181
Brain Research, 546 (1991) 181-189 t~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391164447 BRES 16444
Research Reports
Autoradiographic localization and pharmacological characterization of [3H]tandospirone binding sites in the rat brain Hiroyasu Tanaka, Hiroshi Shimizu, Yoshino Kumasaka, Akira Hirose, Tohru Tatsuno and Mitsutaka Nakamura Research Laboratories, Sumitomo Pharmaceuticals Co., Osaka (Japan) (Accepted 23 October 1990) Key words: Tandospirone; Anxiolytic; SerotoninlA receptor; Hippocampus; Quantitative autoradiography; 8-OH-DPAT; Fludiazepam; Rat brain
The regional distribution and pharmacological properties of [3H]tandospirone binding sites in the rat brain were investigated using quantitative autoradiography. [3H]Tandospirone binding was notably high in the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus. The distribution profiles of [3I-I]tandospirone binding sites significantly correlated with that of serotonin (5-HT)I A receptors identified using [3H]8-OH-DPAT. In competitive binding studies, [3H]tandospirone binding was inhibited by 5-HT, 8-OH-DPAT, pindolol, buspirone and N-(a,a,a-tdfluoro-m-tolyl)-pipera~lne. The potencies of these ligands correlated with their affinities for 5-HT1A receptors. In addition, there was no significant difference in the dissociation constant of [3H]tandospirone binding between the dentate gyms, CA1 area, dorsal raphe nucleus, lateral septum and entorhinal cortex (about 10 nM) suggesting that [3H]tandospirone binds to 5-HTtA receptors with same affinities in these brain structures. The distribution pattern of binding sites for [3H]tandospirone was also compared with that of benzodiazepine receptors identified using [3H]fludiazepam to find common effector sites for different types of anxiolytics. Some similarities were observed. It is evident in the hippocampal formation that an overlap of intense binding occurred. 5-HT~A receptors in the hippocampus may participate in the anxiolytic effects of tandospirone. INTRODUCTION The serotonin (5-HT) system has long been thought to be involved in anxiety. This mainly arose from observations of the activity of 5-HT antagonist in conflict models 4 as well as from the association between reduction in the turnover of 5-HT and the anxiolytic effects of benzodiazepines (BZs) 15. Recently, piperazinyl derivatives such as buspirone, ipsapirone and gepirone have been reported to possess anxiolytic effects 5'8'9'22'34 and high affinities for 5-HT1A receptors 13'31. However, c o m m o n features in the mechanisms of the anxiolytic effects of these drugs have not been observed 2'3'1°'16'34'38. Tandospirone (formerly SM-3997), a piperazinyl derivative, is a clinically effective anxiolytic and in animal models of anxiety 35 it has been shown to be as effective as diazepam, a typical BZ-anxiolytic. Binding studies with homogenized tissue 36"a7 have shown that tandospirone had no direct effect on the G A B A receptor/Cl channel complex which is the site of action of B Z and binds to 5-HT~A receptors with high affinity in the hippocampus suggesting the possibility that the anxiolytic action of tandospirone is partially or fully mediated by
5-HT1A receptors and not by the G A B A receptor/Cl channel complex. To clarify the mechanisms of the anxiolytic effect of tandospirone, it is very important to understand its distribution and characteristics of the sites of action in the central nervous system. Therefore, we examine the autoradiographic localization and pharmacological properties of [3H]tandospirone binding sites in the rat brain. In addition, the distribution of [3H]tandospirone and [3H]fludiazepam binding sites, a tritiated ligand of a BZ-anxiolytic which is used clinically, are compared in order to find a region which is affected by both types of anxiolytics. MATERIALS AND METHODS Tissue preparation Male Sprague-Dawley rats weighing 250-350 g were decapitated and the brains quickly frozen using ethanol and dry ice. Coronal and sagittal sections (20 ~m thick) were thaw-mounted onto gelatincoated slides and stored at -25 °C until use (within 2 weeks). [3H]Tandospirone binding assay Brain sections were preincubated at room temperature for 30 rain in 0.17 M Tris-HCl buffer, pH 7.4, and incubated at 25 °C with
Correspondence: H. Tanaka, Research Laboratories, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98, Kasugadenaka, Konohana-ku, Osaka, 554 Japan.
182 various concentrations of [3H]tandospirone (3.64 TBq/mmol; Amersham), 0.5/~M clomipramine (Ciba-Geigy), a 5-HT reuptake inhibitor, and 4 mM CaCI2. Non-specific binding was measured by addition of 10 /~M tandospirone (Sumitomo). In the inhibition studies, 0.1/~M of neurotransmitters or drugs were included in the incubation medium. Incubations were terminated by 4 consecutive 15-s rinses in fresh ice-cold buffer followed by immersion in ice-cold distilled water. Sections were either transferred from the slide into scintillation vials in order to measure the radioactivity or dried with cold nitrogen gas in preparation for autoradiography.
highest specific binding ratio ( 8 1 % ) was observed following 4 consecutive 15-s rinses (data not shown). [3H]Tandospirone binding showed tissue linearity within a section thickness range of 5 - 3 0 / ~ m (data not shown). Based on these results, the assay conditions were established as followed: section thickness 20 ~ m ; preincubation time 30 min; incubation time 1 h; wash condition 4 x 15 s.
[JH]8-OH-DPATbindingassay [3H]8-OH-DPAT binding was carried out according to the procedure of Rainbow et al.33 with minor modifications. In brief, preincubation was performed in the same manner as [3H]tandobinding. The incubation buffer additionally contained 2 nM ]8-OH-DPAT (6.77 TBq/mmol; Amersham), 0.5 /~M clomipramine and 4 mM CaCI2. Non-specific binding was assessed following the addition of 1/~M 8-OH-DPAT (Sumitomo). Tissues were incubated for 1 h at 25 °C, washed twice for 5 min in ice-cold preincubation buffer, immersed in ice-cold distilled water and dried with cold nitrogen gas.
[3H]Fludiazepambindingassay Sections were preincubated for 5 min in 0.17 M Tris-HCl buffer, pH 7.4, at room temperature, then incubated at 0 *C with 2 nM [3H]fludiazepam (3.23 TBq/mmol; NEN) in the same buffer for 2 h. Non-specific binding was assessed following the addition of 1/~M fludiazepam (Sumitomo). Slices were washed 4 times for 1 rain, immersed in ice-cold distilled water and'dried with cold nitrogen gas.
Autoradiography Labelled sections and [3H]miero-seale (Amersham), a tritiated plastic standard, were placed on Hyper-film[aH] (Amersham) at 4 *C. Films were developed after 9 weeks exposure to [3H]tandospirone-labelied sections, 3 weeks exposure to [3H]8-OH-DPATlabelled sections and 2 weeks exposure to [3H]fiudiazepam-labelled sections. Autoradiograms were quantified using a computer-assisted image-analysis system (UHG-1; Unique Medical). Autoradiographic images were converted into pseudo-color images according to the optical densities. A region where the highest [3H]ligand binding was found was represented as red and the color scale shown on the left side of each image indicated decreasing binding densities from red to purple. All data in tables and figures represented the values after subtraction of non-specific binding.
