Biochemical and behavioural alterations following 5,6-dihydroxytryptamine administration into brain

BIOCHEMICAL AND BEHAVIOURAL ALTERATIONS FOLLOWING 5,6_DIHYDROXYTRYPTAMINE ADMINISTRATION INTO BRAIN G. R. BREESE,B. R. COOPER.L. D. GRANTand R. D. SMITH Departments of Pharmacology, Psychiatry and Anatomy, Biological Sciences Research Center, Child Development Institute. UNC School of Medicine, Chapel Hill. North Carolina 27514

(Accepted

9 Septenlher

1973)

Summary-Administration of 75 pg 5,6_dihydroxytryptamine (5,6-DHT) intracisternally caused a prolonged reduction of brain serotonin with little effect on brain catecholamines. This effect was moderately potentiated by administering an additional dose of 5,6-DHT or pretreating animals with pargyline before injection of 5,6-DHT. Injections of 5,6-DHT into the rapht nuclei also caused reductions in brain serotonin, but caused severe tissue damage in some rats as well. 5,6-dihydroxytryptamine (40 pg) given intracisternally to 7-day-old neonatal rats produced only a 30% reduction of serotonin. Reduction of tryptophan hydroxylase activity following 5,6-DHT treatment supports the view that the depletion of brain serotonin may be the result of fibre degeneration. In spite of moderate effects of 5,6-DHT on brain serotonin, treatments caused behavioural alterations including enhanced muricide and facilitated acquisition of an active avoidance task.

5,6_Dihydroxytryptamine (5,6-DHT) has recently been shown to cause a long lasting depletion of 5-hydroxytryptamine (serotonin) in rat brain and spinal cord after intraventricular injection (BAUMGARTEN, BJ~RKLUND. LACHENMAYER, NOBIN and STENEVI, 1971; BAUMGAKTEN, BJGRKLUND, HOLSTEIN and NOBIN. 1972a; BAUMGARTEN, EVETTS, HOLMAN, IVERSEN, VOGT and WILSON, 1972b; BAUMGARTEN and LACHENMAYER, 1972a, b). Ultrastructural analysis of effects of 5,6-DHT on serotonin fibres in the central nervous system (BAUMGARTEN et hl., 1972a; BAUMGARTEN and LACHENMAYER, 1972~) strongly suggests that this drug exerts a cytotoxic action similar to that exerted by 6-hydroxydopamine on central cetecholamine neurones after injection into the brain (BLOOM, ALGERI, GROPPETTI, REVUELTA and COSTA, 1969; BREESEand TRAYLOR, 1970, 1971). One purpose of the present research was to obtain additional evidence for a cytotoxic effect of 5,6-DHT on serotonin neurones by demonstrating that a prolonged reduction of brain tryptophan hydroxylase activity accompanies the decrease of serotonin after injection of the drug into brain. In addition, the effects of several different methods of treatment were explored as a means to obtain greater reductions of brain serotonin than currently reported in the literature. Since pretreatment of animals with pargyline has been shown to potentiate the destructive action of 6-hydroxydopamine on central dopaminergic neurones (BREESEand TRAYLOR, 1970), the ability of pargyline to enhance the depleting effects of 5,6-DHT on brain serotonin was also assessed. Other methods examined were multiple injections of 5,6-DHT, administration of 5,6-DHT to neonatal rats and injection of 5,6DHT directly into midbrain raphl nuclei, which have a high concentration of serotonincontaining cell bodies. Finally, efforts were made to determine if behavioural and physiological effects could be demonstrated as a consequence of 5,6-DHT administration. 177

178

G.

