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Changes during growth in the volume and surface area of cortical neurons in the rabbit
EXPERIMENTAL
NEUROLOGY
2, 158-178
(1960)
Changes during Growth in the Volume and Surface Area of Cortical Neurons in the Rabbit J. P. Division
of Biology,
SCHAD~
California
and Central Institute
for
Received
C. F.
AND
Institute Brain
BAXTER~
of Technology, Pasadena, California, Research, Amsterdam, Holland
January
18, 1960
A method was developed to measure the surface area of cortical nerve cells. It consists of measuring the proportional volume of the constituents of cortical neurons and their diameter. A modification of the method of Chalkley is developed to determine the volume and surface area of the dendrites. This measurement is accomplished by cementing four tungsten microelectrodes to the diaphragm of the ocular. An attempt has been made to find parallels between quantitative histological measurements and biochemical composition in a specific area of the developing rabbit brain cortex. The proportional volume and surface area of the perikaryal membranes decreased during the first 5 days after birth and then remained practically constant during development. The proportional volume and surface area of the apical and basal dendrites increased markedly from 5 to 30 days after birth. The change in the levels of glutamic acid decarboxylase, y-aminobutyric acid, and some other amino acids have been measured as a function of age. The largest changes in glutamic acid decarboxylase coincided with the most rapid phase in the development of the surface area of the dendrites. Surface area measurements of neurons in the cerebral cortex may be of value to both the physiologist and the biochemist. For the physiologist the surfaces of the perikarya and dendrites form the receptive part of the neuron. Any interpretation of ion movements and potential differences must take into account the extent of these surfaces. The biochemist might find these measurements a basis for a comparison of enzyme systems associated with cytoplasmic membranes. Introduction
Studies which attempt to correlate chemical composition with histological structure are hampered by the complexity of the neuronal elements 1 This investigation was supported in part by Research Grants from the National Institute of Neurological Diseases and Blindness (B 340) and from the National Science Foundation (G 4029). The authors are indebted to Dr. A. Van Harreveld and Dr. E. Roberts for their constructive criticism, and to Mrs. Ruth Estey and Mrs. Nancy Kennedy for histological preparations. The address of Dr. Baxter is Department of Biochemistry, Medical Research Institute, City of Hope Medical Center, Duarte, California, U.S.A.; that of Dr. Schade, Central Institute for Brain Research, Mauritskade, Amsterdam, Holland. 158
VOLUME
AND
SURFACE
AREA
OF NEURONS
159
in this area. In the cerebral cortex processes of the nerve cells are exceedingly long. The basal dendrites in the rabbit attain a length of 250 to 300 p (36). In the cat basal dendrites of 600 p (34) and apical dendrites as long as 1500 p have been reported. The cytoplasmic volume and surface area may be many times greater than that of the nerve cell bodies. Fundamental data about the chemical composition of the nerve cell body have been obtained by applying microchemical techniques to single isolated nerve cell bodies (7, 8, 15-20). However, the application of these micromethods to whole nerve cells can yield only approximate results, since in practice the isolation (for chemical analysis) of all the dendritic processes associated with a single nerve cell body inevitably is incomplete. The chemical architectonics of a number of brain structures in several species have been investigated using these microtechniques (16-20, 22, 23, 25, 26) and in some cases, yielded results which indicated that the activity of enzymes, such as acetylcholine esterase and Ca-adenosinetriphosphatase was more closely associated with the cell processes than with the cell bodies (22, 23). An alternative method of correlating chemical composition with histological structure was adopted by Flexner and his associates (9, 10, 11, 2 1) in their classical studies on biochemical and physiological differentiation during morphogenesis. They investigated the frontal cortex of the guinea pig (9, 10, 11, 2 1) paying special attention to the enzyme development in conjunction with the morphological maturation of the cerebral cortex. In this study an attempt has been made to find parallels between quantitative histological measurements and biochemical composition in a specific area of the developing rabbit brain cortex. The levels of y-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were chosen as biochemical parameters because of the unique presence of GAD and GABA in the central nervous system. Particular attention has been focused upon the development of total volume and total surface area of neuronal constituents in the cerebral cortex. Material
and
Methods
Histological Methods. Rabbits were used exclusively. To measure the dimensions of nerve cells and dendrites, histological preparations had to be made in which the fluid distribution between neuronal and extraneuronal compartments of the cerebral cortex was represented as faithfully as possible.
160
SCHADk
AND
BAXTER
The dorsal aspect of both hemispheres was widely exposed under ether narcosis leaving a small bony edge to protect the sagittal sinus. A skin cup was formed by sewing the skin surrounding the wound to a steel ring. The preparations were then immobilized with Intocostrin (5 units/kg/hr) and artificial respiration was applied with a Palmer pump. The state of activity in the cerebral cortex was checked with a four channel Offner electroencephalograph. Isopentane cooled to its freezing point (approximately -160” C) with liquid nitrogen was poured onto the cortex at a time when cortical activity was normal. The animal was decapitated and the head immersed in cold isopentane (-70 to --SO” C). The isopentane was rinsed off with absolute alcohol at the same temperature. The head was then placed in a beaker with cold absolute alcohol (-40” C) and stored at -25” C for 10 days in a refrigerator. After fixation, a slice of
FIG. 1. Dorsal view of the cerebral cortex of the rabbit. The longitudinal sulcus is indicated. Left hemisphere: the two vertical lines indicate the slice which was taken for histological examination. The arrow indicates the area in which measurements were made. The square (area Z-3) indicates the portion of cerebral cortex which was excised for biochemical measurements ; 1, frontal cortex; 2, motor cortex; 3, sensory cortex; 4, retrosplenial area; 5 and 7, visual cortex; 6, auditory cortex.
cortex was removed from an area as indicated in Fig. 1. The slice was put in methylbenzoate, embedded in paraffin, sectioned, and stained. For the study of cell bodies a gallocyanin stain was used and for dendrites method B of Ram&-r y Cajal (4). 1Measurements. The method employed for the determination of cell density of the cortices has been described (28).
VOLUME
AND
SURFACE
AREA
OF NEURONS
161
The proportional volume of nerve cell bodies (total volume per cubic millimeter of cerebral cortex) and of apical and basal dendrites were determined according to a modification of the method of Chalkley (5, 6) with the corrections of Haug (12, 13). According to this method the relative volume occupied by a specific constituent of a tissue is proportional to the number of times a point, placed at random throughout the tissue, will hit the constituent of the tissue. This measurement is accomplished by cementing four tungsten microelectrodes with points of 1 to 2 u to the diaphragm of the ocular (32). The wires appear in the focal plane. A section of cortex is moved at random under the objective of the microscope. A contact of the image of the point of the microelectrode with the clearly observed image of the tissue constituent is counted as a hit. The number of hits made in this way on nerve cell bodies, apical, and basal dendrites are recorded. Branches of apical dendrites are counted in the slides as basal dendrites, because they are very difficult to distinguish from basal dendrites when viewed under oil immersion. Observations were made using a 20 X ocular and a 2-mm oil immersion objective. A camera lucida was used to measure the diameter of the cell bodies and the dendrites. The magnification of this system was 3000x. Whenever a constituent was hit the diameter of this element was measured on the camera lucida screen. Cell bodies and dendrites were subdivided on the basis of measured diameters, each subdivision representing a difference of l/3 u (equivalent to 1 mm on the camera lucida screen). Twelve histological sections (25 p thick) from each animal were investigated. Four series of animals (littermates) were investigated. After measuring the standard deviation of the first series of samples it was found that 2000 to 3000 hits per slice of cortex gave a sufficient degree of accuracy. Cahdations. The total surface area of cell bodies and dendrites per unit volume was calculated as follows: Nerve cell bodies, 3v A=[II
i-
in which A = the surface area per cubic millimeter of cerebral cortex, V = the percentage of cortex which is nerve cell body, measured by the “hit” method, and P = the mean radius of the cell bodies. In this formula the cell bodies are treated as spheres. As an independent check, individual nerve cell bodies were measured and the surface was calculated by treating the cell bodies as prolate spheroids by using the formula
162
SCHADI?
