Acta neuropath. (Berl.) 34, 137-- 148 (1976)
Acta Neuropathologica
9 by Springer-Verlag 1976
Autoradiographic Localisation of 8H-Uridine in Spinal Ganglion Neurones of the Rat and the Effects of Methyl Mercury Poisoning N. Carmichael and J. B. Cavanagh M. R. C. Research Group in Applied Neurobiology, Institute of Neurology, Queen Square, London WC1N 3AR
Summary. The uptake of 3H-uridine into rat spinal ganglion neurones has been followed by autoradiography for up to 48 h after its intravenous injection. Labelling of nucleoli and of nuclei reached a peak within 1 h and then declined. Nuclear labelling returned to background levels by 24 h, but nucleolar labelling was still significant after 48 h. Animals dosed with methyl mercury chloride (7.5 mg/kg daily) showed no change in labelling rate in nucleolus or nucleus after 1, 2, or 4 doses. After 8 doses there was severe reduction in labelling in both nucleolus and nucleus; this amount causes extensive loss of axons, loss of some cell bodies and a marked reduction in amino acid incorporation into proteins. On recovery after a further 8 days, labelling levels returned to normal. It is concluded that at the time when loss of ribosomes occurs from the cytoplasm methyl mercury is more likely to be directly disturbing ribosomal structure than R N A synthesis, for methyl mercury causes marked changes in ribosomal organisation after 4 doses, but disturbances to R N A synthesis do not occur until the 8th dose. Key words: Autoradiography -- aH Uridine -- Methyl mercury intoxication -Spinal ganglion neurones.
INTRODUCTION In earlier studies it has been shown that after 8 daily doses of methyl mercury salts there is a marked depression of the capacity of spinal ganglion neurones to incorporate amino acids into proteins (Yoshino et al., 1966; Cavanagh and Chen, 1971). Thus, after 5 mg/kg/day • 8 the uptake is reduced to 61.5 % of normal, and after 7.5 mg/kg/day • 8 it is reduced to 55.8 % (Chen, 1973). Recent ultrastructural studies have shown, moreover, that the earliest abnormal feature in these cells is focal disorganization of rough endoplasmic reticulum and some loss of attached ribosomes (Jacobs et al., 1975). These changes may be visible after only two daily 10"
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N. Carmichael and J. B. Cavanagh
oral doses of 7.5 mg/kg of methyl mercury chloride, but they become more marked subsequently. They always precede by several days any degeneration in axons. The mode of action of methyl mercury compounds is not known, nor is it certain whether it is the methyl mercury component or the free mercury ion that is responsible for the metabolic lesion. It has been suggested that vacuolation of the cell body is an important early change (Chang and Hartmann, 1972a), but we have not found confirmation of this nor have we found any evidence to support the suggestion that the axon is the primary structure affected (Miyakawa et al., 1970; Chang and Hartmann, 1972b) and in our experience changes in the cell body precede any axonal alterations by several days. Furthermore, Chang et al. (1972) found changes in the base ratios of R N A in the spinal ganglia in poisoned animals which they considered to be of important pathogenetic significance, but precisely how this may happen is not clear. The question that concerns us in this study is whether the failure of protein synthesis and the structural disorganisation of the ribosomes noted above are preceded by a failure of R N A synthesis or not. I f not, then it is possible that the ribosomal changes may be the primary event and directly due to interaction with mercury in some form. The results of the present study indicate that a failure of R N A synthesis probably occurs at the same time as the failure in protein synthesis and therefore follows, not precedes, ribosomal damage.
MATERIALS AND METHODS Specific-pathogen-free male CFHB rats (Carworth, Europe) from 70--110 g were used. For the normal studies, the animals were injected intravenously into the tail vein with 5,6-aH-uridine (Radiochemical Centre, Amersham: 49 Ci/m tool specific activity). The dose given was 300 ~Ci/100 g body weight in a concentration of 1 mCi/ml. Particular care was taken to be certain that the intravenous injection was successful, and if there were any doubts about this the animal was discarded. This was an important precaution learnt from earlier studies, in which it was found that the labelling rate was critically dependent upon having a sharp rise and fall of precursor in the circulation to approximately similar levels in each instance. This observation is confirmed by the two studies of Pakkenberg and Fog (1972, 1973) which compared intravenous and intraperitoneal injections of uridine in mice. At 0.5, 1, 2, 4, 24 and 48 h after injection the animals were anaesthetised with chloroform and rapidly perfused through a cannula inserted into the aorta with fixative consisting of 10~ formalin containing 1 ~ acetic acid.
Preparation of the Autoradiographs The fixed carcass was stored for 24 h in a polythene bag and then the cervical spinal ganglia, C 5 and C 6, were dissected out and fixed for a further 24 h. The ganglia were lightly stained with eosin to make them more readily visible, dehydrated in graded alcohols, cleared in propylene oxide and embedded in araldite (TAAB). The eosin was useful in orientating the block for sectioning and unlike toluidine blue was not found to cause chemical reduction of the silver halide in the autoradiographs (Rogers, 1973). Sections of 1 ~z thickness were mounted on glass slides. In the dark, Ilford K 2 emulsion was melted at 45 ~C and diluted 1 : 1 (vol:
aH-Uridine Uptake into Spinal Ganglion Neurones
139
vol) with distilled water; it was transferred to a coplin jar, kept at 45~ in a water bath and the slides were dipped slowly three times over a period of 10 s. The slides were dipped in a random order, air dried for 3 h in a vertical position, and packed in tight boxes with silica gel. They were exposed for 21 days at 4 ~C. For development the slides were allowed to warm to room temperature and then developed in freshly filtered D19b (Kodak) developer for 10 rain at 20~ in a thermostatically controlled water bath. They were then rinsed in 2 ~ acetic acid and fixed for 5 min in Amfix (May and Baker Ltd.) diluted 1 in 7. After hardening in 1 0 ~ formalin for 10rain (Richter and King, 1972) the slides were dried at 40~ Racks of slides were finally stained with toluidine blue at 60~ (Grimley et al., 1965), differentiated in 5 0 ~ acetone and dried at 40~ Coverslips were mounted with araldite identical to the embedding medium while standing on a hot plate at 50 ~C.
