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Brain somatostatin depletion by cysteamine attenuates the penile erection induced by serotonergic and dopaminergic, but not by cholinergic, activation in rats
BRAIN RESEARCH ELSEVIER
Brain Research 729 (! 996) 132-136
Short communication
Brain somatostatin depletion by cysteamine attenuates the penile erection induced by serotonergic and dopaminergic, but not by cholinergic, activation in rats Nobuya Matsuoka *, Noriaki Maeda, Mayako Yamazaki, Isamu Yamaguchi Basic Research Gl~up, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba. lbaraki, 300-26, J~q~an
Accepted 7 May 1996
Abstract
To clarify the role of brain somatostatin in the expression of penile erection, the effects of cysteamine, a somatostatin depletor, on the penile erection induced by serotonergic, cholinergic and dopaminergic stimulants were investigated in rats. Fenfluramine (0. I-10 mg/kg, i.p.), pilocarpine (0.032-3.2 mg/kg, i.p.) and apomorphine (0.01-1 mg/kg, i.p.) induced penile erection in rats, with bell-shaped dose-response curves. Pretreatment with cysteamine (200 mg/kg, s.c.) significantly attenuated the penile erection induced by fenfluramine and apomorphine, but scarcely affected that induced by pilocarpine. Neurochemical measures revealed that cysteamine pretreatment significantly reduced the somatostatin content in all brain regions examined. These results provide the first pharmacological evidence that the brain somatostatin may play an important role in drug-induced penile erection. Keywords: Penile erection; Somatostatin; Cysteamine; Cholinergic; Serotonergic; Dopaminergic
The hippocampus and its afferents have been demonstrated to have a crucial part in the expression of penile erection in rats [14]. This brain area is densely innervated by cholinergic nerve fibers derived from cells in the medial septum [21], leading to the view that the cholinergic nerve activity in the hippocampus functions in the expression of penile erection [15]. On the other hand, the hippocampal formation also receives dense serotonergic projections from the midbrain raphe nuclei [22]. Our previous studies and those of others showed that serotonergic stimulants such as serotonin (5-HT) re-uptake inhibitors or receptor agonists induce penile erection in rats [1,5,17], suggesting that an activation of the central serotonergic systems is involved. Dopaminergic stimulants such as apomorphine also induce penile erection in male rats [3,11,15]. We have previously investigated the effects of hippocampal lesions on the penile erection induced by fenfluramine, pilocarpine and apomorphine in rats, and found that the hippocampus is one of the important anatomical loci necessary for the penile erection by serotonergic, cholinergic, and dopaminergic activation and the
* Corresponding author. Fax: + 81 (298) 47-1536.
septo-hippocampal cholinergic system exerts a powerful modulatory role in d o w n s t r e a m to the serotonergic or dopaminergic mechanisms in the expression of penile erection [15-17]. Somatostatin, tetradecapeptide, is currently believed to be an important neuromodulator in the central nervous system for several reasons [9]. Somatostatin is highly concentrated in the cerebral cortex, amygdala and hippocampus [12], and several lines of evidence have confirmed that somatostatin affects the neuronal activity in these brain regions [8]. We have already shown that the role of hippocampal somatostatin is crucial to the mnemonic process; that is, somatostatin directly activates the synaptic function in the hippocampus [18] and causes an enhancement of memory function [19]. Findings from our lab taken together with those of others suggested that modulation of the septo-hippocampal cholinergic pathway is involved in the physiological action of somatostatin in the hippocampus. Cysteamine (2-mercaptoethylamine; NH2-CH2-CH2-SH) has been found to provoke a marked specific decrease of endogenous brain somatostatin [26,27]. Furthermore, cysteamine-induced depletion of somatostatin has been shown to affect physiological processes mediated by somatostatin (e.g. mnemonic responses [20] or ther-
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006- 8993(96)005 60-4
N. Matsuoka et al. / Brain Research 729 (1996) 132-136