Distribution of [3H]tandospirone binding The distribution of [3H]tandospirone binding is shown in Figs. 1 and 2 and Table I. A high degree of binding was observed in the limbic region such as the dentate gyrus and CA1 area of the hippocampus, lateral septum and cingulate cortex. Intense labelling was also observed in the dorsal raphe nucleus, entorhinal cortex and interpeduncular nucleus. Moderate labelling occurred in the hypothalarnus, amygdala, frontal cortex and C A 2 - 3 areas of the hippocampus. Low level binding occurred in the caudate putamen. There was only a little or even undetectable binding in the substantia nigra, choroid plexus and cerebellum.
Comparison with the 5-HT1A receptor distribution 8 - O H - D P A T has been shown to selectively bind to 5-HT1A receptors 26. The autoradiographic localization of specific [3H]8-OH-DPAT binding is shown in Fig. 2 E - H . The binding sites were concentrated in the dentate gyrus and CA1 area of the hippocampus, dorsal raphe nucleus, entorhinal cortex, lateral septum and interpeduncular nucleus. The distribution profiles of 5-HTIA receptors labelled with [3H]8-OH-DPAT and [3H]tandospirone binding sites were very similar (Fig. 3) and there was a significant correlation between them (r = 0.96, P < 0.001).
Inhibition of [3H]tandospirone binding RESULTS
Assay conditions for [3H]tandospirone binding Coronal sections (A = - 5 . 6 to - 6 . 0 m m from the bregma) were used. After incubation with [3H]tandospirone, sections were removed from slides and the radioactivity was counted. Preincubation of sections in buffer enhanced specific [3H]tandospirone binding due to the reduction of endogenous 5-HT. This effect was at its maximum after 30-40 min (data not shown). [3H]Tandospirone binding to sections reached an equilibrium state after 60 min incubation (data not shown). After incubation, washing sections in the ice-cold preincubation buffer within 2 min did not significantly decrease specific binding and the
To clarify the pharmacological properties of [3H]tandospirone binding sites, we performed competitive binding experiments. In the presence of 0.1 ~ M 5-HT, [3H]tandospirone binding in the dentate gyrus markedly decreased, but other neurotransmitters (dopamine, norepinephrine, acetylcholine and G A B A ) did not affect the binding (Table II and Fig. 4). A m o n g the 5-HTergic drugs, 8 - O H - D P A T was the most potent inhibitor of [3H]tandospirone binding, followed by pindolol, buspirone and N-(a,a,a-trifluoro-m-tolyl)-piperazine (TFMPP) ( 8 - O H - D P A T = 5-HT > pindolol = buspirone > T F M P P ; Table II), and ketanserin, a potent 5-HT 2 antagonist, had no effect. Benzodiazepine ligands, R o 15-1788 and fludiazepam, also had no effects on the specific [3H]tandospirone binding. Similar results were observed in the CA1 area of the hippocampus, dorsal
EC
CA1
4
8-OH-OPAT (0.1 pM)
5--I'IT (0.1
Control
Ro-1¢,--17U
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Fig. 1. Autoradiographic localization of specific [3H]tandospirone binding sites in the rat brain. Adjacent sagittal sections taken at L = 0.4 mm (A), 1,4 mm (B) and 4.2 mm (C) were incubated with 8 nM [3H]tandospirone. Non-specific binding was assessed following the addition of 10/~M tandospirone. Autoradiograms were obtained following 9 weeks exposure and converted into pseudo-color images by computer-assisted image analyzer (see 'Materials and Methods'). The color scale shown on the left side of each image indicates decreasing binding densities from red to purple. Illustrations are from ref. 29. CA1, CA1 area of Ammon's horn; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; Hip, hippocampus; IP, interpeduncular nucleus; LS, lateral septum. Fig. 4. Effects of neurotransmitters and drugs on specific [3H]tandospirone binding in the rat brain. Adjacent coronal sections taken at A = -3.3. mm from the bregma were incubated with [3H]tandospirone in the presence of 0.1/~M 5-HT (B), 8-OH-DPAT (C), norepinephrine (D), dopamine (E) or Ro 15-1788 (F).
DG
LS
Hip
IP
DR
O0
184
Fig. 2. Autoradiographic localization of specific [3H]tandospirone binding sites and specific [3H]8-OH-DPAT binding sites in the rat brain. Adjacent coronal sections were taken at A = +0.7 mm (A,E), -3.3 mm (B,F), -5.8 mm (C,G) and -7.8 mm from the bregma (D,H). Sections were incubated with 8 nM [3H]tandospirone (A-D) or 2 nM [3H]8-OH-DPAT (E-H). Autoradiograms were obtained following 9 weeks exposure (A-D) or 3 weeks exposure (E-H) and converted into pseudo-color images by computer-assisted image analyzer (see 'Materials and Methods'). The region showing highest binding of [3H]ligand in the brain areas tested is represented in red. Hence, a true comparison can be made using Fig. 3 (see text). Illustrations are from ref. 29. CA1, CA1 area of Ammon's horn; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; Hip, hippocampus; IP, interpeduncular nucleus; LS, lateral septum.
raphe nucleus, lateral septum and entorhinal cortex (Table II).
