Behavioral and neurochemical alterations caused by diet restriction - the effect of tyrosine administration in mice

RESEARCH Brain Research 732 (1996) 133-144

Research report

Behavioral and neurochemical alterations caused by diet restriction – the effect of tyrosine administration in mice Yosefa Avraham a, Omer Bonne b, Elliot M. Berry a’* ‘ Department of Human Nutrition and Metabolism, Hebrew Uniuersi&Hadassah Medical School, POB 12272, Jerusalem 91120, Israel b Department of Psychiatry, Hebrew Uniuersitj-Hadassah Medical School, Jerusalem, Israel Accepted 23 April 1996

Abstract We have investigated the effect of tyrosine administration on the cognitive and neurochemical alterations caused by diet restriction (DR) in mice, as a possible model for some of the behavioral symptoms of patients with anorexia nervosa. Young female mice were fed

to 100, 60, and 40% of the calculated daily nutritional requirements for a period of up to 18 days. Cognitive function was evaluated using a modified eight-arm maze with water as a reward. Animals fed to 60% of controls showed significantly improved maze performance while this was impaired in animals on DR to 40Y0. However, in these animals, injections of tyrosine (100 mg/kg/day) restored performance. Improved maze performance in the 60% DR and 40% DR + tyrosine animals was related to increased beta: alpha tone in the hippocampus – an area, together with the septum, responsible for spatial learning. This was associated with changes in ci- and ~-receptor density (B~=), without affecting affinity (K,J; and increased notepinephrine (NE)inthe40% DR + Wosinegroup, andmethwWmxyphenylglycol (MHPG) in both groups. In the hypothalamus, the brain area responsible for energy metabolism, there was a progressive increase in alpha:beta tone with increasing DR associated with changes in B~=. Tyrosine treatment reversed these alterations. Tyrosine improves some of the neurobiological disturbances of DR without causing an increase in body weight. Such a strategy might have important implications for the possible treatment of patients with anorexia nervosa. Keywords:Adrenergicreceptor; Hypothalamus;Septal-hippocampalpathway;Tyrosine;Cognitivefunction; Mouse; Eight-arm maze; Diet restriction; Anorexia nervosa

1. Introduction In nature there are examples of voluntary starvation of animals in the presence of an adequate food supply as in bird migration, hibernation or territorial defense [47]. Attempts have been made to develop an animal model for self-induced weight loss utilizing exercise with food restriction [57], and stress-induced either by immobilization [61] or by tail-pinching [39]. None of these models is considered a true replica of the human disease [64], but they are useful for developing ideas and experimental techniques with which to study the problem. Work in rats has shown that catecholamine pathways in the hypothalamus are involved in feeding behavior [38]. Alpha-adrenergic stimulation, in the area of the paraventricular nucleus of the hypothalamus, induces feeding, while ~-adrenergic stimulation is inhibitory [37]. Dopa-de-

“ Corresponding author. Fax: +972 (2) 43-1105.

pleted animals may stop all operant behavior, and actually starve themselves to death [22]. This may be analogous to the chronically malnourished patient with anorexia nervosa (AN) in whom appetite may be suppressed if central noradrenergic pathways have insufficient NE and/or if dopa levels are chronically depleted. In the cerebrospinal fluid of subjects with AN, levels of tyrosine, NE and metabolizes are all decreased [17,28]. While most of these values return to normal with weight restoration, abnormalities in NE metabolism may persist even after long-term weight recovery [17]. Thus, studying the effects of undernutrition on catecholarnine regulation and cognitive function might enable a more comprehensive understanding of the patho-physiology of AN in man, and may lead to a more rational approach to its treatment. Semistarvation per se can be accompanied by mental changes as shown by the experience of doctors in the Warsaw Ghetto [69] and the experiments of Keys and co-workers on semistarvation – diet restriction (DR) to 50Y0– in conscientious objectors [31]. In AN, after the

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Y.Auraham et al./Brain Research 732 (1996) 133-144

initial psychologically-induced weight loss, part of the stubborn refusal to eat may be a psychological concomitant of neurochemical dysfunction caused by malnutrition. In order to investigate this hypothesis, we have used DR in mice to study its effects on cognitive behavior and the catecholarninergic system. The possible neurochemical alterations in brain function after DR have been evaluated and correlated with behavioral function. We have focused particularly on alterations in specific brain regions – the hippocampus and septum – which are responsible for cognitive function [8,42], and on the hypothalamus, an area critically involved in the regulation of systemic energy balance [38,51]. This paper describes changes in behavior and neurochemical status induced by DR in mice and the restitutive effects of tyrosine administration.

