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Gastrointestinal motility and body weight gain in rats after brain serotonin depletion by 5,7-dihydroxytryptamine
GASTROINTESTINAL MOTILITY AND BODY WEIGHT GATN IN RATS AFTER BRAIN SEROTONIN DEPLETION BY 5,7_DIHYDROXYTRYPTAMINE* C. F. SALLER and Psychobiology
Program.
E. M. STRICKER~
Departments of Life Sciences and Psychology, Pittsburgh. Pennsylvania 15260. U.S.A. (Accepted
University
of Pittsburgh.
3 Ocroher1977)
Summary-Intraventricular injections of 5.7-dihydroxytryptamine. combined with systemic administration of the norepinephrine uptake blocker desmethylimipramine. produced large depletions of brain serotonin without affecting norepinephrine levels in 42 rats. Twelve animals showed enhanced body weight gain. in some 25-4@‘,, above control values. Most of these rats (7;12) were handled extensively before and during the 4-5 month period of observation. The other 30 rats had normal body weights. but a marked depression of gastrointestinal motility led to a considerable accumulation of food and wastes in the lower intestines. Most of these rats (26/30) were not handled extensively. It is proposed that the intestinal dysfunctions resulted from an exaggerated sympathoadrenal response to stress. and that handling tended to ameliorate this effect, In contrast. desmethylimipramine apparently potentiates the decrease in gastrointestinal motility. since this effect did not occur in rats when the drug either was withheld or was inexplicably ineffective, However. these latter animals did not show enhanced body weight gain despite apparently normal intestinal function and comparable losses of brain serotonin. perhaps because substantial depletions of norepinephrine occurred as well.
Administration of 5,7_dihydroxytryptamine (5,7DHT) into the cerebral ventricles of rats. following treatment with desmethylimjpramine (DMI), produces large and apparently sefective depletions of 5-hydroxytryptamine (5-HT) in the brain (Bjiirklund, Baumgarten and Rensch, 1975; Gerson and Baidessarini, 1975). Recently the present authors reported that juvenile male rats given this treatment maintained body weight gains of 5-6 g per day into aduithood and grew much larger than control rats. Hyperphagia did not develop despite comparable losses of 5-HT when the pretreatment was withheld, perhaps because substantial depletion of norepinephrine (NE) occurred as well (Sailer and Stricker, 1976a). The experiment reported here confirms those findings but focuses on a new factor that can prevent enhanced growth despite large and selective depletions of brain 5-HT. This factor became evident in the early phases of the present work, when it was found that many rats given 5,7-DHT and DMI did not have body weights that were significantly above control values 4 months after treatment. Each of these animals had intestines that were greatly distended, a dysfunction that had not occurred previously. Since the only procedural difference was that now rats were being handled only for 3 days before they received 5.7-DHT and DMI, whereas in the initial investigation, rats were handled daily for 2-3 weeks prior to treatment, it was decided to pursue this issue by examining the effects of extensive handling on gastro*Partially supported by U.S. Public Health Service (NIMH) Grants MH-20620 and MH-25140. *To whom all correspondence should be addressed.
intestinal motility and body weight gain in rats foilowing the administration of 5,7-DHT and DMI.
METHODS
Male Sprague-Dawley rats (Zivic-Miller Laboratories. Pittsburgh. PA.) initially weighing 190-2 IS g were housed and tested singly in wire mesh cages in a room maintained at 23’C. with fluorescent lighting from 0600 to I800 hr. All the rats had continuous access to Purina Lab Chow pellets (placed on the cage Roar) and tapwater (presented in bottles with metal drinking nozzles). except as noted.
Rats were pretreated with DMI (Norpramin@. Lakeside Labs, 25 mg/kg i.p.) (n = 54) or 0.9”,, NaCl solution (n = 10). Fifty minutes later, with the rats under ether anaesthesia, a 20 ~1 solution of 0.9”; NaCl and O.l”, ascorbate containing 15Opg (n = 16) or 2OOpg (n = 38) of 5,7-DHT creatinine sulphate (Regis, dose expressed as free base) was injected into the right lateral ventricle (Noble. Wurtman and Axeirod, 1967). Sixteen rats were given a second injection of 5,7-DHT (15Opg, into the contralateral ventricle). following DMI treatment, 3 days later. To prevent sodium pentobarbital (Nembut~~f~. convulsions, Abbott Laboratories, 35 m&kg i.p.) was given immediately after the intraventricuiar injections (Baumgarten and Lachenmayer. 1972). and was suppiemented when necessary by ether anaesthesia. Additional animals received DMI once (n = 14) or twice (n = 4). or 0.9, NaCi solution (n = 5). followed by
C.
500
F.
SALLER and
intraventricular injections of the 5.7-DHT vehicle solution. and served as the control groups. Thirteen rats given a single injection of 5,7-DHT and DMI. and the 10 rats given 5,7-DHT without DMI, were handled daily for 2 weeks prior to treatment and weighed (+ 2 g) each day thereafter. Control rats given the 5.7-DHT vehicle with (n = 4) or without DMI pretreatment (n = 5) were treated similarly. The remaining animals were handled for only 3 days before treatment. and were weighed every third day subsequently. Rats were sacrificed either 120 or 150 days after the intraventricular injections. Brain monoamine levels were determined in each animal. and gastrointestinal motility was assessed in selected animals from each subgroup, as below. Monoamine
assays
Animals were killed by decapitation. Each brain was separated from the spinal cord at the level of the foramen magnum and was rapidly removed from the skull. The pineal glands were discarded. and the remaining brain tissue was frozen on dry ice. stored at -70 C for not more than 1 week. and later analysed fluorometrically for 5-HT. NE and dopamine (DA) using minor modifications of methods that have been described elsewhere (Atack. 1973; Atack and Lindqvist, 1973; Haggendal, 1963). Trace amounts of radioactive amines (New England Nuclear) were added to the samples to permit determinations of recovery. Measurements
OJyastrointesrinal
motilit~~
Gastrointestinal motility was measured. by a modification of previous methods (Bridges. Dent and Johnson, 1976). in 21 rats given 5,7-DHT. none of which displayed abdominal distention. Food and water were removed 1 hr prior to an intragastric intubation of water containing the non-absorbable marker polyethylene 1,2-[“‘Cl-glycol (PEG) (0.5 nCi in I ml. New England Nuclear). Three hours after the intubation. the rats were sacrificed and the stomach, 6 equal segments of small intestine, and the colon were removed. The tissues were immersed in IOml of a solution containing 2 N NaOH, methanol, and Triton-X-405 (60:30:10) for 12 hr. following which 10ml of water were added and the tissues were homogenized. The homogenate was adjusted to pH 7.0 with concentrated nitric acid. and an 0.5 ml aliquot was analysed for [‘“Cl-PEG by liquid scintillation spectrometry. Counting efficiency was monitored using [‘JC]-PEG as an internal standard. A relative index of gastrointestinal motility in the 5-HT-depleted rats was computed by assigning the numbers 0 through 7 to the 8 successive sections of gastrointestinal tract (i.e. stomach. 6 segments of small intestine, and colon), multiplying those numbers by the percentage of total recovered radioactivity that was found in each section, and dividing the sum of these products by the mean value obtained from com-