moregulation [6]). Therefore, the present study was undertaken to investigate the functional status of somatostatin as an endogenous substance modulating penile erection. We examined the effects of central somatostatin depletion by cysteamine on drug-induced penile erection in rats and discussed a possible interplay between somatostatin and the limbic serotonergic, cholinergic, and dopaminergic links in relation to penile erection. Male Fischer-344 rats, aged 9 weeks, were purchased from Charles River (Atsugi, Japan) at least one week before the experiments. All animals were housed 6 to a stainless mesh cage (28 × 38 × 17 cm) in a temperaturecontrolled room (22 _+ I°C) under a 12:12 light/dark cycle with lights on at 8:00 h, 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. All animals were handled 3 rain a day for three successive days before the behavioral tests. The rats were tested in groups of 5 - 6 ; various doses of fenfluramine, apomorphine and pilocarpine were given in semi-randomized order. Immediately alter the drug injection, each rat was placed in a Perspex box (25 × 25 × 35 cm). 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 obserwltion of the animal. A penile erection was defined as repeated pelvic thrusts immediately followed by an upright position presenting an emerging, engorged penis, that the rat proceeded to lick. The frequency of penile erections was near zero in the rats who did not receive any injections. Cysteamine hydrochloride was injected 2 h before the behavioral observation. For neurochemical measurement, the changes in the brain contents of somatostatin, catecholamines, and choline acetyltransferase (CHAT) activities were determined 2 h
A. F e n f l u r a m i n e 8
(n=5)
after the injection of cysteamine (200 m g / k g , s.c.). Each rat was killed and the brain was rapidly removed and dissected on ice into the following regions: hippocampus, striatum, anterior part of the cortex including the prefrontal cortex, and posterior part of the cortex including the parietal, occipital and temporal cortex. The tissue was stored at - 8 0 ° C until assay. Tissue contents of somatostatin were determined by radioimmunoassay. Extracts were made by dropping frozen tissue samples into boiling 2 M acetic acid for 5 min. Samples were homogenized after cooling. Alter centrifugation at 27 000 × g for 15 min at 4°C, an aliquot of the supernatant was lyophilized. The specimens were then reconstituted in 1 ml of 1% BSAborate buffer. Immunoreactive somatostatin was determined using a commercial radioimmunoassay kit (INCSTAR, MA, USA: Catalog no. 20100). Dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography with an electrochemical detector (HPLC-ECD, Eicom, Kyoto, Japan). The tissues were homogenized in 0.2 M PCA and 100 p,M EDTA (2Na) at 0.5 m l / 1 0 0 mg tissue, and the homogenates were centrifuged at 20000 × g for 15 rain in 0°C. To adjust the pH, 1 ml of supernatant was vortexed with 400 p~l of 1 M CH3COONa. Fifty pJ of sample was injected into a HPLC-ECD for assays of DA, 5-HT and metabolites. Isoproterenol (10 m g / m l ) was used as an internal standard. A M A - 5 0 D S column (Eicompak, 4.6 × 150 ram: Eicom) was used. The mobile phase consisted of 0.1 M citric acid-sodium acetate containing 5 mg/1 EDTA (2Na), 100 m g / l sodium octyl sulfate and 15% methanol which was filtered and degassed prior to use. The flow rate was 1 m l / m i n . Monoamines were detected using ~n ECD-100
B. Pilocarpine
##
4
7
6 ¢._o 5 "5 4
133
C. A p o m o r p h i n e 4
(n=6)
~t-3
t-
.o_ "5
O
.
~2
uJ 3
(n=6)
3 2
IJJ
d3
"E 1
,'- 2 a_ 1
Q.
~1 0
0.1 0.32
3.2
~
n
0 10
0 0.032 0.1 0.32 1
3.2
Pilocarpine (mg/kg)
Fenfluramine (mg/kg) I
I Saline
~
0
0 0.010.0320.1 0.32 1 Apomorphine (mg/kg)
Cysteamine (200 mg/kg)
Fig. I. Effects of cysteaminc pre-treatment on penile erections induced by fenfluramine (A), pilocarpine (B) or apomorphine (C). Each column and bar
represent the mean _+S.E.M. Each ordinate represents the mean number of penile erections during 1 h following i.p. administration of each drug. * P < 0.05, * ' P < 0.01; statistically significant compared with vehicle control in saline-treated animals. * P < 0.05, ** P < 0.01: compared with vehicle control group in the cysteamine-treated animals. ##P < 0.01; compared with each corresponding dosed group in saline-treated animals (by Mann-Whitney U-test). The number of rats tested is given in parentheses. Cysteamine (200 mg/kg, s.c.) was administered 2 h prior to the behavioral observations.
134
N. Matsuoka et al. / Brain Research 729 (19961 132-136
