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Histochemical study of the cerebral cortex in rats thyroidectomised at birth
BRAIN RESEARCH
351
H1STOCHEMICAL STUDY OF THE CEREBRAL CORTEX IN RATS T H Y R O I D E C T O M I S E D AT BIRTH
N. ROBINSON ANDJ. T. EAYRS Departments of Chemical Pathology, Institute of Neurology and of Neuroendocrinology, hlstitute of Psychiatry, University of London, London (Great Britain)
(Accepted January 19th, 1968)
INTRODUCTION Developmental defects in mammals resulting from a deficiency of thyroid hormone early in life are now well established. Changes in bodily configuration have long been recognised 3°,33, and more recently attention has been paid to the influence of the thyroid on the maturation of the central nervous system. In this context irreversible behavioural impairment, associated with changes in the electrical activity of the brain4, ~ and with structural modifications involving the size of cortical perikarya al, the growth of neuropil v, the patterning of the capillary bed 6 and the deposition of myelin 3 has been shown to arise when thyroid deficiency is present during a critical period of development s. The biochemical correlates of these phenomena have, until recently, attracted little attention. Changes in the DNA/RNA ratio consistent with the earlier neurohistological findings have been reported2,15 while evidence has been provided indicating modification in the activities of several enzymes'~,16, 21. The aim of the present study has been to determine whether any changes in the localisation and activity of some of the enzymes controlling important metabolic pathways could be identified by use of histochemical techniques. The enzymes investigated have been those concerned with the capacity of the cerebral cortex for oxidation-reduction processes, energy transforrnation, nervous transmission, regulation of phosphates and amine oxidation. MATERIALSAND METHODS Albino rats of the Sprague-Dawley strain were used and one pair oflittermates from each of 6 litters provided the material for the histochemical observations. One animal of each pair was thyroidectomized on the day of birth by giving a single injection of 150-200/~C 131I (ref. 18). The other, acting as control, received the vehicle only. The rats were killed by decapitation at 30 days of age at which time the 6 thyroidectomized rats weighed 37-44 g and the controls 83-95 g. The brain was rapidly removed and instantly frozen to --70°C. Fresh frozen sections were cut between Brain Research, 9 (1968) 351-362
I"-J
~D
2" NORMAL
AND HYPOTHYROID
RAT CEREBRAL CORTEX
a-Glycerophosphate dehydrogenase D NADH~-diaphorase dehydrogenase D Succinate dehydrogenase W ~ = Glucose 6-phosphate dehydrogenas¢ D ..... Acid phosphatase P---~- ~ Alkaline phosphatase Mg~+-ATPase d Ca~+-ATPase 5-nucleofidase D Thiamine pyrophosphatase P Monoamine oxidase W Acetylcholinesterase W
N
N
d d
~
D WD-
d D÷ ~
d D-! ~-+
D P W W
d
d
d
d d
D
H
d
d
D
D
H
Neuropil
Nerve cells cytoplasm
P D
W
W d
W
N
Glial cells
P D
W
W d
W
H
D
D
D D--.
D
W
N
Nerve fibres
D
D
D
D
W
H
W
D p---P D P
W
P
W
D p~ P D P
W
P
Blood vessels and capillaries N H
N, normal cortex; H, hypothyroid cortex; P, prominent reaction, showing continuous heavy staining; D, distinct reaction, moderate well-defined staining; W, weak reaction, indistinct cytoarchitecture; d, diffuse reaction, homogeneous distribution, localisation poor. No symbols indicate reaction undetectable. The plus signs ( ) indicate relative intensities between normal and hypothyroid tissue. Where no plus signs, differences were undetectable.
LOCALISAT1ON AND RELATIVE INTENSITIES OF ENZYME ACTIVITIES IN THE 30-DAY-OLD
TABLE I
7-
2-
m
> z
Z
©
z
L/I b,9
HISTOCHEMISTRYOF CEREBRALCORTEX
353
10 and 14 # thick on a Pearse-Slee freezing microtome and the following enzymes were demonstrated histochemically: NADH2-diaphorase, a-glycerophosphate (menadione added), succinate (menadione added) and glucose 6-phosphate dehydrogenases34; acid phosphatase2°; alkaline phosphataselg; CaZ+-adenosine triphosphatase '~9; Mg2~-adenosine triphosphatase 3a; 5-nucleotidase z6; thiamine pyrophosphatase27; monoamine oxidasel7; and acetylcholinesterase 2a. Nucleic acids (gallocyanin-chromalum) and proteins (ninhydrin) were also stained. Sections from normal and thyroidectomised rats were simultaneously incubated in the same substrate medium and subsequently treated in the same staining medium to enable a direct comparison of the intensity of enzyme activity to be made. The cofactors in the dehydrogenase incubation media and the substrates for all other enzymes were omitted in the blanks. RESULTS The distribution and relative intensities of the enzyme activities in the cerebral cortex of normal and thyroidectomised rats are summarised in Table 1. The detailed findings are set out below. Oxidatire enzymes. The histochemical concentration of oxidative enzymes in the cytoplasm of nerve cells in the cerebral cortex of normal rats decreased in order NADH2-diaphorase, glucose 6-phosphate, succinate and (t-glycerophosphate dehydrogenase. In agreement with Thomas and Pearse 34 there was no detectable reaction in the nucleus or nucleolus (Fig. 1). A moderate reaction for these enzymes was observed in the neuropil and a-glycerophosphate dehydrogenase also exhibited a strong reaction in the capillaries. In the thyroidectomised animals the normal
Fig. I. NADH2-diaphorase in cortical nerve cell cytoplasm and dendrite of normal 30-day-old rat. No counterstain. × 400. Brain Research, 9 (1968) 351-362
354
y. ROBINSON AND J, I, I!AX~RS
<
Fig. 2. Same enzyme as Fig. 1 but showing the characteristic absence of a reaction ill the dendritcs of cortical nerve cells of 30-day-old rats thyroidectomised at birth. No coumerstain. 400. o r d e r o f staining intensity was retained and the nerve cells exhibited only a slight reduction in the staining reaction o f oxidative enzymes, seen mainly in lhe glucose 6 - p h o s p h a t e and succinate d e h y d r o g e n a s e activities. A strong reaction f o r N A D H e d i a p h o r a s e in the neuropil was present in both n o r m a l a n d experimental tissues but only in the n o r m a l were dendrites detectable by this m e t h o d (ty'~ Figs. i a n d 2). Acid phosphatase. Enzyme activity was distributed t h r o u g h o u t the c y t o p l a s m
Fig. 3. Acid phosphatase activity in the cerebral cortex ofthe normal 30-day-olcl rat. Nocounterstain.
