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Possible involvement of the septo-hippocampal cholinergic and raphe-hippocampal serotonergic activations in the penile erection induced by fenfluramine in rats
BRAIN RESEARCH ELSEVIER
Brain Research 652 (1994) 181-189
Research report
Possible involvement of the septo-hippocampal cholinergic and raphe-hippocampal serotonergic activations in the penile erection induced by fenfluramine in rats Noriaki Maeda, Nobuya Matsuoka *, Isamu Yamaguchi Basic Research Group, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3, Tokodai, Tsukuba, Ibaraki, 300-26, Japan Accepted 12 April 1994
Abstract
Fenfluramine (0.1-10 mg/kg, i.p.) induced penile erection in naive rats with a bell-shaped dose response curve. The response to fenfluramine (1 mg/kg) was antagonized by scopolamine (0.032-1 mg/kg) but not by methyl-scopolamine (0.032-1 mg/kg) pre-treatment. The septo-hippocampal cholinergic deafferentations by medial septum lesion or fimbria-fornix transection also attenuated the penile erection induced by fenfluramine, whereas dopaminergic blockade by sulpiride (3.2-100 mg/kg) had hardly any effect. Pindolol (0.1-3.2 mg/kg), a 5-HT 1 antagonist, and ICS205-930, a 5-HT3 antagonist, but not ketanserin, a 5-HT 2 antagonist, inhibited the penile erection induced by fenfluramine. Depletion of 5-HT not only by systemic injections of p-chlorophenylalanine (150 mg/kg i.p. at 72, 48 and 24 h before the test) but also by the injections of 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, into the median- and dorsal-raphe nuclei significantly attenuated the fenfluramine-induced penile erections. Neurochemical analyses revealed that the raphe-lesion significantly reduced the contents of serotonin (5-HT) and its major metabolite, 5-hydroxyindole acetic acid (5-HIAA) but not choline acetyltransferase (CHAT) activities in all the cortical and subcortical regions examined. The results suggest that fenfluramine facilitates the expression of penile erection in rats through an activation of the septo-hippocampal cholinergic pathway as a consequence of excitation of the raphe-hippocampal serotonergic pathway in which 5-HTI and/or 5-HT 3 receptors appear to play a regulatory role. Key words: Fenfluramine; Penile erection; Cholinergic; Serotonergic; Dopaminergic; Hippocampus; Medial septum; Fimbria-fornix; Raphe nucleus
I. Introduction
Electrical stimulation of various brain areas has demonstrated that the hippocampus has a crucial role in expressing penile erection [24]. The brain area is densely innervated by cholinergic nerve fibers derived from cells in the medial septum [28,42]. In addition, the median and dorsal midbrain raphe nuclei project serotonergic nerve terminals directly to the hippocampus [2,19,30]. These evidence favor the view that interactions between cholinergic and serotonergic nerves may underlie the mechanism of penile erection. Serotonergic stimulants reportedly induce penile erection in rats [3,5] and in primates [40], and the
* Corresponding author. Fax: (81) 298-47-8610. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 4 6 5 - 0
effect is attenuated by drugs with 5-HT l antagonist activity such as propranolol or pindolol [5]. The 5-HT l receptor is now divided into several subtypes, and Berendsen et al. suggested that central 5-HTIc receptors play a crucial role in the expression of the penile erections [6]. Interestingly, Baraldi et al. reported that fenfluramine, a serotonergic stimulant, induced penile erection in a manner antagonizable by cholinergic antagonists [3]. On the other hand, both direct and indirect dopaminergic stimulants also induce penile erection in rats [4,16]. We have recently demonstrated that penile erections induced by apomorphine and amantadine were almost completely canceled by surgical deafferentations of the septo-hippocampal cholinergic pathway, which, however, had little effect on those induced by pilocarpine [25]. It is thus speculated that the septo-hippocampal cholinergic pathway modulated
N. Maeda et al./Brain Research 652 (1994) 181-189
182
by the central dopaminergic neurons might play an important role in the expression of penile erection. In the present paper, we examined the effects of neuronal antagonist pre-treatment and surgical deafferentations of the hippocampal serotonergic and cholinergic systems on penile erection induced by fenfluramine in rats. The possible role of an interplay between the limbic cholinergic, serotonergic and dopaminergic pathways in penile erections is discussed.
2. Materials and methods 2.1. Animals Male Fischer 344 rats at the age of 9 weeks were purchased from Charles River Inc. (Atsugi, Japan) at least 1 week before experiments. All animals were housed 6 to a stainless mesh cage (28 x 38 × 17 cm) in a temperature controlled room (22+ I°C) under a 12:12 h light/dark cycle with lights on at 8:00, and given food and water ad libitum. Experiments were carried out between 13:30 and 19:00 h in the room where animals were housed.
Medial-septum lesion
,,,,,;r-,
AP 0.8
2. 2. Behal'ioral procedures All animals were handled 3 rain a day for 3 successive days before the behavioral tests. The rats were tested in groups of six and various doses of fenfluramine or each antagonist were given in semi-randomized order. Immediately after the drug injection, each rat was placed in a perspex box ( 2 5 × 2 5 × 3 5 cm) and its behavior was observed for 60 min, during which time the number of penile erections was counted. A mirror was situated behind each box to facilitate observation of the animal. A penile erection was defined as repeated pelvic thrusts immediately followed by an upright position presenting an emerging, engorged penis which the rat proceeds to lick. Antagonists were injected 15 rain before the behavioral observation. Lesioned and sham-operated rats were assigned to receive all doses throughout a series of test sessions. Briefly, each group of six rats was administered with vehicle or doses of a drug in the first session. In the next session, each rat received a dose of the drug in ascending order, and those which had received the largest dose were given the vehicle. In this way, the test sessions were repeated the same number of times as the total number of vehicle and drug doses. At least 3 days intervened between each session, and a series of tests was finished within 4 weeks at the longest. In the experiment with 5-HT depletion by p-chlorophenylalanine (PCPA), the animals were treated with PCPA 150 m g / k g i.p. 72, 48
Fimbria-fornix lesion
t
P -2.0
Raphe lesion Median raphe
~
'
Dorsal raphe
AP -8.3
AP -8.7
Fig. 1. Schematic drawings of the position of medial septum (MS), fimbria-fornix (FF), median-raphe and dorsal raphe lesions in a coronal section approximately at the level of 0.8 mm anterior, 2.0 mm posterior, 8.3 mm posterior and 8.7 mm posterior from bregma, respectively, according to the atlas of K6nig and Klippel. The stippled areas indicate the representative extent and placement of each lesion.
N. Maeda et al. /Brain Research 652 (1994) 181-189 and 24 h before the test. A parallel group was treated three times with the vehicle.
2.3. Surgical procedures The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and fixed on a stereotaxic apparatus with the incisor bar set 2.3 mm below the intra-aural line. In all lesions, stereotaxic coordinates were positioned according to the atlas of K6nig and Klippel [20]. In each lesion, the body temperature of rats was maintained at 37°C until they recovered from anesthesia after the operations. At the end of the experiments the placement and size of the lesions were checked by histological examination on 10 # m thick slices subjected to Nissl staining. The location and size of MS, FF or raphe lesion are shown schematically in Fig. i.
2.3.1. Medial-septum (MS) or fimbria-fornix (FF) lesion The procedures for lesioning of the septo-hippocampal pathway used in the present study were similar to those used in the previous study [25]. In brief, for MS lesion, an electrode (0.7 mm in diameter and insulated except for the top 0.2 mm) was placed at 0.8 mm anterior from bregma on the midline and 7.0 mm ventral from the surface of the skull. A radiofrequency current was delivered from a lesion generator (Radionics, Burlington, USA: model RFG-4A) for 60 s through the electrode. The temperature was set at 60°C. Sham operation was done in the same way except delivery of the radiofrequency current. For FF lesion, a micro-surgery knife (Feather, Japan: No.715) mounted on a stereotaxic apparatus was positioned at 4.0 mm lateral to the midline, 2.0 mm posterior to bregma and lowered 5.0 mm below the surface of the skull. The FF was transected bilaterally by driving the microknife 8.0 mm laterally and retrieved at this position. For the sham lesions, only the sagittal sinus was transected. The behavioral test was started 7 days after each operation.
23,2. Dorsal- and median-rapke lesion Specific lesions of serotonergic neurons were made by stereotaxically placed local injections of 5,7-dihydroxytryptamine (5,7-DHT; in 1 tzl of 0.1% ascorbic acid-saline solution; 0.2 /zl/min) through a 26-gauge cannula connected by polyethylene tubing to a gear-driven Hamilton syringe. Sham animals were injected with the vehicle. Injections into the median raphe nucleus were made at the coordinates of 8.3 mm posterior to bregma, on the midline, 8.0 mm ventral from the skull surface; injections into the dorsal raphe nucleus were made at the coordinates of 8.7 mm posterior to bregma, on the midline, 6.3 mm ventral from the skull surface. Both nuclei were injected with 2 /xg of 5,7-DHT in a volume of 1 /xl and the rats were pretreated with desipramine (25 mg/kg, i.p.) 30-45 min prior to the local injections in order to protect noradrenergic neurons from the lesion [8j. The behavioral test was started 14 days after the operation.
2.4. Neurochernical analyses Brain regional monoamine contents and the choline acetyltransferase (CHAT) activity were measured in order to verify the effect of raphe lesion on brain neurochemistry. Two weeks after the lesioning, rats were decapitated and the whole brain and spinal cord were removed, and the brain was rapidly dissected into the following regions on ice: the hippocampus, the striatum, anterior part of the cortex including prefrontal cortex and posterior part of the cortex including parietal, occipital and temporal cortex. The tissue was stored at - 8 0 ° C until assay.