GC/lCJl”Ul

R. BREESL,B. R. C’oww,

L. D.

C&AN 1‘ and

R, D. SMITH

METHODS

Male Sprague-Dawley rats (Holtzman Co., Madison, Wisconsin) were used for all experiments. In one series of experiments, developing rats and young adult rats (150-180 g) received intracisternal injections of the drug. In another set of experiments. mature adult rats (300-350 g) had 5,6-DHT stereotaxically injected via a 27 gauge needle into their midbrain raphl nuclei, the site of origin of the majority of serotonin fibres innervating forebrain regions. In the first series of experiments, rats weighing 15&180 g were given 75 pg of the free base of the 5.6DHT intra~isternally. The compound was dissolved in sterile saline containing Q.5 mg,/ml of ascorbic acid. The total volume administered was 25 /ll (SC‘HANRERG, SCHILDKKAUT, BREESEand KOPIN, 1968; BREESEand TRAYLOR,1970). Some animals were pretreated with pargyline (50 mg/kg) 30 min before the administration of the 5,fLDHT. A second injection of 5,6-DHT (75 pg) was administered 7 days later to animals from each of the above treatment groups. Control animals received intracisternal injections of the saline-ascorbic acid vehicle and pargyline treatment where appropriate. Seven-day-old rat pups were injected intracisternally with 40 pg of 5,6-DHT in 10 ,LJof vehicle (see BREESE and TRAYLOK,1972). Other litter-mate animals received vehicle by the same route of administration. In the second set of studies with 30&350 g rats, utilizing a system described by UNGtKSTEDT(1971). S-10 pg of 5,6-DHT dissolved in 4-5 ~1 saline with ascorbic acid was infused at the rate of I ,@min (2 pgimin) into the dorsal raphe nucieus and then into the median raph@ nucleus. An infusion pump obtained from Sage Instrument Co. (White Plains. NY) was used to infuse all solutions. Some of these animals were pretreated with 30 mg/kg pargyline (i.p.) 30 min before injection of the 5,6-DHT. Thus, each subject received a total of 16-20 ~18of 5,6-DHT administered into the midbrain raphC nuclei. Sham-operated control animals received comparable infusions of the saline vehicle into the raphi nuclei. The accuracy of all raphC injections was assessed histologically after sacrifice, as described below. For animals in which 5,6-DHT or saline was injected into the raphi, the brainstem was separated from forebrain by a cut extending from the mammillary nuclei to the superior colliculi. The brainstem was used to evaluate the location of injection sites and to assess histological changes due to 5,6-DHT or injection of the saline vehicle alone. After each brainstem was embedded in paraffin, 16 pm sections were taken through the area containing the cannula tip and were stained according to the method of KL~~VERand BAKRERA (1953) for microscopic examination of the injection site. Bi~~~~~~illic~ll procedure.7

For the measurement of brain concentrations of biogenic amines, tyrosine hydroxylase and tryptophan hydroxylase, animals were killed by cervical fracture and decapitated at various time periods after the injection of the 5,6-DHT. Brains were removed and divided by a midsagital cut. One-half of the brain was homogenized in 0.4 N perchloric acid. Determinations of norepinephrine and dopamine were carried out on the supernatant of the perchloric acid homogenate as previously described (BREESEand TRAYLOR,1970, 1971). The other half was frozen and later homogenized in 0.1 N hydrochloric acid containing O.OS’/, ascorbic acid. Serotonin was determined in this homogenate according to the method of BOGDANSKI, PLETSCHEK, BRODIEand UDENFRIENU (1956). In other animals, onehalf of the brain was used to determine the effects of 5,6-DHT on tryptophan hydroxylase

5,6-DHT intracerebrally

and behaviour

179

activity (ICHIYAMA, NAKAMURA, NISHIZUKA and HAYAISHI, 1968). The fresh brain tissue was homogenized in 5 vol of a buffer (pH 7.5) containing 0.32 M sucrose, 0.25 M phosphate, 0.05 M mercaptoethanol and 0.02% ascorbic acid (SPAM). After homogenization, the homogenates were spun at 3000 rev/min for 5 min. An aliquot of 100 or 200 ~1 supernatant was then added to 200 ~1 of a reaction mixture containing 75 ~1 water, 50 ~1 1.728 x 10e4~ tryptophan-1C’4 (sp ccl‘fic radioactivity 9.44 mCi/mmol, New England Nuclear) 50 ~1 1.28 M sucrose, and 25 ~1 2 M phosphate buffer (pH 7.5). Samples were incubated at 37 ‘C for 10 min and the reaction stopped by adding 0.75 ml of a 20% citric acid solution. Blanks were run with SPAM buffer and reaction mixture. 14C0, was trapped on an NaOH impregnated wick. The wick was added to a scintillation vial containing 04% 2.5 diphenxyloxazole in toluene and triton X-100 in a 1 : 1 mixture. Samples were counted at 5°C in a Packard scintillation counter. The other half was frozen and later homogenized in distilled water as described by MUSACCHIO, JULOU, KETY and GLOWINSKI (1969) to permit the isolation of tyrosine hydroxylase from brain. Enzyme activity was determined using a modification of the procedure described by NAGATSU, LEVITT and UDENFRIEND (1964). Norepinephrine, dopamine and serotonin content in the forebrain portions were assayed immediately after sacrifice. In this case, the brain was homogenized in 0.2 N hydrochloric acid containing ascorbic acid. Following the removal of an aliquot from the homogenate for serotonin analysis, perchloric acid was added to make a final concentration of 04 N; brain catecholamines were determined in the supernatant following centrifugation of the homogenate in a Sorvall refrigerated centrifuge at 15,000 g (BREW and TRAYLOR, 1970). Physiological