× 250.
Brain Research, 9 (1968) 351-362
HISTOCHEMISTRYOF CEREBRALCORTEX
355
"4
p
Fig. 4. Reduced activity in cell processes and poor cytoplasmic localisation of acid phosphatase in the same cortical region of a 30-day-old rat thyroidectomised at birth. No counterstain, x 250. and larger processes of the nerve cells in normal tissue; activity was not present in the cell nuclei. Within each individual nerve cell the localisation of enzyme activity showed some cytoplasmic variation but the reaction from cell to cell was generally consistent for all cortical nerve cells. The enzyme activity of this enzyme in the neuropil was undetectable but oligodendrocytes frequently exhibited a moderate granulation. Some enzyme activity was observed along nerve fibres (Fig. 3). Acid phosphatase activity was of similar intensity to controls in the cortical nerve cell bodies of thyroidectomised rats but the distribution along the processes appeared markedly less consistent and intense (Fig. 4). Alkaline phosphatase. The activity of this enzyme in normal cortex was exclusively within the blood vessels and capillaries as reported by Friede 14 (Fig. 5). Fewer vessels containing enzyme activity were seen in the cortex of thyroidectomised rats and in many the staining was markedly less intensive (Fig. 6). CaZ+-adenosine triphosphatase. High activity of the Ca" ~-activated enzyme was distributed t h r o u g h o u t the blood vessels and capillaries of normal rats with a low diffuse extracellular enzyme activity elsewhere in the cortex; this finding is in agreement with that of N a i d o o and Pratt 2s and of Hess and Pope "2. Blood vessels and capillaries in the cortex of thyroidectomised rats showed less intensity o f staining but the reaction was less diffuse. Nerve cell bodies in the thyroidectomised rats were more easily identified than in normal cortex by an unreactive pericellular space surrounding a feeble cytoplasmic reaction. Mgg+-adenosine triphosphatase. In the neuropil of normal tissue this enzyme was moderately active and more easily detectable than the Ca'~+-activated enzyme. The cytoplasm of nerve cells exhibited a weak enzyme activity surrounding an unreactive nucleus. The capillaries showed a moderate activity. The relative activities
Brain Research, 9 (1968) 351 362
356
N. ROBINSON AND
~'. 45",'rl,~,
Fig. 5. Alkaline phosphatase actNit~ within the blood ~,essels of the cerebral corw~ ,~t a normal 30-day-old rat. No counterstain. 40. o f the Ca ~'- and Mg"T-activated enzymes were in general ag r eem en t ~ i t h thos,~recorded by Hess and PopeZL T h e cortical nerve cells, neuropil and capillaries o f t h y r o i d e c t o m i s e d rats exhibited a normal response.
5-Nucleotidase. T h e c y t o p l a s m o f normal cortical nerve cells exhibited a strong 5-nucleotidase activity. O l i g o d e n d r o c y t e s and nerve fibres also exhibited the m o d e r a t e activity previously reported by N a i d o o e6. but the neuropil showed no reaction.
Fig. 6. A similar intensity of alkaline pbosphatase activity in fewer blood vessels in the .,;ame cortical region of a 30-day-old rat thyroidectomised at birth. No counterstain. ~, 40.
Brain Research, 9 (1968) 351-362
357
HISTOCHEMISTRY OF CEREBRAL CORTEX
Fig. 7. Thiamine pyrophosphatase activity in the cerebral cortex ofa 30-day-oldratthyroidectomised at birth. Activity is depleted in capillaries but is normal in nerve cells. No counterstain. 250.