183
2. 4.1. Monoamine contents Levels of 5-HT, 5-HIAA, NE, DA and DOPAC were measured in the stored tissues of each of the rat using a reverse-phase highperformance liquid chromatography (HPLC) method with electrochemical detector. Briefly, brain tissues were homogenized in 0.2 M perchloric acid (PCA) containing isoproterenol (10 n g / m l ) as a internal standard, and centrifuged at 20,000× g for 15 min at 4°C. The pH of the supernatant was adjusted to 3.0 with 1 M CH3COONa, and 50 ml of sample was injected into a Eicompak MA-5 ODS liquid chromatography system (Eicom Co.; Kyoto, Japan). Amines were detected using an Eicom ECD-100 electrochemical detector (Eicom Co.) with a glossy carbon electrode held at + 750 mV vs. an A g / A g C I reference. The flow rate was maintained at l ml/min. The mobile phase consisted of 0.1 M acetate-citrate buffer (pH 3.5) containing 1 mM sodium octane sulfonate as ion pair reagent, 0.01 mM EDTA and 17% methanol. The detection limit was approximately 5 pg for each standard amine. Standard sample of each amine was run at the concentration of 10 ng/ml. 2.4.2. ChAT actit,ity To characterize the changes in the integrity of brain cholinergic neurons, the ChAT activity was determined in the brain areas of raphe lesioned rats. The activity was measured by the method of Fonnum [12], using [14C]acetyl coenzyme A (New England Nuclear), and expressed as nmol A C h / h / m g protein. Proteins were measured by the method of Lowry [23]. 2.5. Drugs The following drugs were used: (+)-fenfluramine hydrochloride (synthesized in our research laboratories); sulpiride hydrochloride (synthesized in our research laboratories); (+_)-pindolol hydrochloride (Sigma Chemical Co., St. Louis, MO, USA); ketanserin tartrate (Research Biochemicals Inc., Natick, MA, USA); ICS205-930 ((3tropanyl)-lH-indole-3-carboxylicacid,ester HCI, synthesized in our research laboratories); scopolamine hydrobromide (Nacalai tesque, Kyoto, Japan); methyl-scopolamine hydrobromide (Nacalai tesque); (_+)-metoprolol (Sigma); 5,7-dihydroxytryptamine creatinine sulfate (5,7-DHT, Sigma); dl-p-chlorophenylalanine methyl ester hydrochloride (PCPA, Sigma). Pindolol and sulpiride were dissolved in 1% tartrate-saline with the pH adjusted to 6.0-7.0. 5,7-DHT was dissolved in sterile saline containing 0.2% ascorbic acid immediately before use and injected into the raphe nuclei. All other drugs were dissolved in physiological saline and given intraperitoneally in a volume of 1 ml/kg. Control animals were injected with an equivalent volume of vehicle. Antagonists and agonists were injected 15 min and just before the start of the behavioral experiments, respectively. The dosages of antagonists were selected based on our previous observations [25].
2.6. Statistics All results were expressed as the mean+_ S.E.M.. In the behavioral experiments, the statistical significance was estimated with Mann-Whitney U-test. In the comparison of brain neurochemistry between the lesioned and sham rats, Student's t-test. P values less than 0.05 were used as the criterion for statistical significance.
3. Results
3.1. Effects of fenfluramine on penile erection in rats Fig. 2 shows the number of penile erections in rats after i.p. injection of fenfluramine. Fenfluramine (0.1-
N. Maeda et al. /Brain Research 652 (1994) 181-189
184 7
(n=ll)
Table 1 Effects of dopaminergic or /3-adrenergic antagonists on the penile erection induced by fenfluramine
**•
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5
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4
**
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°
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11
1
-
Antagonist dose ( m g / k g , i.p.)
Fenfluramine (1 m g / k g / , i.p.)
n
Penile e r e c t i o n s / h
Saline Saline Sulpiride
Saline Fenfluramine 1 1 1 1 1
6 6 6 6 6 6
0.50_+0.22 ** 6.00_+0.58 5.33 _+0.42 6.00_+0.93 7.33 _+ 1.63 4.00_+0.82
Saline Fenfluramine 1 1 1 I 1
6 6 6 6 6 6
0.67_+0.33 ** 4.50_+ 0.34 4.17_+0.48 4.83 _+0.70 5.17_+0.48 4.17_+0.31
0 0
0.1
0.32
1
3.2
10
Fenfluramine (mg/kg) Fig. 2. Effect of fenfluramine on penile erection in rats. Each ordinate represents the mean n u m b e r of penile erections in 1 h following i.p. administration of fenfluramine. ** P < 0.01, *** P < 0.001; statistically significant compared with saline-treated group (by M a n n - W h i t n e y U-test). Each column and bars represents the mean + S.E.M. The n u m b e r of rats tested is given in parenthesis.
10 mg/kg) increased the number of penile erection with a bell-shaped dose-response curve with statistically significance (P < 0.01 by Mann-Whitney U-test), with the maximum increase above spontaneous values being observed at 1 mg/kg of the drug. Based on these effects, fenfluramine 1 mg/kg was employed in parts of the following studies. No other signs of abnormal behavior except penile erection were observed in rats dosed with fenfluramine up to 3.2 mg/kg, however, the highest dose (10 mg/kg) of the drug caused a persistent backwards movement which lasted throughout behavioral observation.
Saline Saline Metoprolol 0.32 1 3.2 10
Antagonists were administered i.p. 15 min before administration of fenfluramine. Each value represents the mean number of penile erections of 6 rats observed in 1 h+_S.E.M. * * P < 0.01; statistically significant compared with saline-fenfluramine treated group (by M a n n - W h i t n e y U-test).
3.2. Effects of cholinergic antagonists on fenfluramineinduced penile erection Fig. 3 summarizes the effects of scopolamine and methyl-scopolamine on penile erections induced by fenfluramine (1 mg/kg). Scopolamine (0.032-1 mg/kg) dose-dependently reduced the number of penile erections induced by fenfluramine (1 mg/kg). Statistically significant changes (P < 0.05) were observed in groups subjected to higher dosages than 0.32 mg/kg of scopolamine, as shown in Fig. 3A. On the contrary, methylscopolamine (0.032-1 mg/kg), a peripheral acting
B. Methyl-scopolamine
A. Scopolamine 7
(n=12)
8ill119In6 I "~- 7
5
~6
-~4
*
3
***
~4
~
1
0
3.2 10 32 100
3
2 1 0
0
0 0.032 0.1 0.32
1
Scopolamine (mg/kg) F e n f l u r a m i n e (1 m g / k g )
0
0 0.032 0.1 0.32
1
Methyl-scopolamine (mg/kg) F e n f l u r a m i n e (1 m g / k g )
Fig. 3. Effects of (A) scopolamine or (B) methyl-scopolamine on the penile erections induced by fenfluramine. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine (1 mg/kg). Each antagonist was administered 15 min prior to behavioral observations. * P < 0.05, * * P < 0.01, * * * P < 0.001; statistically significant compared with control (saline-pretreated) groups treated with fenfluramine alone (by M a n n - W h i t n e y U-test). Each column and bar represents the mean _+ S.E.M. The number of rats tested is given in parentheses.
N. Maeda et al. /Brain Research 652 (1994) 181-189 B. F i m b r i a - f o r n i x l e s i o n
A.Medial septum lesion # 9 t-.
8
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##
(n=6)
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1
0
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0.1 0.32 3.2 Fenfluramine (mg/kg)
0
0.1 0.32 1 3.2 Fenfluramine (mg/kg)
Fig. 4. Effects of medial-septum lesion (A) or fimbria-fornix lesion (B) on the penile erections induced by fenfluramine. Each neurosurgery was performed as described in the Materials and methods. Seven days after surgery, behavioral tests were started and repeated as each rat experienced all dosages of the drug with at least 3 days intervals. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine. * P < 0.05, ** P < 0.01; statistically significant compared with vehicle control in sham animals. * P < 0.05, • * P < 0.01; compared with vehicle control group in lesioned animals. ~ P < 0.05, ## P < 0.01; compared with each corresponding dosed group in sham animals (by M a n n - W h i t n e y U-test). Each column and bar represents the mean _+ S.E.M. The number of rats tested is given in parentheses.
muscarinic antagonist, had little effect on the responses induced by fenfluramine (Fig. 3B). Furthermore, pretreatment of sulpiride (3.2-100 mg/kg), a dopamine D 2 blocker, hardly affected the responses induced by fenfluramine as shown in Table 1.
3.3. Effects of septo-hippocampal cholinergic denervation on penile erection by fenfluramine The effects of fenfluramine on penile erection in medial septum-lesioned rats are shown in Fig. 4A. The overall dose-response in the sham operated rats did not differ from that in the above-mentioned intact rats, i.e. statistically significant ( P < 0.01) responses occurred at doses of 0.32 and 1 m g / k g and the maximum response of about 7 penile e r e c t i o n s / h was observed at 1 mg/kg. The penile erections induced by the drug were fewer in the medial septum-lesioned rats than in
A. Pindolol
3.4. Effects of serotonergic receptor antagonists on fenfluramine-induced penile erection The effects of serotonergic receptor antagonists on the penile erections produced by fenfluramine were investigated (Fig. 5 and Table 1). Pindolol (0.1-3.2 mg/kg), a 5-HT 1 and /3-adrenoceptor antagonist [31], dose-dependently and significantly abolished the penile
B. Ketanserin
.c 5
C. ICS205-930
.o
2 3 "~ 2 0
the sham rats, and the difference between the two was statistically significant ( P < 0.05) at the dose of 1 mg/kg. Also in the fimbria fornix-lesioned rats, the maximum response at 1 m g / k g of fenfluramine was significantly ( P < 0.01) reduced compared with that in the sham rats (Fig. 4B), and the change was more pronounced than in the medial septum-lesioned rats (Fig. 4A).
**
0
0
0.1 0.32
1
3.2
Pindolol ( m g / k g ) Fenfluramine (1 m g / k g )
.o
~ .~
** 0
0
'~ 2
0
0.1 0.32
1
3.2
0
**
0
0
0.01 0.1
1
10
Ketanserin ( m g / k g )
1CS205-930 ( m g / k g )
Fenflurarnine (1 m g / k g )
Fenfluramine (1 m g / k g )
Fig. 5. Effects of (A) pindolol, (B) ketanserin or (C) ICS205-930 on the penile erections induced by fenfluramine. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine (1 mg/kg). Each 5-HT antagonist was administered 15 min prior to behavioral observations. * P < 0.05, ** P < 0.01; statistically significant compared with control (saline-pretreated) groups treated with fenfluramine alone (by M a n n - W h i t n e y U-test). Each column and bar represents the mean + S.E.M. The number of rats tested is given in parentheses.
N. Maeda et al. /Brain Research 652 (1994) 181-189
186
A. R a p h e l e s i o n
B. P C P A - t r e a t m e n t
## 9
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[""7 Saline PCPA
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0
0.1 0.32
3.2
0
10
Fenfluramine (mg/kg)
0.1 0.32
3.2
10
Fenfluramine (mg/kg)
Fig. 6. Effects of central 5-HT d e p l e t i o n by (A) r a p h e lesion and (B) P C P A p r e - t r e a t m e n t on the penile e r e c t i o n s i n d u c e d by fenfluramine. Medial- and d o r s a l - r a p h e lesioning was p e r f o r m e d as d e s c r i b e d in the M a t e r i a l s and m e t h o d s . B e h a v i o r a l tests were s t a r t e d 14 days after surgery, and r e p e a t e d as each rat e x p e r i e n c e d all dosages of the drug with at least 3 days intervals. P C P A (150 m g / k g ) was a d m i n i s t e r e d i.p. 72, 48 and 24 h before the test. E a c h o r d i n a t e r e p r e s e n t s the m e a n n u m b e r of penile e r e c t i o n s in 1 h following i.p. a d m i n i s t r a t i o n of fenfluramine. * P < 0.05, • * P < 0.01, *** P < 0.001; statistically significant c o m p a r e d with vehicle control g r o u p in s h a m animals. * P < 0.05, ** P < 0.01; c o m p a r e d with vehicle control g r o u p in r a p h e - l e s i o n e d animals. # P < 0.05, ## P < 0.01, ### P < 0.001; c o m p a r e d with each c o r r e s p o n d i n g d o s e d g r o u p in sham a n i m a l s (by M a n n - W h i t n e y U-test). E a c h c o l u m n and b a r r e p r e s e n t s the m e a n 4- S . E M . The n u m b e r of rats t e s t e d is given in p a r e n t h e s e s .
erections induced by 1 m g / k g of fenfluramine (Fig. 5A), whereas metoprolol, a /3-adrenoceptor-selective antagonist without 5-HT antagonistic activity, failed to affect the response (Table 1). Ketanserin, a 5-HT 2 antagonist, also failed to affect the response (Fig. 5B). On the other hand, ICS205-930 (0.01-10 mg/kg), a specific 5-HT 3 antagonist [34], dose-dependently attenuated the response by fenfluramine, as shown in Fig. 5C.
