Uridine uptake pattern in the cerebral cortex of grivet monkey

Neurochem. Int. Vol. 22, No. 4, pp. 385-393, 1993 Printed in Great Britain. All rights reserved

0197-0186/9356.00+0.00 Copyright © 1993 Pergamon Press Ltd

U R I D I N E UPTAKE PATTERN IN THE CEREBRAL CORTEX OF GRIVET MONKEY H. PAKKENBERG Neurological Research Laboratory, Bartholin Instituttet, Kommunehospitalet, Copenhagen, DK- 1399 Denmark (Received 10 April 1992 : accepted 23 September 1992) Abstract--Assumingthat uridine uptake is correlated to RNA synthesis, and thereby to nerve cell function, the distribution of 5-[H3]-uridine-labellednerve cell nuclei in the cerebral cortex of three hemispheres from two grivet monkeys was examined by microautoradiography. The labelling pattern for 50 cells in layers 2-6 of 10 cortical locations were different in many locations, but in each location they were generally similar. The precentral areas had relatively high labelling, while the motor cortex relatively low, parallelling the bloodflow in these regions. The labelling of layer 4 and 6 was lower than in the other layers of cortex. These measurements provide basic information about the pattern of uridine labelling on the cellular level in primate cortex from animals moving freely in the cage.

The metabolism of nerve cells can be examined in many different ways. In most cases it is the function of rather large groups of cells which are measured, e.g. by measuring 02 uptake or glucose metabolism (Raichle, 1975 ; Risberg and Ingvar, 1973 ; Roland et al., 1982, 1989; Sokoloff, 1975, 1981). Determination of activity in small groups of nerve cells is more diffic u l t - i n some cases it is necessary to evaluate the activity of individual cells. For this purpose, single cell electrodes can only be used in a very limited number of cells, and indicate electrical activity only (Brown and Flaming, 1986). As several studies have shown that nerve cell activity in primate cortex differs from area to area, e.g. concerning 02 uptake, glucose metabolism and blood flow, a determination of uptake of uridine for RNA synthesis by single cells might increase our knowledge about the complex pattern of cortical activity. Microautoradiography using [H3]-uridine provides an impression of R N A synthesis at the time of injection of precursor and the following minutes. Previously we measured uridine labelling in several regions of a grivet monkey brain (Pakkenberg and Fog, 1983). The aim of the present study was to compare the uridine labelling in the cerebral cortex in 10 locations in 3 hemispheres of the same species.

maintained under standard laboratory conditions prior to use in these experiments. The experiment was approved by the Danish Ethical Committee of Animal Experiments. In monkey 1, wt 3.7 kg, the left hemisphere was used for other examinations (Pakkenberg and Fog, 1983). The weight of monkey 2 was 2.5 kg. The animals were observed for 6 months to ensure that they were normal and healthy. One h prior to death (9.00 a.m.) monkey 1 was injected intravenously with 30 mCi 5-[H3]-uridine (specific activity 1.10 TBq/mmol, Amersham), monkey 2 with 20 mCi 5[H3]-uridine (specific activity 1.07 TBq/mmol), 8 mCi/kg animal. The [3H]-uridine was given in 5 ml distilled water. To avoid the use of anaesthetics the animals were caught in a squeeze-back cage by an animal technician wearing protective clothing including heavy leather gloves. During injection in the small saphenous vein the monkey was held in the prone position on a table by the technician. After injection the monkey was released into the cage. One h later the monkey was injected intramuscularly with 25 mg ketamine (Ketalar Vet 50 mg/ml, Parke-Davis) followed by 6% pentobarbital sodium, 0.3 ml intracardially. When anaesthetized the monkey was transcardially perfused through the left ventricle of the heart with about 300 ml of a 0.9% solution of sodium chloride, then with around 500 ml of 4% formaldehyde solution (Lillies fixative). During perEXPERIMENTAL PROCEDURES fusion the descending aorta was closed with hemoTwo adult male Ethiopian grivet monkeys (cer- static forceps. After decapitation, the head of the copithecus aethiops, obtained from Ethiopia) were monkey was placed in 4% formaldehyde solution for 385