E. M.
STRICKER
parable computations usmg tissue from 7 control animals. A less exact index of gastrointestinal motility was obtained in other animals by simply measuring the amount of food that was present in the tract during arl lihiturn feeding. Upon sacrifice, the stomach. small intestine and colon were separated, their ends were clamped, and they were placed in preweighed glass beakers. Wet tissue weights were recorded before heating them at IO@110-C to dryness. after which they were reweighed. Wet and dry weights of tissue segments from 5 untreated control rats were obtained for purposes of comparison. RESULTS
Animals injected intraventricularly with 5,7-DHT vehicle solution whether pretreated once with DMI or with 0.9”, NaCl solution, had similar body weight gains and brain monoamine levels and consequently they were combined into a single group (Group A). Rats given a single injection of 5.7-DHT all had substantial depletions of brain 5-HT. and whole brain DA levels (M i S.E.M. = 0.98 f 0.03 pg/g wet tissue) that were unchanged. However. such clear differences in their body weights and brain NE levels were apparent that four groups were formed for further analysis. as follows. Group B. given DMI pretreatment and showing no depletion of brain NE. contained the only rats whose body weights were above the control range; Group C. given DMI and similarly showing no depletion of brain NE. contained rats whose body weights fell within the control range; Group D. also given DMI. contained rats that nevertheless showed moderate depletions of brain NE: and Group E, not given DMI, contained rats that had large depletions of brain NE, as expected. The partition of these animals is indicated in Table 1. along with the mean +S.E.M. levels of brain 5-HT and NE and body weight increases. while individual body weight gains are presented in Figure I. Depletions of brain 5-HT in Group B were not uniform but ranged from 67 to SS”,,. Individual values are presented in Figure 2. in association with the body weight increases of each animal. Note that the increase in body weight was imersely proportional to residual 5-HT levels (r = -0.76, P < 0.01). and that the two heaviest animals were the only ones whose depletions of 5-HT exceeded 85”,,. The enhanced body weight of rats in Group B usually did not begin immediately after 5.7-DHT treatment but instead became prominent 45560 days later, when the growth rate of the controls began to diminish. One exceptional animal. however. began to gain weight at an accelerated rate within the first postoperative week and remained heavier than the others throughout the 4-month period of observation (Fig. 3). This rat and the two other heaviest animals clearly were larger in size than the controls. Autopsy revealed no unusual accumulation of adipose tissue
Increased Table
501
in rats after 5.7-DHT
I. Whole brain monoamine levels and body weight increases in rats after intraventricular 5.7-DHT and parenteral DMI
Treatment
n
Controls 5.7-DHT/DMl 5.7-DHTI’DMI 5,7-DHT/DMI 5.7-DHT
(19) (12) (21) (5) (10)
Group A B c D E
growth
NE @g/g wet weight 0.44 0.56 0.50 0.29 0.17
+ + + + +
0.03 0.06 0.10 0.05* 0.02*
Body weight increase (g t S.E.M.)
5-HT _t S.E.M.) 0.88 0.20 0.24 0.20 0.17
k * + f *
0.06 0.02* 0.09* 0.0.5* O.OSf
* P < 0.01. by r-test. in comparison with control animals which In = 14) or 0.9”,. NaCl solution (n = 51 followed by intraventricular 5.7-DHT vehicle solution. ’
within the abdominal cavity of these rats or any of the others in Group B. Handling had a significant effect on body weight gain in rats with specific depletions of brain S-HT: 7 of the 12 rats in Group B were handled extensively, whereas 17 of the 21 rats in Group C were not (x2 = 3.68, P < 0.05, one-tailed test). In contrast. despite extensive handling, body weight gain was not enhanced in 2 rats in Group D or in 10 rats in Group E, all of which had sizeable depletions of brain NE. Gastrointestinal motility was measured in 21 rats given 5.7-DHT (circles, Fig. 1). Rats in Groups B, D and E had normal motility, whereas rats in Group C had reductions of 1P35”f0 (Fig. 4; Table 2). Similar findings were obtained when 17 of the other 27 rats 600 0
A
550
120 days
369.5 463.9 355.7 340.2 339.5
L- 5.5 f 15.8* * 15.7 I 6.7 i 6.9
had received DMI injections of the
given a single injection of 2OOpg 5.7-DHT were autopsied (triangles, Fig. 1). None of the remaining 14 rats in Groups B, D and E showed g~trointestinal dysfunctions upon inspection, whereas large increases in intestinal material were present in three of the rats in Group C (the only animals in which abdominal distention was conspicuous before they were sacrificed). Comparable findings also were obtained in 16 rats given 2 intraventricular injections of 150 pg 5,7-DHT following DMI treatment, none of which were handled extensively (Table 3). Four animals died during the 5-month period of observation, each displaying pronounced abdominal distention. None of the remaining 12 rats had body weights above control values. Three of them had substantial depletions of NE as well as .5-HT. and in each rat gastrointestinal tract weights were normal. The other 9 rats, with selective depletions of S-HT, had total gastrointestinal tract weights that were significantly greater than con0
0
500 0 OA 450
0
$
\
OM
c?
0
P
400
0
0
___ ____ ____ F \
oa
0
0
_-e---
350
*
______
__
______
__-_
X
x A
A l
300
x
x
Y __I___________-----
0 _____--
____--I__-
\ A
B
C
D
E
Fig. I. Individual increases in body weight 120 days after rats were given injections of 5,7-DHT (2OOpg. intraventricular) and DMI (25mg/kg, i.p.). Groups A-E as in Table I. Gastrointestinal motility was assessed in 21 rats by passage of the non-absorbable marker PEG. and was found to be normal (0) or depressed (0). Intestinal accumulation of food and wastes was observed at autopsy in 24 other rats, and was found to be normal (A) or elevated (A). No such information was obtained in the remaining 22 animals ( x ).
1,
0
1.
a2 5-HT
1 0.4 LEVELS
”
’
0.6
0.8
c
c
(ilglgl
Fig. 2. Individual increases in body weight 120 days after rats were given injections of 5.7-DHT (2oOpg. intraventricular) and DMI (25 mg/kg, i.p.1, as a function of residual 5-HT levels in whole brain (0). The horizontal dashed lines represent the range of body weight increases in 19 control rats. and x represents the mean values for both variables in those animals. The regression line is ?’ = -653.3 Y + 594.6.
C. F. SALLER and E. M. STRICKER
DAYS
POST
INJECTION
Fig. 3. Body wetghts of 5 rats that showed enhanced growth during the first 120 days after injections of 5.7-DHT (200 pg. intraventricular) and DMI (25 mgikg. i.p.) (0). Gastrointestinal motility had been assessed in each ammal by passage of PEG (see Fig. I. Group B). Not shown are 7 additional rats given 5.7-DHT and DMI which showed comparable rates of growth. Transient decreases in body weight during the first week after the injections also are not shown. Two rats given the vehicle solution (0). which represent the extremes of the control group. are shown for purposes of comparison.
trol values. Two rats. weighing 541 g and 546g. were remarkable in this regard because their gastrointestinal tracts plus contents weighed 303 g and 235 g. respectively; comparable v*alues in control rats with similar body weights were only 30 to 5Og. Most of the extra weight was found in the distal portion of the small intestine and the colon. Although none of the seven other 5-HT-depleted rats had distended abdomens. their gastrointestinal tracts nevertheless were found Table
to be enlarged
2. Whole
brain
monoamine
Treatment
Group A B c D E
substantially
Controls 5.7-DHT, DMI 5.7-DHT,DMI 5.:-DHT.‘DMI 5.7-DHT
when
3. Whole
brain
Treatment Untreated controls DMI-treated controls 5.7-DHT/DMI 5.7-DHT/DMI
DISCUSSION
The present work confirms previous observations that intraventricular injection of 5,7-DHT, given after systemic treatment with DMI. enhances growth in juvenile male rats. and that this phenomenon does not occur when central NE losses accompany 5-HT
wet and
levels. body weight increases. and gastromtestmal intraventricular 5.7-DHT and parenteral DMI
NE (~g;.g wet weight
n (7) (5) (81 (3) (5)
0.43 0.56 0.46 0.26 0.19
* P < 0.01. by f-test. in comparison with control injection of the 5.7-DHT vehicle solution. Table
dry weights were determined (by 53”, and 71”,, respectively. above control values).
+ + k f *
animals
0.01 0.08 0.1 I 0.09* 0.04* whtch
+ f + + f
0.06 0.03* 0.08* 0.08* 0.06*
had received
369.6 476.0 370.3 340.1 339.0
DMI
II (5) (4) (9) (3)
_~ 0.41 ) 0.01 0.44 + 0.03 0.27 f 0.08*
* P < 0.01. by r-test, in comparison with control injection of the 5.7-DHT vehicle solution.
5-HT +_S.E.M.)
0.84 rfr 0.02 0.19 i_ 0.03” 0.18 f 0.041 animals
+ + + * +
followed
monoamine levels. body weights. and gastrointesttnal tract weights treatments with intraventricular 5.7-DHT and parenteral DMI
NE (pg;g wet weight
in rats
Body weight increase (g f S.E.M.)
5-HT _tS.E.M.) 0.85 0.20 0.22 0.21 0.18
motility
120 days
after
Gastrointestinal motility (“,, control)
6.0 3l.V 9.7 18.8 31.7
100.0 104.1 73.8’ 94.9 96.7
by an intraventricular
of rats
150 days
after
Body weight (g + S.E.M.)
Gastrointestinal tract Wet weight Dry weight (g f S.E.M.)