(P < 0.05) and 1 m g / k g (P < 0.01) of fenfluramine gave statistically significant responses in the sham rats. In the raphe lesioned rats, significant erections were still observed at the dose of 1 m g / k g of the drug, however, the maximum response was significantly (P < 0.01) attenuated compared to that in the sham-operated rats. Pretreatment with 150 m g / k g of PCPA hardly affected spontaneous penile erection (drug 0 in Fig. 6B), but markedly reduced the penile erection produced by fenfluramine, and the changes were statistically significant ( P < 0 . 0 5 ) at 0.1, 1 and 3.2 m g / k g of fenfluramine, as shown in Fig. 6B.
3.5. Effects of 5-HT depletion by lesioning of the raphe nuclei or systemic PCPA administration on fenfluramine-induced penile erection
3.6. Brain neurochemistry
Fig. 6 shows the effects of 5-HT depletion by raphe lesioning or systemic treatment of PCPA on penile erection induced by fenfluramine. The doses of 0.32
Brain regional monoamines content after medianand dorsal-raphe lesioning are presented in Table 2.
Table 2 C h a n g e s in m o n o a m i n e s c o n t e n t s in the brain a r e a s of r a p h e - l e s i o n e d rats Brain region
Monoamine contents (ng/mg protein) NE
DOPAC
Hippocampus Sham Lesion
11.65 4- 0.23 10.56 + 0.38 *
0.413 + 0.040 0.390 + 0.061
Cortex anter. Sham Lesion
8.64 _+ 1.89 10.73 + 0.38
4.240 _+ 1.060 4.370 _+ 0.412
C o r t e x poster. Sham Lesion
7.570 + 1.26 6.700 + 1.38
Striatum Sham Lesion
7.590 + 0.57 7.090 + 0.34
0.957 4- 0.204 0.711 + 0.122
24.05 24.75
4- 0.712 + 3.630
DA 1.580 _+ 0.508 1.690 _+ 0.629
25.96 22.43
_+ 6.670 _+ 4.630
6.450 4- 1.060 4.800 4- 0.929
230.6 226.2
4- 9.36 4- 21.09
5-HIAA
5-HT
11.85 3.74
13.19 _+ 0.67 3.53 _+ 1.45 ***
4- 0.81 + 1.10 ***
10.10 _+ 2.25 4.74 4- 1.27
8.640 4- 1.48 2.670 4- 0.57 **
20.50 10.11
4- 1.36 + 1.34 ***
13.37 _+ 2.98 5.26 _+ 1.76 *
11.12 + 2.28 3.17 4- 0.78 *
14.47_+ 0.84 6.61 _+ 1.10 ***
E a c h value r e p r e s e n t s m e a n + S.E.M.. Brain m o n o a m i n e s c o n t e n t s w e r e d e t e r m i n e d 2 w e e k s following r a p h e lesion. Six a n i m a l s w e r e e m p l o y e d in each group. * P < 0.05, ** P < 0.01, *** P < 0.001; statistically significant c o m p a r e d to c o r r e s p o n d i n g region of s h a m a n i m a l s by S t u d e n t ' s t-test.
N. Maeda et al. /Brain Research 652 (1994) 181-189
The lesion significantly reduced the 5-HT and 5-HIAA levels in all the cortical and subcortical brain areas but not in the spinal cord (data not presented). The most striking effect was observed in the hippocampus where 5-HT and 5-HIAA levels were decreased by 73 and 68% of those of the sham rats, respectively. Other catecholamines and their metabolites in the brain areas were hardly changed in the lesioned rats compared with the sham control. The ChAT activity in the brain regions was also determined in raphe lesioned rats. However, no differences in the activities were observed in the areas between two groups as follows: Sham 48.5 + 1.0, raphe lesion 48.3 + 0.9 in the hippocampus; sham 41.3 + 0.5, raphe lesion 41.9 + 0.9 in the anterior cortex; sham 43.9 + 0.5, raphe lesion 44.9 _+0.3 in the posterior cortex; sham 106.5 + 4.6, raphe lesion 104.9 + 2.8 in the striatum (nmol ACh/h/mg protein, n = 6 in each group).
4. Discussion
Confirming and extending the previous finding by Baraldi et al. [3], we observed that scopolamine but not methyl-scopolamine attenuated the penile erection induced by fenfluramine. In addition, the fenfluramineinduced response was attenuated in rats subjected to MS or FF lesioning. It has been widely accepted that the cells in the medial septum (MS) and diagonal band of Broca (DB) send cholinergic nerve fibers to the hippocampus through the fimbria-fornix (FF) [28], and their activities play an important role in the regulation of penile erection [24]. These results favor the view t h a t . a n activation of septo-hippocampal cholinergic nerves is responsible for the penile erection by fenfluramine. However, we have previously shown that the FF lesion almost completely depleted hippocampal ChAT activity [27], and that 1 mg/kg of scopolamine completely abolished the penile erection induced by pilocarpine and apomorphine [25]. In the present study, however, neither FF lesioning nor scopolamine completely abolished the fenfluramine-induced penile erection. Other mechanisms in addition to septo-hippocampal cholinergic activation may also be involved in the response by fenfluramine. The present study demonstrated that pindolol, a /3-adrenoceptor antagonist with 5-HT 1 blocking activity [31], but not metoprolol, a /3-adrenoceptor antagonist without 5-HT 1 blocking activity [17], dose-dependently attenuated the penile erection induced by fenfluramine. Furthermore, it is a novel finding that ICS205930, a 5-HT 3 antagonist [34], but not ketanserin, a 5-HT 2 antagonist [22] attenuated the fenfluramine-induced penile erection. These results in combination with the observations that fenfluramine enhances 5-HT
187
release and reduces 5-HT re-uptake [9,14,15] suggest that an activation of 5-HT~ and 5-HT 3 receptors is involved in the fenfluramine-induced penile erection. While it has been speculated that central 5-HT~B a n d / o r 5-HT~c receptors activities play a crucial role in sexual behavior [5,6], the present result is the first evidence implicating that 5-HT 3 receptors mediate the expression of penile erection. The growing body of evidence indicates that not only 5-HT~ but also 5-HT 3 receptors are densely distributed within the limbic areas including the amygdala, cerebral cortex and hippocampus [18,29]. Furthermore, it is interesting to note that stimulation of the 5-HT~ receptors enhances ACh release in the hippocampus [32,33]. An activation of intra-hippocampal 5-HT 3 receptors are also postulated to enhance ACh release via the enhancement of 5-HT transmission [26]. We speculate that fenfluramine activates the central 5-HT 1 and 5-HT 3 receptors to cause ACh release which in turn leads to penile erection. However, as mentioned above, scopolamine as well as septo-hippocampal cholinergic deafferentations induced incomplete attenuation of the fenfluramine-induced penile erection, while the largest dose (3.2 mg/kg) of pindolol almost completely abolished it. These results prefer the view that an activation of the 5-HT receptors by fenfluramine causes penile erection through mechanisms with and without cholinergic mediation. Another important finding of the present study is that PCPA-treatment as well as 5,7-DHT injections in the raphe nuclei significantly attenuated the fenfluramine-induced penile erection. The dose regime of PCPA-treatment reportedly depletes brain 5-HT levels by more than 90% [39], whereas the present neurochemical analysis revealed that the lesioning of the raphe nuclei by 5,7-DHT injection specifically depleted 5-HT and its major metabolite 5-HIAA in the brain. The latter observation coincides with the previous findings that serotonergic nerve terminals derived from the median- and dorsal-raphe nuclei are widely distributed in the brain areas such as the cerebral cortex, septum and hippocampus [2,19]. It has been widely accepted that the raphe-hippocampal serotonergic pathway exerts a powerful infuence on the neuronal activity of the hippocampus [1,36,41]. In addition, recent electrophysiological studies have revealed that the septo-hippocampal pathway is negatively regulated by hippocampal GABA-ergic interneurons [10], and that the raphe-hippocampal serotonergic pathway modulates the septo-hippocampal neuronal excitability by disinhibiting these inhibitory interneurons [13,38]. These findings together with the above discussion suggest that an activation of the raphe-serotonergic neurons by fenfluramine, at least partly, leads to an activation of septo-hippocampal cholinergic neurons resulting in the expression of penile erection (the possible mechanisms
188
AIO
N. Maeda et al. /Brain Research 652 (1994) 181-189
$~~t~um
/~ Raphe I ~Fenfluramine
Hippocampus Penile erection Fig. 7. Schematic drawing for the possible mechanisms of the limbic cholinergic, serotonergic and dopaminergic link modulating the expression of penile erection in rats.