und hehaaioural

tntmuwmwts

In certain groups, the effects of 5,6-DHT were examined on temperature regulation, mouse-killing behaviour and acquisition of an active avoidance response. Rectal temperatures were obtained via a 6.4 cm probe connected to a telethermometer (Yellow Springs Instrument Company). Readings were obtained prior to treatments and at 30-min intervals thereafter. All experiments were performed at room temperature (23-24”C), with each animal placed in an individual plastic cage (12 x 12 x 6 inches). Only intracisternally treated rats were tested. The effect of 5,6-DHT on muricidal behaviour was evaluated 7 days after treatment by placing a single adult male mouse in the cage of each treated and saline-control animal for I hr. At the end of this time period, the number of rats that killed mice was determined. The effect of intracisternally administered 5,6-DHT on acquisition of the shuttle-box avoidance response was determined using a modified automated shuttle-box (LeHigh Valley, Inc.), which has been described previously(C~~~~~, BREESE,HOWARD and GRANT, 1972; COOPER, BREESE, GRANT and HOWARD, 1973). A single loo-trial session was used to examine acquisition. One minute after the rat was placed in the shuttle-box, the session was initiated with the lighting of a lamp (conditioned stimulus). The light remained on until the animal crossed to the opposite compartment, thereby avoiding shock, or for a maximal interval of 10 sec. If the animal did not cross to the other compartment, it received a continuous electric shock of 0.8 mA which was terminated when it escaped into the other compartment of the shuttle-box or at the end of 5 sec. Each avoidance response, escape response or failure to respond was followed by a 30-set interval before initiation of the next trial. Performance on each successive block of 25 trials during the loo-trial acquisition test was automatically recorded by electromechanical programming equipment.

G. R. BREESE, B. R. COOPER, L. D. GRANT and R. D. SMITH

180

Drugs The 5,6_dihydroxytryptamine (5,6-DHT) creatinine sulphate dihydrate was furnished by Dr. A. A. Manian, Psychopharmacology Service Center, National Institute of Mental Health and synthesized by Regis Chemical Company, Chicago, Illinois, under a research contract. The pargyline was furnished by Abbott Laboratories. RESULTS EfSect of pargyline pretreatment and an additional injectiolz levels after intracisternally administered 5,6-DHT

qf 5,6-DHT

on brain monoamine

To determine the effects of pargyline pretreatment and of multiple injections on the depletion produced by 5,6-DHT, amines were determined 14 and 30 days after treatment. A single intracisternal injection of 5,6-DHT (75 pg) was found to reduce brain serotonin by 37 + 2% when measured 30 days after administration. Neither norepinephrine nor dopamine in the brain were significantly different from the control. Pargyline treatment prior to the administrationof 5,6-DHT intracisternally caused a small but significantly greater depletion of serotonin. In this case, serotonin was reduced by 56 f 4%, while the content of norepinephrine and dopamine in brain were not significantly changed. Additional injections of 5,6-DHT were then given to determine if even greater depletions would occur. In animals treated with 5,6-DHT alone, a second injection of 75 pg produced a 49 + 3% reduction of serotonin when measured 30 days later. This depletion was significantly greater than that produced by the single 5,6-DHT treatment. After two injections of 5,6-DHT, norepinephrine and dopamine were again not significantly affected. Fourteen days following the second treatment with 5,6-DHT, serotonin was reduced by 55 k 4%, which was not significantly different from the concentration observed at 30 days. Administration of a second-injection of 5,6-DHT one week after the pargyline plus 5,6DHT treatment reduced serotonin by 58 ? 4%. The content of norepinephrine and dopamine in whole brain was not altered. This reduction of serotonin, however. did not differ from that obtained in animals treated with a single injection of 5,6-DHT plus pargyline. Again, data obtained 14 days after the second injection were essentially like those obtained at 30 days. EfSect

qf 5,6-DHT

on tryptophan

hydroxylase

activity

Following central administration of 5,6-DHT, BAUMGARTEN and LACHENMAYER(1972~) have provided evidence that degeneration of nerve terminals accompanies the depletion of serotonin. To obtain further evidence for a cytotoxic action of 5,6-DHT on serotonergic fibres, tryptophan hydroxylase activity in brain was measured following certain 5,6-DHT treatments. Thirty days after treatment with two doses of 5,6-DHT (5,6-DHT-2X) tryptophan hydroxylase activity was 73 f 5.3% of control. Following administration of two doses of 5,6-DHT, the first dose given 30 min after pargyline (P + 5,6-DH,T-2X), tryptophan hydroxylase was reduced to 60 f 3% of control. However it should be noted that in both cases the degree of reduction of tryptophan hydroxylase produced by the 5,6-DHT treatments was not as great as the reduction of brain serotonin. Whether this infers that the extent of destruction was not as great as the serotonin reduction might suggest will require further study. Tyrosine hydroxylase activity was not altered after 5,6-DHT administration supporting the view that catecholamine-containing fibres were not affected by these treatments.