Kinetics of [3H]tandospirone binding The affinities of [3H]tandospirone in various brain
regions were examined. Equilibrium binding analysis in a concentration range of 2-20 n M (Fig. 5A) yielded a dissociation constant (Kd) of 12.1. n M and maximal n u m b e r of binding sites (Bmax) of 114 fmol/mg tissue in the dentate gyrus (Fig. 5B). The K~ and B .... values are
185 shown in Table III. There was no significant difference in K d value in all brain regions examined. Comparison with benzodiazepine receptor distribution We examined the distribution of BZ receptors, the sites of action of typical anxiolytics, and compared it with that of [3H]tandospirone binding sites. BZ receptors were identified using [3H]fludiazepam and the highest level of [3H]fludiazepam labelling was found in the cerebral cortex, hippocampus, substantia nigra and medial septum (Fig. 6). The distribution profile of [3H]fludiazepam binding sites was quite different from that of [3n]tandospirone binding sites but an overlap of intense binding occurred in the hippocampal formation (Fig. 6). DISCUSSION The purpose of the present study was to clarify the distribution of the sites of action of tandospirone and their pharmacological properties in the central nervous system and to deduce its mechanism of anxiolytic action. The binding sites for [3H]tandospirone in the rat brain are predominantly located in the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus (Figs. 1, 2 and Table I) which are areas closely associated with 5-HT pathways. Anatomical studies 21 has shown that the dorsal raphe nucleus and interpeduncular nucleus contain 5-HT neuronal cell bodies and parts of the limbic system, especially the hippocampus, septum and entorhinal cortex, are densely
innervated with 5-HTergic projections. However, [3a]tandospirone binding was practically absent in the substantia nigra and choroid plexus (Table I), areas also associated with 5-HT pathways and a high density of 5-HTIB43 and 5-HTIc 3° receptors, respectively. The distribution profile of [3H]tandospirone binding sites significantly corresponded with that of 5-HT1A receptors identified using [3H]8-OH-DPAT (Figs. 2 and 3). In the quantitative determination of binding properties in the structures where highest [3H]tandospirone binding was found (the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex and dorsal raphe nucleus) [3H]tandospirone binding was strongly inhibited by only 5-HT of 5 neurotransmitters tested (Table III). In addition, the inhibition potencies of 5-HT receptor ligands (8-OH-DPAT = 5-HT > pindolol = buspirone > TFMPP > > ketanserin) correlated well with their affinities for 5-HT1A receptors as described in the literature 2°'32. The conclusion drawn from the results of the distribution and inhibition study is that tandospirone preferentially binds to 5-HT1A receptors in all brain structures. However, there is little difference between the distribution pattern of [3H]tandospirone and [3H]8-OH-DPAT binding sites which is evident in the caudate putamen (Fig. 2A,E). Low level [3H]tandospirone binding was detected but [3H]8-OH-DPAT binding was not observed in this region. Unfortunately, [3H]tandospirone binding in the caudate putamen was too low to allow quantitation
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Regional distribution of specific f H]tandospirone binding in the rat brain
C A 1
Data are mean + S.E.M. of 3-4 independent experiment, n.d., not detectable. Brain structure
Specific [SH]tandospirone binding (fraol/mgtissue)
Dentate gyrus Entorhinal cortex Dorsal raphe nucleus Lateral septum Interpeduncular nucleus CA1 area Cingulate cortex Cortical amygdaloidnucleus CA3 area Frontal cortex CA2 area Medial septum Median raphe nucleus Central amygdaloidnucleus Caudate putamen Substantia nigra Choroid plexus
56.1 + 5.1 48.0 + 2.4 45.6 + 5.2 38.3 + 2.2 37.9 + 3.1 37.0 + 2.9 20.6 + 1.0 16.3 + 1.6 16.2 + 1.3 14.0 + 1.4 13.9 + 1.8 11.9 + 0.9 10.0 + 1.7 7.0 + 0.7 6.4 + 0.6 0.9 + 0.4 n.d.
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Fig. 3. Comparison of the regional distribution of specific [3H]tandospirone binding sites and specific [3H]8-OH-DPATbinding sites. Data are the mean + S.E.M. of 3-4 independent experiments. CA1-3, CA1-3 area of Ammon's horn; CeA, central amygdaloid nucleus; ChP, choroid plexus; Cg, cingulate cortex; CoA, cortical amygdaloid nucleus; CP, caudate putamen; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; FC, frontal cortex; IP, interpeduncular nucleus; LS, lateral septum; MR, median raphe nucleus; MS, medial septum; SN, substantia nigra.
186 in the pharmacological investigations. From considerations of results of binding studies in homogenized tissue 36'37, the distribution of [3H]tandospirone binding in the caudate putamen seems to correspond with dopamine 2 receptors. A behavioral study35 suggested that the action of tandospirone on dopamine2 receptors is antagonistic but very weak. From the results of the present experiments, the efficacy of tandospirone as a 5-HT~A agonist cannot be estimated although tandospirone has already been reported to have agonistic activities with high efficacy. For example, the effect of GTP, which is thought to selectively decrease the affinity of agonists, was observed in [3H]tandospirone binding study with hippocampal membrane preparations 37. Furthermore, tandospirone induced 5-HT behavioral syndrome (flat body posture), inhibited the activity of forskolin-stimulated adenylate cyclase39, and decreased the neuronal activities of the hippocampal pyramidal cells 19 and 5-HTergic neuronal cells in the dorsal raphe nucleus 14. These actions were blocked by 5-HT1A antagonists. In the Vogel conflict test, a simple and reliable method that can estimate the clinical potency of compounds as anxiolytic44, tandospirone showed a positive action which was not antagonized by Ro 15-1788 (a BZ antagonist), haloperidol (a potent dopamine 2 antagonist) or ketanserin (a selective 5-HT 2 antagonist), but was antagonized by spiperone (dopamine2, 5-HT 2 and 5-HTlA mixed antagonist)a< Based on the results of the present autoradiographic study in which tandospirone preferentially bound to 5-HT~A receptors and from the Vogel
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conflict test, we conclude that the anxiolytic effect of tandospirone is at least partially mediated by central 5-HT~A receptors. It has been reported that 5-HT~A receptors in the central nervous system consist of two functionally diverse types. One found at high density in the hippocampus and septum located on postsynaptic neurons of 5-HTergic terminals ('postsynaptic receptor') ~1'43 and the other found at high density in the dorsal raphe nucleus located on the 5-HT neuronal cell bodies and which regulate the activities of 5-HT neurons ('autoreceptor') 6'4°. 5-HT1A agonists at the 'autoreceptors' will inhibit the firing of 5-HT neurons and decrease the release of 5-HT from nerve terminals, whilst at the 'postsynaptic receptors' it will mimic endogenous 5-HT and decrease the firing of neurons 19'23"25. It is of interest to determine which type of 5 - H T I A receptor contributes to the anxiolytic effect of tandospirone. In the conflict test using rats, a 5-HT neuronal lesion induced by 5,7-dihydroxytryptamine, which inhibits the action of 5-HTIA agonists for 'autoreceptors', failed to decrease the anti-conflict activity of tandospirone 38. We think that 'postsynaptic receptors' play a more important role in the anxiolytic effect of tandospirone rather than the 'autoreceptors'. Electrophysiological evidence supports this hypothesis; the sustained administration of tandospirone TM and gepirone 2 in rats produced desensitization of 'autoreceptors' in the dorsal raphe nucleus but
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Fig. 5. Saturation analysis and Scatchard plot of [3H]tandospirone binding to the dentate gyms. Adjacent coronal sections taken at A = -3.3 mm from the bregma were incubated with various concentrations (2-20 nM) or [3H]tandospirone. Data comprise typical results from 4 independent experiments. A, saturation analysis, O, total binding; A, non-specific binding; O, specific binding; B, Scatchard plot.