of the limbic cholinergic a n d s e r o t o n e r g i c link m o d u l a t ing the expression of p e n i l e e r e c t i o n s is p r e s e n t e d in Fig. 7). O u r s p e c u l a t i o n is f u r t h e r s t r e n g t h e n e d by the o b s e r v a t i o n s that f e n f l u r a m i n e i n c r e a s e d the excitability of the rat h i p p o c a m p u s in a m a n n e r a n t a g o n i z a b l e by P C P A p r e - t r e a t m e n t [35], a n d t h a t systemic a d m i n istration of p - c h l o r o a m p h e t a m i n e , a 5 - H T releaser, i n c r e a s e d h i p p o c a m p a l A C h r e l e a s e in naive but not in r a p h e - l e s i o n e d rats [32]. A l t h o u g h M a c L e a n , after a series of e l e c t r o p h y s i o logical studies, c o n c l u d e d that the h i p p o c a m p u s a n d its afferents play a crucial role in the expression o f p e n i l e e r e c t i o n [24], the e f f e r e n t p a t h w a y s are still to be clarified. O n e of the e f f e r e n t s could be the subspinal d e s c e n d i n g s e r o t o n e r g i c system t h a t has b e e n sugg e s t e d to be involved in sexual b e h a v i o r [7]. O u r present o b s e r v a t i o n that slight r e s p o n s e s to f e n f l u r a m i n e still r e m a i n e d in the animals s u b j e c t e d to r a p h e - l e s i o n ing may b e explained, at least in part, by an activation of the b u l b - s p i n a l s e r o t o n e r g i c system by f e n f l u r a m i n e . However, similar r e s p o n s e s w e r e o b s e r v e d in P C P A t r e a t e d rats, which m a y a r g u e against the possibility. It is thus t e m p t i n g to s p e c u l a t e that s p a r e d s e r o t o n e r g i c nerves after r a p h e - l e s i o n i n g a n d P C P A - t r e a t m e n t is r e s p o n s i b l e for the residual responses. It has b e e n r e p o r t e d previously that p e n i l e e r e c t i o n s i n d u c e d by direct and indirect d o p a m i n e r g i c stimulants are also m e d i a t e d by s e p t o - h i p p o c a m p a l cholinergic activation [25]. D o p a m i n e r g i c cell b o d i e s in the ventral t e g m e n t a l a r e a (A10) a n d s u b s t a n t i a nigra (A9) send p r o j e c t i o n s to the r a p h e nuclei which is b e l i e v e d to control s e r o t o n e r g i c n e u r a l activity in the nuclei [11,21]. In addition, t h e r e is e v i d e n c e that d o p a m i n e r g i c neurons in the A 1 0 might play a positive m o d u l a t o r y role in the s e p t o - h i p p o c a m p a l cholinergic p a t h w a y in drug i n d u c e d p e n i l e e r e c t i o n in rats [28,37]. T h e s e findings c o m b i n e d with o u r p r e s e n t ones p r o v i d e a question of interest in t e r m s of the m o d e of i n t e r a c t i o n b e t w e e n the d o p a m i n e r g i c a n d s e r o t o n e r g i c p a t h w a y s m o d u l a t ing p e n i l e erection. T h e p r e s e n t o b s e r v a t i o n that sulpiride failed to b l o c k the p e n i l e e r e c t i o n s by fenflu-
r a m i n e a d d r e s s the issue. T h e s e e v i d e n c e t a k e n tog e t h e r suggest that d o p a m i n e r g i c s t i m u l a t i o n c o m e s down to h i p p o c a m p a l s e r o t o n e r g i c a n d c h o l i n e r g i c activation finally to cause p e n i l e erection. In conclusion, the p r e s e n t results d e m o n s t r a t e the c e n t r a l n e u r a l m e c h a n i s m s u n d e r l y i n g the p e n i l e erection p r o d u c e d by f e n f l u r a m i n e , a n d p r o v i d e novel findings r e g a r d i n g the limbic A C h - 5 - H T i n t e r p l a y which exerts an i m p o r t a n t role in d r u g - i n d u c e d p e n i l e erection in rats.
References [1] Assaf, S.Y. and Miller, J.J., The role of raphe serotonin system in the control for septal unit activity and hippocampal desynchronization, Neuroscience, 3 (1978) 539-550. [2] Azmitia, E.C. and Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. Comp. Neurol., 179 (1978) 641-668. [3] Baraldi, M., Benassi-Benelli, A. and Lolli, M., Penile erections in rats after fenfluramine administration, Ret,. Farmacol. Ther., 8 (1977) 375-379. [4] Benassi-Benelli, A., Ferrani, F. and Pelegrini-Quarantotti, B., Penile erection induced by apomorphine and N-n-propylnorapomorphine in rats, Arch. Int. Pharmacodyn. Ther., 241 (1979) 128-134. [5] Berendsen, H.H.G. and Broekkamp, C.L.E., Drug-induced penile erections in rats: indications of serotonin m receptor mediation, Eur. J. Pharmacol., 135 (1987) 279-287. [6] Berendsen, H.H.G., Jenck, F. and Broekkamp, C.L.E., Involvement of 5-HTlc-receptors in drug-induced penile erections in rats, Psychopharmacology, 101 (1990) 57-61. [7] Bitran, D. and Hull, E.M., Pharmacological analysis of male rat sexual behavior, Neurosci. Biobehav. Rev., 11 (1987)365-389. [8] Bj6rklund, A., Baumgarten, H.G. and Rensch, A., 5,7-Dihydroxytryptamine: Improvement of its selectivity for serotonin neurons in the CNS by pretreatment with desipramine, J. Neurochem., 24 (1975) 833-835. [9] Costa, E., Groppetti, A. and Revuelta, A., Action of fenfluramine on monoamine stores of rat tissues, Br. J. Pharmacol., 41 (1971) 57-64. [10] Curtis, D.R., Felix, D. and McLennan, H., GABA and hippocampal inhibition, Br. J. Pharmacol., 40 (1970) 881-883. [11] Ferre, S. and Artigas, F., Dopamine D 2 receptor-mediated regulation of serotonin extracellular concentration in the dorsal raphe nucleus of freely moving rats, J. Neurochem., 61 (1993) 772-775. [12] Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975)407409. [13] Freund, T.F., Gulyas, A.I., Acsady, L., Gorcs, T. and Toth, K., Serotonergic control of the hippocampus via local inhibitory interneurons, Proc. Natl. Acad. Sci. USA, 87 (1990) 8501-8505. [14] Garattini, S., Buczko, W., Jori, A. and Saminin, R., The mechanism of action of fenfluramine, Postgrad. Med. J., 51 (Suppl. 1) (1975) 27-35. [15] Garattini, S., Mennini, T., Bendotti, C., Invernizzi, R. and Samanin, R., Neurochemical mechanism of action of drugs which modify feeding via the serotonergic system, Appetite, 7 (1986) 15-38. [16] Gower, A.J., Berendsen, H.H.G., Proncen, M.M. and Broekkamp, C.L.E., The yawning-penile erection syndrome as a model for putative dopamine autoreceptor activity, Eur. J. Pharmacol., 103 (1984) 81-89.
N. Maeda et al. /Brain Research 652 (1994) 181-189 [17] Hjorth, S. and Carlsson, A., (-)-Pindolol stereospecifically inhibits rat brain serotonin (5-HT) synthesis, Neuropharmacology, 24 (1985) 1143-1146. [18] Kilpatrick, G.J., Jones, B.J. and Tyers, M.B., Identification and distribution of 5-HT 3 receptors in rat brain using radioligand binding, Nature, 330 (1987) 746-748. [19] K6hler, C. and Steinbusch, H., Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain, Neuroscience, 7 (1982) 951-975. [20] K6nig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. [21] Lee, E.H.Y. and Geyer, M.A., Dopamine autoreceptor mediation of the effects of apomorphine on serotonin neurons, Pharmacol. Biochem. Behat~., 21 (1984)301-311. [22] Leysen, J.E., Awouters, F., Kennis, L., Laduron, P.M., Vandenberk, J. and Janssen, P.A.J., Receptor binding profile of R41 468, a novel antagonist at 5-HT 2 receptors, Life Sci., 28 (1981) 1015-1022. [23] Lowry, O.H., Rosenberg, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [24] MacLean, P.D., Brain mechanisms of primal sexual functions and related behavior. In M. Sandier and G.L. Gessa (Eds.), Sexual BehaL~ior: Pharmacology and Biochemistry, Raven, New York, 1975, pp. 1-11. [25] Maeda, N., Matsuoka, N. and Yamaguchi, I., Septohippocampal cholinergic pathway and penile erections induced by dopaminergic and cholinergic stimulants, Brain Res., 537 (1990) 163-168. [26] Martin, K.F., Hannon, S., Phillips, I. and Heal, D.J., Opposing roles for 5-HT m and 5-HT 3 receptors in the control of 5-HT release in rat hippocampus in vivo, Br. J. Pharmacol., 106 (1992) 139-142. [27] Matsuoka, N., Maeda, N., Yamazaki, M., Ohkubo, Y. and Yamaguchi, I., Effect of FR121196, a novel cognitive enhancer, on the memory impairment of rats in passive avoidance and radial maze tasks, J. Pharmacol. Exp. Ther., 263 (1992) 436-444. [28] Mesulam, M-M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. [29] Mitchell, E.A. and Pratt, J.A., Neuroanatomical structures involved in the action of the 5-HT 3 antagonist ondansetron: a 2-deoxyglucose autoradiographic study in the rat, Brain Res., 538 (1991) 289-294.
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[30] Moore, R.Y., Halaris, A.E. and Jones, B.E., Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol., 180 (1978) 417-438. [31] Nahorski, S.R. and Willcocks, A.R., Interaction of 3-adrenoceptor antagonists with 5-hydroxytryptamine receptor binding in rat cerebral cortex, Br. J. Pharmacol., 78 (1983) 107-111. (Abstract) [32] Nilsson, O.G., Leanza, G. and Bj6rklund, A., Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis, Brain Res., 584 (1992) 132-140. [33] Ohue, T., Koshimura, K., Akiyama, Y., Ito, A., Kido, T., Takagi, Y. and Miwa, S., Regulation of acetylcholine release in vivo from rat hippocampus by monoamines as revealed by novel column-switching HPLC with electrochemical detection, Brain Res., 572 (1992) 340-344. [34] Richardson, B.P., Engel, G., Donatsch, P. and Stadler, P.A., Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs, Nature, 316 (1985) 126-131. [35] Richter-Levin, G. and Segal, M., Effects of serotonin releasers on dentate granule cell excitability in the rat, Exp. Brain Res., 82 (1990) 199-207. [36] Richter-Levin, G. and Segal, M., Grafting of midbrain neurons into the hippocampus restores serotonergic modulation of hippocampal activity in the rat, Brain Res., 521 (1990) 1-6. [37] Robinson, S.E., Malthe-S0renssen, D. and Wood, P.L., Dopaminergic control of the septo-hippocampal cholinergic pathway, J. Pharmacol. Exp. Ther., 208 (1979) 476-479. [38] Segal, M., Serotonin attenuates a slow inhibitory postsynaptic potential in rat hippocampal neurons, Neuroscience, 36 (1990) 631-641. [39] Stoessl, A.J., Dourish, C.T. and Iversen, S.D., Pharmacological characterization of the behavioural syndrome induced by the NK-3 tachykinin agonist senktide in rodents: evidence for mediation by endogenous 5-HT, Brain Res., 517 (1990) 111-116. [40] Szele, F.G., Murphy, D.L. and Garrick, N.A., Effects of fenfluramine, m-chlorophenylpiperazine, and other serotonin-related agonists and antagonists on penile erections in nonhuman primates, Life Sci., 43 (1988) 1297-1303. [41] Vanderwolf, C.H., Cerebral activity and behavior, Int. ReL,. Neurobiol., 30 (1988) 225-240. [42] Wenk, H., Bigl, V. and Meyer, U., Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Re~., 2 (1980) 295-316.
