THE QUANTITATIVE HISTOCHEMISTRY OF BRAIN

III. AMMON’S HORN*

BY OLIVER H. LOWRY, NlRA R. ROBERTS, KATHERINE Y. LEINER, MEI-LING WU, A. LEWIS FARR, AND R. WAYNE ALBERS

(From the Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri)

(Received for publication, July 13, 1953)

Ammon’s horn is a region of the cerebral cortex (allocortex) which is organized in such a manner as to invite quantitative histochemical study. It is divided into six sharply defined layers or zones, several of which are histologically simple in comparison to the brain as a whole (Table I, Fig. 1). One of the six layers (pyramidalis) is tightly packed with most of the cell bodies of the single predominant type of nerve cell present (a small pyramidal cell). Another layer (radiata) consists of very nearly pure den- drites.

Samples of 5 to 20 y of each of the six zones can be isolated quite easily from frozen-dried sections. Such samples have been subjected to analysis for dry weight, Cl, inorganic P, acid-soluble P, lipide P, nucleic acid and residual P, cholesterol, lecithins, cephalins, sphingomyelins, riboflavin, acid and alkaline phosphatase, adenosinetriphosphatase, cholinesterase, aldolase, and fumarase.

Since it would require an unreasonably large number of determinations to study many different brains in this histochemical manner, fifteen whole rabbit brains were homogenized and analyzed for the constituents listed to determine the amount of variation which occurs from animal to animal.

Material and Methods

The method of obtaining suitable frozen-dried tissue for analysis and most of the microchemical procedures have been described (l-3). Choles- terol was determined by a fluorometric procedure.’ Lecithins, cephalins, and sphingomyelins were measured by procedures of Robins et aL2 The samples analyzed mere not whole sections but small fragments of histo- logically distinct layers dissected from dry sections under a microscope.

The analyses of fifteen whole rabbit brains were carried out on a some- what larger scale than the histochemical measurements, but the same

* Supported in part by a grant from the American Cancer Society through the Committee on Growth of the National Research Council.

1 Albers, R. W., and Lowry, 0. H., to be published. 2 Robins, E., McCaman, R. E., and Lowry, 0. H., to be published.

39

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40 HISTOCHEMISTRY OF BRAIN. III

methods were used. These brains were from albino rabbits of the same type and age group as those of the histochemical studies.

Results

Variation between Different Whole Rabbit Brains-There was found to be remarkably little range in the concentration of fourteen constituents in

-- - - __--- -

Layer Approxi-

mate thickness

FIG. 1. Histological layers of Ammon’s horn (alveus, oriens, pyramidalis, radiata, lacunosum, and molecularis). Also shown is the molecular layer of the adjacent fascia dentata (M. F. D.). Serial sections stained with toluidine blue (Nissl) and osmic acid.

TABLE I

Structure of Ammon’s Horn

Major components

Alveus. .._...... Oriens. Pyramidalis. .’

Radiata Lacunosum Molecularis

P

200 Myelinated fibers (with cell bodies in pyramidalis) 250 Non-myelinated axons and dendrites

50 Densely packed cell bodies which give rise to nearly all fibers in other layers

400 Dendrites, closely packed 150 “ crossed by myelinated fibers 200 Terminal arborizations of dendrites and communi-

cating axons together with prominent vessels of pia mater

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LOWRY, ROBERTS, LEINER, WU, FARR, AND ALBERS 41

fteen rabbit brains (Table II). The coefficients of variation were only bout 1 per cent for protein and total P, and were approximately 5 per :nt for most of the other constituents. The effects of age were negligible, xcept for a 5 per cent (statistically significant) increase in the proportion f dry weight in the older group. r Because of the small dispersion of these data for whole brain, it is to be xpected that a given zone of the brain will also be rather constant in com-

TABLE II

Composition of Fifteen Rabbit Brains

Values based on wet weight.

Age Rabbit Brain weight weight

, Dry weight ! Protein

I gm. per kg. gvn. per kg

211 97.8 3.4 /

/ 1.3

1.2 0.4 223 ! 97.4

4.0 0.9 2.3 0.4

horganic Acid-

P* soluble organic P

Total P

ZM per kg

123.8 1.2 0.4

124.7 1.8 0.7

.‘n

19.2 0.3 0.1 /

18.3 1

8-11 S.d. S.e.m.