5.6-DHTintracerebrally Effects of intracerebral irzjections of 5,6-DHTiJzto pinephrine arld dopamilze

181

and behaviour

raphe’ nuclei oJz forebrairz

serotoJ?iJz, Jzore-

Two major groups of cell bodies which contain serotonin are found in the midbrain raphi (DAHLSTR~~M and FUXE,1964). Studies were initiated to determine if injection of 5,6DHT into these areas of brain would reduce serotonin in the forebrain of treated rats. As shown in Table 1, this treatment reduced forebrain serotonin by approximately 387& Pargyline pretreatment moderately enhanced the depletion produced by injecting 5,6-DHT into the raphC (53% of control). These treatments did not significantly alter forebrain catecholamine content. Table

1. Effect of 5,6-DHT

Treatment Control (saline) Sham 5,6-DHT P + 5,6-DHT

administered brain serotonin

n 15 8 17 7

into raphe

Serotonin (nmol/g 5.27 5.03 3.28 2.83

nuclei

on fore-

in forebrain + S.E.M.) f + k k

0.23 0.29 0.09* O.ll*

5,6-DHT was injected into the raphC nuclei as described in Methods. P + 5,6-DHT refers to animals that received pargyline (30 mg/kg, i.p.) 30 min before receiving 5,6-DHT into the raphe nuclei. n refers to the number of animals in each group. * P i 0.01 when compared with control.

When injection areas for 5,6-DHT-treated rats were examined histologically, two types of results were observed. While a few rats showed no readily discernible histological evidence of tissue destruction beyond the track caused by the infusion cannula, most animals after 5,6-DHT infusion showed more extensive damage, i.e. gross lesions centred in the region of the midbrain raphC nuclei (Fig. 1). However, irrespective of the type of histological finding, serotonin was consistently reduced in the forebrain of the 5,6-DHT treated animals. Sham control subjects showed no evident damage beyond the track left by the infusion cannula. &ficts

of admiJ?isteriJzg 5,6_DHTintracisterJzal/y

to neonate rats

Following the injection of 40 pg of 5,6-DHT to seven-day-old rats, young animals showed a continuous running motion of the legs. Mortality was high in the seven litters injected, with about half of the animals being dead within 4 hr after injection. Animals that survived were sacrificed at 30 days of age. However, brain serotonin was reduced by only 3 1% following this treatment (Table 2). EfSect of ~,~-DHToJI rectal temperature

aJ?d gerzeral behaviour

After pretreatment with pargyline, most of the animals that received 5,6-DHT salivated profusely. Since this reaction suggested that 5,6-DHT might be influencing systems controlling body temperature, rectal temperatures were monitored in animals that received 5,6-DHT alone and in animals that received pargyline prior to 5,6-DHT administration.

182

G. R. BKEESE,B. R. COOPF.K,L. D. GRANT and R. D. SMITH Table 2. Serotonin

Treatment Control (saline) 5.6-DHT (40 peg)

levels in brains of seven-day-old 5,6-DHT intracisternally

n 16 24

rats treated

with

Brain serotonin (nmol.;g f S.E.M.) 5.39 * 0.14 3.70 * @OS*

Developing seven-day-old rats were injected intracisternally with 40 ng 5.6-DHT. Brain serotonin levels were determined in 30-day-old rats. A total of 56 animals were injected with 5,6-DHT and of that total. 30 animals survived the treatment. * P < @Ol when compared with control.

Fig. 1. Photomicrograph illustrating tissue damage produced by injection of 5,6-DHT directly into the dorsal and median raphe nuclei of the midbrain. Sections were cut at I6 pm and stained by the KL~VER-BARRERA(1953) method. The arrows indicate the area of lesioning.