Brain Research 652 (1994) 181-189
Research report
Possible involvement of the septo-hippocampal cholinergic and raphe-hippocampal serotonergic activations in the penile erection induced by fenfluramine in rats Noriaki Maeda, Nobuya Matsuoka *, Isamu Yamaguchi Basic Research Group, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3, Tokodai, Tsukuba, Ibaraki, 300-26, Japan Accepted 12 April 1994
Abstract
Fenfluramine (0.1-10 mg/kg, i.p.) induced penile erection in naive rats with a bell-shaped dose response curve. The response to fenfluramine (1 mg/kg) was antagonized by scopolamine (0.032-1 mg/kg) but not by methyl-scopolamine (0.032-1 mg/kg) pre-treatment. The septo-hippocampal cholinergic deafferentations by medial septum lesion or fimbria-fornix transection also attenuated the penile erection induced by fenfluramine, whereas dopaminergic blockade by sulpiride (3.2-100 mg/kg) had hardly any effect. Pindolol (0.1-3.2 mg/kg), a 5-HT 1 antagonist, and ICS205-930, a 5-HT3 antagonist, but not ketanserin, a 5-HT 2 antagonist, inhibited the penile erection induced by fenfluramine. Depletion of 5-HT not only by systemic injections of p-chlorophenylalanine (150 mg/kg i.p. at 72, 48 and 24 h before the test) but also by the injections of 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, into the median- and dorsal-raphe nuclei significantly attenuated the fenfluramine-induced penile erections. Neurochemical analyses revealed that the raphe-lesion significantly reduced the contents of serotonin (5-HT) and its major metabolite, 5-hydroxyindole acetic acid (5-HIAA) but not choline acetyltransferase (CHAT) activities in all the cortical and subcortical regions examined. The results suggest that fenfluramine facilitates the expression of penile erection in rats through an activation of the septo-hippocampal cholinergic pathway as a consequence of excitation of the raphe-hippocampal serotonergic pathway in which 5-HTI and/or 5-HT 3 receptors appear to play a regulatory role. Key words: Fenfluramine; Penile erection; Cholinergic; Serotonergic; Dopaminergic; Hippocampus; Medial septum; Fimbria-fornix; Raphe nucleus
I. Introduction
Electrical stimulation of various brain areas has demonstrated that the hippocampus has a crucial role in expressing penile erection [24]. The brain area is densely innervated by cholinergic nerve fibers derived from cells in the medial septum [28,42]. In addition, the median and dorsal midbrain raphe nuclei project serotonergic nerve terminals directly to the hippocampus [2,19,30]. These evidence favor the view that interactions between cholinergic and serotonergic nerves may underlie the mechanism of penile erection. Serotonergic stimulants reportedly induce penile erection in rats [3,5] and in primates [40], and the
* Corresponding author. Fax: (81) 298-47-8610. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 4 6 5 - 0
effect is attenuated by drugs with 5-HT l antagonist activity such as propranolol or pindolol [5]. The 5-HT l receptor is now divided into several subtypes, and Berendsen et al. suggested that central 5-HTIc receptors play a crucial role in the expression of the penile erections [6]. Interestingly, Baraldi et al. reported that fenfluramine, a serotonergic stimulant, induced penile erection in a manner antagonizable by cholinergic antagonists [3]. On the other hand, both direct and indirect dopaminergic stimulants also induce penile erection in rats [4,16]. We have recently demonstrated that penile erections induced by apomorphine and amantadine were almost completely canceled by surgical deafferentations of the septo-hippocampal cholinergic pathway, which, however, had little effect on those induced by pilocarpine [25]. It is thus speculated that the septo-hippocampal cholinergic pathway modulated
N. Maeda et al./Brain Research 652 (1994) 181-189
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by the central dopaminergic neurons might play an important role in the expression of penile erection. In the present paper, we examined the effects of neuronal antagonist pre-treatment and surgical deafferentations of the hippocampal serotonergic and cholinergic systems on penile erection induced by fenfluramine in rats. The possible role of an interplay between the limbic cholinergic, serotonergic and dopaminergic pathways in penile erections is discussed.
2. Materials and methods 2.1. Animals Male Fischer 344 rats at the age of 9 weeks were purchased from Charles River Inc. (Atsugi, Japan) at least 1 week before experiments. All animals were housed 6 to a stainless mesh cage (28 x 38 × 17 cm) in a temperature controlled room (22+ I°C) under a 12:12 h light/dark cycle with lights on at 8:00, and given food and water ad libitum. Experiments were carried out between 13:30 and 19:00 h in the room where animals were housed.
Medial-septum lesion
,,,,,;r-,
AP 0.8
2. 2. Behal'ioral procedures All animals were handled 3 rain a day for 3 successive days before the behavioral tests. The rats were tested in groups of six and various doses of fenfluramine or each antagonist were given in semi-randomized order. Immediately after the drug injection, each rat was placed in a perspex box ( 2 5 × 2 5 × 3 5 cm) and its behavior was observed for 60 min, during which time the number of penile erections was counted. A mirror was situated behind each box to facilitate observation of the animal. A penile erection was defined as repeated pelvic thrusts immediately followed by an upright position presenting an emerging, engorged penis which the rat proceeds to lick. Antagonists were injected 15 rain before the behavioral observation. Lesioned and sham-operated rats were assigned to receive all doses throughout a series of test sessions. Briefly, each group of six rats was administered with vehicle or doses of a drug in the first session. In the next session, each rat received a dose of the drug in ascending order, and those which had received the largest dose were given the vehicle. In this way, the test sessions were repeated the same number of times as the total number of vehicle and drug doses. At least 3 days intervened between each session, and a series of tests was finished within 4 weeks at the longest. In the experiment with 5-HT depletion by p-chlorophenylalanine (PCPA), the animals were treated with PCPA 150 m g / k g i.p. 72, 48
Fimbria-fornix lesion
t
P -2.0
Raphe lesion Median raphe
~
'
Dorsal raphe
AP -8.3
AP -8.7
Fig. 1. Schematic drawings of the position of medial septum (MS), fimbria-fornix (FF), median-raphe and dorsal raphe lesions in a coronal section approximately at the level of 0.8 mm anterior, 2.0 mm posterior, 8.3 mm posterior and 8.7 mm posterior from bregma, respectively, according to the atlas of K6nig and Klippel. The stippled areas indicate the representative extent and placement of each lesion.
N. Maeda et al. /Brain Research 652 (1994) 181-189 and 24 h before the test. A parallel group was treated three times with the vehicle.
2.3. Surgical procedures The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and fixed on a stereotaxic apparatus with the incisor bar set 2.3 mm below the intra-aural line. In all lesions, stereotaxic coordinates were positioned according to the atlas of K6nig and Klippel [20]. In each lesion, the body temperature of rats was maintained at 37°C until they recovered from anesthesia after the operations. At the end of the experiments the placement and size of the lesions were checked by histological examination on 10 # m thick slices subjected to Nissl staining. The location and size of MS, FF or raphe lesion are shown schematically in Fig. i.
2.3.1. Medial-septum (MS) or fimbria-fornix (FF) lesion The procedures for lesioning of the septo-hippocampal pathway used in the present study were similar to those used in the previous study [25]. In brief, for MS lesion, an electrode (0.7 mm in diameter and insulated except for the top 0.2 mm) was placed at 0.8 mm anterior from bregma on the midline and 7.0 mm ventral from the surface of the skull. A radiofrequency current was delivered from a lesion generator (Radionics, Burlington, USA: model RFG-4A) for 60 s through the electrode. The temperature was set at 60°C. Sham operation was done in the same way except delivery of the radiofrequency current. For FF lesion, a micro-surgery knife (Feather, Japan: No.715) mounted on a stereotaxic apparatus was positioned at 4.0 mm lateral to the midline, 2.0 mm posterior to bregma and lowered 5.0 mm below the surface of the skull. The FF was transected bilaterally by driving the microknife 8.0 mm laterally and retrieved at this position. For the sham lesions, only the sagittal sinus was transected. The behavioral test was started 7 days after each operation.
23,2. Dorsal- and median-rapke lesion Specific lesions of serotonergic neurons were made by stereotaxically placed local injections of 5,7-dihydroxytryptamine (5,7-DHT; in 1 tzl of 0.1% ascorbic acid-saline solution; 0.2 /zl/min) through a 26-gauge cannula connected by polyethylene tubing to a gear-driven Hamilton syringe. Sham animals were injected with the vehicle. Injections into the median raphe nucleus were made at the coordinates of 8.3 mm posterior to bregma, on the midline, 8.0 mm ventral from the skull surface; injections into the dorsal raphe nucleus were made at the coordinates of 8.7 mm posterior to bregma, on the midline, 6.3 mm ventral from the skull surface. Both nuclei were injected with 2 /xg of 5,7-DHT in a volume of 1 /xl and the rats were pretreated with desipramine (25 mg/kg, i.p.) 30-45 min prior to the local injections in order to protect noradrenergic neurons from the lesion [8j. The behavioral test was started 14 days after the operation.
2.4. Neurochernical analyses Brain regional monoamine contents and the choline acetyltransferase (CHAT) activity were measured in order to verify the effect of raphe lesion on brain neurochemistry. Two weeks after the lesioning, rats were decapitated and the whole brain and spinal cord were removed, and the brain was rapidly dissected into the following regions on ice: the hippocampus, the striatum, anterior part of the cortex including prefrontal cortex and posterior part of the cortex including parietal, occipital and temporal cortex. The tissue was stored at - 8 0 ° C until assay.
183
2. 4.1. Monoamine contents Levels of 5-HT, 5-HIAA, NE, DA and DOPAC were measured in the stored tissues of each of the rat using a reverse-phase highperformance liquid chromatography (HPLC) method with electrochemical detector. Briefly, brain tissues were homogenized in 0.2 M perchloric acid (PCA) containing isoproterenol (10 n g / m l ) as a internal standard, and centrifuged at 20,000× g for 15 min at 4°C. The pH of the supernatant was adjusted to 3.0 with 1 M CH3COONa, and 50 ml of sample was injected into a Eicompak MA-5 ODS liquid chromatography system (Eicom Co.; Kyoto, Japan). Amines were detected using an Eicom ECD-100 electrochemical detector (Eicom Co.) with a glossy carbon electrode held at + 750 mV vs. an A g / A g C I reference. The flow rate was maintained at l ml/min. The mobile phase consisted of 0.1 M acetate-citrate buffer (pH 3.5) containing 1 mM sodium octane sulfonate as ion pair reagent, 0.01 mM EDTA and 17% methanol. The detection limit was approximately 5 pg for each standard amine. Standard sample of each amine was run at the concentration of 10 ng/ml. 2.4.2. ChAT actit,ity To characterize the changes in the integrity of brain cholinergic neurons, the ChAT activity was determined in the brain areas of raphe lesioned rats. The activity was measured by the method of Fonnum [12], using [14C]acetyl coenzyme A (New England Nuclear), and expressed as nmol A C h / h / m g protein. Proteins were measured by the method of Lowry [23]. 2.5. Drugs The following drugs were used: (+)-fenfluramine hydrochloride (synthesized in our research laboratories); sulpiride hydrochloride (synthesized in our research laboratories); (+_)-pindolol hydrochloride (Sigma Chemical Co., St. Louis, MO, USA); ketanserin tartrate (Research Biochemicals Inc., Natick, MA, USA); ICS205-930 ((3tropanyl)-lH-indole-3-carboxylicacid,ester HCI, synthesized in our research laboratories); scopolamine hydrobromide (Nacalai tesque, Kyoto, Japan); methyl-scopolamine hydrobromide (Nacalai tesque); (_+)-metoprolol (Sigma); 5,7-dihydroxytryptamine creatinine sulfate (5,7-DHT, Sigma); dl-p-chlorophenylalanine methyl ester hydrochloride (PCPA, Sigma). Pindolol and sulpiride were dissolved in 1% tartrate-saline with the pH adjusted to 6.0-7.0. 5,7-DHT was dissolved in sterile saline containing 0.2% ascorbic acid immediately before use and injected into the raphe nuclei. All other drugs were dissolved in physiological saline and given intraperitoneally in a volume of 1 ml/kg. Control animals were injected with an equivalent volume of vehicle. Antagonists and agonists were injected 15 min and just before the start of the behavioral experiments, respectively. The dosages of antagonists were selected based on our previous observations [25].