2. Materials and methods 2.1. Establishing model of DR Female Sabra mice were assigned at random to different groups of 10 mice to a cage. All cages contained wood-chip bedding and were placed in a temperature-controlled room at 22°C on a 12-h light/dark cycle (lights on at 0700). The mice had access to water 24 h a day. The food provided was Purina chow, containing carbohydrate 54.9Y0,protein 21.1%, fat 4.7%, humidity 12.0%, ash 5.2% (K= 0.79%, P = 0.57%, Ca = 0.60%, Na = 0.22Y0Cl = 0.35%) cellulose 2.64%. Amino acid content: 0.50’%.methionine, 0.33% cysteine, 1.1590lysine, 1.25Y0arginine, 0.27% tryptophan, l.78’%oleucine, 0.92% isoleucine, 1.03% valine, 1.03% phenylalanine, 0.74Y0tyrosine, 0.77% threonine and 0.5% histidine. The food was given between 1000 and 1100. Two groups of control mice received a diet of 95 kCal/week\mouse (3.6 g/day/mouse) as suggested by Ingram et al. [24]. This amount was considered to be 100% of the daily nutritional requirements. Two groups received a diet of 57 kCal/week/mouse (2.16 g/day/mouse) as 60% of the requirements. Three groups received only 40% of the requirements, a diet of 38 kCal/week/mouse (1.44 g/day/mouse). DR was carried out for 18 days. The duration of the study was not predetermined and the dietary schedule was in relation to the weight of the mice. DR was continued until either weight plateaued (100 and 60Yo)or reached 25 g or less. Previous experiments showed a very high mortality below this weight. Thereafter behavioral testing was performed with the eight arm spatial maze. During the behavioral tests half of the animals in each group were injected with tyrosine (100 mg/kg/day) and the other half with saline. Mice were killed after the behavioral tests and the hippocarnpus, hypothalamus and septum were frozen at –70”C. The hypothalamus and hippocampus were evaluated for ~ ~- and ~-adrenergic receptors, and the septum

and hypothalamus for measurement of catecholamine synthesis and turnover. 2.2. Eight arm spatial maze The radial eight arm spatial maze was similar to that developed for rats by Olton and Samuelson [52], but has been scaled down for mice [73]. Our method for the maze differed from the original technique because the reward used was water instead of food. Use of the radial arm maze to test the spatial learning ability of undernourished rats is problematical [7]. A food reward is required in order to induce the rats to perform the test. In order to accomplish this, the rats have to be hungry at the time of testing and this is obviously a complicating factor in experiments where malnutrition or undernutrition is the factor under investigation in the first place. Bedi and other investigators used the Morris water maze because it offers the advantage that an appetitive reward is not required. However, we considered that this maze demanded too much physical strength for the 40Y0 DR mice, which did not have the same stamina as the control or 60~0 DR-fed animals. For these reasons we used water as the reward in the radial arm maze. Mice were put on a water deprivation schedule for 6 days before the start of the experiment, and the animals received water for 30 min each day. On the seventh day they were introduced to the radial maze for 10 min and the first 16 entries to the arms recorded. On this day no reward was offered. On the next day 50 p,l water was presented in each arm. The animals were observed until they made entries to all eight arms or until they completed 16 entries (whichever camefirst). The animals were then taken out of the apparatus. The number of correct entries (once to each of the eight arms) within the 16 trials (maximum possible = 8) was recorded and the test was repeated for 5 days. Tyrosine (100 mg/kg\day) or normal saline was administered to control and DR animals during the behavioral tests. 2.3. Biochemistry of adrenergic receptors 2.3.1. Brain extraction Brain was extracted according to the procedure of Atlas et al. [4], with minor modifications. Female Sabra mice (approximately 30 g) were killed by decapitation. The brains were rapidly removed and the hypothalamus, hippocarnpus and septum were dissected out and placed in 20 volumes of ice-cold buffer TME (0.05M tris/HCi pH 7.5, 0.002 M Mg(Ac)2 and 0.002 M EDTA). The tissue was homogenized in a glass tube by 15 strokes of a motor driven Teflon pestle rotating at 1000 r.p.m. The homogenate was then centrifuged for 5 min at 500g at 4“C. The pellet which consisted of nuclei and other debris was discarded. The supernatant fraction was centrifuged at

Y. Auraham et al, /Brain Research 732 (1996) 133-144

135

50000g for 20 tin at 4“C. The supernatant fraction was then discarded and the pellet was suspended in cold buffer TME and centrifuged once more as before. The washed pellet was suspended in buffer TME at approximately 3 mg protein/ml. Protein was determined using a commercial protein assay kit, based on the method of Bradford (BioRad, Munich, Germany). 2.4. a- and ~-adrenergic receptors 220L-..---––.-.-—.4-------