The only difference seen in the thyroidectomised rats was a more intense reaction within capillaries, other organelles showing a normal reaction. Thiamine pyrophosphatase. In the cortex of normal rats a strong enzyme reaction, seen as coarse granules, was apparent in the nerve cell cytoplasm, sometimes extending into the processes; surrounding neuropil showed a weak reaction but gliat cells were identified by a course cytoplasmic granulation. Capillaries and blood vessels exhibited an intense reaction. The thyroidectomised tissue showed a normal nerve cell activity but fewer capillaries exhibiting a normal reaction were seen (Fig. 7). Monoamine oxidase. Activity of this enzyme in the cortex of the normal rat was most prominent along the nerve fibres which presented a beaded appearance. Nerve cells showed the strongest intensity adjacent to the nuclear membrane but the cytoplasmic reaction was somewhat masked by a strong and diffuse staining within the neuropil (Fig. 8). Thyroidectomised rats showed a poor localisation of m o n o a m i n e oxidase activity in the nerve fibres the normal beaded appearance showing as independent nodules (Fig. 9). Nerve cells exhibited a normal cytoplasmic reaction but the neuropil was less intensely stained than in the normal tissues. Acetylcholinesterase. This enzyme was located mainly within the cytoplasm of the nerve cells of normal cortex usually terminating a short way along prominent processes. A faint reaction was evident along nerve fibres but none in neuropiI. These observations were in general agreement with those of Foldes e t a / . 12 and A d a m s et al. 1. Thyroidectomised rats showed no significant differences from the normal.
Brain~2Research,~9 ( 1968)351-362
358
N. ROBINSON ANI) ;. ;, LAYR.'-,
Fig. 8. M o n o a m i n e oxidase activity localised along nerve fibres in the cortex of a normal 30-day-old rat. N o counterstain. ~ 250.
DISCUSSION
It has become clear from histological evidence that important alterations in cortical structure are associated with the behavioural deficiencies characteristic of experimentally induced cretinism (for review see Eayrsg). There is good reason to believe that an impairment of the mechanisms underlying the synthesis of protein is an important factor mediating these changes and it is therefore likely that corresponding alterations may exist in some important enzymic proteins which control the metabolism of cortical tissue. This possibility is rendered more likely by the recent finding that, although there is close correspondence between the degree of behavioural and morphological impairment engendered by thyroidectomy undertaken at different stages of development, restoration of cortical structure as a result of replacement therapy is not always associated with behavioural recovery thus suggesting that enzymological factors which are not revealed by standard histological techniques are also involved 1°. Among the relatively few studies in this field the quantitative estimations of Brain Research, 9 (1968) 351-362
H1STOCHEMISTRYOF CEREBRALCORTEX
359
Fig. 9. Loss in localisation of nerve fibre monoamine oxidase activity in the same cortical region of a 30-day-old rat lhyroidectomised at birth. No counterstain, x 250.
aldolase, succinate dehydrogenase, cytochrome oxidase and acetylcholinesterase reported by Hamburgh and Flexner 21 indicated that the activities of succinate dehydrogenase and acetylcholinesterase in the 30-day-old rat thyroidectomised at birth had respectively decreased by 29 ~ and 11 ~ , the former effect being irreversible when therapy was delayed beyond the 15th postnatal day. These findings have since been confirmed and extended to include cholinesterase by Geel and Timiras 16 and reductions in the activities of glutamate dehydrogenase and of glutamate decarboxylase have likewise been reported by Balazs et aLL Although differences in activity of some of the 12 enzymes examined in 30-day-old hypothyroid rats were observed in the present study there was no one enzyme that demonstrated a gross abnormality suggestive of a principle factor involved in the metabolism of cortical nerve cells or other cortical elements of thyroidectomised rats (see Table I). In cortical nerve cells of the thyroidectomised rats small departures from the normal in localisation and intensity of the oxidative enzymes were apparent but the oxidation-reduction capacity of the cells may well be regarded as sufficient to maintain normal metabolic processes in view of the excess of enzyme capacity of the cells above Brain Research, 9 (1968) 351-362
360
,~. ROBINSONAN~ !
