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

ACKNOWLEDGEMENT ••••••••••••••••••••••••••••.••••

LIST OF TABLES •••• · ••••••••.•••••••••••••.••••••••

LIST OF FIGURES ••••••••••••••••••••••••••••••••

ABSTRACT •••••••••••••••••••••••••••••••••••••••

INTRODUCTION •••••••••••••••••••••••••••••••••••

MATERIALS AND METHODS ••••••••••••••••••••••••••

RESULTS ••••••••••••••••••••••••••••••••••••••••

DISCUSSION •••••••••••••••••••••••••••••••••••••

LITERATURE CITED •••••••••••••••••••••••••••••••

APPENDIX 1 •••••••••••••••••••••••••••••••••••••

APPENDIX 2 •••••••••••••••••••••••••••••••••••••

APPENDIX 3 •••••••••••••••••••••••••••••••••••••

APPENDIX 4 •••••••••••••••••••••••••••••••••••••

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18

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LIST OF TABLES

Table Page

1. Cellular Populations and Ratios of Glial to Neuronal Cells.................. 7

2. Cellular Populations and Ratios of Glial to Neuronal Cells.................. 8

3~ Mean Values for K1 and K2.....•.••..•...... 11

4. A Comparison of Regional Respiratory Rates for Different Parameters........... 12

5. Respiration Rates in Normal Ringers and in Excess K+-Ringers................. 14

6. Respiration Rates in Na+-Free Ringers and in 20 mM Na+-Ringers................. 16

v

LIST OF FIGURES

Figure Page

1. Number of Glia/mg Wet Weight vs. Percent Change in Respiration............. 13

2. 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 hear­

ring, 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 para­

meters 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 under­

standing the relationship between these parameters and

function. Also, these studies should lead to more insight

1

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.

2

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 coordina­

tion and equilibrium of the body (8). The olfactory bulb

is associated with sensory function. 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 respira­

tion 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.

3

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. No piece

was greater than 1 mm 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,

4

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 homo­

genizing fluid (Appendix 1) and a magnetic bar, for 23 -

102 minutes. The mean homogenizing time was about 80

minutes. 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

5

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, approximate­

ly 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.

6

Forebrain

Midbrain

Hindbrain

Forebrain

Midbrain

Hindbrain

Table 1

Cellular Populations and Ratios of Glial to Neuronal Cells

Total Cells Per Brain

Total Cell Population

X 106

4.36±1.06(8)

4.02±0.38(9)

1.28±0.13(9)

Glial Population

X 106

4.03±1.00(8)

3.69±0.34(9)

1.20±0.09(9)

Cells Per mg Wet Weight

Neuronal Population

X 106

0.28±0.04(9)

0.27±0.05(9)

0.14±0.02(8)

Total Cell Population/ mg Wet Wt.

Glial Population/ mg Wet Wt.

Neuronal Population/ mg Wet Wt. Ratio

. Glial Neuron 4

X 10 X 104 X 10 4

6.32±1.71(8) 5.83±1.61(8) 0.40±0.07(9) 14.61

5.61±0.59(9) 5.14±0.52(9) 0.39±0.08(9) 13.51

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) .

7

.· ..

Forebrain

Midbrain

Hindbrain

Forebrain

Midbrain

Hindbrain

Table 2

Cellular Populations and Ratios of Glial to Neuronal Cells

Total Cells Per Brain

Total Cell Population

Glial Population

Neuronal Population

X 106

4.52±0.60

5.52±0.40

1.79±0.19

X 106

4.19±0.65

5.49±0.38

1.63±0.19

X 106

0.43±0.07(11)

0.62±0.09(10)

0.20±0.03 (9)

Cells Per mg Wet Weight

Total Cell Population/ mg Wet Wt.

X 104

6.46±0.99

7.53±0.56

3.48±0.30

Glial Population/ mg Wet Wt.