527.4 539.2 514.5 508.0
38.5 42.6 110.6 33.1
f i + +
10.1 36.4 12.0 8.3
which twice had received
+ + + f
DMI followed
5.9 6.1 3l.l* 5.4
10.0 Il.0 27.1 8.0
+ * f +
two
I.1 0.9 5.7* I.5
by an intraventricular
Increased
growth
503
in rats after 5.7-DHT
5
6
7
Fig. 4. Percentage of total radioactivity recovered from the stomach, six segments of small intestine. and colon of rats given injections of 5.7-DHT (200 pg. intraventricular) and DMI (25 mg/kg. i.p.) (n = 8) or the vehicle solutions (n = 7) (Groups C and A. respectively. from Table 2). Bars represent mean values.
depletions (Saller and Stricker, 1976a). In addition, it has now been found that despite large and selective depletions of brain S-HT, enhanced body weight gain also fails to occur in rats which show a pronounced decrease in gastrointestinal motility after 5.7-DHT administration, and that extensive handling of rats decreases the probability that such intestinal dysfunctions will develop. Each of these findings will now be considered in turn. Enhanced
growth
Twelve rats in the present experiment showed enhanced body weight gain after treatment with 5,7-DHT and DMI (Group B, Table 1). None of these animals had depletions of central NE, but all showed a substantial loss of whole brain 5-HT. In a previous study, regional analyses of brains from identically treated rats had revealed that S-HT losses were not uniform but were much greater in the telencephalon than in the diencephalon or brain stem (Saller and Stricker, 1976a). It is not known in which brain area the loss of S-HT is critical for enhancing growth. Nevertheless, if it is assumed that this depletion occurs in parallel with the loss of whole brain 5-HT. it would appear from Figure 2 that a considerable destruction of central serotonergic neurones is required before an effect on growth is obtained, presumably because compensatory alterations in residual serotonergic neurones serve to maintain normal function. Moreover, once some threshold value is reached, further 5-HT depletions appear to be inversely related to body weight gain after the injection. These findings are consistent with many reports that cyproheptadine. a 5-HT receptor blocker, increases food consumption and weight gain in human subjects (e.g. Silverstone and Schuyler, 1975; however, see also Clineschmidt, Hanson, McGufin, Lotti, Scriabine and Stone, 1976). They also complement \P 177-g
observations that feeding is decreased following the administration of various drugs that presumably act by increasing serotonergic transmission. These include 5-hydroxytryptophan. the precursor of 5-HT, fluoxetine (Lilly 110140), a 5-HT uptake blocker, and fenfluramine. a drug which increases 5-HT release (e.g. Blundell and Leshem, 1975; Garattini, Buczko, Jori and Samanin, 1975: Goudie, Thornton and 1976). Moreover, cyproheptadine and Wheeler, lesions of 5-HT-containing cells in the brain have both been reported to block fenfluramine-induced anorexia (Samanin. Ghezzi, Valzelli and Garattini. 1972; Clineschmidt. 1973; Kruk, 1973). Collectively, these results suggest that central 5-HT-containing neurones are involved in the control of food intake. Destruction of central serotonergic neurones may increase the initiation of hunger signals from the periphery (Friedman and Stricker, 1976; Stricker, Rowland, Saller and Friedman, 1977), due to an increased activity in the sympathetic nervous system (see below). Alternatively, the loss of 5-HT-containing neurones may lead to some defect in a central satiety system (see review by Blundell, 1977). However, it seems unlikely that an increased food intake that resulted from either of these possibilities was primarily responsible for the enhanced body weight gain of 5,7-DHT-treated rats because, as noted previously (Saller and Stricker, 1976a). these animals did not become obese but simply grew larger than did controls. These observations suggest that some change in peripheral metabolism also resulted from the central 5-HT depletions, and that the induced hyperphagia might be secondary, or at least complementary. to an alteration in the secretion. metabolism or effectiveness of growth hormone. In this regard, it is interesting to note that surgical isolation of the basomedial hypothalamus of rats, by knife cuts of areas through which serotonergic neurones might be
504
C. F. SALLER and E. M. PRICKER
expected to ascend, have been reported to increase food intake, longitudinal growth and circulating levels of growth hormone (Palka, Liebelt and Critchlow, 1971; Mitchell, Hutchins, Schindler and Critchlow. 1973). NE/.5-HT
inteructions
Ten rats given intraventricular injections of 5,7-DHT without DMI pretreatment showed depletions of both NE and 5-HT (Group E. Table 1). as expected (Baumgarten and Lachenmayer. 1972; Baumgarten, Bjiirklund. Lachenmayer and Nobin. 1973). So too did 8 rats given 5.7-DHT together with DMI, in which the DMI evidently had not totally blocked uptake of the neurotoxin into noradrenergic neurones (Tables 1 and 3). Not one of these 18 animals showed enhanced growth, despite losses of 5-HT that were comparable to those of rats in Group B (Table 1). Depletions of whole brain NE were 6@70”,, in some animals (Group E) but were only 20-25”,, in others (Group D) with similar body weights (see Fig. 1). The mechanism by which damage to central noradrenergic neurones retards body weight gain in 5-HTdepleted rats is not certain. Although decreases in growth are usually obtained when NE-containing neurones are destroyed by intracerebral injections of 6-hydroxydopamine, normal growth curves have been reported in some rats with greater than 90”,, depletions of telencephalic NE (Stricker and Zigmond. 1974). Thus, it seems unlikely that the small NE losses in the present experiment were directly responsible for the failure of 5,7-DHT-treated rats to show enhanced growth. In the light of the demonstrated interaction between the activities of central noradrenergic and serotonergic neurones (e.g. Jouvet. 1972; Blondaux. Juge. Sordet, Chouvet. Jouvet and Pujol. 1973: Kostowski, Samanin. Bareggi, Marc. Garattini and Valzelli, 1974). it seems possible instead that the NE loss modified the activity of residual serotonergic neurones and thereby decreased their effect on food intake or growth hormo.ne secretion. Accompanying NE depletions may have interfered with the development of hyperphagia following other treatments which are commonly used to reduce brain 5-HT levels in rats, such as electrolytic lesions of the midbrain raphe nuclei or repeated intraperitoneal injections of p-chlorophenylalanine (PCPA) (e.g. Lorens. Sorensen and Yunger, 1971; Tagliamonte. Tagliamonte, Corsini, Mereu and Gessa. 1973; Panksepp and Nance. 1974; Coscina and Stancer. 1977). In contrast. depletions of central 5-HT by highly selective lesions of the median raphe nucleus in juvenile male rats have been found to produce significant increases in body weight (Geyer. Puerto. Dawsey. Knapp. Bullard and Mandell, 1976). Similarly, intraventricular injections of PCPA producing specific depletions of brain 5-HT have been reported to increase food intake in adult male and female rats (Breisch. Zemlan and Hoebel. 1976). On the other
hand, hyperphagia has been reported following knife cuts in midbrain tegmentum which depleted both 5-HT and NE in the forebrain (Grossman, Grossman and Halaris, 1977). It remains to be determined if the hyperphagia observed in the present study and that described by Grossman and his colleagues have the same neurological basis. Gastrointestinal
mofilir~
