Brain Research, 131 (1977) 259-269 © Elsevier/North-Holland Biomedical Press

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T H E EFF EC T OF I S O L A T I O N ON C A T E C H O L A M I N E C O N C E N T R A T I O N A N D T U R N O V E R IN DISCRETE AREAS OF T H E RAT BRAIN

NGUYEN B. THOA, YOUSEF TIZABI* and DAVID M. JACOBOWITZ**

Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.)

(Accepted November llth, 1976)

SUMMARY

In normal grouped rats there are large variations in the rate of decline of cate- cholamines (CA) in 23 brain regions following inhibition of tyrosine hydroxylase. This suggests that regardless of the neuronal origin there are variations in turnover activity of CA terminals in different brain areas. Following tyrosine hydroxylase inhibition, the largest reductions of norepinephrine (NE) and dopamine (DA) were observed in the cell body areas. Furthermore, there is no distinction in turnover between regions innervated by the dorsal or ventral noradrenergic pathways. After 13 weeks of isola- tion, a significant decrease in the steady-state NE concentration was observed in the hippocampus, nucleus amygdaloideus centralis, while an increase was seen in the entorhinal cortex. A decrease in steady-state DA concentration was found in the a- mygdaloid centralis, while an increase was noted in the olfactory tubercle. Therefore, both a rise and fall in steady-state levels of amines occur in a few discrete regions of the brain. A decrease in turnover of NE was observed in 6 of 23 areas (hippocampus, amygdaloid centralis, cingulate cortex, caudate, nucleus interstitialis stria terminalis, paraventricular nucleus) following alpha methyl-p-tyrosine treatment. A decrease in turnover of DA was seen in the amygdaloid centralis.

In prolonged isolation, it appears that the reduction of external stimuli results in a decrease in catecholaminergic activity in some limbic areas which may correlate with increased levels of emotionality and aggression.

INTRODUCTION

The external environment plays a critical role in the maintenance of a normal

* Post-doctoral Fellow, Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, D.C. 20007, U.S.A. Present address: Dept. Pharmacology, Howard Univ., Wash. D.C. ** To whom requests for reprints should be addressed.

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emotional state. Deprivation of social stimulation and interaction by prolonged isola- tion has been shown to induce aggressive behavior in monkeys 12, rats ~8 and mice1,38, 45.

In the latter species, isolation produces what has been termed 'the isolation syndrome' (ref. 41), a composite behavior characterized by increases of general reactivity, re-

sponses to painful stimuli, vocalization, irritability and development of a compulsive aggressive behavior.

Changes in biogenic amine dynamics have been shown to occur, both peripheral- ly and centrally, following isolation. In mice, a significant reduction in adrenal cate- cholamine synthesizing enzymes 3, a decrease in brain turnover rate of norepinephrine (NE) 4z-44 and a decrease in brain synthesis rate of NE and dopamine (DA) but not of serotonin 3° have been observed. In rats, prolonged isolation caused an increase in brain NE levels 1°.

In a previous study 39 we have shown that young Sprague-Dawley rats, isolated for 13 weeks, have higher steady-state concentrations of NE in the entorhinal cortex and lower levels of NE in the amygdaloid centralis nucleus. In this study, NE and DA

concentrations are measured in 23 different brain areas of grouped and isolated rats following administration of alpha-methyl-p-tyrosine (methyl ester; AMPT), an in- hibitor of tyrosine hydroxylase (TH) 5,11, the rate limiting enzyme in the biosynthetic pathway of DA and NE 2a. The rate of decline of both amines after AMPT will be used as an index for measuring their turnover rate 2°.

METHODS

Animals

Thirty-two male Sprague-Dawley rats (Zivic-Miller, Allison Park, Pa.), 24-28

days old, weighing 40-50 g, were divided into two groups: rats from one group were kept in metallic isolation cages (20 cm × 29 cm × 17.5 cm) and the cages were kept on a rack, which faced a blank wall so that the animals did not have visual contact with each other. Animals from the second group were housed in the same rack, 6 to a cage (40 cm × 29 cm × 17.5 cm). All animals were kept in a temperature controlled room (24-25 °C), with light cycle of 6: 00 a.m. to 6: 00 p.m., with food and water ad lib.