AND
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a6 sin-l e i21 e in which A z the surface area of the individual cell body, a = half of the major axis of the nerve cell body, b = half of the minor axis, and A=2?rb2+2r----
e = \/ 1 Dendrites,
(b2 / a2). A=-
2v
[31 iin which A = the surface area of the dendrites per cubic millimeter of cerebral cortex, V = the percentage which is occupied by the apical or basal dendrites (branches of apical dendrites included) measured by the “hit” method i- = the mean radius of the apical or basal dendrites. In this formula the surface is calculated as the curved surface of a cylinder. Biochemical Methods. A section of pallium (cerebral cortex with underlying white matter) corresponding to the dotted area in Fig. 1 was excised from the brain of normal animals immediately after decapitation. Rabbits 30 days or older were lightly etherized. Excised areas were weighed and processed as follows: For the determination of GABA, samples were dropped into cold 75 per cent ethanol and usually left for a few days before extraction. For the determination of GAD, samples were homogenized in water and two aliquots placed into incubation tubes containing ice cold glutamic acid, pyridoxal phosphate, and phosphate buffer pH 6.3. One aliquot was deproteinized immediately with ethanol. The second aliquot was incubated for 30 min at 37” C and then deproteinized with ethanol. The difference in GABA content of the two samples was an index of GAD activity. The method for the extraction and processing of tissue and homogenate samples has been described ( 1). The total GABA in 40-mg equivalents of fresh tissue and GAD in 5 mg of tissue were measured using an enzymatic technique (14) adopted for GABA and GAD in tissue preparations (1). Two dimensional chromatographic assays were made using the same tissue extracts as were used for the enzymatic GABA assay. Relative amounts of glutamic acid, aspartic acid, and ethanolamine phosphate were determined semiquantitatively. Histological
Findings
The results are given in Tables 1, 2, and 3 and in Figs. 2 through 7, inclusive. Examples of the counts (average values with standard devia-
VOLUME
AND
SURFACE
AREA
163
OF NEURONS
tions) and the measurements of the diameter of the dendrites are shown; range values of four series of experiments are plotted in graphs against age. Percentage of Cortex Occupied by Nerve Cell Body, Apical, and Basal Dendrites. In newborn rabbits between 12.9 and 15.2 (range values) per cent of the cerebral cortex is occupied by nerve cell bodies (Fig. 2).
0
I
I
I
I
I
5
10
15
20
25
I 30
A YAGE
FIG. 2. The points
Changes indicate
300 lilA YSI
during growth in the proportional volume of the nerve cell bodies. the mean values; the small vertical lines the range values.
Dendrites are hardly countable at this age. The volume of the nerve cell bodies decreasesduring the first 5 days after birth from an average of 14.2 per cent to an average value of 7.5 per cent. This change may be the result of the fast decrease in cell density (28, 30). During later stagesof the development the volume occupied by the nerve cell bodies remains practically constant and in adult animals an average value of 6.7 per cent is found (Table 1, and Fig. 2). At 5 days of age the total volume occupied by the dendrites in the cortex is between 1.2 and 1.6 per cent. The volume of the dendrites increasessharply during the next 15 days (Tables 2 and 3, Figs. 3 and 4).
30 30 30 30 average
Grand
Grand
CORTEX
7.1 5.6 5.4 7.2 6.3
A B C D 6.7 6.4 7.0 6.8 6.7
7.1 6.8 5.9 7.2 6.8
A B C D
BY
Proportional volume
OCCUPIED
A B C D
Animal
OF
NERVE
CELL
TABLE
1
0.56 0.55 0.57 0.56
0.57 0.52 0.50 0.58
0.47 0.46 0.43 0.48
SD.8
BODIES
AND
Mean
CALCULATED
7.0 6.5 6.4 6.1
6.5 6.1 7.1 6.8
5.5 5.0 6.0 5.4
radius (PL)
THEIR
2.9 3.0 3.3 3.3 3.1
0.67 0.53 0.74 0.80
=
=
b S,D.
c SD.
dy.
--2‘2 12
“XT
m which
p is the percentage
and n the number
of hits.
of 0, 5, 15, 20, 25, 45,
3.3 2.8 2.3 3.2 2.9
0.73 0.84 0.69 0.82
Surface area per mm3 of cortex t x 107 $9 3.9 4.1 3.0 4.8 3.8
AREAS
0.41 0.34 0.42 0.38
S.D.c
SURFACE
a To avoid lengthy tables only the data of three “key” age groups are given. The data of the groups and 65 days of age are omitted. The range values for all age groups are given in the figures.
300 300 300 300 average Grand
10 10 10 10 average
Age (days)
PERCENTAGE
5
VOLUME
AND
SURFACE
AREA
OF
NEURONS
165
In the adult animals between 6.6 and 7.7 per cent of the cortex is occupied by apical dendrites and between 7.5 and 8.4 per cent by basal dendrites (and branches of the apical dendrites). In living tissue, however, this percentage of apical and basal dendrites may be much higher, as the smaller terminals of the basal dendrites and branches of the apical dendrites cannot be observed with light microscopy and probably may not be stained. Even at birth a number of neurons show apical dendrites; they can be observed in Golgi-Cox preparations as thin threads. Most striking however, is the growth of the basal dendritic plexuses after birth. The development of the basal dendritic plexuses seem to occur first in the deeper layers, as seen in the Cajal preparations. The tangential fibers appear first in layers V and VI, and then successively in the more superficial ones. To check the validity of the results obtained by the “hit” method the axes of eight hundred nerve cells were measured in the same preparations. The percentage of cortex occupied by the nerve cell bodies was calculated on the basis of these measurements and the previously determined packing density of the cells. Results are shown in Table 4 and are in close agreement with those shown in Table 1. The slightly different values in Table 4 may be the result of subjective errors in measuring the length of the axes of the nerve cell bodies. The difference in values obtained by the “hit” method and by axes measurements was also observed by Peters and Flexner (21) in studying the frontal cortex of the guinea pig. With a different method, the length and diameter of the apical dendrites of 1359 pyramidal cells, in a known volume of cerebral cortex, was determined (Schade and Van Backer, to be published). Consequently the proportional volume of the apical dendrites could be calculated. A value of 7.5 + 0.9 (SD.) was found which is in close concordance with the results obtained by the “hit” method. Comparison of the results obtained by the two different methods indicate that the “hit” method seems a reliable technique to determine the relative volume of cortex occupied by nerve cell constituents. The Surface Area of Cell Bodies and Dendrites per Unit Volume. In newborn animals an average surface area for the nerve cell bodies of 12.7 X lo7 p2 was calculated per 1 mm3 of cerebral cortex. The total surface area of the cell bodies per unit volume of cerebral cortex decreases very fast from 0 to 5 days after birth (Fig. 5). The surface area of the apical and basal dendrites increases markedly
30 30 30 30 average
Grand
Grand
300 300 300 300 Grand average
10 average
10
10 10
Age (days)
PERCENTAGE
A B C D
A B C D
A B C D
Animal
OF CORTEX
7.1 6.9 6.6 7.7 7.1
6.2 5.9 6.7 6.1 6.2
1.7 2.4 1.9 2.3 2.1
Proportional volume
OCCUPIED
BY
APICAL
0.57 0.57 0.55 0.60
0.54 0.53 0.56 0.54
0.24 0.28 0.25 0.27
SD.
TABLE 2 DENDRITES AND
Mean
0.94 0.97 0.83 0.93
0.88 0.86 0.93 0.84
0.71 0.64 0.69 0.73
(p)
CALCULATED
radius
THEIR
14.1 14.2
15.9 16.5 15.4
0.11
0.19 0.09
14.2 0.17
0.14 0.09
0.11
0.13
6.0 14.1 13.7 14.4 14.4
$ ?Ji El
5
1.
5.5 6.3 0.10 0.11
z
Surface area per mms of cortex (X 10’ P2) 4.8 7.5
AREA
0.11 0.09
S.D.
SURFACE
30 30 30 30 average
Grand
Grand
300 300 300 300 Grand average
10 10 10 10 average
Age (days)
PERCENTAGE
A B C D
A B C D
A B C D
Animal
OF CORTEX
7.9 8.4 8.1 7.5 8.0
6.9 8.3 6.7 7.5 7.3
1.9 2.3 2.7 1.8 2.2
Proportional volume
OCCUPIED
0.60 0.62 0.61 0.59
0.5 7 0.62 0.56 0.59
0.25 0.27 0.30 0.24
S.D.
TABLE 3 BY BASAL DENDRITES
0.3 1 0.45 0.44 0.38
0.39 0.43 0.40 0.41
0.24 0.32 0.28 0.31
(+)
CALCULATED
radius
THEIR
Mean
AND
0.12 0.09 0.08 0.11
0.09 0.08 0.09 0.10
0.08 0.06 0.09 0.10
S.D.
SURFACE
AREA
51.0 37.3 36.8 39.5 41.2
35.4 38.6 35.5 36.6 36.0
15.8 14.4 16.8 11.6 14.7
Surface area per mm3 of cortex tx 107 I.4
3 3 kY/ c/J
s
$ ?
F 8
2
5
2 m
s
168
SCHADI?
