Enzyme histochemistry of glutamate dehydrogenase in ageing rat cerebellar cortex

Mechanisms o f Ageing and Development, 47 (1989) 199--205


Elsevier Scientific Publishers Ireland Ltd.


L A U R A FELICI% E L E N A B R O N Z E T T I a and F R A N C E S C O A M E N T A a,b aDipartimento di Scienze Neurlogiche, Universita "'La Sapienza ""and bDipartimento di Sanit~ Pubblica e Biologia Cellulare, Universitk "Tor Vergata "" Roma (Italy)

(Received February 6th, 1988) (Revision receivedAugust 10th, 1988)

SUMMARY The influence o f ageing on glutamate dehydrogenase activity was studied in the cerebellar cortex o f 3-month-old (young), 12-month-old (adult) and 26-month-old (aged) male Sprague--Dawley rats by using an enzyme histochemical technique. In young rats the enzyme reactivity was observed in the neuropil of the molecular layer as well as in the perikarya of basket cells and of stellate cells; within the cytoplasm of Purkinje neurons and in synaptic glomeruli of the granular layer. Glutamate dehydrogenase activity was significantly increased in the cerebellar cortex of adult rats and decreased in old animals. The synaptic glomeruli of the granular layer were the structures of the cerebellar cortex more remarkably affected by age-related changes. The possibility that decreased glutamate catabolism occurring in the ageing cerebellar cortex may result in an excess of the amino acid and may contribute to the nerve cell loss occurring in the cerebellum o f old rats is discussed.

K e y w o r d s : Ageing; Cerebellar cortex; Glutamate dehydrogenase; Microphotome-

try; Rat INTRODUCTION Increasing electrophysiological and neurochemical evidence indicates that L-glutamate may represent an excitatory transmitter used by afferent inputs and interneutons in the central nervous system (for a review see [1]). Moreover L-glutamate plays an important role in cerebral metabolism. In fact the amino acid is involved in cereAddress all correspondence to: Dr. Francesco Amenta, Dipartimento di Scienze Neurologiche, Via A. Borelli, 50, 00161 Rome, Italy. 004%6374/89/$03.50 Printed and Published in Ireland

© 1989Elsevier Scientific Publishers Ireland Ltd.

200 bral energy metabolism via tricarboxylic acid and a-ketoglutarate pathways [2,3]; it is incorporated into both neuronal and glial proteins and peptides [4,5]; it is a precursor of gamma-aminobutyric acid (GABA) [6]. Finally, the glutamate-glutamine system regulates the ammonia levels in the brain [7]. Age-related changes in glutamate metabolism have been described by some authors who have found decreased levels of glutamic acid [8] and decreased glutamate dehydrogenase (GDH) activity [9] in the brain of aged rats. These data, however were only partly supported by more recent investigations reporting a moderate decrease in glutamate levels in brain of 30-month-old in comparison with 3- and 12month-old rats and no important age-related changes in cerebral metabolism of Lglutamate [10]. Therefore increased release of the amino acid induced by depolarization has been reported in the brain of aged subjects and animals [11]. The cerebellar cortex, which is a brain structure impaired in old age [12], is rich in L-glutamate that seems to be the main excitatory neurotransmitter of granule cells [13]. It has been demonstrated that increasing levels, systemic or local injection of excitatory amino acids such as L-glutamate induce brain lesions and nerve cell loss or impairment [14,15]. We have therefore analyzed whether GDH activity, the main enzyme involved in L-glutamate catabolism [16], was changed in the cerebellar cortex of ageing rat. MATERIALSAND METHODS Male Sprague--Dawley rats of 3 months of age (considered to be young), of 12 months of age (considered to be adult) and of 26 months of age (considered to be old) were used in the present study. The average life span of the rat colony used was 25 months. Ten animals for each age group were killed by decapitation under ether anaesthesia. The brain was removed and the brain-stem and over-lying cerebellum were detached from the rest of the brain. The right cerebellar hemisphere was dissected by a cut in the frontal plane between lobules VII--VIII. Blocks comprising lobule II--VII samples were frozen in a solid CO2-acetone mixture after embedding in cryoprotectant (OCT compound, Ames, U.S.A.). Serial 10/am thick sections were cut using a - 2 0 ° C microtome cryostat and mounted on cover slips. The sections were then air dried and processed for the demonstration of GDH (E.C. according to the technique proposed by Andersen and Contestabile [17] using nicotinamide-adenine-dinucleotide as coenzyme and nitro blue tetrazolium (NBT grade III, Sigma U.S.A.) as the tetrazolium salt. The specificity of the histochemical reaction was tested by incubating some sections in blanks in which the coenzyme or NBT were omitted, Parallel sections not processed for GDH histochemistry were stained with toluidine blue to verify microanatomical details. The intensity of GDH staining within the neuropil of the molecular layer, the cytoplasm of Purkinje neurons and the synaptic glomeruli of the granular layer was assessed microphotometrically according to the technique described in earlier papers [18--21]. Briefly, GDH activity was measured with a Fluoval Photometric unit at a wavelength of 550 nm. The