i~}~(1

t ]. ]'AKKENBERG

24 h, ihen the brain was removed, and immersion tixed in formaldehyde for 8 days. The brain was then cut into blocks, rinsed in water, dehydrated in alcohol. cleared in chloroform and embedded in paraffin. Microlome sections were cut in 4 / m l thickness and placed on glass slides. Autoradiographs were produced by dipping the sections in Ilford Nuclear Research Emulsion K-5 followed by exposure for 48 h and development in Amidol for 4 min at 1 8 C. The sections were stained with gallocyanine-chrome alum for 24 h. Grains were counted in 100 nerve cell nuclei in each of layers 2 6 in samples taken from 10 different cortical locations (Fig. 1) in monkey 1. In monkey 2 grains covering only 50 nerve cell nuclei were counted as the statistical evaluation of the counts from monkey I showed that the variance was increased by at mosl 25% by counting grains in 50 cells only. The grains were only counted in cell nuclei with a visible nucleolus (Fig. 2). The starting point for counting in each layer was random and all countings were carried out "blind" on renumbered sections to prevent bias. The diameter of all cell nuclei was measured by an ocular micrometer.

Some of the nuclei are oval. In these cases both diameters are measured and the average is used. The region in which all measurements were made was about 400/~m in each location multiplied by the thickness of the layers. As this thickness was about 200 itm (layer 2,5, and 6), 300/~m (layer 4), and 400 Hm (layer 3), the counting areas were between 80,000 and 160,000/~m ~. To differentiate between nerve cells and glial cells was generally no problem as the number of grains was three or more in nerve cells, but only a few glial cells were labelled (Fig. 2) and with one or two grains only. The background in the autoradiograms was low (short exposure time). It was 1-2 grains per 10,000 Itm 2. A precise description of the "locations" in which the counting has been perforined is difficult. However, the brains of the two grivet monkeys concerning sulcus pattern looked very much like maeaca monkeys. Therefore the terminology of Bonin and Bailey (1947) is used, see legend for Fig. I. A high dose of [3H]-uridine was used for 2 reasons. The exposure time could be short (2 days), which gives

Fig. 1. The locations in which the grain counting was carried out. The terminology is from Bonin and Bailey (1947). 1, superior frontal gyrus (FB) ; 2, middle frontal gyrus, dorsal part (FD) ; 3, inferior frontal gyrus (ventral part of FD) ; 4, anterior central gyrus (dorsal part of FA) ; 5, anterior central gyrus (ventral part of FA) ; 6, superior temporal gyrus (TA) ; 7, superior parietal lobule (PE) ; 8, middle temporal gyrus (TE); 9, occipital lobe, dorsal part (OC); 10, occipital lobe, ventral part (OC).

Fig. 2(a,b)--legend overleaf

...j

Fig. 2. Microautoradiographs

from location

1000 x(a) Layer 2. (b) Layer 3. (c) Layer 5. (d) White wlxtancc. I. frontal lobe. Magnification nerve cells. The glial cells have no grains.

Fig. 2(c. d) frontal

lohe

g.

<
cell\

: I: Smali

Uridine uptake in monkey cortex a very low background. A high dose of precursor gives a constant precursor specific activity (Scornik, 1974). The 1 h experimental time is chosen on the background of earlier observations (Pakkenberg and Fog, 1972) showing that maximal labelling is obtained 1/21 h after injection in mice. Statistical methods Variation in grain counts within each cortical layer at each location was studied. Counts were found to follow Poisson distributions with means proportional to the square of the cell nucleus diameters. The results of analyses are present.ed as mean counts, K, per cell nucleus adjusted to a standard cell nucleus diameter of 10 #m. For each location (numbers 1-10), a twoway analysis of variance (hemisphere × layer) was carried out. The average residual variance was estimated as SD 2 = 0.65000 = 0.8062 including a variance due to Poisson variation less than 0.16. The standard errors given in Table 1 are calculated using SD 2 = 0.6500. RESULTS

As nuclear profile area in the section determines labelling intensity, grain counts in all measured cells

389

were corrected using a standard diameter. In all nuclei with a diameter deviating from 10 #m the number of grains has been regulated down in nuclei greater than 10/~m, or up in nuclei smaller than 10/~m. Thus all cell counts express the labelling of standard nuclei of 10/~m diameter. This permits a direct comparison of counts throughout the cortex. Table 1 shows that significant differences occurred in labelling at the different locations and in the different layers. The frontal areas (location 1 and 2) showed a significantly higher labelling than average, while the motor areas (location 4 and 5) have lower than average labelling. All the remaining locations have almost the same labelling, with the exception of the low grain count in the ventral visual area (location 10). Furthermore, layers 4 and 6 have obviously lower labelling than the other layers while layer 3 labelling was slightly higher. Layer 4 is missing in motor areas (location 1, 4, and 5). The labelling pattern in the three hemispheres is demonstrated in Fig. 3. The differences in shape of the curves are not greater between the right hemisphere of monkey 1 and that of monkey 2, than between the two hemispheres of monkey 2. While there is a significant labelling of the nucleus very few grains are found in cytoplasm and neuropil (Fig. 2). As shown earlier