14 S.d. S.e.m.

10

5

2.2 6.76 0.2 0.45 0.1 0.14 4.6 7.66 0.6 0.63 0.3 0.32

0.6 1 0.3

11.2 0.5 0.1

11.8 0.3 0.1

Acid phos-

phatase

i ATPase Cholin-

esterase FUllXl-

IBX

?%M per 9n.w peu WADI per ?nM per VIZ‘?4 geu naM gev kgi per

I kyy kc!er kgiry kgirter kyfer

271 64 1252 388 675 2800 12 12 92 14 26 140 4 4 32 4 9 45

250 54 1013 398 675 2720 19 5 200 42 28 79

9 3 89i 19, 12 35

Acid-

As Lipide P insoluble non-lipide

P

Nucleic acid I’

mos. ?nM per m‘w ger kg.

ll~.w per kg kg.

a-11 59.0 15.2 6.80 Rd. 1.4 0.6 0.29 S.e.m. 0.4 0.2 0.09

14 59.7 16.6 6.69 S.d. 2.0 0.8 0.15 S.e.m. 0.9 0.4 0.07

* Inorganic plus labile P t Data for cholinesterase and aldolase obtained with samples from a second set

of brains.

position from rabbit to rabbit. Therefore, analyses of various zones of only a few brains would seem to have general validity. Of course it is not possible t’o conclude that all brain constituents will prove to be as constant as these that have been measured.

Homogen&y withtin Each Zone of Amman’s Horn-It has been found as a rule that each zone is quite constant in composition throughout, whereas abrupt transitions in values usually occur between one zone and the next. This is illustrated by typical individual dnta for lipide P (Fig. 2) and adeno- sinetriphosphatase (Fig. 3). These data also serve t,o confirm the sharp-

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42 IIISTOCIIEMISTRY OF BRAIN. III

ness of dissection. Although homogeneity within a given layer has bee generally true, in several instances minor differences have been observe between the inner and outer portions of the wide zona radiata. The po! tion of this zone which is adjacent to the pyramidal cell layer contain large packed dendrites which have not yet undergone much branching.

Amman’s Horn, Proximate Composition (Table III)--The most strikin contrast in over-all composition of the layers of Ammon’s horn is betwee the myelinated alveus and the layer of packed cell bodies, pyramidalis The total dry weight is almost twice as great per unit volume in alveu as in pyramidalis. The dry weight data have been confirmed rather re markably by Dr. Holger Hyden, using a completely independent means o

I 0 I I 12- 0 8

1.2x-AI.’ ’ ’ ’ ’ ’ ’ ’ - 0

2 0 0 0

x x s ̂ cJo x xxx ” - 00 00

.9-ix 10-z Ra.X x

L,a. - 0 oo or. xx

or, xxi x O0 .E 2 * x xxno .s 0 o. Ra.

,6 -; --:o, x wxxix x -$+ MO. o’. - MF 8-g La.& Z.Q 6 xxx 0

o ** :. ““;x&jm~

y” x .,PY. o oMo.

a SPY. x 1 0 Al. .

.3-g -Jh - 6-= z Distance from Surface ey X Dlstance from Surface

o.p, I ’ ’ ’ I I ’ I I I I

.2 .4 .6 mm. 1.0 1.2 1.4 1.6 .3 .6 mm. .9 1.2

Fig. 2 Fig. 3

FIG. 2. Distribution of lipide phosphorus in six layers of Ammon’s horn and the molecular layer of fascia dentata (M. F.).