At the end of 13 weeks, both groups of animals were weighed and each group divided into two subgroups, one receiving AMPT (Sigma) 430 mg/kg (i.p.) dissolved in 4 ml of warm saline and the control group received 4 ml of warm saline, i.p. The rats were alternatively decapitated 2 h after drug or saline injections.

Biochemical determinations

Brain NE and DA. The brains were quickly removed and frozen on dry ice. Brain slices (frontal plane section) of 300 #m thickness were cut in a cryostat at - -7 °C. The frozen sections were placed on cold slides, slightly thawed and quickly refrozen on dry ice. The slides were placed on a cold plate under a stereomicroscope. Microdissection of nuclei and other brain regions were performed by the 'punch' procedure using small stainless steel cannulae 34. The coordinates for the various dissected regions are in-

TABLE I

Area Number of Size Approximate punches cannula (mm) coordinates (#m)

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Frontal cortex slice A12,000 N. accumbens 4 0.5 A9650; A9410 Caudate nucleus 2 1.0 A8920 Olfactory tubercle 4 0.5 A9650; A9410 N. interstitialis stria terminalis

(dorsal and ventral) 4 0.5 A6860 Cingulate cortex 4 1.0 A6860; A6570 Piriform cortex 4 0.5 A6860; A6570 Preoptic medialis 2 1.0 A6860 Supraoptic nucleus 4 0.5 A6360; A5910 Paraventricular nucleus 2 0.5 A5660 Periventricular nucleus 3 0.5 A6570; A6280 Arcuate nucleus 6 0.3 A4890 to A3750 Median eminence 3 0.5 A4890 to A3750 N. dorsomedialis 2 0.5 A4110 Hippocampus 4 0.75 A4380; A4110 Dorsal bundle 4 0.5 A3180; A2790 Median forebrain bundle (MFB) 2 0.75 A3430 Entorhinal cortex 4 0.5 A2420; A2180 N. Amygdaloideus centralis 4 0.5 A4380; A4110 Substantia nigra compacta 4 0.5 A2180 Ventromedial tegmentum (A10) 2 0.5 AI800 Habenula 4 1.0 A3990; A3750 Locus coeruleus 2 0.5 P2800

dicated in Table I and correspond to the atlases o f K6nig and Klippe122, Jacobowitz and Palkovits 16 and Palkovits and Jacobowitz 35. The size o f the cannulae used for

dissection o f various regions is also shown in Table I. For assay o f D A and NE, samples f rom both right and left brain regions o f each

animal were blown f rom the dissecting cannulae into 100 #1 ice-cold 0.1 N perchloric

acid and homogenized by sonification as previously described 17. Ten /A homogenate

aliquots were removed for protein determination 26, the remaining samples were frozen

and stored at - - 8 0 °F until assayed. After the frozen samples were thawed, vortexed

and centrifuged (8000 × g for 30 sec), 25 #1 of the supernatant was assayed for D A and N E by means o f a sensitive enzymatic isotopic method 6. Pooled tissue supernatant

f rom each region was used for determination o f internal standards. The results are reported as nanograms of catecholamine per mg of protein.

Statistics

Compar ison between groups was made by the Student 's t-test.

RESULTS

Body weight

The body weight o f isolated rats (530 d: 13 g; n = 16) was significantly higher than those o f grouped rats (439 q- 14 g; n = 16).