2.6. Statistics All results were expressed as the mean+_ S.E.M.. In the behavioral experiments, the statistical significance was estimated with Mann-Whitney U-test. In the comparison of brain neurochemistry between the lesioned and sham rats, Student's t-test. P values less than 0.05 were used as the criterion for statistical significance.
3. Results
3.1. Effects of fenfluramine on penile erection in rats Fig. 2 shows the number of penile erections in rats after i.p. injection of fenfluramine. Fenfluramine (0.1-
N. Maeda et al. /Brain Research 652 (1994) 181-189
184 7
(n=ll)
Table 1 Effects of dopaminergic or /3-adrenergic antagonists on the penile erection induced by fenfluramine
**•
6 ""
5
}
4
**
"/ 2
°
~"
11
1
-
Antagonist dose ( m g / k g , i.p.)
Fenfluramine (1 m g / k g / , i.p.)
n
Penile e r e c t i o n s / h
Saline Saline Sulpiride
Saline Fenfluramine 1 1 1 1 1
6 6 6 6 6 6
0.50_+0.22 ** 6.00_+0.58 5.33 _+0.42 6.00_+0.93 7.33 _+ 1.63 4.00_+0.82
Saline Fenfluramine 1 1 1 I 1
6 6 6 6 6 6
0.67_+0.33 ** 4.50_+ 0.34 4.17_+0.48 4.83 _+0.70 5.17_+0.48 4.17_+0.31
0 0
0.1
0.32
1
3.2
10
Fenfluramine (mg/kg) Fig. 2. Effect of fenfluramine on penile erection in rats. Each ordinate represents the mean n u m b e r of penile erections in 1 h following i.p. administration of fenfluramine. ** P < 0.01, *** P < 0.001; statistically significant compared with saline-treated group (by M a n n - W h i t n e y U-test). Each column and bars represents the mean + S.E.M. The n u m b e r of rats tested is given in parenthesis.
10 mg/kg) increased the number of penile erection with a bell-shaped dose-response curve with statistically significance (P < 0.01 by Mann-Whitney U-test), with the maximum increase above spontaneous values being observed at 1 mg/kg of the drug. Based on these effects, fenfluramine 1 mg/kg was employed in parts of the following studies. No other signs of abnormal behavior except penile erection were observed in rats dosed with fenfluramine up to 3.2 mg/kg, however, the highest dose (10 mg/kg) of the drug caused a persistent backwards movement which lasted throughout behavioral observation.
Saline Saline Metoprolol 0.32 1 3.2 10
Antagonists were administered i.p. 15 min before administration of fenfluramine. Each value represents the mean number of penile erections of 6 rats observed in 1 h+_S.E.M. * * P < 0.01; statistically significant compared with saline-fenfluramine treated group (by M a n n - W h i t n e y U-test).
3.2. Effects of cholinergic antagonists on fenfluramineinduced penile erection Fig. 3 summarizes the effects of scopolamine and methyl-scopolamine on penile erections induced by fenfluramine (1 mg/kg). Scopolamine (0.032-1 mg/kg) dose-dependently reduced the number of penile erections induced by fenfluramine (1 mg/kg). Statistically significant changes (P < 0.05) were observed in groups subjected to higher dosages than 0.32 mg/kg of scopolamine, as shown in Fig. 3A. On the contrary, methylscopolamine (0.032-1 mg/kg), a peripheral acting
B. Methyl-scopolamine
A. Scopolamine 7
(n=12)
8ill119In6 I "~- 7
5
~6
-~4
*
3
***
~4
~
1
0
3.2 10 32 100
3
2 1 0
0
0 0.032 0.1 0.32
1
Scopolamine (mg/kg) F e n f l u r a m i n e (1 m g / k g )
0
0 0.032 0.1 0.32
1
Methyl-scopolamine (mg/kg) F e n f l u r a m i n e (1 m g / k g )
Fig. 3. Effects of (A) scopolamine or (B) methyl-scopolamine on the penile erections induced by fenfluramine. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine (1 mg/kg). Each antagonist was administered 15 min prior to behavioral observations. * P < 0.05, * * P < 0.01, * * * P < 0.001; statistically significant compared with control (saline-pretreated) groups treated with fenfluramine alone (by M a n n - W h i t n e y U-test). Each column and bar represents the mean _+ S.E.M. The number of rats tested is given in parentheses.
N. Maeda et al. /Brain Research 652 (1994) 181-189 B. F i m b r i a - f o r n i x l e s i o n
A.Medial septum lesion # 9 t-.
8
"~
7
185
##
(n=6)
8
(n=6)
['---] Sham Lesion
~1
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t.,
5 4
4
3
~a
2
•~ 2
3
1
0
0
0
0.1 0.32 3.2 Fenfluramine (mg/kg)
0
0.1 0.32 1 3.2 Fenfluramine (mg/kg)
Fig. 4. Effects of medial-septum lesion (A) or fimbria-fornix lesion (B) on the penile erections induced by fenfluramine. Each neurosurgery was performed as described in the Materials and methods. Seven days after surgery, behavioral tests were started and repeated as each rat experienced all dosages of the drug with at least 3 days intervals. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine. * P < 0.05, ** P < 0.01; statistically significant compared with vehicle control in sham animals. * P < 0.05, • * P < 0.01; compared with vehicle control group in lesioned animals. ~ P < 0.05, ## P < 0.01; compared with each corresponding dosed group in sham animals (by M a n n - W h i t n e y U-test). Each column and bar represents the mean _+ S.E.M. The number of rats tested is given in parentheses.
muscarinic antagonist, had little effect on the responses induced by fenfluramine (Fig. 3B). Furthermore, pretreatment of sulpiride (3.2-100 mg/kg), a dopamine D 2 blocker, hardly affected the responses induced by fenfluramine as shown in Table 1.
3.3. Effects of septo-hippocampal cholinergic denervation on penile erection by fenfluramine The effects of fenfluramine on penile erection in medial septum-lesioned rats are shown in Fig. 4A. The overall dose-response in the sham operated rats did not differ from that in the above-mentioned intact rats, i.e. statistically significant ( P < 0.01) responses occurred at doses of 0.32 and 1 m g / k g and the maximum response of about 7 penile e r e c t i o n s / h was observed at 1 mg/kg. The penile erections induced by the drug were fewer in the medial septum-lesioned rats than in
A. Pindolol
3.4. Effects of serotonergic receptor antagonists on fenfluramine-induced penile erection The effects of serotonergic receptor antagonists on the penile erections produced by fenfluramine were investigated (Fig. 5 and Table 1). Pindolol (0.1-3.2 mg/kg), a 5-HT 1 and /3-adrenoceptor antagonist [31], dose-dependently and significantly abolished the penile
B. Ketanserin
.c 5
C. ICS205-930
.o
2 3 "~ 2 0
the sham rats, and the difference between the two was statistically significant ( P < 0.05) at the dose of 1 mg/kg. Also in the fimbria fornix-lesioned rats, the maximum response at 1 m g / k g of fenfluramine was significantly ( P < 0.01) reduced compared with that in the sham rats (Fig. 4B), and the change was more pronounced than in the medial septum-lesioned rats (Fig. 4A).
**
0
0
0.1 0.32
1
3.2
Pindolol ( m g / k g ) Fenfluramine (1 m g / k g )
.o
~ .~
** 0
0
'~ 2
0
0.1 0.32
1
3.2
0
**
0
0
0.01 0.1
1
10
Ketanserin ( m g / k g )
1CS205-930 ( m g / k g )
Fenflurarnine (1 m g / k g )
Fenfluramine (1 m g / k g )
Fig. 5. Effects of (A) pindolol, (B) ketanserin or (C) ICS205-930 on the penile erections induced by fenfluramine. Each ordinate represents the mean number of penile erections in 1 h following i.p. administration of fenfluramine (1 mg/kg). Each 5-HT antagonist was administered 15 min prior to behavioral observations. * P < 0.05, ** P < 0.01; statistically significant compared with control (saline-pretreated) groups treated with fenfluramine alone (by M a n n - W h i t n e y U-test). Each column and bar represents the mean + S.E.M. The number of rats tested is given in parentheses.
N. Maeda et al. /Brain Research 652 (1994) 181-189
186
A. R a p h e l e s i o n
B. P C P A - t r e a t m e n t
## 9
8 ~
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(n=6)
['-'-'3 Sham Raphe lesion
7
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8 7 6 5 4 3
[""7 Saline PCPA
#
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1 o
0
0.1 0.32
3.2
0
10
Fenfluramine (mg/kg)
0.1 0.32
3.2
10
Fenfluramine (mg/kg)
Fig. 6. Effects of central 5-HT d e p l e t i o n by (A) r a p h e lesion and (B) P C P A p r e - t r e a t m e n t on the penile e r e c t i o n s i n d u c e d by fenfluramine. Medial- and d o r s a l - r a p h e lesioning was p e r f o r m e d as d e s c r i b e d in the M a t e r i a l s and m e t h o d s . B e h a v i o r a l tests were s t a r t e d 14 days after surgery, and r e p e a t e d as each rat e x p e r i e n c e d all dosages of the drug with at least 3 days intervals. P C P A (150 m g / k g ) was a d m i n i s t e r e d i.p. 72, 48 and 24 h before the test. E a c h o r d i n a t e r e p r e s e n t s the m e a n n u m b e r of penile e r e c t i o n s in 1 h following i.p. a d m i n i s t r a t i o n of fenfluramine. * P < 0.05, • * P < 0.01, *** P < 0.001; statistically significant c o m p a r e d with vehicle control g r o u p in s h a m animals. * P < 0.05, ** P < 0.01; c o m p a r e d with vehicle control g r o u p in r a p h e - l e s i o n e d animals. # P < 0.05, ## P < 0.01, ### P < 0.001; c o m p a r e d with each c o r r e s p o n d i n g d o s e d g r o u p in sham a n i m a l s (by M a n n - W h i t n e y U-test). E a c h c o l u m n and b a r r e p r e s e n t s the m e a n 4- S . E M . The n u m b e r of rats t e s t e d is given in p a r e n t h e s e s .
erections induced by 1 m g / k g of fenfluramine (Fig. 5A), whereas metoprolol, a /3-adrenoceptor-selective antagonist without 5-HT antagonistic activity, failed to affect the response (Table 1). Ketanserin, a 5-HT 2 antagonist, also failed to affect the response (Fig. 5B). On the other hand, ICS205-930 (0.01-10 mg/kg), a specific 5-HT 3 antagonist [34], dose-dependently attenuated the response by fenfluramine, as shown in Fig. 5C.