Fig. 6. Comparison of the regional distribution of specific [3H]tandospirone binding sites and specific [3H]fludiazepam binding sites in the rat brain. Adjacent coronal sections were incubated with 2 nM [3H]fludiazepam. Data are the mean --- S.E.M. of 3-4 independent experiment. CA1-3, CA1-3 area of Ammon's horn; CeA, central amygdaloid nucleus; Cg, cinguiate cortex; CoA, cortical amygdaloid nucleus; CP, caudate putamen; DG, dentate gyms; DR, dorsal raphe nucleus; EC, entorhinal cortex; FC, frontal cortex, IP, interpeduncular nucleus; LS, lateral septum; MR, median raphe nucleus; MS, medial septum; SN, substantia nigra; VH, ventral hypothalamus.
187 TABLE II
TABLE III
Effect of neurotransmitters and drugs on specific [3H]tandospirone binding in the rat brain
Dissociation constants and maximal number of binding sites of [3H]tandospirone in the rat brain
Coronal sections taken at A = +0.7 mm (LS), -3.3 mm (DG, CA1) and -7.8 mm (EC, DR) from the bregma were used. Data are the mean + S.E.M. of 4 independent experiments. NE, norepinephrine; DA, dopamine; DG, dentate gyrus; CA1, CA1 area of Ammon's horn; DR, dorsal raphe nucleus; EC, entorhinal cortex.
Coronal sections taken at A = +0.7 mm (lateral septum), -3.3 ram (dentate gyms, CA1 area) and -7.8 mm (entorhinal cortex, dorsal raphe nucleus) from the bregma were incubated with various concentrations of [3H]tandospirone (2-20 nM). Data are the mean -+ S.E.M. of 4 independent experiments.
Drugs (O.11tM)
[3H]tandospirone binding (% of specific binding)
Brain structure
Kd (nM)
DG
CA1
DR
EC
LS
B,,~ (fraollmg tissue)
NE DA 5-HT ACh GABA 8-OH-DPAT Pindolol Buspirone TFMPP Ketanserin Ro 15-1788 Fludiazepam
106+8 107 + 5 5+3 103 + 5 93+6 4+2 16 + 4 14 + 5 47 + 12 97 + 6 102+11 96 + 8
105+4 102 + 4 6+3 105 + 3 98+7 6_+3 21 + 7 15 _+7 49 _+7 101 _+3 96+4 103 + 6
113+14 98 + 3 8+4 99 + 2 95-+7 6+4 8 -+ 5 15 -+ 3 62 _+7 94 _+6 95-+6 90 -+ 4
102+2 93 + 4 5+2 102 -+ 7 108+11 8_+4 12 + 3 15 + 3 59 + 6 99 -+ 10 91-+7 92 _+6
100+3 98 + 1 13+6 104 + 5 101_+3 4+2 25 -+ 10 32 -+ 5 68 + 6 94 + 4 100+3 96 _+5
Dentate gyrus CA1 area Lateral septum Dorsal raphe nucleus Entorhinal cortex
12.1 + 0.6 11.4 + 2.0 8.9 + 2.0 12.0 + 3.2 11.2 + 2.7
114 + 16 70.0 + 11.2 74.4 + 11.2 76.7 + 13.9 119 + 26
did not influence the sensitivity of 'postsynaptic receptors'. However, the anti-conflict activity of tandospirone was not suppressed by repeated administration 35. M a n y investigators ~°'42 maintained that the anxiolytic effects of 5-HTIA agonists (buspirone, ipsapirone and 8 - O H - D P A T ) involved actions on 'autoreceptors'. Durish 7 pointed out that BZs also suppress 5-HT neuronal activities, and the anxiolytic effects of buspirone, ipsapirone and 8 - O H - D P A T are produced at similar doses to those inducing hyperphagic effects which are thought to be mediated by 'autoreceptors '12. In addition, 8-OH-DPAT, buspirone and ipsapirone applied with very small iontophoretic currents markedly depress firing of 5-HT neurons in the raphe nuclei 6'4°. That is, this hypothesis based on the theory that 'autoreceptors' are more sensitive to the action of agonists than 'postsynaptic receptors' and the inhibition of 5-HTergic impulse flow induced by both B Z and 5-HTIA agonist represent an important c o m m o n pathway in their anxiolytic activity. Basse I has observed that 5-HTIA agonists administrated systemically at a lower dose than that which effects 5-HT neurons in the dorsal raphe nucleus decreased firing of some kind of neurons in the hippocampus. In our experiments, the dissociation constant of [3H]tandospirone binding was uniform in all brain structures examined (Table III), suggesting that tandospirone binds to 'autoreceptors' and 'postsynaptic receptors' with the same affinities. It is important to interpret the actions of 5-HTIA agonists as net effects on functions regulated by
5-HTergic neurons in the brain. Wree 45 has observed that ipsapirone administered systemically at a dose which induces anxiolytic effects suppresses neuronal activities not only in the dorsal raphe nucleus but also in the hippocampus and septum. This effect in the hippocampus and septum is due to the direct action of ipsapirone on the 'postsynaptic receptors', suggesting that in the areas where 'postsynaptic receptors' exist at high density the agonistic action on 'postsynaptic receptors' dominates that on 'autoreceptors'. Certain brain regions have been suggested to be associated with anxiety. Papez 28 first proposed that the hypothalamus, anterior thalamic nuclei, cingulate gyms, hippocampus, and their interconnections constitute a neuroanatomical circuit involved in anxiety. Subsequent behavioral and pharmacological studies in animals and positron emission tomography scanning studies in humans have implicated many brain structures 24. It appears that key neuroanatomical substrates of anxiety include the limbic regions and other areas that may modulate limbic regions 17'24. Many brain structures where [3H]tandospirone binding sites exist at high density belong to these areas (e.g the hippocampus, lateral septum, entorhinal cortex and cingulate cortex), that probably participate in the anxiolytic effect of tandospirone. Especially, the hippocampus contains both 5-HT1A receptors and B Z receptors at high density (Fig. 6), and 5-HT1A agonists 6' 19.4o and B Z 18 commonly suppress neuronal activities in this region. Furthermore, Miyazaki 27 has shown that microinjection of tandospirone into the hippocampus induces anti-conflict effects in rats. Therefore, the agonistic action on the postsynaptic 5-HT1A receptors in the hippocampus could play the most important role in the anxiolytic effects of tandospirone.
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40 41
42 43
SM-3997: a comparative study with some 5-HT~A related anxiolytics, Jpn. J. Pharmacol., 49 (Suppl.) (1989) 227P. Sprouse, J.S. and Aghajanian, G.K., (-)Propranolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5-HT~A selective agonist, Eur. J. Pharmacol., 128 (1986) 295-298. Tatsuno, T., Shimizu, H., Hirose, A., Kumasaka, Y., Nakamura, M. and Katsube, J., The mechanism of anti-conflict action of SM-3997: involvement of the 5-HTIA system (in Japanese), Jpn. J. Psychopharmacol., 8 (1988) 33-34. Traber, J. and Glaser, T., 5-HTIA receptor-related anxiolytics, TIPS, 8 (1987) 432-437. Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., Mesti-
kawy, S.EI., Gozian, H. and Hamon, M., Quantitative autoradiography of multiple 5-HT~ receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats, J. Neurosci., 6 (1986) 3474-3482. 44 Vogel, J.R., Beer, B. and Clody, D.E., A simple reliable conflict procedure for testing antianxiety agents, Psychopharmacology, 21 (1971) 1-7. 45 Wree, A, Zilles, K., Schleicher, A., Horvath, E. and Traber, J., Effect of the 5-HT1A receptor agonist ipsapirone on the local cerebral glucose utilization of rat hippocampus, Brain Research, 436 (1987) 283-290.
