European Journal of Pharmacology, 216 (1992) 385-392
385
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52496
Influence of stress and antidepressant treatment on 5-HT-stimulated phosphoinositide hydrolysis in rat brain T o m i k o K a w a n a m i a, S h i g e r u M o r i n o b u a, S h i r o h T o t s u k a a a n d M a s a o E n d o h b a Department of Neuro-Psychiatry and b Department of Pharmacology, School of Medicine, Yamagata University, lida-Nishi-2-2-2, Yamagata 990-23, Japan Received 3 February 1992, revised MS received 17 March 1992, accepted 24 March 1992
The aim was to elucidate the role of 5-hydroxytryptamine (5-HT)-stimulated phosphoinositide (PI) metabolism in stress situations and in the behavioral improvement produced by chronic antidepressant treatment. Rat cerebral cortex slices were used for the purpose. Forced swimming for 15 min and longer induced changes in behavioral activities of rats associated with a significant reduction of 5-HT-stimulated PI metabolism, without any changes in density and affinity of 5-HTz receptors. This suggests that modulation of the receptor coupling process but not of the 5-HT2 receptor binding characteristics may be responsible for the significant reduction of 5-HT-stimulated PI metabolism in stress situations. Chronic antidepressant treatment tended to reduce 5-HT-stimulated PI metabolism. This treatment improved significantly the behavioural activities during forces swimming, and prevented the forced swimming-induced reduction of 5-HT-stimulated PI metabolism. It is postulated that chronic antidepressant treatment may improve behavioral activities in relation to PI metabolism in stress situations. Phosphoinositide hydrolysis; 5-HT2 receptors; Forced swimming test; Mianserin; Imipramine; Affective disorders
I. Introduction
Affective disorders are one of the representative mental disorders, but the cause is unknown. Investigating the cellular a n d / o r subcellular metabolic changes in the central neuronal transduction systems could yield useful information about the mechanism involved in causing affective disorders. While lithium carbonate has been reported to be useful in the treatment of patients with affective disorders, the mechanism involved in this therapeutic effect is still unknown. Since it is known that lithium (Li ÷) inhibits the enzyme involved in one of the intracellular messenger transduction systems, phosphoinositide (PI) metabolism, it has been supposed that PI metabolism may be responsible for the subcellular mechanism of affective disorders (Berridge, 1983; H~illcher and Sherman, 1980). It is recognized that the activation of certain receptor systems is closely linked to the induc-
Correspondence to: T. Kawanami, Department of Neuro-Psychiatry, School of Medicine, Yamagata University, Iida-Nishi-2-2-2, Yamagata 990-23, Japan. Tel. 81.236.33 1122.
tion of PI hydrolysis and increased inositol phosphate (IP) accumulation. It has been shown that, in rat cerebral cortex, the 5-hydroxytryptamine (5-HT-induced IP accumulation may be mediated by 5-HT 2 receptors (Conn and Sanders-Bush, 1984, 1985, 1986; Kendall and Nahorsky, 1985). A number of antidepressants such as mianserin and imipramine as well as lithium carbonate are used in the treatment of patients with affective disorders. Most of these antidepressants inhibit the uptake of 5-HT and facilitate the 5-HT-mediated signal transduction process in the central nervous system (CNS). In the course of treatment of patients with depression, psychiatrists find that patients with affective disorders have often been in the stress situations. Further, it is well known that various stresses induce metabolic changes in neurotransmitters included 5-HT in rat cerebral cortex (Farska et al., 1988; Morgan et al., 1975; Mueller et al., 1976; Oomagari, 1989). These observations imply the involvement of 5-HT in stress situations. We therefore investigated the changes of PI metabolism linked to 5-HT receptors in the CNS in a stress situation and the influence of antidepressants on the stress-induced changes.
386
Rats were forced to swim for 5, 10, 15 and 20 min in a vertical cylinder (height: 45 cm, diameter: 25 cm) containing 25 cm of water maintained at 25°C for 5, 10, 15 and 20 min. A rat was judged to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position, its head just above the surface, according to Porsolt's criteria (Porsolt et al., 1978). A rat was judged to be in the escape-directed behavior whenever it positively tried to jump out of the cylinder, or swim around or dive to find the exit, according to Kitada's criteria (Kitada et al., 1981). The duration of the immobility and the escape directed behavior was measured. When antidepressants were injected, the forced swimming test was performed 18 h after the final injection.
(1984) with minor modifications. The slices were washed extensively and incubated in 250 /zl of KH buffer containing 2 /xCi [3H]myo-inositol and 7.5 mM LiC1 per slice at 37°C for 90 min under the same oxygenation condition. Labeled slices were washed and incubated with the agonist, 5-HT, in a final volume of 270 /zl of KH buffer containing 7.5 mM LiC1 and 10 /zM pargyline. The slices were incubated at 37°C for 45 min under the same oxygenation conditions. The reactions were terminated by adding 1 ml of 5% TCA. The reaction mixtures were centrifuged at 2500 rpm for 10 min and 750 /zl of the upper phase was prepared for assay of [3H]inositol phosphates. The slices were weighed and homogenized with 1 ml of TCA and centrifuged at 2000 rpm for 2 min. An aliquot of 750/xl of the upper phase was removed and added to the previously collected 750 /zl. TCA was removed from the collected supernatant solution by washing three times with three volumes of diethylether. The pellets were used for the measurement of tissue proteins by the method of Lowry et al. (1951), and for the estimation of [3H]myo-inositol incorporation into phospholipids. Samples were added to columns containing 750 #l of Dowex-1 resin (100-200 mesh: ×8, formate form) and phosphate esters were extracted by stepwise addition of formate solutions of increasing strength. The columns were washed through with 10 ml of distilled water to wash out all free inositol Glycerophosphoinositol and inositol 1 : 2 cyclic phosphate were then eluted with 5 mM sodium t e t r a b o r a t e / 6 0 mM sodium formate. Inositol-l-monophosphate (IP 0 was eluted with 200 mM ammonium f o r m a t e / 1 0 0 mM formate, inositol1,4-bisphosphate (IP 2) was eluted with 450 mM ammonium formate/100 mM formate and inositol-l,4,5-trisphosphate (IP 3) with 1 M ammonium f o r m a t e / 1 0 0 mM formate. Samples (8 ml) of the extracts were added to 8 ml of scintilator (ACS-II) and their radioactivity was acounted. All data were expressed as the percentage stimulation over the basal [3H]IP level in the absence of 5-HT.
2.4. Tissue preparation and [3H]IP accumulation
2.5. [ 3H]Ketanserin binding
The rats were decapitated immediately after the forced swimming test, their brains were rapidly removed and cross-chopped slices (350 /zm) were prepared from cerebral cortex (mainly frontal). The slices were preincubated for 60 min in a modified K r e b s Henseleit (KH) buffer (in mM: 118 NaCI, 4.7 KCI, 2.55 CaCI2, 1.18 K H 2 P O 4, 1.18 MgSO 4, 24.88 N a H C O 3, 11.1 glucose), equilibrated with 95% O z - 5 % CO 2 at 37°C (pH 7.4). [3H]Inositol phosphates were assayed by using the methods of Berridge et al. (1983) and Brown et al.
After decapitation, the frontal cortex was dissected and weighed. It was homogenized in 25 volumes of a 50 mM Tris-HC1 buffer (pH 7.4) by using a Polytron PT-10/35. The homogenates were centrifuged for 20 min at 40 000 × g at 4°C. The supernatants were discarded and the pellets were suspended in the same buffer then homogenized and recentrifuged. The final pellets were immediately used as crude membrane preparations for the receptor binding study. Tissue protein was assayed by using the method of Lowry et al. Proteins amounted to ca. 0.3-0.5 mg per tube.
