Neurochemical changes of long-term adrenalectomy in rat brain: Effects on neurotransmitter amino acids

0736-5748/92 $5.00+0.00 Pergamon Press Ltd. © 1992 ISDN

Int. J. Devl. Neuroscience, Vol. 10, No. 5, pp. 439-445, 1992

Printed in Great Britain.

N E U R O C H E M I C A L C H A N G E S OF L O N G - T E R M A D R E N A L E C T O M Y IN R A T B R A I N : EFFECTS ON N E U R O T R A N S M I T T E R AMINO ACIDS N. ZONTA,* P. FERRARIO,A. M. DI GIULIO, L. ZECCA,t A. GORIOand P. MANTEGAZZA Dipartimento di Farmacologia,Chemioterapiae TossicologiaMedica,Universit~di Milano,via Vanvitelli32, 20129 Milano,Italyand tCNR--ITBA, via Amp&e 56, 20131 Milano, Italy (Received 30 April 1992; revised 13 July 1992;accepted 23 July 1992) Abstraet-- The levels of five amino acids together with glutamine synthetase activity, were measured in brain regions of rats with bilateral adrenalectomy, performed in newly weaning rats on postnatal day 22 and sacrificed 3 months later. Adrenalectomy caused a general decrease of glutamine concentration in three hippocampal regions (CA1-CA2, CA3, CA4-dentate gyrus), in hypothalamus, striatum and cerebellum. This reduction, which was particularly significant in hippocampus and cerebellum, was paralleled by a decrease of glutamine synthetase activity. Treatment with corticosterone reversed the effect of adrenalectomy. Little or no change was observed in the tissue levels of taurine, aspartic, glutamic or gamma-amino butyric acids.

Glucocorticoids (GCs) play a variety of roles, including neuro- and gliogenesis, in the CNS of developing and adult r a t s . 1'7'9'1°'16'29 A major neural target of steroid hormones is the hippocampal formation. This region, where high concentrations of both type I and type II adrenal steroid receptors are present, 6'2° is known to be involved in the processes of learning, memory and neuroendocrine function. 17,25,28 It is well-known that chronic exposure to corticosteroid hormone can damage neurons and exacerbate neurotoxic insults to hippocampus. 24 On the other hand, a prolonged exposure to reduced levels of GCs delays the age related loss of hippocampal neurons. 12 These data are apparently non-coherent with some recent reports suggesting that endogenous steroid hormones enhance the survival of specific hippocampal neurons.8'13'27 Sloviter and co-workers observed that adrenalectomy of adult rats causes a massive neuronal loss in the dentate gyrus (DG) of hippocampal formation at 3 months. 27 To study possible biochemical and morphological changes associated with this neuronal cell death and to evaluate the CNS development in rats with bilateral adrenalectomy, performed at weaning age, we have measured the levels of neurotransmitter amino acids (AAs), taurine (TAU), glutamine (GLN), aspartic (ASP), glutamic (GLU) and gamma-amino butyric (GABA) acids, in three hippocampal regions (CA1-CA2, CA3 and CA4-dentate gyrus), in hypothalamus, striatum and cerebellum of rats sacrificed 3 months after surgery. In the same brain areas, we have also determined glutamine synthetase (GS) activity, since GCs have been reported to regulate the activity of this enzyme in the developing rat brain. 18 EXPERIMENTAL PROCEDURES Animals and treatment

Young male Sprague-Dawley rats (Charles River) were used. Under barbiturate anaesthesia (Nembutal 35 mg/kg i.p.) postnatal day 22 rats underwent bilateral adrenalectomy (ADX group) or sham operation (sham group), via the dorsal approach. Completeness of surgery was verified by autopsy at the end of the experiment. One group of rats served as control and did not undergo any kind of treatment (control group). After surgery, animals were individually housed at a constant temperature (22-23°C), standard pellet food and water were provided ad libitum. *Author to whom correspondence should be addressed at: Dip. Farmacologia, Chemioterapia e Tossicoiogia Medica via Vanvitelli 32, 20129 Milano, Italy. Abbreviations: AAs, amino acids; ADX, adrenalectomized; ASP, aspartic acid; CB, cerebellum; CORT, corticosterone-21 acetate; DG, dentate gyrus; G A B A , gamma-amino butyric acid; GCs, glucocorticoids; GLN, glutamine; GLU, glutamic acid; GS, glutamine synthetase; HI, hippocampus; HY, hypothalamus; ST, striatum; TAU, taurine.