Quantitative Methods Counts were done by two different observers in each case and the results collated. Each observer measured and counted 20 cells on separate sections. In about half the animals two separate spinal ganglia (C 5 and 6) were counted, but it was found that there was never any significant difference between ganglia in any one animal, so the results from separate ganglia in individual animals were finally pooled. Thus, between 40 and 80 ganglion cells from each animal were measured and their overlying silver grains counted. Grain counts were made from nucleoli, nuclei and cytoplasm of each cell containing all three features. No particular selection of cells was done, for, as will be shown, there is normally no difference in the counts per unit area from cell to cell, whatever their size. Sizes of nucleolus, nucleus and cytoplasm were computed from measurements of the maximum and minimum diameters of these three features, deriving the areas of these, and subtracting nucleolar from nuclear area and total nuclear from cytoplasmic area. From these data grain counts per unit area for nucleolus, nucleus and cytoplasm were calculated for ganglia from each animal. These figures, therefore, represent the concentration of grains over the total nucleolar, nuclear and cytoplasmic areas measured rather than the mean counts/unit area over individual cells. The population ("n") for statistical purposes was, thus, not numbers of cells, but numbers of animals. Background counts were assessed by counting grains over tissue-free areas of the section using a graticule of known area. All counts and measurements were made using • 100 oil immersion objectives.
Experimental Series The first series of normal animals were killed at 0.5, 1, 2, 4, 24 and 48 h after intravenous injection of 3H-uridine. In addition methyl mercury dosed animals (7.5 mg/kg/daily) were killed in pairs at day 2 (24 h after the second oral dose) and on day 8 (24 h after the eighth oral dose), each animal having been given radioactive uridine intravenously 24 h before death.
140
N. Carmichael and J. B. Cavanagh
The second series of animals were all killed 1 h after intravenous 3H-uridine (300 [xCi/100 g). Normal animals were now compared with animals poisoned daily with methyl mercury chloride (7.5 mg/kg) for I, 2, 4 or 8 doses: two further animals were treated similarly on day 16 having been given methyl mercury for 8 daily doses in the same way. The methyl mercury chloride (R. Emmanuel, Middlesex) was made up in 50 % polyethylene glycol 400 and administered by gastric intubation under light ether anaesthesia.
RESULTS Determination of the areas of normal spinal ganglion cells, their nuclei, and their nucleoli in these I Ot thick plastic sections showed there to be a significant linear correlation between these three parameters. Such relationships are well known for the cells of the cerebral cortex (Bok, 1959) and are to be expected here, too. It was of interest also to look at grain counts per unit area in individual large cells (above mean size values) and in small cells (below mean size values). This was done in six ganglia 1 h following intravenous ~H-uridine. No statistically significant difference was found in the mean grain counts per unit area from each group. Moreover, no linear correlation of the grain concentration with size of nucleoli or of nuclei (correlation coefficient "r" was < 0.1) was found when sought for among individual cells. It must be concluded, therefore, that the radioactive uridine uptake occurred in all cells at a rate that was directly related to their size, a conclusion of some practical value to the counting procedure as it therefore did not matter whether cells of any particular size were chosen.
Distribution of grains over nucleoli and nuclei at different times after uridine injection As shown in Figure 1 and Table 1, nucleolar grain concentration was consistently about 10 times that of nuclei, while the latter was about 10 times the background count taken over cell cytoplasm or elsewhere. Counts at 0.5 h were little different from those at 2 h, while at 4 h there was perhaps some slight indication of a downward trend in nucleolar counts. At 24 h counts per bt2 in both structures were about half earlier levels, but at 48 h, while nucleolar counts were still appreciably raised, nuclear counts were definitely at background levels. The figure at 1 h derives from the control animals in the second experiment and the mean figure of these four animals suggests that the peak uptake may be at this time. While only two animals were studied at each of the other points, the figures of each pair are fairly close to one another. The figure for each animal is derived from counts and measurements upon 40 to 80 ganglion cells. Moreover, the general shape of the curve is very similar to that for uridine uptake into mouse brain cells found by Pakkenberg and Fog (1972) although these authors did not correct for variation in nuclear size nor did they take nucleolar labelling into account.
aH-Uridine Uptake into Spinal Ganglion Neurones
1
50 g
. ~ /
i ~,
141
$ nucleolus 0 nucleus
[ \
301
x
%
10{
0:5
"~
~
hours { l o g
4
1'0
2'4
4~
scale)
Fig. 1. Incorporation of ~H-uridine by dorsal root ganglion cells: grains per unit area following I. V. injection Table 1. Grain counts per ~ • 10 3 over nucleoli, nuclei and cytoplasm of spinal ganglion cells at different times after intravenous aH-uridine (300 ~zCi/100 g). All values are median except t h where they are mean values i standard deviations Time after 3H-uridine injection
Number of animals
Nucleolus grains/ ~2 • 10-3
0.5 1 2 4 24 48
2 4 2 2 2 2
247 493• 275 181 72 48
~
Nucleus grains/ ~.2 • lO-a
Cytoplasm grains/ [z2• 10-~
48 70~19 ~ 47 45 21 17
21 16 17 21 18 26
Mean background: 18 ~ 5 grains/~z 2 10-a Mean values 5_: S.D.
