CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
REGIONAL CELL NUMBERS AND RESPIRATION
\\
IN FROG BRAIN
A thesis submitted in satisfaction of the requirements
for the degree of Master of Science in
Biology
by
Roy Roger Yeaman
_....
January 1979
The Thesis of Roy Roger Yeaman is approved:
Marvin H. Cantor, Ph.D.
Richard L. Potter, Ph.D.
California State University, Northridge
ii
ACKNOWLEDGEMENT
I would like to give special recognition to Dr.
Richard L. Potter for his patience, moral support and
belief in me.
Special thanks go to Dr. Marvin H. Cantor
for his special advice and training.
I am grateful to Dr.
Jim W. Dole for his willingness to read and suggest ideas
in the writing of the thesis.
I would also like to thank
Drs. Moore, Kuhn and Pollock for the generous use of their
equipment.
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ••••••••••••••••••••••••••••.••••
iii
LIST OF TABLES •••• ·••••••••.•••••••••••••.••••••••
v
LIST OF FIGURES ••••••••••••••••••••••••••••••••
vi
ABSTRACT •••••••••••••••••••••••••••••••••••••••
vii
INTRODUCTION •••••••••••••••••••••••••••••••••••
1
MATERIALS AND METHODS ••••••••••••••••••••••••••
4
RESULTS ••••••••••••••••••••••••••••••••••••••••
6
DISCUSSION •••••••••••••••••••••••••••••••••••••
18
LITERATURE CITED •••••••••••••••••••••••••••••••
32
APPENDIX 1 •••••••••••••••••••••••••••••••••••••
38
APPENDIX 2 •••••••••••••••••••••••••••••••••••••
44
APPENDIX 3 •••••••••••••••••••••••••••••••••••••
45
APPENDIX 4 •••••••••••••••••••••••••••••••••••••
46
iv
LIST OF TABLES
Table
1.
2.
3~
4.
5.
6.
Page
Cellular Populations and Ratios of
Glial to Neuronal Cells..................
7
Cellular Populations and Ratios of
Glial to Neuronal Cells..................
8
Mean Values for K1 and K .....•.••..•......
2
A Comparison of Regional Respiratory
Rates for Different Parameters...........
11
12
Respiration Rates in Normal Ringers
and in Excess K+-Ringers.................
14
Respiration Rates in Na+-Free Ringers
and in 20 mM Na+-Ringers.................
16
v
LIST OF FIGURES
Page
Figure
1.
2.
Number of Glia/mg Wet Weight vs.
Percent Change in Respiration.............
13
Number of Neurons/mg Wet Weight vs.
Respiration Rate in Na+-Free
Ringers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17
vi
ABSTRACT
REGIONAL CELL NUMBERS AND RESPIRATION
IN FROG BRAIN
by
Roy Roger Yeaman
Master of Science in Biology
Regional brain functions may be related to
characteristic histological and physiological parameters.
Regions of the bullfrog brain, with known functions, were
characterized by their cellular population and respiration
rates.
Regional cellular counts were determined using a
homogenization technique.
Oxygen electrodes recorded the
regional respiratory rates of regional brain minces.
The
percent change in respiratory rate due to the addition of
excess K+ to the incubation medium, is proportional to the
number of glia per mg wet weight.
The tissue respiratory
rate per wet weight in Na+-free Ringers is proportional to
the number of neurons per mg wet weight.
Ratios of 9 - 15
glia to one neuron were found for the three brain regions.
vii
-
-
"
-
The hindbrain respires at 1/4 of the rate of the forebrain
and midbrain in Na+-free Ringers.
The addition of 20 mM
Na+ to Na+-free Ringers enhanced the respiration of the
hindbrain almost twice as much as for the more rostral
regions.
This may indicate that glia have a higher
respiratory rate in the hindbrain than the glia in the
other two regions.
The experimental data suggests that
tissue respiratory rates under the previously described
conditions can be used to estimate the relative numbers of
glia and neurons in brain tissues.
Differences in regional
respiratory rates are at least partly due to differences in
cellular populations and possibly due to differences in
respiratory rates per cell.
viii
INTRODUCTION
Brains have always been partitioned into discreet
regions.
This has been done in the belief that these
observable areas are different in function, i.e., one area
is the site for vision, another area is the site for hearring, and another is the site for the control of voluntary
muscle action.
If these areas are functionally different,
then they may also differ histologically with respect to
glial and neuronal cell numbers;·and in physiological parameters such as respiration rate.
In the bullfrog brain,
there is incomplete physiological and histological data on
specific areas.
Understanding the relationship between
histological and physiological parameters and regional
functions in the bullfrog brain may provide a model for
the mammalian brain.
The measurement of cellular densi-
ties in the various brain regions and the measurement of
the changes in oxygen consumption of the various brain
regions by manipulation of the concentration of K+ and Na+
ions in the Ringers solution will add more data for understanding the relationship between these parameters and
function.
Also, these studies should lead to more insight
1
2
into the structural organization of the brain.
The enhancement of respiration of brain tissue
slices by the addition of K+ to Ringers solution bathing
the tissue has been well documented {1, 2, 3, 4).
Hertz
(5) demonstrated that the increased respiration, caused by
the addition of K+ was due to an increased respiration in
gl:ial cells and not in neuronal cells.
This is supported
by the work of Haljamae and Hamberger (6).
The percent
increase in respiration rate may be proportional either to
the number or to the volume of glial cells.
The former is
more likely because the relative contribution of glial
processes to the cell's total respiration rate is small
( 7) •
According to-Hertz (5), glial respiration is halted
or considerably reduced when brain tissue slices are
incubated in Na+-free Ringers, which is kept iso-osmotic
with sucrose; only neuronal elements respire.
Respiration
rate of tissue in Na+-free Ringers should be proportional
to the number of neuronal cells or to the volume of
neuronal cells.