Binding to the az-adrenergic receptors was studied using phenyl-4-[3H]clonidine hydrochloride (Amersham, specific activity = 22 Ci/mmol) according to the procedure of U’prichard et al. [67] with minor modifications. Washed membranes were incubated in triplicates using varying concentrations of ligand and preparations from both treated and control mice. The mice were assayed on the same day using the same amount of tissue per assay tube for each group. Binding reaction mixtures contained in a final volume of 0.3 ml were: 40–60 pg protein per assay in 50 rnM tris/HCl, pH 7.4, 2 mM Mg(Ac)z and 2 mM EDTA; and 0.5–10 nM clonidine hydrochloride in the case of cx-receptors. Tissue from mice was pooled to provide adequate quantities to develop saturation isotherms using six ligand concentrations. @adrenergic receptors were evaluated using antagonists such as [3H]dihydro-alprenolol (Amersham, specific activity= 59 Ci/mmol) by the same procedure. Nonspecific binding was measured by using 10 I.LMphentolamine for IXreceptors, and alprenolol for ~ receptors. Reaction mixtures were incubated for 30 tin at 25°C for a receptors and 20 tin at 30”C for ~ receptors. Five ml of ice-cold buffer was added to each tube of reaction rrtixture and immediately filtered onto Whatman GF/C filters under reduced pressure. Each filter was immediately washed three times with 5 ml of the same buffer diluted 1:10 and counted in scintillation fluid. Results of binding experiments were evaluated using Scatchard plot and linear-regression analysis to determine total binding (B~,X) and dissociation constant (Kd). 2.5. Catecholamine synthesis and turnover The assays for dopamine, DOPAC (3,4-dihydroxyphenylacetic acid), NE and MHPG (methoxyhydroxyphenylglycol) were performed by standard alumina extraction, and HPLC/EC separation and detection using dehydroxybenzylamine as an internal standard [29,73].

In general, data are compiled as means and standard deviations. Results were evaluated first by a global analysis of variance with multiple levels and repeated measures when appropriate. Homogeneity of variances of the different groups was then assessed by Bartlett’s test. Post-hoc testing was only performed if the overall P value was less

.. . . .. D

Fig. 1. Weight curves of mice fed different dietary reshiction regimens of 100, 60 and 40%. Bars represent the S.E.M.

than 0.05 and was carried out using the Tukey–Kramer multiple comparisons procedure [13,63,68]. Results were analyzed as Scatchard plots, with resolution typically to models involving one affinity state using the appropriate computer analysis. For Scatchard analysis, every score (B~,X or K~) represents n of 1. Possible differences between control and treated mice in receptor numbers were analyzed by comparing the average B~.X values of the individual experiments (t-test).

3. Results 3.1. Weight loss Fig. 1 describes the weight changes following the different degrees of DR, 100, 60 and 40%. The body weight of control mice did not change significantly during 18 days of experiment being 30.4 + 2.1 g at the beginning and 32.1 + 2.6 g at the end. For the mice on 60% DR, weight changed significantly from 30.0 + 2.3 g to 26.2 + 2.4 g (mortality 4%) and for the 40% DR group from 30.7+ 2.6 g to 23.1 + 2.3 g at the end of the experiment (mortality 36%). Weights of the control groups were significantly higher than the 40% groups (P< 0.05) (Fig. 1). Mice held on DR to 60’%lost approximately 12.69i0of their weight on average, while mice fed DR to 40% lost on average 24.79Z0 Table 1 Number of trials (mean+ S.D.) required to enter, after 5 days of learning, all eight arms of maze in controls and animals fed diet restriction (DR) to 60 and 40% Group

2.6. Statistical analysis

10 ~aya

N

N

–Tyrosine

20 13.70t0.58 a,b Control DR to 60% 18 10.05iO.52 a * * *,C of controls DR to 40% 13 14.42*0.52 b * * ,C * of controls

+Tyrosine

20 13.25+0.57 15 10.25~0.46 “ y

,d 17 11.53+0.29 d * * *

‘ P<0.05,‘ “P<0.01,* ** P <0.001. a–d represent the pairs of values which are statistically different from each other.

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Y.Auraham et al. /Brain Research 732 (1996) 133-144

H-l.I jj .E

I

HI

3

n

n.d. 0.70+0.05

2.05 tO.55 a ‘ * “,b * ‘ *,d 3.63 +0.40 d * * *

8 9

40%

n.d, Not detectable. * * P <0.05, * “ P <0.01, “ “ * P <0.001. a–j represent the vafues which are statistically different.