Estimations of cytoplasmic radioactivity was not the aim of this study and the grain concentrations were too low, with the exposures used, for accurate estimates of this parameter. However, it should be noted that cytoplasmic counts per unit area were apparently at b a c k g r o u n d levels except at 48 h when there was a probable increase in cytoplasmic grain concentration. Further studies are clearly needed to decide the significance of this increase. Of more interest to the present problem was the occurrence of nucleolar grain concentrations that were significantly above b a c k g r o u n d levels at b o t h 24 h and 48 h. After two doses of methyl mercury chloride, nucleolar grain concentrations estimated at 24 h after aH-uridine injection were the same as the control figures,
142
N. Carmichael and J. B. Cavanagh
Table 2. Grain counts per ~z2• 10-3 over nucleoli, nuclei and cytoplasm 24 h after intravenous 3H-uridine in spinal ganglia of normal rats and in those after 2 doses and after 8 doses of methyl mercury chloride (7.5 mg/kg/day) by mouth. Median figures only
Normal animals , Methyl mercury 2 days Methyl mercury 8 days
Number of animals
Nucleolus
Nucleus
Cytoplasm
2
72
21
18
2
75
18
20
2
31
23
19
Mean background: 18 grains/~ 2 • 10 -3
500
x
30(
\ 100-
'
'
'
'
'
'
'
1'6
days
Fig.2. 3H-uridine incorporated into dorsal root ganglion cells in animals dosed for /, 2, 4 and 8 days with 7.5 mg/kg/day methyl mercuric chloride. Animals at day 16 were dosed for the first 8 days only whereas after 8 days o f dosing n u c l e o l a r c o n c e n t r a t i o n s were a b o u t h a l f the c o n t r o l figures. G r a i n c o n c e n t r a t i o n s over nuclei, however, were n e a r to or at b a c k g r o u n d levels for b o t h times (Table 2). I n F i g u r e 2 and in T a b l e 3 are shown the grain c o n c e n t r a t i o n s over nucleoli, nuclei, a n d c y t o p l a s m at 1 h after i n t r a v e n o u s injection o f ~H-uridine at various
3H-Uridine Uptake into Spinal Ganglion Neurones
143
Table 3. Grain counts/~ 2 • 10 a over nucleoli, nuclei and cytoplasm of spinal ganglion cells of rats poisoned with daily dosing (up to • 8) of 7.5 mg/kg methyl mercury chloride, compared with control animals. In each case ~H-uridine (300 ~Ci/100 g) was given intravenously 1 h before killing. Note return of grain count towards normal in 16 day animals, 8 days after the last dose Days in experiment
0 (controls) 1 2 4 8 16
Number of animals
4 4 3 3 4 2
Nucleolus grains/
Nucleus grains/
~2 • 10-~
Ix~ • 10 ~
Cytoplasm grains/ ~2 • lO-a
482 i 111 3 9 9 ~ 54 470 4- 121 476 4- 133 9 9 • 61 c 383 ~
50 " 48460 458 418• 53 ~
5.4 4- 1.1 5.9~0.5 7.0 4- 1.4 7,6 ~- 1.5 7.0_+1.6 4.5 ~
19 7 9 17 5b
Mean background: 5.7 • 2.1 • 10 -a grains/~ 2 Median values b P < 0.01 c P < 0.001
times after beginning an eight day course of methyl mercury (7.5 mg/kg) by mouth. It will be seen that there is no real difference from control figures at 1, 2 and 4 days after beginning the dosing, a time which is of critical importance because it is then that the polysomes and the ribosomes of the rough endoplasmic reticulum show dispersion. The capacity of these cells to incorporate labelled uridine into nuclear and nucleolar R N A seems, however, to be unimpaired when these striking ultrastructural changes are taking place. However, at eight days there was a considerable reduction in grain concentration to about a quarter of control figures (Figs. 3, 4 and 5). Of the two animals studied at 16 days (i. e. 8 days after the final dose), one showed a return to normal levels of grain concentrations. There was some doubt about the completeness of the intravenous injection in the other animal. Earlier experience had shown that in such an instance low grain counts might be expected. It is possible, therefore, that there might be some overshoot during the recovery phase of R N A synthesis, but further studies of this phase of the system are clearly needed.
DISCUSSION It is now well known that ribosomal R N A (r-RNA) is made in the nucleolus of the cell whereas messenger R N A (m-RNA) and transfer R N A (t-RNA) are products of the cell's nucleus (Darnell, 1968; Weinberg, 1972; Ashworth, 1973). While most of the r - R N A passes to the cytoplasm after it is made, only a small proportion, apparently, of the nuclear R N A finds its way to the cytoplasm as m - R N A and as t - R N A ; the fate and significance of the remainder is uncertain. There is some autoradiographic and experimental evidence also to indicate that the flow of t - R N A and probably also of m - R N A is in some way under the influence of the
144
N. Carmichael and J. B. Cavanagh
m
Fig. 3. Autoradiograph showing a dorsal root ganglion cell from a control rate 1 h after injection of 3H-uridine. Arrows show position of nuclear membrane. (Grains visible over nucleolus 6, nucleus 22, cytoplasm 10.) Araldite 1 ~m section stained with toluidine blue and photographed through a • 4 blue filter to show silver grains. Mag. x 1750
nucleolus (Sidebottom and Harris, 1969). Certainly destruction of the nucleolus seems to lead to a striking diminution of the arrival of the t - R N A into the cytoplasm (Deak, 1973). Since our present problem centred upon the metabolic state of the cytoplasmic ribosomes, both free and membrane-bound, it was essential for us to concentrate firstly upon the nucleolus and secondly upon the nucleus, in order to determine whether there was any evidence of changes in the precursor uptake in these structures before the onset of ribosomal dispersion. Previous autoradiographic studies upon the uptake of SH-uridine into neurones and its transfer from nucleus to cytoplasm were unhelpful to this problem. The studies of Watson (1965), Pakkenberg and Fog (1972, 1973), Shimada and N a k a m u r a (1969) and of Chang, Martin and H a r t m a n n (1972) are not free from criticism for a number of reasons. Thus, none of these authors have considered correction of their estimate of grain counts to cover changes in nuclear size, and none of them have expressed their results in terms of grain concentration. This is essential since from our findings the grain concentration appears to be constant over nuclei of greatly differing sizes. This is also particularly important for nucleoli that may vary in diameter from less than I ~t to more than 5 ix. Shimada and N a k a m u r a (1966) estimated nucleolar grains and found that they were always less than 50
4
Figs. 4 and 5. Autoradiographs of dorsal root ganglion cells from rats dosed with 8 daily doses of 7.5 mg/kg CHaHgC1. Technical details as Fig. 3. (Fig. 4, grains visible: nucleolus 1, nucleus 4, cytoplasm 7; Fig. 5, grains visible: nucleolus 0, nucleus 3, cytoplasm 7)
146
N. Carmichael and J. B. Cavanagh
than those of nuclei. In our normal material grain concentration over nucleoli is consistently ten times that over nuclei, while that over nuclei is consistently ten times the background figure. In the first few hours after injection cytoplasmic grain concentration is that of the background, as was also found by Pakkenberg and Fog (1972). It is thus obvious that when dealing with the wide range of sizes of these structures, such as are found in the nervous system, meaningful comparisons can only be made when grain concentrations are considered. Furthermore, in no other study have thin, I ~t, sections been used, and unless this is done resolution will be too poor to allow sufficient accuracy of localisation in deciding whether the grains are over the nucleolus or the nucleus. If nucleoli of > 1 ~z to > 5 ~ are contained within a conventional 5 ~ paraffin section there is little likelihood of more than a few nucleoli in a given section being close enough to the halide emulsion for this to be reached by the t3 particles, whose path with tritium is generally considered to be about 1 ~m. Serious underestimates of the radioactivity present will therefore result. Route of administration of" the precursor is also an important technical point if consistent results are to be obtained. Earlier efforts to get high labelling rates used the subarachnoid route (Watson, 1965; Hogenhuis and Spaulding, 1967) but diffusion is patchy with this route and it is not suitable for comparative studies of this sort. Intraperitoneal injections have been used widely, but here, too, there is a major disadvantage for the study of relative uptakes. The peak labelling rate after intraperitoneal injection in mice is at about 18 h after injection (Pakkenberg and Fog, 1973), due presumably to the slow release of the precursor from this site. By contrast, Pakkenberg and Fog (1972) found in mice that the peak after intravenous injection was at half to 2 h and that by 24 h the labelling was already declining. From practical experience we constantly found that the grain concentration was always much lower in those animals in which doubt was expressed about the completeness of the injection at the time. Although this was not explored further by us, we would stress the need for attention to this detail if consistency is to be obtained. Taking these factors into account, therefore, the effects of giving methyl mercury salts on the labelling of nuclei and nucleoli with radioactive uridine is striking and clear-cut. The important point is that the depression in the labelling follows the structural changes affecting the ribosomes by several days, and therefore the latter cannot be due to failure of the normal replacement mechanisms. Impairment of labelling could, however, be caused from the failure of the protein synthetic mechanisms due to deprivation of some essential protein with a short halflife. Alternatively the methyl mercury may cause a reduction in some precursor which is necessary both for the synthesis of protein and of RNA. This hypothesis has, however, yet to be tested. The fact that ribosomal disorganisation is an early event in the sequence that follows poisoning with methyl mercury suggests that this substance in some way has a direct effect upon ribosomal structure within a short while of entering the cell. One possible reason for this may be the importance o f - - S - - S - - bonding to the structure of ribosomes. The mammalian ribosome seems to have rather more than 50 - - S H groups, according to Steinert et al. (1974) and conformational changes take place during the process of peptide synthesis that involve these structural elements. Moreover, it is also established for bacterial ribosomes that blockage o f - - S H
aH-Uridine Uptake into Spinal Ganglion Neurones
147
groups with p-chloromercuribenzoate alters b o t h the structure and their function (Garrett and Wittmann, 1973). Whether in methyl mercury poisoning the methyl radicle is important in these hypothetical considerations is uncertain. It should however be noted that similar, but less extensive and complete disorganisation and dispersion of b o t h polysomes and m e m b r a n e - b o u n d ribosomes is f o u n d in sensory ganglion cells after chronic feeding of mercuric chloride to rats (Jacobs et al., 1975).