FIG. 3. Distribution of adenosinetriphosphatase in the layers of Ammon’s horn. It will be noted that in several instances samples from two different layers were dis- sected from the same tissue section.

measurement (x-ray absorption). His data for the first five layers are 370, 220, 160, 220, and 260 gm. per liter.3 Although the value for alveus is higher than that in Table III, samples of Ammon’s horn have been found in the present study which averaged 330 gm. dry weight per liter in the myelinated layer. ,

The cell body layer contains relatively little lipide (hexane- and chloro- form-soluble material). Although the cell bodies are Gghtly packed, there is some admixture of fibers as they approach their cell bodies. Therefore the cell bodies are undoubtedly even lower in lipide than the figures indi- cate. The two layers adjacent to pyramidalis, which consist almost en- tirely of dendrites and naked axons, contain a rather surprising amount of lipide considering the absence of any visible myelinaCon. The effect of

3 Personal communication.

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LOWRY, ROBERTS, LEINER, WU, FARR, .4r\‘D ALRERS 43

myelinated fibers in lacunosum on its composition is evident, in that the values lie between those for alveus and radiata.

It is to be seen that the amount of protein is relatively constant on a volume basis. (The lower value in alveus has not been borne out in other series.) This relative constancy is an analytical convenience, since any

TABLE III

Dry Weight, Protein, l’ofal Lipides, and Chloride of Amman’s Horn

All values recorded as per cent of dry weight unless indicated. Three different sets of samples were used for these data (Columns 1 to 4, 5 to 8, and 9 respectively).

Alveus. . S.e.m.*.

Oriens S.e.m..

Pyramidalis.. S.e.m..

Radiata. S.e.m..

Lacunosum S.e.m..

Molecularis. S.e.m..

/ x!$L

(1)

303 11

204 8

170

: i 3

233 12

271 7

206

.j 8

Dry weight removed by successive extractions

with

Hex- ane

(2)

31.0 26.9 3.9 38.3 30.8 7.5 93 0.4 1.5 0.9 0.3 1.8 1.6 6

21.5 11.7 9.7 59.4 51.0 8.4 104 0.4 0.7 1.0 0.8 1.4 1.0 6 9.7 9.7 6.0 78.0 66.0 12.0 112 1.6 1.5 t 1.5 1.3 0.7 6

18.7 12.1 9.3 62.1 54.2 7.7 126 1.0 0.9 1.4 1.1 1.7 1.1 8

26.2 17.2 8.6 49.2 43.7 5.5 118 0.7 0.6 0.3 1.8 2.1 1.0 6

19.4 12.0 7.8 61.3 51.6 9.7 107 0.7 1.0 0.5 0.5 1.0 1.8 5

(

1 Proteir Non-

xotein after

:thanol I ‘rot&

(5) (6) (7) (8)

&TM. per 1.

I

P

1

Cl

(9)

n&q. w kg.

dry might

195 4

189 7

238 2

216 5

183 6

192 2

* Standard error of the mean. Most values are the average of five or six deter- minations.

t Average of only two values.

data which are based on protein will approximately parallel data based on wet weight or volume.

If chloride is entirely extracellular, it can be used as a measure of extra- cellular fluid. When the values in Table III are recalculated as millimoles per liter they become 59, 39, 40, 50, 50, and 40 respectively. Assuming the extracellular chloride concentration to be about 110 InM per liter these data suggest that 35 to 45 per cent of the non-myelinated layers and about 55 per cent of alveus consist of extracellular fluid.

Phosphorus and Lipide Fractions-In view of the differences in proximate composition among the layers of Ammon’s horn, it is surprising to find

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44 HISTOCHEMISTRY OF RR.Z11\;. III

almost the same amounts of inorganic and acid-soluble P in all layers Ta ble IV). These values are based on protein and therefore indicate approx imate constancy on a volume or wet weight basis. The residual P also is found to be constant except in alveus, and here it is possible that failure to extract the last traces of lipide may give a falsely high value. (Residua P is that left after extracting successively with trichloroacetic acid, alcohol

TABLE IV

Phosphorus and Lipide Fractions oj Amman’s Horn

All values recorded as millimoles per kilo of protein. Data from three rabbita (Columns 1 to 3, 4 to 5, and 6 to 9 respectively). In most instances each value is the average of five to seven single values.

Alveus .............. S.e.m ... ..........

Oriens .............. S.e.m ............

Pyramidalis. .......... S.e.m ...............

Radiata ............... S.e.m ...............

