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Intracisternal administration of 200 µg of 5,7-dihydroxytryptamine (5,7-DHT) caused a prolonged reduction of brain serotonin which was accompanied by a depletion of brain norepinephrine. The depletion of norepinephrine was found to be antagonized by agents that inhibit uptake of norepinephrine as well as by several monoamine oxidase inhibitors. Intracisternal injections of 5,7-DHT (75 or 100 µg) to 7-day-old neonatal rats reduced brain serotonin and norepinephrine and produced a significant reduction of adult body weight. As in adults, pretreatment of neonatal rats with pargyline or desipramine prevented 5,7-DHT induced depletion of norepinephrine without affecting depletion of serotonin. Behaviorally, treatment of adult rats with 5,7-DHT facilitated acquisition of an active avoidance task and enhanced muricidal behavior. 5,7-DHT treatment was also found to enhance the depressant effects of 5-hydroxytryptophan on a fixed-ratio barpress response, suggesting that 5,7-DHT treated rats are supersensitive to serotonin in the central nervous system.
In 1971, Baumgarten et al.3, reported that 5,6-dihydroxytryptamine destroyed serotonergic fibers. However, extensive investigations of this compound revealed that 5,6-dihydroxytryptamine also caused non-specific damage to myelinated neurons when administered in doses in excess of 75 µg2,7. Such limitations on this compound led to the examination of other dihydroxytryptamine (DHT) derivatives. One of these analogues, 5,7-dihydroxytryptamine (5,7-DHT), has been found to produce a marked reduction of serotonin4–6,9 and tryptophan hydroxylase8. However, 5,7-DHT was found to reduce norepinephrine content in whole brain, indicating that destruction of noradrenergic fibers accompanied the effects of 5,7-DHT on serotonergic netlrons4,23.
Several compounds have been shown to reduce the cytotoxic effects of 6-hydroxydopamine on noradrenergic fibers, presumably by preventing its uptake11,15 Therefore, it was felt that such drugs might be used to reduce the effects of 5,7-DHT on noradrenergic fibers, thereby increasing the cytotoxic specificity of 5,7-DHT for serotonergic neurons. Since pretreatment of animals with pargyline has been shown to potentiate the destructive effects of 6-hydroxydopamine on dopaminergic fibers14,15, the ability of pargyline to alter the actions of 5,7-DHT was also examined. In other experiments, the effect of 5,7-DHT was studied in developing rats.
Male Sprague-Dawley rats obtained from Zivic Miller laboratories were used for all experiments. Adult rats (170–190 g) were given 200 µg of 5,7-DHT intracisternally, dissolved in sterile saline containing 0.5% ascorbic acid14. Some animals were pretreated with various drugs before 5,7-DHT was injected intracisternally to determine what effect they would have on the monoamine changes produced by 5,7-DHT. These drugs included pargyline (50 mg/kg), iproniazid (100 mg/kg), tranylcypromine (10 mg/kg), pheniprazine (15 mg/kg), chlorpromazine (25 mg/kg), benztropine (5 mg/kg), p-chlorophenylalanine (2 × 150 mg/kg), brompheniramine (25 mg/kg), reserpine (2 × 2.5 mg/kg), chlorimipramine (25 mg/kg), fenfluramine (10 mg/kg), desipramine (25 mg/kg) or L-110140 (15 mg/kg, 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine hydrochloride). All adult animals received 5,7-DHT (200 µg) intracisternally. Some animals received a second injection of 5,7-DHT (150 µg) 10 days after the first. Five to ten minutes prior to treatment, animals received pentobarbital sodium (20–30 mg/kg) or phenobarbital sodium (30 mg/kg) intraperitoneally to prevent seizures6. Surgical anesthesia was induced by ether. Control animals received intracisternal injections of the saline vehicle or drug treatments where appropriate. Drugs were injected 15 min (L-110140, brompheniramine and benztropine), 30 min (fenfluramine, pargyline, pheniprazine, tranylcypromine, iproniazid) or 60 min (chlorpromazine, desipramine, chlorimipramine) prior to 5.7-DHT. Reserpine and p-chlorophenylalanine were administered 48 and 24 h before the intracisternal injection of 5,7-DHT.
In another series of experiments, 7- and 14-day-old rat pups received 75 or 100 µg of 5,7-DHT intracisternally in 10 µl of vehicle27. Some of the animals were pretreated with desipramine (20 mg/kg) or pargyline (40 mg/kg) before receiving 5,7-DHT. Neonatal rats were also treated with pentobarbital (10–20 mg/kg) to prevent hyperactivity and convulsions.
In order to measure the content of biogenic amines, animals were killed by cervical fracture and decapitated at various time periods after injection of 5,7-DHT. In some cases, brains were removed and divided by a midsagittal cut. One half was immediately frozen on dry ice and stored (−76 °C) until homogenized in 0.1 N HCI for analysis of brain serotonin according to the method of Bogdanski et al.10. The remaining half of brain was homogenized in 0.4 N perchloric acid and kept frozen until it could be analyzed for catecholamine content within 24–48 h14,15,22. The brains from other animals treated with 5,7-DHT were dissected into specific brain areas including hypothalamus, striatum, brain stem and rest of brain as previously described18.