+Tyr

11 11

60% 60% +Tyr

bo~o

0.21 +0.29 e 0.92+0.04 e “ “ *

3.54~0.70 b 4.33 * 1.35

11 11

100% 100?4o +Tyr

n.d. 2.04+0.95

4.07 +0.49 a,c 2.86~0.37 C * * *

NNE

Diet

MHPG

,J 9.38 *2.25 1.* **..** 9.82+0.17

17.64+2.39 h ‘ * *,j 11.19*9.94

31.67* 12.27 f “ * 26.23 + 13.09 21.40+ 7.00 g * * * 23.90~ 3.70

24.66*5.26 h,i 16.46+3.10

DOPAC

58.49 +21.28 f,g 42.25 ~ 15.30

Dopamine

0.192

0.059 0.212 (x3.6)

0.713

Turnover MHPG/Nf3

Table 2 The amount of norepinepbrirre(NE) and doparnine (ng/mg protein, mean+ S.D.) in the septum of control animafs and those fed DR to 60 and 40%

0.438 0.410 (X O.93)

0.557 0.427 (X O.77)

0.422 0.389 (xo.92)

Turnover DOPAC/dopamine

6 6

6 6

6 6

100% 100% +Tyr

60% 60% +Tyr

40% 40% +Tyr

0.85 ~0.21 b * ‘ ‘ 2.25 ~0.20

0.65 &O.05C 3.43 + 1.89 C * ‘

0.748 +0.891 2.28 ~ 1.14

0.335+0.009 0.757 * 1.071

1.29+0.20 a 2.07 +0.58

5.81 + 1.50a * *,b 8.67+ 1.49

MHPG

NE

0.51 ~0.22 e * * ‘ ,f ‘ * *,h 1.90+0.25 h * * “

2.79&0.03 d “ * *,f g 0.89+0.51 g

1.08+0.09 d,e 1.97*o.15

Doparnine

Sample sizes in parentheses. Norepinepbrine and dopamine measured by HPLC with an electrochemical detector. * P <0.05, ‘ ‘ P <0.01, * “ * P <0.001. a–j represent the values which are statistically different.

N

Diet

0.69+0.33 j ‘ * 1.21+0.35

0.95 iO.19 i * 0.89+0.03

1.42&0.35 i,j 2.16+ 1.25

DOPAC

0.764 1.524 (X2.0)

0.128 0.263 (X2.0)

0.260 0.365 (x 1.4)

Turnover MHPG/NE

Table 3 The amount of norephinephrine (NE) and doparnine (ng\mg protein, mean+ S.D.) in the hypothalamus of control animals and those fed DR to 60 and 40%

1.352 0.636 (XO.47)

0.340 1.000 (x2.9)

1.315 1.096 (x O.83)

Turnover DOPAC\dopamine

~ ~ n Y b ~ =. z % $ D 2 s ~ b g g ~ k k UJ ~ ‘& a

? $

Y. Auraham et al. /Brain Research 732 (1996) 133-144

of their weight and at a much faster rate. Multiple food dishes were used in each cage to ensure equal access to food. Tyrosine supplementation did not affect the weight of the mice (data not shown).

139

40% DR-treated mice (P< 0.011). Injections of tyrosine caused a variable response with decreases in the receptors in the IOOYoDR (P < O.Olj) and 40$Z0DR (P < 0.05m), but increase in the 60% DR mice which did not quite reach significance (P= 0.07).

3.2. Behavior Table 1 shows the results of three different experiments of the effects of DR on maze performance after 5 days of learning. Mice held on DR to 60% significantly improved their performance (needing less number of trials) (P< 0.001a), while mice on DR to 40% showed impaired performance in relation to the controls (P< O.Olb). Administration of 100 mg/kg/day of tyrosine to animals fed on DR to 40Yosignificantly improved their performance in the eight-arm maze (P < 0.001d) while mice fed on DR to 60% and controls showed no change in performance in response to tyrosine. 3.3. Neurobiochemistry 3.3.1. az-Receptors Receptor binding experiments showed varying changes in the levels of a z-receptors according to the degree of DR and cerebral localization (Fig. 2). In the hypothalamus (Fig. 2A), the increases were very significant for both 60% DR. (P < O.OIC), and 40?70DR-(F’ < 0.05d) treated mice. Tyrosine significantly reduced the number of receptors in the hypothalamus in all the experimental groups – 100% DR (P< O.Ole), 60% DR (P< O.Olg), and 40% DR (F’ < 0.05h). In the hippocampus (Fig. 2B), there was a decrease in the 60% DR (P< O.Oli) and increase in the