5.6.DHT

intracerebrally

183

and behaviour

5,6_Dihydroxytryptamine alone produced a significant hypothermia 30 min after injection. Following the administration of 5,6-DHT to animals pretreated with pargyline, however, an initial hypothermia was followed by a marked hyperthermia. This hyperthermic response reached a peak of 104.1 f 0.5”F 240 min after injection of the drug. In some of the animals, body temperature rose to a point in excess of 107”F, causing several deaths. Two weeks later, rectal temperatures of animals surviving this treatment were not significantly different from those of control rats. When placed together in a cage after intracisternal injections, male animals were found to show frequent episodes of fighting behaviour, as well as increased frequency of mounting behaviour when observed 2448 hr following treatment. After this observation, animals were individually caged. It was also noticed that animals initially lost body weight following injection. This was particularly significant 14 days after a second injection of 5,6-DHT (Table 3). By 30 days, body weights seemed to be reduced slightly in all groups, but severe weight loss observed at 14 days in some groups was no longer evident (Table 3). Table

3. Effect of intracisternally administered S,6-DHT weight, 14 and 30 days after treatment

Treatment Control 5,6-DHT 5,6-DHT-2X P + 5,6-DHT P + 5,6-DHT-2X

n 20 5 20 10 15

on body

Body weight (grams) 14 Days 30 Days 272 247 188 234 167

k + + i i

6 16 14* 13 I2*

400 353 336 347 332

* rt f * i

17 18 12t 12: 16t

Treatments are the same as those described in Figure 1. n refers to number of rats included in each group at 14 and 30 days after treatment. * P < 0,001 when compared with controls. t P -c 001 when compared with controls. z P i 0.05 when compared with controls.

Following intracisternal injections of 5,6-DHT into pargyline treated rats, skin lesions often developed about the head of the animals and especially around the eyes. Although we have administered numerous compounds intracisternally, in our experience this is the first time such lesions have been observed. cfict

of 5,6-DHTon

mouse-killing

hehaviour

Depletion of serotonin with p-chlorophenylalanine or following midbrain raphC lesions has previously been shown to induce muricidal behaviour (SHEARD.1969; GRANT, COSCINA. GROSSMANand FKEEDMAN, 1973). In the present study, experiments were performed using both intracisternally treated rats and rats with specific injections into the raphC nuclei to determine if 5,6-DHT might have a similar action. A single 75 pg dose of 5,6-DHT was found to cause a significant increase in muricidal behaviour one week after treatment (Table 4). None of the control rats killed mice, while nine out of 19 treated rats (47%) killed after a single treatment with 5,6-DHT. Pretreatment with pargyline markedly potentiated mouse-killing after 5,6-DHT injection (Table 4).

184

G. R. BREW, B. R. COOPER, L. D. GRANT and R. D. SMITH Table 4. Effect of 5,6-DHT

Treatment

groups

A. Intracisternal injection Control 5,6-DHT P + 5,6-DHT B. Raph& injection”” Sham 5,6-DHT P + 5,6-DHT

treatments

on muricide

induction

in rats ‘io

Behavioural results (No. killers/No. tested)

Killers

O/38 9/19t 17,‘25t

0 47 68

O/8 4/11* 12/35*

0 36 34

(a)

(a1Male rats (150-180 g) received either saline (control) or a single 75 pg dose of 5,6-DHT intracisternally. Some of the animals were pretreated with pargyline (50 mg/kg) 30 min before injection (P + 5,6-DHT). Control animals that received pargyline (n = 10) did not differ from untreated rats and are included in the control group. All animals were tested 7 days after treatment. lb) Male rats (3Os-350 g) received two 10 pg injections, one into the dorsal and the other into the median rapht nuclei as described in Methods. Sham-operated controls received saline into the raphC nuclei. Some animals received pargyline pretreatment (i.p.) before injection of the 5,6-DHT. * P co.05 by the Fisher Exact Probability Test when compared with control. t P < 0.01 by the Fisher Exact Probability Test when compared with control.

Similar studies were also performed after destruction of serotonin cell bodies in the midbrain raphC with injections of 5,6-DHT. Following this treatment, four out of 11 animals (367;) were found to kill mice, while none of the control rats killed. Similarly, 5,6-DHT administration into the raph& in pargyline pretreated rats also induced muricidal beha>-

)-

>-

I-

e--O 5,6 DHT-2X (n=8) Mcontrol (n=7) O

1 I

I 2

I

I

3 4 Successive periods of 25 trials

Fig. 2. Effect of intracisternally administered 5,6-DHT on the acquisition of the shuttle-box avoidance response. The group 5,6-DHT-2X received two 75 pg doses of 5,6-DHT, one with and the other without pargyline pretreatment (50 mg/kg) as described in Methods. Animals were tested 30 days after the last injection of the drug. All periods are significantly different from control (P < 0.01) except for the value for the last 25 trial period. Vertical bars indicate the SE. of the mean.