detector (Eicom) with a glossy carbon electrode held at + 750 mV vs. an A g / A g C 1 reference. The detection limit was approximately 5 pg for each standard amine. Standard samples of each amine were run at a concentration of 10 n g / m l . Brain ChAT activities were measured by the method of F o n n u m [10], using [HC]acetyl coenzyme A (New England Nuclear, Boston, MA), and expressed as nmol A C h / h / m g protein. In each experiment, protein was measured by the method of Lowry et al. [13]. The drugs used were cysteamine hydrochloride (2mercaptoethylamine; Sigma Chemical, St. Louis, MO), ( + ) - f e n f l u r a m i n e hydrochloride (synthesized in our research laboratories), pilocarpine hydrochloride (Sigma), and apomorphine hydrochloride (Sigma). Cysteamine was dissolved in physiological saline and the pH was adjusted to 7.0 with 1 N NaOH. It was administered s.c. in a volume of 2 m l / k g . All other drugs were dissolved in physiological saline and given intraperitoneally in a volume of 1 m l / k g . Control animals were injected with the equivalent volumes of saline. All results were expressed as the mean _+ S.E.M. In the behavioral experiments, the statistical significance was estimated with M a n n - W h i t n e y U-test by considering a P-value less than 0.05 as statistically significant. Statistical significance of neurochemical changes was calculated by Student's t-test (two-tailed) and compared with those of the corresponding control animal groups. The effects of cysteamine pre-treatment on drug-induced penile erection are shown in Fig. 1. Fenfluramine ( 0 . 1 - 1 0 m g / k g ) increased the number of penile erections with a bell-shaped dose-response curve, with statistically significant ( P < 0.05) responses at doses of 0.32 and 1 m g / k g and maximum response at 1 m g / k g . The number of penile erections induced by the drug were much fewer in the cysteamine-treated rats compared with the control rats, and the difference was statistically significant ( P <
0.01) at the dose of 1 m g / k g (Fig. 1A). On the other hand, pilocarpine (0.032-3.2 m g / k g ) facilitated the penile erection with a bell-shaped dose-response in rats with or without cysteamine pre-treatment. Apomorphine (0.01-1 m g / k g ) increased the number of penile erections with a bell-shaped dose-response curve, and the effect was statistically significant ( P < 0.05) at 0.032-0.1 m g / k g of drug in the control rats, but the drug failed to affect the response in cysteamine-treated rats (Fig. I C). The neurochemical effects of cysteamine treatment are presented in Table 1. Cysteamine (200 m g / k g ) led to the depletion of somatostatin from all brain regions of the treated rats when determined 2 h after administration. These changes were statistically significant ( P < 0.01) when compared with the value in each corresponding brain region of the saline-treated rats. DA levels were significantly higher in the hippocampus and lower in the striatum of the cysteamine (200 m g / k g , s.c.)-treated rats compared with the controls. Similarly, cysteamine treatment significantly increased DOPAC levels in the hippocampus and the cortex, and tended to increase those in the striatum but hardly changed 5-HT levels in the brain areas of either cysteamine-treated rats or the control, although 5-HIAA levels were slightly but significantly higher in the former. Cysteamine treatment hardly affected ChAT activities in these brain regions. Cysteamine rapidly and selectively depletes the endogenous somatostatin contents in different organs [26,27]. Cysteamine action in the brain is known to be neuropeptide specific, and this specificity of cysteamine in decreasing the brain somatostatin has been demonstrated by the lack of effect of this treatment on the contents of other neuropeptides such as vasoactive intestinal peptide, enkephalin, cholecystokinin, vasopressin, substance P [24] or neuropeptide Y [7], although this agent at high concentrations is also an inhibitor of dopamine-13-hydroxylase
Table I Changes in the somatostatin, monoaminescontents and ChAT activities in the brain areas of cysteamine-and saline-treatedrats Brain region Somatostincontent
Monoamine content (ng/mg protein)
ChAT activity
(ng/mg protein)
(n)
DOPAC
DA
5-HIAA
5-HT
(n)
(nmolACh/ b/mg protein)
(n)
7.75 _+0.68 4.44 ± 0.34 * *
(6) (6)
0.082± 0.018 0.393± 0.024 * * *
0.86 ± 0.14 1.49 ± 0.15 *
5.1)5 ± 0.69 6.78 ± 0.26
9.99 ± 1.32 I 1.2 ± 0.39
(5) (7)
46.7_+0.4 47.6± 1.0
(5) (5)
5.46 ± 0.42 3.83 ± 0.27 * " *
(6) (6)
2.97± 0.19 3.78_+0.39 * * ~
24.9 ± 2.24 22.6 _+2.88
4.23 _+0.09 5.09 ± 0.26
13.4 ± 0.84 12.9 ±//.66
(6) (6)
62.3_+ 1.2 59.2± 0.8
(5) (5)
4.64 ± 0.24 3.25 ± 0.18 * ~
(6) (6)
0.49± 0.05 (/.70± 0.04 * * *
7.92 ± 0.55 7.87 ± 0.67
4.76 _+0.09 5.19 ± 0.17 .....
10.9 _+0.12 I 1.4 ± 0.64
(5) (7)
47.7_+0.8 46.7+ (1.5
(5) (5)
8.17+0.80 3.79_+0.43 * *
(6) (6)
16.3 ± 0 . 6 3 17.3 ± 0 . 6 6
150.3±5.04 129.7±5.37 ***
9.04+_0.3I 10.5+0.65 * **
11.9_+0.62 (6) 11.8_+ 1.15 (7)
117.1_+3.4 117.1+2.8
(5) (5)
Hippocampus
Saline Cysteamine Cortex anterior
Saline Cysteamine
Cortex posterior
Saline Cysteamine Striatum
Saline Cysteamine
Brain somatostatin and monoaminescontents and ChAT activity were determined 2 h alter administrationof cysteamine(200 mg/kg, s.c.). Each value represents the mean ±S.E.M. Number of rats is shown in parentheses. * P < 0.05, * * P < 0.01, ~ * * P < 0.001, Statisticallysignificantcompared to corresponding region of control by Student's t-test.