3.3.2. @Receptors Fig. 2C,D shows the binding experiments for ~ receptors. In the hypothalamus (Fig. 2C) there was a gradual decrease in the 60% DR- and 40% DR- (P< 0.05a) treated animals; in the hippocampus (Fig. 2D) there was a significant increase in the 60% DR (P < 0.05c), with no significant change in the 40’%0DR animals. In the hypothalamus (Fig. 2C), administration of tyrosine caused a decrease in the 100% DR almost no change in the 60% DR and increase in the 4090 DR mice (P < 0.001b). In the hippocampus (Fig. 2D), there was a decrease in the 100% DR animals which was not statistically significant, almost no change in the 60’%0DR ones, and a non-significant increase in the 40’%0 DR animals. The K~ for the Q- and ~-receptors in the hypothalamus and hippocampus, as measured by binding to clonidine and dihydropranolol, respectively, remained within the normal range in all the experimental groups. 3.3.3. Catecholamines; septum Table 2: The amount of NE in the septum was reduced progressively in the 60 and 40% DR-treated mice (P< 0.001a,b). Administration of tyrosine reduced the amount in the 100YoDR (P < 0.00Ic) mice, with no change in the 60% DR ones, and an increase in the 40Y0 DR mice (P< 0.001d).

Table 4 The effect of diet restriction on maze performance catecholamines level, and brain adrenoreceptor tone Diet

Maze

Alpha 100% 100% + Tyr

Beta

-—

60% 60% + Tyr

+++ n.c.

40% 40% + Tyr

+++

-——

Hippocampus

Adrenoeeptors

—— (++ ++ —

)

Beta:alpha

n.c.

2,01 2.81

++ n.c.

6.27 2.58

(+) (i- + )

1.79 12.1

Catecholamines in the septum NE

MHPG

Dopamine

DOPAC

n.c. n.c.

++ +

—n.c.

n.c.

-—

n,c, ++

——

——

n.c.

n.c.

+++

—

Hypothalamus 100% 100% + Tyr 60% 60% + Tyr

40% 40% + Tyr

n,c.

-—

n.c

0.776 0.394

+++

++

(-)

3.287

n.c.

——

n.c.

+++ ——

———

——— +++

+++

0.530 19.6 0.520

++

(+)

++

—

n.c.

(+)

--

n.c.

——

n.c. ++

—-

—

++

+

+++

Values at 60 and 40% are compared to 100%, the changes after tyrosine are compared to the same DR group. n.c, no change; bracketed vatues show a trend which is not significant.

.—

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Y.Auraham et al. /Brain Research 732 (1996) 133-144

MHPG was not detectable in the 100% DR and 40% DR mice. Administration of tyrosine elevated the amount of MHPG in all treated animals. The amount of dopamine was reduced gradually in the 60% DR (P < O.Olf) and the 407. DR groups (P< 0.001g); administration of tyrosine did not change the concentrations in any of the experimental groups. The amount of DOPAC was reduced in the 60% DR (P< 0.001h) and 40% DR mice (P< 0.001i); administration of tyrosine did not lead to any significant changes. Thus, in the septum, concentrations of NE, dopamine and DOPAC were all progressively reduced in response to diet restriction. When the amount of NE was reduced there was less turnover to MHPG. Tyrosine administration increased the turnover of MHPG/NE by a factor of 3.6 in the 60% DR-treated animals, while decreasing that of DOPAC/dopamine. The latter turnover was unaffected by tyrosine in the 100YoDR and 40?Z0DR animals. 3.3.4. Catecholamines; hypothalamus Table 3: The concentrations of NE and dopamine were increased on 60?t0 DR and decreased on 409. DR in relation to the control animals; the concentrations of DOPAC were progressively and significantly reduced with increasing DR, with no significant changes in MHPG. Administration of tyrosine significantly altered the concentrations of dopamine – decreasing it in the 60Y0DR mice and increasing it in the 409Z0DR ones – and increased MHPG in the 40% DR animals. The level of MHPG/NE turnover was reduced in the 60?koDR mice and elevated in the 40% DR group. Administration of tyrosine increased the level of turnover in 100% DR (X 1.4), 60% DR (x2.0) and 40% DR (x2.0) mice. The level of DOPAC/dopamine turnover was reduced in the 60% DR and elevated in the 40% DR mice. Administration of tyrosine reduced the level of turnover in 100% DR ( X 0.83) increased it in the 60% DR ( X 2.9) and reduced it in the 4090 (X0.47) DR animals. In the septum, the effect of tyrosine was more pronounced on the levels of NE than on those of doparnine; in the hypothalamus both were affected. 3.4. Summary of results Table 4 is an attempt to summarize the findings in terms of changes in alpha:beta ‘tone’ in the different brain regions. The ‘tone’ was derived by dividing the relevant B~~, values from Fig. 2. The values are in comparison to the DR 1009ZO group, and the tyrosine effects are compared to the DR category whether 100, 60 or 4070. The responses of the hippocarnpus and the hypothalamus to DR were generally in opposite directions, such that &z-receptor density was raised in the hypothalamus and lowered in the hippocampus in 6090 DR. The changes in w-receptors in the hypothalamus increased with the degree of DR. Beta-receptors were decreased in the hypothalamus