Combined administration of 5.7-DHT and DMI was successful in producing large depletions of brain 5-HT without affecting NE levels in 42 rats. However, in contrast with a previous report (Sailer and Stricker. 1976a), only twelve of these animals showed enhanced growth. Gastrointestinal motility was examined in 20 of the 30 rats which did not show this phenomenon, and in every animal there was either a decreased passage of the non-absorbable marker PEG or an increased accumulation of food and wastes in the intestines. These dysfunctions occasionally were so severe that a grotesque abdominal distention ensued in which the gastrointestinal contents amounted to approximately 50”. of the animal’s body weight. Indeed, in 6 such animals (4 of which did not survive the 5-month period of observation) autopsy revealed that adipose tissue was largely absent from the peritoneal cavity. In contrast to these observations, not a single animal that showed enhanced growth exhibited intestinal dysfunctions (Fig. 1). nor did any of the 8 such rats in a previous study (Saller and Stricker. 1976a). It seems likely that the alterations in gastrointestinal motility interfered with the enhanced growth that otherwise would result following treatment with 5,7-DHT and DMI, especially in light of the following observations made recently. Two rats were hyperphagic and showed enhanced growth for 76 days following treatment with 5.7-DHT (1 x 2OOpg, intraventricular) and DMI (25 mg/kg, i.p.), at which point food was inadvertently withheld from them for 3 days. Upon its return, rats were hypophagic. body weights decreased and then levelled off within the control range. and gastrointestinal motility (measured by passage of PEG) was markedly depressed. Since these effects never happened in control rats after food deprivation, it would appear that 5-HT-depleted rats have an increased potential for developing intestinal dysfunctions which. when they occur, preclude enhanced growth. Central 5-HT depletion may depress gastrointestinal motility by increasing sympathoadrenal activity. In support of this hypothesis are observations that serotonergic fibres from the medullary raphe nuclei project down the spinal cord and synapse on or near preganglionic sympathetic neurones (Dahlstrom and Fuxe. 1965). In addition. there are numerous reports indicating that activity in the raphe nuclei, or in central serotonergic neurones. inhibits sympathoadrenal function (e.g. Ito and Schanberg, 1972; Coote and MacLeod. 1974; Neumayr. Hare and Franz, 1974;
Increased
growth
Quik and Sourkes. 1977). Thus, the stress associated with intraventricular injections of 5,7-DHT may produce an unusually large sympathoadrenal response in rats as the 5-HT-containing neurones are destroyed. It is interesting to consider the effects of DMI from the perspective of this hypothesis. By blocking reuptake of NE. DMI would be expected to potentiate peripheral sympathetic activity and. in fact. it has been shown to decrease gastrointestinal motility (Bridges t’r a/.. 1976). It is therefore noteworthy that DMI pretreatment was given to every animal in which gastrointestinal motility was depressed (indeed. more severe abdominal distention was observed in rats given two treatments of 5,7-DHT and DMI than in rats given a single injection of the drugs). whereas intestinal dysfunctions did not occur when DMI was withheld or was inexplicably ineffective. Thus, the decrease in gastrointestinal motility described here probably should be attributed to an elevated sympathoadrenal response resulting both from the depletion of brain 5-HT and from the DMI pretreatment. This hypothesis could explain how extensive handling prevented the intestinal dysfunctions in 5,7-DHTtreated rats. in that it may have reduced the stress associated with the procedures used. Conversely, an exaggerated responsiveness of the sympathoadrenal system could explain how the stress of prolonged food deprivation might have prevented enhanced growth from continuing in the two rats mentioned above. Finally. this hypothesis predicts that the intestinal dysfunctions would be self-perpetuating if they are stressful to the rats: if not. it is possible that some alteration in potassium balance resulted from the initial sympathetic response which thereafter served to maintain the paralytic ileus (cf. Streeten and Williams. 1952). In this regard, the present authors have recently observed that gastrointestinal motility also is reduced in rats depleted of brain 5-HT by intraperitoneal injections of PCPA, and that administration of KC1 solutions attenuates that effect (Sailer and Stricker, 1976b). To summarize. it now seems clear that several conditions must be met for the growth of juvenile male rats to be enhanced following intraventricular injection of 5.7-DHT. First, the 5-HT depletions must be substantial. At present. it is estimated that enhanced growth is associated with at least 65”;, depletions of whole brain 5-HT, with the largest effects seen when 5-HT losses exceed 85”;. Second, the 5-HT depletions must be specific, at least with regard to central NEcontaining neurones. It has been found that enhanced growth does not occur when NE depletions are only 2&25”.. and even smaller depletions may be sufficient to block the effect. Third, gastrointestinal motility must not be impaired. Although such impairments can lead to pronounced abdominal distention that is conspicuous before the peritoneal cavity is exposed, more subtle intestinal dysfunctions are apparently still caoable of oreventine enhanced growth even when
in rats after S.7-DHT
505
5-HT depletions are large and specific. Finally. time must be allowed for the phenomenon to develop. With the exception of those rats with the largest 5-HT depletions, the present authors have found that it takes 2-3 months after the 5.7-DHT treatment before differences in body weight are statistically reliable. However,
because
this
delay
probably
reflects
the
for the rapid rate of growth in control male animals to diminish. it may be avoided by using adult animals, which have stopped growing rapidly. as experimental subjects (cf. Saller and Stricker. 1976a).
time
required
Acknow/edyr,ncnrs-The authors thank M. Bianco, D. Greenstone and M. Sippel for their technical assistance. and Dr Michael J Zigmond for his advice. encouragement and support.
REFERENCES
Atack. C. V. (1973).
The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay. Br. J. Pharmac. 48: 699-714. Atack. C. V. and Lindqvist, M. (1973). Conjoint native and orthopthaldialdehyde-condensate assays for the fluorimetric determination of 5-hydroxyindoles in brain. Naunyn-Schmirdrhrrgs Arch. cj.xp. Path. Pharmak. 279: 267-284. Baumgarten. H. G. and Lachenmayer. L. (1972). S.7-dihydroxytryptamine: improvement in chemical lesioning of indoleamine neurons in the mammalian brain. Z. Zcll,forsch. mikrosk. Anat. 135: 399414. Baumgarten. H. G.. BjGrklund. A.. Lachenmayer. L. and Nobin. A. (1973). Evaluation of the effects of S.7-dihydroxytryptamine on serotonin and catecholamine neurons in the rat CNS. Acra ph?sio/. stand. Suppl. 391: 3-19. Bjijrklund. A.. Baumgarten. H. G. and Rensch. A. (1975). S,7-dihydroxytryptamine: improvement of its selectivity for serotonin neurons in the CNS by pretreatment with desipramine. J. Neurochrm. 24: 833~ 835. Blondaux, C.. Juge. A.. Sordet. F.. Chouvet. G., Jouvet. M. and Pujol. J! F. (1973). Modification du metabolisme de la serotonine (S-HT) cerebrale induite chez le rat oar administration de 6-hydroxydopamine. Brain Rcs. ‘50: 101-l 14. Blundell. J. E. (1977). Is there a role for serotonin (S-hydroxytryptamine) in feeding’? Inr. J. Ohrsity I: I s-12. Blundell. J. E. and Leshcm. M. B. (1975). The cffcct of S-hydroxytryptophan on food intake and on the anorexic action of amphetamine and fenfluramine. J. Pharm. Pharmac. 27: 31 37. Breisch. S. T.. Zemlan. F. P. and Hoebel. B. G. (1976). Hyperphagia and obesity following serotonin depletion by intraventricular p-chlorophenylalanine. Sciew 192: 382-385. Bridges. J. W., Dent, J. G. and Johnson, P. (1976). The effects of some pharmacologically active amines on the rate of gastric emptying in rats. I& Sci. 18: 97-108. Clineschmidt, B. V. (1973). 5.6-Dihydroxytryptamine: suppression of the anorexigenic action of fenfluramine. Eur. J. Pharmac. 24: 405409. Clineschmidt. B. V., Hanson, H. M.. McGuffin. J. C., Lotti. V. J.. Scriabine. A. and Stone. C. A. (1976). Appetite stimulant activity of 3-carboxy-IO.1 I-dihydrocyproheptadine. Archs inr. Pharmacodyn. Thir. 223: 287-300.
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F.