N. Matsuoka et al. / Brain Research 729 (19961 132 136
[28]. Confirming these findings, the present study showed a marked reduction in the somatostatin levels in all brain regions in the rats receiving cysteamine, and these rats showed slight increases in DA and its metabolite D O P A C in some brain areas. The present study confirmed previous observations [ 1 3,1 1,15-17], that fenfluramine, pilocarpine and apomorphine induced penile erection in naive rats. Extending these findings, however, the present studies showed that cysteamine significantly and totally prevented penile erection induced by fenfluramine, suggesting strongly that endogenous somatostatin plays a role in the expression of penile erection produced by this drug. This finding provides, for the first time, pharmacological evidence that somatostatin may be involved in penile erection produced by serotonergic activation. We previously demonstrated that penile erection induced by fenfluramine was dose-dependently attenuated not only by 5-HT1 antagonists, but also by scopolamine [17]. Also, the lesioning of the septohippocampal cholinergic pathways significantly attenuated the penile erection induced by fenfluramine, suggesting that fenfluramine indirectly activates the septo-hippocampal cholinergic neurons to induce penile erection [17]. On the other hand, several lines of evidence suggest that 5-HT may interact with somatostatinergic neurons in the brain; e.g. there is evidence that 5-HT stimulates release of somatostatin in the hippocampus and frontal cortex [4]. Taken together with these findings, the present results indicate that intra-hippocampal somatostatin might be activated by raphe-hippocampal serotonergic pathways by fenfluramine resulting in cholinergic activation in the hippocampus to elicit penile erection. Failure of cysteamine to block the penile erection by pilocarpine may rule out the involvement of somatostatin in the cholinergically-mediated penile erection. Because we have previously shown that the penile erection by pilocarpine was attenuated by cholinergic blockade by scopolamine but not by the septo-hippocampal lesions [15], it is likely that pilocarpine could produce the response as a consequence of the direct stimulation of post-synaptic muscarinic cholinergic receptors existing in the hippocampus. The lack of the changes in brain ChAT activities by cysteamine treatment would not contradict this view. Another important finding in the present study was that cysteamine markedly attenuated the penile erection induced by apomorphine, suggesting the involvement of somatostatin in the penile erection produced by dopaminergic activation. In vivo microdialysis studies by Nilsson et al. revealed that systemic administration of apomorphine increases hippocampal ACh release that is specifically blocked by a 6-hydroxydopamine lesion of the ventral tegmental area (AI0) [23], thus suggesting that the septohippocampal cholinergic pathway is under control of the dopaminergic nerve fibers derived from the cell bodies in the A I0 [25]. Confirming this fact, we previously have shown that septo-hippocampal lesions canceled apomor-
135
phine-induced penile erection. Bi-directional communications between central dopaminergic and somatostatinergic systems have been suggested, and there is a finding that an activation of cortical DA receptors enhances somatostatin secretion in vitro [4]. Given these findings, it would be reasonable to assume that intra-hippocampal somatostatin might be activated by A10-septal dopaminergic pathways resulting in cholinergic activation in the hippocampus. In conclusion, the present studies have shown that brain somatostatin depletion by cysteamine significantly attenuated the penile erection induced by fenfluramine or apomorphine without affecting that by pilocarpine, suggesting that endogenous somatostatin plays an important role in the expression of penile erection produced by fenfluramine and apomorphine. These findings provide the first pharmacological evidence, to our knowledge, that somatostatin-associated mechanisms, possibly in the: hippocampus, may be involved in the penile erection induced by serotonergic or dopaminergic stimulation.
Acknowledgements We thank Dr. Thomas G. Aigner (National Institute on Drug Abuse, MD, USA) for his helpful comments on the manuscript.
References [1] Baraldi, M., Benassi-Benetli, A. and Lolli, M.. Penile erections m rats after fenfluramine administration, Ri;. Farmacol. Ther., 8 (1977) 375-379. [2] Baraldi, M. and Bertolini, A., Penile erections induced by amantadine in male rats, Lifo Sci., 14 (19741 1231-1235. [3] Benassi-Benelli, A., Ferrani, F. and Pelegrini-Quarantotti, B., Penile erection induced by apomorphine and N-n-propyl-norapomorphine in rats, Arch. Int. Pharmaco&'n. Ther., 241 ( 19791 128-134. [4] Bennett. G.W., Edwardson, J.A., Marcano de Cotte. D., Berelowilz. M., Pimstone, B.L. and Kronheim. S., Release of somatostatin from rat brain synaptosomes, J. Neurochem.. 32 ( 19791 1127 1130. [5] Berendsen, H.H.G., Jenck, F. and Broekkamp, C.L.E., lnw)lvemem of 5-HT~c-receptors in drug-induced penile erections in rats. Psvchopharmacolo. W, 101 (1990) 57 61. [6] Brown, M., Fisher, L., Mason, R.T.. Rivier, ,I. and Vale, W.. Neurobiological actions of cysteamine. Fed. Proc., 44 (1985) 25562560. [7] Chattha, G.K. and Beal, M.F., Effect of cysteamine on somatostatin and neuropeptide Y in rat striatum and cortical synaptosomes, Brain Res., 401 (1987) 359 364. [8] Dodd, J. and Kelly, J.S., Is somatostatin an excitatory transmiuer in the hippocampus?Nature, 273 (1978) 674-675. [9] Epelbaum, J.+ Somatostatin in the central nervous system: physiology and pathological modifications, t'rov. Neurot:.iol., 27 (1986) 63-1 (10. [10] Fonnum, F., A rapid radiochemical method for the determination of choline aeetyltransferase, J. Neupz~chem.. 24 (1975) 407 409. [11] 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 activit,x, Eur..l. Pharmacol., 103 (19841 81-89.