but raised in the hippocampus on both 40 and 60% DR. In the hippocarnpus, 40Y0 DR did not lead to a further increase in ~ receptors. These data suggest that cognitive function may correlate in the septum-hippocampus with an increase in beta:alpha tone and maze performance in the 609. DR animals and a decrease in both in the 40% DR group. The improvement in maze performance in the 609. DR and 407. DR + tyrosine groups was associated with an increase in the beta:alpha tone (6.27 and 12.1, respectively). In the septum this was associated with an increase in MHPG levels. Tyrosine administration elevated beta to alpha tone in the hippocampus of the 40% DR animals, and increased the amounts of NE and MHPG with no changes in dopamine and DOPAC. In the hypothalamus the responses to DR were characterized by a progressive increase in alpha:beta tone. The tone increased from 0.776 to 3.287 to 19.6 in response to progressive DR from 100 to 60 to 409.. This was accompanied by a decrease in DOPAC concentrations. After administration of tyrosine there was a progressive decrease (normalization) in alpha:beta tone (100% DR 0.394; 60% DR – 0.530; 40% DR – 0.520) which was associated with an increase in catecholamines, in particular MHPG and dopamine.

4. Discussion The objectives of this research were to study the effects of dietary restriction (DR) on maze performance by mice and correlate the behavior with autonomic system activity. This was measured by the biochemistry of ~- and (3-receptor function and catecholarnine levels in specific brain regions – in particular the hippocampus and septum which are involved in spatial learning, and the hypothalamus – an area crucial for the regulation of systemic energy homeostasis. In addition, we studied whether tyrosine administration could affect cognitive function as a possible therapeutic modality for some aspects of behavior in patients with anorexia nervosa. The results of the a and ~ adrenergic binding studies (Bm,X and K~ calculations) as well as the catecholamine determinations are consistent with the values in mice which have been published in the literature [16,26,50,74]. The point at issue is at what stage does ‘benign’ undernutrition (equivalent to 60Y0 DR) become ‘malignant’ malnutrition (? 4070 DR), and how does this apply to the cognitive disturbances in anorexia. It is postulated that part of the latter is caused by a depletion of neurotransmitter precursors, particularly tyrosine. Tyrosine is the physiological precursor for catecholamine synthesis [33], and its administration in experimental animals, can increase the rates at which brain neurons synthesize both dopa and NE [18]. In our experiments we injected 100 mg/kg tyrosine 2 h before behavioral testing. According

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Y. Auraham et al. /Brain Research 732 (1996) 133-144