Lacunosum ........... S.e.m ... ......

Molecularis. ...... , .e.m s .......

lo Ill-

rganir P .;;

(1) I- 162

7 185

5 187

8 185

3 166

5 171

5

Acid- mluble xganic

P

Lipide P

Nu- cleic acid P*

Resid- ual P

:epha- Leci- lins thins

;phin- ,omye-

lins Chol- .sterol

(2) (3) (4) (5) (6) (7) (8) (9)

142 1033 8 37

140 627 8 28

128 297 5 21

127 563 5 20

145 630 6 25

131 515 8 15

23 7

191 22 29

5 27

5 33

2

85 492 361 140 841 11 33 23 10 23 47 269 285 58 308

7 12 14 4 18 50 162 173 29 93 16 t t t 10 43 246 283 52 272

2 7 9 4 8 44 373 370 124 453

5 22 7 18 34 53 339 337 80 313

2 28 7 6 10

* Values for nucleic acid based on absorption at X 260 rnp are in reasonable agree- ment with these values. Thus with a different brain average values were obtained of 40, 222, 31, and 33 millimoles of nucleotide per kilo of protein in oriens, pyra- midalis, radiata, and lacunosum plus molecularis respectively.

t Based on only two values.

and hot perchloric acid.) The nucleic acid dist’ribution emphasizes the sharp localization of cell bodies in pyramidalis.

The large differences in lipide P are borne out by the data for individual phospholipides. The cell body layer is low in all lipides, but is especially poor in sphingomyelin and cholesterol (Table IV). It seems possible that all the cholesterol found in pyramidalis is present in contaminating fibers rather than in cell bodies. In agreement with Brante (4) and others sphingomyelin and cholesterol are found to be far more abundant in white matter than elsewhere, and lecithins are relatively less important in white matter than in gray. It will be noted that there is about a 1: 1 ratio of

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LOU-RY, ROBERTS, LMNER, WU, FARR, AXD ALBERS 45

oephalins to lecithins in all layers except alveus. In most of the layers the four lipide fractions measured account for about 90 per cent of the weight of the extracted lipides of Table III. However, in alveus only 60 per cent l,f the lipides were accounted for. Most of the discrepancy may be attrib- uted to cerebrosides (not measured), which others have found to be espe- zially prominent in white matter (4-6).

Attention is drawn to the fact that the three fiber layers, oriens, radiata, and molecularis, although free of myelinated fibers, are nevertheless quite rich in lipides, especially lecithins and cephalins.

Enzymes and RiboJlavin (Table V)-The true significance of the presence of a certain enzyme at a certain concentration is difficult to establish. However, until proved otherwise, it may be reasonable to assume that the enzymes measured are functionally significant and are present in propor- tion to their actual activity in the living tissue. According to this assump- tion fumarase would be indicative of the level of metabolism of dicar- boxylic acids and probably of the Krebs cycle and aldolase would be a measure of glucose metabolism whether glycolytic or oxidative. Cholines- terase is a presumed measure of cholinergic fibers, particularly of their finer arborizations and synapses (7). The significance of adenosinetri- phosphatase is unknown, but it is tempting to suggest that it is related to total adenosinetriphosphate (ATP) metabolism and therefore a measure of total energy production. Acid and alkaline phosphatase are without es- tablished significance in the brain. Riboflavin, as a probable measure of total flavoproteins, is indicative of a large group of the oxidative enzymes.

Three of the six enzymes measured, acid phosphatase, aldolase, and ATPase, are found to be distributed according to a somewhat similar pat- tern in the six layers of Ammon’s horn. Within each layer the ratios of these three enzymatic activities approximate 1: 1.5: 2 respectively. In general all three enzymes are high in the three non-myelinated fiber and dendrite layers (oriens, radiata, and molecularis). The molecular layer, however, is relatively poor in aldolase. In the myelinated layer, alveus, the ratios between the three enzymes differ from those of other layers; there are only about half as much acid phosphatase and two-thirds a,s much ATPase as might be expected from the amount of aldolase present.