AND
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5 days after birth. The surface of the apical dendrites increases particularly from 10 to 15 days after birth (Fig. 6). The surface area of the basal dendrites and branches of the apical dendrites increases in a 2.5day period (5 to 30 days after birth) more than tenfold (Figs. 7 and 8). Table 5 shows calculations for the surface area of the average nerve cell body and dendritic plexus. The data were obtained by dividing the
0 0
I 5
I 10
I 15
I
20
I
25
I
30
A e6:
300 AGE (DAYSI
FIG. 3. Changes during growth in the proportional volume of the apical dendrites. The points indicate the mean values; the small vertical lines the range values. Apical and basal dendrites were measured together in preparations, 5 days of age. The dotted line indicates the interpolated value for the apical dendrites, if we consider an even distribution of apical and basal dendrites.
data of Tables 2 and 3 (percentage values) by the packing density of the cells in area 2-3 of the rabbit cortex. In this way a value for the surface area of an average neuron in a particular field of the rabbit cerebral cortex can be obtained. The results of the Table 5 show that the main part of the receptive zone of the neuron (perikaryon and dendrites) is formed by the surface area of the basal dendrites.
VOLUME
AND
SURFACE
Biochemical
AREA
OF NEURONS
169
Findings
Levels of GABA and GAD during Development of th Cerebral Cortex. Levels of GABA and GAD in area 2-3 of the developing rabbit cerebral cortex are compared in Fig. 9. A threefold increase in GABA levels was observed between the first and the twentieth days after birth. Approxi-
20 10 0
/ 0
I 5
I 10
I 15
FIG. 4. Changes during growth in The points indicate the mean value; and basal dendrites were measured dotted line indicates the interpolated even distribution of apical and basal
I
20
I
25
I
fi
30
AGE (DAYS) the proportional volume of the basal dendrites. the small vertical lines the range values. Apical together in preparations, 5 days of age. The value for the basal dendrites, if we consider an dendrites.
mately two-thirds of this increase was achieved in the first 10 days after birth. Adult GABA levels were achieved by 20 days. In contrast, GAD apoenzymeincreasedalmost ninefold in the first 20 days of life and did not attain adult levels until 30 days after birth. It is noteworthy that GAD increased fourfold between 10 and 20 days after birth, a period in which GABA levels rose only by about one-third. In all probability this difference indicates that GABA-a-ketoglutaric acid trans-
170
SCHADl?
AND
BAXTER
aminase (1, 3) also increased in activity during this period (this problem is under investigation at the moment). The effect of such changes would involve inevitably an increased turnover rate of GABA. TABLE PERCENTAGE
OF CEREBRAL
CALCULATED
ON THE
Volume Age (days)
Animal
300 300 300 300
A B C D
5 the 0 and
CORTEX BASIS
4 OCCUPIED
BY NERVE
OF AXES
average nerve body (~~1
1423 1318 1298 1411
BODIES
cell
prolate spheroids
spheres
CELL
MEASUREMENTS
1257 1128 1075 1287
Proportional volume (19
W)
8.25 7.64 7.52 8.18
7.29 6.54 6.23 7.46
This value is obtained by multiplying the packing density of the nerve cells and volume of the average nerve cell body (treated as a sphere). This value also obtained by multiplying the packing density of the nerve cells the volume of the average nerve cell body (treated as a prolate spheroid).
FIG.
as Fig.
5. 2.
Changes
during
growth
in surface
area
of nerve
cell bodies.
Same legend
VOLUME
AND
SURFACE
AREA
171
OF NEURONS
Changes in the Amino Acid Patterns during Development of the Cerebral Cortex. The .over-all changes in the amino acid patterns of area 2-3 during maturation from one to 30 days are shown in Fig. 10 and Table 6. Semiquantitative estimates of the amino acid levels presented by spots on the chromatograms are given in Table 6. In addition to VOLUME
TABLE 5 AND SURFACE AREA OF THE AVERAGE NERVE CELL AND THE AVERAGE DENDRITIC PLEXUS Volume
Age (days)
average nerve cell body (p3)
Animal
BODY
Surface average nerve cell body WY
average dendritic plexus w
area average dendritic plexus WY
1155 1103
2586
500
114OOa
300
A B
2637
517
8880
300
C
1206
2534
568
9090
300
D
1173
2620
568
9660
300
a On the basis of these values surface area could be calculated. C-1:16.0, D-1:17.0.
40
, 0
FIG. 6. Fig. 3.
I 5
I 10
1 15
Changes
during
growth
the ratio cell body surface area:dendritic This ratio is respectively for A-1:22.8,
I 20
in surface
2sI
30I
area of apical
plexus B-1:17.2,
/\ Yb:oo AGE fOA YSI
dendrites.
Same legend
as
172
SCHADI?
AND
BAXTER
TABLE CHANGES
Amino
IN AMINO
ACID
6
OF CORTICAL
CONTENT
Acid
(0 days)
AREA
2-3 DURING
Per cent of 30&y (IO days)
3s 28
y-Aminobutyric acid Glutamic Acid Aspartic acid Ethanolamine phosphate
DEVELOPMENT level (20 days)
85 73
46
85
198
210
100 85 100 112
GABA, the level of glutamic acid increased almost fourfold and the level of aspartic acid doubled during the first 30 days of life. In studies with whole brains of various speciesonly GABA was found to show progressiveincreaseswith development (24). Discussion
Surface area measurementsof neurons in the cerebral cortex may be of value to both the physiologist and the biochemist. For the physiologist
I 5 FIG.
as Fig.
7. Changes 4.
I 10
I 15
during
growth
I 20
I 25
in surface
I 30
area
Ir
of basal
Y-
300’ AGE (DAYS)
dendrites.
Same
legend
VOLUME
AND
SURFACE
AREA
OF NEURONS
173
the ssurfaces of the perikarya and the dendrites form the receptive IIart of the r neuron. Any interpretation of ion movements and potential diffc :rences must take into account the extent of these surfaces. The biochemist might find these measurements a basis for a comparison of the acti\ rity of
FIG . 8.
Photomicrographs of cerebral cortex preparations: A, lo-day-old Cajal-stained; bottom, gallocyanin-stained) ; B, 30-day-old animal (top, staine d; bottom, gallocyanin-stained). Note the decrease in packing density nerve cell bodies and the increase in density of the dendrites. Magnification (top,
animal Cajalof the 600 x.
174
SCHADk
AND
BAXTER
enzyme systems associated with cytoplasmic membranes and with the total surface area of nerve cell componentsper unit volume. Recently a number of investigators have reported values for the volume occupied by nerve cell bodies in the cerebral cortex. Using the Chalkley technique (5, 6)) Peters and Flexner (21) found this value to be about
1.00
l-
FIG. 9. A comparison of levels of GABA and GAD apoenzyme in area 2-3 of the developing rabbit brain cortex. Assay conditions are described in text. The small vertical lines indicate range values.
12 per cent in the frontal cortex of the guinea pig. By a modified Chalkley technique, Haug (12, 13) calculated the “gray cell coefficient” for the frontal cortex of the rabbit as 0.045 (i.e., 4.5 per cent of the frontal cortex is occupied by cell bodies). Values for the percentage of cortex occupied by cell bodies in a number of other mammalsrange from about 2 to 20 per cent (12, 13, 33). Sholl (34, 35) in his excellent studies of the organization in the cerebral cortex of the cat, reported the surprisingly high figure of 2.5 to 35 per
VOLUME
AND
SURFACE
AREA
OF NEURONS
175
cent for the volume occupied by nerve cell bodies in the visual cortex. However, his technique of preparing and staining the tissue did not prevent considerable swelling of the perikarya (37). Our estimates of the percentage of cortex which consists of cell body are generally in good agreement with those of Haug ( 12), but somewhat lower than those of Peters and Flexner (2 1). This discrepancy may be a reflection of a higher cell density in the frontal cortex of the guinea pig, as compared to the area investigated in the rabbit.