to Wurtrnan et al. [72], the administration of a low dose (5O mg/kg) of tyrosine caused an 81% increase in brain tyrosine after 45 min. 4.1. Diet restriction, learning and hippocampalfinction We found that animals fed DR to 60% of controls significantly improved maze performance, while performance was impaired after DR to 40% (Table 1). Maze performance related to increased beta:alpha tone in the hippocampus (Table 4), and the concentrations of NE in the septum. In both the DR 6090 treated mice and the 40% + tyrosine group, the beta:alpha tone was increased over the 100% levels. These results agree with the basic concept of Masoro [43,44] and Ingram [24], who claimed that the effects of DR upon cognitive and behavioral function depended on the degree of restriction. Ingram et al. [24] found that diet restriction (25 to 6090 from ad libitum level in most studies) benefited learning and motor performance in aged mice. This was found also in early and mid-life [9,23,49,75]. DR to 40% has not been discussed in the literature, perhaps because of its deleterious effects and relatively high mortality ( = 36Y0in our experiments). The use of such extreme restriction is justified by the fact that the human disease of AN has an appreciable mortality. The eight arm spatial maze behavior is often studied in animals [1] as an indicator of spatial memory and discrimination. Obviously, no behavior is entirely controlled by a single brain structure and other inputs clearly influence maze behavior [48]. Nevertheless, this behavior appears to be more dependent on intact septum and hippocampus than are other behaviors, such as spontaneous alternations [12], long regarded as a typical ‘hippocampal behavior’ [55]. Spatial learning is also known to be affected by the balance between adrenergic and cholinergic tone [19]. Lesions within the septo-hippocampal system produce severe impairment of maze behavior, which may be reversed by transplantation of septal cholinergic cells into the hippocampus [8,42]. Diet restriction may also affect rotational behavior and striatal dopamine levels in aged rats [27]. The relation between nutritional status and the adrenergic and cholinergic systems has not been thoroughly discussed in the literature. However, some of the relevant evidence is as follows: rats undernourished for a relatively short period during early postnatal life, exhibit alterations in the morphological structure of the dentate gyms which is involved in the control of spatial memory [3]. Hippocampal-associated spatial memory deficits have been found in previously undernourished animals as well as altered activity and exploration levels. The cholinergic parasympathetic system has been repeatedly shown to be affected by nutritional factors [21]. Structural damage in the CA3 cholinergic-rich area has been demonstrated in rats born to dams who had undergone low protein diet (670 casein instead of 25?ZO)through pregnancy and/or while

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nursing [15]. Lack of response to cholinergic inhibition (N-methylscopolamine) was observed in postnatal underfed mice [54]. Reversible behavioral impairment related to hippocampal integrity was found in rats who were subjected to 60% DR and 40% DR of that eaten by well-fed controls until 30 days after birth [7]. Selective alterations in neurotransrnitter synthetic enzyme activity such as tyrosine hydroxylase, and cholinergic receptor concentrations in brains of diet restricted rats have been reported [40]. In future experiments we propose to measure cholinergic activity in this model of DR. 4.2. Tyrosine, catecholamines and behavioral function Tyrosine improved maze performance of the 40~0 DR mice but did not change the performance of the control or 60% DR animals. These results may be related to an increase in septal concentrations of NE after tyrosine supplementation of the 4070 DR mice. The results support the observation that catecholaminergic neurons are affected by an increase in tyrosine only when they have been active or stressed as might be expected after severe DR [25,34,58,65,66], as shown also by changes in corticosterone production (see below). Tyrosine administration elevated concentrations of NE and MHPG (its breakdown product) in the septum and hypothalamus but did not change concentrations of dopamine in the septum, yet elevated them in the hypothalamus. These metabolic precursor-product relationships suggest that under the conditions of severe DR, tyrosine availability was a limiting factor in the biosynthesis and metabolism of NE. The mechanism by which the physiological activity of catecholaminergic neurons is coupled to their ability to respond to supplementary tyrosine probably involves the activation of tyrosine hydroxylase which uncouples the relation between the enzyme and its tetrahydrobiopterin cofactor (whose availability maybe rate limiting for catecholamine biosynthesis under basal conditions [30,41,71]), and decreases the enzyme’s susceptibility to end-product inhibition [56]. A continuous increase in tyrosine utilization within a catecholaminergic nerve terminal, as during DR, could also lead to a relative local depletion of the amino acid which would make the terminal more responsive to supplementation with tyrosine [34]. According to Schweiger et al. [59], acute starvation, as well as 3 weeks of semi-starvation with low protein high carbohydrate or high protein low carbohydrate diet, decreased NE turnover significantly. That catecholamine synthesis is dependent on tyrosine metabolism was shown by the study of Schoenfeld and Seiden [60], where w-methyltyrosine (an inhibitor of tyrosine hydroxylase) caused a decrease in brain levels of doparnine and NE as well as impaired operant behavior. DR may also lead to cognitive impairment through its effects on cerebral hypoglycemia. According to Gold [19], modest increases in circulating glucose concentrations en-

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hance learning and memory processes in rodents and humans, and glucose may attenuate opiate inhibition of acetylcholine release in the hippocampus. NE release in particular, is an important contributor to the process by which memory formation is regulated. Injections of either epinephrine or glucose near the time of training can enhance learning and memory in aged rats and mice [19]. The balance between ti and (3 catecholamine tone may also influence glucose homeostasis through modulation of pancreatic insulin secretion, and peripheral tissue and brain glucose metabolism [45]. Glucose acts within the medial septum which modulates the activity of neuron projections including those from the septum to the hippocampus. The improvement observed in maze performance after tyrosine, may also be related to potentiation of long lasting memory [11]. This requires activation of &adrenergic receptors which initiate reactions that underline potentiation or depression of neural activation in the hippocampus [10].