microphotometer was calibrated taking as " z e r o " the value of control sections incubated without co-factors. The microphotometric unit contained an adjustable circular diaphragm which permitted optical separation of the structure to be measured for transmittance from its surroundings. Each measurement was performed using a 40 x / 0 . 9 5 immersion objective and a 12.5 x ocular. The determinations were made in a circular area of 3 - - 6 / ~ m in diameter de-lineated by measuring the diaphragm at the centre of an ocular. For measurements o f the neuropil of the molecular layer a fixed size field o f 3/~m in diameter was used. Other determinations were made by regulating the measuring diaphragm to cover the cytoplasm o f a Purkinje neuron or the area of a synaptic glomerulus at a specific time. Analysis o f variance (ANOVA) and two-tailed Student's t-test for unpaired data were used to assess significance o f differences a m o n g the three age groups. RESULTS

In the cerebellar cortex o f young animals a weak G D H activity was found in the neuropil of the molecular layer (Table I). A slightly more elevated activity was seen in the perikarya of stellate and basket cells. Weak to moderate activity was observed in the cytoplasm of Purkinje neurons. In the granular layer a strong activity was seen within synaptic glomeruli o f the granular layer (Table I). No G D H activity was found in the white matter. The pattern of G D H localization was the same in the cerebellar cortex of adult rats (Fig. la and Table I). The G D H activity in 12-month-old rats was significantly higher than in young animals o f about 19°70 (P < 0.05) in the molecular layer, of about 17o70 ( P < 0.05) in Purkinje neurons and of about 21o70 ( P < 0.01) in synaptic glomeruli of the granular layer (Figs. 1, 2 and Table I). In the cerebellar cortex o f old rats there was a significant loss of G D H activity in comparison with adult animals (Fig. lb), of about 24070 (P < 0.01) in the molecular layer, of about 19o70 (P < 0.05) in Purkinje neurons and o f about 3207o (P < 0.001) in synaptic glomeruli o f the granular layer (Figs. 1b, 2 and Table I). TABLE I M I C R O P H O T O M E T R I C D E T E R M I N A T I O N OF G L U T A M A T E D E H Y D R O G E N A S E ACTIVITY W I T H I N D I F F E R E N T LAYERS OF R A T C E R E B E L L A R C O R T E X

Molecular layer Purkinjeneuron Granular layer




(n = 10)

(n = 10)

(n = 10)

23.00 _ 2.57 19.07 ± 1.01 28.02 _+ 3.21

28.64 ± 2.28" 22.31 ± 2.02" 35.68 ± 3.13b

21.77 ± 2.04c 18.14 ± 2.17d 24.24 ± 2.68'

' P < 0.05 vs. young, b p < 0.01 VS. young, c p < 0.01 vs. adult, dp<~ 0.05 vs. adult, ¢P< 0.001 vs. adult. The values represent the intensity of glutamate dehydrogenase reaction and are expressed in arbitrary units (see Material and Methods). The values are the m e a n s + S.D. of 50 r a n d o m microphotometric determinations per animal for the neuropil of the molecular layer, for the cytoplasm o f Purkinje neurons and for the synaptic glomeruli o f the granular layer. N u m b e r o f animals (n) is given in brackets.


b Fig. 1. Rat cerebeliar cortex. GDH histochemistry, a = 12-month-old rat; b = 26-month-old rat. I h c enzyme reactivity is localized in the neuropil of the molecular layer (m), in the cytoplasm of Purkinje neurons (located between the molecular and the granular layers) and in the glomeruli (arrows) of the granular layer (g). Note the remarkable decrease of enzyme reactivity occurring within the various layers of the cerebellar cortex of 26-month-old rats and primarily in correspondence of the glomeruli of the granular layer. ( × 600). DISCUSSION

The main age-related changes occurring within the cerebellar cortex consist in the reduction in the number and in the width of the dendritic tree of Purkinje neu-


% 40








Fig. 2. Rat cerebellar cortex. Histograms o f the distribution of the intensity o f G D H reactivity (abscissae) within glomeruli of the granular layer. Y = 3-month-old; A = 12-month-old; O = 26-month-old. Ordinate: percentages o f the glomeruli (see Materials and Methods) examined. As can be seen, the intensity of enzymatic reactivity o f synaptic glomeruli in the three age groups examined is not the same, with areas showing a stronger reactivity than others.