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Adrenalectomized rats were maintained for 3 months with isotonic saline solution, in place of the usual drinking water provided to the control and sham groups. To prevent the death that can occur in A D X rats immediately after adrenalectomy, corticosterone-21 acetate (CORT) was added to the saline drinking solution at a final concentration of 3 mg/l of corticosterone free base. C O R T concentration was progressively halved every 2 days until the 6th day, when C O R T treatment was suspended. Treatment with the hormone at the same concentration of 3 mg/l was continued for the duration of the experiment in adrenalectomized rats ( A D X + C O R T group), to determine the effect of C O R T replacement. Survival, behaviour, body weight and drinking solution intake were monitored every other day to ascertain the real oral dose of C O R T treatment. This dose was derived as the mean of the daily drinking solution intake normalized for body weight. Biochemical assays Determination o f neurotransmitter amino acids'. AAs were quantified by the H P L C method of Zecca and Ferrario 31 with minor modifications. Three months after surgery, between 1.00 and 5.30 p.m., 10 rats from each group were killed by exposing their heads for 7 sec to high-energy microwave radiations (oven: 2.0 kW, 2.45 GHz, 75 W/cm2; Medical Engineering Consultants, U.S.A.). The striatum, hippocampus, hypothalamus and cerebellum were dissected, placed on an ice-cold Petri dish, weighed, frozen on dry ice and stored at -80°C until assayed. The hippocampus was unfolded along the septotemporal axis and divided into three parts containing the C A 1 - C A 2 pyramidal neurons, the CA3 and the D G regions. The tissue was homogenized with an ultrasonicator (Soniprep 150, MSE) with 30-15-15-10 ml/g of 80% methanol, containing ascorbic acid (0.4 mM) as antioxidant and 5-amino-valeric acid (300-100 mM) as internal standard for AAs assay. Homogenate was centrifuged at 1500 g for 10 min and the supernatant stored at -80°C. A supernatant aliquot of 100 Ixl of each sample was introduced into a 10 ml screw-capped tube and evaporated under nitrogen flow, then derivatized with 35 M of NaHCO3 0.1 M and 80 pA of dansyl chloride 0.5% in acetone at 90°C for 15 min. A Series 10 Perkin-Elmer liquid chromatograph with a variable wavelength UV detector LC/ 250 was employed. The injection valve was a model 7125 (Rheodyne, Berkeley, CA, U.S.A.). The system was connected to a CR4A Shimadzu integrator. A C8 Pecosphere column (3 Ixm particle size; 8 cm x 46 mm I.D.) from Perkin-Elmer was operated at room temperature. The mobile phase was acetonitrile (19%) in acetate buffer (0.25 mM sodium acetate). The buffer was adjusted to pH = 3.25 with orthophosphoric acid. The flow rate was 1.8 ml/min and the eluted dansyl derivatives were monitored at 254 nm. Determination of glutamine synthetase activity. Ten rats from each group were sacrificed by decapitation. The brains were immediately removed and washed with ice-cold saline solution. Hippocampus, hypothalamus, striatum and cerebellum were dissected out at 2-4°C. Tissue was weighed, frozen on dry ice and stored at - 8 0 ° C until GS assay. Tissue was homogenized with a I K A - U T homogenizer with 10 ml/g of ice-cold glass distilled water and centrifuged at 1500 g for 10 min at room temperature. The determination of GS activity was carried out by the method of Rowe et al. z2 A 100 Ixl aliquot of homogenate was incubated for 15 rain at 37°C with 900 ~xl of solution at p H = 7.2 containing: imidazole-HC1 (55.5 mM), MgClz (22.2 mM), 2-mercaptoethanol (27.8 mM), sodium glutamate (55.5 mM), hydroxylamine-HC1 (111 mM) and A T P (11.1 mM). The reaction was stopped with 1.5 ml of ferric chloride reagent (FeCI3 0.37 M, T C A 0.20 M, HCI 0.67 M). To remove the precipitated protein, the tube contents were centrifuged at 1500 g for 10 min at room temperature and the absorbancy of 535 nm of the supernatant was read against a reagent blank from which A T P was omitted. A calibration curve was prepared with known standards of gamma-glutamyl hydroxamic acid. Enzyme activity was expressed as micro moles per gramme of wet weight per hour of gammaglutamyl hydroxamate formed. Histology Eight rats from each group were sacrificed by decapitation. The brain was removed and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at pH = 7.4 for 24 h. After 30% sucrose

Neurochemical changes of long-term adrenalectomy in rat brain

441

immersion, tissues were frozen at - 2 0 ° C and 20 ixm-thick sections were cut with a cryotome (Frigocut 2700 Reichert-Jung). Sections were stained with 0.1% cresyl violet for 10 min.