2. Materials and methods
2.1. Drugs The following drugs were kindly provided by the companies indicated: mianserin (Organon) and imipramine (Ciba-Geigy). 5-HT creatinine sulphate, pargyline HC1, serum bovine albumin were purchased from Sigma Chemical Co.; sodium tetraborate, sodium formate, formate, ammonium formate, trichloracetic acid (TCA) and all other chemicals and drugs were from Wako Chemical Co. [3H]Myo-inositol (18.3 C i / m m o l ) was purchased from Amersham Radiolabeled Chemicals and [3H]ketanserin HCI (64.1 C i / m m o l ) from New England Nuclear.
2.2. Animals and treatment Male standard Wistar rats (weighing ca. 220 g) were housed four or five per cage with food and water available ad libituml For the chronic treatment with antidepressants, the rats were injected intraperitoneally (i.p.) with mianserin or imipramine, 10 m g / k g daily for 14 days. A control group of rats was injected with saline.
2.3. Forced swimming test
387
5-HT 2 binding was assayed using the methods of Battaglia et al. (1983) and Bennett and Snyder (1975) with minor modifications. The final volume in each glass assay tube was 1 ml after addition of 0.1 ml of [3H]ketanserin and receptor preparations to the tube already containing 0.8 ml of the 50 mM Tris-HCl buffer (pH 7.4) for total binding and 0.7 ml of the buffer and 0.1 ml of 5-HT (100/zM) for determination of non-specific binding. The reaction mixtures were incubated for 30 min at 25°C. The assay was terminated by rapid filtration using a Brandel automated cell harvester (3 × 3 ml ice-cold Tris-HC1 buffer, pH 7.4, Whatman G / C filter). The filters were transferred to counting vials containing 5 ml of ACS-II and their radioactivity was then counted by means of a liquid scintillation counter.
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Fig. 2. The 5-HT-induced [3HIIPI and [3H]IFs accumulation in rat cerebral cortex slices. Slices were incubated with 10 g,M pargyline and 5-HT in various concentration for 45 rain, The data were expressed as the percentage stimulation over the basal [3H]IP level in the absence of 5-HT. Each point is the mean _+S.E.M. of six separate experiments, which were done in triplicate.
3. Results
3.1. The relation between 5-HT-stimulating time and [ 3H]IP accumulation
2.6. Data analysis The concentration of the agonist that produced half-maximal stimulation (ECs0) was estimated graphically. The significance of differences between groups were tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values. The differences between mean values were considered to be significant when a P value was smaller than 0.05.
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5-HT (10/zM) increased [3H]IP] and [3H]IPs (IP 1 + IP 2 + IP 3) accumulation in a time-dependent manner, reaching a plateau level after 30 rain (fig. 1). The maximal levels of [3H]IP 1 and [3H]IPs were 202 + 21% at 60 min and 202 + 15% at 45 min, respectively. In contrast, the increase in accumulation of [3H]IP2 and [3H]IP3 was transient. [3H]IP 2 accumulation attained a maximum at 10 min (200 + 30%) then decreased with time up to 60 min after the stimulation. The maximal accumulation of [3H]IP3 was achieved at 1 min (200 + 34%) and the level then decreased gradually with time. Subsequent experiments for determination of the concentration-response relation for the accumulation of
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Fig. 3. The influence of forced swimming for various periods on 5-HT-stimulated [3H]IP1 ad [3H]IP s accumulation in rat cerebral cortex slices. Rats were forced to swim for 5, 10, 15 and 20 min, the groups being designated as $5, S10, $15 and $20, respectively. The basal radioactivities of [3H]IP1 accumulation were 920 + 125 dpm/mg protein for control, 9125:102 d p m / m g protein for $5, 9645:175 dpm/mg protein for S10, 930_+128 dpm/mg protein for $15 and 9835:208 d p m / m g protein for $20. The basal radioactivities of [3H]IPs accumulation were 12945:225 dpm/mg protein for control, 12414-196 d p m / m g protein for $5, 1456 5:345 d p m / m g protein for $10, 1426_+269 d p m / m g protein for $15 and 13295:217 dpm/mg protein for $20. The significance of differences between groups was tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values (* P < 0.05, ** P < 0.01).
388 [3H]IP] and [3H]IPs in response to 5-HT were performed at 45 min after the administration of 5-HT.
3.2. The concentration-response relation of [ 3H]IP1 and [3H]IPs accumulations induced by 5-HT 5-HT increased [3H]IP1 and [3H]IPs accumulation in a concentration-dependent manner (fig. 2). [3H]IP1 accumulation reached a plateau at 10 /xM 5-HT. The maximal accumulation of [3H]IP t and [3H]IPs was 188 + 1 3 % (at 100 ~ M ) and 2 0 1 + 2 7 % (at 100 ~M), respectively. The ECs0 values of 5-HT were 0.3 ~ M for [3H]IPt accumulation and 0.47/xM for [3H]IPs accumulation. Subsequent experiments were carried out with 10/xM 5-HT as the agonist, since this concentration was suitable to produce an adequate response and higher concentrations could cause non-specific responses.
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TABLE 1 The influence of forced swimming (15 min) on [3H]ketanserin binding. Rats were formed to swim for 15 rain (designated as S15) and the frontal cortex was removed immediately thereafter for the preparation of crude membranes. T h e data are the m e a n s + S.E.M.
Control S15
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Rats were submitted to forced swimming for 5, 10, 15 and 20 min of swimming time, the groups being designated as $5 (n = 10), S10 (n = 5), S15 (n = 6) and $20 (n = 5), respectively. The changes in 5-HT (10 /.~M)-stimulated [3H]IPI and [3H]IPs accumulation in rat cerebral cortex slices isolated from each group were assessed (fig. 3). [3H]Myo-inositol incorporation into phospholipids was not affected by any of the forced swimming periods (data not shown). The basal levels of [3H]IP1 and [3H]IPs prior to stimulation by 5-HT were not affected by forced swimming as shown in the legend to fig. 3. [3H]IP1 accumulation in the control group not submitted to forced swimming (n = 7) was 187 + 21%. A significant reduction of [3H]IPt accumulation was produced in groups S15 (134 + 7%) and $20 (132 + 8%). While there was a clear tendency to a reduction of [3H]IPI accumulation in groups $5 (173 + 12%) and S10 (164 + 6%), the changes were statistically not significant. Substantially identical changes in [3H]IPs accumulation were induced by forced swimming: IP~ accumulation in the control group was 192 + 19%, a significant reduction being found in S15 (127 + 8%), $20 (130 + 6%) and $5 (157 + 9%). These results indicate that
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3.3. Changes in 5-HT-stimulation [3H]IP accumulation after forced swimming
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$15 $15 S~5 [n=7} (n-5~ {n-4l ,,~0~s,,pc~,l
Fig. 4. Changes in behavioral activities - immobility (IMO) and the escape-directed behavior (EDB) - of rats forced to swim for 5, 10, 15 and 20 min (upper) and the influence of pretreatment with mianserin (MIA) and imipramine (IMP) on the changes in behavioral activities during forced swimming for 15 min (lower). The significance of differences between groups was tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values (* P < 0.05, * * P < 0.01).
forced swimming reduces 5-HT-stimulated [3H]IPs accumulation in a time-dependent manner, and the change appears to reach a steady level in the groups forced to swim 15 min and longer.