X 104 c •.• • •

6.01±1.05

7.46±0.47

3.16±0.32

Neuronal Population/ mg Wet Wt.

X 10 4

Ratio Glial Neuron

0.61±0.10 (11) 9. 75

0.84±0.12(10) 8.91

0.38±0.05(9) 8.31

Numbers indicate mean ± standard error of the mean (SEM} , and (N) .

8

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

9

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 K1 values for .neuronal cells are

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 K2 values also are somewhat larger

than glial K2 values, indicating some difference between

glial and. neuronal nuclear membranes but this difference

is not as great as that existing between their plasma

membranes (Appendix 2).

10

Forebrain

Midbrain

Hindbrain

Forebrain

.Midbrain

Hindbrain

Table 3

Mean Values for K1

and K2

Glial Cells

0.148±0.179(8)

0.096±0.055(9)

0.162±0.084(9)

0.016±0.010(8)

0.016±0.011(9)

0.016±0.009(9)

Neuronal Cells

0.218±0.234(9)

0.133±0.076(9)

0.214±0.211(8)

0.026±0.011(9)

0.022±0.007(8)

0.029±0.013(8)

The rate constants are given. Numbers indicate mean± standard deviation and (N).

11

Table 4

A Comparison of Regional Respiratory Rates for Different Parameters

(Potter, 1977)

ul o2/hr/cell ul 02 /hr/ ul o2 /hr/ -6 DNA Wet Wt. :X 10 ug mg

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 02 /hr/ug DNA but not with ul o2 ;

hr/mg wet weight. 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

12

(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

% change in respira­tion rate

40

20

0

(2.35, 21)

4 6 No. Glia/mg Wet Wt. (x 10 4 )

56)

Data were used from Table 1 and Potter (11). Line was drawn by least square method. F = Forebrain, M = Midbrain, H = Hindbrain

13

Brain Region

Olfactory lobes

Ventral cerebrum

Dorsal cerebrUm

Dien-cephal on

Me sen-cephalon

Cere-bellum

Table 5

Respiration Rates in Normal Ringers and in Excess K+-Ringers

Control2 rnM K+ ul 02 /hr/mg

Wet Wt.

0.36±0.07

0.30±0.04

0.24±0.04

0.26±0.03

0.23±0.05

0.15±0.04

40 - 59 rnM K+ ul o2/hr/mg

Wet Wt.

0.42±0.06

0.48±0.05

0.49±0.05

0.42±0.05

0.39±0.09

10.84±7.08

% Change

25.43±11.92(9)

67.00±14.79(9)

137.11±34.37(9)

62.89±7.24(9)

72.44±10.30(9)

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

14

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. The data

show just the opposite. 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

15

Table 6

Respiration Rates in Na+-Free Ringers and in 20 mM.Na+-Ringers

+ Na -Free 20 mM Na+ + Na -Free 20mMNa+ u1 o2 /hr/ ul 02 /hr/ u1 o2 /hr/ u1 02 /hr/ Rate %

Brain Region mg Wet Wt. mg Wet Wt. ug DNA ug DNA Change

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)

Numbers indicate mean± SEM and (N). DNA measurements come from Potter, unpublished data.

1-' 0'\

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 02 /hr/mg wet wt.

0.080

0.040

(0.40, 0.083) (0.386, 0.080)

(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.

17

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

18

-5 3.88 x 10 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 hind­

brain 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 fore-

and 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

19

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

20

.. ,-;·,

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. Not

all neuclei stained blue. Some were glassy in appearance

with granules. These had to be qualitatively distin­

guished from debris. Some nuclei were in the process of

destruction. 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. There were

various stages of destruction. This made their

21

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 K1 was greater than K2 . The larger

K1

was relative to K2 , the better was the estimation of

cell numbers (15). Brizzee et al. (15) had not mentioned

the relative sizes of their K1 's and K2 's.