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[12] Johansson, O., H6kfelt, T. and Elde, R.P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat, Neuroscience, 13 (1984) 265-339. [13] Lowry, O.H., Rosenherg, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [14] MacLean, P.D., Brain mechanisms of primal sexual functions and related behavior. In M. Sandler and G.L. Gessa (Eds.), Sexual Behaeior: Pharmacology and Biochemistry, Raven Press, New York, 1975, pp. 1-11. [15] 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. [16] Maeda, N., Matsuoka, N. and Yamaguchi, I., Role of dopaminergic, serotonergic and cholinergic link in the expression of penile erection in rats, Jpn. J. Pharmacol., 66 (1994) 59-66. [17] Maeda, N., Matsuoka, N. and Yamaguchi, I., Possible involvement of the septo-hippocampal cholinergic and raphe-hippocampal serotonergic activations in the penile erection induced by fenfluramine in rats, Brain Res., 652 (1994) 181-189. [18] Matsuoka, N., Kaneko, S. and Satoh, M., Somatostatin augments long-term potentiation of the mossy fiber-CA3 system in guinea-pig hippocampal slices, Brain Res., 553 (1991) 188-194. [19] Matsuoka, N., Maeda, N., Yamaguchi, I. and Satoh, M., Possible involvement of brain somatostatin in the memory formation of rats and the cognitive enhancing action of FR121196 in passive avoidance task, Brain Res,, 642 (1994) 11-19. [20] Matsuoka, N., Yamazaki, M. and Yamaguchi, I., Changes in brain
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somatostatin in memory deficient rats: comparison with cholinergic markers, Neuroscience, 66 (1995) 617-626. 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. Moore, R.Y., Halaris, A.E. and Jones, B.E., Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol., 18[] (1978) 417-438. Nilsson, O.G., Leanza, G. and BjiSrklund, A., Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis, Brain Res., 584 (1992) 132-140. Palkovits, M., Brownstein, M.J., Eiden, L.E., Beinfeld, M.C., Arimura, A. and Szabo, S., Selective depletion of somatostatin in rat brain by cysteamine, Brain Res., 240 (1982) 178-180. Robinson, S.E., Malthe-Sorenssen, D. and Wood, P.L., Dopaminergic control of the septo-hippocampal cholinergic pathway, J. Pharmacol. Exp. Ther., 208 (1979) 476-479. Sagar, S.M., Landry, D., Millard, W.J., Badger, T.M., Arnold, M.A. and Martin, J.B., Depletion of somatostatin-like immunoreactivity in the rat central nervous system by cysteamine, J. Neurosci., 2 (1982) 225-231. Szabo, S. and Reichlin, S., Somatostatin in rat tissue is depleted by cysteamine administration, Endocrinology, 109 (1981) 2255-2257. Terry, L.C. and Craig, R., Cysteamine effects on monoamines, dopamine-beta-hydroxylase and the hypothalamic-pituitary axis, Neuroendocrinology, 41 (1985) 467-475.
Brain Research 729 (! 996) 132-136
Short communication
Brain somatostatin depletion by cysteamine attenuates the penile erection induced by serotonergic and dopaminergic, but not by cholinergic, activation in rats Nobuya Matsuoka *, Noriaki Maeda, Mayako Yamazaki, Isamu Yamaguchi Basic Research Gl~up, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba. lbaraki, 300-26, J~q~an
Accepted 7 May 1996
Abstract
To clarify the role of brain somatostatin in the expression of penile erection, the effects of cysteamine, a somatostatin depletor, on the penile erection induced by serotonergic, cholinergic and dopaminergic stimulants were investigated in rats. Fenfluramine (0. I-10 mg/kg, i.p.), pilocarpine (0.032-3.2 mg/kg, i.p.) and apomorphine (0.01-1 mg/kg, i.p.) induced penile erection in rats, with bell-shaped dose-response curves. Pretreatment with cysteamine (200 mg/kg, s.c.) significantly attenuated the penile erection induced by fenfluramine and apomorphine, but scarcely affected that induced by pilocarpine. Neurochemical measures revealed that cysteamine pretreatment significantly reduced the somatostatin content in all brain regions examined. These results provide the first pharmacological evidence that the brain somatostatin may play an important role in drug-induced penile erection. Keywords: Penile erection; Somatostatin; Cysteamine; Cholinergic; Serotonergic; Dopaminergic
The hippocampus and its afferents have been demonstrated to have a crucial part in the expression of penile erection in rats [14]. This brain area is densely innervated by cholinergic nerve fibers derived from cells in the medial septum [21], leading to the view that the cholinergic nerve activity in the hippocampus functions in the expression of penile erection [15]. On the other hand, the hippocampal formation also receives dense serotonergic projections from the midbrain raphe nuclei [22]. Our previous studies and those of others showed that serotonergic stimulants such as serotonin (5-HT) re-uptake inhibitors or receptor agonists induce penile erection in rats [1,5,17], suggesting that an activation of the central serotonergic systems is involved. Dopaminergic stimulants such as apomorphine also induce penile erection in male rats [3,11,15]. We have previously investigated the effects of hippocampal lesions on the penile erection induced by fenfluramine, pilocarpine and apomorphine in rats, and found that the hippocampus is one of the important anatomical loci necessary for the penile erection by serotonergic, cholinergic, and dopaminergic activation and the
* Corresponding author. Fax: + 81 (298) 47-1536.