FIG. 10. Changes in the free amino acid pattern of area 2-3 cerebral cortex. Point of origin is marked by +, solvent systems (horizontal), lutidine-Hz0 (vertical).
of the developing were phenol-H20
Apart from a preliminary communication by Sholl (3.5), no other recent detailed studies of surface area measurements have been reported. Although Sholl’s absolute values are considerably higher than those reported here, his observation that the dendritic surface forms approximately 90 to 95 per cent of the receptive surface of the neuron, is in remarkably good agreement with our estimates. It was found (Table 5) that for the average neuron the dendritic surface contributed to 94 to 96 per cent of the receptive surface of the neuron. In attempting to correlate the biochemical composition of the cerebral cortex with histological parameters, a number of criteria might be used. For example, no relationship could be demonstrated in the cerebral cortex
176
SCHADk
AND
BAXTER
between the number of cell bodies per unit volume and levels of GABA and GAD (2). Those histological measurementswhich appeared to bear some kind of relationship to the biochemical constituents which were measuredare listed below: The greatest increase in the level of GABA in area 2-3 coincided in time with the sharpest decreasein the proportional volume of the nerve cell bodies and an increase in the proportional volume of the dendrites. The rapid increase of GABA levels also marked the beginning of growth of basal dendritic plexuses and branches of the apical dendrites. According to earlier studies it was also correlated with the appearance of Nissl bodies (24, 30). The time at which adult GABA levels were observed coincided with the morphological maturation of the cell body nucleus and the attainment of adult EEG patterns (28, 29). The levels of free or easily extractable glutamic and aspartic acids and GABA increasein a parallel manner while the level of ethanol amine phosphate in area 2-3 dropped precipitously after the first 10 days of life. In the present study the greatest increase of GAD activity coincided most closely with the fastest growth of the surface area of the dendrites. However a numerical correlation between this surface area and the activity of the apoenzyme could not be demonstrated. It might be anticipated that the activity of enzyme systems associated with cytoplasmic membranes and the cytoplasm of nerve cells will be correlated most closely with the measurementsof proportional surface area and volume. The activity of particulate bound enzyme systems such as those associatedwith mitochondria, might be expected to correlate more closely with the proportional surface area and volume of particulate structures. References 1. 2.
3. 4. 5.
BAXTER, C. F., and E. ROBERTS, The y-aminobutyric acid-a-ketoglutaric acid transaminase of beef brain. J. Biol. Chem. 233: 1135-1139, 1958. BAXTER, C. F., J. P. SHAD& and E. ROBERTS, Maturational changes in cerebral cortex. II. Levels of glutamic acid decarboxylase, y-aminobutyric acid and some related amino acids. In “Inhibition in the nervous system and GABA,” E. Roberts (ed.) 1960. (In press.) BESSMAN, S. P., J. ROSSEN, and E. C. LAYNE, y-Aminobutyric acid glutamic acid transamination in brain. /. Biol. Chem. 201: 385-391, 1953. CAJAL, S. RAM~N Y, “Degeneration and regeneration in the nervous system,” R. M. May (ed.), London. 1936. CHALKLEY, H. W., Method for the quantitative morphologic analysis of tissues. J. Nat. Cancer Inst. 4: 47-53, 1943.
VOLUME
6.
AND
SURFACE
AREA
OF NEURONS
177
H. W., A method for the measurement of average nuclear volumes of shape. Anat. Rec. 103: 433, 1949. EDSTR~M, J. E., Ribonucleic acid mass and concentration in individual nerve cells-a new method for quantitative determinations. Biockim. biophys. acta, Amt. 12: 361-386, 1953. EDSTR~M, J. E., and H. HYDEN, Ribonucleotide analysis of individual nerve cells. Nature, Lond. 174: 128, 1954. FLEXNER, L. B., E. L. BELKNAP, and J, B. FLEXNER, Cytochrome oxidase, succinic dehydrogenase and succinoxidase in the developing cerebral cortex and liver of the fetal guinea pig. J. CeZZuZ. Physiol. 42: 151-162, 1953. FLEXNER, L. B., and J. B. FLEXNER, Compounds of phosphorus in the developing cerebral cortex and liver of the fetal guinea pig. J. CeZZuZ. Physiol. 36:
CHALKLEY,
independent
7.
8. 9.
10.
351-368,
11. 12. 13. 14. 15.
16. 17. 18.
19.
20. 21. 22.
23.
24.
1950.
FLEXNER, L. B., and J. B. FLEXNER, The nucleic acids of the developing cerebral cortex and liver of the fetal guinea pig. J. Cellul. PhysioZ. 38: I-16, 1951. HAUG, H., Der Grauzellkoeffizient des Stirnhirnes der Mammalia in einer pbylogenetischen Betrachtung. Acta anat., BaseZ 19: 239-270, 1953. HAUG, H., Remarks on the determination and significance of the gray cell coefficient. J. Camp. New. 104: 473-492, 1956. JAKOBY, W. B., and E. M. SCOTT, Aldehyde oxidation. III. Succinic semialdehyde dehydrogenase. J. BioZ. Chew 234: 937-940, 1959. LOWRY, 0. H., The quantitative histochemistry of brain. Histological sampling. J. Histochem. Cytochem. 1: 420, 1953. LOWRY, 0. H., N. R. ROBERTS, and M. L. CHANG, The analysis of single cells. J. BioZ. Chews. 22: 97-107, 1956. LOWRY, 0. H., N. R. ROBERTS, and J, I. KAPPHARN, The fluorometric measurements of pyridine nucleotides. J. BioZ. Chem. 224: 1047-1064, 1957. LOWRY, 0. H., N. R. ROBERTS, K. Y. LEINER, M. L. WV, and A. L. FARR, The quantitative histochemistry of brain. I. Chemical methods. J. BioZ. Chem. 207: l-17, 1954. LOWRY, 0. H., N. R. ROBERTS, K. Y. LEINER, M. L. WV, A. L. FARR, and R. W. ALBERS, The quantitative histochemistry of brain. III. Ammon’s horn. J. BioZ. Chew 207: 39-49, 1954. LOWRY, 0. H., N. R. ROBERTS, and C. LEWIS, The quantitative histochemistry of the retina. J. Biol. Chem. 220: 879-892, 1956. PETERS, V. B., and L. B. FLEXNER, Quantitative morphologic studies on developing cerebral cortex of the fetal guinea pig. Al%. J. Anat. 86: 133-157, 1950. POPE, A., and H. H. HESS, Cytochemistry of neurones and neuroglia. In “Metabolism of the nervcus system, ” D. Richter, (ed.), New York, Pergamon Press, 1957 (ref. pp. 72-86). POPE, A., Studies on the biochemical architecture of the isocortex of man. In: “Structure and function of the cerebral cortex,” (D. B. Tower and J. P. SchadC, (eds.) Amsterdam, Elsevier, 1960. (In press.) ROBERTS, E., P. J. HARMAN, and S. FRANKEL, y-Aminobutyric acid content and glutamic decarboxylase activity in developing mouse brain, PYOC. Sot. Exp. Biol., N. Y. 78: 799-808, 1951.
178 25.
26.
27. 28. 29. 30. 31.
32. 33. 34. 35. 36. 37. 38.
SCHADk
AND
BAXTER
ROBINS, E., N.R. ROBERTS, K. M. EYDT, 0. H. LOWRY, and D. E. SMITH, Microdetermination of a-keto-acids with special reference to malic, lactic and glutamic dehydrogenases in brain. J. Sol. Chem. 218: 897-909, 1956. ROBINS, E., D. E. SMITH, and E. R. MCCAMAN, Microdetermination of purine nucleoside phosphorylase activity in brain and its distribution within the monkey cerebellum. J. Biol. Chem. 204: 927-937, 1953. SCHAD~, J. P., Development of EEG and spreading depression in the rabbit. Electroencephalography, Montreal 10: 359-360, 1958. SCHADI?, J. P., A histological and histochemical analysis of the developing cerebral cortex. Proc. R. Acad. SC. Amst. 62: 445-460, 1959. SCHADB, J. P., Maturational aspects of EEG and spreading depression in rabbit. J. Neurophysiol. 22: 245-257, 1959. SCHAD$, J. P., Differential growth of nerve cells in cerebral cortex. Growth, Phila. 23: 159-168, 1959. SCHAD~, J. P., and C. F. BAXTER, Maturational changes in cerebral cortex. I. Volume and surface determinations of nerve cell components. In “Inhibition in the nervous system and GABA,” E. Roberts (ed.), 1960. (In press.) SCHAD~, J. P., and I. J. WEILER, Electroencephalographic patterns of the goldfish (Carassius Auratus L.). J. Exp. Biol. 36: 435-452, 1959. SHARIFF, G. A., Cell counts in the primate cerebral cortex. J. Camp. New. 98: 381-400, 1953. SHOLL, D. A., Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat., Lond. 87: 387-406, 1953. SHOLL, D. A., The surface area of cortical neurons. J. Anat., Lond. 89: 571572, 1955. VAN DER Loos, H., Dendro-dendritische verbindingen in de schors der grote hersenen. Thesis, University of Amsterdam, 1959. VAN HARREVELD, A., Changes in volume of cortical neuronal elements during asphyxiation. Am. J. Physiol. 191: 233-242, 1957. VAN HARREVELD, A., and J. P. SCHAD~, Chloride movements in cerebral cortex after circulatory arrest and during spreading depression. J. Cellul. Physiol. 1959. (In press.)