] "~ t~S
their maximum requirements for normal function ~4. Paucity of an axonal reaction probably reflects the morphological changes associated with cretinism 7` Acid phosphatase and 5-nucleotidase are known to show a decrease in nerve cell act~v~t~ in somL" pathological conditions 31.:3' and in this study this enzyme showed a markedly lower response in the nerve cell processes of thyroidectomised rat cortex compared with thal seen in normal cortex. Other depbosphorylating enzymes showed no significant differences within the nerve cells. The decrease in acetylcholinesterasc acttwtv. previously estimated by biochemical methods ~'t. was not detectable with confidence in the present study which showed a normal Iocalisation of the enzyme. The enzyme reactions tn glia showed no evidence of hyperactive astrocvte~ observed in several human pathological conditions 1:~.:~1.3'~.3~; and in rat+ followilqg experimentally produced local brain injury `'~. The alteration in physical texture of the neuropil did not appear to be reflected by any significant changes of enz3.me activit'v. Nerve fibres in the thyroidectomised rat cortex exhibited a loss of acid phosphatase activity and poor localisation of monoamine oxidase which normally appears as a line of interconnected nodules of formazan de posits along the fibre. Both of these alterations are thought to be related to the structural modifications seen histologically and may be important factors in a disturbance of the function of cortical neurone+. Two enzymes, alkaline phosphatase and Cae+-adenosine triphosphatase. localised within the vascular walls 14,~s were of interest in view of the increase in size of blood vessels and depletion of capillaries in the cortex of thyroidectomised rats. In the present study fewer vessels exhibited the normal localisation and activity. This may be related to the evidence of Eayrs <+who demonstrated hypoplasia and morphological alterations in the blood vessels of the cortex suggesting a lower level of metabolism. The lower level of adenosine Lriphosphatase activity in the th\ roidectomised rat brain would be compatible with the lower energy requirements ot impaired mobility. SUMMARY The localisation and activity of enzymes concerned in important metabolic pathways of the central nervous system have been examined in the cerebral cortex of 30-day-old rats thyroidectomised at birth and in their normal littermates. Absence of thyroid hormone from the time of birth gave rise to no gross abnormality affecting any one enzyme involved in the metabolism of cortical neurones or other cortical elements. On the other hand, several enzymes showed changes in localization and activity which have been compared with alterations occurring in other pathological and experimental conditions. ACKNOWLEDGMENT This work was supported by a grant from the Research Fund of the Bethlem Royal and Maudsley Hospitals which is gratefully acknowledged. The authors also wish to thank Mr. J. Mills, Institute of Neurology for the photographic work. Braht Research, 9 (1968) 351-362
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361
REFERENCES 1 ADAMS,C. W. M., DAVISON, A. N., AND GREGSON, N. A., Enzyme inactivity in myelin; histochemical and biochemical evidence, J. Neurochem., 10 (1963) 383--395. 2 BALAZS, R., KOVACS, S., TEICHGR~BER, P., COCKS, W., AND EAYRS, J. T., Effects of neonatal thyroid deficiency on the developing brain, Proc. First int. Neurochem. Syrup., (1967) abstr. 12. 3 BARRNETT, R. J., Quoted in G. PINCUS AND K. V. TmMANN (Eds.), The Hormones: Physiology, Chemistry and Applications, VoL 2, Academic Press, New York, 1948, p. 235. 4 BRADLEY, P. B., EAYRS, J. T., GLASS, A., AND HEATH, R. W., The maturation and metabolic consequences of neonatal tbyroidectomy upon the recruiting response in the rat, Electroenceph. olin. Neurophysiol., 13 (1961) 557-586. 5 BRADLEY, P. B., EAYP,S, J. T., AND SCHMALBACIt, K., The electroencephalogram of normal and thyroidectomised rats, Electroenceph. clin. Neurophysiol., 12 (1960) 467 477. 6 EAYRS,J. T., The vascularity of the cerebral cortex in normal and cretinous rats, J. Anat. (Lond.), 88 (1954) 164 173. 7 EAYRS, J. T., The cerebral cortex of normal and hypothyroid rats, Acta anat. (Basel), 25 (1955) 160 183. 8 EAYRS, J. T., Age as a factor determining the severity and reversibility of the effects of thyroid deprivation in the rat, J. Endocr., 22 (1961) 409 419. 9 EAYRS, J. T., Thyroid and central nervous development. In: Scientific Basis o f Medicine Annual Reviews, Ch. XIX, Athlone, London, 1966, pp. 317 339. 10 EAYRS, J. T., La influencia de la hormona tiroidea sobre el desarrollo del sistema nervioso, Rer. Nettro-psiquiat., 30 (1967) 117-136. 1 I EAYRS,J. T., AND TAYLOR, S. H., The effect of thyroid deficiency induced by methyl thiouracil on the maturation of the central nervous system, J. Anat. (Lond.), 85 (I 951) 350-358. 12 FOLDES F. F., ZSIGMOND, E. K., FOLDES, V. M., AND ERDOS, E. G., The distribution of acetylcholinesterase and butyrylcholinesterase in human brain, J. Nearochem., 9 (1962) 559-572. 13 FRIEDe R. L., The cytochemistry of normal and reactive astrocytes, J. Neuropath. exl). Nearol., 21 (1962) 471 478. 14 FRIEDE R. L., An enzyme histochemical study of cerebral arteriosclerosis, Acta neuropath. (Berl.), 2 (1962) 58 72. 15 BELL, S. E., AND ]'IMIRAS, P. S., The influence of neonatal hypothyroidism and thyroxine on the ribonucleic and deoxyribonucleic acid concentrations of rat cerebral cortex, Brain Research, 5(1967) 135 142. 16 GeEL, S. E., AND TIMIRAS, P. S., Influence of neonatal hypothyroidism and of thyroxine on the acetylcholinesterase and cholinesterase activities in the developing central nervous system of the rat, Endocrinology, 80 (1967) 1069 1074. 17 GLENNER,G. C., BURTNER, H. J., AND BROWN, G. W., The histochemical demonstration of monoamine oxidase activity by tetrazolium salts, J. Histochem. Cvtochem., 5 (1957) 591-600. 18 GOLI)BERG, R. C., AND CHA1KOFF,1. L., A simplified procedure for thyroidectomy of the newborn rat without concomitant parathyroidectomy, Endocrinology, 45 (1949) 64-70. 19 GOMORI, G., A new histochemical test for glycogen and mucin, Amer. J. olin. Path., 16, Tech. Sect. 7 (1946) 177 179. 20 GoMom, G., An improved histochcmical technique for acid phosphatase, Stahl TechnoL, 25 (1956) 81 87. 21 HAMBUR(;H, M., AND FLEXNER, L. B., Biochemical and physiological differentiation during morphogenesis. XXI., J. Neurochem., I (1957) 279-288. 22 Hess, H. H., AND POPE, A., Intralaminar distribution of adenosine triphosphatase activity in rat cerebral cortex, J. Neurochem., 3 (1959) 287-299. 23 KOeLLE, G. B., AND FRIEDENWALD, J. S., A histochemical method for localization of cholinesterase activity, Proc. Soc. exp. Biol. (N. Y.), 70 (1949) 617-625. 24 MCILWAIN, H., Biochemistry and the Central Nervotts System, 2nd ed., Churchill, London, 1959, p. 41. 25 MOSSAKOWSKI,M. J., 'The activity of succinic dehydrogenase in the reactive glia, Acta neuropath. (BerL), 3 (1963) 282-290. 26 NMDOO, D., The activity of 5-nucleotidase determined histochemically in the developing rat brain, J. Histochem. Cytochem., 10 (1962) 421 434. 27 NMDOO, D., Thiamine pyrophosphatase: a histochemical study, J. Histochern. Cvtochem., 10 (1962) 580-59 I.