septo-hippocampal cholinergic system exerts a powerful modulatory role in d o w n s t r e a m to the serotonergic or dopaminergic mechanisms in the expression of penile erection [15-17]. Somatostatin, tetradecapeptide, is currently believed to be an important neuromodulator in the central nervous system for several reasons [9]. Somatostatin is highly concentrated in the cerebral cortex, amygdala and hippocampus [12], and several lines of evidence have confirmed that somatostatin affects the neuronal activity in these brain regions [8]. We have already shown that the role of hippocampal somatostatin is crucial to the mnemonic process; that is, somatostatin directly activates the synaptic function in the hippocampus [18] and causes an enhancement of memory function [19]. Findings from our lab taken together with those of others suggested that modulation of the septo-hippocampal cholinergic pathway is involved in the physiological action of somatostatin in the hippocampus. Cysteamine (2-mercaptoethylamine; NH2-CH2-CH2-SH) has been found to provoke a marked specific decrease of endogenous brain somatostatin [26,27]. Furthermore, cysteamine-induced depletion of somatostatin has been shown to affect physiological processes mediated by somatostatin (e.g. mnemonic responses [20] or ther-
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006- 8993(96)005 60-4
N. Matsuoka et al. / Brain Research 729 (1996) 132-136
moregulation [6]). Therefore, the present study was undertaken to investigate the functional status of somatostatin as an endogenous substance modulating penile erection. We examined the effects of central somatostatin depletion by cysteamine on drug-induced penile erection in rats and discussed a possible interplay between somatostatin and the limbic serotonergic, cholinergic, and dopaminergic links in relation to penile erection. Male Fischer-344 rats, aged 9 weeks, were purchased from Charles River (Atsugi, Japan) at least one week before the experiments. All animals were housed 6 to a stainless mesh cage (28 × 38 × 17 cm) in a temperaturecontrolled room (22 _+ I°C) under a 12:12 light/dark cycle with lights on at 8:00 h, 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. All animals were handled 3 rain a day for three successive days before the behavioral tests. The rats were tested in groups of 5 - 6 ; various doses of fenfluramine, apomorphine and pilocarpine were given in semi-randomized order. Immediately alter the drug injection, each rat was placed in a Perspex box (25 × 25 × 35 cm). 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 obserwltion of the animal. A penile erection was defined as repeated pelvic thrusts immediately followed by an upright position presenting an emerging, engorged penis, that the rat proceeded to lick. The frequency of penile erections was near zero in the rats who did not receive any injections. Cysteamine hydrochloride was injected 2 h before the behavioral observation. For neurochemical measurement, the changes in the brain contents of somatostatin, catecholamines, and choline acetyltransferase (CHAT) activities were determined 2 h
A. F e n f l u r a m i n e 8
(n=5)
after the injection of cysteamine (200 m g / k g , s.c.). Each rat was killed and the brain was rapidly removed and dissected on ice into the following regions: hippocampus, striatum, anterior part of the cortex including the prefrontal cortex, and posterior part of the cortex including the parietal, occipital and temporal cortex. The tissue was stored at - 8 0 ° C until assay. Tissue contents of somatostatin were determined by radioimmunoassay. Extracts were made by dropping frozen tissue samples into boiling 2 M acetic acid for 5 min. Samples were homogenized after cooling. Alter centrifugation at 27 000 × g for 15 min at 4°C, an aliquot of the supernatant was lyophilized. The specimens were then reconstituted in 1 ml of 1% BSAborate buffer. Immunoreactive somatostatin was determined using a commercial radioimmunoassay kit (INCSTAR, MA, USA: Catalog no. 20100). Dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography with an electrochemical detector (HPLC-ECD, Eicom, Kyoto, Japan). The tissues were homogenized in 0.2 M PCA and 100 p,M EDTA (2Na) at 0.5 m l / 1 0 0 mg tissue, and the homogenates were centrifuged at 20000 × g for 15 rain in 0°C. To adjust the pH, 1 ml of supernatant was vortexed with 400 p~l of 1 M CH3COONa. Fifty pJ of sample was injected into a HPLC-ECD for assays of DA, 5-HT and metabolites. Isoproterenol (10 m g / m l ) was used as an internal standard. A M A - 5 0 D S column (Eicompak, 4.6 × 150 ram: Eicom) was used. The mobile phase consisted of 0.1 M citric acid-sodium acetate containing 5 mg/1 EDTA (2Na), 100 m g / l sodium octyl sulfate and 15% methanol which was filtered and degassed prior to use. The flow rate was 1 m l / m i n . Monoamines were detected using ~n ECD-100
B. Pilocarpine
##
4
7
6 ¢._o 5 "5 4
133
C. A p o m o r p h i n e 4
(n=6)
~t-3
t-
.o_ "5
O
.
~2
uJ 3
(n=6)
3 2
IJJ
d3
"E 1
,'- 2 a_ 1
Q.