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and E. M. STRICKER
Coote. J. H. and MacLeod. V. H. (1974). The influence of bulbospinal monoaminergic pathways on sympathetic nerve activity. J. P~~~s~o~. 241: 453-475. Coscina, D. V. and Stancer. H. C. (1977). Selective blockade of hypothalamic hyperphagia and obesity in rats by serotonin-depleting midbrain lesions. Science 19% 416419. Dahlstrom. A. and Fuxe. K. (1965). Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems. Acra physiol. wand. Suppl. 247, 64: I-36. Friedman. M. I. and Stricker. E. M. (1976). The physiological psychology of hunger: a physiological perspective. Psychoi. Ret’. 83: 409113 1. Garattini. S., Buczko. W., Jori, A. and Samanin. R. (1975). The mechanism of action of fenfluramine. Pastgrad. med. J. Suppl t. 51: 27-35. Gerson. S. and Baldessarini. R. J. (1975). Selective destruction of serotonin terminals in rat forebrain by high doses of 5.7-dihydroxytryptamine. Brain Res. 85: 140-145. Geyer, M. A.. Puerto. A., Dawsey, W. J.. Knapp. S., Builard. W. P. and Mandell. A. J. (1976). Histologic and enzymatic studies of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 241-256. Goudie. A. 1.. Thornton. E. W. and Wheeler, T. J. (t976). Effects of Lilly 110140. a specific inhibitor of 5-hydroxytryptamine uptake. on food intake and on 5-hydroxytryptophan-induced anorexia. Evidence for serotoninergic inhibition of feeding. .I. Pharm. Pharmac. 28: 318-320. Grossman, S. P.. Grossman. L. and Halaris. A. (1977). Effects on hypothalamic and telencephalic NE and S-HT of tegmental knife cuts that produce hyperphagia and hyperdipsia in the rat. Pharmac. Biachem. Behac. 6: 101-106. Haggendal, J. (1963). An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues. Acra physiol. stand. 59: 242-254. Ito. A. and Schanberg. S. M. (1972). Central nervous system mechanisms responsible for blood pressure elevation induced by p-chlorophenylalanine. J. Pharmac. exp. Ther. 181: 6574. Jouvet, M. (1972). The role of monoamines and acetylcholine-containing neurons in the regulation of the sleepwaking cycle. Erg&. Physiol. 61: 166307. Kostowski, W.. Samanin, R., Bareggi, S. R., Marc. V., Carattini. S. and Valzelli, L. (1974). Biochemical aspects of the interaction between midbrain raphe and locus coeruleus in the rat. Brain Res. 82: 1788182. Kruk, Z. I.,. (1973). Dopamine and 5-hydroxytryptamine inhibit feeding in rats. Nature New Bioi. 246: 52-53. Lorens, S. A.. Sorensen, J. P. and Yunger. L. M. (1971). Behavioral and neurochemical effects of lesions in the
raphe system of the rat. J. camp. physiol. Psychol. 77: 48-52.
Mitchell. J. A., Hutchins, M., Schindler. W. J. and Critchlow. V. (1973). Increases in plasma growth hormone concentration and naso-anal length in rats following isolation of the medial basal hypothalamus. Neuroendocrinology 12: 161-173. Neumayr, R. J.. Hare. B. D. and Franz, D. N. (1974). Evidence for bulbospinal control of sympathetic pregangIionic neurons by monoaminergic pathways. L{fp Sci. t4: 793-806. Noble. E. P., Wurtman. R. J. and Axelrod. J. (1967). A simple and rapid method for injecting H3-norepinephrine into the lateral ventricle of the rat brain. L$? Sei. 6: 281-291. Palka. Y., Liebelt, R. A. and Critchlow, V. (1971). Obesity and increased growth following partial or complete isolation of ventromedial hypothalamus. Physiol. B&au. 7: 187-194. Panksepp. J. and Nance. D. M. (1974). Effects of parachlorophenylalanine on food intake in rats. Phrsiol. Psychol. 2~ 360-364. Quik, M. and Sourkes. T. I_,.(1977). Central dopaminergic and serotoninergic systems in the regulation of adrenal tyrosine hydroxylase. 3. Neurochem. 28: 137- 147. Sailer, C. F. and Snicker, E. M. (1976a). Hyperphagia and increased growth in rats after intraventricular injection of 5,7-dihydroxytryptamine. Science 192: 385-387. Sailer, C. F. and Stricker, E. M. (1976b). Decreased gastrointestinal motility following central serotonin depletions. Neurosci. hstr. 2: 91.Samanin. R.. Ghezzi. D.. Valzelli. L. and Garattini. S. (1972). The effects of selective lesioning of brain serotonin or catecholamine-containing neurones on the anorectic activity of fenfluramine and amphetamine. Eur. .I. Pharmac. 19: 318-322. Silverstone. T. and Schuyler. D. (1975). The effect of cyproheptadine on hunger, caloric intake and body weight in man, Psychopharmacology 40: 335-340. Streeten, D. H. P. and Williams, E. M. V. (1952). Loss of cellular potassium as a cause of intestinal paralysis in dogs. .I. Physiol. 118: 1499170. Stricker, E. M. and Zigmond. M. J. (1974). EIIects on homeostasis of intraventricular injection of &hydroxydopamine in rats. J. camp. physiol. Psycho!. 86: 973-994. Stricker. E. M.. Rowland. N.. Saller. C. F. and Friedman M. I. (1977). Homeostasis during hypoglycemia: central control of adrenal secretion and peripheral control of feeding. Science 196: 79-81. Tagliamonte, A., Tagliamonte. P.. Corsini, G. U.. Mereu, G. P. and Gessa. G. L. (1973). Decreased conversion of tyrosine to catechoiamines in the brain of rats treated with p-chlorophenylalanine. J. Pharm. Pharmac. 25: lOI--103.
Program.
E. M. STRICKER~
Departments of Life Sciences and Psychology, Pittsburgh. Pennsylvania 15260. U.S.A. (Accepted
University
of Pittsburgh.
3 Ocroher1977)
Summary-Intraventricular injections of 5.7-dihydroxytryptamine. combined with systemic administration of the norepinephrine uptake blocker desmethylimipramine. produced large depletions of brain serotonin without affecting norepinephrine levels in 42 rats. Twelve animals showed enhanced body weight gain. in some 25-4@‘,, above control values. Most of these rats (7;12) were handled extensively before and during the 4-5 month period of observation. The other 30 rats had normal body weights. but a marked depression of gastrointestinal motility led to a considerable accumulation of food and wastes in the lower intestines. Most of these rats (26/30) were not handled extensively. It is proposed that the intestinal dysfunctions resulted from an exaggerated sympathoadrenal response to stress. and that handling tended to ameliorate this effect, In contrast. desmethylimipramine apparently potentiates the decrease in gastrointestinal motility. since this effect did not occur in rats when the drug either was withheld or was inexplicably ineffective, However. these latter animals did not show enhanced body weight gain despite apparently normal intestinal function and comparable losses of brain serotonin. perhaps because substantial depletions of norepinephrine occurred as well.
Administration of 5,7_dihydroxytryptamine (5,7DHT) into the cerebral ventricles of rats. following treatment with desmethylimjpramine (DMI), produces large and apparently sefective depletions of 5-hydroxytryptamine (5-HT) in the brain (Bjiirklund, Baumgarten and Rensch, 1975; Gerson and Baidessarini, 1975). Recently the present authors reported that juvenile male rats given this treatment maintained body weight gains of 5-6 g per day into aduithood and grew much larger than control rats. Hyperphagia did not develop despite comparable losses of 5-HT when the pretreatment was withheld, perhaps because substantial depletion of norepinephrine (NE) occurred as well (Sailer and Stricker, 1976a). The experiment reported here confirms those findings but focuses on a new factor that can prevent enhanced growth despite large and selective depletions of brain 5-HT. This factor became evident in the early phases of the present work, when it was found that many rats given 5,7-DHT and DMI did not have body weights that were significantly above control values 4 months after treatment. Each of these animals had intestines that were greatly distended, a dysfunction that had not occurred previously. Since the only procedural difference was that now rats were being handled only for 3 days before they received 5.7-DHT and DMI, whereas in the initial investigation, rats were handled daily for 2-3 weeks prior to treatment, it was decided to pursue this issue by examining the effects of extensive handling on gastro*Partially supported by U.S. Public Health Service (NIMH) Grants MH-20620 and MH-25140. *To whom all correspondence should be addressed.
intestinal motility and body weight gain in rats foilowing the administration of 5,7-DHT and DMI.
METHODS
Male Sprague-Dawley rats (Zivic-Miller Laboratories. Pittsburgh. PA.) initially weighing 190-2 IS g were housed and tested singly in wire mesh cages in a room maintained at 23’C. with fluorescent lighting from 0600 to I800 hr. All the rats had continuous access to Purina Lab Chow pellets (placed on the cage Roar) and tapwater (presented in bottles with metal drinking nozzles). except as noted.
Rats were pretreated with DMI (Norpramin@. Lakeside Labs, 25 mg/kg i.p.) (n = 54) or 0.9”,, NaCl solution (n = 10). Fifty minutes later, with the rats under ether anaesthesia, a 20 ~1 solution of 0.9”; NaCl and O.l”, ascorbate containing 15Opg (n = 16) or 2OOpg (n = 38) of 5,7-DHT creatinine sulphate (Regis, dose expressed as free base) was injected into the right lateral ventricle (Noble. Wurtman and Axeirod, 1967). Sixteen rats were given a second injection of 5,7-DHT (15Opg, into the contralateral ventricle). following DMI treatment, 3 days later. To prevent sodium pentobarbital (Nembut~~f~. convulsions, Abbott Laboratories, 35 m&kg i.p.) was given immediately after the intraventricuiar injections (Baumgarten and Lachenmayer. 1972). and was suppiemented when necessary by ether anaesthesia. Additional animals received DMI once (n = 14) or twice (n = 4). or 0.9, NaCi solution (n = 5). followed by
C.