Brain Research, 546 (1991) 181-189 t~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391164447 BRES 16444
Research Reports
Autoradiographic localization and pharmacological characterization of [3H]tandospirone binding sites in the rat brain Hiroyasu Tanaka, Hiroshi Shimizu, Yoshino Kumasaka, Akira Hirose, Tohru Tatsuno and Mitsutaka Nakamura Research Laboratories, Sumitomo Pharmaceuticals Co., Osaka (Japan) (Accepted 23 October 1990) Key words: Tandospirone; Anxiolytic; SerotoninlA receptor; Hippocampus; Quantitative autoradiography; 8-OH-DPAT; Fludiazepam; Rat brain
The regional distribution and pharmacological properties of [3H]tandospirone binding sites in the rat brain were investigated using quantitative autoradiography. [3H]Tandospirone binding was notably high in the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus. The distribution profiles of [3I-I]tandospirone binding sites significantly correlated with that of serotonin (5-HT)I A receptors identified using [3H]8-OH-DPAT. In competitive binding studies, [3H]tandospirone binding was inhibited by 5-HT, 8-OH-DPAT, pindolol, buspirone and N-(a,a,a-tdfluoro-m-tolyl)-pipera~lne. The potencies of these ligands correlated with their affinities for 5-HT1A receptors. In addition, there was no significant difference in the dissociation constant of [3H]tandospirone binding between the dentate gyms, CA1 area, dorsal raphe nucleus, lateral septum and entorhinal cortex (about 10 nM) suggesting that [3H]tandospirone binds to 5-HTtA receptors with same affinities in these brain structures. The distribution pattern of binding sites for [3H]tandospirone was also compared with that of benzodiazepine receptors identified using [3H]fludiazepam to find common effector sites for different types of anxiolytics. Some similarities were observed. It is evident in the hippocampal formation that an overlap of intense binding occurred. 5-HT~A receptors in the hippocampus may participate in the anxiolytic effects of tandospirone. INTRODUCTION The serotonin (5-HT) system has long been thought to be involved in anxiety. This mainly arose from observations of the activity of 5-HT antagonist in conflict models 4 as well as from the association between reduction in the turnover of 5-HT and the anxiolytic effects of benzodiazepines (BZs) 15. Recently, piperazinyl derivatives such as buspirone, ipsapirone and gepirone have been reported to possess anxiolytic effects 5'8'9'22'34 and high affinities for 5-HT1A receptors 13'31. However, c o m m o n features in the mechanisms of the anxiolytic effects of these drugs have not been observed 2'3'1°'16'34'38. Tandospirone (formerly SM-3997), a piperazinyl derivative, is a clinically effective anxiolytic and in animal models of anxiety 35 it has been shown to be as effective as diazepam, a typical BZ-anxiolytic. Binding studies with homogenized tissue 36"a7 have shown that tandospirone had no direct effect on the G A B A receptor/Cl channel complex which is the site of action of B Z and binds to 5-HT~A receptors with high affinity in the hippocampus suggesting the possibility that the anxiolytic action of tandospirone is partially or fully mediated by
5-HT1A receptors and not by the G A B A receptor/Cl channel complex. To clarify the mechanisms of the anxiolytic effect of tandospirone, it is very important to understand its distribution and characteristics of the sites of action in the central nervous system. Therefore, we examine the autoradiographic localization and pharmacological properties of [3H]tandospirone binding sites in the rat brain. In addition, the distribution of [3H]tandospirone and [3H]fludiazepam binding sites, a tritiated ligand of a BZ-anxiolytic which is used clinically, are compared in order to find a region which is affected by both types of anxiolytics. MATERIALS AND METHODS Tissue preparation Male Sprague-Dawley rats weighing 250-350 g were decapitated and the brains quickly frozen using ethanol and dry ice. Coronal and sagittal sections (20 ~m thick) were thaw-mounted onto gelatincoated slides and stored at -25 °C until use (within 2 weeks). [3H]Tandospirone binding assay Brain sections were preincubated at room temperature for 30 rain in 0.17 M Tris-HCl buffer, pH 7.4, and incubated at 25 °C with
Correspondence: H. Tanaka, Research Laboratories, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98, Kasugadenaka, Konohana-ku, Osaka, 554 Japan.
182 various concentrations of [3H]tandospirone (3.64 TBq/mmol; Amersham), 0.5/~M clomipramine (Ciba-Geigy), a 5-HT reuptake inhibitor, and 4 mM CaCI2. Non-specific binding was measured by addition of 10 /~M tandospirone (Sumitomo). In the inhibition studies, 0.1/~M of neurotransmitters or drugs were included in the incubation medium. Incubations were terminated by 4 consecutive 15-s rinses in fresh ice-cold buffer followed by immersion in ice-cold distilled water. Sections were either transferred from the slide into scintillation vials in order to measure the radioactivity or dried with cold nitrogen gas in preparation for autoradiography.
highest specific binding ratio ( 8 1 % ) was observed following 4 consecutive 15-s rinses (data not shown). [3H]Tandospirone binding showed tissue linearity within a section thickness range of 5 - 3 0 / ~ m (data not shown). Based on these results, the assay conditions were established as followed: section thickness 20 ~ m ; preincubation time 30 min; incubation time 1 h; wash condition 4 x 15 s.
[JH]8-OH-DPATbindingassay [3H]8-OH-DPAT binding was carried out according to the procedure of Rainbow et al.33 with minor modifications. In brief, preincubation was performed in the same manner as [3H]tandobinding. The incubation buffer additionally contained 2 nM ]8-OH-DPAT (6.77 TBq/mmol; Amersham), 0.5 /~M clomipramine and 4 mM CaCI2. Non-specific binding was assessed following the addition of 1/~M 8-OH-DPAT (Sumitomo). Tissues were incubated for 1 h at 25 °C, washed twice for 5 min in ice-cold preincubation buffer, immersed in ice-cold distilled water and dried with cold nitrogen gas.
[3H]Fludiazepambindingassay Sections were preincubated for 5 min in 0.17 M Tris-HCl buffer, pH 7.4, at room temperature, then incubated at 0 *C with 2 nM [3H]fludiazepam (3.23 TBq/mmol; NEN) in the same buffer for 2 h. Non-specific binding was assessed following the addition of 1/~M fludiazepam (Sumitomo). Slices were washed 4 times for 1 rain, immersed in ice-cold distilled water and'dried with cold nitrogen gas.