The effect of the various 5,7-DHT treatments on muricidal behavior was evaluated 7 days after treatment by placing a single adult male mouse in the cage of each treated and saline-control animal for 1 h. At the end of this time period, the number of rats that killed mice was determined12. The effects of intracisternally administered 5,7-DHT on acquisition of a shuttle-box avoidance response was determined using a modified automated shuttle-box which has been described previously18,27.
In order to determine if 5,7-DHT treated rats would show an enhanced response to 5-hydroxytryptophan (5-HTP), 5-HTP was administered to control and 5,7-DHT treated animals and the depressant effect of 5-HTP1 on operant behavior was examined using a fixed ratio-20 schedule of food reinforcement. Rats used to examine operant behavior were maintained on a 23 h schedule of food deprivation.
Various treatment groups were compared with the use of Dunnett’s t-test. The Fisher exact probability test was used to compare the effects of the various treatments on muricidal behavior.
In accordance with previous findings5, administration of 5,7-DHT was found to cause a significant reduction of brain serotonin content (Table I). A significant decline in norepinephrine content was found to accompany the reduction of serotonin, but no effect on brain dopamine concentration was observed. Reduction of serotonin content produced by 5,7-DHT was found to persist 270 days after treatment (Table I). Norepinephrine content seemed to recover gradually with time. A second injection of 5,7-DHT produced only a small additional increment in depletion of serotonin (Table II). Following a single treatment with 5,7-DHT, reduction of brain serotonin was most marked in the striatum with less effect on content in brain stem, hypothalamus, and rest of brain (Table IV).
Since pargyline was found to enhance the effects of intracisternally administered 6-hydroxydopamine on dopaminergic neurons14, animals were pretreated with pargyline to determine what effect inhibition of monoamine oxidase would have on the actions of 5,7-DHT. While this treatment did not enhance the effects of 5,7-DHT on serotonin-containing fibers, pargyline was unexpectedly found to block the effects of this neurocytotoxic agent on noradrenergic fibers (Table III). As previously described, an additional injection of 5,7-DHT produced only a moderate increase in the depletion of serotonin in pargyline treated rats. The reduction of brain serotonin in various brain areas when 5,7-DHT was administered with pargyline was similar to that observed when 5,7-DHT was injected alone (Table IV).
This finding prompted examination of several other monoamine oxidase inhibitors to determine if they might also reduce the depletion of norepinephrine produced by 5,7-DHT. As shown in Table V, iproniazid, pheniprazine and tranylcypromine were all found to prevent the action of 5,7-DHT to reduce norepinephrine, while having no effect on the ability of 5,7-DHT to reduce serotonin.
In this series of experiments (Table VI), it was found that neither reserpine nor p-chlorophenylalanine, which deplete serotonin content, antagonized the effect of 5,7-DHT to reduce brain serotonin and norepinephrine. Desipramine and chlorimipramine, which have been shown to decrease uptake of both norepinephrine and serotonin into monoaminergic neurons16,26, were found to reduce the effects of 5,7-DHT on noradrenergic neurons without affecting 5,7-DHT depletion of serotonin. Chlorpromazine, which has been shown to reduce the actions of 6-hydroxydopamine on noradrenergic neurons15, was also found to decrease the depletion of norepinephrine content in brain produced by 5,7-DHT. L-110140, a compound found to inhibit uptake of serotonin25,28, did not alter the action of 5,7-DHT on serotonin-containing fibers.
When rats 7 or 14 days of age were injected with 5,7-DHT, a marked depletion of brain serotonin and an accompanying depletion of norepinephrine was observed when rats were killed 60 days after treatment (Table VII). Brain dopamine content was not significantly affected by 5,7-DHT treatment. Survival rate of rats injected with 5,7-DHT (100 µg) at 7 days of age was 27 %. Some 60 % of the animals survived if 5,7-DHT (100 µg) was administered on day 14. At the time rats were killed (80 days of age), body weight of the group injected at 7 days was severely reduced compared with the body weight of control rats. Observation of the infant rats suggested that the apparent reason for the growth retardation was a reduction in suckling by the rat pups. Administration of pargyline to developing rats protected the animals against death caused by administration of 100 µg of 5,7-DHT (85% survived) and limited the growth retardation. Furthermore, this treatment prevented the effects of 5,7-DHT on noradrenergic fibers (Table VII). Desipramine was also found to prevent the depletion of norepinephrine produced by 5,7-DHT. However, pretreatment of animals with desipramine at 7 days of age did not reduce the mortality in treated animals (30% survived) nor did it eliminate the reduction in growth (Table VII).