It appears that alteration of this physic-
logical parameter, the respiration rate, under the above
described conditions, could be used as an index of the
relative number of glial or neuronal cells in different
parts of the brain.
Different respiration rates in different areas of
the brain might be indicative of their different functions.
3
The loci of functions have been demonstrated by brain
lesions.
Decerebrate anurans were nonspontaneous,
especially when observed in their natural environment.
However, this condition was less marked if the basal
cerebral ganglia were left intact.
Nerve tracts
originating in these ganglia extend into the diencephalon
.and mesencephalon (8).
This suggests that the ventral
halves of the cerebral hemispheres have functions different
from the dorsal halves of the hemispheres.
In the bull-
frog, the cerebellum is the regulator of muscle coordination and equilibrium of the body (8).
is associated with sensory function.
The olfactory bulb
The diencephalon,
which includes the thalamus, is associated with sensory
integration.
The mesencephalon is associated with visual
sensory integration.
Part of this study was undertaken to test the
hypotheses:
1) the percent increase in respiration rate
by the addition of K+ is proportional to glial cell number,
2) neuronal cell numbers are proportional to the respiration rate of tissue in Na+-free Ringers.
Another aim of
this study was to determine whether discreet anatomical
structures of the brain have different respiration rates
and to determine whether differences in respiration rate
in normal and high K+ Ringers could be correlated with the
known functions of these discreet anatomical structures of
the bullfrog brain.
4
MATERIALS AND METHODS
Adult Rana catesbeiana were used.
All respiration
rates were measured polarographically using a. YSI Model 53
oxygen electrode.
The Ringers were that of Heilbrunn (9).
Air was bubbled into the Ringers for at least 10 minutes to
fully saturate it with
o2 .
The solubility of oxygen in
Ringe_rs and Na +-free Ringers was taken from Table 1 in
"Macro- and Micro-Oxygen Electrode Technique for Cell
Measurements"
(10).
A Kelby air purifier and equalizer was
used within the air lines.
For the measurement of respira-
tion rate, the brain regions were dissected apart under a
dissecting microscope.
Each region of the brain was
finely sliced on a slide using a razor blade.
was greater than 1 mm
No piece
in-di-amete-r~--
Respiration rates of the three regions - forebrain,
midbrain and hindbrain- as described by Potter (11), were
measured in Na+-free Ringers with equimolar sucrose
replacing the Na+.
Afterwards, Na+ was added to the
bathing solution to bring the final Na+ concentration to
20 mM, and the rates were measured again.
This final
concentration was chosen because it was shown that the
respiration rate in 20 mM Na+-Ringers was minimally
reduced compared to normal Ringers.
This tissue was
incubated in the Na+-free Ringers for 2 - 4.5 hours in an
ice bath before the rate was measured (12).
The respiration rates of the olfactory bulb,
5
cerebellum, diencephalon, mesencephalon, dorsal and
ventral cerebrums of the bullfrog were measured in Ringers.
KCl was added to the bathing solution to bring the final
K+ concentration to 40 mM, and the rates were measured
again.
However, for the cerebellum, two measurements were
made at 40 mM, three at 59 mM and one at 48 mM.
The procedure for counting cell types was as
follows.
The brains were removed and divided into three
parts as described by Potter (11) .
The enumeration of
different cell types in each of the three regions of the
frog brain was based upon the procedure of Nurnberger and
Gordon (13)
(Appendix 1).
However, a few modifications in
the procedure were made because of the minute amount of
tissue used.
The tissue, 40 - 80 mg wet weight, was
homogenized on a magnetic stirrer set at 3 - 4 in a 25 ml
flask, containing 25 five mm glass beads, 4 ml of homogenizing fluid (Appendix 1) and a magnetic bar, for 23 102 minutes.
minutes.
The mean homogenizing time was about 80
One forebrain was homogenized for 255 minutes on
a heavy duty agitator instead of on a magnetic stirrer.
Ten lambda aliquots of suspension were removed at time
intervals and mixed with 10 lambda of methylene blue stain
on a slide with a round depression.
Then, two hemo-
cytometers were filled with aliquots of this 1:2 diluted
stained suspension.
Discrimination of neuronal and glial
nuclei was based upon the descriptions of Nurnberger and
6
Gordon (13) and Kongsmark (14).
A Zeiss microscope at 500X
magnification was used.
The raw data were treated by two methods to
generate the results - one by Nurnberger and Gordon (13)
and the other by Brizzee et al.
(15).
The two results were
then compared.
RESULTS
Table 1 lists cellular counts generated by the
Brizzee et al. method.
The fore- and midbrain have
approximately three times the number of cells, approximately three times more glial cells and about twice as many
neuronal cells as the hindbrain.
The total cells per mg
wet weight of the fore- and midbrain are about 2.4 times
greater than the hindbrain, whereas the glial cells per
mg wet weight of the fore- and midbrain are more than
twice that of the hindbrain and their neuronal cells per
mg wet weight are only approximately 1.4 times greater.
,,
7
Table 1
Cellular Populations and Ratios of Glial
to Neuronal Cells
Total Cells Per Brain
Total Cell
Population
6
X 10
Glial
Population
Forebrain
4.36±1.06(8)
4.03±1.00(8)
0.28±0.04(9)
Midbrain
4.02±0.38(9)
3.69±0.34(9)
0.27±0.05(9)
Hindbrain
1.28±0.13(9)
1.20±0.09(9)
0.14±0.02(8)
X
10
6
Neuronal
Population
6
X 10
Cells Per mg Wet Weight
Total Cell
Population/
mg Wet Wt.
4
X 10
Glial
Population/
mg Wet Wt.
X
10
4
Neuronal
Population/ Ratio
mg Wet Wt.