Nurnberger and Gordon (13) had low standard

deviations for their measurements. Brizzee et al. (15)

gave no S.D.'s. 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

K1 was greater than K2 , the accuracy in the cellular

estimation fluctuated (15).

Kemali and Braitenberg (29) reported cell counts

22

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 Q02 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 o2

/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 o2 /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

23

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 respira­

tion, 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. + Na -free Ringers

appears to inhibit glial respiration. 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

24

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·~ ·

34) • + In fact, Na was the only ion capable of performing

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

+ d N + . d' . . + d + K an no a 1n a me 1um conta1n1ng Na an no K • 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 whenever

+ + + Na and K were absent (38, 39). Only Na stimulated the

ATPase and not K+; and Mg2+ 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). K+-increased respiration

depended on intact membranes. Probably, ATPase regulated

respiration rate in brain cortex through ADP production

( 3 2) •

In summary, Na+ was required for ATPase activity

25

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 + t . 1ncrease K concen rat1on. 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 glycoly­

sis (47, 48). Excess K+ increased glucose metabolism (32).

dd .. f + d . d + A 1t1on o excess K le to an 1ncrease K

concentration in slices (6, 34). Slices incubating inK+

free medium lost K+ (38) . 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

26

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. K+

was actively taken up by the tissue (52) . 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 or

concentration when K+ was added. 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.

27

KCl and Na+-glutamate lowered the resting membrane

potentials of cells ·in brain slices. Potassium even

depolarized them at times (51, 54). But the type of cells

depolarized were not specified. 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). Na+ did not lower cells' resting potentials

(51). 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

28

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-

+ 2+ + ting of ATP was catalyzed by a Na -Mg -ATPase. Na and

Mg2+ 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 non­

radioactive {cold) K+, there was a loss of hot K+. But at

+ the same time, there was an increase in cold K concentra-

tion in the slices due beyond normal diffusion rate {53).

How can this be explained?

external membrane surface.

The cold K+ bound to the·

+ 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.

29

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).

Ouabain has been shown to increase the free Ga2+ level in

the cell. This added free ca2+, increased mitochondrial

respiration (58). Ouabain inhibited more than just ATPase

enzymes. Increased or decreased respiration depended on

the additive effect of ouabain on all the systems combined

and under what conditions. The effect of ca2+ on respira-

tion was also confusing (2, 47). Because of the diverse

roles ca2+ has in the cell, further studies on the role of

C 2+ . 1 . . . . f h . d + a 1n re at1on to resp1rat1on 1s urt er requ1re • K

also stimulated the release of neurotransmitters and thus

increased respiration (47, 48). K+ may affect the level

of free ca2+ 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

30

rates of all systems of the brain slice. Chemical

additions to the tissue will affect many systems of the

tissue.

31

LITERATURE CITED

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2. Ruscak, M., and R. Whittam, 1967, The metabolic response of brain slices to agents affecting the sodium pump. J. Physiol., 190: 595-610.

3. Hertz, L., and T. Clausen, 1963, Effects of potassium and sodium on respiration: their specificity to slices from certain brain regions. Biochem. J., 89: 526-533.

4. Ridge, J. W., 1967, Resting and stimulated respiration in vitro in the central nervous system. Biochem. J., 105: 831-835.

5. Hertz, L., 1966, Neurological location of potassium and sodium effects on respiration in brain. J. Neurochem., 13: 1373-1387.

6. Haljamae, H., and A. Hamberger, 1971, Potassium accumulation by bulk prepared neuronal and glial cells. J. Neurochem., 18: 1903-1912.

7. Dittman, L., M. Sensenbrenner, L. Hertz, and P. Mandel, 1973, Respiration by cultivated astrocytes and neurons from the cerebral hemispheres. J. Neurochem., 21: 191-198.

8. Jorgensen, Barker C., 1974, Integrative Function of the Brain, in Physiology of the Amphibia (Vol. 2), Brian Lofts Editor, Academic Press, New York, 1-52.