REFERENCES Ashworth, J. M. (Ed.): In: Cell differentiation, pp. 43--47. London: Chapman and Hall 1973 Bok, S. T. : Histonomy of the cerebral cortex. Amsterdam: Elsevier 1959 Cavanagh, J. B., Chen, F. C. K. : The effects of methyl mercury dicyandiamide on the peripheral nerves and spinal cord of rats. Acta neuropath. (Berl.) 19, 208--215 (1971) Chang, L. W., Desnoyers, P. A., Hartmann, H. A. : Quantitative cytochemical studies of R N A in experimental mercury poisoning. I. Changes in R N A content. J. Neuropath. exp. Neurol. 31, 489--501 (1972) Chang, L. W., Hartmann, H. A.: Ultrastructural studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta neuropath. (Berl.) 20, 122--138 (1972a) Chang, L. W., Hartmann, H. A. : Ultrastructural studies of the nervous system after mercury intoxication. II. Pathological changes in the nerve fibres. Acta neuropath. (Bed.) 20, 316--334 (1972b) Chang, L. W., Martin, A. H., Hartmann, H. A. : Quantitative autoradiographic study on the R N A synthesis in the neurons after mercury intoxication. Exp. Neurol. 37, 62--67 (1972) Chen, F. C. K. : Studies in neurotoxicity. P h . D . Thesis, University of London (1973) Darnell, J. E. : Ribonucleic acids from animal cells. Bact. Rev. 32, 262--290 (1968) Deak, I. I. : Further experiments on the role of the nucleolus in the transfer of R N A from the nucleus to cytoplasm. J. Cell Sci. 13, 395--401 (1973) Garrett, R. A., Wittmann, H. G. : The structure of bacterial ribosomes. Advanc. Protein Chem. 27, 277--347 (1973) Grimley, P. M., Albrecht, J. M., Michelitch, H. J. : Preparation of large epoxy sections for light microscopy as an adjunct to fine structure studies. Stain Technol. 40, 357--366 (1965) Hogenhuis, L. A. H., Spaulding, S. W. : Autoradiography of long-term R N A metabolism in rabbit neurones. Nature. (Lond.) 215, 281 --283 (1967) Jacobs, J. M., Carmichael, N., Cavanagh, J. B. : Ultrastructural changes in the dorsal root and trigeminal ganglia of rats poisoned with methyl mercury. Neuropath. appl. Neurobiol. 1, 1--19 (1975) Jacobs, J. M., Cavanagh, J. B., Carmichael, N. : The effect of chronic dosing with mercuric chloride on dorsal root and trigeminal ganglia of rats. Neuropath. appl. Neurobiol. 1,321--337 (1975) Miyakawa, T., Deshimaru, H., Sumiyoshi, S., Teraoka, A., Udo, M., Haltore, E., Tatetsu, S. : Experimental organic mercury poisoning--Changes in peripheral nerves. Acta neuropath. (Berl.) 15, 45--55 (1970) Pakkenberg, H., Fog, R. : Kinetics of 3H-5-uridine incorporation in brain cells of the mouse. Exp. Neurol. 36, 405--410 (1972) Pakkenberg, H., Fog, R. : Kinetics of the incorporation of some tritiated nucleic acid precursors and (3H) lysine into mouse brain cells following intraperitoneal injection. J. Neurochem. 21, 841--848 (1973) Richter, C. B., King, C. S. : Formalin as a hardener of photographic emulsions to facilitate staining of epoxy sections after autoradiography. Stain Technol. 47, 268--269 (1972)
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Rogers, A. W. : Techniques of autoradiography, p. 115. Amsterdam: Elsevier 1973 Shimada, M., Nakamura, T. : R N A synthesis in the neurons of the brain of mouse and kitten as visualized by autoradiography after injection of 3H-uridine. J. Neurochem. 13, 391--396 (1966) Sidebottom, E., Harris, H. : The role of the nucleolus in the transfer of R N A from nucleus to cytoplasm. J. Cell Sci. 5, 351--364 (1969) Steinert, P. M., Baliga, B. S., Munro, H. N. : Available sulphydryl groups of mammalian ribosomes in different functional states. J. molec. Biol. 88, 895--911 (1974) Watson, W. E. : An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve regeneration. J. Physiol. (Lond.) 180, 741--753 (1965a) Watson, W. E. : An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve stimulation. J. Physiol. (Lond.) 180, 745--765 (1965b) Weinberg, R. A. : Nuclear R N A metabolism. Ann. Rev. Biochem. 42, 329--354 (1972) Yoshino, Y., Mozai, T., Nakao, K. : Biochemical changes in the brain in rats poisoned with an alkyl mercury compound with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurochem. 13, 1223--1230 (1966)
Received September 15, 1975; Accepted November 13, 1975 Professor J. B. Cavanagh M. R. C. Research Group in Applied Neurobiology Institut of Neurology 8/1 Queen Square London WCI England
Acta Neuropathologica
9 by Springer-Verlag 1976
Autoradiographic Localisation of 8H-Uridine in Spinal Ganglion Neurones of the Rat and the Effects of Methyl Mercury Poisoning N. Carmichael and J. B. Cavanagh M. R. C. Research Group in Applied Neurobiology, Institute of Neurology, Queen Square, London WC1N 3AR
Summary. The uptake of 3H-uridine into rat spinal ganglion neurones has been followed by autoradiography for up to 48 h after its intravenous injection. Labelling of nucleoli and of nuclei reached a peak within 1 h and then declined. Nuclear labelling returned to background levels by 24 h, but nucleolar labelling was still significant after 48 h. Animals dosed with methyl mercury chloride (7.5 mg/kg daily) showed no change in labelling rate in nucleolus or nucleus after 1, 2, or 4 doses. After 8 doses there was severe reduction in labelling in both nucleolus and nucleus; this amount causes extensive loss of axons, loss of some cell bodies and a marked reduction in amino acid incorporation into proteins. On recovery after a further 8 days, labelling levels returned to normal. It is concluded that at the time when loss of ribosomes occurs from the cytoplasm methyl mercury is more likely to be directly disturbing ribosomal structure than R N A synthesis, for methyl mercury causes marked changes in ribosomal organisation after 4 doses, but disturbances to R N A synthesis do not occur until the 8th dose. Key words: Autoradiography -- aH Uridine -- Methyl mercury intoxication -Spinal ganglion neurones.