. Glial
4
X 10
Neuron
Forebrain
6.32±1.71(8) 5.83±1.61(8) 0.40±0.07(9) 14.61
Midbrain
5.61±0.59(9) 5.14±0.52(9) 0.39±0.08(9) 13.51
Hindbrain
2.49±0.20(9) 2.35±0.15(9) 0.28±0.05(8)
8.71
Numbers indicate mean ± standard error of the mean
(SEM) , and (N) .
8
Table 2
Cellular Populations and Ratios of Glial
to Neuronal Cells
.·
..
Total Cells Per Brain
Total Cell
Population
6
X 10
Glial
Population
6
X 10
Neuronal
Population
Forebrain
4.52±0.60
4.19±0.65
0.43±0.07(11)
Midbrain
5.52±0.40
5.49±0.38
0.62±0.09(10)
Hindbrain
1.79±0.19
1.63±0.19
0.20±0.03 (9)
X
10
6
Cells Per mg Wet Weight
Total Cell
Population/
mg Wet Wt.
4
X 10
Glial
Population/
mg Wet Wt.
X 10 4
Forebrain
6.46±0.99
Midbrain
Hindbrain
Neuronal
Population/
mg Wet Wt.
4
Ratio
Glial
Neuron
6.01±1.05
0.61±0.10 (11)
9. 75
7.53±0.56
7.46±0.47
0.84±0.12(10) 8.91
3.48±0.30
3.16±0.32
0.38±0.05(9)
c •.• •
•
X
10
8.31
Numbers indicate mean ± standard error of the mean
(SEM} , and (N) .
9
Cellular counts generated by the Nurnberger and
Gordon method are shown in Table 2.
The fore- and midbrain
have about 2. 7 times more total cells, 3 times as many
~­
glial cells, and 2.5 times more neurons than the hindbrain.
Cells per mg wet weight of the fore- and midbrain are
approximately twice that of the hindbrain.
The glial
cells per mg wet weight in the hindbrain is about 1/2 of
the other two regions, while the neuronal cells per mg wet
weight in the forebrain is 1.6 times and in the midbrain
2.2 times that in the hindbrain.
Tables 1 and 2 show that the cellular populations
in the forebrain and midbrain are greater than those of
the hindbrain, but not as great when computed as cells per
mg wet weight.
Comparison of the two tables shows that
even though the numbers differ, both indicate the same
pattern for the three regions of the bullfrog brain.
The
fore- and midbrain are similar in the relative numbers of
each cell type and both have several times more cells than
the hindbrain.
However, there is a difference between the
two methods in estimating the neuronal populations.
The
neuronal populations are estimated to be higher by the
Nurnberger and Gordon method, especially for the fore- and
midbrain.
This method gives a higher count for both
cellular type populations, but gives a larger count for the
neuronal poulations.
Both methods for practical purposes are about the
10
same in reproducibility.
However, Table 1 tends on the
average to have smaller SEM's than Table 2, but this
desirable attribute might be compensated by the latter
method being simpler and faster in estimating cell numbers.
Mean nuclear release and destruct rate contants
for each region, determined by the Brizzee method, are
shown in Table 3.
The K values for .neuronal cells are
1
higher than those for glia indicating that the rate of
destruction 6f neuronal plasma membrane is more rapid than
is that of glial plasma membrane.
This suggests a
structural difference between the glial and neuronal plasma
membranes.
Neuronal K values also are somewhat larger
2
than glial K values, indicating some difference between
2
glial and. neuronal nuclear membranes but this difference
is not as great as that existing between their plasma
membranes (Appendix 2).
11
Table 3
Mean Values for K and K2
1
Glial Cells
Forebrain
0.148±0.179(8)
0.016±0.010(8)
Midbrain
0.096±0.055(9)
0.016±0.011(9)
Hindbrain
0.162±0.084(9)
0.016±0.009(9)
Neuronal Cells
Forebrain
.Midbrain
Hindbrain
0.218±0.234(9)
0.026±0.011(9)
0.133±0.076(9)
0.022±0.007(8)
0.214±0.211(8)
0.029±0.013(8)
The rate constants are given. Numbers indicate
mean± standard deviation and (N).
12
Table 4
A Comparison of Regional Respiratory Rates
for Different Parameters
(Potter, 1977)
ul
o2 /hr/cell
:X 10
-6
o2 /hr/
ul 0 2 /hr/
ug DNA
mg Wet Wt.
ul
Forebrain
3.32
0.09
0.21
Midbrain
3.74
0.11
0.21
Hindbrain
7.23
0.15
0.18
The cellular rates were calculated from cell counts
in Table 1 and from Potter's regional brain respiration data.
Table 4 shows the respiration rate per cell in the
hindbrain to be approximately twice that for the other two
regions.
These figures are only averages and do not take
into account differences in respiration rate between
neuronal and glial cells.
Because there are more neurons
per glia in the hindbrain than in the other two regions
(Table 1) , this probably led to the higher value of ul
o2 /hr/cell
for the hindbrain.
The cellular rates corres-
pond quite closely to ul 0 /hr/ug DNA but not with ul
2
hr/mg wet weight.
o2 ;
This confirms the assumption that DNA
measurements are proportional to cell numbers (Appendix 3).
For the fore- mid- and hindbrain, a correlation
value of r
= +0.99
exists between percent change in
respiration rate and the number of glia/mg wet weight
13
(Figure_l).
The percent change in respiration is presumed
to indicate the relative number of glia/rng wet weight; the
greater the percent change is, the greater is the glia per
,· mg wet weight.
Figure 1
Number of Glia/mg Wet Weight vs. Percent
Change in Respiration
60
56)
% change
in respiration rate
40
20
0
(2.35, 21)
4
No. Glia/mg Wet Wt.