(P < 0.05) and 1 m g / k g (P < 0.01) of fenfluramine gave statistically significant responses in the sham rats. In the raphe lesioned rats, significant erections were still observed at the dose of 1 m g / k g of the drug, however, the maximum response was significantly (P < 0.01) attenuated compared to that in the sham-operated rats. Pretreatment with 150 m g / k g of PCPA hardly affected spontaneous penile erection (drug 0 in Fig. 6B), but markedly reduced the penile erection produced by fenfluramine, and the changes were statistically significant ( P < 0 . 0 5 ) at 0.1, 1 and 3.2 m g / k g of fenfluramine, as shown in Fig. 6B.
3.5. Effects of 5-HT depletion by lesioning of the raphe nuclei or systemic PCPA administration on fenfluramine-induced penile erection
3.6. Brain neurochemistry
Fig. 6 shows the effects of 5-HT depletion by raphe lesioning or systemic treatment of PCPA on penile erection induced by fenfluramine. The doses of 0.32
Brain regional monoamines content after medianand dorsal-raphe lesioning are presented in Table 2.
Table 2 C h a n g e s in m o n o a m i n e s c o n t e n t s in the brain a r e a s of r a p h e - l e s i o n e d rats Brain region
Monoamine contents (ng/mg protein) NE
DOPAC
Hippocampus Sham Lesion
11.65 4- 0.23 10.56 + 0.38 *
0.413 + 0.040 0.390 + 0.061
Cortex anter. Sham Lesion
8.64 _+ 1.89 10.73 + 0.38
4.240 _+ 1.060 4.370 _+ 0.412
C o r t e x poster. Sham Lesion
7.570 + 1.26 6.700 + 1.38
Striatum Sham Lesion
7.590 + 0.57 7.090 + 0.34
0.957 4- 0.204 0.711 + 0.122
24.05 24.75
4- 0.712 + 3.630
DA 1.580 _+ 0.508 1.690 _+ 0.629
25.96 22.43
_+ 6.670 _+ 4.630
6.450 4- 1.060 4.800 4- 0.929
230.6 226.2
4- 9.36 4- 21.09
5-HIAA
5-HT
11.85 3.74
13.19 _+ 0.67 3.53 _+ 1.45 ***
4- 0.81 + 1.10 ***
10.10 _+ 2.25 4.74 4- 1.27
8.640 4- 1.48 2.670 4- 0.57 **
20.50 10.11
4- 1.36 + 1.34 ***
13.37 _+ 2.98 5.26 _+ 1.76 *
11.12 + 2.28 3.17 4- 0.78 *
14.47_+ 0.84 6.61 _+ 1.10 ***
E a c h value r e p r e s e n t s m e a n + S.E.M.. Brain m o n o a m i n e s c o n t e n t s w e r e d e t e r m i n e d 2 w e e k s following r a p h e lesion. Six a n i m a l s w e r e e m p l o y e d in each group. * P < 0.05, ** P < 0.01, *** P < 0.001; statistically significant c o m p a r e d to c o r r e s p o n d i n g region of s h a m a n i m a l s by S t u d e n t ' s t-test.
N. Maeda et al. /Brain Research 652 (1994) 181-189
The lesion significantly reduced the 5-HT and 5-HIAA levels in all the cortical and subcortical brain areas but not in the spinal cord (data not presented). The most striking effect was observed in the hippocampus where 5-HT and 5-HIAA levels were decreased by 73 and 68% of those of the sham rats, respectively. Other catecholamines and their metabolites in the brain areas were hardly changed in the lesioned rats compared with the sham control. The ChAT activity in the brain regions was also determined in raphe lesioned rats. However, no differences in the activities were observed in the areas between two groups as follows: Sham 48.5 + 1.0, raphe lesion 48.3 + 0.9 in the hippocampus; sham 41.3 + 0.5, raphe lesion 41.9 + 0.9 in the anterior cortex; sham 43.9 + 0.5, raphe lesion 44.9 _+0.3 in the posterior cortex; sham 106.5 + 4.6, raphe lesion 104.9 + 2.8 in the striatum (nmol ACh/h/mg protein, n = 6 in each group).
4. Discussion
Confirming and extending the previous finding by Baraldi et al. [3], we observed that scopolamine but not methyl-scopolamine attenuated the penile erection induced by fenfluramine. In addition, the fenfluramineinduced response was attenuated in rats subjected to MS or FF lesioning. It has been widely accepted that the cells in the medial septum (MS) and diagonal band of Broca (DB) send cholinergic nerve fibers to the hippocampus through the fimbria-fornix (FF) [28], and their activities play an important role in the regulation of penile erection [24]. These results favor the view t h a t . a n activation of septo-hippocampal cholinergic nerves is responsible for the penile erection by fenfluramine. However, we have previously shown that the FF lesion almost completely depleted hippocampal ChAT activity [27], and that 1 mg/kg of scopolamine completely abolished the penile erection induced by pilocarpine and apomorphine [25]. In the present study, however, neither FF lesioning nor scopolamine completely abolished the fenfluramine-induced penile erection. Other mechanisms in addition to septo-hippocampal cholinergic activation may also be involved in the response by fenfluramine. The present study demonstrated that pindolol, a /3-adrenoceptor antagonist with 5-HT 1 blocking activity [31], but not metoprolol, a /3-adrenoceptor antagonist without 5-HT 1 blocking activity [17], dose-dependently attenuated the penile erection induced by fenfluramine. Furthermore, it is a novel finding that ICS205930, a 5-HT 3 antagonist [34], but not ketanserin, a 5-HT 2 antagonist [22] attenuated the fenfluramine-induced penile erection. These results in combination with the observations that fenfluramine enhances 5-HT
187
release and reduces 5-HT re-uptake [9,14,15] suggest that an activation of 5-HT~ and 5-HT 3 receptors is involved in the fenfluramine-induced penile erection. While it has been speculated that central 5-HT~B a n d / o r 5-HT~c receptors activities play a crucial role in sexual behavior [5,6], the present result is the first evidence implicating that 5-HT 3 receptors mediate the expression of penile erection. The growing body of evidence indicates that not only 5-HT~ but also 5-HT 3 receptors are densely distributed within the limbic areas including the amygdala, cerebral cortex and hippocampus [18,29]. Furthermore, it is interesting to note that stimulation of the 5-HT~ receptors enhances ACh release in the hippocampus [32,33]. An activation of intra-hippocampal 5-HT 3 receptors are also postulated to enhance ACh release via the enhancement of 5-HT transmission [26]. We speculate that fenfluramine activates the central 5-HT 1 and 5-HT 3 receptors to cause ACh release which in turn leads to penile erection. However, as mentioned above, scopolamine as well as septo-hippocampal cholinergic deafferentations induced incomplete attenuation of the fenfluramine-induced penile erection, while the largest dose (3.2 mg/kg) of pindolol almost completely abolished it. These results prefer the view that an activation of the 5-HT receptors by fenfluramine causes penile erection through mechanisms with and without cholinergic mediation. Another important finding of the present study is that PCPA-treatment as well as 5,7-DHT injections in the raphe nuclei significantly attenuated the fenfluramine-induced penile erection. The dose regime of PCPA-treatment reportedly depletes brain 5-HT levels by more than 90% [39], whereas the present neurochemical analysis revealed that the lesioning of the raphe nuclei by 5,7-DHT injection specifically depleted 5-HT and its major metabolite 5-HIAA in the brain. The latter observation coincides with the previous findings that serotonergic nerve terminals derived from the median- and dorsal-raphe nuclei are widely distributed in the brain areas such as the cerebral cortex, septum and hippocampus [2,19]. It has been widely accepted that the raphe-hippocampal serotonergic pathway exerts a powerful infuence on the neuronal activity of the hippocampus [1,36,41]. In addition, recent electrophysiological studies have revealed that the septo-hippocampal pathway is negatively regulated by hippocampal GABA-ergic interneurons [10], and that the raphe-hippocampal serotonergic pathway modulates the septo-hippocampal neuronal excitability by disinhibiting these inhibitory interneurons [13,38]. These findings together with the above discussion suggest that an activation of the raphe-serotonergic neurons by fenfluramine, at least partly, leads to an activation of septo-hippocampal cholinergic neurons resulting in the expression of penile erection (the possible mechanisms
188
AIO
N. Maeda et al. /Brain Research 652 (1994) 181-189
$~~t~um
/~ Raphe I ~Fenfluramine
Hippocampus Penile erection Fig. 7. Schematic drawing for the possible mechanisms of the limbic cholinergic, serotonergic and dopaminergic link modulating the expression of penile erection in rats.