INTRODUCTION In earlier studies it has been shown that after 8 daily doses of methyl mercury salts there is a marked depression of the capacity of spinal ganglion neurones to incorporate amino acids into proteins (Yoshino et al., 1966; Cavanagh and Chen, 1971). Thus, after 5 mg/kg/day • 8 the uptake is reduced to 61.5 % of normal, and after 7.5 mg/kg/day • 8 it is reduced to 55.8 % (Chen, 1973). Recent ultrastructural studies have shown, moreover, that the earliest abnormal feature in these cells is focal disorganization of rough endoplasmic reticulum and some loss of attached ribosomes (Jacobs et al., 1975). These changes may be visible after only two daily 10"
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N. Carmichael and J. B. Cavanagh
oral doses of 7.5 mg/kg of methyl mercury chloride, but they become more marked subsequently. They always precede by several days any degeneration in axons. The mode of action of methyl mercury compounds is not known, nor is it certain whether it is the methyl mercury component or the free mercury ion that is responsible for the metabolic lesion. It has been suggested that vacuolation of the cell body is an important early change (Chang and Hartmann, 1972a), but we have not found confirmation of this nor have we found any evidence to support the suggestion that the axon is the primary structure affected (Miyakawa et al., 1970; Chang and Hartmann, 1972b) and in our experience changes in the cell body precede any axonal alterations by several days. Furthermore, Chang et al. (1972) found changes in the base ratios of R N A in the spinal ganglia in poisoned animals which they considered to be of important pathogenetic significance, but precisely how this may happen is not clear. The question that concerns us in this study is whether the failure of protein synthesis and the structural disorganisation of the ribosomes noted above are preceded by a failure of R N A synthesis or not. I f not, then it is possible that the ribosomal changes may be the primary event and directly due to interaction with mercury in some form. The results of the present study indicate that a failure of R N A synthesis probably occurs at the same time as the failure in protein synthesis and therefore follows, not precedes, ribosomal damage.
MATERIALS AND METHODS Specific-pathogen-free male CFHB rats (Carworth, Europe) from 70--110 g were used. For the normal studies, the animals were injected intravenously into the tail vein with 5,6-aH-uridine (Radiochemical Centre, Amersham: 49 Ci/m tool specific activity). The dose given was 300 ~Ci/100 g body weight in a concentration of 1 mCi/ml. Particular care was taken to be certain that the intravenous injection was successful, and if there were any doubts about this the animal was discarded. This was an important precaution learnt from earlier studies, in which it was found that the labelling rate was critically dependent upon having a sharp rise and fall of precursor in the circulation to approximately similar levels in each instance. This observation is confirmed by the two studies of Pakkenberg and Fog (1972, 1973) which compared intravenous and intraperitoneal injections of uridine in mice. At 0.5, 1, 2, 4, 24 and 48 h after injection the animals were anaesthetised with chloroform and rapidly perfused through a cannula inserted into the aorta with fixative consisting of 10~ formalin containing 1 ~ acetic acid.
Preparation of the Autoradiographs The fixed carcass was stored for 24 h in a polythene bag and then the cervical spinal ganglia, C 5 and C 6, were dissected out and fixed for a further 24 h. The ganglia were lightly stained with eosin to make them more readily visible, dehydrated in graded alcohols, cleared in propylene oxide and embedded in araldite (TAAB). The eosin was useful in orientating the block for sectioning and unlike toluidine blue was not found to cause chemical reduction of the silver halide in the autoradiographs (Rogers, 1973). Sections of 1 ~z thickness were mounted on glass slides. In the dark, Ilford K 2 emulsion was melted at 45 ~C and diluted 1 : 1 (vol:
aH-Uridine Uptake into Spinal Ganglion Neurones
139
vol) with distilled water; it was transferred to a coplin jar, kept at 45~ in a water bath and the slides were dipped slowly three times over a period of 10 s. The slides were dipped in a random order, air dried for 3 h in a vertical position, and packed in tight boxes with silica gel. They were exposed for 21 days at 4 ~C. For development the slides were allowed to warm to room temperature and then developed in freshly filtered D19b (Kodak) developer for 10 rain at 20~ in a thermostatically controlled water bath. They were then rinsed in 2 ~ acetic acid and fixed for 5 min in Amfix (May and Baker Ltd.) diluted 1 in 7. After hardening in 1 0 ~ formalin for 10rain (Richter and King, 1972) the slides were dried at 40~ Racks of slides were finally stained with toluidine blue at 60~ (Grimley et al., 1965), differentiated in 5 0 ~ acetone and dried at 40~ Coverslips were mounted with araldite identical to the embedding medium while standing on a hot plate at 50 ~C.