3.4. Influence of forced swimming for 15 min on [ 3H]ketanserin binding There were no significant differences in either B m a x or K d values between the control and S15, indicating that forced swimming for 15 min did not affect the density and affinity of 5-HT 2 receptors in the rat cerebral cortex (table 1).
3.5. Behavioral activities of rats submitted to forced swimming The changes in behavioral activities were assessed from the duration of immobility and escape-directed behavior (fig. 4, upper). The total duration of immobility was prolonged in a time-dependent manner: 50 + 16 s in $5, 2 4 2 + 18 s in S10, 3 9 0 + 76 s in S15 and 560 + 118 s in $20. While the total duration of escapedirected behavior showed no clear time-dependent prolongation, there was a significant difference between $5 (172 + 20 s) and S15 (236 + 31 s). These
389
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reduce the 5-HT-stimulated [3H]IP1 accumulation (160 + 19%) and [3H]IPs accumulation (163 + 23%). These findings suggest that the chronic antidepressant pretreatment tended to reduce the 5-HT-stimulated [3H]IPt and [3H]IPs accumulation, but these reductions were not statistically significant.
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3. 7. Influence of the chronic treatment with antidepressants on [3H]IP1 ad [3H]IPs accumulation after 15 min forced swimming ($15)
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Fig. 5. The influence of pretreatments with mianserin (MIA) and imipramine (IMP) on the 5-HT-stimulated [3H]IPI and [3H]IPs accumulation (upper) and on the reduced 5-HT-stimulated [3H]IP1 and [3H]IPs accumulation after forced swimming for 15 min (B). The basal radioactivities of [3H]IP1 accumulation were 893 + 127 d p m / m g protein for control (SAL), 949 + 129 d p m / m g protein for SAL + S15, 1084+213 d p m / m g protein for MIA, 1027_+ 167 d p m / m g protein for MIA+S15, 980_+132 d p m / m g protein for IMP and 975+132 d p m / m g protein for I M P + S15. The basal radioactivities of [3H]IPs accumulation were 1309-+ 246 d p m / m g protein for control (SAL), 1379 + 276 d p m / m g protein for SAL + S15, 1504-+ 311 d p m / m g protein for MIA, 1626+301 d p m / m g protein for MIA+S15, 1617_+334 d p m / m g protein for IMP and 1500_+305 d p m / m g protein for IMP +S15. The significance of differences between groups were tested using a one-way analysis of variance, and differences between individual groups were determined using Student's t-test for unpaired values (* P < 0.05, ** P < 0.01).
results indicate that forced swimming for 15 min induces significant changes in both immobility and escape-directed behavior.
3.6. Influence of chronic treatment with antidepressants on [3HIIP1 and [3H11Ps accumulation Rats were pretreated with mianserin (n = 5), imipramine (n = 5) (10 m g / k g i.p. each) or saline (n = 5) for 14 days, and were decapitated 18 h after the final injection. [3H]Myo-inositol incorporation into phospholipids was not affected by the treatment with either of the agents (data not shown). The basal levels of [3H]IPI and [3H]IP~ prior to stimulation by 5-HT were also not affected by the treatment with the two agents as shown in the legend to fig. 5. Mianserin pretreatment tended to reduce the 5-HT-stimulated [3H]IPI accumulation (152 _ 18%) and the [3H]IP~ accumulation (144 + 8%) (fig. 5, upper). Imipramine pretreatment also tended to
As a significant reduction of 5-HT-stimulated [3H]IP1 and [3H]IPs accumulation was induced by forced swimming for 15 min, it was examined whether the chronic treatment with mianserin and imipramine would modulate the reduction. The rats were pretreated with mianserin (n = 5), imipramine (n = 4) or saline (n = 5) for 14 days, and were forced to swim for 15 min 18 h after the final injection. The 5-HT-stimulated [3H]IP] accumulation in cerebral cortex slices was 148 + 10% in the mianserin-pretreated group and 154 + 17% in the imipramine-pretreated group (fig. 5, lower). While a significant reduction of 5-HT-stimulated [3H]IP1 accumulation was induced by forced swimming in the control group (from 185 + 21 to 135 + 7%), no significant reduction was induced in the mianserin-pretreated group (from 152 + 18 to 148 + 10%) and in the imipramine-pretreated group (from 160 + 19 to 154 + 17%). These observations indicate that the antidepressants used in these experiments antagonized the forced swimming-induced reduction of 5-HTstimulated [3H]IP1 accumulation. The 5-HT-stimulation [3H]IPs accumulation in slices isolated from rats pretreated with mianserin or imipramine was 132 + 12 or 148 + 13%, respectively, and these values were not significantly different from those for the mianserin- or imipramine-pretreated group not submitted to forced swimming. These results suggest that the chronic pretreatment with antidepressants prevents the reduction of 5-HT-stimulated [3H]IPs accumulation in cerebral cortex slices of rats forced to swim for 15 min.
3.8. Influence of the chronic treatment with antidepressants on behavioral activities during forced swimming ($15) Pretreatment with mianserin tended to prolong the total duration of escape-directed behavior (322 + 52 s) and shorten the total duration of immobility (303 + 50 s), but the changes were not significantly different from the respective control values (239 + 28 s for escape-directed behavior and 396 + 71 s for immobility) (fig. 4B). The pretreatment with imipramine significantly prolonged the total duration of escape-directed behavior to 386 + 63 s, and significantly shortened the total
390 duration of immobility to 154 + 54 s. It is apparent from these results that the chronic treatment with either mianserin or imipramine effectively prolonged the escape-directed behavior and shortened immobility, indicating the enhancement of behavioral activities during forced swimming for 15 min.