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264

Brain NE levels: decline with AMPT (Tabh" 11) In grouped rats, 2 h after AMPT, there was a non-uniform decline in NE in most

of the 23 brain areas studied. The most pronounced reduction was found in the locus coeruleus (79 ~ decrease). In the nucleus interstitialis stria terminalis (N IST), cingulate cortex, arcuate nucleus, nucleus dorsomedialis, hippocampus, dorsal bundle and a- mygdala centralis, a reduction of 50-70 ~ was observed. In the caudate nucleus, pyri- form, frontal and entorhinal cortices, preoptic medialis, paraventricular supraoptic nuclei and the median forebrain bundle (MFB), a reduction of 30-50 o~ was observed. Areas that showed little or no change included the nucleus accumbens, olfactory tu- bercle, periventricular nucleus, median eminence, habenula, substantia nigra compacta (SNC) and ventromedial tegmentum (ATV) (AI0).

In isolated rats, the steady state NE concentration was significantly higher than in grouped rats in the entorhinal cortex and lower in the hippocampus and amygda- loid centralis. After AMPT, the decline in NE levels in the entorhinal cortex was not significantly different than grouped animals, but was significantly lower in the hippo- campus, amygdaloid centralis, cingulate cortex, caudate, N IST and the paraventricular nucleus.

Brain DA levels: decline after AMPT (Table 1II) Some areas have DA levels just above the limit of the methods sensitivity

(< twice blank reading) and were therefore excluded from the table. These include the pyriform, hippocampus, entorhinal and frontal cortices and the MFB. In grouped rats, after AMPT, there was a 60-70~o reduction of DA in the ventromedial tegmental area, SNC, arcuate nucleus, amygdaloid centralis and nucleus accumbens. In the median eminence, olfactory tubercle and caudate there was a decline of 40-45~. Other areas, with a low DA concentration, such as the NIST, cingulate cortex, pre- optic medialis, nucleus dorsomedialis, supraoptic nucleus, periventricular nucleus and the locus coeruleus, were reduced by 40-75 ~o following AMPT. The DA in some of these regions probably constitute precursor quantities in noradrenergic nerves. In habenula and dorsal bundle, little reduction was seen.

In the isolated rats, a lower steady state level of DA was observed in the amyg- daloid centralis and a higher level in the olfactory tubercle. After AMPT, a lower turn- over was found in the former area and a higher decline in the latter area.

DISCUSSION

After 13 weeks of isolation, rats showed a decrease in motility, and upon hand- ling, isolated rats showed higher reactivity. The incidences of squealing, escaping and glove biting are more frequent than with grouped rats. Increased emotionality, mani- fested as irritable or 'agressive' behavior after prolonged isolation, is well documented < 12,18,19,30,38,46. In this study, young rats isolated for 13 weeks showed increased emotionality, since they vocalized, attempted to escape and glove-bit more than grouped rats. These reactions seemed to stem more from fear than aggressiveness since isolated rats remain quiescent and do not fight other isolated rats that are placed in the cage.

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Isolated rats ate more and gained more weight than grouped rats (ref. 4 and the present study), probably because of the availability of food and the paucity of any other motor activity.

Prolonged isolation is known to be associated with changes in biogenic amine dynamics in whole brain. Geller et al. 1° found that young rats, 20 days old, isolated for one month, showed an increase in whole brain NE content. Welch and Welch 42 found a decrease in mice brain CA turnover rate 14 weeks after the beginning of isola- tion. Modigh 3° found that, in mice isolated for 6-8 weeks, brain CA synthesis rate was decreased but that of serotonin was unaltered.

In a previous study 39 it was shown that young rats isolated for 13 weeks have lower NE levels in the nucleus amygdaloideus centralis and higher levels in cingulate and entorhinal cortices. In this study, the effect of prolonged isolation on CA turnover rate in 23 brain areas were examined. It is possible to estimate catecholamine turnover rates in brain nuclei by using an inhibitor of tyrosine hydroxylase. Kizer et al. 2° observed that after administration of AMPT (400 mg/kg i.p.) to rats at 0 and 3 h, the rate of decline of NE and DA in various hypothalamic nuclei was constant from 1 to 5 h after enzyme inhibition. Measure of CA during that time period would reveal changes in turnover rates and, therefore, provide an index of the functional activity of the a- minergic neurons in these nuclei.