This may be because ~z-receptors have a low affinity for NE and that the concentration of epinephrine in the brain is relatively low [5]. The physiological responses to DR lead to modulation of the autonomic nervous system tone in order to conserve energy homeostasis [32] and stimulate appetite in the hypothalamus [37]. Thus, the sympathetic tone in the hypothalamus was characterized by an increase in alpha:beta tone, whereas in the hippocampus the changes were in the opposite direction, and cognitive function correlated with an increase in beta:alpha tone, as related to Bmax. There was practically no change in the K~ of the uand ~-receptors. Tyrosine administration reversed these changes: it decreased a ~-receptors and elevated ~ concentrations. This suggests a partitioning of neurotransmitters according to metabolic, physiologic and behavioral function in response to DR. 4.5. Possible relevance for patients with anorexia neruosa

4.3. The neuroendocrine responses to the stress of DR One of the features of neuroendocrine changes caused by starvation is low catecholarnine turnover in peripheral organs and in the brain [53]. Evidence points to a central mediation of low peripheral catecholamine turnover. Corticosterone stimulates tyrosine aminotransferase which is the main catabolic enzyme for tyrosine [6,30], and levels of corticosterone are higher in DR-treated animals [53]. Pirke and Spyra [53] also presented evidence that noradrenergic turnover is reduced in acute starvation. ACTH secretion which governs corticosterone secretion is generally believed to be under inhibitory noradrenergic control [14]. Starvation and semi-starvation are associated with low catecholarnine turnover in peripheral organs [32] and in the brain of rats [53,59]. Anorectic patients also exhibit signs of reduced central NE turnover [28]. Tyrosine availability is only operant in situations in which noradrenergic neurons fire at an increased rate [34]. A decreased activity of noradrenergic neurons may be responsible for the corticosterone increase in the plasma of starved rats. Noradrenergic and dopaminergic neurons are among other factors involved in the hypothalamic regulation of gonadotrophins and ACTH [53]. Animal studies support the concept that the hypothalamic monoamine systems which regulate appetite dopamine and 5-HT may also affect behavior [37]. 4.4. Regional dl~erences in adrenergic receptors in response to DR NE has a role in the regulation of a2-adrenergic receptors. According to Minneman et al. [46], the number of ci~and a ~-receptors increased after the noradrenergic neurons in the brain had been destroyed by injections of 6-hydroxydopamine; the number of ~l-receptors increasedmarkedly but no change occurred in the number of ~z-receptors.

It can be concluded that DR in mice affects cognitive behavior and adrenoceptor status, and its effects may be partially reversed by the administration of tyrosine. Since there is no available brain storage tissue, the level of nutrition is considered to influence cognitive processes [70,71]. According to this assumption, part of the refusal to eat in patients with anorexia may be a psychological concomitant to neurochemical dysfunction in eating behavior caused by the lack of neurotransrnitters (dopaminergic and catecholaminergic), connected to tyrosine metabolism [35,36]. This idea is supported by the observation that levels of tyrosine and NE in the plasma and cerebrospinal fluid are reduced in anorectic patients in comparison with normal subjects, and remain so even after partial recovery [28]. Since the problem of eating disorders in humans is still not well understood, psychotherapy and medical treatment of patients with AN cannot be successful until weight is regained – but these patients refuse to eat and deny that they are thin. In human subjects, the administration of tyrosine to patients with Parkinson’s disease caused increases in plasma tyrosine, catecholarnine excretion [2], and CSF tyrosine, dopa and homovanillic acid [20]. Tyrosine may also improve memory performance in man under conditions of experimental stress [62]. Tyrosine, therefore, may be beneficial for the treatment of cognitive disturbance in women with AN by supplying substrates for noradrenergic neurons whose impaired activity may be caused by lack of tyrosine precursor for NE synthesis. This knowledge and the results of our data (despite being performed on an animal model), give preliminary support to the idea that tyrosine supplementation may protect against both the neurobiochemical and the behavioral effects of DR. It may normalize sympathetic nervous system activity and cognitive function in patients with the severe dietary restriction associated with anorexia nervosa before weight is gained.

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If this supposition is substantiated, then tyrosine may provide a novel therapeutic intervention for these patients.

Acknowledgements We thank Professor Yossi Yanai and Professor David Mostofsky for valuable help and encouragement in this project, and Dr. Michael Newman and Dr David Berry for reviewing the manuscript. This research was supported in part by a grant from the Hebrew University (Sir Zelman Cowan Universities Trust – 034-4250) and from the United States - Israel Binational Science Foundation (9400140).

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