4
(x 10 )
6
Data were used from Table 1 and Potter (11). Line
was drawn by least square method. F = Forebrain,
M = Midbrain, H = Hindbrain
14
Table 5
Respiration Rates in Normal Ringers
and in Excess K+-Ringers
Control2 rnM K+
ul 0 2 /hr/mg
Brain
Region
Wet Wt.
40 - 59 rnM K+
ul o /hr/mg
2
Wet Wt.
% Change
Olfactory
lobes
0.36±0.07
0.42±0.06
25.43±11.92(9)
Ventral
cerebrum
0.30±0.04
0.48±0.05
67.00±14.79(9)
Dorsal
cerebrUm
0.24±0.04
0.49±0.05
137.11±34.37(9)
Diencephal on
0.26±0.03
0.42±0.05
62.89±7.24(9)
Me sencephalon
0.23±0.05
0.39±0.09
72.44±10.30(9)
Cerebellum
0.15±0.04
10.84±7.08
9171. 50±5180. 32
(6)
Numbers indicate mean± SEM and (N).
In Table 5, the cerebellum has the largest percent
change, indicating the highest glia/ mg wet weight.
In
support of this, it has been reported that glial cells have
a lower respiration rate than neuronal cells (5, 7), and
the cerebellar respiratory rate is lower than any other
region, thus indicating a high glia/mg wet weight.
Accord-
ing to a Duncan's Range test, a significant difference
exists between the cerebellar rate and that of the other
brain regions, and also between the olfactory lobes and the
15
other brain regions at the 0.05% confidence level.
The
respiratory rate and the percent change in rate inversely
correlated with each other for all regions except for the
dorsal cerebrum, i.e., the lower the respiratory rate is,
the greater is the percent change in rate.
This supports
the notion that each region has a different rate, which is
due to variations in the glial population.
The elevated
respiratory rates after K+ addition lasted on the average
4 ± 2 (S.D.) minutes, than the rates decreased below the
basal rates.
Table 6 shows that the fore- andmidbrain rates
are generally similar to each other while that of the
hindb~ain
differs considerably.
This patter is consistent
with the other histological and physiological data on the
three brain regions.
If respiration in Na+-free Ringers
is due solely to neuronal elements, the percent increase
in respiratory rate is not easily explained.
In Table 1,
there are more glia per mg wet weight for the fore- and
midbrain than for the hindbrain.
These two regions should
have a greater percent increase than the hindbrain if the
addition of Na+ reactivates glial respiration.
show just the opposite.
The data
The glial cells may have been
damaged by the lack of Na + , or because the glia in the
hindbrain respired at a higher rate than the ones in the
other two regions (Table 4}.
But, according to a Duncan's
Range test, there is no significant difference among the
Table 6
Respiration Rates in Na+-Free Ringers and in 20 mM.Na+-Ringers
Brain Region
+
Na -Free
u1 o /hr/
2
mg Wet Wt.
20 mM Na+
ul 0 2 /hr/
mg Wet Wt.
+
Na -Free
u1 o /hr/
2
ug DNA
20mMNa+
u1 0 /hr/
2
ug DNA
Forebrain
0.083±0.008
0.156±0.014
0.034±0.003
0.065±0.006
85.91±16.04(11)
Midbrain
0.080±0.006
0.135±0.012
0.040±0.003
0.067±0.006
69.06±14.35(9)
Hindbrain
0.022±0.003
0.636±0.010
0.019±0.003
0.054±0.009
131. 88±38. 34 (9)
Rate %
Change
Numbers indicate mean± SEM and (N).
DNA measurements come from Potter, unpublished data.
1-'
0'\
17
means of the percent change at the 0.05% confidence level.
Unlike the transient K+ effect, the elevated rates after
Na+ addition remained as long as the recordings were taken
(2 - 21 minutes) .
There was no indication of a decrease
in respiration rate after the higher rate was reached.
A correlation value "r" between number of neurons
per mg wet weight and tissue respiration rate in Na+-free
Ringers is +0.9995 (Figure 2).
This confirms the hypo-
thesis that only neuronal elements respire in Na +-free
Ringers.
Figure 2
Number of Neurons/mg Wet Weight vs. Respiration
Rate in Na+-Free Ringers
ul 0 /hr/mg wet wt.
2
0.080
(0.40, 0.083)
(0.386, 0.080)
0.040
(0.28, 0.022)
Data was used from Tables 1 and 6. Line was drawn
by least square method. F = Forebrain, M =
Midbrain, H = Hindbrain.
18
DISCUSSION
These experiments confirmed the findings of Hertz
(5) and Haljamae and Hamberger (6).
By a direct counting
procedure, it was shown that the percent increase in
respiration rate due to the addition of potassium was
directly proportional to the number of glial cells per mg
wet weight.
In addition, the respiration rate per mg wet
weight in Na+-free Ringers paralleled the number of
neuronal cells per mg wet weight.
Respiration rate had
been used to estimate the percentage of respiration
associated with Na+-K+- ATPase activity and the effects of
drugs on this activity (16).
Respiration rate can also be
used to estimate the relative numbers of glial and neuronal
cells in the brain.
The ratios of glial to neuronal cells differed
considerably between Tables 1 and 2, but the respiratory
data of Potter (11) tended to support the ratios in Table
1.
He found that the percent increases in respiration
rate, after K+ addition, were 56, 54.6, and 21% for the
fore-, mid- and hindbrain respectively.
This correlated
well with the ratios in Table 1, and not with the ratios
in Table 2.
Ris and Mirsky (17) calculated 1.53 x 10-S ug DNA/
nucleus for the frog brain.
Using the data in Table 1 and
the amount of DNA for each region (Potter's unpublished
data) , the amount of DNA per cell was calculated to be
19
3.88 x 10
-5 ug DNA/cell.