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28 NAIDOO,D., AND PRATT, O. E., The effect of magnesium and calcium ions on adenosine tripilo~phatase in the nervous and vascular tissue of the brain, Biochem. J., 62 (1956) 465 46~). 29 N1LES, N. R., CHAYEN, J., CUNNINGHAM, G. J., AND BITENSKY, L., The histochcmical demonstration of adenosine triphosphatase activity in myocardium, J. Histochem. Cytochem , I 2 (1964) 740-743. 30 RAY, R. D., SIMPSON, M. E., LI, C. H., ASLI,",G, C. W., AND EVANS, H. M., Effects of the pituitar) growth hormone and of thyroxin on growth and differentiation of the skeleton of ~he rat thyroidectomized at birth, Amer. J. Anat., 80 (1950) 479-576. 3 l ROBINSON,N., Friedreich's ataxia: a histochemical and biochemical study, ll. Hydrolytic enzymes. Acta neuropath. (Berl.), 6 (1966) 35-45. 32 ROBINSON, N., A histochemical study of motor neurone disease, Acta neuropath. ([¢crl, L 7 (1966) 101-110.
33 Scow, R. O., AND SIMPSON, M. E., Thyroidectomy in the newborn rat, Anat. Rec.. 91 (1945) 209-226. 34 THOMAS, E., ANO PEARSE, A. G. E., The fine localisation of dehydrogenases in the nervous system, Histochemie, 2 (1961) 266-282. 35 WACHSTHN, M., ANt) MEISEL, E., Histochemistry of hepatic phosphatases at a ph)siological pH with special reference to the demonstration of bile canaliculi, Amer. J. clin. Path., 27 (1957) 13 -23. 36 WALLACE, B. J., VOLK, B. W., AND LAZARUS, S. S., Glial cell enzyme alterations in infantile amaurotic family idiocy (Tay-Sachs disease), J. Neurochem., 10 (1960) 439-446.
Brain Research, 9 (1968) 351-362
351
H1STOCHEMICAL STUDY OF THE CEREBRAL CORTEX IN RATS T H Y R O I D E C T O M I S E D AT BIRTH
N. ROBINSON ANDJ. T. EAYRS Departments of Chemical Pathology, Institute of Neurology and of Neuroendocrinology, hlstitute of Psychiatry, University of London, London (Great Britain)
(Accepted January 19th, 1968)
INTRODUCTION Developmental defects in mammals resulting from a deficiency of thyroid hormone early in life are now well established. Changes in bodily configuration have long been recognised 3°,33, and more recently attention has been paid to the influence of the thyroid on the maturation of the central nervous system. In this context irreversible behavioural impairment, associated with changes in the electrical activity of the brain4, ~ and with structural modifications involving the size of cortical perikarya al, the growth of neuropil v, the patterning of the capillary bed 6 and the deposition of myelin 3 has been shown to arise when thyroid deficiency is present during a critical period of development s. The biochemical correlates of these phenomena have, until recently, attracted little attention. Changes in the DNA/RNA ratio consistent with the earlier neurohistological findings have been reported2,15 while evidence has been provided indicating modification in the activities of several enzymes'~,16, 21. The aim of the present study has been to determine whether any changes in the localisation and activity of some of the enzymes controlling important metabolic pathways could be identified by use of histochemical techniques. The enzymes investigated have been those concerned with the capacity of the cerebral cortex for oxidation-reduction processes, energy transforrnation, nervous transmission, regulation of phosphates and amine oxidation. MATERIALSAND METHODS Albino rats of the Sprague-Dawley strain were used and one pair oflittermates from each of 6 litters provided the material for the histochemical observations. One animal of each pair was thyroidectomized on the day of birth by giving a single injection of 150-200/~C 131I (ref. 18). The other, acting as control, received the vehicle only. The rats were killed by decapitation at 30 days of age at which time the 6 thyroidectomized rats weighed 37-44 g and the controls 83-95 g. The brain was rapidly removed and instantly frozen to --70°C. Fresh frozen sections were cut between Brain Research, 9 (1968) 351-362
I"-J
~D
2" NORMAL
AND HYPOTHYROID
RAT CEREBRAL CORTEX
a-Glycerophosphate dehydrogenase D NADH~-diaphorase dehydrogenase D Succinate dehydrogenase W ~ = Glucose 6-phosphate dehydrogenas¢ D ..... Acid phosphatase P---~- ~ Alkaline phosphatase Mg~+-ATPase d Ca~+-ATPase 5-nucleofidase D Thiamine pyrophosphatase P Monoamine oxidase W Acetylcholinesterase W
N
N
d d
~
D WD-
d D÷ ~
d D-! ~-+
D P W W
d
d
d
d d
D
H
d
d
D
D
H
Neuropil
Nerve cells cytoplasm
P D
W
W d
W
N
Glial cells
P D
W
W d
W
H
D
D
D D--.