Table 1. M e a n grain counts for three hemispheres Layer Location

2

3

4

5

6

Average (excl. 1.4)

10.83 6.29 7.23 9.89 8.79 7.74 9.13 8.12 7.97 6.75

9.36 7.75 8.20 9.60 8.95 8.05 9.20 7.59 8.4l 6.97

0.0 0.0 0.0 6.80 8.61 7.27 7.34 6.40 7.12 6.73

8.12 7.37 7.39 8.89 7.78 7.86 7.96 7.98 8.50 7.96

8.08 6.04 6.70 9.22 8.02 7.16 8.05 7.58 7.84 7.48

9.10 6.86 7.38 9.40 8.38 7.70 8.59 7.82 8.18 7.29

8.27

8.41

7.98

7.62

8.07

--0.09

--0.45 xx~

no.

1 4 5 2 3 6 7 8 9 10 Average 1 10 Deviationt Average 2,3,6-10 Deviation{

0.21

0.34 '~

* Average for location total average (8.07) : SE = 0.22. t Average for layer-total average (8.07) : SE = 0.13. }7.18 8.19 SE = 0.15. ' P < 0.05, " P < 0.01, ' " P < 0.001.

7.18 _

1.0t

8.19 -,~,

Deviation*

1.03 ~'x 1.21'"" - 0.69~ 1.33 x~ 0.31 0.37 0.52 ~ -0.25 0.1 I 0.78'"

(a) 12--

10

Location I

B

B A..'.

- -

s -

(b) 12 --

I0

""'::'~...... A A C ........ , .... C - ~ C q

c

Location 3

-

j c C B

_

~ B x / B~ "C~

C

~ ~'C

"'""S

6 -

6 -

I

I

I

I

I

I

I

I

I

I

2

3

4

5

6

2

3

4

5

6

Layer

Layer

12 --

Location 2 B

10

12 -

10

Location 4

--

C.....

8 -

B ....

". . . . . . . . C ~

c/~ ..........B~c

s

A. . . . . . . . . .

C

6 9

B

....\

I

I

I

l

I

I

I

I

I

AI

2

3

4

5

6

2

3

4

5

6

Layer

Layer

(d)

(c) 12

Location 5

12 --

Location 7

B

10- ~~!~'~c-~_

I0

B

B-. ....

s -- C - - ~ A

.......

Bx

~c-..-...--.: .... ~__~.~ ~-.... ~-----2~ I

I

I

I

I

I

I

I

I

I

2

3

4

5

6

2

3

4

5

6

Layer

Layer

12 --

Location 6

12

-

Location 8

10

I0

c -

c.

c

~~.~_~~

A

-- A ~.,...,..~A X C

/

A.~.

A

~ A ~ A ~ A

I

I

I

I

I

I

[

I

I

[

2

3

4

5

6

2

3

4

5

6

Layer

Layer F i g 3(a d)

l
Uridine uptake in monkey cortex

(e) Location 9

12 -

10

B

/.&._c

B

A

I

I

I

I

I

2

3

4

5

6

Layer

12

L o c a t i o n 10

10

8 --

6

c/

C

A / A

C

~c

B

A

I

I

I

I

I

2

3

4

5

6

Layer Fig. 3. Diagrams showing the mean grain counts in microautoradiograms in 10 locations for the three hemispheres. Layer 4 is missing in location 1, 4, and 5. The right hemisphere from monkey 1 is called A, the right hemisphere from monkey 2 is B, and the left from monkey 2 is C.

(Pakkenberg and Fog, 1972), the labelling of the cytoplasm starts about 6-9 h after uridine-injection and decrease again after 12 days. The neuropil labelling is low and increases after 12 h, only to decreases again after 4 days. In the present experiment the labelling of both cytoplasm and neuropil is negligeable (Fig. 2).