4. Discussion Lithium, a useful therapeutic agent in the treatment of affective disorders, has been shown to inhibit the activity of inositol-l-phosphatase within the therapeutic range (H~illcher and Sherman, 1980; Berridge, 1983). On the other hand, a number of antidepressants inhibit the uptake of 5-HT, and 5-HT z receptors are shown to be linked to PI metabolism (Conn and Sanders-Bush, 1984, 1985, 1986; Janowski et al., 1984; Kendall and Nahorski, 1985). This evidence suggests that the function of 5-HT 2 receptors coupled to PI metabolism may play an important role in the pathogenesis of affective disorders. Certain kinds of stress induce metabolic changes in the signal transduction process triggered by neurotransmitters. Metabolic changes in 5-HT-mediated mechanisms have been reported to occur during stress such as immobilization stress (Farska et al., 1988; Morgan et al., 1975; Mueller et al., 1976; Oomagari, 1989). Raskovsky and Medina (1989) have indicated that forced swimming for 15 min enhanced rat cerebral cortex PI metabolism assessed by injection of [3H]myo-inositol into the cerebral ventricles. Our findings (fig. 3) show that forced swimming for 15 min induced a significant reduction of 5-HT-stimulated [3H]IP1 accumulation in the rat cerebral cortex, which is not consistent with the results of Raskovsky and Medina. Since the reduction of 5-HT-stimulated IP~ and IPs accumulation was time-dependent, developing gradually within 15 min, we believe that our findings concern 5-HT receptor-linked subcellular processes induced by the stress. However, while the reason for the differences is unclear, different experimental procedures were used in the two studies. It has been demonstrated that repeated forced swimming for 5 min significantly decreases the density of 5-HT 1 receptors (Segawa et al., 1982). To our knowledge, there have been only few reports on changes in 5-HT 2 receptors-mediated signal transduction processes in stress situations. The present results (table 1) showing that a single forced swimming of 15-min period changed neither the affinity nor the density of 5-HT 2 receptors indicate that a reduction of 5-HTstimulated PI metabolism may be unrelated to 5-HT 2 receptor binding characteristics. Recent molecular biological studies of 5-HT receptors have indicated that GTP-binding protein (Gp pro-
tein) is involved in the coupling process between the activation of 5-HT receptors and that of phospholipase C (Battaglia et al., 1984; Lyon et al., 1986; Branchek et al., 1990). On the other hand, it has been demonstrated that protein kinase C (PKC) exerts negative feedback control over various points of the subcellular signaling process (Nishizuka, 1988). The mechanism involved in the reduction of 5-HT-stimulated PI metabolism has not yet been clarified, but the modulation of the subcellular signal transduction system, including the activation of PKC or inactivation of G-protein is a potential pathophysiological mechanism for stress-induced funtional changes. Mianserin is one of the tetracyclic antidepressants, a blocker of presynaptic a 2- and 5-HT 2 receptors. Imipramine, on the other hand, is one of the tricyclic antidepressants, known as a blocker of both norepinephrine and 5-HT uptake. Our results (fig. 5, upper) show that mianserin or imipramine treatment tended to reduce 5-HT-stimulated IP 1 and IPs accumulation, but the changes were not statistically significant. The 5-HT-stimulated IP~ accumulation was about 61% of the control in the mianserin group and 71% of the control in the imipramine group. The effect of chronic treatment with antidepressants on PI metabolism is still rather controversial: Conn and Sanders-Bush (1986) reported that mianserin treatment (5 mg/kg) for 10 days causes a significant reduction of 5-HTstimulated IP 1 accumulation (51% of the control). Kendall and Nahorsky (1985) reported that imipramine treatment for 21 days (10 mg/kg) causes a significant reduction of 5-HT-stimulated IP 1 accumulation (58%) but Mikuni et al. (1987) found no significant change in hippocampal slices with 15 m g/ kg of imipramine for 10 days. The evidence that 5-HT-stimulated PI metabolism in hippocampus is mediated not only by the 5-HT 2 receptor but also by another type of 5-HT receptor (5-HTIc receptor) may be of interest in resolving the controversy (Janowsky et al., 1984; Liibbert et al., 1987; Mikuni et al., 1987, 1988; Julius et al., 1988; Mizuta and Segawa, 1989). It has been shown that chronic mianserin or imipramine treatment reduces the density of 5-HT binding sites (e.g. to 52% of the control, with mianserin treatment) (Conn and Sanders-Bush, 1986). Thus the percentage reduction of 5-HT-stimulated PI metabolism in the present study appears to agree with that of the 5-HT 2 binding sites and therefore may be due to the down-regulation of 5-HT 2 receptors induced by chronic treatment with antidepressants. The forced swimming test has been proposed as useful for evaluating the behavioral effect not only of acute but also of chronic treatment with antidepressants (Matsubara et al., 1985). The treatment of rats with these antidepressants for 14 days enhanced behavioral activity (fig. 4B). Nevertheless there were no significant differences in 5-HT-stimulated IP accumu-
391
lation between the groups forced to swim for 15 min for antidepressant-pretreated rats (designated as group MIA + S15 or IMP + S15 in fig. 5B) and te control (saline-pretreated) rats (SAL + S15). While a significant reduction of 5-HT-stimulated PI metabolism was found between the control (SAL) not forced to swim and the group forced to swim (SAL + $15), no such significant reduction was found for chronic antidepressant-pretreated rats (MIA, IMP versus MIA + S15, IMP + $15). That is, chronic treatment with antidepressants could suppress the reduction of 5-HT-stimulated PI accumulation during stress. These results imply that suppression by chronic antidepressant treatment of the forced swimming-induced reduction of 5-HT-stimulated PI metabolism may be important for the improvement of behavioral activity. We postulate that forced swimming may act on a subcellular signal transduction system included PKC or G-protein. The forced swimming-induced changes were modulated by chronic treatment with antidepressants. The mechanism of this suppression effect of antidepressants is unclear but it is suggested that antidepressants may also act on some points of subcellular signal transduction. Mikuni and coworkers (1988) have recently reported that some antidepressants as well as lithium carbonate inhibit the r e c e p t o r - e f f e c t o r coupling process due to their actions on the G protein. Further study is necessary to elucidate the subcellular signal transduction system during stress and the effectiveness of treatment with antidepressants in relation to the cause of affective disorders, keeping in mind that down-regulation of 5-HT 2 receptors is induced by chronic treatment with antidepressants.
Acknowledgements We would like to thank T. Hiramoto M.D. for technical advice, F. Nakamura and K. Katoh for their assistance with the analysis of data.
References Battaglia, G., M, Shannon and M. Titeler, 1983, Modulation of brain S 2 serotonin receptors by lithium, sodium and potassium chrolide, Life Sci. 32, 2597. Battaglia, G., M. Shannon and M. Titeler, 1984, Guanyl nucleotide and divalent cation regulation of cortical S 2 serotonin receptors, J. Neurochem. 43, 1213. Bennett, J.P. and S.H. Snyder, 1975, Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to postsynaptic serotonin receptors, Mol. Pharmacol. 12, 373. Berridge, M.J., 1983, Rapid accumulation of inositol triphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol, Biochem. J. 212, 849. Berridge, M.J., R.M.C. Dawson, C.P. Downes, J.P. Heslop and R.F. Irvine, 1983, Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides, Biochem. J. 212, 473.
Branchek, T., N. Adham, M. Macchi, H.-T. Kao and P.R. Hartig, 1990, [3 H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H]ketanserin label two affinity states of the cloned human 5-hydroxytryptamine 2 receptor, Mol. Pharmacol. 38, 604. Brown, E., D.A. Kendall and S.R. Nahorski, 1984, Inositol phospholipid hydrolysis in rat cerebral cortical slices: 1. Receptor characterization, J. Neurochem. 42, 1379. Conn, P.J. and E. Sanders-Bush, 1984, Selective 5HT-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex, Neuropharmacology 23, 993. Conn, P.J. and E. Sanders-Bush, 1985, Serotonin-stimulated phosphoinositide turnover: Mediation by the S 2 binding site in rat cerebral cortex but not in subcortical regions, J. Pharmacol. Exp. Ther. 234, 195. Conn, P.J. and E. Sanders-Bush, 1986, Regulation of serotoninstimulated phosphoinositide hydrolysis: Relation to the serotonin 5-HT-2 binding site, J. Neurosci. 6, 3669. Farska, 1., R. Krulik and D. Sliva, 1988, Effect of immobilization stress on tricyclic antidepressant binding and serotonin uptake in rats, Eur. J. Pharmacol. 149, 363. H~illcher, L.M. and W.R. Sherman, 1980, The effects of lithium ion and other agents on the activity of myo-inositol-l-phosphatase from bovine brain, J. Biol. Chem. 255, 10896. Janowsky, A., R. Labarca and S.M. Paul, 1984, Characterization of neurotransmitter receptor-mediated phosphatidylinositol hydrolysis in the rat hippocampus, Life Sci. 35, 1953. Julius, D., A.B. MacDermott, R. Axel and T.M. Jessell, 1988, Molecular characterization of a functional cDNA encoding the serotonergic lc receptor, Science 241, 558. Kendall, D.A. and S.R. Nahorski, 1985, 5-Hydroxytryptamine-stimulated inositol phospholipid hydrolysis in rat cerebral cortex slices: Pharmacological characterization and effects of antidepressants, J. Pharmacol. Exp. Ther. 233, 473. Kitada, Y., T. Miyauchi, A. Satoh and S. Satoh, 1981, Effects of antidepressants in the rat forced swimming test, Eur. J. Pharmacol. 72, 145. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Liibbert, H., T.P. Snutch, N. Dascal, H.A. Lester and N. Davidson, Rat brain 5-HTlc receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes, 1987, J. Neurosci. 7, 1159. Lyon, R.A., H.D. Katharine and M. Titeler, 1986, 3H-DOB (4bromo-2,5-dimethoxyphenylisopropylamine) labels a guanyl nucleotide-sensitive state of cortical 5-HT 2 receptors, Mol. Pharmacol. 31, 194. Matsubara, S., M. Mikuni, T. Kazawa, M. Yoshida and I. Yamashita, 1985, Effect of antidepressant agents on behavior of rats in forced swimming test and on monoaminergic receptors in cerebral cortex, Ann. Rep. Pharmacopsychiat. Res. Found. 16, 146. Mikuni, M., I. Kusumi, T. Nishikawa and K. Takahashi, 1987, Studies on serotonin-stimulated phosphatidylinositol hydrolysis coupled to serotonin-2 receptors in the human platelets from depressed patients and in the hippocampal slices of rats treated with PCPA, Division of Mental Disorder Research, NCNP 25. Mikuni, M., Y. Kuroda, A. Kagaya, H. Yamamoto, I. Kusumi, T. Nishikawa and K. Takahashi, 1988, Study on the function of serotonergic receptor and transmembrane signal control in the affective disorders, Division of Mental Disorder Research, NCNP 59. Mizuta, T. and T. Segawa, 1989, Chronic effects of imipramine and lithium on 5-HT receptor subtypes in rat frontal cortex, hippocampus and choroid plexes: Quantitative receptor autoradiographic analysis, Jap. J. Pharmacol. 50, 315. Morgan, W.W., P.K. Rudeen and K.A. Pfeil, 1975, Effect of immobilization stress on serotonin content and turnover in regions of the rat brain, Life Sci. 17, 143.