In contrast to previous studies of the turnover of CA following stress, where large brain regions were investigated, the present study reveals considerable variations in the decline of CA in 23 brain regions of normal grouped rats following inhibition of TH. The range of changes observed varied from essentially none to a marked depletion of CA. This would suggest that regardless of the neuronal origin (e.g., locus coeruleus), there are variations in turnover activity of catecholaminergic terminals in different brain areas.

It would be appropriate to attempt to establish the possibility of a correlation of the degree of amine turnover with the known noradrenergic and dopaminergic systems. Two major ascending noradrenergic systems, the dorsal and ventral pathways, are derived from distinct cell groups in the hindbrain which give rise to extensive projec- tions to the forebrain2,7,s,13-16,2x,24,25,27,2s,32,3a,35,a7, 4°. The dorsal noradrenergic

bundle is derived primarily from the locus coeruleus (A6) cell bodies and the ventral noradrenergic pathway is derived from cell groups located in the pons-medulla (AI-A7), nomenclature of Dahlstr6m and FuxeL The dorsal noradrenergic system mostly innervates all the cortices of the brain in addition to other regions. The ventral system is believed to project axons to the spinal cord, mesencephalon, hypothalamus and preoptic area. Much of the DA in the brain concentrates within 3 groups of neu- rons: the nigrostriatal system which originates in the substantia nigra (zona compacta, cell group A9) and terminates in the striatum and amygdala; the mesolimbic system which begins in the ventral tegmental area (cell group A 10) and projects mainly to the nucleus accumbens and olfactory tubercle; and the arcuatoinfundibular hypothalamic system which originates in the arcuate nucleus (cell group A12) and terminates in the median eminenoea,9,16,35, 40.

Following TH inhibition, the largest reductions in NE or DA were observed in

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the cell body areas. A 79 ~o reduction in NE was observed in the locus coeruleus, and a 60-70 ~ decrease in DA was seen in the substantia nigra compacta, ventromedial tegmental area and the arcuate nucleus.

A comparison of changes in turnover in the NE-containing areas did not reveal a distinction between regions innervated by the dorsal or ventral noradrenergic path- ways. A relatively large reduction of NE (50-60 9/00) was observed in the cingulate cor- tex, hippocampus, and amygdala which belong to the dorsal NE system as well as in the NIST, and nucleus dorsomedialis which receives innervation from the ventral system. Reductions of 30-50 ~ were also found in areas belonging to both systems: pyriform, frontal, entorhinal cortices (dorsal bundle) and preoptic medialis and para- ventricular nucleus (ventral bundle). NE reduction was lowest (5-25 ~o) in predomi- nantly DA-containing areas such as the substantia nigra compacta, ventromedial tegmental area, olfactory tubercle, nucleus accumbens and the median eminence. A relatively large reduction (40-60 ~o) was observed in the caudate nucleus, amygdala centralis and arcuate nucleus.

Comparatively large reductions (40-60 ~ ) in DA concentrations were observed in regions innervated by the three dopaminergic systems (nigrostriatal, mesolimbic, arcuato-infundibular) following an injection of AMPT. An exceptionally large reduc- tion of DA, comparable to that observed in the cell body areas, was seen in the nucleus amygdaloideus centralis.

Following prolonged isolation, a significant decrease in the steady-state NE concentration was observed in the hippocampus and nucleus amygdaloideus centralis while an increase was seen in the entorhinal cortex. Such changes in a few small areas of the brain would probably not have been detected in analysis of large brain regions. Although the three regions that contain significant changes in NE receive their nora- drenergic innervation via the dorsal bundle, not all areas derived from this pathway (e.g., frontal cortex, piriform cortex, habenula) show changes in the steady-state levels of this neurotransmitter. Furthermore, the direction of change is not uniform in that both a rise and a fall in steady-state levels was observed.