~1 0
0.1 0.32
3.2
~
n
0 10
0 0.032 0.1 0.32 1
3.2
Pilocarpine (mg/kg)
Fenfluramine (mg/kg) I
I Saline
~
0
0 0.010.0320.1 0.32 1 Apomorphine (mg/kg)
Cysteamine (200 mg/kg)
Fig. I. Effects of cysteaminc pre-treatment on penile erections induced by fenfluramine (A), pilocarpine (B) or apomorphine (C). Each column and bar
represent the mean _+S.E.M. Each ordinate represents the mean number of penile erections during 1 h following i.p. administration of each drug. * P < 0.05, * ' P < 0.01; statistically significant compared with vehicle control in saline-treated animals. * P < 0.05, ** P < 0.01: compared with vehicle control group in the cysteamine-treated animals. ##P < 0.01; compared with each corresponding dosed group in saline-treated animals (by Mann-Whitney U-test). The number of rats tested is given in parentheses. Cysteamine (200 mg/kg, s.c.) was administered 2 h prior to the behavioral observations.
134
N. Matsuoka et al. / Brain Research 729 (19961 132-136
detector (Eicom) with a glossy carbon electrode held at + 750 mV vs. an A g / A g C 1 reference. The detection limit was approximately 5 pg for each standard amine. Standard samples of each amine were run at a concentration of 10 n g / m l . Brain ChAT activities were measured by the method of F o n n u m [10], using [HC]acetyl coenzyme A (New England Nuclear, Boston, MA), and expressed as nmol A C h / h / m g protein. In each experiment, protein was measured by the method of Lowry et al. [13]. The drugs used were cysteamine hydrochloride (2mercaptoethylamine; Sigma Chemical, St. Louis, MO), ( + ) - f e n f l u r a m i n e hydrochloride (synthesized in our research laboratories), pilocarpine hydrochloride (Sigma), and apomorphine hydrochloride (Sigma). Cysteamine was dissolved in physiological saline and the pH was adjusted to 7.0 with 1 N NaOH. It was administered s.c. in a volume of 2 m l / k g . All other drugs were dissolved in physiological saline and given intraperitoneally in a volume of 1 m l / k g . Control animals were injected with the equivalent volumes of saline. All results were expressed as the mean _+ S.E.M. In the behavioral experiments, the statistical significance was estimated with M a n n - W h i t n e y U-test by considering a P-value less than 0.05 as statistically significant. Statistical significance of neurochemical changes was calculated by Student's t-test (two-tailed) and compared with those of the corresponding control animal groups. The effects of cysteamine pre-treatment on drug-induced penile erection are shown in Fig. 1. Fenfluramine ( 0 . 1 - 1 0 m g / k g ) increased the number of penile erections with a bell-shaped dose-response curve, with statistically significant ( P < 0.05) responses at doses of 0.32 and 1 m g / k g and maximum response at 1 m g / k g . The number of penile erections induced by the drug were much fewer in the cysteamine-treated rats compared with the control rats, and the difference was statistically significant ( P <
0.01) at the dose of 1 m g / k g (Fig. 1A). On the other hand, pilocarpine (0.032-3.2 m g / k g ) facilitated the penile erection with a bell-shaped dose-response in rats with or without cysteamine pre-treatment. Apomorphine (0.01-1 m g / k g ) increased the number of penile erections with a bell-shaped dose-response curve, and the effect was statistically significant ( P < 0.05) at 0.032-0.1 m g / k g of drug in the control rats, but the drug failed to affect the response in cysteamine-treated rats (Fig. I C). The neurochemical effects of cysteamine treatment are presented in Table 1. Cysteamine (200 m g / k g ) led to the depletion of somatostatin from all brain regions of the treated rats when determined 2 h after administration. These changes were statistically significant ( P < 0.01) when compared with the value in each corresponding brain region of the saline-treated rats. DA levels were significantly higher in the hippocampus and lower in the striatum of the cysteamine (200 m g / k g , s.c.)-treated rats compared with the controls. Similarly, cysteamine treatment significantly increased DOPAC levels in the hippocampus and the cortex, and tended to increase those in the striatum but hardly changed 5-HT levels in the brain areas of either cysteamine-treated rats or the control, although 5-HIAA levels were slightly but significantly higher in the former. Cysteamine treatment hardly affected ChAT activities in these brain regions. Cysteamine rapidly and selectively depletes the endogenous somatostatin contents in different organs [26,27]. Cysteamine action in the brain is known to be neuropeptide specific, and this specificity of cysteamine in decreasing the brain somatostatin has been demonstrated by the lack of effect of this treatment on the contents of other neuropeptides such as vasoactive intestinal peptide, enkephalin, cholecystokinin, vasopressin, substance P [24] or neuropeptide Y [7], although this agent at high concentrations is also an inhibitor of dopamine-13-hydroxylase
Table I Changes in the somatostatin, monoaminescontents and ChAT activities in the brain areas of cysteamine-and saline-treatedrats Brain region Somatostincontent
Monoamine content (ng/mg protein)
ChAT activity
(ng/mg protein)
(n)
DOPAC
DA
5-HIAA
5-HT
(n)
(nmolACh/ b/mg protein)
(n)
7.75 _+0.68 4.44 ± 0.34 * *
(6) (6)
0.082± 0.018 0.393± 0.024 * * *
0.86 ± 0.14 1.49 ± 0.15 *
5.1)5 ± 0.69 6.78 ± 0.26
9.99 ± 1.32 I 1.2 ± 0.39
(5) (7)
46.7_+0.4 47.6± 1.0
(5) (5)
5.46 ± 0.42 3.83 ± 0.27 * " *
(6) (6)
2.97± 0.19 3.78_+0.39 * * ~
24.9 ± 2.24 22.6 _+2.88
4.23 _+0.09 5.09 ± 0.26
13.4 ± 0.84 12.9 ±//.66
(6) (6)
62.3_+ 1.2 59.2± 0.8
(5) (5)
4.64 ± 0.24 3.25 ± 0.18 * ~
(6) (6)
0.49± 0.05 (/.70± 0.04 * * *
7.92 ± 0.55 7.87 ± 0.67
4.76 _+0.09 5.19 ± 0.17 .....