Materials Corticosterone-21 acetate, taurine, glutamine, aspartic acid, glutamic acid, G A B A , 5-amino valeric acid, dansyl chloride, sodium heptanesulphonate, gamma-glutamyl hydroxamate were obtained from S I G M A (St Louis, M O , U.S.A.). All other chemical reagents and solvents were of chromatography and analytical grade (Farmitalia-Carlo Erba, Milan, Italy).

Statistical Statistical evaluation of results was carried out by the analysis of variance, and comparisons between groups were p e r f o r m e d using the Duncan test. 4 RESULTS All adrenalectomized rats survived for 3 months although they appeared apathetic and hypotonic at manipulation.

Body and brain weight A d r e n a l e c t o m y caused a reduction in body growth of the A D X rats. Three months after surgery, on the day of sacrifice, this reduction was - 2 0 . 7 % ( P - 0 . 0 0 1 vs controls and sham) as reported in Table 1 and Fig. 1. The retardation in body growth was significantly reversed (P-<0.001 vs A D X ) by the chronic replacement of C O R T in A D X + C O R T group. Considering the C O R T concentration of drinking solution and its daily intake, normalized for body weight, the effective mean oral dose of C O R T replacement was 1 mg/kg/day over the 3 month survival period. Table 1. Body weight, brain and cerebellar wet weight in adrenalectomized rats (M + SEM) Final body weight (g) (n = 20) Controls Sham ADX ADX+ CORT

Brain weight (g) (n = 10)

487.2 _+10.5 486.7 -+9.7 386.1 -+15.5"** 464.2 _--+11.8000

Cerebellar weight (mg) (n = 10)

1.466 _+0.032 1.487 ---0.015 1.627-+ 0.038*** 1.493 -+0.0200

261.3 ---5.9 274.7 -+7.4 317.8---4.0"** 298.2 _+5.200

Duncan test: ***P-<0.001 vs sham; 0P---0.05; 00P-<0.01; 000P_<0.001 vs ADX. GROWTH

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In contrast brain weight was enhanced by adrenalectomy. Compared to sham and control groups, ADX rats had significantly higher mean wet weights for both brain and cerebellum (+ 10%, P-<0.01). CORT replacement at the oral dose of 1 mg/kg/day restored brain weight to the level of the control and sham groups. Hippocampus No significant changes in TAU, ASP, GLU and GABA contents were observed, while GLN tissue levels were markedly reduced (Table 2) in all hippocampal regions of ADX rats, compared to those in the sham group (CA1-CA2: - 3 2 % , P-<0.01; Ca3 -37.8')0, P-<0.01; DG ....27.8°/, P-<0.01). This decrease was completely reversed in the A D X + C O R T group. The reduction of GLN content in hippocampal formation of ADX rats was correlated with a decrease in GS activity (-24.7% vs sham; P-<0.01) (Fig. 2). The CORT replacement therapy in the A D X + C O R T group restored normal values of GS activity, comparable to those in controls. Hypothalamus The hypothalamic GLN (Table 3) and GS activity levels (Fig. 2) in ADX rats were also decreased ( - 1 7 . 0 ° , P-<0.001 and -22.5%; P_<0.01 vs sham), but the CORT treatment, at the Table 2. Hippocampal neurotransmitter amino acid levels (micromoles per gramme) in adrenalectomizcd rats (M _+SEM; n = 10) Taurine

Glutamine

Glutamate

Aspartate

GABA

2.03 -+ 0.08 1.87_+0.05 1.71 _+0.06 1.60_+0.09

1.74 _+0.08 1.78_+(I.05 1.77+-(I.08 1.65_+0.04

CAllA2

Controls Sham ADX ADX+CORT

5.36 +_0.14 5.36_+0.13 5.52_+0.18 4.90+-0.11

4.26 _+0.27 4.52_+0.16 3.06-+0.13"* 3.97 +0.1900

9,16 -+ 0.41 9.03+-0.16 9,46_+0.23 9.47+0.15

CA3

Controls Sham ADX ADX+CORT

4.20_+0.14 4.40-+0.23 4.64 _~0.20 4.57-+0.12

4.61-+0.17 4.55+0.19 2.83_+0.10"* 4.14 -+ 0.1400

11~04_+ 0.60 11.24-+(I.25 11.36_+0.31 10.98-+0.43

1.98_+0.11 1.93-+(I.(t4 2.10_+0.16 1.97-+0.04 1.81+0.15 2.12-+0.09 2.01 -+11.16 2.00-+(I.04