500
F.
SALLER and
intraventricular injections of the 5.7-DHT vehicle solution. and served as the control groups. Thirteen rats given a single injection of 5,7-DHT and DMI. and the 10 rats given 5,7-DHT without DMI, were handled daily for 2 weeks prior to treatment and weighed (+ 2 g) each day thereafter. Control rats given the 5.7-DHT vehicle with (n = 4) or without DMI pretreatment (n = 5) were treated similarly. The remaining animals were handled for only 3 days before treatment. and were weighed every third day subsequently. Rats were sacrificed either 120 or 150 days after the intraventricular injections. Brain monoamine levels were determined in each animal. and gastrointestinal motility was assessed in selected animals from each subgroup, as below. Monoamine
assays
Animals were killed by decapitation. Each brain was separated from the spinal cord at the level of the foramen magnum and was rapidly removed from the skull. The pineal glands were discarded. and the remaining brain tissue was frozen on dry ice. stored at -70 C for not more than 1 week. and later analysed fluorometrically for 5-HT. NE and dopamine (DA) using minor modifications of methods that have been described elsewhere (Atack. 1973; Atack and Lindqvist, 1973; Haggendal, 1963). Trace amounts of radioactive amines (New England Nuclear) were added to the samples to permit determinations of recovery. Measurements
OJyastrointesrinal
motilit~~
Gastrointestinal motility was measured. by a modification of previous methods (Bridges. Dent and Johnson, 1976). in 21 rats given 5,7-DHT. none of which displayed abdominal distention. Food and water were removed 1 hr prior to an intragastric intubation of water containing the non-absorbable marker polyethylene 1,2-[“‘Cl-glycol (PEG) (0.5 nCi in I ml. New England Nuclear). Three hours after the intubation. the rats were sacrificed and the stomach, 6 equal segments of small intestine, and the colon were removed. The tissues were immersed in IOml of a solution containing 2 N NaOH, methanol, and Triton-X-405 (60:30:10) for 12 hr. following which 10ml of water were added and the tissues were homogenized. The homogenate was adjusted to pH 7.0 with concentrated nitric acid. and an 0.5 ml aliquot was analysed for [‘“Cl-PEG by liquid scintillation spectrometry. Counting efficiency was monitored using [‘JC]-PEG as an internal standard. A relative index of gastrointestinal motility in the 5-HT-depleted rats was computed by assigning the numbers 0 through 7 to the 8 successive sections of gastrointestinal tract (i.e. stomach. 6 segments of small intestine, and colon), multiplying those numbers by the percentage of total recovered radioactivity that was found in each section, and dividing the sum of these products by the mean value obtained from com-
E. M.
STRICKER
parable computations usmg tissue from 7 control animals. A less exact index of gastrointestinal motility was obtained in other animals by simply measuring the amount of food that was present in the tract during arl lihiturn feeding. Upon sacrifice, the stomach. small intestine and colon were separated, their ends were clamped, and they were placed in preweighed glass beakers. Wet tissue weights were recorded before heating them at IO@110-C to dryness. after which they were reweighed. Wet and dry weights of tissue segments from 5 untreated control rats were obtained for purposes of comparison. RESULTS
Animals injected intraventricularly with 5,7-DHT vehicle solution whether pretreated once with DMI or with 0.9”, NaCl solution, had similar body weight gains and brain monoamine levels and consequently they were combined into a single group (Group A). Rats given a single injection of 5.7-DHT all had substantial depletions of brain 5-HT. and whole brain DA levels (M i S.E.M. = 0.98 f 0.03 pg/g wet tissue) that were unchanged. However. such clear differences in their body weights and brain NE levels were apparent that four groups were formed for further analysis. as follows. Group B. given DMI pretreatment and showing no depletion of brain NE. contained the only rats whose body weights were above the control range; Group C. given DMI and similarly showing no depletion of brain NE. contained rats whose body weights fell within the control range; Group D. also given DMI. contained rats that nevertheless showed moderate depletions of brain NE: and Group E, not given DMI, contained rats that had large depletions of brain NE, as expected. The partition of these animals is indicated in Table 1. along with the mean +S.E.M. levels of brain 5-HT and NE and body weight increases. while individual body weight gains are presented in Figure I. Depletions of brain 5-HT in Group B were not uniform but ranged from 67 to SS”,,. Individual values are presented in Figure 2. in association with the body weight increases of each animal. Note that the increase in body weight was imersely proportional to residual 5-HT levels (r = -0.76, P < 0.01). and that the two heaviest animals were the only ones whose depletions of 5-HT exceeded 85”,,. The enhanced body weight of rats in Group B usually did not begin immediately after 5.7-DHT treatment but instead became prominent 45560 days later, when the growth rate of the controls began to diminish. One exceptional animal. however. began to gain weight at an accelerated rate within the first postoperative week and remained heavier than the others throughout the 4-month period of observation (Fig. 3). This rat and the two other heaviest animals clearly were larger in size than the controls. Autopsy revealed no unusual accumulation of adipose tissue
Increased Table
501
in rats after 5.7-DHT
I. Whole brain monoamine levels and body weight increases in rats after intraventricular 5.7-DHT and parenteral DMI
Treatment
n
Controls 5.7-DHT/DMl 5.7-DHTI’DMI 5,7-DHT/DMI 5.7-DHT
(19) (12) (21) (5) (10)
Group A B c D E
growth
NE @g/g wet weight 0.44 0.56 0.50 0.29 0.17
+ + + + +
0.03 0.06 0.10 0.05* 0.02*
Body weight increase (g t S.E.M.)
5-HT _t S.E.M.) 0.88 0.20 0.24 0.20 0.17
k * + f *
0.06 0.02* 0.09* 0.0.5* O.OSf
* P < 0.01. by r-test. in comparison with control animals which In = 14) or 0.9”,. NaCl solution (n = 51 followed by intraventricular 5.7-DHT vehicle solution. ’
within the abdominal cavity of these rats or any of the others in Group B. Handling had a significant effect on body weight gain in rats with specific depletions of brain S-HT: 7 of the 12 rats in Group B were handled extensively, whereas 17 of the 21 rats in Group C were not (x2 = 3.68, P < 0.05, one-tailed test). In contrast. despite extensive handling, body weight gain was not enhanced in 2 rats in Group D or in 10 rats in Group E, all of which had sizeable depletions of brain NE. Gastrointestinal motility was measured in 21 rats given 5.7-DHT (circles, Fig. 1). Rats in Groups B, D and E had normal motility, whereas rats in Group C had reductions of 1P35”f0 (Fig. 4; Table 2). Similar findings were obtained when 17 of the other 27 rats 600 0
A
550
120 days
369.5 463.9 355.7 340.2 339.5
L- 5.5 f 15.8* * 15.7 I 6.7 i 6.9
had received DMI injections of the
given a single injection of 2OOpg 5.7-DHT were autopsied (triangles, Fig. 1). None of the remaining 14 rats in Groups B, D and E showed g~trointestinal dysfunctions upon inspection, whereas large increases in intestinal material were present in three of the rats in Group C (the only animals in which abdominal distention was conspicuous before they were sacrificed). Comparable findings also were obtained in 16 rats given 2 intraventricular injections of 150 pg 5,7-DHT following DMI treatment, none of which were handled extensively (Table 3). Four animals died during the 5-month period of observation, each displaying pronounced abdominal distention. None of the remaining 12 rats had body weights above control values. Three of them had substantial depletions of NE as well as .5-HT. and in each rat gastrointestinal tract weights were normal. The other 9 rats, with selective depletions of S-HT, had total gastrointestinal tract weights that were significantly greater than con0
0
500 0 OA 450
0
$
\
OM
c?
0
P
400
0
0
___ ____ ____ F \
oa
0
0
_-e---
350
*
______
__
______
__-_
X
x A
A l
300
x
x
Y __I___________-----
0 _____--
____--I__-
\ A
B
C
D
E
Fig. I. Individual increases in body weight 120 days after rats were given injections of 5,7-DHT (2OOpg. intraventricular) and DMI (25mg/kg, i.p.). Groups A-E as in Table I. Gastrointestinal motility was assessed in 21 rats by passage of the non-absorbable marker PEG. and was found to be normal (0) or depressed (0). Intestinal accumulation of food and wastes was observed at autopsy in 24 other rats, and was found to be normal (A) or elevated (A). No such information was obtained in the remaining 22 animals ( x ).