Quantitative Methods Counts were done by two different observers in each case and the results collated. Each observer measured and counted 20 cells on separate sections. In about half the animals two separate spinal ganglia (C 5 and 6) were counted, but it was found that there was never any significant difference between ganglia in any one animal, so the results from separate ganglia in individual animals were finally pooled. Thus, between 40 and 80 ganglion cells from each animal were measured and their overlying silver grains counted. Grain counts were made from nucleoli, nuclei and cytoplasm of each cell containing all three features. No particular selection of cells was done, for, as will be shown, there is normally no difference in the counts per unit area from cell to cell, whatever their size. Sizes of nucleolus, nucleus and cytoplasm were computed from measurements of the maximum and minimum diameters of these three features, deriving the areas of these, and subtracting nucleolar from nuclear area and total nuclear from cytoplasmic area. From these data grain counts per unit area for nucleolus, nucleus and cytoplasm were calculated for ganglia from each animal. These figures, therefore, represent the concentration of grains over the total nucleolar, nuclear and cytoplasmic areas measured rather than the mean counts/unit area over individual cells. The population ("n") for statistical purposes was, thus, not numbers of cells, but numbers of animals. Background counts were assessed by counting grains over tissue-free areas of the section using a graticule of known area. All counts and measurements were made using • 100 oil immersion objectives.
Experimental Series The first series of normal animals were killed at 0.5, 1, 2, 4, 24 and 48 h after intravenous injection of 3H-uridine. In addition methyl mercury dosed animals (7.5 mg/kg/daily) were killed in pairs at day 2 (24 h after the second oral dose) and on day 8 (24 h after the eighth oral dose), each animal having been given radioactive uridine intravenously 24 h before death.
140
N. Carmichael and J. B. Cavanagh
The second series of animals were all killed 1 h after intravenous 3H-uridine (300 [xCi/100 g). Normal animals were now compared with animals poisoned daily with methyl mercury chloride (7.5 mg/kg) for I, 2, 4 or 8 doses: two further animals were treated similarly on day 16 having been given methyl mercury for 8 daily doses in the same way. The methyl mercury chloride (R. Emmanuel, Middlesex) was made up in 50 % polyethylene glycol 400 and administered by gastric intubation under light ether anaesthesia.
RESULTS Determination of the areas of normal spinal ganglion cells, their nuclei, and their nucleoli in these I Ot thick plastic sections showed there to be a significant linear correlation between these three parameters. Such relationships are well known for the cells of the cerebral cortex (Bok, 1959) and are to be expected here, too. It was of interest also to look at grain counts per unit area in individual large cells (above mean size values) and in small cells (below mean size values). This was done in six ganglia 1 h following intravenous ~H-uridine. No statistically significant difference was found in the mean grain counts per unit area from each group. Moreover, no linear correlation of the grain concentration with size of nucleoli or of nuclei (correlation coefficient "r" was < 0.1) was found when sought for among individual cells. It must be concluded, therefore, that the radioactive uridine uptake occurred in all cells at a rate that was directly related to their size, a conclusion of some practical value to the counting procedure as it therefore did not matter whether cells of any particular size were chosen.
Distribution of grains over nucleoli and nuclei at different times after uridine injection As shown in Figure 1 and Table 1, nucleolar grain concentration was consistently about 10 times that of nuclei, while the latter was about 10 times the background count taken over cell cytoplasm or elsewhere. Counts at 0.5 h were little different from those at 2 h, while at 4 h there was perhaps some slight indication of a downward trend in nucleolar counts. At 24 h counts per bt2 in both structures were about half earlier levels, but at 48 h, while nucleolar counts were still appreciably raised, nuclear counts were definitely at background levels. The figure at 1 h derives from the control animals in the second experiment and the mean figure of these four animals suggests that the peak uptake may be at this time. While only two animals were studied at each of the other points, the figures of each pair are fairly close to one another. The figure for each animal is derived from counts and measurements upon 40 to 80 ganglion cells. Moreover, the general shape of the curve is very similar to that for uridine uptake into mouse brain cells found by Pakkenberg and Fog (1972) although these authors did not correct for variation in nuclear size nor did they take nucleolar labelling into account.
aH-Uridine Uptake into Spinal Ganglion Neurones
1
50 g
. ~ /
i ~,
141
$ nucleolus 0 nucleus
[ \
301
x
%
10{
0:5
"~
~
hours { l o g
4
1'0
2'4
4~
scale)
Fig. 1. Incorporation of ~H-uridine by dorsal root ganglion cells: grains per unit area following I. V. injection Table 1. Grain counts per ~ • 10 3 over nucleoli, nuclei and cytoplasm of spinal ganglion cells at different times after intravenous aH-uridine (300 ~zCi/100 g). All values are median except t h where they are mean values i standard deviations Time after 3H-uridine injection
Number of animals
Nucleolus grains/ ~2 • 10-3
0.5 1 2 4 24 48
2 4 2 2 2 2
247 493• 275 181 72 48
~
Nucleus grains/ ~.2 • lO-a
Cytoplasm grains/ [z2• 10-~
48 70~19 ~ 47 45 21 17
21 16 17 21 18 26
Mean background: 18 ~ 5 grains/~z 2 10-a Mean values 5_: S.D.