CA4-DG

Controls Sham ADX ADX+CORT

4.16_+0.17 4.54_+0.06 4.59_+0.13 4.54-+0.08

3.90+_0.26 4.02-+0.15 2.91-+0.12 ** 3.84-+0.1600

11.66_+0.50 12.94_+0.62 12.39_+0.28 12.58_+0.51

2.11 -+0.10 2.29-+0.18 2.34_+0.09 2.36-+0.14

1.86_+0.03 1.95-+0.02 2.05_+0.08 1.90+0.02

D u n c a n test: **:P-<0.01 vs sham; 00:P-<0.01 vs A D X .

- -1 Controls ADX

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HY

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Fig. 2. GS activity (micromoles per g r a m m e of wet weight per hour) in hippocampus (HI), hypothalamus (HY), striatum (ST) and cerebellum (CB) of adrenalectomized rats. (*P-<0.05, **P--<0.01; ***P--<0.001 vs Sham; 00P-<0.01,000P-<0.001 vs A D X ; D u n c a n test).

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Table 3. Hypothalamic (HY), striatal (ST) and cerebeUar (CB) neurotransmitter amino acid levels (micromoles per gramme) in adrenalectomized rats (M - SEM; n = 10) Taurine

Glutamine

Glutamate Aspartate

GABA

HY

Controls Sham ADX ADX+CORT

3.11 -+0.08 3.06-+0.10 3.28-+0.14 2.93±0.08

8.20 -+0.26 8.49-+0.18 6.96-+0.24*** 7.63-+0.29

8.75 ± 0.56 9.02-+0.22 8.72-+0.58 8.66-+0.40

3.00-+0.08 2.95-+0.09 2.80-+0.10 2.93-+0.12

3.92 -+0.12 4.13-+0.14 3.76-+0.13 4.02-+0.16

ST

Controls Sham ADX ADX+CORT

7.22 - 0.17 6.93-+0.20 7.88+-0.17 ** 7.24-+0.210

6.41 -+0.20 6.07---0.11 5.39-+0.18"* 5.90-+0.170

9.56 ± 0.32 9.29-+0.30 9.40-+0.34 9.21---0.19

1.57-+0.04 1.59-+0.08 1.39±0.03" 1.55-+0.070

2.17 -+0.07 2.37-+0.07 2.34-+0.15 2.36-+0.15

CB

Controls Sham ADX ADX+CORT

4.31-+0.12 4.23-+0.09 4.68---0.14" 4.35-+0.060

8.96-+0.10 9.24-+0.22 6.48±0.66** 8.76-+0.180

11.22---0.28 10.71--_0.19 10.93-+0.34 10.63±0.10

1.77-+0.07 1.77±0.05 1.55-+0.04"* 1.83-+0.0500

1.67-+0.08 1.83-+0.04 1.79-+0.14 1.90---0.02

Duncan test: *:P--<0.05; **:P-<0.01; ***:P-<0.001 vs sham; 0 :P-<0.05; 00:P~0.01 vs ADX. m e a n oral dose of 1 mg/kg/day, did not completely restore the levels to those of the control and sham groups. The hypothalamic concentrations of the other analyzed A A s were not significantly changed by adrenalectomy.

Striatum Striatal G L N (Table 3) and GS activity levels (Fig. 2) in A D X rats were significantly lower than those of sham and control animals ( - 1 1 . 2 % , P-<0.01 and - 1 6 . 8 % , P-<0.01 vs sham). The levels of ASP also appeared to be decreased ( - 1 1 . 9 % , P-<0.05 vs sham), while T A U content seemed to be increased ( + 1 3 . 5 % , P-<0.01). The C O R T replacement, restored the striatal levels of T A U , G L N and ASP in the A D X + C O R T rats.

Cerebellum The cerebellum of A D X rats showed a m a r k e d decrease in the levels of G L N (Table 3: - 2 9 . 9 % , P-<0.01) and GS activity (Fig. 2: - 2 4 . 7 % , P-<0.01 vs sham), which was accompanied by a decrease of ASP ( - 1 2 . 4 % , P-<0.01 vs sham) and an increase in T A U levels ( + 1 0 . 4 % , P-<0.05 vs sham). The oral administration of C O R T totally restored the levels of these A A s and GS activity to those of the control and sham groups.