D
W
N
Nerve fibres
D
D
D
D
W
H
W
D p---P D P
W
P
W
D p~ P D P
W
P
Blood vessels and capillaries N H
N, normal cortex; H, hypothyroid cortex; P, prominent reaction, showing continuous heavy staining; D, distinct reaction, moderate well-defined staining; W, weak reaction, indistinct cytoarchitecture; d, diffuse reaction, homogeneous distribution, localisation poor. No symbols indicate reaction undetectable. The plus signs ( ) indicate relative intensities between normal and hypothyroid tissue. Where no plus signs, differences were undetectable.
LOCALISAT1ON AND RELATIVE INTENSITIES OF ENZYME ACTIVITIES IN THE 30-DAY-OLD
TABLE I
7-
2-
m
> z
Z
©
z
L/I b,9
HISTOCHEMISTRYOF CEREBRALCORTEX
353
10 and 14 # thick on a Pearse-Slee freezing microtome and the following enzymes were demonstrated histochemically: NADH2-diaphorase, a-glycerophosphate (menadione added), succinate (menadione added) and glucose 6-phosphate dehydrogenases34; acid phosphatase2°; alkaline phosphataselg; CaZ+-adenosine triphosphatase '~9; Mg2~-adenosine triphosphatase 3a; 5-nucleotidase z6; thiamine pyrophosphatase27; monoamine oxidasel7; and acetylcholinesterase 2a. Nucleic acids (gallocyanin-chromalum) and proteins (ninhydrin) were also stained. Sections from normal and thyroidectomised rats were simultaneously incubated in the same substrate medium and subsequently treated in the same staining medium to enable a direct comparison of the intensity of enzyme activity to be made. The cofactors in the dehydrogenase incubation media and the substrates for all other enzymes were omitted in the blanks. RESULTS The distribution and relative intensities of the enzyme activities in the cerebral cortex of normal and thyroidectomised rats are summarised in Table 1. The detailed findings are set out below. Oxidatire enzymes. The histochemical concentration of oxidative enzymes in the cytoplasm of nerve cells in the cerebral cortex of normal rats decreased in order NADH2-diaphorase, glucose 6-phosphate, succinate and (t-glycerophosphate dehydrogenase. In agreement with Thomas and Pearse 34 there was no detectable reaction in the nucleus or nucleolus (Fig. 1). A moderate reaction for these enzymes was observed in the neuropil and a-glycerophosphate dehydrogenase also exhibited a strong reaction in the capillaries. In the thyroidectomised animals the normal
Fig. I. NADH2-diaphorase in cortical nerve cell cytoplasm and dendrite of normal 30-day-old rat. No counterstain. × 400. Brain Research, 9 (1968) 351-362
354
y. ROBINSON AND J, I, I!AX~RS
<
Fig. 2. Same enzyme as Fig. 1 but showing the characteristic absence of a reaction ill the dendritcs of cortical nerve cells of 30-day-old rats thyroidectomised at birth. No coumerstain. 400. o r d e r o f staining intensity was retained and the nerve cells exhibited only a slight reduction in the staining reaction o f oxidative enzymes, seen mainly in lhe glucose 6 - p h o s p h a t e and succinate d e h y d r o g e n a s e activities. A strong reaction f o r N A D H e d i a p h o r a s e in the neuropil was present in both n o r m a l a n d experimental tissues but only in the n o r m a l were dendrites detectable by this m e t h o d (ty'~ Figs. i a n d 2). Acid phosphatase. Enzyme activity was distributed t h r o u g h o u t the c y t o p l a s m
Fig. 3. Acid phosphatase activity in the cerebral cortex ofthe normal 30-day-olcl rat. Nocounterstain.
× 250.