Autoradiography Labelled sections and [3H]miero-seale (Amersham), a tritiated plastic standard, were placed on Hyper-film[aH] (Amersham) at 4 *C. Films were developed after 9 weeks exposure to [3H]tandospirone-labelied sections, 3 weeks exposure to [3H]8-OH-DPATlabelled sections and 2 weeks exposure to [3H]fiudiazepam-labelled sections. Autoradiograms were quantified using a computer-assisted image-analysis system (UHG-1; Unique Medical). Autoradiographic images were converted into pseudo-color images according to the optical densities. A region where the highest [3H]ligand binding was found was represented as red and the color scale shown on the left side of each image indicated decreasing binding densities from red to purple. All data in tables and figures represented the values after subtraction of non-specific binding.
Distribution of [3H]tandospirone binding The distribution of [3H]tandospirone binding is shown in Figs. 1 and 2 and Table I. A high degree of binding was observed in the limbic region such as the dentate gyrus and CA1 area of the hippocampus, lateral septum and cingulate cortex. Intense labelling was also observed in the dorsal raphe nucleus, entorhinal cortex and interpeduncular nucleus. Moderate labelling occurred in the hypothalarnus, amygdala, frontal cortex and C A 2 - 3 areas of the hippocampus. Low level binding occurred in the caudate putamen. There was only a little or even undetectable binding in the substantia nigra, choroid plexus and cerebellum.
Comparison with the 5-HT1A receptor distribution 8 - O H - D P A T has been shown to selectively bind to 5-HT1A receptors 26. The autoradiographic localization of specific [3H]8-OH-DPAT binding is shown in Fig. 2 E - H . The binding sites were concentrated in the dentate gyrus and CA1 area of the hippocampus, dorsal raphe nucleus, entorhinal cortex, lateral septum and interpeduncular nucleus. The distribution profiles of 5-HTIA receptors labelled with [3H]8-OH-DPAT and [3H]tandospirone binding sites were very similar (Fig. 3) and there was a significant correlation between them (r = 0.96, P < 0.001).
Inhibition of [3H]tandospirone binding RESULTS
Assay conditions for [3H]tandospirone binding Coronal sections (A = - 5 . 6 to - 6 . 0 m m from the bregma) were used. After incubation with [3H]tandospirone, sections were removed from slides and the radioactivity was counted. Preincubation of sections in buffer enhanced specific [3H]tandospirone binding due to the reduction of endogenous 5-HT. This effect was at its maximum after 30-40 min (data not shown). [3H]Tandospirone binding to sections reached an equilibrium state after 60 min incubation (data not shown). After incubation, washing sections in the ice-cold preincubation buffer within 2 min did not significantly decrease specific binding and the
To clarify the pharmacological properties of [3H]tandospirone binding sites, we performed competitive binding experiments. In the presence of 0.1 ~ M 5-HT, [3H]tandospirone binding in the dentate gyrus markedly decreased, but other neurotransmitters (dopamine, norepinephrine, acetylcholine and G A B A ) did not affect the binding (Table II and Fig. 4). A m o n g the 5-HTergic drugs, 8 - O H - D P A T was the most potent inhibitor of [3H]tandospirone binding, followed by pindolol, buspirone and N-(a,a,a-trifluoro-m-tolyl)-piperazine (TFMPP) ( 8 - O H - D P A T = 5-HT > pindolol = buspirone > T F M P P ; Table II), and ketanserin, a potent 5-HT 2 antagonist, had no effect. Benzodiazepine ligands, R o 15-1788 and fludiazepam, also had no effects on the specific [3H]tandospirone binding. Similar results were observed in the CA1 area of the hippocampus, dorsal
EC
CA1
4
8-OH-OPAT (0.1 pM)
5--I'IT (0.1
Control
Ro-1¢,--17U
DA I
NE (0.1 laM)
IIII
Fig. 1. Autoradiographic localization of specific [3H]tandospirone binding sites in the rat brain. Adjacent sagittal sections taken at L = 0.4 mm (A), 1,4 mm (B) and 4.2 mm (C) were incubated with 8 nM [3H]tandospirone. Non-specific binding was assessed following the addition of 10/~M tandospirone. Autoradiograms were obtained following 9 weeks exposure and converted into pseudo-color images by computer-assisted image analyzer (see 'Materials and Methods'). The color scale shown on the left side of each image indicates decreasing binding densities from red to purple. Illustrations are from ref. 29. CA1, CA1 area of Ammon's horn; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; Hip, hippocampus; IP, interpeduncular nucleus; LS, lateral septum. Fig. 4. Effects of neurotransmitters and drugs on specific [3H]tandospirone binding in the rat brain. Adjacent coronal sections taken at A = -3.3. mm from the bregma were incubated with [3H]tandospirone in the presence of 0.1/~M 5-HT (B), 8-OH-DPAT (C), norepinephrine (D), dopamine (E) or Ro 15-1788 (F).
DG
LS
Hip
IP
DR
O0
184
Fig. 2. Autoradiographic localization of specific [3H]tandospirone binding sites and specific [3H]8-OH-DPAT binding sites in the rat brain. Adjacent coronal sections were taken at A = +0.7 mm (A,E), -3.3 mm (B,F), -5.8 mm (C,G) and -7.8 mm from the bregma (D,H). Sections were incubated with 8 nM [3H]tandospirone (A-D) or 2 nM [3H]8-OH-DPAT (E-H). Autoradiograms were obtained following 9 weeks exposure (A-D) or 3 weeks exposure (E-H) and converted into pseudo-color images by computer-assisted image analyzer (see 'Materials and Methods'). The region showing highest binding of [3H]ligand in the brain areas tested is represented in red. Hence, a true comparison can be made using Fig. 3 (see text). Illustrations are from ref. 29. CA1, CA1 area of Ammon's horn; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; Hip, hippocampus; IP, interpeduncular nucleus; LS, lateral septum.
raphe nucleus, lateral septum and entorhinal cortex (Table II).