9. Heilbrunn, L. V., 1948, An OUtline of General Physiology. W. B. Saunders, Philadelphia.

10. Lessler, Milton A., 1972, Macro- and Micro-Oxygen & Electrode Techniques for Cell Measurements, in Methods in Cell Physiology, Vol. 5. David M. Prescott Editor, Academic Press, New York, 199-218.

32

11. Potter, R. L., 1977, Regional differences of respira­tion in the bullfrog brain based on cell density. J. Neurobiology, 8: 133-139.

12. Hertz, L., and M. Schou, 1962, Univalent cations and the respiration of brain cortex slices. Biochem. J. 1 85: 93-104.

13. Nurnberger, J. I., and M. W. Gordon, 1957, The cell density of neural tissues: direct counting method and possible applications as a biological referent. Ultrastructure and Cellular Chemistry of Neural Tissue, in Progress in Neurobiology, Vol. II. H. W. Waelsch, Editor, New York, Haeber, 100~138.

14. Konigsmark, B. W., 1970, Methods for the counting of Neurons, in Contemporary Research Methods in Neuroanatomy. w. J. H. Nauta and S. 0. E. Ebbesson Editors, Springer Verlag, New York, 315-380.

15. Brizzee, K. R., J. Vogt, and X. Kharetchko, 1964, Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat, in Progress in Brain Research, Vol. 4, Growth and Maturation of the Brain. Dominick P. Purpura and J. P. Schade Editors, Elsevier Publishing Company, New York, 136-149.

16. Gubitz, R. H., T. Akers, and T. M. Brody, 1977, Control of brain slice respiration by (Na + K)­activated adenosine triphosphatase and the effect of enzyme inhibitors. Biochem. Biophys. Acta, 459: 263-277.

17. Ris, H., and A. E. Mirsky, 1949, Quantitative cytochemical determination of deoxyribonucleic acid with the Feulgen nucleal reaction. J. Gen. Physiol., 33: 125-146.

18. Watson, W. E., 1976, Cell Biology of Brain, Chapman and Hall Ltd., London.

19. del Rio Hortega, P., 1932, Microglia, in Cytology and Cellular Pathology of the Nervous System, Vol. 2. Wilder Penfield Editor, Hafner Publishing Company, New York, 1965, 481-534.

20. Penfield, W., 1928, Neuroglia and Microglia: The interstitial tissue of the Central Nervous System, in Special Cytology, Vol. 3. Edmund Cowdry

33

Editor, Hafner Publishing Company Inc., New York, 1963, 1447-1482.

21. Rappoport, D. A., P. Maxcy Hr., and H. F. Daginawala, 1969, Nuclei, in Handbook of Neurochemistry, Vol. 2, Structural Neurochemistry. Abel Lajtha Editor, Plenum Press, New York, 241--254.

22. Silver, M. L., 1942, Glial elements of the frog. J. Comp. Neural., 77: 41-47.

23. Kling, J. S., 1966, Comparative investigation of neuroglia in representative vertebrates: a silver carbonate study. J. Morph., 119: 435-466.

24. Schonbach, C., 1969, The neuroglia in the spinal cord of the newt Triturus viridescens. J. Comp. Neural., 135: 93-120.

25. Silver, M. L., 1942, Glial elements of the spinal cord of the frog. J. Comp. Neural., 71: 41-47.

26. Kappers, C. U. A., G. C. Huber, and E. C. Crosby, 1960, The Comparative Anatomy of the Nervous System of Vertebrates, including Man, Vol. 1. Hafner Publishing Company, New York, 1-134.

27. Hyden, H., 1960, The Neuron, in The Cell, Vol. 4. Jean Brachet and Alfred E. Mirsky Editors, Academic Press New York, 215-324.