NEUROLOGY
2, 158-178
(1960)
Changes during Growth in the Volume and Surface Area of Cortical Neurons in the Rabbit J. P. Division
of Biology,
SCHAD~
California
and Central Institute
for
Received
C. F.
AND
Institute Brain
BAXTER~
of Technology, Pasadena, California, Research, Amsterdam, Holland
January
18, 1960
A method was developed to measure the surface area of cortical nerve cells. It consists of measuring the proportional volume of the constituents of cortical neurons and their diameter. A modification of the method of Chalkley is developed to determine the volume and surface area of the dendrites. This measurement is accomplished by cementing four tungsten microelectrodes to the diaphragm of the ocular. An attempt has been made to find parallels between quantitative histological measurements and biochemical composition in a specific area of the developing rabbit brain cortex. The proportional volume and surface area of the perikaryal membranes decreased during the first 5 days after birth and then remained practically constant during development. The proportional volume and surface area of the apical and basal dendrites increased markedly from 5 to 30 days after birth. The change in the levels of glutamic acid decarboxylase, y-aminobutyric acid, and some other amino acids have been measured as a function of age. The largest changes in glutamic acid decarboxylase coincided with the most rapid phase in the development of the surface area of the dendrites. Surface area measurements of neurons in the cerebral cortex may be of value to both the physiologist and the biochemist. For the physiologist the surfaces of the perikarya and dendrites form the receptive part of the neuron. Any interpretation of ion movements and potential differences must take into account the extent of these surfaces. The biochemist might find these measurements a basis for a comparison of enzyme systems associated with cytoplasmic membranes. Introduction
Studies which attempt to correlate chemical composition with histological structure are hampered by the complexity of the neuronal elements 1 This investigation was supported in part by Research Grants from the National Institute of Neurological Diseases and Blindness (B 340) and from the National Science Foundation (G 4029). The authors are indebted to Dr. A. Van Harreveld and Dr. E. Roberts for their constructive criticism, and to Mrs. Ruth Estey and Mrs. Nancy Kennedy for histological preparations. The address of Dr. Baxter is Department of Biochemistry, Medical Research Institute, City of Hope Medical Center, Duarte, California, U.S.A.; that of Dr. Schade, Central Institute for Brain Research, Mauritskade, Amsterdam, Holland. 158
VOLUME
AND
SURFACE
AREA
OF NEURONS
159
in this area. In the cerebral cortex processes of the nerve cells are exceedingly long. The basal dendrites in the rabbit attain a length of 250 to 300 p (36). In the cat basal dendrites of 600 p (34) and apical dendrites as long as 1500 p have been reported. The cytoplasmic volume and surface area may be many times greater than that of the nerve cell bodies. Fundamental data about the chemical composition of the nerve cell body have been obtained by applying microchemical techniques to single isolated nerve cell bodies (7, 8, 15-20). However, the application of these micromethods to whole nerve cells can yield only approximate results, since in practice the isolation (for chemical analysis) of all the dendritic processes associated with a single nerve cell body inevitably is incomplete. The chemical architectonics of a number of brain structures in several species have been investigated using these microtechniques (16-20, 22, 23, 25, 26) and in some cases, yielded results which indicated that the activity of enzymes, such as acetylcholine esterase and Ca-adenosinetriphosphatase was more closely associated with the cell processes than with the cell bodies (22, 23). An alternative method of correlating chemical composition with histological structure was adopted by Flexner and his associates (9, 10, 11, 2 1) in their classical studies on biochemical and physiological differentiation during morphogenesis. They investigated the frontal cortex of the guinea pig (9, 10, 11, 2 1) paying special attention to the enzyme development in conjunction with the morphological maturation of the cerebral cortex. In this study an attempt has been made to find parallels between quantitative histological measurements and biochemical composition in a specific area of the developing rabbit brain cortex. The levels of y-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were chosen as biochemical parameters because of the unique presence of GAD and GABA in the central nervous system. Particular attention has been focused upon the development of total volume and total surface area of neuronal constituents in the cerebral cortex. Material
and
Methods
Histological Methods. Rabbits were used exclusively. To measure the dimensions of nerve cells and dendrites, histological preparations had to be made in which the fluid distribution between neuronal and extraneuronal compartments of the cerebral cortex was represented as faithfully as possible.
160
SCHADk
AND
BAXTER
The dorsal aspect of both hemispheres was widely exposed under ether narcosis leaving a small bony edge to protect the sagittal sinus. A skin cup was formed by sewing the skin surrounding the wound to a steel ring. The preparations were then immobilized with Intocostrin (5 units/kg/hr) and artificial respiration was applied with a Palmer pump. The state of activity in the cerebral cortex was checked with a four channel Offner electroencephalograph. Isopentane cooled to its freezing point (approximately -160” C) with liquid nitrogen was poured onto the cortex at a time when cortical activity was normal. The animal was decapitated and the head immersed in cold isopentane (-70 to --SO” C). The isopentane was rinsed off with absolute alcohol at the same temperature. The head was then placed in a beaker with cold absolute alcohol (-40” C) and stored at -25” C for 10 days in a refrigerator. After fixation, a slice of
FIG. 1. Dorsal view of the cerebral cortex of the rabbit. The longitudinal sulcus is indicated. Left hemisphere: the two vertical lines indicate the slice which was taken for histological examination. The arrow indicates the area in which measurements were made. The square (area Z-3) indicates the portion of cerebral cortex which was excised for biochemical measurements ; 1, frontal cortex; 2, motor cortex; 3, sensory cortex; 4, retrosplenial area; 5 and 7, visual cortex; 6, auditory cortex.
cortex was removed from an area as indicated in Fig. 1. The slice was put in methylbenzoate, embedded in paraffin, sectioned, and stained. For the study of cell bodies a gallocyanin stain was used and for dendrites method B of Ram&-r y Cajal (4). 1Measurements. The method employed for the determination of cell density of the cortices has been described (28).
VOLUME
AND
SURFACE
AREA
OF NEURONS
161
The proportional volume of nerve cell bodies (total volume per cubic millimeter of cerebral cortex) and of apical and basal dendrites were determined according to a modification of the method of Chalkley (5, 6) with the corrections of Haug (12, 13). According to this method the relative volume occupied by a specific constituent of a tissue is proportional to the number of times a point, placed at random throughout the tissue, will hit the constituent of the tissue. This measurement is accomplished by cementing four tungsten microelectrodes with points of 1 to 2 u to the diaphragm of the ocular (32). The wires appear in the focal plane. A section of cortex is moved at random under the objective of the microscope. A contact of the image of the point of the microelectrode with the clearly observed image of the tissue constituent is counted as a hit. The number of hits made in this way on nerve cell bodies, apical, and basal dendrites are recorded. Branches of apical dendrites are counted in the slides as basal dendrites, because they are very difficult to distinguish from basal dendrites when viewed under oil immersion. Observations were made using a 20 X ocular and a 2-mm oil immersion objective. A camera lucida was used to measure the diameter of the cell bodies and the dendrites. The magnification of this system was 3000x. Whenever a constituent was hit the diameter of this element was measured on the camera lucida screen. Cell bodies and dendrites were subdivided on the basis of measured diameters, each subdivision representing a difference of l/3 u (equivalent to 1 mm on the camera lucida screen). Twelve histological sections (25 p thick) from each animal were investigated. Four series of animals (littermates) were investigated. After measuring the standard deviation of the first series of samples it was found that 2000 to 3000 hits per slice of cortex gave a sufficient degree of accuracy. Cahdations. The total surface area of cell bodies and dendrites per unit volume was calculated as follows: Nerve cell bodies, 3v A=[II
i-
in which A = the surface area per cubic millimeter of cerebral cortex, V = the percentage of cortex which is nerve cell body, measured by the “hit” method, and P = the mean radius of the cell bodies. In this formula the cell bodies are treated as spheres. As an independent check, individual nerve cell bodies were measured and the surface was calculated by treating the cell bodies as prolate spheroids by using the formula
162
SCHADI?