Estimations of cytoplasmic radioactivity was not the aim of this study and the grain concentrations were too low, with the exposures used, for accurate estimates of this parameter. However, it should be noted that cytoplasmic counts per unit area were apparently at b a c k g r o u n d levels except at 48 h when there was a probable increase in cytoplasmic grain concentration. Further studies are clearly needed to decide the significance of this increase. Of more interest to the present problem was the occurrence of nucleolar grain concentrations that were significantly above b a c k g r o u n d levels at b o t h 24 h and 48 h. After two doses of methyl mercury chloride, nucleolar grain concentrations estimated at 24 h after aH-uridine injection were the same as the control figures,
142
N. Carmichael and J. B. Cavanagh
Table 2. Grain counts per ~z2• 10-3 over nucleoli, nuclei and cytoplasm 24 h after intravenous 3H-uridine in spinal ganglia of normal rats and in those after 2 doses and after 8 doses of methyl mercury chloride (7.5 mg/kg/day) by mouth. Median figures only
Normal animals , Methyl mercury 2 days Methyl mercury 8 days
Number of animals
Nucleolus
Nucleus
Cytoplasm
2
72
21
18
2
75
18
20
2
31
23
19
Mean background: 18 grains/~ 2 • 10 -3
500
x
30(
\ 100-
'
'
'
'
'
'
'
1'6
days
Fig.2. 3H-uridine incorporated into dorsal root ganglion cells in animals dosed for /, 2, 4 and 8 days with 7.5 mg/kg/day methyl mercuric chloride. Animals at day 16 were dosed for the first 8 days only whereas after 8 days o f dosing n u c l e o l a r c o n c e n t r a t i o n s were a b o u t h a l f the c o n t r o l figures. G r a i n c o n c e n t r a t i o n s over nuclei, however, were n e a r to or at b a c k g r o u n d levels for b o t h times (Table 2). I n F i g u r e 2 and in T a b l e 3 are shown the grain c o n c e n t r a t i o n s over nucleoli, nuclei, a n d c y t o p l a s m at 1 h after i n t r a v e n o u s injection o f ~H-uridine at various
3H-Uridine Uptake into Spinal Ganglion Neurones
143
Table 3. Grain counts/~ 2 • 10 a over nucleoli, nuclei and cytoplasm of spinal ganglion cells of rats poisoned with daily dosing (up to • 8) of 7.5 mg/kg methyl mercury chloride, compared with control animals. In each case ~H-uridine (300 ~Ci/100 g) was given intravenously 1 h before killing. Note return of grain count towards normal in 16 day animals, 8 days after the last dose Days in experiment
0 (controls) 1 2 4 8 16
Number of animals
4 4 3 3 4 2
Nucleolus grains/
Nucleus grains/
~2 • 10-~
Ix~ • 10 ~
Cytoplasm grains/ ~2 • lO-a
482 i 111 3 9 9 ~ 54 470 4- 121 476 4- 133 9 9 • 61 c 383 ~
50 " 48460 458 418• 53 ~
5.4 4- 1.1 5.9~0.5 7.0 4- 1.4 7,6 ~- 1.5 7.0_+1.6 4.5 ~
19 7 9 17 5b
Mean background: 5.7 • 2.1 • 10 -a grains/~ 2 Median values b P < 0.01 c P < 0.001
times after beginning an eight day course of methyl mercury (7.5 mg/kg) by mouth. It will be seen that there is no real difference from control figures at 1, 2 and 4 days after beginning the dosing, a time which is of critical importance because it is then that the polysomes and the ribosomes of the rough endoplasmic reticulum show dispersion. The capacity of these cells to incorporate labelled uridine into nuclear and nucleolar R N A seems, however, to be unimpaired when these striking ultrastructural changes are taking place. However, at eight days there was a considerable reduction in grain concentration to about a quarter of control figures (Figs. 3, 4 and 5). Of the two animals studied at 16 days (i. e. 8 days after the final dose), one showed a return to normal levels of grain concentrations. There was some doubt about the completeness of the intravenous injection in the other animal. Earlier experience had shown that in such an instance low grain counts might be expected. It is possible, therefore, that there might be some overshoot during the recovery phase of R N A synthesis, but further studies of this phase of the system are clearly needed.
DISCUSSION It is now well known that ribosomal R N A (r-RNA) is made in the nucleolus of the cell whereas messenger R N A (m-RNA) and transfer R N A (t-RNA) are products of the cell's nucleus (Darnell, 1968; Weinberg, 1972; Ashworth, 1973). While most of the r - R N A passes to the cytoplasm after it is made, only a small proportion, apparently, of the nuclear R N A finds its way to the cytoplasm as m - R N A and as t - R N A ; the fate and significance of the remainder is uncertain. There is some autoradiographic and experimental evidence also to indicate that the flow of t - R N A and probably also of m - R N A is in some way under the influence of the
144
N. Carmichael and J. B. Cavanagh
m
Fig. 3. Autoradiograph showing a dorsal root ganglion cell from a control rate 1 h after injection of 3H-uridine. Arrows show position of nuclear membrane. (Grains visible over nucleolus 6, nucleus 22, cytoplasm 10.) Araldite 1 ~m section stained with toluidine blue and photographed through a • 4 blue filter to show silver grains. Mag. x 1750
nucleolus (Sidebottom and Harris, 1969). Certainly destruction of the nucleolus seems to lead to a striking diminution of the arrival of the t - R N A into the cytoplasm (Deak, 1973). Since our present problem centred upon the metabolic state of the cytoplasmic ribosomes, both free and membrane-bound, it was essential for us to concentrate firstly upon the nucleolus and secondly upon the nucleus, in order to determine whether there was any evidence of changes in the precursor uptake in these structures before the onset of ribosomal dispersion. Previous autoradiographic studies upon the uptake of SH-uridine into neurones and its transfer from nucleus to cytoplasm were unhelpful to this problem. The studies of Watson (1965), Pakkenberg and Fog (1972, 1973), Shimada and N a k a m u r a (1969) and of Chang, Martin and H a r t m a n n (1972) are not free from criticism for a number of reasons. Thus, none of these authors have considered correction of their estimate of grain counts to cover changes in nuclear size, and none of them have expressed their results in terms of grain concentration. This is essential since from our findings the grain concentration appears to be constant over nuclei of greatly differing sizes. This is also particularly important for nucleoli that may vary in diameter from less than I ~t to more than 5 ix. Shimada and N a k a m u r a (1966) estimated nucleolar grains and found that they were always less than 50
4
Figs. 4 and 5. Autoradiographs of dorsal root ganglion cells from rats dosed with 8 daily doses of 7.5 mg/kg CHaHgC1. Technical details as Fig. 3. (Fig. 4, grains visible: nucleolus 1, nucleus 4, cytoplasm 7; Fig. 5, grains visible: nucleolus 0, nucleus 3, cytoplasm 7)
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N. Carmichael and J. B. Cavanagh
than those of nuclei. In our normal material grain concentration over nucleoli is consistently ten times that over nuclei, while that over nuclei is consistently ten times the background figure. In the first few hours after injection cytoplasmic grain concentration is that of the background, as was also found by Pakkenberg and Fog (1972). It is thus obvious that when dealing with the wide range of sizes of these structures, such as are found in the nervous system, meaningful comparisons can only be made when grain concentrations are considered. Furthermore, in no other study have thin, I ~t, sections been used, and unless this is done resolution will be too poor to allow sufficient accuracy of localisation in deciding whether the grains are over the nucleolus or the nucleus. If nucleoli of > 1 ~z to > 5 ~ are contained within a conventional 5 ~ paraffin section there is little likelihood of more than a few nucleoli in a given section being close enough to the halide emulsion for this to be reached by the t3 particles, whose path with tritium is generally considered to be about 1 ~m. Serious underestimates of the radioactivity present will therefore result. Route of administration of" the precursor is also an important technical point if consistent results are to be obtained. Earlier efforts to get high labelling rates used the subarachnoid route (Watson, 1965; Hogenhuis and Spaulding, 1967) but diffusion is patchy with this route and it is not suitable for comparative studies of this sort. Intraperitoneal injections have been used widely, but here, too, there is a major disadvantage for the study of relative uptakes. The peak labelling rate after intraperitoneal injection in mice is at about 18 h after injection (Pakkenberg and Fog, 1973), due presumably to the slow release of the precursor from this site. By contrast, Pakkenberg and Fog (1972) found in mice that the peak after intravenous injection was at half to 2 h and that by 24 h the labelling was already declining. From practical experience we constantly found that the grain concentration was always much lower in those animals in which doubt was expressed about the completeness of the injection at the time. Although this was not explored further by us, we would stress the need for attention to this detail if consistency is to be obtained. Taking these factors into account, therefore, the effects of giving methyl mercury salts on the labelling of nuclei and nucleoli with radioactive uridine is striking and clear-cut. The important point is that the depression in the labelling follows the structural changes affecting the ribosomes by several days, and therefore the latter cannot be due to failure of the normal replacement mechanisms. Impairment of labelling could, however, be caused from the failure of the protein synthetic mechanisms due to deprivation of some essential protein with a short halflife. Alternatively the methyl mercury may cause a reduction in some precursor which is necessary both for the synthesis of protein and of RNA. This hypothesis has, however, yet to be tested. The fact that ribosomal disorganisation is an early event in the sequence that follows poisoning with methyl mercury suggests that this substance in some way has a direct effect upon ribosomal structure within a short while of entering the cell. One possible reason for this may be the importance o f - - S - - S - - bonding to the structure of ribosomes. The mammalian ribosome seems to have rather more than 50 - - S H groups, according to Steinert et al. (1974) and conformational changes take place during the process of peptide synthesis that involve these structural elements. Moreover, it is also established for bacterial ribosomes that blockage o f - - S H
aH-Uridine Uptake into Spinal Ganglion Neurones
147
groups with p-chloromercuribenzoate alters b o t h the structure and their function (Garrett and Wittmann, 1973). Whether in methyl mercury poisoning the methyl radicle is important in these hypothetical considerations is uncertain. It should however be noted that similar, but less extensive and complete disorganisation and dispersion of b o t h polysomes and m e m b r a n e - b o u n d ribosomes is f o u n d in sensory ganglion cells after chronic feeding of mercuric chloride to rats (Jacobs et al., 1975).