28. Peters, A., S. c. Paley, and H. Webster, 1976, The Fine Structure of the Nervous System: The Neurons and Supporting Cells. W. B. Saunders Company, Philadelphia.

29. Kemali, M., and V. Braitenberg, 1969, Atlas of the Frog's Brain, Springer-Verlag, New York.

30. Depaoli, J. R., E. T. Segura and Elena C. Lascano, 1973, Effects of acclimation of oxygen consump­tion (Q0 2 ) of the brain and its different areas in two genera of anurans, Comp. Biochem. Physiol., 46A: 725-733.

31. Van Harreveld, A., 1966, Brain Tissue Electrolytes. Butterworths, Washington.

32. Piras, M. M., and J. A. Zadunaisky, 1964, Effect of Potassium and ouabain on glucose metabolism in frog brain. J. Neurochem., 12: 657-661.

34

33. Elliot, K. A. C., and F. Bilodeau, 1962, Influence of potassium. on respiration and glycolysis by brain slices. 84: 421-428.

34. Whittam, R., 1962, The dependence of the respiration of brain cortex on active cation transport. Biochem. J., 82: 205-212.

35. Gonda, 0., and J. H. Quastel, 1962, Effects of ouabain on cerebral metabolism and transport mechanisms in vitro. Biochem. J., 84: 394-405.

36. Glynn, I. M., 1962, Activation of adenosine triphos­phatase activity in cell membrane by external potassium and internal sodium. J. Physiol., 160: 18p.

37. Whittam, R., 1962, Asymmetrical stimulation of a membrane adenosine triphosphatase in relation to active cation transport. Biochem. J., 84: 110-118.

38. Hosie, J. A., 1965, Localization of adenosine triphosphatases in morphologically characteristic subcellular fractions of guinea-pig brain. Bi~chem. J., 96: 404-412.

39. Deuel, D. H., and H. Mcilwain, 1961, Cerebral adeno~ sine triphosphatases differing in cellular location and cation activation. Biochem. J., 80: 19p.

40. Jarnfelt, J., 1961, Sodium-stimulated adenosinetri­phosphatase in microsomes from rat brain. Biochim. Biophys. Acta, 48: 104-110.

41. Whittam, R., and D. M. Blond, 1964, Respiration control by an adenosine triphosphatase involved in active transport in brain cortex. Biochem. J., 92: 147-158.

42. Hess, H. H., and A. Pope, 1959, Intralaminar distribution of adenosine-triphosphatase activity in rat cerebral cortex. J. Neurochem., 3: 287-299.

43. Lewin, E., and H. H. Hess, 1964, Intralaminar distribution of Na+-K+ adenosine triphosphatase in rat cortex. J. Neurochem., 11: 473-481.

44. Torack, R. M., and R. J. Barnett, 1964, Nucleoside phosphatase activity in membranous fine structures

35

of neurons and glia. J. Histochem. Cytochem., 11: 763-772.

45. Cummins, J., and H. Hyden, 1962, Adenosine triphos­phatase levels and adenosine triphosphatases in neurons, glia and neuronal membranes of the vestibular nucleus. ·. Biochim. Biophys. Acta, 60: 271-283.

46. Durell, J., and P. J. Heald, 1961, The effects of potassium ion concentration on phosphate metabolism in cerebral slices. J. Neurochem., 9: 71-79.

47. Bull, R. J., and J. T. Cummins, 1973, Influence of potassium on the steady-state redox potential of the electron transport chain in slices of rat cerebral cortex and the effect of ouabain. J. Neurochem., 21: 923-937.

48. Bull, R. J., and S. D. Lutkhenhoff, 1973, Early changes in respiration, aerobic glycolysis and cellular NAD(P)H in slices of rat cerebral cortex exposed to elevated concentrations of potassium. J. Neurochem., 21: 913-922.

49. Woodman, R. J., and H. Mcilwain, 1961, Glutamic acid, other amino acids and related compounds as substrates for cerebral tissues: their effects on tissue phosphates. Biochem. J., 81: 83-93.