This was 2.4 times larger than
the value of Ris and Mirsky (Appendix 4).
Using the data from Potter (11) and Table 6, and
assuming the Na+-free respiration represents only neurons,
60 percent of the respiration was accounted for by glial
cells in the forebrain, 62 percent in the midbrain and 88
percent in the hindbrain.
Since the ratio of glial to
neuronal cells was lower in the hindbrain than in the other
two regions, it can be concluded that in the hindbrain
either the glia respired at a higher rate or that the
neuronal elements respired at a lower rate or both.
If
glia respired at a higher rate in the hinbrain, this
could explain the greater percent increase for the hindbrain in Table 6 upon restoring the Na+ concentration, and
explain the higher rate per cell for the hindbrain than
for the cells in the other two regions in Table 4.
Also,
regional variations in glial structure are well known
(18), and it likely reflects metabolic differences as well.
The data confirmed that the respiration rate per cell was
not constant throughout the brain.
The hindbrain had a
higher mean rate/cell than the other two regions.
This can
explain why Potter (11) found the rate/ug DNA for the foreand midbrain was lower than the hindbrain (micrograms of
DNA are proportional to cell number) , and then why the
hindbrain rate was lower than the fore- and midbrain rates
when calculated upon mg wet weight (wet weight represents
20
volume of cytoplasm rather than numbers or cells per mg
wet weight) •
From Table 5, it is apparent that mapping the brain
based on anatomical criteria means that these discreet
anatomical areas differ in respiration rate and cellular
populations.
These same areas have been described with
different functions.
Therefore, physiological parameters
may indicate different functions.
The cerebellum was a frag±le brain region, because
several times, it was not possible to obtain a respiration
reading from it.
Whereas, Potter (11) minced his tissue
samples with scissors, in these respiration experiments
the tissue was finely sliced.
This yielded higher
respiration rates at a lower temperature with the oxygen
electrodes than Potter's data yielded by manometry.
It has
been reported that respiration rates decrease when the
tissue is finely sliced but this was not confirmed in
these experiments.
Neuronal and glial cells in mammalian brain have
been well described (19, 20, 21).
There are fewer
descriptions of neuronal and glial cells in amphibians.
Amphibian neuronal and glial cells are similar in structure
to their mammalian counterparts (22, 23, 24, 25).
Although
descriptions of cell types by different authors may
conflict (19, 20), it is generally accepted that the method
of counting neurons may be based upon the assumption that
..
,-;·,
21
each neuron has only one nucleus and one nucleolus.
Occasionally, a neuron may have more than one nucleolus
(21, 26, 27, 28).
Glial cells have one nucleus with no
nucleolus, or one, nucleolus with one or more chromatin
bodies (14).
Authors' descriptions, especially in regard
to·nuclei, were based on small samples.
Therefore, the
assumption that neurons have one nucleus and one nucleolus
is tenuous.
More descriptions of neuronal ahd glial cells
and their nuclei in the bullfrog are required.
Probably, some small neuronal nuclei were not
identified as such and were counted as glial cells (13).
Their nucleoli were not resolvable under the microscope at
500x magnification.
Therefore, the count, presented in
Tables 1 and 2 were underestimations of the neuronaL ·..
population and an overestimation of the glial population.
This led to a higher glial to neuronal cell ratios.
all neuclei stained blue.
with granules.
Some were glassy in appearance
These had to be qualitatively distin-
guished from debris.
destruction.
Not
Some nuclei were in the process of
Such nuclei had broken nuclear membranes and
their nucleoplasm were partially extruded into the
surrounding medium.
This was evident because the nuclear
mernbrane.was visible and the stained blue nucleoplasm was
partially extruded but still coagulated.
These partially
destroyed nuclei were usually crescent shaped.
various stages of destruction.
This made their
There were
22
descrimination doubtful, and the experimenter had to decide
whether to count them as nuclei or not and of which kind.
In summary, the experimenter at times had to make quali··tative judgements whether to count nuclei as neuronal or
glial nuclei.
The equations in Appendix 1 were derived based on
the assumption that K was greater than K . The larger
2
1
K was relative to K , the better was the estimation of
2
1
cell numbers (15).
Brizzee et al.
(15) had not mentioned
the relative sizes of their K 's and K 's.
1
2
Nurnberger and Gordon (13) had low standard
deviations for their measurements.
gave no S.D.'s.
Brizzee et al.
(15)
There were two main reasons why Tables 1
and 2 had high SEM' s.
One was due t.o the ..small amount of
tissue used which meant a small number of nuclei to be
counted in the hemocytometers.
The small counts were
more prone to random fluctuations than were higher counts
as in the former cases.
This resulted in the line draw-
ings on the graphs for cellular estimation to differ
appreciably from one brain to the next, ad and thus to
the high SEM's.
The other reason was due to the
fluctuation in size differences between K1 and K2 (Appendix
2 and Table 3).
As these differences fluctuated, where
K was greater than K , the accuracy in the cellular
1
2
estimation fluctuated (15).
Kemali and Braitenberg (29) reported cell counts
23
for Rana esculenta.
The telencephalon has about
7, 2000, 000 cells.
The tectum, diencephalon and
mesencephalon, which corresponds to Potter's midbrain,
have a combined cell count of about 1, 676, 000 cells.
The
cerebellum, rhombencephalon, and spinal cord have about
6, 936, 000 cells.
The grand total for the central
nervous system is 15, 800, 000 cells.
By making a
comparison of these values with those in Tables 1 and 2,
it can be concluded that there are differences in cell
populations between species within the genus Rana for
corresponding brain regions.
However, the total counts
between the two species are relatively close (9 - 12
million for R. catesbeiana and 16 million for R. esculenta).
DePaoli et al.
(3·0} reported the Q0
2
for the
telencephalon, mesencephalon and medulla plus the spinal
cord, for Bufo arenarum Hensel and Leptodactylus
ocellatus.