It is a little surprising to find the cell body layer somewhat low in most of the six enzymes. It is, however, rich in fumarase. Molecularis is an- other layer with more than average fumarase. Since it also contains the greatest amount of riboflavin, it may be particularly dependent on oxi- dative processes. Except for the higher level in molecularis, riboflavin is rather evenly distributed in ilmmon’s horn.

Cholinesterase is low in all layers compared to a,verage brain. In this respect Ammon’s horn resembles cortex in general (8). The myelinated

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TABL

E V

Six

Enzy

mes

an

d Ri

bofla

vin

of

Amm

on’s

Horn

The

num

ber

of

sam

ples

an

alyze

d is

in

dica

ted

in

pare

nthe

ses.

All

enzy

me

value

s re

cord

ed

as

mol

es

of

subs

trate

sp

lit

or

hydr

ated

per

kilo

of

prot

ein

per

hour

. Ri

bofla

vin

as

mg.

pe

r kil

o of

pr

otein

.

Alkalin

e ph

osph

atase

La

yers

Acid

phos

phata

se

A’s”

euw.

~;

: : :

: : :

: : :

: : :

: : :)

;:6

; (6

)

Orien

s. _’

S.

e.m

.. ._

.__.

.I

4.58

(1

9)

0.05

Py

ram

idalis

. (

3.25

(7

) S.

e.m

.. ._

......

......

. j

0.13

Ra

diata

.. /

3.94

(1

1)

S.e.

m..

.._.

._.

._.i

0.09

La

cuno

sum

I

2.97

(6

)t S.

e.m

.. ,_

.___

_..._

....~

0.

17

Mole

cular

is .I

S.e.

m

._...

._...

......

. ~

3.81

(9

)t 0.

10

Aver

age

brain

.. I

2.6

0.23

8 (6

) 0.

020

0.25

2 (1

2)

0.00

4 0.

228

(7)

0.01

3 0.

288

(19)

0.

007

0.32

5 (6

) 0.

011

0.33

9 (7

) 0.

003

0.60

ATPa

se

5.53

(1

1)

0.10

10

.62

(17)

0.

27

7.61

(9

) 0.

26

9.56

(1

6)

0.20

8.48

(1

1)

0.27

10.3

Cholin

ester

ase

Aldola

se

Fum

arase

Ri

bofla

vin

2.47

(5

) 0.

17

2.98

(9

) 0.

08

3.12

(7

) 0.

18

2.45

*(6)

0.10

2.

60

(3)

0.40

3.

95

(4)

0.17

4.

8

5.30

(5

) 0.

65

7.15

(1

0)

0.17

5.

33

(7)

0.24

7.

86

(14)

0.

29

6.60

(8

) 0.

31

4.74

(1

0)

0.28

7.

0

20.6

(6

) 1.

1 27

.9

(10)

1.

3 38

.7

(6)

1.9

28.0

(1

2)

1.5

30.2

(7

) 2.

5 40

.2

(9)

3.1

30

26.8

(6

) 0.

9 36

.1

(9)

0.2

35.8

(6

) 0.

8 34

.9

(17)

0.

6 30

.9

(10)

0.

8 49

.6

(8)

1.6

42

* Lo

wer

part

of

radia

ta

(furth

est

from

py

ram

idalis

) av

erag

ed

2.08

f

0.6

mol

es

per

kilo

per

hour

. t

From

an

othe

r ra

bbit

than

th

e fir

st

four

st.

rata

. Th

e va

lue

foun

d for

th

e ac

id

phos

phat

ase

in

the

radia

ta

of

this

seco

nd

rabb

it wa

s 3.

55

f 0.

12

mol

es

ner

kilo

per

hour

.

-

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LOWRY, ROBERTS, LEINER, WU, FARR, ASD ALBERS 47

ayer contains more cholinesterase activity than expected, since Nachman- lohn found only one-twentieth as much activity in white matter as in gray tortex (8). The molecularis is the most active of the six layers. This is ,easonable in view of the abundance of arborizing fibers. Pope (9) found rhe molecular layer of rat cortex to be richer in cholinesterase than any jther cortical layer.