DISCUSSION

The relation between nerve cell function and their metabolism is well-known (Yarowsky and Ingvar, 1981). Uridine labelling is correlated to R N A syn-

391

thesis in nerve cells (Koenig, 1958, Engel and Morrell, 1970). Increased function in nerve cells generally increase the content of R N A (Hyden, 1967 ; Pevzner, 1965; Jarlstedt, 1966; Berry, 1969), but more prolonged function decrease the cytoplasmic R N A (Pakkenberg and Thomsen, 1964; Einarson and Krogh, 1955). Slight electrical stimulation increases incorporation of labelled uridine and cytidine into the R N A of cell R-2 in aplysia (Peterson and Kernell, 1970). This synthesis of R N A is a rather slow process, at the level of minutes, even in such extreme cases as during convulsions in mice. The uridine labelling is decreased by 50% in cortex returning to normal by 1 h (Pakkenberg et al., 1985). [3H]-uridine is supposed to label all types of RNA. However, the messenger (m) and ribosomal (r) R N A represent 97 % of RNA synthesis (Alberts et al., 1983), and as the turnover time for r R N A is about 12 days (Von Hungen et al., 1968) and 30 min to 2 h for m R N A (Kimberlin, 1967), I suppose that m R N A is taking up the main part of the injected [3H]-uridine. The origin of uridine for RNA synthesis in the adult brain is not known in details. However, there is an indirect evidence to suggest that most uridine is taken up from the blood: the brain uses uridine but not orotic acid in the synthetic pathways; the amount of enzymes for the novo synthesis of pyrimidine bases is very low in brain ; and the uptake of uridine in brain is fast and maximal after 30 min (Mac Donnell et al., 1980; Pakkenberg and Fog, 1972). Uridine and cytidine are cleared from the blood with T~/2 of less than 5 min (Moyer et al., 1985 ; Pakkenberg and Fog, 1972). This means that in spite of an ever changing pool size, membrane transport, etc. the main part of uridine injected is probably caught in the nuclei by phosphorylation. When the animal is perfused 1 h later, only the "free" unlabelled uridine is removed. This explanation is in accordance with the unchanged--maximal--labelling between 30 min and 12 h after uridine injection in mice (Pakkenberg and Fog, 1972). In the present paper the "hyperflow" of Ingvar (1979), in the frontal region ("hyperfrontality"), which is a more permanent phenomenon, is in accordance with the high grain counts in location 1 and 2. The opposite is seen in the precentral motor cortex (locations 4 and 5) where low grain counts are seen in parallel with relatively low regional cerebral blood flow (Ingvar, 1979 ; Risberg and Ingvar, 1973 ; Roland and Larsen, 1976). The most characteristic finding is that the curves are parallel or have the same slope through most of their course (in 90 out of 102 cases). It is possible,

392

H. PAKKt NI~t'R(;

however, to recognize some more specific similarities, e.g. between the curves of location 2 and location 7 and between location 6 and 10. The Functional significance is unknown. There are obvious differences between the mean grain counts in the different layers, suggesting lk~r example that the general synthetic activity o f layers 4 and 6 is less than that o f the other layers. Blood flow, glucose metabolism, and 02 uptake are measured in rather large cortical areas but it appears from the present study, that synthetic activity in layers 2, 3 and 5 is dominating (Folbergrova et al., 1970). The similarity of the curves for the three hemispheres suggests that the functional pattern of R N A synthesis is different in the 10 locations since uridine uptake is supposed to be an expression of cortical synthetic activity during the first minutes after uridine injection. These data provide basic information about uridine labelling in monkey cortex, and should be c o m p a r e d with other parameters o f nerve cell function in the cerebral cortex, e.g. protein synthesis. Acknowledgements--A donation from the Hartmann Foundation is gratefully acknowledged. The assistance of veterinary Lis Bruun, Statens Seruminstitut, in the handling of animals was very valuable.

REFERENCES

Alberts B., Bray D., Lewis J., Roth M., Roberts K. and Watson J. (1983) Molecular Biology o1" the Cell pp. 406 427. Garland, New York. Berry R. (1969) Ribonucleic acid metabolism of a single neuron: correlation with electrical activity. Science 166, 1021-1023. Bonin G. and Bailey P. (1947) The neocortex of macaca mulatta. The University of Illinois Press. Brown K. and Flaming D. (1986) Advanced micropipette techniques for cell physiology. John Wiley, Chichester, 1986. Einarson L. and Krogh E. (1955) Variations in the basophilia of nerve cells associated with increased cell activity and functional stress. J. Neurol. Neurosurg. Psychiat. 18, 1 16. Engel Jr. J. and Morrell F. (1970) Turnover of RNA in normal and secondarily epileptogenic rabbit cortex. Exp. Neurol. 22, 221-.238. Folbergrova J., Lowry G. and Passonneau J. (1970) Changes in metabolites of the energy reserves in individual layers of mouse cerebral cortex and subjacent white matter during ischaemia and anaesthesia. J. Neurochem. 17, 1155 1162. Hyden, H. (1967) RNA in brain cells. In : The Neurosciences (Quarton, G. Melnechuk, T. Schmitt, F. eds), pp. 248266. Rockefeller University Press, N.Y. lngvar D. (1979) "Hyperfrontal" distribution of the cerebral grey matter flow in resting wakefulness ; on the functional anatomy of the conscious state. Acta Neur. Stand. 60, 289- 297. Jarlstedt J. (1966) Functional localization in the cerebellar