Assuming dry weight is 20% of the wet weight,
the respiratory rate for B. arenarum Hensel is 0.49, 0.58,
and 0.46 ul
o 2 /hr/mg
wet weight for the telencephalon,
mesencephalon and medualla plus the spinal cord,
respectively.
And for L. ocellatus, the values are 0.49,
0.52 and 0.49 ul
o 2 /hr/mg
wet weight for the three brain
regions, respectively, at 30° C.
These values are much
higher than those in Table 5, column one, due to more
than a ten degree higher incubation temperature.
There
appears to be a difference in respiratory rates between
24
anuran species for corresponding brain regions, which is
probably due to differences in the composition of
cellular populations as well as differences in cellular
rates.
It has been suggested that glia can form a high
Na+ and low K+ compartment.
Glia can have a membrane
potential of the same magnitude as neuronal elements (31).
It would not be surprising then that excess K+ can
depolarize glial cells leading to an increase in respiration, while Na+-free Ringers allows only neuronal elements
to respire.
The K+-effect reported in the literature h~s been
explained as a heightened activity in the Na +-K+-ATPase
pump in neuronal·Cells (2, 4).
The addition of Na+to
Na+-free Ringers increases respiration but the effect is
not transient.
A transient effect would be expected if a
pump is involved.
The K+-effect appears to be due to
heightened glial metabolic activity.
appears to inhibit glial respiration.
Na +-free Ringers
A neuronal
Na +-K+ -ATPase pump theory does not easily explain these
phenomena.
A possible theory for the K+ and Na +-effects
is given below based on many items of evidence in the
literature.
Ashford and Dixon noticed an increase in
respiration in brain slices when the K+ concentration of
the incubation medium was increased to 30 mM.·
The maximum
25
respiration rate occurred in a K+ concentration of 40
60mM (32, 12, 33).
Respiration of brain slices decreased
when ouabain was added (2, 34, 35).
Na+ was required for
the increased respiration rate in excess K+ medium (12, 3·~ ·
In fact, Na + was the only ion capable of performing
34) •
this function (12).
Small changes in Na+ concentration
had little effect on respiration rate (3).
A Na +-K +-ATPase
pump was hypothesized to be activated by the addition of
K+, which led to an increase in respiration to replenish
the depleted ATP pool.
The works using red blood cell
ghosts and ouabain gave the basis for this theory (36, 37).
ATPase activity increased when the r.B.C. ghosts contained
. a me d'1um conta1n1ng
. .
Na + an d no K+ •
K+ an d no Na + 1n
This
activity was inhibited by ouabain.
ATPase enzyme activity increased with the addition
+ .
of K
+
and Na .
Ouabain inhibited this increased activity.
However, ouabain did not inhibit the activity
Na
+
+
and K were absent (38, 39).
Only Na
+
whenever
stimulated the
2
ATPase and not K+; and Mg + was required for activity
(40).
Na+ was required for ATPase activity (41).
ATPases
were localized in plasma membranes (42, 43, 44) on the
inside of the membrane (45).
depended on intact membranes.
K+-increased respiration
Probably, ATPase regulated
respiration rate in brain cortex through ADP production
( 3 2) •
In summary, Na+ was required for ATPase activity
26
and increased respiration; K+ increased respiration and not
ATPase activity; ouabain inhibited ATPase and respiration.
Changes in the K+ concentration affected inorganic
phosphate (Pi) and phosphate compound concentrations.
Pi
concentration increased (in cerebral cortex slices) with
.
d K+ concen t rat1on.
.
1ncrease
This was not due to an influx
of Pi into the slices from the medium, but, probably, due
to the splitting of ATP.
This led to an increased
incorporation of Pi into phospholipids and proteins (46).
The effect on the electron transport system by K+ was
observed.
With addition of excess K+ to brain slices,
there was an initial oxidation of the ETS carriers which
occurred at peak
o2
consumption.
And the slower reductive
phase preceded an increase in the rate of aerobic glycolysis (47, 48).
Excess K+ increased glucose metabolism (32).
..
.
d K+
Add 1t1on
o f excess K+ le d to an 1ncrease
concentration in slices (6, 34).
free medium lost K+ (38) .
Slices incubating inK+
Ouabain inhibited K+ accumula-
tion in tissue slices (34, 47, 6).
The removal of Na +
from the incubating medium had the same effect as adding
ouabain.
This reduced K+ accumulation (34), and reduced
respiration (5).
MoreK+ accumulated in glial than in
neuronal cells; and this increase depended on the presence
of Na + (6).
Hertz (5) showed that increased respiration
was due to glial cells and excess K+ had no effect on
neurons.
Also, removal of Na+ from the medium decreased
27
glial and not neuronal respiration.
It appeared that
the increased respiration in glial cells wad due to an
active uptake of K+ ions, and neuronal respiration was not
affected by the excess K+.
This K+-uptake required the
presence of Na + .
Addition of glutamic acid increased respiration
and decreased the phosphocreatine level in slices (49).
The respiration rate was higher with glutamate and glucose
in the incubating medium than with glutamate alone (33).
Addition of glutamate increased the K+ concentration in
brain slices (SO, 51).
L-glutamate was required for the
maintenance of high K+ concentration in brain slices.
was actively taken up by the tissue (52) .
K+
It was shown
that K+ uptake by tissue slices increased when using
glutamate and glucose in the incubating medium rather than
just one of the them (SO, 52).
Loss of K+ into the
incubating medium was stopped by the addition of glutamate
and glucose to the medium (52) .
Hertz (53) showed that the
addition of excess K+ led to an increase in influx and
efflux of K+ leading to an increase K+ concentration in
brain slices.
Glutamate efflux increased in slices when
K+ was added.