1,
0
1.
a2 5-HT
1 0.4 LEVELS
”
’
0.6
0.8
c
c
(ilglgl
Fig. 2. Individual increases in body weight 120 days after rats were given injections of 5.7-DHT (2oOpg. intraventricular) and DMI (25 mg/kg, i.p.1, as a function of residual 5-HT levels in whole brain (0). The horizontal dashed lines represent the range of body weight increases in 19 control rats. and x represents the mean values for both variables in those animals. The regression line is ?’ = -653.3 Y + 594.6.
C. F. SALLER and E. M. STRICKER
DAYS
POST
INJECTION
Fig. 3. Body wetghts of 5 rats that showed enhanced growth during the first 120 days after injections of 5.7-DHT (200 pg. intraventricular) and DMI (25 mgikg. i.p.) (0). Gastrointestinal motility had been assessed in each ammal by passage of PEG (see Fig. I. Group B). Not shown are 7 additional rats given 5.7-DHT and DMI which showed comparable rates of growth. Transient decreases in body weight during the first week after the injections also are not shown. Two rats given the vehicle solution (0). which represent the extremes of the control group. are shown for purposes of comparison.
trol values. Two rats. weighing 541 g and 546g. were remarkable in this regard because their gastrointestinal tracts plus contents weighed 303 g and 235 g. respectively; comparable v*alues in control rats with similar body weights were only 30 to 5Og. Most of the extra weight was found in the distal portion of the small intestine and the colon. Although none of the seven other 5-HT-depleted rats had distended abdomens. their gastrointestinal tracts nevertheless were found Table
to be enlarged
2. Whole
brain
monoamine
Treatment
Group A B c D E
substantially
Controls 5.7-DHT, DMI 5.7-DHT,DMI 5.:-DHT.‘DMI 5.7-DHT
when
3. Whole
brain
Treatment Untreated controls DMI-treated controls 5.7-DHT/DMI 5.7-DHT/DMI
DISCUSSION
The present work confirms previous observations that intraventricular injection of 5,7-DHT, given after systemic treatment with DMI. enhances growth in juvenile male rats. and that this phenomenon does not occur when central NE losses accompany 5-HT
wet and
levels. body weight increases. and gastromtestmal intraventricular 5.7-DHT and parenteral DMI
NE (~g;.g wet weight
n (7) (5) (81 (3) (5)
0.43 0.56 0.46 0.26 0.19
* P < 0.01. by f-test. in comparison with control injection of the 5.7-DHT vehicle solution. Table
dry weights were determined (by 53”, and 71”,, respectively. above control values).
+ + k f *
animals
0.01 0.08 0.1 I 0.09* 0.04* whtch
+ f + + f
0.06 0.03* 0.08* 0.08* 0.06*
had received
369.6 476.0 370.3 340.1 339.0
DMI
II (5) (4) (9) (3)
_~ 0.41 ) 0.01 0.44 + 0.03 0.27 f 0.08*
* P < 0.01. by r-test, in comparison with control injection of the 5.7-DHT vehicle solution.
5-HT +_S.E.M.)
0.84 rfr 0.02 0.19 i_ 0.03” 0.18 f 0.041 animals
+ + + * +
followed
monoamine levels. body weights. and gastrointesttnal tract weights treatments with intraventricular 5.7-DHT and parenteral DMI
NE (pg;g wet weight
in rats
Body weight increase (g f S.E.M.)
5-HT _tS.E.M.) 0.85 0.20 0.22 0.21 0.18
motility
120 days
after
Gastrointestinal motility (“,, control)
6.0 3l.V 9.7 18.8 31.7
100.0 104.1 73.8’ 94.9 96.7
by an intraventricular
of rats
150 days
after
Body weight (g + S.E.M.)
Gastrointestinal tract Wet weight Dry weight (g f S.E.M.)
527.4 539.2 514.5 508.0
38.5 42.6 110.6 33.1
f i + +
10.1 36.4 12.0 8.3
which twice had received
+ + + f
DMI followed
5.9 6.1 3l.l* 5.4
10.0 Il.0 27.1 8.0
+ * f +
two
I.1 0.9 5.7* I.5
by an intraventricular
Increased
growth
503
in rats after 5.7-DHT
5
6
7
Fig. 4. Percentage of total radioactivity recovered from the stomach, six segments of small intestine. and colon of rats given injections of 5.7-DHT (200 pg. intraventricular) and DMI (25 mg/kg. i.p.) (n = 8) or the vehicle solutions (n = 7) (Groups C and A. respectively. from Table 2). Bars represent mean values.
depletions (Saller and Stricker, 1976a). In addition, it has now been found that despite large and selective depletions of brain S-HT, enhanced body weight gain also fails to occur in rats which show a pronounced decrease in gastrointestinal motility after 5.7-DHT administration, and that extensive handling of rats decreases the probability that such intestinal dysfunctions will develop. Each of these findings will now be considered in turn. Enhanced
growth
Twelve rats in the present experiment showed enhanced body weight gain after treatment with 5,7-DHT and DMI (Group B, Table 1). None of these animals had depletions of central NE, but all showed a substantial loss of whole brain 5-HT. In a previous study, regional analyses of brains from identically treated rats had revealed that S-HT losses were not uniform but were much greater in the telencephalon than in the diencephalon or brain stem (Saller and Stricker, 1976a). It is not known in which brain area the loss of S-HT is critical for enhancing growth. Nevertheless, if it is assumed that this depletion occurs in parallel with the loss of whole brain 5-HT. it would appear from Figure 2 that a considerable destruction of central serotonergic neurones is required before an effect on growth is obtained, presumably because compensatory alterations in residual serotonergic neurones serve to maintain normal function. Moreover, once some threshold value is reached, further 5-HT depletions appear to be inversely related to body weight gain after the injection. These findings are consistent with many reports that cyproheptadine. a 5-HT receptor blocker, increases food consumption and weight gain in human subjects (e.g. Silverstone and Schuyler, 1975; however, see also Clineschmidt, Hanson, McGufin, Lotti, Scriabine and Stone, 1976). They also complement \P 177-g
observations that feeding is decreased following the administration of various drugs that presumably act by increasing serotonergic transmission. These include 5-hydroxytryptophan. the precursor of 5-HT, fluoxetine (Lilly 110140), a 5-HT uptake blocker, and fenfluramine. a drug which increases 5-HT release (e.g. Blundell and Leshem, 1975; Garattini, Buczko, Jori and Samanin, 1975: Goudie, Thornton and 1976). Moreover, cyproheptadine and Wheeler, lesions of 5-HT-containing cells in the brain have both been reported to block fenfluramine-induced anorexia (Samanin. Ghezzi, Valzelli and Garattini. 1972; Clineschmidt. 1973; Kruk, 1973). Collectively, these results suggest that central 5-HT-containing neurones are involved in the control of food intake. Destruction of central serotonergic neurones may increase the initiation of hunger signals from the periphery (Friedman and Stricker, 1976; Stricker, Rowland, Saller and Friedman, 1977), due to an increased activity in the sympathetic nervous system (see below). Alternatively, the loss of 5-HT-containing neurones may lead to some defect in a central satiety system (see review by Blundell, 1977). However, it seems unlikely that an increased food intake that resulted from either of these possibilities was primarily responsible for the enhanced body weight gain of 5,7-DHT-treated rats because, as noted previously (Saller and Stricker, 1976a). these animals did not become obese but simply grew larger than did controls. These observations suggest that some change in peripheral metabolism also resulted from the central 5-HT depletions, and that the induced hyperphagia might be secondary, or at least complementary. to an alteration in the secretion. metabolism or effectiveness of growth hormone. In this regard, it is interesting to note that surgical isolation of the basomedial hypothalamus of rats, by knife cuts of areas through which serotonergic neurones might be
504
C. F. SALLER and E. M. PRICKER
expected to ascend, have been reported to increase food intake, longitudinal growth and circulating levels of growth hormone (Palka, Liebelt and Critchlow, 1971; Mitchell, Hutchins, Schindler and Critchlow. 1973). NE/.5-HT
inteructions