AND
BAXTER
a6 sin-l e i21 e in which A z the surface area of the individual cell body, a = half of the major axis of the nerve cell body, b = half of the minor axis, and A=2?rb2+2r----
e = \/ 1 Dendrites,
(b2 / a2). A=-
2v
[31 iin which A = the surface area of the dendrites per cubic millimeter of cerebral cortex, V = the percentage which is occupied by the apical or basal dendrites (branches of apical dendrites included) measured by the “hit” method i- = the mean radius of the apical or basal dendrites. In this formula the surface is calculated as the curved surface of a cylinder. Biochemical Methods. A section of pallium (cerebral cortex with underlying white matter) corresponding to the dotted area in Fig. 1 was excised from the brain of normal animals immediately after decapitation. Rabbits 30 days or older were lightly etherized. Excised areas were weighed and processed as follows: For the determination of GABA, samples were dropped into cold 75 per cent ethanol and usually left for a few days before extraction. For the determination of GAD, samples were homogenized in water and two aliquots placed into incubation tubes containing ice cold glutamic acid, pyridoxal phosphate, and phosphate buffer pH 6.3. One aliquot was deproteinized immediately with ethanol. The second aliquot was incubated for 30 min at 37” C and then deproteinized with ethanol. The difference in GABA content of the two samples was an index of GAD activity. The method for the extraction and processing of tissue and homogenate samples has been described ( 1). The total GABA in 40-mg equivalents of fresh tissue and GAD in 5 mg of tissue were measured using an enzymatic technique (14) adopted for GABA and GAD in tissue preparations (1). Two dimensional chromatographic assays were made using the same tissue extracts as were used for the enzymatic GABA assay. Relative amounts of glutamic acid, aspartic acid, and ethanolamine phosphate were determined semiquantitatively. Histological
Findings
The results are given in Tables 1, 2, and 3 and in Figs. 2 through 7, inclusive. Examples of the counts (average values with standard devia-
VOLUME
AND
SURFACE
AREA
163
OF NEURONS
tions) and the measurements of the diameter of the dendrites are shown; range values of four series of experiments are plotted in graphs against age. Percentage of Cortex Occupied by Nerve Cell Body, Apical, and Basal Dendrites. In newborn rabbits between 12.9 and 15.2 (range values) per cent of the cerebral cortex is occupied by nerve cell bodies (Fig. 2).
0
I
I
I
I
I
5
10
15
20
25
I 30
A YAGE
FIG. 2. The points
Changes indicate
300 lilA YSI
during growth in the proportional volume of the nerve cell bodies. the mean values; the small vertical lines the range values.
Dendrites are hardly countable at this age. The volume of the nerve cell bodies decreasesduring the first 5 days after birth from an average of 14.2 per cent to an average value of 7.5 per cent. This change may be the result of the fast decrease in cell density (28, 30). During later stagesof the development the volume occupied by the nerve cell bodies remains practically constant and in adult animals an average value of 6.7 per cent is found (Table 1, and Fig. 2). At 5 days of age the total volume occupied by the dendrites in the cortex is between 1.2 and 1.6 per cent. The volume of the dendrites increasessharply during the next 15 days (Tables 2 and 3, Figs. 3 and 4).
30 30 30 30 average
Grand
Grand
CORTEX
7.1 5.6 5.4 7.2 6.3
A B C D 6.7 6.4 7.0 6.8 6.7
7.1 6.8 5.9 7.2 6.8
A B C D
BY
Proportional volume
OCCUPIED
A B C D
Animal
OF
NERVE
CELL
TABLE
1
0.56 0.55 0.57 0.56
0.57 0.52 0.50 0.58
0.47 0.46 0.43 0.48
SD.8
BODIES
AND
Mean
CALCULATED
7.0 6.5 6.4 6.1
6.5 6.1 7.1 6.8
5.5 5.0 6.0 5.4
radius (PL)
THEIR
2.9 3.0 3.3 3.3 3.1
0.67 0.53 0.74 0.80
=
=
b S,D.
c SD.
dy.
--2‘2 12
“XT
m which
p is the percentage
and n the number
of hits.
of 0, 5, 15, 20, 25, 45,
3.3 2.8 2.3 3.2 2.9
0.73 0.84 0.69 0.82
Surface area per mm3 of cortex t x 107 $9 3.9 4.1 3.0 4.8 3.8
AREAS
0.41 0.34 0.42 0.38
S.D.c
SURFACE
a To avoid lengthy tables only the data of three “key” age groups are given. The data of the groups and 65 days of age are omitted. The range values for all age groups are given in the figures.
300 300 300 300 average Grand
10 10 10 10 average
Age (days)
PERCENTAGE
5
VOLUME
AND
SURFACE
AREA
OF
NEURONS
165
In the adult animals between 6.6 and 7.7 per cent of the cortex is occupied by apical dendrites and between 7.5 and 8.4 per cent by basal dendrites (and branches of the apical dendrites). In living tissue, however, this percentage of apical and basal dendrites may be much higher, as the smaller terminals of the basal dendrites and branches of the apical dendrites cannot be observed with light microscopy and probably may not be stained. Even at birth a number of neurons show apical dendrites; they can be observed in Golgi-Cox preparations as thin threads. Most striking however, is the growth of the basal dendritic plexuses after birth. The development of the basal dendritic plexuses seem to occur first in the deeper layers, as seen in the Cajal preparations. The tangential fibers appear first in layers V and VI, and then successively in the more superficial ones. To check the validity of the results obtained by the “hit” method the axes of eight hundred nerve cells were measured in the same preparations. The percentage of cortex occupied by the nerve cell bodies was calculated on the basis of these measurements and the previously determined packing density of the cells. Results are shown in Table 4 and are in close agreement with those shown in Table 1. The slightly different values in Table 4 may be the result of subjective errors in measuring the length of the axes of the nerve cell bodies. The difference in values obtained by the “hit” method and by axes measurements was also observed by Peters and Flexner (21) in studying the frontal cortex of the guinea pig. With a different method, the length and diameter of the apical dendrites of 1359 pyramidal cells, in a known volume of cerebral cortex, was determined (Schade and Van Backer, to be published). Consequently the proportional volume of the apical dendrites could be calculated. A value of 7.5 + 0.9 (SD.) was found which is in close concordance with the results obtained by the “hit” method. Comparison of the results obtained by the two different methods indicate that the “hit” method seems a reliable technique to determine the relative volume of cortex occupied by nerve cell constituents. The Surface Area of Cell Bodies and Dendrites per Unit Volume. In newborn animals an average surface area for the nerve cell bodies of 12.7 X lo7 p2 was calculated per 1 mm3 of cerebral cortex. The total surface area of the cell bodies per unit volume of cerebral cortex decreases very fast from 0 to 5 days after birth (Fig. 5). The surface area of the apical and basal dendrites increases markedly
30 30 30 30 average
Grand
Grand
300 300 300 300 Grand average
10 average
10
10 10
Age (days)
PERCENTAGE
A B C D
A B C D
A B C D
Animal
OF CORTEX
7.1 6.9 6.6 7.7 7.1
6.2 5.9 6.7 6.1 6.2
1.7 2.4 1.9 2.3 2.1
Proportional volume
OCCUPIED
BY
APICAL
0.57 0.57 0.55 0.60
0.54 0.53 0.56 0.54
0.24 0.28 0.25 0.27
SD.
TABLE 2 DENDRITES AND
Mean
0.94 0.97 0.83 0.93
0.88 0.86 0.93 0.84
0.71 0.64 0.69 0.73
(p)
CALCULATED
radius
THEIR
14.1 14.2
15.9 16.5 15.4
0.11
0.19 0.09
14.2 0.17
0.14 0.09
0.11
0.13
6.0 14.1 13.7 14.4 14.4
$ ?Ji El
5
1.
5.5 6.3 0.10 0.11
z
Surface area per mms of cortex (X 10’ P2) 4.8 7.5
AREA
0.11 0.09
S.D.
SURFACE
30 30 30 30 average
Grand
Grand
300 300 300 300 Grand average
10 10 10 10 average
Age (days)
PERCENTAGE
A B C D
A B C D
A B C D
Animal
OF CORTEX
7.9 8.4 8.1 7.5 8.0
6.9 8.3 6.7 7.5 7.3
1.9 2.3 2.7 1.8 2.2
Proportional volume
OCCUPIED
0.60 0.62 0.61 0.59
0.5 7 0.62 0.56 0.59
0.25 0.27 0.30 0.24
S.D.
TABLE 3 BY BASAL DENDRITES
0.3 1 0.45 0.44 0.38
0.39 0.43 0.40 0.41
0.24 0.32 0.28 0.31
(+)
CALCULATED
radius
THEIR
Mean
AND
0.12 0.09 0.08 0.11
0.09 0.08 0.09 0.10
0.08 0.06 0.09 0.10
S.D.
SURFACE
AREA
51.0 37.3 36.8 39.5 41.2
35.4 38.6 35.5 36.6 36.0
15.8 14.4 16.8 11.6 14.7
Surface area per mm3 of cortex tx 107 I.4
3 3 kY/ c/J
s
$ ?
F 8
2
5
2 m
s
168
SCHADI?