rons [12,22,24,25], in a significant loss o f synapses in the molecular and granular layers [12,26], in the reduction of some enzymatic activities related to energy transduction [21] and in the loss o f granular neurons (unpublished results). In contrast, much less information is available as to the possible factors determining these changes. In view o f the recognized neurotoxic action o f an excess of L-glutamate [14,15], we investigated whether G D H undergoes age-dependent changes. The most recent studies on the ageing o f the glutamate system were centered primarily on rat brain where conflicting data were reported (see introduction). In the cerebellum L-glutamate levels were unchanged in four groups o f rats ranging from 2 to 30 months o f age [27]. G D H activity behaviour in senescence is debated, with reports describing a reduction [9,28] or no changes of the enzyme activity in subcellular fractions o f old rat whole brain [29]. In the present study a quantitative histochemical technique was used to assess age-related changes o f G D H in the different kinds of nerve cells of the cerebellar

204 cortex. O u r results d e m o n s t r a t e , in a g r e e m e n t with earlier findings o f o t h e r a u t h o r s [30,31], the highest G D H activity w i t h i n the s y n a p t i c g l o m e r u l i o f the g r a n u l a r layer f o l l o w e d , in o r d e r , b y the n e u r o p i l o f the m o l e c u l a r layer a n d b y P u r k i n j e n e u r o n s (Table I). T h e e n z y m e a c t i v i t y s i g n i f i c a n t l y increases in the d i f f e r e n t layers o f the cerebellar c o r t e x o f a d u l t rats a n d decreases in o l d a n i m a l s below levels f o u n d in y o u n g rats ( T a b l e I). L - G l u t a m a t e levels have b e e n r e p o r t e d to be u n c h a n g e d with age in rat c e r e b e l l u m [27]. H e n c e the increase in G D H activity f o u n d in a d u l t rats m a y b e related to the d e c r e a s e in s t r e n g t h o f the n e u r o n a l signals m e d i a t e d b y L-glutam a t e b y a m o r e efficient c a t a b o l i s m o f the a m i n o acid. In c o n t r a s t , in the cerebellar c o r t e x o f a g e d rats G D H activity is significantly r e d u c e d . This argues for i m p a i r e d t g l u t a m a t e d e g r a d a t i n g m e c h a n i s m s in the cerebellar c o r t e x o f o l d rats. In view o f the n e u r o t o x i c a c t i o n o f a n excess o f L - g l u t a m a t e on nerve cells [14], it c a n n o t be e x c l u d e d t h a t r e d u c e d G D H activity in the cerebellar c o r t e x o f 2 6 - m o n t h - o l d rats m a y allow to tissue stores o f i n a c t i v a t e d L - g l u t a m a t e to serve as an e x c i t o t o x i n [11] in ageing cerebellar cortex. T h u s m i c r o s t r u c t u r a l changes o c c u r r i n g in the cerebellar cortex in o l d age m a y b e due also to the n e u r o t o x i c a c t i o n o f n o t fully inactivated e n d o g e n o u s L-glutamate, as has been d e s c r i b e d f o r d e g e n e r a t i v e n e u r o l o g i c a l disorders in G D H - d e f i c i e n t p a t i e n t s [32]. ACKNOWLEDGMENTS T h e p r e s e n t s t u d y was s u p p o r t e d b y a g r a n t o f the U n i v e r s i t y " L a S a p i e n z a " ( P r o g e t t i di F a c o l t h ) . REFERENCES 1 D.R. Curtis and S.A.R. Johnston, Amino acid transmitters in the mammalian central nervous system. Ergeb. Physiol., 69 (1974) 97--188. 2 C.J. Van den Berg, P. Mela and H. Waelsch, On the contribution of the tricarboxylic acid cycle to the synthesis of glutamate, glutamine and aspartate in brain. Biochem. Biophys. Res. Commun., 23 (1966) 479--484. 3 C.J. Van den Berg, L.J. Krtalic, P. Mela and H. Waelsch, Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain. Biochem. J., 113 (1969) 281--290. 4 K.L. Reichelt and E. Kvamme, Acetylated and peptide bound glutamate and aspartate in brain. J. Neurochem., 14 (1967) 987--996. 5 C. Blomstrand and A. Hamberger, Amino acid incorporation in vitro into proteins of neuronal and gliai cell-enriched fractions. J. Neurochem., 17 (1970) 1187-- 1195. 6 N. Seiler and G. Wagner, NAD-dependent formation of y-aminobutyrate (GABA) from glutamate. Neurochem. Res., 1 (1976) 113-- 131. 7 S. Berl, G. Takagaki, D.D. Clarke and H. Waelsch, Metabolic compartments in vivo. Ammonia and glutamic acid metabolism in brain and liver. J. Biol. Chem., 23 7 (1962) 2562--2569. 8 J. Davis and W.A. Himwich, Neurochemistry of the developing and aging mammalian brain. In J.M. Ordy and K.R. Brizee (eds.), Adv. Behav. Biol. (Neurobiology o f Aging) Plenum Press, New York, 1975, pp. 329--357. 9 M.S. Kanungo and G. Kaur, Regulatory changes in enzymes as a function of age of the rat. Proc. 8th Intern. Congr. Geront., 1 (1969) 356--359.