50. Pappius, H. M., and K. A. C. Elliot, 1956, Factors affecting the potassium content of incubated brain slices. Canad. J. Biochem. Physiol., 34: 1053-1067.

51. Hillman, H. H., and H. Mcilwain, 1961, Membrane potentials in mammalian cerebral tissues in vitro: dependence on ionic environment. J. Physiol., 157: 263-278.

52. Terner, C., L. V. Eggleston, and H. A. Krebs, 1950, The role of glutamic acid in the transport of potassium in brain and retina. Biochem. J., 47: 139-149.

53. Hertz, L., 1968, Potassium effects on ion transport in brain. J. Neurochem., 15: 1-16.

54. Gibson, I. M., and H. Mcilwain, 1965, Continuous recording of changes in membrane potential in mammalian cerebral tissues in vitro; recovery after

36

depolarization by added substances. J. Physiol., 176: 261-283.

55. Cummins, J. T., and H. Mcilwain, 1961, Electrical pul~es and the potassium and other ions of isolated cerebral tissues. Biochem. J., 79: 330-341.

56. Mcilwain, H., and M. B. R. Gore, 1951, Actions of electrical stimulation and 2:4-dinitrophenol on the phosphates in sections of mammalian brain in vitro. Biochem. J., 50: 24-28. --

57. LeFevre, M. E., 1973, Effects of ouabain and high K+ on respiration of turtle brain and urinary bladder in vitro. Comp. Biochem. Physiol., 45A: 283-292.

58. Tower, D. B., 1968, Ouabain and the distribution of calcium and magnesium in cerebral tissues in vitro. Exp. Brain Res., 6: 273-283.

37

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

CaC12

·H20 0.0015 M

KCl 0.20 M

KH 2Po4 0.0083 M

K2HP0 4 0.0016 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

CaC12 ·H20 0.0015 M

KCl 0.20 M

KH 2Po4 0.0083 M

K2HP0 4 0.0016 M

methylene blue 0.1 % chloride

PH 6.5

38

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.

39

Neuronal or Glial or Total Cell Count

240

# nuclei/vel.

tt- ~

\~ I \

1/ 2

~

Time/min.

Figure 2

0

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.

40

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. B1 and B2 are

arbitrary points on the tangent line at their times, t 1 and t 2 respectively.

K2 = The rate constant for nuclear destruction.

The above equation gave K2

, the rate constant, for

nuclear destruction. K2 was then substituted into the

following equation:

41

B

K -2

= Time of maximum number of free nuclei (Figure 3)

= Y-intercept

The equation gave B, which was the y-intercept of a straight line on the graph ln K1 vs K1 as shown in Figure

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 K1 :

1 tm ln K1 + B K = 1

K1 = Rate constant for the release of nuclei.

42

Once K1 and K2 were determined, they were substi­

tuted into the following equation:

= Kl - K2 IT

Kl e

D

-e

D = Any point on the graph at time t, greater than t m

n = Number for cell count.

e = 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

K2 • If it was not 1.4 times larger than K2 , then the

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 .

43

APPENDIX 2

The mean K1 and K2 values for the total cell

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 o2/hr/cell ul o2/hr/mg ul o2/hr/mg X 10-G DNA 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

Midbrain

Hindbrain

2.41±0.05

2.00±0.09

1.18±0. 06

( 8)

(8)

(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. (15).

Method 2 = Nurnberger and Gordon (13).

Forebrain Midbrain Hindbrain

Forebrain Midbrain Hindbrain

Method 1

ug DNA/cell x 10-5

3.81 3.56 4.74

3.73 2.66 3.39

Method 2

Ris and Mirsky (17) ug DNA/cell

-5 1.57 X 10

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.

Method 1 Method 2

ug DNA/cell x 10-5

3.88 3.20

46