AND
BAXTER
5 days after birth. The surface of the apical dendrites increases particularly from 10 to 15 days after birth (Fig. 6). The surface area of the basal dendrites and branches of the apical dendrites increases in a 2.5day period (5 to 30 days after birth) more than tenfold (Figs. 7 and 8). Table 5 shows calculations for the surface area of the average nerve cell body and dendritic plexus. The data were obtained by dividing the
0 0
I 5
I 10
I 15
I
20
I
25
I
30
A e6:
300 AGE (DAYSI
FIG. 3. Changes during growth in the proportional volume of the apical dendrites. The points indicate the mean values; the small vertical lines the range values. Apical and basal dendrites were measured together in preparations, 5 days of age. The dotted line indicates the interpolated value for the apical dendrites, if we consider an even distribution of apical and basal dendrites.
data of Tables 2 and 3 (percentage values) by the packing density of the cells in area 2-3 of the rabbit cortex. In this way a value for the surface area of an average neuron in a particular field of the rabbit cerebral cortex can be obtained. The results of the Table 5 show that the main part of the receptive zone of the neuron (perikaryon and dendrites) is formed by the surface area of the basal dendrites.
VOLUME
AND
SURFACE
Biochemical
AREA
OF NEURONS
169
Findings
Levels of GABA and GAD during Development of th Cerebral Cortex. Levels of GABA and GAD in area 2-3 of the developing rabbit cerebral cortex are compared in Fig. 9. A threefold increase in GABA levels was observed between the first and the twentieth days after birth. Approxi-
20 10 0
/ 0
I 5
I 10
I 15
FIG. 4. Changes during growth in The points indicate the mean value; and basal dendrites were measured dotted line indicates the interpolated even distribution of apical and basal
I
20
I
25
I
fi
30
AGE (DAYS) the proportional volume of the basal dendrites. the small vertical lines the range values. Apical together in preparations, 5 days of age. The value for the basal dendrites, if we consider an dendrites.
mately two-thirds of this increase was achieved in the first 10 days after birth. Adult GABA levels were achieved by 20 days. In contrast, GAD apoenzymeincreasedalmost ninefold in the first 20 days of life and did not attain adult levels until 30 days after birth. It is noteworthy that GAD increased fourfold between 10 and 20 days after birth, a period in which GABA levels rose only by about one-third. In all probability this difference indicates that GABA-a-ketoglutaric acid trans-
170
SCHADl?
AND
BAXTER
aminase (1, 3) also increased in activity during this period (this problem is under investigation at the moment). The effect of such changes would involve inevitably an increased turnover rate of GABA. TABLE PERCENTAGE
OF CEREBRAL
CALCULATED
ON THE
Volume Age (days)
Animal
300 300 300 300
A B C D
5 the 0 and
CORTEX BASIS
4 OCCUPIED
BY NERVE
OF AXES
average nerve body (~~1
1423 1318 1298 1411
BODIES
cell
prolate spheroids
spheres
CELL
MEASUREMENTS
1257 1128 1075 1287
Proportional volume (19
W)
8.25 7.64 7.52 8.18
7.29 6.54 6.23 7.46
This value is obtained by multiplying the packing density of the nerve cells and volume of the average nerve cell body (treated as a sphere). This value also obtained by multiplying the packing density of the nerve cells the volume of the average nerve cell body (treated as a prolate spheroid).
FIG.
as Fig.
5. 2.
Changes
during
growth
in surface
area
of nerve
cell bodies.
Same legend
VOLUME
AND
SURFACE
AREA
171
OF NEURONS
Changes in the Amino Acid Patterns during Development of the Cerebral Cortex. The .over-all changes in the amino acid patterns of area 2-3 during maturation from one to 30 days are shown in Fig. 10 and Table 6. Semiquantitative estimates of the amino acid levels presented by spots on the chromatograms are given in Table 6. In addition to VOLUME
TABLE 5 AND SURFACE AREA OF THE AVERAGE NERVE CELL AND THE AVERAGE DENDRITIC PLEXUS Volume
Age (days)
average nerve cell body (p3)
Animal
BODY
Surface average nerve cell body WY
average dendritic plexus w
area average dendritic plexus WY
1155 1103
2586
500
114OOa
300
A B
2637
517
8880
300
C
1206
2534
568
9090
300
D
1173
2620
568
9660
300
a On the basis of these values surface area could be calculated. C-1:16.0, D-1:17.0.
40
, 0
FIG. 6. Fig. 3.
I 5
I 10
1 15
Changes
during
growth
the ratio cell body surface area:dendritic This ratio is respectively for A-1:22.8,
I 20
in surface
2sI
30I
area of apical
plexus B-1:17.2,
/\ Yb:oo AGE fOA YSI
dendrites.
Same legend
as
172
SCHADI?
AND
BAXTER
TABLE CHANGES
Amino
IN AMINO
ACID
6
OF CORTICAL
CONTENT
Acid
(0 days)
AREA
2-3 DURING
Per cent of 30&y (IO days)
3s 28
y-Aminobutyric acid Glutamic Acid Aspartic acid Ethanolamine phosphate
DEVELOPMENT level (20 days)
85 73
46
85
198
210
100 85 100 112
GABA, the level of glutamic acid increased almost fourfold and the level of aspartic acid doubled during the first 30 days of life. In studies with whole brains of various speciesonly GABA was found to show progressiveincreaseswith development (24). Discussion
Surface area measurementsof neurons in the cerebral cortex may be of value to both the physiologist and the biochemist. For the physiologist
I 5 FIG.
as Fig.
7. Changes 4.
I 10
I 15
during
growth
I 20
I 25
in surface
I 30
area
Ir
of basal
Y-
300’ AGE (DAYS)
dendrites.
Same
legend
VOLUME
AND
SURFACE
AREA
OF NEURONS
173
the ssurfaces of the perikarya and the dendrites form the receptive IIart of the r neuron. Any interpretation of ion movements and potential diffc :rences must take into account the extent of these surfaces. The biochemist might find these measurements a basis for a comparison of the acti\ rity of
FIG . 8.
Photomicrographs of cerebral cortex preparations: A, lo-day-old Cajal-stained; bottom, gallocyanin-stained) ; B, 30-day-old animal (top, staine d; bottom, gallocyanin-stained). Note the decrease in packing density nerve cell bodies and the increase in density of the dendrites. Magnification (top,
animal Cajalof the 600 x.
174
SCHADk
AND
BAXTER
enzyme systems associated with cytoplasmic membranes and with the total surface area of nerve cell componentsper unit volume. Recently a number of investigators have reported values for the volume occupied by nerve cell bodies in the cerebral cortex. Using the Chalkley technique (5, 6)) Peters and Flexner (21) found this value to be about
1.00
l-
FIG. 9. A comparison of levels of GABA and GAD apoenzyme in area 2-3 of the developing rabbit brain cortex. Assay conditions are described in text. The small vertical lines indicate range values.
12 per cent in the frontal cortex of the guinea pig. By a modified Chalkley technique, Haug (12, 13) calculated the “gray cell coefficient” for the frontal cortex of the rabbit as 0.045 (i.e., 4.5 per cent of the frontal cortex is occupied by cell bodies). Values for the percentage of cortex occupied by cell bodies in a number of other mammalsrange from about 2 to 20 per cent (12, 13, 33). Sholl (34, 35) in his excellent studies of the organization in the cerebral cortex of the cat, reported the surprisingly high figure of 2.5 to 35 per
VOLUME
AND
SURFACE
AREA
OF NEURONS
175
cent for the volume occupied by nerve cell bodies in the visual cortex. However, his technique of preparing and staining the tissue did not prevent considerable swelling of the perikarya (37). Our estimates of the percentage of cortex which consists of cell body are generally in good agreement with those of Haug ( 12), but somewhat lower than those of Peters and Flexner (2 1). This discrepancy may be a reflection of a higher cell density in the frontal cortex of the guinea pig, as compared to the area investigated in the rabbit.