In isolated rats a significant decrease in the turnover of NE was observed in 6 of the 23 regions studied following AMPT treatment. Again, the areas of change receive aminergic innervation from either the dorsal bundle (e.g., cingulate cortex, hippocam- pus, amygdala) or the ventral pathway (e.g., NIST, paraventricular nucleus). Further- more, the turnover of NE is not changed in all the regions investigated, which is of interest in view of prior studies that report decreases in turnover in large brain areas in isolated rodents31, 42.

A decrease in both steady-state and turnover of NE was observed in the hippo- campus and the nucleus amygdaloideus centralis. An explanation for this is not at hand.

A decrease in NE turnover was observed in the NIST, paraventricular nucleus and caudate nucleus without a corresponding change in the steady-state levels follow- ing chronic isolation. It is also of interest that no change in the turnover rate was ob- served in the locus coeruleus following isolation. The greatest turnover of NE was observed in this cell body region in both the grouped and isolated rats.

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Changes in the steady-state dopamine concentration in isolated rats were ob- served in only two regions. A 50 % increase was seen in the olfactory tubercle while a 35 % decrease in DA was noted in the amygdala centralis. These changes seemed to correlate with the turnover rates following AMPT treatment, i.e., an increase in turn- over in the olfactory tubercle and a reduction in the amygdala centralis. The signif- icance of an increased turnover coupled to an elevated steady-state level in the olfac- tory tubercle is unknown. It would seem that the synthesis of DA in this area proceeds at a greater rate than release in isolated rats.

Although there was no uniformity in the response of any single NE system (dorsal or ventral) to isolation, the observed changes occurred mainly in areas belong- ing to the limbic system. Thus following chronic isolation, the entorhinal cortex, hip- pocampus, amygdaloid centralis and NIST are all limbic brain structures that are accompanied by a decrease in noradrenergic neuronal activity which is manifested as a decrease in turnover rate.

In dopaminergic regions, the reduction in turnover in the amygdaloid centralis correlates with NE changes in that area and also in other limbic structures. It is curious that an increase in turnover was observed in the olfactory tubercle of isolated rats. We have no explanation for the exceptional nature of this important limbic structure.

The limbic system is generally thought to play a critical role in emotional be- havior 29,a6 and it is tempting to speculate that catecholamines modulate neuronal activity in limbic structures. In prolonged isolation, Jt appears that the reduction of external stimuli results in a decrease in catecholaminergic activity in some limbic areas which may correlate with increased levels of emotionality and aggression 1,4,a°,3a.

In summary, in normal grouped rats there are large variations in the rate of decline of CA in 23 brain regions following inhibition of tyrosine hydroxylase. This suggests that, regardless of the neuronal origin, there are variations in turnover activity of CA terminals in different brain areas. Following tyrosine hydroxylase inhibition, the largest reduction of NE and DA was observed in the cell body areas. Furthermore, there is no distinction in turnover between regions innervated by the dorsal or ventral noradrenergic pathways. After 13 weeks of isolation, a significant decrease in the steady-state NE concentration was observed in the hippocampus, amygdaloid centra- lis, while an increase was seen in the entorhinal cortex. A decrease in steady-state DA concentration was found in the amygdaloid centralis while an increase was noted in the olfactory tubercle. Therefore both a rise and fall in steady-state levels of amines occur in a few discrete regions of the brain. A decrease in turnover of CA was observed in 6 of 23 areas following AMPT treatment.

Further studies are required in order to definitively reveal a functional relation- ship between biogenic amines in discrete areas of the brain and emotional behavior following social isolation.

A C K N O W L E D G E M E N T S

The expert technical assistance of Mr. Eric A. Muth and Mr. Robert P. Mc- Devitt is gratefully acknowledged.

Dr. Tizabi was supported by National Institute of Mental Health Grant MH 2544A.~01, 02.

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