5.6-DHT

intracerebrally

185

and behaviour

viour, but killing was not significantly increased over that observed in raphi injected subjects not pretreated with pargyline. EfSect of intracisternal

treatment with 5,6-DHTou

shuttle-box

avoidance

behaviour

Since the serotonin depleting agent p-chlorophenylalanine has been found to facilitate acquisition of active avoidance (TENEN, 1967), this behaviour was also examined after treatment with 5,6-DHT. Following two injections of 75 pg of 5,6-DHT, animals were tested in a shuttle-box avoidance task. As shown in Figure 2, treated animals were found to make significantly more avoidance responses than controls. This may be related to motor activity, since treated rats made significantly more crosses during the intertrial interval when compared with controls (41.5 f 14 crosses for control rats, 125 + 20 crosses for 5,6-DHT treated rats; P < 0.001). DISCUSSION In agreement with earlier reports (BAUMGARTENet al., 1971), intracisternally administered 5,6-DHT produced a long lasting reduction of brain serotonin. This effect was moderately potentiated by administering an additional dose of 5,6-DHT or by pretreating animals with pargyline. From electron microscopic studies, BAUMGARTEN et al. (1972a) have also reported that 5,6-DHT causes changes characteristic of fibre degeneration. Such observations have been offered as evidence that 5,6-DHT produces a chemical degeneration of indoleamine-containing nerve terminals. In the present study, the finding that a reduction of tryptophan hydroxylase activity accompanied the serotonin depleting effects of 5,6-DHT administration into brain adds further support for this view. While some recovery of brain serotonin content has been reported following a single injection of 5,6-DHT (BAUMGARTEN et al., 1971), there was no evidence of recovery 30 days after two injections. Data obtained 30 days after injection indicates that catecholamine fibres are not chronically altered by 5,6-DHT treatment. In spite of these favourable findings with regard to the specificity of 5,6-DHT after intracisternal administration, the inability to achieve destruction of a higher percentage of serotonergic fibres may be a major factor limiting its use. Serotonin depletions in brain of greater than 80% are obtained after the administration of p-chlorophenylalanine (KOE and WEISSMAN,1966), whereas depletions of not greater than 55% were obtained after 5,6-DHT. Recent studies, utilizing 6-hydroxydopamine to evaluate the role of catecholamine-containing fibres in brain, indicate that drastic depletions of the catecholamines may be required to reveal certain changes in behaviour after its administration (COOPERet al., 1972; 1973). By analogy, this may also hold true for 5,6-DHT even though some behavioural changes are shown after treatment with 5,6-DHT. In an effort to overcome this problem, 5,6-DHT was administered into the raphl nuclei so as to bypass any anatomical barrier to the entrance of 5,6-DHT into serotonin neurones in brain. While this procedure was found effective, depletion of serotonin was only moderate, and in some cases 5,6-DHT injected directly into brain tissue was found to cause tissue necrosis at the site of injection. BAUMGARTEN and LACHENMAYER (1972a) recently indicated that inhibition of monoamine oxidase prior to the administration of 5,6-DHT caused acute behavioural disturbances and considerable mortality. Data in this report are in general agreement with their findings. The marked hyperthermia observed may, at least in part, be responsible for the deaths of some of the animals. It will be of interest in future studies to determine if this reaction is related to a direct effect of 5,6-DHT or to the release of endogenous serotonin.