Brain Research, 9 (1968) 351-362
HISTOCHEMISTRYOF CEREBRALCORTEX
355
"4
p
Fig. 4. Reduced activity in cell processes and poor cytoplasmic localisation of acid phosphatase in the same cortical region of a 30-day-old rat thyroidectomised at birth. No counterstain, x 250. and larger processes of the nerve cells in normal tissue; activity was not present in the cell nuclei. Within each individual nerve cell the localisation of enzyme activity showed some cytoplasmic variation but the reaction from cell to cell was generally consistent for all cortical nerve cells. The enzyme activity of this enzyme in the neuropil was undetectable but oligodendrocytes frequently exhibited a moderate granulation. Some enzyme activity was observed along nerve fibres (Fig. 3). Acid phosphatase activity was of similar intensity to controls in the cortical nerve cell bodies of thyroidectomised rats but the distribution along the processes appeared markedly less consistent and intense (Fig. 4). Alkaline phosphatase. The activity of this enzyme in normal cortex was exclusively within the blood vessels and capillaries as reported by Friede 14 (Fig. 5). Fewer vessels containing enzyme activity were seen in the cortex of thyroidectomised rats and in many the staining was markedly less intensive (Fig. 6). CaZ+-adenosine triphosphatase. High activity of the Ca" ~-activated enzyme was distributed t h r o u g h o u t the blood vessels and capillaries of normal rats with a low diffuse extracellular enzyme activity elsewhere in the cortex; this finding is in agreement with that of N a i d o o and Pratt 2s and of Hess and Pope "2. Blood vessels and capillaries in the cortex of thyroidectomised rats showed less intensity o f staining but the reaction was less diffuse. Nerve cell bodies in the thyroidectomised rats were more easily identified than in normal cortex by an unreactive pericellular space surrounding a feeble cytoplasmic reaction. Mgg+-adenosine triphosphatase. In the neuropil of normal tissue this enzyme was moderately active and more easily detectable than the Ca'~+-activated enzyme. The cytoplasm of nerve cells exhibited a weak enzyme activity surrounding an unreactive nucleus. The capillaries showed a moderate activity. The relative activities
Brain Research, 9 (1968) 351 362
356
N. ROBINSON AND
~'. 45",'rl,~,
Fig. 5. Alkaline phosphatase actNit~ within the blood ~,essels of the cerebral corw~ ,~t a normal 30-day-old rat. No counterstain. 40. o f the Ca ~'- and Mg"T-activated enzymes were in general ag r eem en t ~ i t h thos,~recorded by Hess and PopeZL T h e cortical nerve cells, neuropil and capillaries o f t h y r o i d e c t o m i s e d rats exhibited a normal response.
5-Nucleotidase. T h e c y t o p l a s m o f normal cortical nerve cells exhibited a strong 5-nucleotidase activity. O l i g o d e n d r o c y t e s and nerve fibres also exhibited the m o d e r a t e activity previously reported by N a i d o o e6. but the neuropil showed no reaction.
Fig. 6. A similar intensity of alkaline pbosphatase activity in fewer blood vessels in the .,;ame cortical region of a 30-day-old rat thyroidectomised at birth. No counterstain. ~, 40.
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Fig. 7. Thiamine pyrophosphatase activity in the cerebral cortex ofa 30-day-oldratthyroidectomised at birth. Activity is depleted in capillaries but is normal in nerve cells. No counterstain. 250.
The only difference seen in the thyroidectomised rats was a more intense reaction within capillaries, other organelles showing a normal reaction. Thiamine pyrophosphatase. In the cortex of normal rats a strong enzyme reaction, seen as coarse granules, was apparent in the nerve cell cytoplasm, sometimes extending into the processes; surrounding neuropil showed a weak reaction but gliat cells were identified by a course cytoplasmic granulation. Capillaries and blood vessels exhibited an intense reaction. The thyroidectomised tissue showed a normal nerve cell activity but fewer capillaries exhibiting a normal reaction were seen (Fig. 7). Monoamine oxidase. Activity of this enzyme in the cortex of the normal rat was most prominent along the nerve fibres which presented a beaded appearance. Nerve cells showed the strongest intensity adjacent to the nuclear membrane but the cytoplasmic reaction was somewhat masked by a strong and diffuse staining within the neuropil (Fig. 8). Thyroidectomised rats showed a poor localisation of m o n o a m i n e oxidase activity in the nerve fibres the normal beaded appearance showing as independent nodules (Fig. 9). Nerve cells exhibited a normal cytoplasmic reaction but the neuropil was less intensely stained than in the normal tissues. Acetylcholinesterase. This enzyme was located mainly within the cytoplasm of the nerve cells of normal cortex usually terminating a short way along prominent processes. A faint reaction was evident along nerve fibres but none in neuropiI. These observations were in general agreement with those of Foldes e t a / . 12 and A d a m s et al. 1. Thyroidectomised rats showed no significant differences from the normal.
Brain~2Research,~9 ( 1968)351-362
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N. ROBINSON ANI) ;. ;, LAYR.'-,
Fig. 8. M o n o a m i n e oxidase activity localised along nerve fibres in the cortex of a normal 30-day-old rat. N o counterstain. ~ 250.
