Brain Research, 131 (1977) 259-269 © Elsevier/North-Holland Biomedical Press 259 T H E E F F E C T OF I S O L A T I O N O N 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 I N D I S C R E T E A R E A S OF T H E R A T B R A I N 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 catecholamines (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 isolation, a significant decrease in the steady-state N E 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 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 N E was observed in 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 260 emotional state Deprivation of social stimulation and interaction by prolonged isolation 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, responses to painful stimuli, vocalization, irritability and development of a compulsive aggressive behavior Changes in biogenic amine dynamics have been shown to occur, both peripherally and centrally, following isolation In mice, a significant reduction in adrenal catecholamine 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 inhibitor 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 A M P T 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, 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 A M P T (Sigma) 430 mg/kg (i.p.) dissolved in ml of warm saline and the control group received ml of warm saline, i.p The rats were alternatively decapitated h after drug or saline injections Biochemical determinations Brain N E 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 - - °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- 261 TABLE I Area Number of punches Size Approximate cannula(mm) coordinates(#m) Frontal cortex N accumbens Caudate nucleus Olfactory tubercle N interstitialis stria terminalis (dorsal and ventral) Cingulate cortex Piriform cortex Preoptic medialis Supraoptic nucleus Paraventricular nucleus Periventricular nucleus Arcuate nucleus Median eminence N dorsomedialis Hippocampus Dorsal bundle Median forebrain bundle (MFB) Entorhinal cortex N Amygdaloideus centralis Substantia nigra compacta Ventromedial tegmentum (A10) Habenula Locus coeruleus slice 4 0.5 1.0 0.5 A12,000 A9650; A9410 A8920 A9650; A9410 4 4 4 4 4 0.5 1.0 0.5 1.0 0.5 0.5 0.5 0.3 0.5 0.5 0.75 0.5 0.75 0.5 0.5 0.5 0.5 1.0 0.5 A6860 A6860; A6570 A6860; A6570 A6860 A6360; A5910 A5660 A6570; A6280 A4890 to A3750 A4890 to A3750 A4110 A4380; A4110 A3180; A2790 A3430 A2420; A2180 A4380; A4110 A2180 AI800 A3990; A3750 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 F o r assay o f D A and NE, samples f r o m both right and left brain regions o f each animal were blown from the dissecting cannulae into 100 #1 ice-cold 0.1 N perchloric acid and homogenized by sonification as previously described 17 T e n / A homogenate aliquots were removed for protein determination 26, the remaining samples were frozen and stored at - - °F until assayed After the frozen samples were thawed, vortexed and centrifuged (8000 × g for 30 sec), 25 #1 o f the supernatant was assayed for D A and N E by means o f a sensitive enzymatic isotopic m e t h o d Pooled tissue supernatant from each region was used for determination o f internal standards The results are reported as nanograms o f catecholamine per mg o f protein Statistics Comparison between groups was made by the Student's t-test RESULTS Body weight The b o d y 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) 262 X E "s ~ q q ~tq~qq{qtqqq~q+t~q~ H~la dddd ~ q q ~ "- ~4~M X q q q ~ q q q ~ ÷ t q ~ q q ~ t q - I ~ SN~ o o~ o~ RqSq 8~S~SS~$~8~8~SS~ SSSS SSS~*~S~66g£~SS 6555 qqqq ~ 4 ~ 4 ~ 5 ~ 5 x q ~ q ~ q ~ t ~ q ~ q q - H q q t ~ O" II & *~ ~z rO q rO~_ O0 9o ~V ~ :~ E < g~ c5 V V -~ -~ i N ~=~e ~ ~- ~~-_ < ~1~-~ u~Z~OZ ~ ~_~ ~ ' , " ~ O o ~, ~ -s o.o oo VV 263 -H-H~ e~ o ,-4~ " -H -H -H ~ ddd ,4 ~ ,,4 -H~-H ~ m ~ Q ~ zoo oooo oo M ~ M ~ M M ~ d d d d d d ~ £ d d M ~ d ~ ~'~ -H~-H o r,i ~4 , -H -H -H d d d N Z d N ~ N d N M d d 8,, H geg ~4r4 ~-H-H m ~ m V V II g~ =,~ < oV V~ 264 Brain NE levels: decline with A M P T (Tabh" 11) In grouped rats, 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 amygdala centralis, a reduction of 50-70 ~ was observed In the caudate nucleus, pyriform, 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 tubercle, 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 amygdaloid 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 hippocampus, amygdaloid centralis, cingulate cortex, caudate, N IST and the paraventricular nucleus Brain DA levels: decline after A M P T (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, preoptic 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 amygdaloid centralis and a higher level in the olfactory tubercle After AMPT, a lower turnover 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 handling, isolated rats showed higher reactivity The incidences of squealing, escaping and glove biting are more frequent than with grouped rats Increased emotionality, manifested 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 not fight other isolated rats that are placed in the cage 265 Isolated rats ate more and gained more weight than grouped rats (ref 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 isolation 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 A M P T (400 mg/kg i.p.) to rats at and h, the rate of decline of NE and DA in various hypothalamic nuclei was constant from to 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 aminergic 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 projections 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 groups of neurons: 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 T H inhibition, the largest reductions in NE or DA were observed in 266 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 pathways A relatively large reduction of NE (50-60 9/00)was observed in the cingulate cortex, 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 paraventricular nucleus (ventral bundle) NE reduction was lowest (5-25 ~o) in predominantly 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 reduction 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 noradrenergic 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 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, hippocampus, amygdala) or the ventral pathway (e.g., NIST, paraventricular nucleus) Furthermore, 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 hippocampus 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 following chronic isolation It is also of interest that no change in the turnover rate was observed 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 267 Changes in the steady-state dopamine concentration in isolated rats were observed 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 A M P T treatment, i.e., an increase in turnover in the olfactory tubercle and a reduction in the amygdala centralis The significance of an increased turnover coupled to an elevated steady-state level in the olfactory 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 N E system (dorsal or ventral) to isolation, the observed changes occurred mainly in areas belonging to the limbic system Thus following chronic isolation, the entorhinal cortex, hippocampus, 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 N E 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 behavior 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 N E 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 centralis, while an increase was seen in the entorhinal 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