IX6

G. R. BREESE. B. R. COOPER, L. D. G~~hirand

R. D. SMI~

Rats that received 5,6-DHT after pargyline treatment were found to show a drastic loss of body weight, although most animals recovered much of this lost weight by 30 days. Whether the early weight loss is the result of some alteration of consummatory behaviour or a specific toxicity is an issue open to further investigation. In 1966, KOE and WEISSMAN (1966) reported that p-chlorophenylalanine caused massive depletion of brain serotonin and increased irritability. Since then, p-chlorophenylalanine has been found to facilitate both muricidal behaviour (SHEARD.1969) and avoidance responding (TENEN,1967). Serotonin depleting raphe lesions have also been found to produce similar changes (GRANT et al., 1973). Treatments with .5,6-DHT, like other serotonin-depleting manipulations, were also found in the present study to enhance muricide and to facilitate acquisition of a shuttle-box avoidance task. Since intracisternaIly injected 5,6-DHT does not alter peripheral serotonergic stores, effects on central serotonin mechanisms may underlie the observed behavioural effects. In our early investigations with 5,6-DHT, it was noted that rats treated with 5.6-DHT caged together showed increased frequency of mounting behaviour. Recently DA PNADA. CARKUBA,O’BKIEN, SANER and PLETSCHEK(1972) have systematically studied sexual behaviour in male rats and have come to the conclusion that 5,6-DHT enhances copulatory behaviour. These workers have suggested that this is due to diminished serotonin content in brain. Such findings would also be in accord with previous studies of facilitated sex behaviour after p-chlorophenylaianine (TAGLIAMONTE,TAGLIAMONTE.GESSA and BRODIE,1969; FERGIJSON,HENRIKSEN,COHEN, MITCHELL, BARCHASand DEMENT.1970; SALISand DEWSBURY,197 1f. In summary, the administration of .X6-DHT into brain was found to cllronicall~~ affect serotonergic fibres and to cause certain behavioural alterations characteristic of serotonin depletions. The advantages of administrating such a compound into brain to destroy serotonergic systems selectively are obvious. The present data suggest that additional studies are warranted to permit the optimal use of 5,6-DHT. The selectivity shown by 5,6-DHT should also prompt studies into the properties of structural congeners, such as 5,7-dihydroxytryptamine or 6,7_dihydroxytryptamine, in search of a less toxic compound still having selective destructive effects on serotonin neurones. Pursuing the latter approach, BAUMGARTEN and LACHENMAYER (1972b) have recently reported that 5,7-dihydroxytryptamine does indeed destroy serotonin-containing fibres, but is less specific than 5,&DHT. Acknowirdgrmrnts-The authors wish to acknowledge the technical assistance of J. FARMER, MARCYX KW.KI.AI) and SUSAN HOLLISTEK. This work was supported by USPHS grants MH-16522.NS-09844and HD-031 IO. B.R.C. is a postdoctoral fellow in the Neurobiology Program (MH-11107). G.R.B. is a USPHS Career Development Awardee (HD-24585). REFERENCES BAUMFARTEN, H. C., BJ~RKLUND, A., HOLSTEIN, A. F. and NOBIN, A. (1972a). Chemical

degeneration of indoleamine axons in rat brain by 5,6_dihydroxytryptamine: an ultrastructural study. Z. Zellforsch. &rosk. ,Irut. 129: 256-211. B,u.MtiARrrh.. H. G., BJ~~RKI.LINI.I, A.. LACHENMAY~R. L., NOISIN,A. nnd ST~N~VI, U. (1971). Long-lasting sclcctivc depletion of brain serotonin by 5,6-dihydroxytryptamine. .Actuph_~~io~. scared. Suppl.373. BALIMGARTIY. H. W.. EVETTS. K. D.. HOLMAN. R. B.. IV~RSEK, L. L,.. VOC;T, M. and WILSON. G. (1972b). Effects of S.6-dihydroxytryptamine on monoaminergic neurones in the central nervous system of the rat. J. i\ietrro&em. 19: 1587.-1597. BAUMGARTEN,H. G. and LACHENMAYER,L. (1972a). Chemically induced degeneration of indo~eamine-containing nerve terminals in rat brain. Braifz Rrs. 38: 228-232. BAL,~~~ART~~. H. G. and LACHENMAYER.L. (1972b). 5,7-Dihydroxytryptamine: improveInent in chemical lesioning of i~doleamine neurons in the mammalian brain. Z. Zeifforsch.tdwo~k. bar. 135: 399-414.