DISCUSSION
It has become clear from histological evidence that important alterations in cortical structure are associated with the behavioural deficiencies characteristic of experimentally induced cretinism (for review see Eayrsg). There is good reason to believe that an impairment of the mechanisms underlying the synthesis of protein is an important factor mediating these changes and it is therefore likely that corresponding alterations may exist in some important enzymic proteins which control the metabolism of cortical tissue. This possibility is rendered more likely by the recent finding that, although there is close correspondence between the degree of behavioural and morphological impairment engendered by thyroidectomy undertaken at different stages of development, restoration of cortical structure as a result of replacement therapy is not always associated with behavioural recovery thus suggesting that enzymological factors which are not revealed by standard histological techniques are also involved 1°. Among the relatively few studies in this field the quantitative estimations of Brain Research, 9 (1968) 351-362
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359
Fig. 9. Loss in localisation of nerve fibre monoamine oxidase activity in the same cortical region of a 30-day-old rat lhyroidectomised at birth. No counterstain, x 250.
aldolase, succinate dehydrogenase, cytochrome oxidase and acetylcholinesterase reported by Hamburgh and Flexner 21 indicated that the activities of succinate dehydrogenase and acetylcholinesterase in the 30-day-old rat thyroidectomised at birth had respectively decreased by 29 ~ and 11 ~ , the former effect being irreversible when therapy was delayed beyond the 15th postnatal day. These findings have since been confirmed and extended to include cholinesterase by Geel and Timiras 16 and reductions in the activities of glutamate dehydrogenase and of glutamate decarboxylase have likewise been reported by Balazs et aLL Although differences in activity of some of the 12 enzymes examined in 30-day-old hypothyroid rats were observed in the present study there was no one enzyme that demonstrated a gross abnormality suggestive of a principle factor involved in the metabolism of cortical nerve cells or other cortical elements of thyroidectomised rats (see Table I). In cortical nerve cells of the thyroidectomised rats small departures from the normal in localisation and intensity of the oxidative enzymes were apparent but the oxidation-reduction capacity of the cells may well be regarded as sufficient to maintain normal metabolic processes in view of the excess of enzyme capacity of the cells above Brain Research, 9 (1968) 351-362
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,~. ROBINSONAN~ !
] "~ t~S
their maximum requirements for normal function ~4. Paucity of an axonal reaction probably reflects the morphological changes associated with cretinism 7` Acid phosphatase and 5-nucleotidase are known to show a decrease in nerve cell act~v~t~ in somL" pathological conditions 31.:3' and in this study this enzyme showed a markedly lower response in the nerve cell processes of thyroidectomised rat cortex compared with thal seen in normal cortex. Other depbosphorylating enzymes showed no significant differences within the nerve cells. The decrease in acetylcholinesterasc acttwtv. previously estimated by biochemical methods ~'t. was not detectable with confidence in the present study which showed a normal Iocalisation of the enzyme. The enzyme reactions tn glia showed no evidence of hyperactive astrocvte~ observed in several human pathological conditions 1:~.:~1.3'~.3~; and in rat+ followilqg experimentally produced local brain injury `'~. The alteration in physical texture of the neuropil did not appear to be reflected by any significant changes of enz3.me activit'v. Nerve fibres in the thyroidectomised rat cortex exhibited a loss of acid phosphatase activity and poor localisation of monoamine oxidase which normally appears as a line of interconnected nodules of formazan de posits along the fibre. Both of these alterations are thought to be related to the structural modifications seen histologically and may be important factors in a disturbance of the function of cortical neurone+. Two enzymes, alkaline phosphatase and Cae+-adenosine triphosphatase. localised within the vascular walls 14,~s were of interest in view of the increase in size of blood vessels and depletion of capillaries in the cortex of thyroidectomised rats. In the present study fewer vessels exhibited the normal localisation and activity. This may be related to the evidence of Eayrs <+who demonstrated hypoplasia and morphological alterations in the blood vessels of the cortex suggesting a lower level of metabolism. The lower level of adenosine Lriphosphatase activity in the th\ roidectomised rat brain would be compatible with the lower energy requirements ot impaired mobility. SUMMARY The localisation and activity of enzymes concerned in important metabolic pathways of the central nervous system have been examined in the cerebral cortex of 30-day-old rats thyroidectomised at birth and in their normal littermates. Absence of thyroid hormone from the time of birth gave rise to no gross abnormality affecting any one enzyme involved in the metabolism of cortical neurones or other cortical elements. On the other hand, several enzymes showed changes in localization and activity which have been compared with alterations occurring in other pathological and experimental conditions. ACKNOWLEDGMENT This work was supported by a grant from the Research Fund of the Bethlem Royal and Maudsley Hospitals which is gratefully acknowledged. The authors also wish to thank Mr. J. Mills, Institute of Neurology for the photographic work. Braht Research, 9 (1968) 351-362
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33 Scow, R. O., AND SIMPSON, M. E., Thyroidectomy in the newborn rat, Anat. Rec.. 91 (1945) 209-226. 34 THOMAS, E., ANO PEARSE, A. G. E., The fine localisation of dehydrogenases in the nervous system, Histochemie, 2 (1961) 266-282. 35 WACHSTHN, M., ANt) MEISEL, E., Histochemistry of hepatic phosphatases at a ph)siological pH with special reference to the demonstration of bile canaliculi, Amer. J. clin. Path., 27 (1957) 13 -23. 36 WALLACE, B. J., VOLK, B. W., AND LAZARUS, S. S., Glial cell enzyme alterations in infantile amaurotic family idiocy (Tay-Sachs disease), J. Neurochem., 10 (1960) 439-446.
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