392 Mueller, G.P., C.P. Twohy, H.T. Chen and J. Meites, 1976, Effects of 1-tryptophan and restraint stress on hypothalmic and brain serotonin turnover, and pituitary TSH and p¥olactin release in rats, Life Sci. 18, 715. Nishizuka, Y., 1988, The role of protein kinase C in cell surface signal transduction and tumor promotion, Nature 334, 661. Oomagari, K., 1989, Effects of morphine and diazepam pretreatment on immobilization stress-induced increase of serotonin metabolism in discrete brain areas of the rat, Fukuoka Med. J. 80, 81.
Porsolt, R.D., G. Anton, N. Blavet and M. Jarfre, 1978, Behavioral despair in rats: A new model sensitive to antidepressants treatments, Eur. J. Pharmacol. 47, 379. Raskovsky, S. and J. Medina, 1989, In vivo changes in brain phosphoinositides breakdown after an acute stress, Twelfth Meeting of the International Society for Neurochemistry, Abstracts, J. Neurochem. 52, s165. Segawa, T., T. Mizuta and M. Uehara, 1982, New Vista in Depression (Pergamon Press, Oxford) p. 3.
385
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52496
Influence of stress and antidepressant treatment on 5-HT-stimulated phosphoinositide hydrolysis in rat brain T o m i k o K a w a n a m i a, S h i g e r u M o r i n o b u a, S h i r o h T o t s u k a a a n d M a s a o E n d o h b a Department of Neuro-Psychiatry and b Department of Pharmacology, School of Medicine, Yamagata University, lida-Nishi-2-2-2, Yamagata 990-23, Japan Received 3 February 1992, revised MS received 17 March 1992, accepted 24 March 1992
The aim was to elucidate the role of 5-hydroxytryptamine (5-HT)-stimulated phosphoinositide (PI) metabolism in stress situations and in the behavioral improvement produced by chronic antidepressant treatment. Rat cerebral cortex slices were used for the purpose. Forced swimming for 15 min and longer induced changes in behavioral activities of rats associated with a significant reduction of 5-HT-stimulated PI metabolism, without any changes in density and affinity of 5-HTz receptors. This suggests that modulation of the receptor coupling process but not of the 5-HT2 receptor binding characteristics may be responsible for the significant reduction of 5-HT-stimulated PI metabolism in stress situations. Chronic antidepressant treatment tended to reduce 5-HT-stimulated PI metabolism. This treatment improved significantly the behavioural activities during forces swimming, and prevented the forced swimming-induced reduction of 5-HT-stimulated PI metabolism. It is postulated that chronic antidepressant treatment may improve behavioral activities in relation to PI metabolism in stress situations. Phosphoinositide hydrolysis; 5-HT2 receptors; Forced swimming test; Mianserin; Imipramine; Affective disorders
I. Introduction
Affective disorders are one of the representative mental disorders, but the cause is unknown. Investigating the cellular a n d / o r subcellular metabolic changes in the central neuronal transduction systems could yield useful information about the mechanism involved in causing affective disorders. While lithium carbonate has been reported to be useful in the treatment of patients with affective disorders, the mechanism involved in this therapeutic effect is still unknown. Since it is known that lithium (Li ÷) inhibits the enzyme involved in one of the intracellular messenger transduction systems, phosphoinositide (PI) metabolism, it has been supposed that PI metabolism may be responsible for the subcellular mechanism of affective disorders (Berridge, 1983; H~illcher and Sherman, 1980). It is recognized that the activation of certain receptor systems is closely linked to the induc-
Correspondence to: T. Kawanami, Department of Neuro-Psychiatry, School of Medicine, Yamagata University, Iida-Nishi-2-2-2, Yamagata 990-23, Japan. Tel. 81.236.33 1122.
tion of PI hydrolysis and increased inositol phosphate (IP) accumulation. It has been shown that, in rat cerebral cortex, the 5-hydroxytryptamine (5-HT-induced IP accumulation may be mediated by 5-HT 2 receptors (Conn and Sanders-Bush, 1984, 1985, 1986; Kendall and Nahorsky, 1985). A number of antidepressants such as mianserin and imipramine as well as lithium carbonate are used in the treatment of patients with affective disorders. Most of these antidepressants inhibit the uptake of 5-HT and facilitate the 5-HT-mediated signal transduction process in the central nervous system (CNS). In the course of treatment of patients with depression, psychiatrists find that patients with affective disorders have often been in the stress situations. Further, it is well known that various stresses induce metabolic changes in neurotransmitters included 5-HT in rat cerebral cortex (Farska et al., 1988; Morgan et al., 1975; Mueller et al., 1976; Oomagari, 1989). These observations imply the involvement of 5-HT in stress situations. We therefore investigated the changes of PI metabolism linked to 5-HT receptors in the CNS in a stress situation and the influence of antidepressants on the stress-induced changes.
386
Rats were forced to swim for 5, 10, 15 and 20 min in a vertical cylinder (height: 45 cm, diameter: 25 cm) containing 25 cm of water maintained at 25°C for 5, 10, 15 and 20 min. A rat was judged to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position, its head just above the surface, according to Porsolt's criteria (Porsolt et al., 1978). A rat was judged to be in the escape-directed behavior whenever it positively tried to jump out of the cylinder, or swim around or dive to find the exit, according to Kitada's criteria (Kitada et al., 1981). The duration of the immobility and the escape directed behavior was measured. When antidepressants were injected, the forced swimming test was performed 18 h after the final injection.