10.9 _+0.12 I 1.4 ± 0.64
(5) (7)
47.7_+0.8 46.7+ (1.5
(5) (5)
8.17+0.80 3.79_+0.43 * *
(6) (6)
16.3 ± 0 . 6 3 17.3 ± 0 . 6 6
150.3±5.04 129.7±5.37 ***
9.04+_0.3I 10.5+0.65 * **
11.9_+0.62 (6) 11.8_+ 1.15 (7)
117.1_+3.4 117.1+2.8
(5) (5)
Hippocampus
Saline Cysteamine Cortex anterior
Saline Cysteamine
Cortex posterior
Saline Cysteamine Striatum
Saline Cysteamine
Brain somatostatin and monoaminescontents and ChAT activity were determined 2 h alter administrationof cysteamine(200 mg/kg, s.c.). Each value represents the mean ±S.E.M. Number of rats is shown in parentheses. * P < 0.05, * * P < 0.01, ~ * * P < 0.001, Statisticallysignificantcompared to corresponding region of control by Student's t-test.
N. Matsuoka et al. / Brain Research 729 (19961 132 136
[28]. Confirming these findings, the present study showed a marked reduction in the somatostatin levels in all brain regions in the rats receiving cysteamine, and these rats showed slight increases in DA and its metabolite D O P A C in some brain areas. The present study confirmed previous observations [ 1 3,1 1,15-17], that fenfluramine, pilocarpine and apomorphine induced penile erection in naive rats. Extending these findings, however, the present studies showed that cysteamine significantly and totally prevented penile erection induced by fenfluramine, suggesting strongly that endogenous somatostatin plays a role in the expression of penile erection produced by this drug. This finding provides, for the first time, pharmacological evidence that somatostatin may be involved in penile erection produced by serotonergic activation. We previously demonstrated that penile erection induced by fenfluramine was dose-dependently attenuated not only by 5-HT1 antagonists, but also by scopolamine [17]. Also, the lesioning of the septohippocampal cholinergic pathways significantly attenuated the penile erection induced by fenfluramine, suggesting that fenfluramine indirectly activates the septo-hippocampal cholinergic neurons to induce penile erection [17]. On the other hand, several lines of evidence suggest that 5-HT may interact with somatostatinergic neurons in the brain; e.g. there is evidence that 5-HT stimulates release of somatostatin in the hippocampus and frontal cortex [4]. Taken together with these findings, the present results indicate that intra-hippocampal somatostatin might be activated by raphe-hippocampal serotonergic pathways by fenfluramine resulting in cholinergic activation in the hippocampus to elicit penile erection. Failure of cysteamine to block the penile erection by pilocarpine may rule out the involvement of somatostatin in the cholinergically-mediated penile erection. Because we have previously shown that the penile erection by pilocarpine was attenuated by cholinergic blockade by scopolamine but not by the septo-hippocampal lesions [15], it is likely that pilocarpine could produce the response as a consequence of the direct stimulation of post-synaptic muscarinic cholinergic receptors existing in the hippocampus. The lack of the changes in brain ChAT activities by cysteamine treatment would not contradict this view. Another important finding in the present study was that cysteamine markedly attenuated the penile erection induced by apomorphine, suggesting the involvement of somatostatin in the penile erection produced by dopaminergic activation. In vivo microdialysis studies by Nilsson et al. revealed that systemic administration of apomorphine increases hippocampal ACh release that is specifically blocked by a 6-hydroxydopamine lesion of the ventral tegmental area (AI0) [23], thus suggesting that the septohippocampal cholinergic pathway is under control of the dopaminergic nerve fibers derived from the cell bodies in the A I0 [25]. Confirming this fact, we previously have shown that septo-hippocampal lesions canceled apomor-
135
phine-induced penile erection. Bi-directional communications between central dopaminergic and somatostatinergic systems have been suggested, and there is a finding that an activation of cortical DA receptors enhances somatostatin secretion in vitro [4]. Given these findings, it would be reasonable to assume that intra-hippocampal somatostatin might be activated by A10-septal dopaminergic pathways resulting in cholinergic activation in the hippocampus. In conclusion, the present studies have shown that brain somatostatin depletion by cysteamine significantly attenuated the penile erection induced by fenfluramine or apomorphine without affecting that by pilocarpine, suggesting that endogenous somatostatin plays an important role in the expression of penile erection produced by fenfluramine and apomorphine. These findings provide the first pharmacological evidence, to our knowledge, that somatostatin-associated mechanisms, possibly in the: hippocampus, may be involved in the penile erection induced by serotonergic or dopaminergic stimulation.
Acknowledgements We thank Dr. Thomas G. Aigner (National Institute on Drug Abuse, MD, USA) for his helpful comments on the manuscript.
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