Kinetics of [3H]tandospirone binding The affinities of [3H]tandospirone in various brain
regions were examined. Equilibrium binding analysis in a concentration range of 2-20 n M (Fig. 5A) yielded a dissociation constant (Kd) of 12.1. n M and maximal n u m b e r of binding sites (Bmax) of 114 fmol/mg tissue in the dentate gyrus (Fig. 5B). The K~ and B .... values are
185 shown in Table III. There was no significant difference in K d value in all brain regions examined. Comparison with benzodiazepine receptor distribution We examined the distribution of BZ receptors, the sites of action of typical anxiolytics, and compared it with that of [3H]tandospirone binding sites. BZ receptors were identified using [3H]fludiazepam and the highest level of [3H]fludiazepam labelling was found in the cerebral cortex, hippocampus, substantia nigra and medial septum (Fig. 6). The distribution profile of [3H]fludiazepam binding sites was quite different from that of [3n]tandospirone binding sites but an overlap of intense binding occurred in the hippocampal formation (Fig. 6). DISCUSSION The purpose of the present study was to clarify the distribution of the sites of action of tandospirone and their pharmacological properties in the central nervous system and to deduce its mechanism of anxiolytic action. The binding sites for [3H]tandospirone in the rat brain are predominantly located in the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus (Figs. 1, 2 and Table I) which are areas closely associated with 5-HT pathways. Anatomical studies 21 has shown that the dorsal raphe nucleus and interpeduncular nucleus contain 5-HT neuronal cell bodies and parts of the limbic system, especially the hippocampus, septum and entorhinal cortex, are densely
innervated with 5-HTergic projections. However, [3a]tandospirone binding was practically absent in the substantia nigra and choroid plexus (Table I), areas also associated with 5-HT pathways and a high density of 5-HTIB43 and 5-HTIc 3° receptors, respectively. The distribution profile of [3H]tandospirone binding sites significantly corresponded with that of 5-HT1A receptors identified using [3H]8-OH-DPAT (Figs. 2 and 3). In the quantitative determination of binding properties in the structures where highest [3H]tandospirone binding was found (the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex and dorsal raphe nucleus) [3H]tandospirone binding was strongly inhibited by only 5-HT of 5 neurotransmitters tested (Table III). In addition, the inhibition potencies of 5-HT receptor ligands (8-OH-DPAT = 5-HT > pindolol = buspirone > TFMPP > > ketanserin) correlated well with their affinities for 5-HT1A receptors as described in the literature 2°'32. The conclusion drawn from the results of the distribution and inhibition study is that tandospirone preferentially binds to 5-HT1A receptors in all brain structures. However, there is little difference between the distribution pattern of [3H]tandospirone and [3H]8-OH-DPAT binding sites which is evident in the caudate putamen (Fig. 2A,E). Low level [3H]tandospirone binding was detected but [3H]8-OH-DPAT binding was not observed in this region. Unfortunately, [3H]tandospirone binding in the caudate putamen was too low to allow quantitation
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TABLE I
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Regional distribution of specific f H]tandospirone binding in the rat brain
C A 1
Data are mean + S.E.M. of 3-4 independent experiment, n.d., not detectable. Brain structure
Specific [SH]tandospirone binding (fraol/mgtissue)
Dentate gyrus Entorhinal cortex Dorsal raphe nucleus Lateral septum Interpeduncular nucleus CA1 area Cingulate cortex Cortical amygdaloidnucleus CA3 area Frontal cortex CA2 area Medial septum Median raphe nucleus Central amygdaloidnucleus Caudate putamen Substantia nigra Choroid plexus
56.1 + 5.1 48.0 + 2.4 45.6 + 5.2 38.3 + 2.2 37.9 + 3.1 37.0 + 2.9 20.6 + 1.0 16.3 + 1.6 16.2 + 1.3 14.0 + 1.4 13.9 + 1.8 11.9 + 0.9 10.0 + 1.7 7.0 + 0.7 6.4 + 0.6 0.9 + 0.4 n.d.
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Fig. 3. Comparison of the regional distribution of specific [3H]tandospirone binding sites and specific [3H]8-OH-DPATbinding sites. Data are the mean + S.E.M. of 3-4 independent experiments. CA1-3, CA1-3 area of Ammon's horn; CeA, central amygdaloid nucleus; ChP, choroid plexus; Cg, cingulate cortex; CoA, cortical amygdaloid nucleus; CP, caudate putamen; DG, dentate gyrus; DR, dorsal raphe nucleus; EC, entorhinal cortex; FC, frontal cortex; IP, interpeduncular nucleus; LS, lateral septum; MR, median raphe nucleus; MS, medial septum; SN, substantia nigra.
186 in the pharmacological investigations. From considerations of results of binding studies in homogenized tissue 36'37, the distribution of [3H]tandospirone binding in the caudate putamen seems to correspond with dopamine 2 receptors. A behavioral study35 suggested that the action of tandospirone on dopamine2 receptors is antagonistic but very weak. From the results of the present experiments, the efficacy of tandospirone as a 5-HT~A agonist cannot be estimated although tandospirone has already been reported to have agonistic activities with high efficacy. For example, the effect of GTP, which is thought to selectively decrease the affinity of agonists, was observed in [3H]tandospirone binding study with hippocampal membrane preparations 37. Furthermore, tandospirone induced 5-HT behavioral syndrome (flat body posture), inhibited the activity of forskolin-stimulated adenylate cyclase39, and decreased the neuronal activities of the hippocampal pyramidal cells 19 and 5-HTergic neuronal cells in the dorsal raphe nucleus 14. These actions were blocked by 5-HT1A antagonists. In the Vogel conflict test, a simple and reliable method that can estimate the clinical potency of compounds as anxiolytic44, tandospirone showed a positive action which was not antagonized by Ro 15-1788 (a BZ antagonist), haloperidol (a potent dopamine 2 antagonist) or ketanserin (a selective 5-HT 2 antagonist), but was antagonized by spiperone (dopamine2, 5-HT 2 and 5-HTlA mixed antagonist)a< Based on the results of the present autoradiographic study in which tandospirone preferentially bound to 5-HT~A receptors and from the Vogel
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conflict test, we conclude that the anxiolytic effect of tandospirone is at least partially mediated by central 5-HT~A receptors. It has been reported that 5-HT~A receptors in the central nervous system consist of two functionally diverse types. One found at high density in the hippocampus and septum located on postsynaptic neurons of 5-HTergic terminals ('postsynaptic receptor') ~1'43 and the other found at high density in the dorsal raphe nucleus located on the 5-HT neuronal cell bodies and which regulate the activities of 5-HT neurons ('autoreceptor') 6'4°. 5-HT1A agonists at the 'autoreceptors' will inhibit the firing of 5-HT neurons and decrease the release of 5-HT from nerve terminals, whilst at the 'postsynaptic receptors' it will mimic endogenous 5-HT and decrease the firing of neurons 19'23"25. It is of interest to determine which type of 5 - H T I A receptor contributes to the anxiolytic effect of tandospirone. In the conflict test using rats, a 5-HT neuronal lesion induced by 5,7-dihydroxytryptamine, which inhibits the action of 5-HTIA agonists for 'autoreceptors', failed to decrease the anti-conflict activity of tandospirone 38. We think that 'postsynaptic receptors' play a more important role in the anxiolytic effect of tandospirone rather than the 'autoreceptors'. Electrophysiological evidence supports this hypothesis; the sustained administration of tandospirone TM and gepirone 2 in rats produced desensitization of 'autoreceptors' in the dorsal raphe nucleus but
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Fig. 5. Saturation analysis and Scatchard plot of [3H]tandospirone binding to the dentate gyms. Adjacent coronal sections taken at A = -3.3 mm from the bregma were incubated with various concentrations (2-20 nM) or [3H]tandospirone. Data comprise typical results from 4 independent experiments. A, saturation analysis, O, total binding; A, non-specific binding; O, specific binding; B, Scatchard plot.