5.6.DHT

intracerebrally

BALJMGARTEN,H. G. and LACHENMAYCR.L. (1972~). Evidence

and behaviour

187

for a degeneration of indoleamine containing nerve terminals in rat brain induced by 5.6-dihydroxytryptamine. 2. Zrllforsch. mikrosk. Amt. 125: 553-569. BLOOM. F. E.. ALC;LRI. S.. GKOPPETTI. A.. RLVU~LTA, A. and COSTA, E. (1969). Lesions of central norepinephrine terminals with 6-OH-dopamine: biochemistry and fine structure. Scirrzce 166: 1284-1286. BOGDANSKI, D. F., PLETSCHER,A., BRODIE, B. B. and UDENFRIEND. S. (1956). Identification and assay of serotonin in brain. J. Pharmac. exp. Thu. 117: 82-88. BREESE,G. R. and TRAYLOR, T. D. (1970). Effect of h-hydroxydopamine on brain norepinephrine and dopamine: evidence for selective degeneration of catecholamine neurons. J. Pkartnc~. up. Thu. 174: 413420. BREESI:.G. R. and TRAYLOR. T. D. (1971). Depletion of brain noradrenaline and dopamine bq 6-hgdroxydopamine. Br. J. Phtrrmc~t 42: XX 99. BREESE, G. R. and TRAYLOR, T. D. (1972). Developmental characteristics of brain catecholamines and tyrosine hydroxylase in the rat: effects of 6-hydroxydopamine. Br. J. Phurntac. 44: 210-222. COOPER, B. R., BREESE,G. R., GRANT, L. D. and HOWARD, J. L. (1973). Effects of 6-hgdroxydopamine treatments on active avoidance responding: evidence for involvement of brain dopamine. J. Phnrmac. erp. Thu. 185: 358370. COOPI.R,B. R.. BR~+sE, G. R.. HOWARII. J. L. and GKAXT. L. D. (1972). Enhanced behavioural depressant effects of reserpine and alphamethyltyrosine after h-hydroxydopamine treatment. Ps!chopharr,lclcologiu 27: 99-110. DAHLSTROM, A. and FUXE, K. (1964). Evidence for the presence of monoamine containing neurons in the central nervous system-l. Demonstration of monoamines in the cell bodies of brain stem neurons. ilctu physiol. stand. 62, Suppl. 232. DA PRADA, M., CARRUBA, M., O’BRIEN. R. A.. SAKER. A. and PLETSCHER, A. (1972). The effect of 5,6-dihydroxytryptamine on sexual behaviour of male rats. Eur. J. Pharmac. 19: 288-390. FERGUSON, J., HENRIKSEN, S.. COHEN. H., MITCHELL, G.. BARCHAS, J. and D~MEN~. W. (1970). “Hypersexuality” and behavioural changes in cats caused by administration of p-chlorophenylalanine. Scirrm 168: 499%501. GRANT, L. D., COSCINA, D. V., GROSSMAX. S. P. and FREEDMAN.D. X. (1973). Muricide after serotonin-depleting lesions of midbrain raphe nuclei. Phamuc. Biochm. Behac. I : 77-80. ICHIYAMA, A., NAKAMURA, S., NISHIZUKA. Y. and HA~AISHI, 0. (1968). Tryptophan-5-hydroxylase in mammalian brain. Adu. Pharrnac. 6A: 5-17. KLijvt% H. and BAKRIZRA.E. (1953). A method for the combined staining of cells and fibs in the nervous system, J. Neuropatlz. exp. Nrwol. 12: 40&402. KOE, B. K. and WEISSMAN,A. (1966). P-Chlorophenylalanine: a specific depletor of brain serotonin. J. Pharmc. exp. Thu. 154: 499-516. MUSA~CHIO. J. M., Jc-LOU. L.. K~TY, S. S. and GLOWINSKI. J. (1969). Increase in rat brain tyrosine hydroxylase activity produced by electroconvulsant shock. Proc. mm. Acud. Sci. U.S.A. 63: I 117-I I 19. NAGATSU, T., LEVITT, M. and UDFNFRILNII. S. (1964). A rapid and simple radioassay for tyrosine hydroxylase activity. Analyt. Biochrm. 9: 122-126. SALIS, P. J. and DEWSBURY, D. A. (1971). p-Chlorophenylalanine facilitates copulatory behaviour in male rats. Naturr, Land. 232: 4OwOl. SCHANBERG, S. M., SCHILIIKRAUT, J. J., BREESE,G. R. and KOPIN, I. J. (1968). Metabolism of normetanephrine in rat brain: identification of conjugated 3-methoxy-4-hydroxy-phenylglycol as the major metabolite. B&hem. Pharmac. 17: 247 254. SHEARD, M. H. (1969). The effect of p-chlorophenylalanine on behaviour in rats: relation to brain serotonin and 5-hydroxyindoleacetic acid. Brain Res. 15: 524-528. TAGLIAMONTE,A., TAGLIAMONTE.P., GESSA, G. L. and BROUIE, B. B. (1969). Compulsive sexual activity induced by p-chlorophenylalanine in normal and pinealectomized rats. Science 166: 1433-1435. TENEN, S. (1967). The effects of p-chlorophenylalanine, a serotonin depletor, on avoidance acquisition, pain sensitivity and related behaviour in the rat. Ps),ckophilrma~ologia 10: 204-219. UNGERSTEDT. U. (1971). Stereotaxic mapping of the monoamine pathways in the rat brain. Acra. physiol. scared. Suppl. 367: I 48.