FIG. 10. Changes in the free amino acid pattern of area 2-3 cerebral cortex. Point of origin is marked by +, solvent systems (horizontal), lutidine-Hz0 (vertical).
of the developing were phenol-H20
Apart from a preliminary communication by Sholl (3.5), no other recent detailed studies of surface area measurements have been reported. Although Sholl’s absolute values are considerably higher than those reported here, his observation that the dendritic surface forms approximately 90 to 95 per cent of the receptive surface of the neuron, is in remarkably good agreement with our estimates. It was found (Table 5) that for the average neuron the dendritic surface contributed to 94 to 96 per cent of the receptive surface of the neuron. In attempting to correlate the biochemical composition of the cerebral cortex with histological parameters, a number of criteria might be used. For example, no relationship could be demonstrated in the cerebral cortex
176
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between the number of cell bodies per unit volume and levels of GABA and GAD (2). Those histological measurementswhich appeared to bear some kind of relationship to the biochemical constituents which were measuredare listed below: The greatest increase in the level of GABA in area 2-3 coincided in time with the sharpest decreasein the proportional volume of the nerve cell bodies and an increase in the proportional volume of the dendrites. The rapid increase of GABA levels also marked the beginning of growth of basal dendritic plexuses and branches of the apical dendrites. According to earlier studies it was also correlated with the appearance of Nissl bodies (24, 30). The time at which adult GABA levels were observed coincided with the morphological maturation of the cell body nucleus and the attainment of adult EEG patterns (28, 29). The levels of free or easily extractable glutamic and aspartic acids and GABA increasein a parallel manner while the level of ethanol amine phosphate in area 2-3 dropped precipitously after the first 10 days of life. In the present study the greatest increase of GAD activity coincided most closely with the fastest growth of the surface area of the dendrites. However a numerical correlation between this surface area and the activity of the apoenzyme could not be demonstrated. It might be anticipated that the activity of enzyme systems associated with cytoplasmic membranes and the cytoplasm of nerve cells will be correlated most closely with the measurementsof proportional surface area and volume. The activity of particulate bound enzyme systems such as those associatedwith mitochondria, might be expected to correlate more closely with the proportional surface area and volume of particulate structures. References 1. 2.
3. 4. 5.
BAXTER, C. F., and E. ROBERTS, The y-aminobutyric acid-a-ketoglutaric acid transaminase of beef brain. J. Biol. Chem. 233: 1135-1139, 1958. BAXTER, C. F., J. P. SHAD& and E. ROBERTS, Maturational changes in cerebral cortex. II. Levels of glutamic acid decarboxylase, y-aminobutyric acid and some related amino acids. In “Inhibition in the nervous system and GABA,” E. Roberts (ed.) 1960. (In press.) BESSMAN, S. P., J. ROSSEN, and E. C. LAYNE, y-Aminobutyric acid glutamic acid transamination in brain. /. Biol. Chem. 201: 385-391, 1953. CAJAL, S. RAM~N Y, “Degeneration and regeneration in the nervous system,” R. M. May (ed.), London. 1936. CHALKLEY, H. W., Method for the quantitative morphologic analysis of tissues. J. Nat. Cancer Inst. 4: 47-53, 1943.
VOLUME
6.
AND
SURFACE
AREA
OF NEURONS
177
H. W., A method for the measurement of average nuclear volumes of shape. Anat. Rec. 103: 433, 1949. EDSTR~M, J. E., Ribonucleic acid mass and concentration in individual nerve cells-a new method for quantitative determinations. Biockim. biophys. acta, Amt. 12: 361-386, 1953. EDSTR~M, J. E., and H. HYDEN, Ribonucleotide analysis of individual nerve cells. Nature, Lond. 174: 128, 1954. FLEXNER, L. B., E. L. BELKNAP, and J, B. FLEXNER, Cytochrome oxidase, succinic dehydrogenase and succinoxidase in the developing cerebral cortex and liver of the fetal guinea pig. J. CeZZuZ. Physiol. 42: 151-162, 1953. FLEXNER, L. B., and J. B. FLEXNER, Compounds of phosphorus in the developing cerebral cortex and liver of the fetal guinea pig. J. CeZZuZ. Physiol. 36:
CHALKLEY,
independent
7.
8. 9.
10.
351-368,
11. 12. 13. 14. 15.
16. 17. 18.
19.
20. 21. 22.
23.
24.
1950.
FLEXNER, L. B., and J. B. FLEXNER, The nucleic acids of the developing cerebral cortex and liver of the fetal guinea pig. J. Cellul. PhysioZ. 38: I-16, 1951. HAUG, H., Der Grauzellkoeffizient des Stirnhirnes der Mammalia in einer pbylogenetischen Betrachtung. Acta anat., BaseZ 19: 239-270, 1953. HAUG, H., Remarks on the determination and significance of the gray cell coefficient. J. Camp. New. 104: 473-492, 1956. JAKOBY, W. B., and E. M. SCOTT, Aldehyde oxidation. III. Succinic semialdehyde dehydrogenase. J. BioZ. Chew 234: 937-940, 1959. LOWRY, 0. H., The quantitative histochemistry of brain. Histological sampling. J. Histochem. Cytochem. 1: 420, 1953. LOWRY, 0. H., N. R. ROBERTS, and M. L. CHANG, The analysis of single cells. J. BioZ. Chews. 22: 97-107, 1956. LOWRY, 0. H., N. R. ROBERTS, and J, I. KAPPHARN, The fluorometric measurements of pyridine nucleotides. J. BioZ. Chem. 224: 1047-1064, 1957. LOWRY, 0. H., N. R. ROBERTS, K. Y. LEINER, M. L. WV, and A. L. FARR, The quantitative histochemistry of brain. I. Chemical methods. J. BioZ. Chem. 207: l-17, 1954. LOWRY, 0. H., N. R. ROBERTS, K. Y. LEINER, M. L. WV, A. L. FARR, and R. W. ALBERS, The quantitative histochemistry of brain. III. Ammon’s horn. J. BioZ. Chew 207: 39-49, 1954. LOWRY, 0. H., N. R. ROBERTS, and C. LEWIS, The quantitative histochemistry of the retina. J. Biol. Chem. 220: 879-892, 1956. PETERS, V. B., and L. B. FLEXNER, Quantitative morphologic studies on developing cerebral cortex of the fetal guinea pig. Al%. J. Anat. 86: 133-157, 1950. POPE, A., and H. H. HESS, Cytochemistry of neurones and neuroglia. In “Metabolism of the nervcus system, ” D. Richter, (ed.), New York, Pergamon Press, 1957 (ref. pp. 72-86). POPE, A., Studies on the biochemical architecture of the isocortex of man. In: “Structure and function of the cerebral cortex,” (D. B. Tower and J. P. SchadC, (eds.) Amsterdam, Elsevier, 1960. (In press.) ROBERTS, E., P. J. HARMAN, and S. FRANKEL, y-Aminobutyric acid content and glutamic decarboxylase activity in developing mouse brain, PYOC. Sot. Exp. Biol., N. Y. 78: 799-808, 1951.
178 25.
26.
27. 28. 29. 30. 31.
32. 33. 34. 35. 36. 37. 38.
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ROBINS, E., N.R. ROBERTS, K. M. EYDT, 0. H. LOWRY, and D. E. SMITH, Microdetermination of a-keto-acids with special reference to malic, lactic and glutamic dehydrogenases in brain. J. Sol. Chem. 218: 897-909, 1956. ROBINS, E., D. E. SMITH, and E. R. MCCAMAN, Microdetermination of purine nucleoside phosphorylase activity in brain and its distribution within the monkey cerebellum. J. Biol. Chem. 204: 927-937, 1953. SCHAD~, J. P., Development of EEG and spreading depression in the rabbit. Electroencephalography, Montreal 10: 359-360, 1958. SCHADI?, J. P., A histological and histochemical analysis of the developing cerebral cortex. Proc. R. Acad. SC. Amst. 62: 445-460, 1959. SCHADB, J. P., Maturational aspects of EEG and spreading depression in rabbit. J. Neurophysiol. 22: 245-257, 1959. SCHAD$, J. P., Differential growth of nerve cells in cerebral cortex. Growth, Phila. 23: 159-168, 1959. SCHAD~, J. P., and C. F. BAXTER, Maturational changes in cerebral cortex. I. Volume and surface determinations of nerve cell components. In “Inhibition in the nervous system and GABA,” E. Roberts (ed.), 1960. (In press.) SCHAD~, J. P., and I. J. WEILER, Electroencephalographic patterns of the goldfish (Carassius Auratus L.). J. Exp. Biol. 36: 435-452, 1959. SHARIFF, G. A., Cell counts in the primate cerebral cortex. J. Camp. New. 98: 381-400, 1953. SHOLL, D. A., Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat., Lond. 87: 387-406, 1953. SHOLL, D. A., The surface area of cortical neurons. J. Anat., Lond. 89: 571572, 1955. VAN DER Loos, H., Dendro-dendritische verbindingen in de schors der grote hersenen. Thesis, University of Amsterdam, 1959. VAN HARREVELD, A., Changes in volume of cortical neuronal elements during asphyxiation. Am. J. Physiol. 191: 233-242, 1957. VAN HARREVELD, A., and J. P. SCHAD~, Chloride movements in cerebral cortex after circulatory arrest and during spreading depression. J. Cellul. Physiol. 1959. (In press.)