cortex studied by quantitative determinations of Purkinlc cell RNA. Acre physiol. Stand. 67, 243 252. Kimberlin R. (1967) RNA synthesis in mouse brain. ,Vcuroclu,mis'trv, 14, 123 134. Koenig tl. (1958) An autoradiographic study of the nucleic acid and protein turnover m tile mammalian neuraxis. ,/ Biophys. hiochem. Cytol. 4, 785 792. Mac Donncll P., Huff K., Grouse L. and Guroff (i. (1980) Bra#~ nuch, ic acids. In Biochemist O" o1' Brain, (Kumar S., ed.), pp. 21 I 240. Pergamon Press. New York. Moyer J., Malinowski N. and Ayens O. (1985) Salvage of circulating pyrimidine nucleosides by tissue of the mouse. J. biol. Chem. 260, 2812 2818. Pakkenberg H. and Fog R. (1972) Kinetics of ~H-5-uridine incorporation in brain cells of the mouse. Exp. Neurol. 36, 405 41(I. Pakkenberg It. and Fog R. (1983) Uridine uptake by nerve cells of the grivet monkey and its relation to cytoplasmic ribonucleic acid concentrations. Neurochem. Int. 5, 553 557. Pakkenberg H. and Thomsen E. (1964) Cytoplasmic basophilia in spiral ganglion cells of the guinea pig following strong acoustic stimulation. Acta Oto-laryng. 58, 299 311. Pakkenberg H., Pakkenberg B. and Fog R. (1985) Effect of electrical convulsions on uridine labelling and activity pattern in nerve cells in mice. Exp. Neurol. 89, 115 122. Peterson R. P. and Kernell D. (1970) Effects of nerve stimulation on the metabolism of RNA in a molluscan giant neurone. J. Neuroehem. 17, 1075 1085. Pevzner L. (1965) Topochemical aspects of nucleic acids and protein metabolism within the neuron-neuroglia unit of the superior cervical ganglion. J. Neuroehem. 12, 993 1005. Raichle M. (1975) Sensori-motor area increase o! oxyqen uptake during contralateral hand exercise. In : Brain Work, (Ingvar D. and Lassen N. A., eds), pp. 372 376. Munksgaard, Copenhagen. Risberg J. and Ingvar D. (1973) Patterns of activation in the grey matter of the dominant hemisphere during memorizing and reasoning. Brain 96, 737 756. Risberg J. and Prohovnik I. (1983) Cortical processing of visual and tactile stimuli studied by non-invasive rCBF measurements. Hum. Neurobiol 2, 5 10. Roland P. and Larsen B. (1976) Focal increase of cerebral blood flow during stereognostic testing in man. Arch. Neur. 33, 551 558. Roland P., Meyer E., Shibasahi T., Yamamoto Y. and Thompson C. (1982) Regional cerebral blood flow changes in cortex and basal ganglia during voluntary movements in normal human volunteers. J. Neurophysio148, 467--480. Roland P., Eriksson L., Widen L. and Stone-Elander S. (1989) Changes in regional cerebral oxidative metabolism induced by tactile learning and recognition in man. Eur. J. Neurosci. 1, I 18. Scornik O. (1974) In rit:o rate of translation by ribosomes ot" normal and regenerating liver. Biol. Chem. 249, 3876 3883. Sokoloff L. (1975) Influence q/junctional activity on local cerehralglucose utilization. In Ingvar D. and Lassen N. A. (eds), Brain Work, p p 386 388. Munksgaard, Copenhagen. Sokoloff L. (1981) Relationships among local functional activity energy metabolism and blood flow in the central nervous system. Fed. Proc. 40, 2311 2316.

Uridine uptake in monkey cortex Spector R. (1985) Uridine transport and metabolism in the central nervous system..I. Neurochem. 44, 1411 1418. Swindale N. (1990) Is the cerebral cortex modular? Trends Neurosci. 13, 487-492. Von Hungen K., Mahler H. and Moore W. (1968) Turnover

393

of protein and ribonucleic acid in synaptic subcellular fractions from rat brain. J. biol. Chemistry 243, 14151423. Yarowsky P. J. and Ingvar D. H. (1981) Neuronal activity and energy metabolism. Fed. Proc. 40, 2353 2362.