Alkaline phosphatase is also low in Ammon’s horn on both a relative and lbsolute basis. The comparatively high value in molecula.ris is in keeping with the finding of Landow et al. that blood vessels in the brain give a strong react>ion for alkaline phosphatase by microscopic techniques (10).

DISCUSSION

Although anatomically distinct layers have been analyzed with resulting simplification in comparison to the brain as a whole, each layer is still rather complex compared, for example, with skeletal muscle. Perhaps the simplest layers are pyramidalis and radiata. The bulk of radiata cer- tainly appears to consist of dendrites. One might assume that this zone has essentially two phases, extracellular fluid making up 40 per cent of the volume, judged from the chloride data, and dendrites making up the rest. However, the histological appearance of frozen-dried material together with the high lipide content suggests the hypothesis that this region is composed of dendritic cytoplasm with relatively low content of total solids, embedded in or coated by a lipide-containing extracellular matrix or cover- ing. Whether or not this is correct, dendrites appear to be associated with lipide which is composed of approximately equal molar ratios of cephalins, lecithins, and cholesterol, with much less sphingomyelin and presumably little cerebroside (since most of the lipide is accounted for). The observed enzymatic activities make it appear that dendrites are very active metabolically. Dr. Jack 12. Strominger (unpublished) has found that radiata is also quite rich in both lactic and malic dehydrogenases, which, together with data for aldolase and fumarase, indicates a high capacity for both glycolytic and oxidative metabolism. It may be pointed out that dendrites probably compose the bulk of non-myelinated parts of the brain, since non-myelinated axons are relatively slender and cell bodies and glia probably make up less than 10 per cent of the mass of gray matter (11, 12). Because of this and their richness in the above enzymes it seems probable that “brain metabolism” is quantitatively chiefly “dendrite me- tabolism.”

Brant.e (4) has suggested that the cytoplasm of the neuron is almost devoid of cholesterol and sphingomyelin. If it is assumed that there is no cholesterol in the cell bodies in pyramidalis, then it may be calculated that this layer consists of about 20 per cent fibers (exclusive of extracellular

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48 IIISTOCHEMISTRP OF BR.AIN. III

fluid), 35 per cent extracellular fluid, and 45 per cent actual cell bodies. Assuming these values, it is possible to correct, for the fiber const’ituent present and roughly calculate the composition of the cell bodies them selves.5 On a wet weight basis such calculation indicates 22 per cent drj weight, 17 per cent protein, 2.5 per cent total lipides (chiefly equal parts o lecithins and cephalins), 1.5 per cent nucleic acid, and 1.7 per cent othe solids. Total acid-soluble P is about 50 rnM per kilo. These values ar not dissimilar from those found for cells of an organ such as the kidney There is no evidence from present enzyme measurements that the eel bodies are more active metabolically than their dendrites. The modes value for acid phosphatase in pyramidalis does not concur with that re ported by Wolf et al. (13). These authors, using a microscopic staining technique (Gomori (14)), found the greatest activity in cell bodies ana axons. The discrepancy may arise from the rather marked instability oj bra,in acid phosphatase (3), which might distort the staining methods.

It is surprising to find the myelinated fiber layer so rich in the enzymes measured. Acid phosphatase is the only enzyme which is not at least half as active in alveus as in any of the other layers. Acid-soluble P, which one associates with active cytoplasm, is about equal in alveus to that in other layers. Tupikova and Gerard made the same observation with respect to cortical gray and white matter (15). These findings raise t’he question whether all of the metabolic activity can be assigned to the axons plus the oligodendroglia and astrocytes. The glial cells, while numerous, must contribute a rather small bulk. Unless the axons are larger than is usually visualized or richer in enzymes than is most cytoplasm, it may be necessary to attribute some of the metabolic activity to the myelin itself, as though the myelin were a special lipide-rich variety of cytoplasm.

In conclusion, the present data on Amman’s horn demonstrate that it is practicable to analyze directly bits of brain or other tissue weighing 10 y or less and thereby to derive concrete evidence as to the chemical compo- sition of some of the individual components of an organ as complex as the brain. By further scaling down methods, a quite practical undertaking,

4 The average cholesterol in the two layers adjacent to the cell body layer is 290 rn~ per kilo of protein. Therefore, 93/290 X 100 = 32 per cent of p>-ramidalis con- sists of fibers and their attendant extracellular fluid. According to the chloride, 40 per cent of this or 12 per cent is extracellular fluid, which leaves a net of 20 per cent actual fibers (dendrites and axons).