205 10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26


28 29 30 31 32

I.F. De Konig-Verest, Glutamate metabolism in ageing rat brain. Mech. Ageing Dev., 13 (1980) 83 --92. D.M. Bowen, C.C.T. Smith and A.N. Davison, Excitotoxicity in aging and dementia. In D.M. Bowen (ed.), Excitotoxins J. Wiley and Sons, New York, 1985, pp. 39--46. J. Rogers, S.F. Zornetzer, F.E. Bloom and R.E. Mervis, Senescent microstructural changes in rat cerebellum. Brain Res., 292 (1984) 23--32. G.E. Fagg and A.C. Forster, Amino acid neurotransmitters and their pathways in the mammalian central nervous system. Neuroscience, 9 (1983) 701--719. J.W. Olney, Excitotoxic mechanism of neurotoxicity. In P.S. Spencer and H.H. Shaumberg (eds.), Experimentaland ClinicaINeurotoxicity, Williams and Wilkins, Baltimore, 1980, pp. 272--294. J.W. Olney, Excitotoxins: an overview. In K. Fuxe, P. Roberts and R. Schwartz (eds.), Excitotoxins, Vol. 39, Werner-Oreen. Int. Symp. Ser., Mac Millan, London, 1985, pp. 82--96. L.S. Salganigoff and E. De Robertis, Subcellular distribution of the enzymes of the glutamic acid, glutamine and gamma-aminobutyric acid cycles in rat brain. J. Neurochem., 12 (1965) 287--309. H. Andersen and A. Contestabile, The pyridine nucleotide and non pyridine nucleotide dependence of L-glutamate dehydrogenase in the histochemical system. Histochemistry, 53 (1977) 117--133. F. Amenta, C. Cavallotti, M. De Rossi, G. Bossoni and C. Carpi, Effect of acetyl-l-carnitine treatment on some behavioural, histochemical and histological parameters of metylazoxymethanol microencephalic rats. Int. J. Tissue React., 8(1986) 513--526. M. Meco, V. Bonifati, W.L. Collier, M.T. Ramacci and F. Amenta, Enzyme histochemistry of monoamine oxidase in the heart of aged rats. Mech. Ageing Dev., 38 (1987) 145--155. F. Ferrante and F. Amenta, Enzyme histochemistry of the choroid plexus in old rats. Mech. Ageing Dev., 41 (1987) 65--72. E. Bronzetti, L. Felici, F. Ferrante and F. Amenta, Age-related changes of the metabolic profile of rat cerebellar cortex: Enzyme bistochemical study. Mech. Ageing Dev., 44 (1988) 277--286. R.S. Ellis, Norms for some structural changes in the human cerebellum from birth to old age. J. Comp. Neurol., 32 (1920) 1--33. J. Rogers, S. Zornetzer and F.E, Bloom, Senescent pathology of cerebellum: Purkinje neurons and their parallel fiber efferents. Neurobiol. Aging, 2 (1981) 15--25. U. Ricken, H. Heinsen and Y.L. Heinsen, Quantitative studies in ageing male and female Chbb: Thom (Wistar) rats. Bibl. Anat., 28(1986) 106--120. R.J. Pentney, Quantitative analysis of dendritic networks of Purkinje neurons during aging. Neurobiol. Aging, 7 (1986) 241--248. C. Bertoni-Freddari, C. Giuli, C. Pieri and D. Pace, Age-related morphological rearrangement of synaptic junctions in the rat cerebellum and hyppocampus. Arch. Gerontol. Geriatr., 5 (1986) 297-304. P.S. Timiras, D.B. Hudson and S. Oklund, Changes in central nervous system free amino acids with development and aging. In D.H. Ford (ed). Neurobiological Aspects o f Maturation and Ageing (Progr. Brain Res. 4), Elsevier, Amsterdam, 1973, pp. 267--275. D.R. Deshmukh and M.S. Patel, Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain. Mech. Ageing Dev., 13 (1980) 75--81. J. Vitorica, A. Andr6s, J. Satrustegui and A. Machado, Age-related quantitative changes in enzyme activities of rat brain. Neurochem. Res., 6(1981) 127--135. R. Martinez Rodriquez, A histochemical study of some aspects of the metabolism of glutamic acid in the cerebellum of mammals. Acta Histochem., 49 (1974) 74--82. A. Contestabile and H. Andersen, Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases. Histochemistry, 56 (1978) 117-- 132. A. Plaitakis, S. Berl and M.D. Yahr, Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder. Science, 216(1982) 193--196.