(1984) with minor modifications. The slices were washed extensively and incubated in 250 /zl of KH buffer containing 2 /xCi [3H]myo-inositol and 7.5 mM LiC1 per slice at 37°C for 90 min under the same oxygenation condition. Labeled slices were washed and incubated with the agonist, 5-HT, in a final volume of 270 /zl of KH buffer containing 7.5 mM LiC1 and 10 /zM pargyline. The slices were incubated at 37°C for 45 min under the same oxygenation conditions. The reactions were terminated by adding 1 ml of 5% TCA. The reaction mixtures were centrifuged at 2500 rpm for 10 min and 750 /zl of the upper phase was prepared for assay of [3H]inositol phosphates. The slices were weighed and homogenized with 1 ml of TCA and centrifuged at 2000 rpm for 2 min. An aliquot of 750/xl of the upper phase was removed and added to the previously collected 750 /zl. TCA was removed from the collected supernatant solution by washing three times with three volumes of diethylether. The pellets were used for the measurement of tissue proteins by the method of Lowry et al. (1951), and for the estimation of [3H]myo-inositol incorporation into phospholipids. Samples were added to columns containing 750 #l of Dowex-1 resin (100-200 mesh: ×8, formate form) and phosphate esters were extracted by stepwise addition of formate solutions of increasing strength. The columns were washed through with 10 ml of distilled water to wash out all free inositol Glycerophosphoinositol and inositol 1 : 2 cyclic phosphate were then eluted with 5 mM sodium t e t r a b o r a t e / 6 0 mM sodium formate. Inositol-l-monophosphate (IP 0 was eluted with 200 mM ammonium f o r m a t e / 1 0 0 mM formate, inositol1,4-bisphosphate (IP 2) was eluted with 450 mM ammonium formate/100 mM formate and inositol-l,4,5-trisphosphate (IP 3) with 1 M ammonium f o r m a t e / 1 0 0 mM formate. Samples (8 ml) of the extracts were added to 8 ml of scintilator (ACS-II) and their radioactivity was acounted. All data were expressed as the percentage stimulation over the basal [3H]IP level in the absence of 5-HT.
2.4. Tissue preparation and [3H]IP accumulation
2.5. [ 3H]Ketanserin binding
The rats were decapitated immediately after the forced swimming test, their brains were rapidly removed and cross-chopped slices (350 /zm) were prepared from cerebral cortex (mainly frontal). The slices were preincubated for 60 min in a modified K r e b s Henseleit (KH) buffer (in mM: 118 NaCI, 4.7 KCI, 2.55 CaCI2, 1.18 K H 2 P O 4, 1.18 MgSO 4, 24.88 N a H C O 3, 11.1 glucose), equilibrated with 95% O z - 5 % CO 2 at 37°C (pH 7.4). [3H]Inositol phosphates were assayed by using the methods of Berridge et al. (1983) and Brown et al.
After decapitation, the frontal cortex was dissected and weighed. It was homogenized in 25 volumes of a 50 mM Tris-HC1 buffer (pH 7.4) by using a Polytron PT-10/35. The homogenates were centrifuged for 20 min at 40 000 × g at 4°C. The supernatants were discarded and the pellets were suspended in the same buffer then homogenized and recentrifuged. The final pellets were immediately used as crude membrane preparations for the receptor binding study. Tissue protein was assayed by using the method of Lowry et al. Proteins amounted to ca. 0.3-0.5 mg per tube.
2. Materials and methods
2.1. Drugs The following drugs were kindly provided by the companies indicated: mianserin (Organon) and imipramine (Ciba-Geigy). 5-HT creatinine sulphate, pargyline HC1, serum bovine albumin were purchased from Sigma Chemical Co.; sodium tetraborate, sodium formate, formate, ammonium formate, trichloracetic acid (TCA) and all other chemicals and drugs were from Wako Chemical Co. [3H]Myo-inositol (18.3 C i / m m o l ) was purchased from Amersham Radiolabeled Chemicals and [3H]ketanserin HCI (64.1 C i / m m o l ) from New England Nuclear.
2.2. Animals and treatment Male standard Wistar rats (weighing ca. 220 g) were housed four or five per cage with food and water available ad libituml For the chronic treatment with antidepressants, the rats were injected intraperitoneally (i.p.) with mianserin or imipramine, 10 m g / k g daily for 14 days. A control group of rats was injected with saline.
2.3. Forced swimming test
387
5-HT 2 binding was assayed using the methods of Battaglia et al. (1983) and Bennett and Snyder (1975) with minor modifications. The final volume in each glass assay tube was 1 ml after addition of 0.1 ml of [3H]ketanserin and receptor preparations to the tube already containing 0.8 ml of the 50 mM Tris-HCl buffer (pH 7.4) for total binding and 0.7 ml of the buffer and 0.1 ml of 5-HT (100/zM) for determination of non-specific binding. The reaction mixtures were incubated for 30 min at 25°C. The assay was terminated by rapid filtration using a Brandel automated cell harvester (3 × 3 ml ice-cold Tris-HC1 buffer, pH 7.4, Whatman G / C filter). The filters were transferred to counting vials containing 5 ml of ACS-II and their radioactivity was then counted by means of a liquid scintillation counter.
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Fig. 2. The 5-HT-induced [3HIIPI and [3H]IFs accumulation in rat cerebral cortex slices. Slices were incubated with 10 g,M pargyline and 5-HT in various concentration for 45 rain, The data were expressed as the percentage stimulation over the basal [3H]IP level in the absence of 5-HT. Each point is the mean _+S.E.M. of six separate experiments, which were done in triplicate.
3. Results
3.1. The relation between 5-HT-stimulating time and [ 3H]IP accumulation
2.6. Data analysis The concentration of the agonist that produced half-maximal stimulation (ECs0) was estimated graphically. The significance of differences between groups were tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values. The differences between mean values were considered to be significant when a P value was smaller than 0.05.
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5-HT (10/zM) increased [3H]IP] and [3H]IPs (IP 1 + IP 2 + IP 3) accumulation in a time-dependent manner, reaching a plateau level after 30 rain (fig. 1). The maximal levels of [3H]IP 1 and [3H]IPs were 202 + 21% at 60 min and 202 + 15% at 45 min, respectively. In contrast, the increase in accumulation of [3H]IP2 and [3H]IP3 was transient. [3H]IP 2 accumulation attained a maximum at 10 min (200 + 30%) then decreased with time up to 60 min after the stimulation. The maximal accumulation of [3H]IP3 was achieved at 1 min (200 + 34%) and the level then decreased gradually with time. Subsequent experiments for determination of the concentration-response relation for the accumulation of
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S15
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Fig. 3. The influence of forced swimming for various periods on 5-HT-stimulated [3H]IP1 ad [3H]IP s accumulation in rat cerebral cortex slices. Rats were forced to swim for 5, 10, 15 and 20 min, the groups being designated as $5, S10, $15 and $20, respectively. The basal radioactivities of [3H]IP1 accumulation were 920 + 125 dpm/mg protein for control, 9125:102 d p m / m g protein for $5, 9645:175 dpm/mg protein for S10, 930_+128 dpm/mg protein for $15 and 9835:208 d p m / m g protein for $20. The basal radioactivities of [3H]IPs accumulation were 12945:225 dpm/mg protein for control, 12414-196 d p m / m g protein for $5, 1456 5:345 d p m / m g protein for $10, 1426_+269 d p m / m g protein for $15 and 13295:217 dpm/mg protein for $20. The significance of differences between groups was tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values (* P < 0.05, ** P < 0.01).
388 [3H]IP] and [3H]IPs in response to 5-HT were performed at 45 min after the administration of 5-HT.
