much research is needed before such an

idea can be put into practice today. However. the use of acridine orange

132

fluorescence microspectrophotometry, in conjunction with dissociated
neuron cultures, can facilitate the realization of this concept in the
future.

Forthwith, biophysical cytochemistry, i.e., the use of fluorescent
molecular probes in conjunction with neuron cultures, can be applied in
the investigation of the effects of alcohol (CZHGO) on the structure
and function of nucleic acids and on the behavioral and electrophysio-
logical functions of the single living and/or fixed neuron; this is a
research proposal of the author. Is the RNA metabolism and the functional
activity of neurons maintained in the presence of alcohol significantly
different compared to neurons not under the influence of alcohol? Is
the correlation between the RNA metabolism and the functions of the
intoxicated neuron different from that of the sober neuron? The response
to these and many more questions concerning the molecular phenomena of
neuronal functions can be facilitated by this technique. The RNA content
in specific neuronal types and ages, as a function of the alcohol con-
centration in the medium of the living neuron, can be determined by
the application of acridine orange fluorescence microspectrophotometry
to single neurons from the cerebral cortex and/or the substantia nigra
of the rat brain. Assuming that the synthesis of RNA in the neuron is
slowed or inhibited by alcohol, neurons maintained in a high alcohol
content environment should have less RNA than neurons of the same age
and type living in an alcohol-free environment. Due to the metabolic
changes of aging, the RNA synthesis of_aged neurons. chronically sub-
jected to high doses of alcohol, should be less than that of younger
neurons under the same conditions. The pulse rate and the RNA content

of a specific neuronal type and age can be determined before, during,

133

and after stimulation of the neuron; the firing behavior correlated to
the RNA content of the alcoholic neuron should be different than that
of the nonalcoholic neuron. EthBr fluorescence microspectrophotometry
may possibly be used to follow the kinetics of polyribosome formation
and structural changes of the chromosomes in neurons influenced by
alcohol. If the correlation between the RNA metabolism and the functions
of neurons, jg_gjgg, are similar to those of neurons in culture, when
subjugated to the same alcohol concentrations, the behavior performance
and synthesis of RNA of the intoxicated animal or neuron may be lower
than that of the sober neuron or animal. The author's application of
biophysical cytochemistry to single neurons in culture represents a
model system that may provide salient answers concerning the molecular

events engaged in neuron-to-neuron interactions.

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