Histology A d r e n a l e c t o m y resulted in a subtotal disappearance of granule cells of D G in 75% of A D X rats. In other words, two rats out of eight did not show cell losses after adrenalectomy. The decrease in cell n u m b e r occurring in the granule cell layer of D G was consistently between 40 and 60% in the A D X animals, a2 A m o n g the surviving granule cells most showed pyknotic nuclei with condensed chromatin. In the A D X rats treated with C O R T there was no apparent loss of granule cells. DISCUSSION Our results show that long-term adrenalectomy, p e r f o r m e d in newly weaned rats, reduces the levels of glutamine and glutamine synthetase activity in hippocampus, hypothalamus, striatum and cerebellum at 3 months after surgery. Moreover, the administration to adrenalectomized rats of corticosterone in drinking water at a m e a n oral dose of I mg/kg/day restores the normal tissue levels of both glutamine and glutamine synthetase activity in all these brain areas, except for the hypothalamus, where the recovery of basal level is incomplete. While the administration of glucocorticoid h o r m o n e s in newborn rats impairs brain and body growth, 16 in agreement with previous studies 3,15 we show that adrenalectomy reduces the rate of body growth and enhances brain and cerebellar wet weights. These effects are likely to be due to increased D N A synthesis rather than to water accumulation, as reported previously. 15,29,30 These changes were prevented

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by corticosterone replacement therapy. In addition, our results are somewhat confirming the reports by other authors that have described a hippocampai neuronal loss after adrenalectomy performed in rats of young and adult a g e . ~A4'~3"26'27 I n fact, we observed that six out of eight ADX rats showed a significant D G granular cell loss, that was prevented by C O R T replacement. Therefore, adrenalectomy causes a marked cell loss in the DG. This result is in agreement with previous reports suggesting that rat adrenalectomy performed in young age causes an cxtensive loss o| D(; n e u r o n s 7"9'13'27'30 that may be smaller at older age. ~'~ A failed stimulation of glutamine synthetase activity during development, following the absence of glucocorticoids produced by ablation of the adrenal glands, may account for the reduced tissue level of glutamine. Our results are in agreement with the observations of others who have reported a regulatory effect of GCs on brain glutamine synthetase activity.18 Patel el al. have shown an increase in the brain level of glutamine synthetase activity during postnatal development of rats, Is which parallels the age-related increase in endogenous glucocorticoids. 24 That study also demonstrated that the administration of corticosterone caused in cerebellum, olfactory bulbs and forebrain, an age-dependent increase in glutamine synthetase activity which was due to an increase in enzyme protein. This study suggested that the elevation of enzyme activity is dependent on the maturational state of the region at the time of the hormone treatment, i.e. the induction of the enzyme protein is higher in younger animals. In a preliminary study in which 50-day-old rats underwent adrenalectomy, we have found a reduction of glutamine levels ( - 2 0 % ) only in the hippocampus (data not published). In the present study adrenalectomy was performed on the 22nd day of age and glutamine concentrations were particularly reduced in the hippocampal formation and cerebellum. Because of its high concentrations of both type I and type II adrenal steroid receptors, ~'2'' the hippocampus is the major neural target of glucocorticoids, and, consequently, its response to the lack of these hormones is likely to be more evident. Cerebellar glutamine synthetase activity has been shown to be affected by corticosterone administration even if the treatment with the hormone was performed after the 20th day of age, suggesting a delayed maturation of this region with respect to other brain areas, such as olfactory bulbs and forebrain, t8 Therefore, cerebellum may be more easily affected by adrenalectomy performed on the 22nd day of age. Although glutamine is the precursor of glutamate and G A B A in neurons and glia, 2'5 the tissue concentration of these two AAs seems not to be affected by the lowered levels of glutamine and glutamine synthetase in A D X rats. In fact, besides the glutamine pathway, neurotransmitter G A B A and glutamate are synthesized in large part from glucose or from 2-oxoglutarate supplied from glial cells. These alternative substrates apparently account for the results of a recent study in which an alteration of G A B A metabolism was observed only when the glutamine brain level was reduced to about 20% of normal levels, t,~ The mechanisms responsible for the striatal decrease in the level of aspartate are not completely clear, although the inhibition of glial glutamine synthetase has been reported to reduce levels and release of aspartate in rat striatum. 2~ In contrast, taurine levels appeared slightly, but significantly, augmented in striatum and cerebellum of A D X rats. 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