of the limbic cholinergic a n d s e r o t o n e r g i c link m o d u l a t ing the expression of p e n i l e e r e c t i o n s is p r e s e n t e d in Fig. 7). O u r s p e c u l a t i o n is f u r t h e r s t r e n g t h e n e d by the o b s e r v a t i o n s that f e n f l u r a m i n e i n c r e a s e d the excitability of the rat h i p p o c a m p u s in a m a n n e r a n t a g o n i z a b l e by P C P A p r e - t r e a t m e n t [35], a n d t h a t systemic a d m i n istration of p - c h l o r o a m p h e t a m i n e , a 5 - H T releaser, i n c r e a s e d h i p p o c a m p a l A C h r e l e a s e in naive but not in r a p h e - l e s i o n e d rats [32]. A l t h o u g h M a c L e a n , after a series of e l e c t r o p h y s i o logical studies, c o n c l u d e d that the h i p p o c a m p u s a n d its afferents play a crucial role in the expression o f p e n i l e e r e c t i o n [24], the e f f e r e n t p a t h w a y s are still to be clarified. O n e of the e f f e r e n t s could be the subspinal d e s c e n d i n g s e r o t o n e r g i c system t h a t has b e e n sugg e s t e d to be involved in sexual b e h a v i o r [7]. O u r present o b s e r v a t i o n that slight r e s p o n s e s to f e n f l u r a m i n e still r e m a i n e d in the animals s u b j e c t e d to r a p h e - l e s i o n ing may b e explained, at least in part, by an activation of the b u l b - s p i n a l s e r o t o n e r g i c system by f e n f l u r a m i n e . However, similar r e s p o n s e s w e r e o b s e r v e d in P C P A t r e a t e d rats, which m a y a r g u e against the possibility. It is thus t e m p t i n g to s p e c u l a t e that s p a r e d s e r o t o n e r g i c nerves after r a p h e - l e s i o n i n g a n d P C P A - t r e a t m e n t is r e s p o n s i b l e for the residual responses. It has b e e n r e p o r t e d previously that p e n i l e e r e c t i o n s i n d u c e d by direct and indirect d o p a m i n e r g i c stimulants are also m e d i a t e d by s e p t o - h i p p o c a m p a l cholinergic activation [25]. D o p a m i n e r g i c cell b o d i e s in the ventral t e g m e n t a l a r e a (A10) a n d s u b s t a n t i a nigra (A9) send p r o j e c t i o n s to the r a p h e nuclei which is b e l i e v e d to control s e r o t o n e r g i c n e u r a l activity in the nuclei [11,21]. In addition, t h e r e is e v i d e n c e that d o p a m i n e r g i c neurons in the A 1 0 might play a positive m o d u l a t o r y role in the s e p t o - h i p p o c a m p a l cholinergic p a t h w a y in drug i n d u c e d p e n i l e e r e c t i o n in rats [28,37]. T h e s e findings c o m b i n e d with o u r p r e s e n t ones p r o v i d e a question of interest in t e r m s of the m o d e of i n t e r a c t i o n b e t w e e n the d o p a m i n e r g i c a n d s e r o t o n e r g i c p a t h w a y s m o d u l a t ing p e n i l e erection. T h e p r e s e n t o b s e r v a t i o n that sulpiride failed to b l o c k the p e n i l e e r e c t i o n s by fenflu-
r a m i n e a d d r e s s the issue. T h e s e e v i d e n c e t a k e n tog e t h e r suggest that d o p a m i n e r g i c s t i m u l a t i o n c o m e s down to h i p p o c a m p a l s e r o t o n e r g i c a n d c h o l i n e r g i c activation finally to cause p e n i l e erection. In conclusion, the p r e s e n t results d e m o n s t r a t e the c e n t r a l n e u r a l m e c h a n i s m s u n d e r l y i n g the p e n i l e erection p r o d u c e d by f e n f l u r a m i n e , a n d p r o v i d e novel findings r e g a r d i n g the limbic A C h - 5 - H T i n t e r p l a y which exerts an i m p o r t a n t role in d r u g - i n d u c e d p e n i l e erection in rats.
References [1] Assaf, S.Y. and Miller, J.J., The role of raphe serotonin system in the control for septal unit activity and hippocampal desynchronization, Neuroscience, 3 (1978) 539-550. [2] Azmitia, E.C. and Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. Comp. Neurol., 179 (1978) 641-668. [3] Baraldi, M., Benassi-Benelli, A. and Lolli, M., Penile erections in rats after fenfluramine administration, Ret,. Farmacol. Ther., 8 (1977) 375-379. [4] Benassi-Benelli, A., Ferrani, F. and Pelegrini-Quarantotti, B., Penile erection induced by apomorphine and N-n-propylnorapomorphine in rats, Arch. Int. Pharmacodyn. Ther., 241 (1979) 128-134. [5] Berendsen, H.H.G. and Broekkamp, C.L.E., Drug-induced penile erections in rats: indications of serotonin m receptor mediation, Eur. J. Pharmacol., 135 (1987) 279-287. [6] Berendsen, H.H.G., Jenck, F. and Broekkamp, C.L.E., Involvement of 5-HTlc-receptors in drug-induced penile erections in rats, Psychopharmacology, 101 (1990) 57-61. [7] Bitran, D. and Hull, E.M., Pharmacological analysis of male rat sexual behavior, Neurosci. Biobehav. Rev., 11 (1987)365-389. [8] Bj6rklund, A., Baumgarten, H.G. and Rensch, A., 5,7-Dihydroxytryptamine: Improvement of its selectivity for serotonin neurons in the CNS by pretreatment with desipramine, J. Neurochem., 24 (1975) 833-835. [9] Costa, E., Groppetti, A. and Revuelta, A., Action of fenfluramine on monoamine stores of rat tissues, Br. J. Pharmacol., 41 (1971) 57-64. [10] Curtis, D.R., Felix, D. and McLennan, H., GABA and hippocampal inhibition, Br. J. Pharmacol., 40 (1970) 881-883. [11] Ferre, S. and Artigas, F., Dopamine D 2 receptor-mediated regulation of serotonin extracellular concentration in the dorsal raphe nucleus of freely moving rats, J. Neurochem., 61 (1993) 772-775. [12] Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975)407409. [13] Freund, T.F., Gulyas, A.I., Acsady, L., Gorcs, T. and Toth, K., Serotonergic control of the hippocampus via local inhibitory interneurons, Proc. Natl. Acad. Sci. USA, 87 (1990) 8501-8505. [14] Garattini, S., Buczko, W., Jori, A. and Saminin, R., The mechanism of action of fenfluramine, Postgrad. Med. J., 51 (Suppl. 1) (1975) 27-35. [15] Garattini, S., Mennini, T., Bendotti, C., Invernizzi, R. and Samanin, R., Neurochemical mechanism of action of drugs which modify feeding via the serotonergic system, Appetite, 7 (1986) 15-38. [16] Gower, A.J., Berendsen, H.H.G., Proncen, M.M. and Broekkamp, C.L.E., The yawning-penile erection syndrome as a model for putative dopamine autoreceptor activity, Eur. J. Pharmacol., 103 (1984) 81-89.
N. Maeda et al. /Brain Research 652 (1994) 181-189 [17] Hjorth, S. and Carlsson, A., (-)-Pindolol stereospecifically inhibits rat brain serotonin (5-HT) synthesis, Neuropharmacology, 24 (1985) 1143-1146. [18] Kilpatrick, G.J., Jones, B.J. and Tyers, M.B., Identification and distribution of 5-HT 3 receptors in rat brain using radioligand binding, Nature, 330 (1987) 746-748. [19] K6hler, C. and Steinbusch, H., Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain, Neuroscience, 7 (1982) 951-975. [20] K6nig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. [21] Lee, E.H.Y. and Geyer, M.A., Dopamine autoreceptor mediation of the effects of apomorphine on serotonin neurons, Pharmacol. Biochem. Behat~., 21 (1984)301-311. [22] Leysen, J.E., Awouters, F., Kennis, L., Laduron, P.M., Vandenberk, J. and Janssen, P.A.J., Receptor binding profile of R41 468, a novel antagonist at 5-HT 2 receptors, Life Sci., 28 (1981) 1015-1022. [23] Lowry, O.H., Rosenberg, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [24] MacLean, P.D., Brain mechanisms of primal sexual functions and related behavior. In M. Sandier and G.L. Gessa (Eds.), Sexual BehaL~ior: Pharmacology and Biochemistry, Raven, New York, 1975, pp. 1-11. [25] Maeda, N., Matsuoka, N. and Yamaguchi, I., Septohippocampal cholinergic pathway and penile erections induced by dopaminergic and cholinergic stimulants, Brain Res., 537 (1990) 163-168. [26] Martin, K.F., Hannon, S., Phillips, I. and Heal, D.J., Opposing roles for 5-HT m and 5-HT 3 receptors in the control of 5-HT release in rat hippocampus in vivo, Br. J. Pharmacol., 106 (1992) 139-142. [27] Matsuoka, N., Maeda, N., Yamazaki, M., Ohkubo, Y. and Yamaguchi, I., Effect of FR121196, a novel cognitive enhancer, on the memory impairment of rats in passive avoidance and radial maze tasks, J. Pharmacol. Exp. Ther., 263 (1992) 436-444. [28] Mesulam, M-M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. [29] Mitchell, E.A. and Pratt, J.A., Neuroanatomical structures involved in the action of the 5-HT 3 antagonist ondansetron: a 2-deoxyglucose autoradiographic study in the rat, Brain Res., 538 (1991) 289-294.
189
[30] Moore, R.Y., Halaris, A.E. and Jones, B.E., Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol., 180 (1978) 417-438. [31] Nahorski, S.R. and Willcocks, A.R., Interaction of 3-adrenoceptor antagonists with 5-hydroxytryptamine receptor binding in rat cerebral cortex, Br. J. Pharmacol., 78 (1983) 107-111. (Abstract) [32] Nilsson, O.G., Leanza, G. and Bj6rklund, A., Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis, Brain Res., 584 (1992) 132-140. [33] Ohue, T., Koshimura, K., Akiyama, Y., Ito, A., Kido, T., Takagi, Y. and Miwa, S., Regulation of acetylcholine release in vivo from rat hippocampus by monoamines as revealed by novel column-switching HPLC with electrochemical detection, Brain Res., 572 (1992) 340-344. [34] Richardson, B.P., Engel, G., Donatsch, P. and Stadler, P.A., Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs, Nature, 316 (1985) 126-131. [35] Richter-Levin, G. and Segal, M., Effects of serotonin releasers on dentate granule cell excitability in the rat, Exp. Brain Res., 82 (1990) 199-207. [36] Richter-Levin, G. and Segal, M., Grafting of midbrain neurons into the hippocampus restores serotonergic modulation of hippocampal activity in the rat, Brain Res., 521 (1990) 1-6. [37] Robinson, S.E., Malthe-S0renssen, D. and Wood, P.L., Dopaminergic control of the septo-hippocampal cholinergic pathway, J. Pharmacol. Exp. Ther., 208 (1979) 476-479. [38] Segal, M., Serotonin attenuates a slow inhibitory postsynaptic potential in rat hippocampal neurons, Neuroscience, 36 (1990) 631-641. [39] Stoessl, A.J., Dourish, C.T. and Iversen, S.D., Pharmacological characterization of the behavioural syndrome induced by the NK-3 tachykinin agonist senktide in rodents: evidence for mediation by endogenous 5-HT, Brain Res., 517 (1990) 111-116. [40] Szele, F.G., Murphy, D.L. and Garrick, N.A., Effects of fenfluramine, m-chlorophenylpiperazine, and other serotonin-related agonists and antagonists on penile erections in nonhuman primates, Life Sci., 43 (1988) 1297-1303. [41] Vanderwolf, C.H., Cerebral activity and behavior, Int. ReL,. Neurobiol., 30 (1988) 225-240. [42] Wenk, H., Bigl, V. and Meyer, U., Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Re~., 2 (1980) 295-316.