When adult male rats treated with 5,7-DHT were caged together, aggressive behavior and fighting was observed within 24 h after treatment, necessitating individual caging of the animals. Tests for mouse killing behavior of singly caged rats 19 days after 5,7-DHT treatments revealed that 5,7-DHT, pargyline + 5,7-DHT and desipramine + 5,7-DHT induced killing in 71, 89 and 40% of the treated rats respectively. None of the control rats killed mice under these conditions.
A single treatment with 5,7-DHT was found to enhance the acquisition of the shuttle-box avoidance response during early trials but not later trials (Table VIII). Similar to the effects obtained after treatment with 5,6-DHT12 marked enhancement of the acquisition of the avoidance response was also observed in rats that were pretreated with pargyline (P + 5,7-DHT). All treatments enhanced the number of intertrial crosses that occurred during the 100 trial session (Table VIII).
In order to examine the possibility that 5,7-DHT treated animals might show an enhanced responsiveness to 5-HTP, the depression of operant responding induced by 5-HTP1 was studied. As shown (in Table IX), there was a marked enhancement of the 5-HTP-induced depression of fixed ratio barpress responding in animals treated with 5,7-DHT.
In accordance with previous results4,6,9, 5,7-DHT was found to produce a pronounced reduction of serotonin content in brain. Since 5,7-DHT has also been found to reduce brain tryptophan hydroxylase activity8, and to produce changes in fibers characteristic of degeneration in areas rich in terminals which contain serotonin6, the reduction of serotonin is believed to be due to destruction of serotonergic nerve terminals. Recent work by Baumgarten et al.5 has shown that brain serotonin content did not recover with time after treatment with 5,7-DHT. This latter finding was confirmed in the present study.
In agreement with other investigators4,20,23, brain norepinephrine content was also found to be reduced by 5,7-DHT. While this latter property of 5,7-DHT could limit its usefulness as a tool to examine the role of serotonin-containing fibers in brain function, two approaches were found to satisfactorily reduce the effects of 5,7-DHT on the noradrenergic system. The first method found effective was to pretreat rats with pargyline or other monoamine oxidase inhibitors. Daly et al.19 have recently found that pargyline also inhibits the action of 5,7-DHT on peripheral noradrenergic fibers. These workers19 have suggested that this action of pargyline may be related to an ability to prevent formation of a metabolite of 5,7-DHT, although this hypothesis has yet to be confirmed.
In addition to pargyline, compounds previously shown to reduce the destructive action of 6-hydroxydopamine on noradrenergic fibers11,15 were also found to reduce the effect of 5,7-DHT on noradrenergic fibers. This likely indicates that 5,7-DHT must be taken up into the noradrenergic neuron to have its destructive action. While several compounds were administered prior to 5,7-DHT that had previously been found to reduce uptake of serotonin16,17,21,24,26,28, none were found to reduce the action of 5,7-DHT to deplete brain serotonin. This latter finding is difficult to understand because previous work has suggested that 5,7-DHT competes for serotonin uptake in vitro, implying that the specificity of this neurocytotoxic compound is related to its specific uptake into serotonin-containing fibers19.
When 5,7-DHT was administered to rats early in development, reduction of serotonin was considerably greater than that observed when given to adult rats. Furthermore, there was little indication that serotonergic fibers recovered from the effects of 5,7-DHT, since animals treated at 7 days of age and killed as long as 240 days after 5,7-DHT showed no recovery of serotonin content in brain (unpublished data). When 5,7-DHT was administered to the developing rat, the mortality was pronounced. At a lower dose (50 µg) than that used in this study, Lytle et al.25 have also reported a prolonged destruction of serotonergic and noradrenergic fibers; however, somewhat less mortality was noted25. As observed in adult rats, treatment with pargyline or desipramine prevented the action of 5,7-DHT on noradrenergic fibers without altering its depletion of serotonin.
Administration of 5,7-DHT to adult rats was also found to alter behavior and increase the sensitivity to 5-HTP. For example, results obtained in a shuttle-box avoidance task after treatment with 5,7-DHT seemed to resemble those previously reported after injection of 5,6-dihydroxytryptamine12. The similarity included a more rapid acquisition of the shuttle-box avoidance response accompanied by a marked increase in intertrial crosses. However, the absence of an accelerated acquisition response after two 5,7-DHT treatments is an inconsistency which should be examined further. In addition, male rats treated with 5,7-DHT when adult showed an increased frequency of mouse-killing. Another property characteristic of 5,7-DHT treated rats was an increased sensitivity to 5-HTP. This seems to be related to the absence of serotonin-containing fibers, because these animals show no enhancement of responding to l-DOPA-induced locomotor activity (Hollister, Breese and Cooper, unpublished data). Whether the change in sensitivity is related to a change in serotonin uptake or in receptor sensitivity has yet to be resolved.
We are grateful to Marcine Kinkead, Susan Hollister and Edna Edwards for their excellent assistance.
This work was supported by USPHS Grants MH-16522 and HD-03110. George R. Breese is a USPHS Career Development Awardee (MH-00013).