REFERENCES Ashworth, J. M. (Ed.): In: Cell differentiation, pp. 43--47. London: Chapman and Hall 1973 Bok, S. T. : Histonomy of the cerebral cortex. Amsterdam: Elsevier 1959 Cavanagh, J. B., Chen, F. C. K. : The effects of methyl mercury dicyandiamide on the peripheral nerves and spinal cord of rats. Acta neuropath. (Berl.) 19, 208--215 (1971) Chang, L. W., Desnoyers, P. A., Hartmann, H. A. : Quantitative cytochemical studies of R N A in experimental mercury poisoning. I. Changes in R N A content. J. Neuropath. exp. Neurol. 31, 489--501 (1972) Chang, L. W., Hartmann, H. A.: Ultrastructural studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta neuropath. (Berl.) 20, 122--138 (1972a) Chang, L. W., Hartmann, H. A. : Ultrastructural studies of the nervous system after mercury intoxication. II. Pathological changes in the nerve fibres. Acta neuropath. (Bed.) 20, 316--334 (1972b) Chang, L. W., Martin, A. H., Hartmann, H. A. : Quantitative autoradiographic study on the R N A synthesis in the neurons after mercury intoxication. Exp. Neurol. 37, 62--67 (1972) Chen, F. C. K. : Studies in neurotoxicity. P h . D . Thesis, University of London (1973) Darnell, J. E. : Ribonucleic acids from animal cells. Bact. Rev. 32, 262--290 (1968) Deak, I. I. : Further experiments on the role of the nucleolus in the transfer of R N A from the nucleus to cytoplasm. J. Cell Sci. 13, 395--401 (1973) Garrett, R. A., Wittmann, H. G. : The structure of bacterial ribosomes. Advanc. Protein Chem. 27, 277--347 (1973) Grimley, P. M., Albrecht, J. M., Michelitch, H. J. : Preparation of large epoxy sections for light microscopy as an adjunct to fine structure studies. Stain Technol. 40, 357--366 (1965) Hogenhuis, L. A. H., Spaulding, S. W. : Autoradiography of long-term R N A metabolism in rabbit neurones. Nature. (Lond.) 215, 281 --283 (1967) Jacobs, J. M., Carmichael, N., Cavanagh, J. B. : Ultrastructural changes in the dorsal root and trigeminal ganglia of rats poisoned with methyl mercury. Neuropath. appl. Neurobiol. 1, 1--19 (1975) Jacobs, J. M., Cavanagh, J. B., Carmichael, N. : The effect of chronic dosing with mercuric chloride on dorsal root and trigeminal ganglia of rats. Neuropath. appl. Neurobiol. 1,321--337 (1975) Miyakawa, T., Deshimaru, H., Sumiyoshi, S., Teraoka, A., Udo, M., Haltore, E., Tatetsu, S. : Experimental organic mercury poisoning--Changes in peripheral nerves. Acta neuropath. (Berl.) 15, 45--55 (1970) Pakkenberg, H., Fog, R. : Kinetics of 3H-5-uridine incorporation in brain cells of the mouse. Exp. Neurol. 36, 405--410 (1972) Pakkenberg, H., Fog, R. : Kinetics of the incorporation of some tritiated nucleic acid precursors and (3H) lysine into mouse brain cells following intraperitoneal injection. J. Neurochem. 21, 841--848 (1973) Richter, C. B., King, C. S. : Formalin as a hardener of photographic emulsions to facilitate staining of epoxy sections after autoradiography. Stain Technol. 47, 268--269 (1972)
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Rogers, A. W. : Techniques of autoradiography, p. 115. Amsterdam: Elsevier 1973 Shimada, M., Nakamura, T. : R N A synthesis in the neurons of the brain of mouse and kitten as visualized by autoradiography after injection of 3H-uridine. J. Neurochem. 13, 391--396 (1966) Sidebottom, E., Harris, H. : The role of the nucleolus in the transfer of R N A from nucleus to cytoplasm. J. Cell Sci. 5, 351--364 (1969) Steinert, P. M., Baliga, B. S., Munro, H. N. : Available sulphydryl groups of mammalian ribosomes in different functional states. J. molec. Biol. 88, 895--911 (1974) Watson, W. E. : An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve regeneration. J. Physiol. (Lond.) 180, 741--753 (1965a) Watson, W. E. : An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve stimulation. J. Physiol. (Lond.) 180, 745--765 (1965b) Weinberg, R. A. : Nuclear R N A metabolism. Ann. Rev. Biochem. 42, 329--354 (1972) Yoshino, Y., Mozai, T., Nakao, K. : Biochemical changes in the brain in rats poisoned with an alkyl mercury compound with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurochem. 13, 1223--1230 (1966)
Received September 15, 1975; Accepted November 13, 1975 Professor J. B. Cavanagh M. R. C. Research Group in Applied Neurobiology Institut of Neurology 8/1 Queen Square London WCI England