3.2. The concentration-response relation of [ 3H]IP1 and [3H]IPs accumulations induced by 5-HT 5-HT increased [3H]IP1 and [3H]IPs accumulation in a concentration-dependent manner (fig. 2). [3H]IP1 accumulation reached a plateau at 10 /xM 5-HT. The maximal accumulation of [3H]IP t and [3H]IPs was 188 + 1 3 % (at 100 ~ M ) and 2 0 1 + 2 7 % (at 100 ~M), respectively. The ECs0 values of 5-HT were 0.3 ~ M for [3H]IPt accumulation and 0.47/xM for [3H]IPs accumulation. Subsequent experiments were carried out with 10/xM 5-HT as the agonist, since this concentration was suitable to produce an adequate response and higher concentrations could cause non-specific responses.
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TABLE 1 The influence of forced swimming (15 min) on [3H]ketanserin binding. Rats were formed to swim for 15 rain (designated as S15) and the frontal cortex was removed immediately thereafter for the preparation of crude membranes. T h e data are the m e a n s + S.E.M.
Control S15
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Rats were submitted to forced swimming for 5, 10, 15 and 20 min of swimming time, the groups being designated as $5 (n = 10), S10 (n = 5), S15 (n = 6) and $20 (n = 5), respectively. The changes in 5-HT (10 /.~M)-stimulated [3H]IPI and [3H]IPs accumulation in rat cerebral cortex slices isolated from each group were assessed (fig. 3). [3H]Myo-inositol incorporation into phospholipids was not affected by any of the forced swimming periods (data not shown). The basal levels of [3H]IP1 and [3H]IPs prior to stimulation by 5-HT were not affected by forced swimming as shown in the legend to fig. 3. [3H]IP1 accumulation in the control group not submitted to forced swimming (n = 7) was 187 + 21%. A significant reduction of [3H]IPt accumulation was produced in groups S15 (134 + 7%) and $20 (132 + 8%). While there was a clear tendency to a reduction of [3H]IPI accumulation in groups $5 (173 + 12%) and S10 (164 + 6%), the changes were statistically not significant. Substantially identical changes in [3H]IPs accumulation were induced by forced swimming: IP~ accumulation in the control group was 192 + 19%, a significant reduction being found in S15 (127 + 8%), $20 (130 + 6%) and $5 (157 + 9%). These results indicate that
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3.3. Changes in 5-HT-stimulation [3H]IP accumulation after forced swimming
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Fig. 4. Changes in behavioral activities - immobility (IMO) and the escape-directed behavior (EDB) - of rats forced to swim for 5, 10, 15 and 20 min (upper) and the influence of pretreatment with mianserin (MIA) and imipramine (IMP) on the changes in behavioral activities during forced swimming for 15 min (lower). The significance of differences between groups was tested using a one-way analysis of variance, and differences between individual groups were evaluated using Student's t-test for unpaired values (* P < 0.05, * * P < 0.01).
forced swimming reduces 5-HT-stimulated [3H]IPs accumulation in a time-dependent manner, and the change appears to reach a steady level in the groups forced to swim 15 min and longer.
3.4. Influence of forced swimming for 15 min on [ 3H]ketanserin binding There were no significant differences in either B m a x or K d values between the control and S15, indicating that forced swimming for 15 min did not affect the density and affinity of 5-HT 2 receptors in the rat cerebral cortex (table 1).
3.5. Behavioral activities of rats submitted to forced swimming The changes in behavioral activities were assessed from the duration of immobility and escape-directed behavior (fig. 4, upper). The total duration of immobility was prolonged in a time-dependent manner: 50 + 16 s in $5, 2 4 2 + 18 s in S10, 3 9 0 + 76 s in S15 and 560 + 118 s in $20. While the total duration of escapedirected behavior showed no clear time-dependent prolongation, there was a significant difference between $5 (172 + 20 s) and S15 (236 + 31 s). These
389
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reduce the 5-HT-stimulated [3H]IP1 accumulation (160 + 19%) and [3H]IPs accumulation (163 + 23%). These findings suggest that the chronic antidepressant pretreatment tended to reduce the 5-HT-stimulated [3H]IPt and [3H]IPs accumulation, but these reductions were not statistically significant.
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Fig. 5. The influence of pretreatments with mianserin (MIA) and imipramine (IMP) on the 5-HT-stimulated [3H]IPI and [3H]IPs accumulation (upper) and on the reduced 5-HT-stimulated [3H]IP1 and [3H]IPs accumulation after forced swimming for 15 min (B). The basal radioactivities of [3H]IP1 accumulation were 893 + 127 d p m / m g protein for control (SAL), 949 + 129 d p m / m g protein for SAL + S15, 1084+213 d p m / m g protein for MIA, 1027_+ 167 d p m / m g protein for MIA+S15, 980_+132 d p m / m g protein for IMP and 975+132 d p m / m g protein for I M P + S15. The basal radioactivities of [3H]IPs accumulation were 1309-+ 246 d p m / m g protein for control (SAL), 1379 + 276 d p m / m g protein for SAL + S15, 1504-+ 311 d p m / m g protein for MIA, 1626+301 d p m / m g protein for MIA+S15, 1617_+334 d p m / m g protein for IMP and 1500_+305 d p m / m g protein for IMP +S15. The significance of differences between groups were tested using a one-way analysis of variance, and differences between individual groups were determined using Student's t-test for unpaired values (* P < 0.05, ** P < 0.01).
results indicate that forced swimming for 15 min induces significant changes in both immobility and escape-directed behavior.
3.6. Influence of chronic treatment with antidepressants on [3HIIP1 and [3H11Ps accumulation Rats were pretreated with mianserin (n = 5), imipramine (n = 5) (10 m g / k g i.p. each) or saline (n = 5) for 14 days, and were decapitated 18 h after the final injection. [3H]Myo-inositol incorporation into phospholipids was not affected by the treatment with either of the agents (data not shown). The basal levels of [3H]IPI and [3H]IP~ prior to stimulation by 5-HT were also not affected by the treatment with the two agents as shown in the legend to fig. 5. Mianserin pretreatment tended to reduce the 5-HT-stimulated [3H]IPI accumulation (152 _ 18%) and the [3H]IP~ accumulation (144 + 8%) (fig. 5, upper). Imipramine pretreatment also tended to
As a significant reduction of 5-HT-stimulated [3H]IP1 and [3H]IPs accumulation was induced by forced swimming for 15 min, it was examined whether the chronic treatment with mianserin and imipramine would modulate the reduction. The rats were pretreated with mianserin (n = 5), imipramine (n = 4) or saline (n = 5) for 14 days, and were forced to swim for 15 min 18 h after the final injection. The 5-HT-stimulated [3H]IP] accumulation in cerebral cortex slices was 148 + 10% in the mianserin-pretreated group and 154 + 17% in the imipramine-pretreated group (fig. 5, lower). While a significant reduction of 5-HT-stimulated [3H]IP1 accumulation was induced by forced swimming in the control group (from 185 + 21 to 135 + 7%), no significant reduction was induced in the mianserin-pretreated group (from 152 + 18 to 148 + 10%) and in the imipramine-pretreated group (from 160 + 19 to 154 + 17%). These observations indicate that the antidepressants used in these experiments antagonized the forced swimming-induced reduction of 5-HTstimulated [3H]IP1 accumulation. The 5-HT-stimulation [3H]IPs accumulation in slices isolated from rats pretreated with mianserin or imipramine was 132 + 12 or 148 + 13%, respectively, and these values were not significantly different from those for the mianserin- or imipramine-pretreated group not submitted to forced swimming. These results suggest that the chronic pretreatment with antidepressants prevents the reduction of 5-HT-stimulated [3H]IPs accumulation in cerebral cortex slices of rats forced to swim for 15 min.