There was no change in Na+ fluxes
concentration when K+ was added.
or
Therefore, in summary,
it appeared that the uptake of K+ in glial cells was
coupled to the extrusion of glutamate and not Na+ as first
hypothesized for neuronal cells.
28
KCl and Na+-glutamate lowered the resting membrane
potentials of cells ·in brain slices.
depolarized them at times (51, 54).
depolarized were not specified.
Potassium even
But the type of cells
Electrical stimulation of
tissue slices led to a loss of K+ (55) .
It also caused a
decrease in phosphocreatine and an increase in Pi in the
slices (56).
(51).
Na+ did not lower cells' resting potentials
Because of Hertz's and Mcilwain's works and others,
it appeared that K+ caused a depolarization of glial cells,
which led to an active uptake of K+ by these glial cells.
This uptake was coupled to an extrusion of glutamate.
This activity was expressed at least by an increase in
respiration rate.
Unfortunately, this author did not run
across any experimental work to verify that glia and not
neurons were depolarized by the addition of K+.
Neuronal and glial processes accounted for a low
percentage of the cell's respiration (7).
Dendritic
processes and white matter accounted for very little
respiration (5) .
Respiration rate of the corpus callosum
had the lowest rate of any other part of the mammalian
brain.
Addition of K+ did not increase respiration rate in
the corpus callosum (4).
These data supported the notion
that respiration occurred mostly in the cell soma.
If
this was true, then it is not surprising to postulate that
the K+ effect occurred around the soma, where most of the
respiration mechanism is located.
The respiration system
29
should be located where it was needed, which is where the
K+-glutamate pump was located.
Further investigations are
definitely required into this hypothesis.
In summary, addition of K+ caused a depolarization
of glial cells.
This caused a decrease in K+ in the cells.
However, this depolarization led to an active uptake of
K+ coupled to glutamate extrusion.
A product of this
event was an increase in respiration.
Neurons were not
affected by adding K+.
The pump led to a net increase in
K+ in the glial cells.
This required energy.
The split-
ting of ATP was catalyzed by a Na + -Mg 2+ -ATPase. Na + and
2
Mg + were required for high ATPase activity. The ATPase
was ouabain sensitive.
The increased ADP level led to
active respiration by the mitochondria.
When slices were loaded with radioactive {hot) K+
and then placed in a medium containing excess nonradioactive {cold) K+, there was a loss of hot K+.
But at
the same time, there was an increase in cold K+ concentration in the slices due beyond normal diffusion rate {53).
How can this be explained?
The cold K+ bound to the·
external membrane surface.
Wherever the K+ was bound,
there was a change in the electron cloud configuration of
the membrane.
This led to a change in permeability.
This
change in permeability involved a depolarization leading
to a loss of hot K+~ which was not bound to the internal
membrane surface.
There was asymmetry to the membrane.
30
The external membrane surface bound K+ and the internal
membrane surface did not.
After depolarization, the
change in membrane configuration activated the K+-gluta-mate-ATPase pump.
The cold bound K+ was actively trans-
ported into the glial cells which reduced the external K+
concentration which kept the glial cells from being
continuously depolarized again.
Reports concerning the effect of ouabain on
respiration have been conflicting.
Ouabain decreased
respiration (34) or increased respiration (57, 75).
2
Ouabain has been shown to increase the free Ga + level in
the cell.
2
This added free ca +, increased mitochondrial
respiration (58).
enzymes.
Ouabain inhibited more than just ATPase
Increased or decreased respiration depended on
the additive effect of ouabain on all the systems combined
and under what conditions.
2
The effect of ca + on respira-
tion was also confusing (2, 47).
Because of the diverse
2
roles ca + has in the cell, further studies on the role of
2 .
.
.
.
. f urt h er requ1re
. d•
re 1 at1on
to resp1rat1on
1s
Ca + 1n
K+
also stimulated the release of neurotransmitters and thus
increased respiration (47, 48).
K+ may affect the level
2
of free ca + and therefore respiration (58) .
ATPase was
at times stimulated and inhibited by the addition of
glutamate (38).
Therefore, the recorded tissue respiration rate
represented the addition of the individual.respiration
31
rates of all systems of the brain slice.
Chemical
additions to the tissue will affect many systems of the
tissue.
LITERATURE CITED
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2.
Ruscak, M., and R. Whittam, 1967, The metabolic
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Hertz, L., and T. Clausen, 1963, Effects of potassium
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11.
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35
33.
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Whittam, R., 1962, The dependence of the respiration
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Biochem. J., 82: 205-212.
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Gonda, 0., and J. H. Quastel, 1962, Effects of ouabain
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404-412.
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Whittam, R., and D. M. Blond, 1964, Respiration
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Torack, R. M., and R. J. Barnett, 1964, Nucleoside
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36
of neurons and glia.
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Cummins, J., and H. Hyden, 1962, Adenosine triphosphatase levels and adenosine triphosphatases in
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Bull, R. J., and J. T. Cummins, 1973, Influence of
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48.
Bull, R. J., and S. D. Lutkhenhoff, 1973, Early
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exposed to elevated concentrations of potassium.
J. Neurochem., 21: 913-922.
49.
Woodman, R. J., and H. Mcilwain, 1961, Glutamic acid,
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37
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--
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APPENDIX 1
A brief description of the enumeration procedure is
in order. The procedure was basically that of Nurnberger
and Gordon (13). The tissue, whose cell types were to be
enumerated, was placed in a 25 ml flask containing 25 5 mm
glass beads, 4 ml homogenizing fluid (given below) and a
magnetic bar for stirring.
Homogenizing Fluid
CaC1 ·H 0
2 2
0.0015 M
KCl
0.20
KH Po
2 4
0.0083 M
K2 HP0
4
0.0016 M
M
PH 6.5
The tissue was homogenized on a magnetic stirrer.