Ten rats given intraventricular injections of 5,7-DHT without DMI pretreatment showed depletions of both NE and 5-HT (Group E. Table 1). as expected (Baumgarten and Lachenmayer. 1972; Baumgarten, Bjiirklund. Lachenmayer and Nobin. 1973). So too did 8 rats given 5.7-DHT together with DMI, in which the DMI evidently had not totally blocked uptake of the neurotoxin into noradrenergic neurones (Tables 1 and 3). Not one of these 18 animals showed enhanced growth, despite losses of 5-HT that were comparable to those of rats in Group B (Table 1). Depletions of whole brain NE were 6@70”,, in some animals (Group E) but were only 20-25”,, in others (Group D) with similar body weights (see Fig. 1). The mechanism by which damage to central noradrenergic neurones retards body weight gain in 5-HTdepleted rats is not certain. Although decreases in growth are usually obtained when NE-containing neurones are destroyed by intracerebral injections of 6-hydroxydopamine, normal growth curves have been reported in some rats with greater than 90”,, depletions of telencephalic NE (Stricker and Zigmond. 1974). Thus, it seems unlikely that the small NE losses in the present experiment were directly responsible for the failure of 5,7-DHT-treated rats to show enhanced growth. In the light of the demonstrated interaction between the activities of central noradrenergic and serotonergic neurones (e.g. Jouvet. 1972; Blondaux. Juge. Sordet, Chouvet. Jouvet and Pujol. 1973: Kostowski, Samanin. Bareggi, Marc. Garattini and Valzelli, 1974). it seems possible instead that the NE loss modified the activity of residual serotonergic neurones and thereby decreased their effect on food intake or growth hormo.ne secretion. Accompanying NE depletions may have interfered with the development of hyperphagia following other treatments which are commonly used to reduce brain 5-HT levels in rats, such as electrolytic lesions of the midbrain raphe nuclei or repeated intraperitoneal injections of p-chlorophenylalanine (PCPA) (e.g. Lorens. Sorensen and Yunger, 1971; Tagliamonte. Tagliamonte, Corsini, Mereu and Gessa. 1973; Panksepp and Nance. 1974; Coscina and Stancer. 1977). In contrast. depletions of central 5-HT by highly selective lesions of the median raphe nucleus in juvenile male rats have been found to produce significant increases in body weight (Geyer. Puerto. Dawsey. Knapp. Bullard and Mandell, 1976). Similarly, intraventricular injections of PCPA producing specific depletions of brain 5-HT have been reported to increase food intake in adult male and female rats (Breisch. Zemlan and Hoebel. 1976). On the other
hand, hyperphagia has been reported following knife cuts in midbrain tegmentum which depleted both 5-HT and NE in the forebrain (Grossman, Grossman and Halaris, 1977). It remains to be determined if the hyperphagia observed in the present study and that described by Grossman and his colleagues have the same neurological basis. Gastrointestinal
mofilir~
Combined administration of 5.7-DHT and DMI was successful in producing large depletions of brain 5-HT without affecting NE levels in 42 rats. However, in contrast with a previous report (Sailer and Stricker. 1976a), only twelve of these animals showed enhanced growth. Gastrointestinal motility was examined in 20 of the 30 rats which did not show this phenomenon, and in every animal there was either a decreased passage of the non-absorbable marker PEG or an increased accumulation of food and wastes in the intestines. These dysfunctions occasionally were so severe that a grotesque abdominal distention ensued in which the gastrointestinal contents amounted to approximately 50”. of the animal’s body weight. Indeed, in 6 such animals (4 of which did not survive the 5-month period of observation) autopsy revealed that adipose tissue was largely absent from the peritoneal cavity. In contrast to these observations, not a single animal that showed enhanced growth exhibited intestinal dysfunctions (Fig. 1). nor did any of the 8 such rats in a previous study (Saller and Stricker. 1976a). It seems likely that the alterations in gastrointestinal motility interfered with the enhanced growth that otherwise would result following treatment with 5,7-DHT and DMI, especially in light of the following observations made recently. Two rats were hyperphagic and showed enhanced growth for 76 days following treatment with 5.7-DHT (1 x 2OOpg, intraventricular) and DMI (25 mg/kg, i.p.), at which point food was inadvertently withheld from them for 3 days. Upon its return, rats were hypophagic. body weights decreased and then levelled off within the control range. and gastrointestinal motility (measured by passage of PEG) was markedly depressed. Since these effects never happened in control rats after food deprivation, it would appear that 5-HT-depleted rats have an increased potential for developing intestinal dysfunctions which. when they occur, preclude enhanced growth. Central 5-HT depletion may depress gastrointestinal motility by increasing sympathoadrenal activity. In support of this hypothesis are observations that serotonergic fibres from the medullary raphe nuclei project down the spinal cord and synapse on or near preganglionic sympathetic neurones (Dahlstrom and Fuxe. 1965). In addition. there are numerous reports indicating that activity in the raphe nuclei, or in central serotonergic neurones. inhibits sympathoadrenal function (e.g. Ito and Schanberg, 1972; Coote and MacLeod. 1974; Neumayr. Hare and Franz, 1974;
Increased
growth
Quik and Sourkes. 1977). Thus, the stress associated with intraventricular injections of 5,7-DHT may produce an unusually large sympathoadrenal response in rats as the 5-HT-containing neurones are destroyed. It is interesting to consider the effects of DMI from the perspective of this hypothesis. By blocking reuptake of NE. DMI would be expected to potentiate peripheral sympathetic activity and. in fact. it has been shown to decrease gastrointestinal motility (Bridges t’r a/.. 1976). It is therefore noteworthy that DMI pretreatment was given to every animal in which gastrointestinal motility was depressed (indeed. more severe abdominal distention was observed in rats given two treatments of 5,7-DHT and DMI than in rats given a single injection of the drugs). whereas intestinal dysfunctions did not occur when DMI was withheld or was inexplicably ineffective. Thus, the decrease in gastrointestinal motility described here probably should be attributed to an elevated sympathoadrenal response resulting both from the depletion of brain 5-HT and from the DMI pretreatment. This hypothesis could explain how extensive handling prevented the intestinal dysfunctions in 5,7-DHTtreated rats. in that it may have reduced the stress associated with the procedures used. Conversely, an exaggerated responsiveness of the sympathoadrenal system could explain how the stress of prolonged food deprivation might have prevented enhanced growth from continuing in the two rats mentioned above. Finally. this hypothesis predicts that the intestinal dysfunctions would be self-perpetuating if they are stressful to the rats: if not. it is possible that some alteration in potassium balance resulted from the initial sympathetic response which thereafter served to maintain the paralytic ileus (cf. Streeten and Williams. 1952). In this regard, the present authors have recently observed that gastrointestinal motility also is reduced in rats depleted of brain 5-HT by intraperitoneal injections of PCPA, and that administration of KC1 solutions attenuates that effect (Sailer and Stricker, 1976b). To summarize. it now seems clear that several conditions must be met for the growth of juvenile male rats to be enhanced following intraventricular injection of 5.7-DHT. First, the 5-HT depletions must be substantial. At present. it is estimated that enhanced growth is associated with at least 65”;, depletions of whole brain 5-HT, with the largest effects seen when 5-HT losses exceed 85”;. Second, the 5-HT depletions must be specific, at least with regard to central NEcontaining neurones. It has been found that enhanced growth does not occur when NE depletions are only 2&25”.. and even smaller depletions may be sufficient to block the effect. Third, gastrointestinal motility must not be impaired. Although such impairments can lead to pronounced abdominal distention that is conspicuous before the peritoneal cavity is exposed, more subtle intestinal dysfunctions are apparently still caoable of oreventine enhanced growth even when
in rats after S.7-DHT
505
5-HT depletions are large and specific. Finally. time must be allowed for the phenomenon to develop. With the exception of those rats with the largest 5-HT depletions, the present authors have found that it takes 2-3 months after the 5.7-DHT treatment before differences in body weight are statistically reliable. However,
because
this
delay
probably
reflects
the
for the rapid rate of growth in control male animals to diminish. it may be avoided by using adult animals, which have stopped growing rapidly. as experimental subjects (cf. Saller and Stricker. 1976a).
time
required
Acknow/edyr,ncnrs-The authors thank M. Bianco, D. Greenstone and M. Sippel for their technical assistance. and Dr Michael J Zigmond for his advice. encouragement and support.
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