3.8. Influence of the chronic treatment with antidepressants on behavioral activities during forced swimming ($15) Pretreatment with mianserin tended to prolong the total duration of escape-directed behavior (322 + 52 s) and shorten the total duration of immobility (303 + 50 s), but the changes were not significantly different from the respective control values (239 + 28 s for escape-directed behavior and 396 + 71 s for immobility) (fig. 4B). The pretreatment with imipramine significantly prolonged the total duration of escape-directed behavior to 386 + 63 s, and significantly shortened the total
390 duration of immobility to 154 + 54 s. It is apparent from these results that the chronic treatment with either mianserin or imipramine effectively prolonged the escape-directed behavior and shortened immobility, indicating the enhancement of behavioral activities during forced swimming for 15 min.
4. Discussion Lithium, a useful therapeutic agent in the treatment of affective disorders, has been shown to inhibit the activity of inositol-l-phosphatase within the therapeutic range (H~illcher and Sherman, 1980; Berridge, 1983). On the other hand, a number of antidepressants inhibit the uptake of 5-HT, and 5-HT z receptors are shown to be linked to PI metabolism (Conn and Sanders-Bush, 1984, 1985, 1986; Janowski et al., 1984; Kendall and Nahorski, 1985). This evidence suggests that the function of 5-HT 2 receptors coupled to PI metabolism may play an important role in the pathogenesis of affective disorders. Certain kinds of stress induce metabolic changes in the signal transduction process triggered by neurotransmitters. Metabolic changes in 5-HT-mediated mechanisms have been reported to occur during stress such as immobilization stress (Farska et al., 1988; Morgan et al., 1975; Mueller et al., 1976; Oomagari, 1989). Raskovsky and Medina (1989) have indicated that forced swimming for 15 min enhanced rat cerebral cortex PI metabolism assessed by injection of [3H]myo-inositol into the cerebral ventricles. Our findings (fig. 3) show that forced swimming for 15 min induced a significant reduction of 5-HT-stimulated [3H]IP1 accumulation in the rat cerebral cortex, which is not consistent with the results of Raskovsky and Medina. Since the reduction of 5-HT-stimulated IP~ and IPs accumulation was time-dependent, developing gradually within 15 min, we believe that our findings concern 5-HT receptor-linked subcellular processes induced by the stress. However, while the reason for the differences is unclear, different experimental procedures were used in the two studies. It has been demonstrated that repeated forced swimming for 5 min significantly decreases the density of 5-HT 1 receptors (Segawa et al., 1982). To our knowledge, there have been only few reports on changes in 5-HT 2 receptors-mediated signal transduction processes in stress situations. The present results (table 1) showing that a single forced swimming of 15-min period changed neither the affinity nor the density of 5-HT 2 receptors indicate that a reduction of 5-HTstimulated PI metabolism may be unrelated to 5-HT 2 receptor binding characteristics. Recent molecular biological studies of 5-HT receptors have indicated that GTP-binding protein (Gp pro-
tein) is involved in the coupling process between the activation of 5-HT receptors and that of phospholipase C (Battaglia et al., 1984; Lyon et al., 1986; Branchek et al., 1990). On the other hand, it has been demonstrated that protein kinase C (PKC) exerts negative feedback control over various points of the subcellular signaling process (Nishizuka, 1988). The mechanism involved in the reduction of 5-HT-stimulated PI metabolism has not yet been clarified, but the modulation of the subcellular signal transduction system, including the activation of PKC or inactivation of G-protein is a potential pathophysiological mechanism for stress-induced funtional changes. Mianserin is one of the tetracyclic antidepressants, a blocker of presynaptic a 2- and 5-HT 2 receptors. Imipramine, on the other hand, is one of the tricyclic antidepressants, known as a blocker of both norepinephrine and 5-HT uptake. Our results (fig. 5, upper) show that mianserin or imipramine treatment tended to reduce 5-HT-stimulated IP 1 and IPs accumulation, but the changes were not statistically significant. The 5-HT-stimulated IP~ accumulation was about 61% of the control in the mianserin group and 71% of the control in the imipramine group. The effect of chronic treatment with antidepressants on PI metabolism is still rather controversial: Conn and Sanders-Bush (1986) reported that mianserin treatment (5 mg/kg) for 10 days causes a significant reduction of 5-HTstimulated IP 1 accumulation (51% of the control). Kendall and Nahorsky (1985) reported that imipramine treatment for 21 days (10 mg/kg) causes a significant reduction of 5-HT-stimulated IP 1 accumulation (58%) but Mikuni et al. (1987) found no significant change in hippocampal slices with 15 m g/ kg of imipramine for 10 days. The evidence that 5-HT-stimulated PI metabolism in hippocampus is mediated not only by the 5-HT 2 receptor but also by another type of 5-HT receptor (5-HTIc receptor) may be of interest in resolving the controversy (Janowsky et al., 1984; Liibbert et al., 1987; Mikuni et al., 1987, 1988; Julius et al., 1988; Mizuta and Segawa, 1989). It has been shown that chronic mianserin or imipramine treatment reduces the density of 5-HT binding sites (e.g. to 52% of the control, with mianserin treatment) (Conn and Sanders-Bush, 1986). Thus the percentage reduction of 5-HT-stimulated PI metabolism in the present study appears to agree with that of the 5-HT 2 binding sites and therefore may be due to the down-regulation of 5-HT 2 receptors induced by chronic treatment with antidepressants. The forced swimming test has been proposed as useful for evaluating the behavioral effect not only of acute but also of chronic treatment with antidepressants (Matsubara et al., 1985). The treatment of rats with these antidepressants for 14 days enhanced behavioral activity (fig. 4B). Nevertheless there were no significant differences in 5-HT-stimulated IP accumu-
391
lation between the groups forced to swim for 15 min for antidepressant-pretreated rats (designated as group MIA + S15 or IMP + S15 in fig. 5B) and te control (saline-pretreated) rats (SAL + S15). While a significant reduction of 5-HT-stimulated PI metabolism was found between the control (SAL) not forced to swim and the group forced to swim (SAL + $15), no such significant reduction was found for chronic antidepressant-pretreated rats (MIA, IMP versus MIA + S15, IMP + $15). That is, chronic treatment with antidepressants could suppress the reduction of 5-HT-stimulated PI accumulation during stress. These results imply that suppression by chronic antidepressant treatment of the forced swimming-induced reduction of 5-HT-stimulated PI metabolism may be important for the improvement of behavioral activity. We postulate that forced swimming may act on a subcellular signal transduction system included PKC or G-protein. The forced swimming-induced changes were modulated by chronic treatment with antidepressants. The mechanism of this suppression effect of antidepressants is unclear but it is suggested that antidepressants may also act on some points of subcellular signal transduction. Mikuni and coworkers (1988) have recently reported that some antidepressants as well as lithium carbonate inhibit the r e c e p t o r - e f f e c t o r coupling process due to their actions on the G protein. Further study is necessary to elucidate the subcellular signal transduction system during stress and the effectiveness of treatment with antidepressants in relation to the cause of affective disorders, keeping in mind that down-regulation of 5-HT 2 receptors is induced by chronic treatment with antidepressants.
Acknowledgements We would like to thank T. Hiramoto M.D. for technical advice, F. Nakamura and K. Katoh for their assistance with the analysis of data.
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