At certain time intervals, ten lambda aliquots of the
suspension were removed using a 10 lambda pipet. Each
sample was mixed with 10 lambda of a methylene blue stain
(given below) on a slide with a round depression.
Staining Fluid
CaC1 ·H 0
2 2
0.0015 M
KCl
0.20
KH 2 Po
M
0.0083 M
4
K2 HP0
4
0.0016 M
methylene blue
chloride
0.1 %
PH 6.5
38
39
The mixing was performed by using the pipet for
swirling the mixture or drawing up the mixture into the
pipet and then blowing it out, and repeating this for at
least one minute. This 20 lambda of solution was then used
to fill two hemocytometers. Free naked nuclei, which were
not surrounded by their cytoplasm (in the five large squares of the hemocytometer) were counted. Most nuclei
were stained blue by the methylene blue. The number of
neuronal nuclei and the number of glial nuclei in the five
squares were recorded and the means between the two
chambers were used as the raw data. Thus, the number of
free nuclei of a particular type in the 4 ml of fluid over
time was determined. These data were plotted and gave a
graph, such as seen in Figure 1.
Neuronal or Glial or Total Cell Count
240
# nuclei/vol.
0
0
Time/min.
Figure 1
Two methods in the treatment of the above graph in
order to achieve an estimate of the total number of cell
types were used. The first, by Nurnberger and Gordon (13)
was simple. Simply, two lines were drawn on the graph as
represented by the dash lines in Figure 2.
40
Neuronal or Glial or Total Cell Count
tt-
\~
240
# nuclei/vel.
~
I \
1/
2
~
0
Time/min.
Figure 2
The first line represented the maximum rate of
release.of nuclei and the second line, the maximum rate of
destruction of nuclei. Their point of intersection gave
the estimated cellular count in the flask and in the whole
tissue.
The second method in the treatment of the data was
described by Brizzee et al. (15). Briefly, it is as
follows:
A tangent line was drawn to the curve as shown in
Figure 3.
41
Neuronal or Glial or Total Cell Count
24
# nuclei/val.
0
m
Time/min.
Figure 3
The slope of this tangent line was multiplied by
the negative reciprocal of the number of nuclei at the
point of tangency (Bt) , which is at a time greater than tm.
1
~t
Slope of the tangent line.
and B are
1
2
arbitrary points on the tangent line at their
times, t
and t
respectively.
2
1
K
2
= The
B
rate constant for nuclear destruction.
The above equation gave K , the rate constant, for
2
nuclear destruction.
K was then substituted into the
2
following equation:
42
K2 -
=
Time of maximum number of free nuclei (Figure
3)
= Y-intercept
B
The equation gave B, which was the y-intercept of
a straight line on the graph ln K1 vs K as shown in Figure
1
4.
15
0
-
•0
Figure 4
The slope of the straight line was 1/t . Two
m
points, A and C, solved the following equation for K :
1
K1
K
1
= Rate
=
1
tm ln K1 + B
constant for the release of nuclei.
43
Once K1 and K were determined, they were substi2
tuted into the following equation:
IT
= Kl
- K2
D
Kl
e
D
= Any
t
-e
point on the graph at time t, greater than
m
n
= Number
e
=
for cell count.
Log function
How these formulas were derived can be found elsewhere (15) . The calculations were performed and a result
obtained only when K1 was at least 1.4 times larger than
K • If it was not 1.4 times larger than K2 , then the
2
results were not used. A graph for neuronal nuclei and
a graph for glial nuclei and a graph for total nuclei were
made and were treated as above. This led to a total of
33 graphs to be analyzed by this method. Not all graphs
were used because K1 was not 1.4 times larger than K2 .
APPENDIX 2
The mean K and K values for the total cell
1
2
population were given below; ±standard deviation (S.D.);
and (N) •
Total
Kl
K2
Forebrain
0.134±0.148
0.018±0.010
(8)
Midbrain
0.087±0.026
0.017±0.009
(9)
Hindbrain
0.134±0.051
0.015±0.007
( 9)
44
APPENDIX 3
,.Method 1 = Brizzee et al.
(15).
Method 2 =Nurnberger and Gordon (13).
Method 2
A Comparison of Regional Respiratory
Rates for Different Parameters
(Potter, 1977)
ul
o 2 /hr/cell
X
ul
10-G
o 2 /hr/mg
DNA
ul
o 2 /hr/mg
wet wt •.
Forebrain
3.25
0.09
0.21
Midbrain
2.79
0.11
0.21
Hindbrain
5.17
0.15
0.18
The cellular rates were calculated from cell counts
in Table 2 and from Potter's regional brain respiration
data.
For Method 1, see Table 4.
45
APPENDIX 4
DNA in the three brain regions in ug DNA/mg wet
weight, and (N) (Potter, unpublished data).
ug DNA/mg wet wt.
Forebrain
2.41±0.05
( 8)
Midbrain
2.00±0.09
(8)
Hindbrain
1.18±0. 06
(8)
Tabulated are the ug DNA/cell for each region
using the data in Tables 1 and 2, and the above values.
Method 1 = Brizzee et al.
Method 2
= Nurnberger
(15).
and Gordon (13).
Method 1
ug DNA/cell x 10- 5
Forebrain
Midbrain
Hindbrain
3.81
3.56
4.74
Ris and Mirsky (17)
ug DNA/cell
1.57 X 10 -5
Method 2
Forebrain
Midbrain
Hindbrain
3.73
2.66
3.39
The following values are obtained if one adds up
all the DNA/mg wet wt. from the three brain regions
(Potter, unpublished data) and uses the total cell counts/
mg wet wt. in Tables 1 and 2.
ug DNA/cell x 10- 5
Method 1
Method 2
3.88
3.20
46