5 For example, the average dry weight in neighboring oriens and radiata is 218 gm. per liter. Therefore fibers and dendrites extending from these la.vers contribute 32 per cent of 218 or 70 gm. to the cell body layer, which leaves 100 gm. associated with the cell bodies and their extracellular fluid. Sfter deducting 1 per cent of the extracellular fluid, this gives 98/45 X 100 = 218 gm. per liter (22 per cent) of dry weight in the cell bodies themselves.

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LOWRY, ROBERTS, LEINER, WU, F.4RR, AND ALBERS 49

nd by broadening the analytical approach, it is evidently possible to assess ne chemical nature of the many types of cell bodies, dendrites, axons, heathes, and glial elements in the nervous system.

SUMMARY

1. By direct microchemical procedures, six histologically distinct layers f Ammon’s horn of the rabbit have been analyzed for dry weight protein, otal lipides, four lipide fractions, five phosphorus fractions, six enzymes, nd riboflavin.

2. The predominant type of cell body present (small pyramidal cell) ppears to be much lower in total lipides than is the rest of the brain. Its :eneral composition resembles that of the cells of an organ such as the :idney.

3. The dendrites are at least as rich in metabolic enzymes as are the cell jodies. They probably account quantitatively for the bulk of brain me- abolism. SubstanGal quantities of lipides are associated with dendrites about equal quantities of cholesterol, lecithins, and cephalins), but the question is raised whether these lipides are not in but around the cytoplasm If the dendrites.

4. As found by others, the lipides of the myelinated fibers are com- paratively rich in sphingomyelin and cholesterol. Because the enzyme activity and content of acid-soluble P in the myelinated layer seem larger than one would expect from the axons and glia, it is suggested that the myelin itself may be metabolically active.

BIBLIOGRAPHY

1. Lowry, 0. H., J. Histochem. and Cytochem., 1, 420 (1953). 2. Lowry, 0. H., Roberts, N. R., Leiner, K. Y., Wu, M.-L., and Parr, A. L., J. Biol.

Chem., 207, 1 (1954). 3. Lowry, 0. H., Roberts, N. R., Wu, M.-L., Hixon, W. S., and Crawford, E. J.,

J. Biol. Chem., 207, 19 (1954). 4. Brante, G., Acta physiol. &and., 18, suppl. 63 (1949). 5. Thudichum, J. L. W., Die chemische Konstitution des Gehirns des Menschen und

der Tiere, Tiibingen (1901). 6. Johnson, A. C., McNabb, A. R., and Rossiter, R. J., Biochem. J., 44, 494 (1949). 7. Anfinsen, C. B., J. Biol. Chem., 152, 267 (1944). 8. Nachmansohn, D., Bull. Sot. chinr. biol., 21, 761 (1939). 9. Pope, A., J. Neurophysiol., 15, 115 (1952).

10. Landow, H., Kabat, E., and Newman, W., Arch. Neural. and Psychiat., 48, 518 (1942).

Il. Donaldson, II. H., J. Nerv. and Merit. Dis., 38, 257 (1911). 12. von Economo, C., Zellaufbau der Grosshirnrinde des Menschcn, Berlin (1927). 13. Wolf, A., Kabat, E. A., and Newman, W., Am. J. Path., 19, 423 (1943). 14. Gomori, G., Arch. Path., 32, 189 (1941). 15. Tupikova, N., and Gerard, Ii.. W., Am. .I. I’hysiol , 119. 414 (3937).

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Wayne AlbersR.Y. Leiner, Mei-Ling Wu, A. Lewis Farr and

Oliver H. Lowry, Nira R. Roberts, KatherineAMMON'S HORN

HISTOCHEMISTRY OF BRAIN: III. THE QUANTITATIVE

1954, 207:39-50.J. Biol. Chem. 

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