Fig. 6. Comparison of the regional distribution of specific [3H]tandospirone binding sites and specific [3H]fludiazepam binding sites in the rat brain. Adjacent coronal sections were incubated with 2 nM [3H]fludiazepam. Data are the mean --- S.E.M. of 3-4 independent experiment. CA1-3, CA1-3 area of Ammon's horn; CeA, central amygdaloid nucleus; Cg, cinguiate cortex; CoA, cortical amygdaloid nucleus; CP, caudate putamen; DG, dentate gyms; DR, dorsal raphe nucleus; EC, entorhinal cortex; FC, frontal cortex, IP, interpeduncular nucleus; LS, lateral septum; MR, median raphe nucleus; MS, medial septum; SN, substantia nigra; VH, ventral hypothalamus.
187 TABLE II
TABLE III
Effect of neurotransmitters and drugs on specific [3H]tandospirone binding in the rat brain
Dissociation constants and maximal number of binding sites of [3H]tandospirone in the rat brain
Coronal sections taken at A = +0.7 mm (LS), -3.3 mm (DG, CA1) and -7.8 mm (EC, DR) from the bregma were used. Data are the mean + S.E.M. of 4 independent experiments. NE, norepinephrine; DA, dopamine; DG, dentate gyrus; CA1, CA1 area of Ammon's horn; DR, dorsal raphe nucleus; EC, entorhinal cortex.
Coronal sections taken at A = +0.7 mm (lateral septum), -3.3 ram (dentate gyms, CA1 area) and -7.8 mm (entorhinal cortex, dorsal raphe nucleus) from the bregma were incubated with various concentrations of [3H]tandospirone (2-20 nM). Data are the mean -+ S.E.M. of 4 independent experiments.
Drugs (O.11tM)
[3H]tandospirone binding (% of specific binding)
Brain structure
Kd (nM)
DG
CA1
DR
EC
LS
B,,~ (fraollmg tissue)
NE DA 5-HT ACh GABA 8-OH-DPAT Pindolol Buspirone TFMPP Ketanserin Ro 15-1788 Fludiazepam
106+8 107 + 5 5+3 103 + 5 93+6 4+2 16 + 4 14 + 5 47 + 12 97 + 6 102+11 96 + 8
105+4 102 + 4 6+3 105 + 3 98+7 6_+3 21 + 7 15 _+7 49 _+7 101 _+3 96+4 103 + 6
113+14 98 + 3 8+4 99 + 2 95-+7 6+4 8 -+ 5 15 -+ 3 62 _+7 94 _+6 95-+6 90 -+ 4
102+2 93 + 4 5+2 102 -+ 7 108+11 8_+4 12 + 3 15 + 3 59 + 6 99 -+ 10 91-+7 92 _+6
100+3 98 + 1 13+6 104 + 5 101_+3 4+2 25 -+ 10 32 -+ 5 68 + 6 94 + 4 100+3 96 _+5
Dentate gyrus CA1 area Lateral septum Dorsal raphe nucleus Entorhinal cortex
12.1 + 0.6 11.4 + 2.0 8.9 + 2.0 12.0 + 3.2 11.2 + 2.7
114 + 16 70.0 + 11.2 74.4 + 11.2 76.7 + 13.9 119 + 26
did not influence the sensitivity of 'postsynaptic receptors'. However, the anti-conflict activity of tandospirone was not suppressed by repeated administration 35. M a n y investigators ~°'42 maintained that the anxiolytic effects of 5-HTIA agonists (buspirone, ipsapirone and 8 - O H - D P A T ) involved actions on 'autoreceptors'. Durish 7 pointed out that BZs also suppress 5-HT neuronal activities, and the anxiolytic effects of buspirone, ipsapirone and 8 - O H - D P A T are produced at similar doses to those inducing hyperphagic effects which are thought to be mediated by 'autoreceptors '12. In addition, 8-OH-DPAT, buspirone and ipsapirone applied with very small iontophoretic currents markedly depress firing of 5-HT neurons in the raphe nuclei 6'4°. That is, this hypothesis based on the theory that 'autoreceptors' are more sensitive to the action of agonists than 'postsynaptic receptors' and the inhibition of 5-HTergic impulse flow induced by both B Z and 5-HTIA agonist represent an important c o m m o n pathway in their anxiolytic activity. Basse I has observed that 5-HTIA agonists administrated systemically at a lower dose than that which effects 5-HT neurons in the dorsal raphe nucleus decreased firing of some kind of neurons in the hippocampus. In our experiments, the dissociation constant of [3H]tandospirone binding was uniform in all brain structures examined (Table III), suggesting that tandospirone binds to 'autoreceptors' and 'postsynaptic receptors' with the same affinities. It is important to interpret the actions of 5-HTIA agonists as net effects on functions regulated by
5-HTergic neurons in the brain. Wree 45 has observed that ipsapirone administered systemically at a dose which induces anxiolytic effects suppresses neuronal activities not only in the dorsal raphe nucleus but also in the hippocampus and septum. This effect in the hippocampus and septum is due to the direct action of ipsapirone on the 'postsynaptic receptors', suggesting that in the areas where 'postsynaptic receptors' exist at high density the agonistic action on 'postsynaptic receptors' dominates that on 'autoreceptors'. Certain brain regions have been suggested to be associated with anxiety. Papez 28 first proposed that the hypothalamus, anterior thalamic nuclei, cingulate gyms, hippocampus, and their interconnections constitute a neuroanatomical circuit involved in anxiety. Subsequent behavioral and pharmacological studies in animals and positron emission tomography scanning studies in humans have implicated many brain structures 24. It appears that key neuroanatomical substrates of anxiety include the limbic regions and other areas that may modulate limbic regions 17'24. Many brain structures where [3H]tandospirone binding sites exist at high density belong to these areas (e.g the hippocampus, lateral septum, entorhinal cortex and cingulate cortex), that probably participate in the anxiolytic effect of tandospirone. Especially, the hippocampus contains both 5-HT1A receptors and B Z receptors at high density (Fig. 6), and 5-HT1A agonists 6' 19.4o and B Z 18 commonly suppress neuronal activities in this region. Furthermore, Miyazaki 27 has shown that microinjection of tandospirone into the hippocampus induces anti-conflict effects in rats. Therefore, the agonistic action on the postsynaptic 5-HT1A receptors in the hippocampus could play the most important role in the anxiolytic effects of tandospirone.
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