Chronic buspirone treatment decreases 5-HT1B receptor densities and the serotonin transporter but increases the density of 5-HT2A receptors in the bulbectomized rat model of depression: an autoradiographic study

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Chronic buspirone treatment decreases 5-HT1B receptor densities and the serotonin transporter but increases the density of 5-HT2A receptors in the bulbectomized rat model of depression: an autoradiographic study Hiroki Sato a,1 , Ivan Skelin a,2 , Mirko Diksic b,⁎ a

Cone Neurosurgical Research Laboratory, Department of Neurology and Neurosurgery, and Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada b Faculty of Medicine Osijek, J. J. Strossmayer University of Osijek, Osijek, Croatia

A R T I C LE I N FO

AB S T R A C T

Article history:

The olfactory bulbectomized (OBX) rat model is an animal model of depression. The

Accepted 18 May 2010

deregulation of the serotonergic (5-HT) system is implicated in the pathophysiology of

Available online 23 May 2010

depression. Buspirone is a partial agonist of 5-HT1A receptors and is used in the treatment of depression and anxiety. The aim of this study was to assess, in OBX rats and sham operated

Keywords:

controls, the effect of chronic buspirone treatment on the densities of 5-HT2A and 5-HT1B

Olfactory bulbectomy

receptors, as well as the 5-HT transporter (5-HTT), which are all important mediators of 5-HT

Serotonin

transmission. Male Sprague-Dawley rats (180-240 g) were used. Two weeks following the

5-HT receptors

surgeries, the rats were assigned into the saline or treatment groups, receiving either saline,

5-HT transporter

or 10 or 20 mg/kg day of buspirone, for 2 weeks by subcutaneous mini pump. Following the

Autoradiography

treatment, the rats were sacrificed. The autoradiographic experiments were performed

Buspirone

ex vivo using [3H]5-HT for the 5-HT1B receptors, [3H]-ketanserin for the 5-HT2A receptors, and [3H]-paroxetine for the 5-HTT binding. The receptors and 5-HTT densities were quantified in 38 brain regions as well as the pineal body. Chronic treatment with buspirone produced the following: 1) a decrease in the 5-HT1B densities, which was more pronounced in the Sham

⁎ Corresponding author. Department of Neurology and Neurosurgery, and Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. Fax: + 1 514 398 8195. E-mail address: [email protected] (M. Diksic). Abbreviations: ANOVA, analysis of variance; OBX, olfactory bulbectomy; OB, olfactory bulbs; 5-HT, serotonin; 5-HTT, serotonin transporter; 5-HIAA, 5-hydroxyindoleacetic acid; Amy-BL, basolateral amygdala; Amy-BM, basomedial amygdala; Amy-Me, medial amygdala; AcN, accumbens nucleus; AONuc, anterior olfactory nucleus; CA1, hippocampus; CA3, hippocampus; CPl, caudate, putamen (lateral); CPm, caudate, putamen (median); CPGP, caudate-putamen and globus pallidus; CxA, auditory cortex; CxCin, cingulate cortex; CxEn, enthorinal cortex; CxF, frontal cortex; CxP, parietal cortex; CX-PL, prefrontal cortex; CxSM, somatosensory cortex; CxV, visual ortex; IC, internal capsul; DS, dorsal subiculum; Hyp, hypothalamus; GP, globus pallidus; LC, locus coeruleus; LG, lateral geniculate; RD-d, dorsal raphe (dorsal part); RD-lat, dorsal raphe (lateral part); RD-v, dorsal raphe (ventral part); MFB, median forebrain bundle; MG, median, geniculate; R-med, median raphe; PB, pineal body; R-mag, raphe magnus; Rpo, raphe pontine; SC, superio colliculus; Scn, suprachiasmatic nucleus; SepN, septal nucleus; SNc, substantia nigra compacta; SNr, substantia nigra reticulata; SPD, Sprague-Dawley; ThD, thalamus (dorsal); ThV, thalamus (ventral); VTA, ventral tegmental area 1 Permanent address: Department of Neurosurgery, University of Yamanashi, 1110 Shimokato Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan. 2 Current address: Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.05.054

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rats; 2) an increase in the 5-HT2A receptor densities, which was more pronounced in the Sham rats; and 3) an decrease in 5-HTT densities in both groups. The results indicate differential effects of chronic antidepressant treatment on the 5-HT system regulation in the OBX model of depression and normal rats. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

The alteration of brain serotonin (5-hydroxytryptamine; 5-HT) neurotransmission has been implicated in the etiology of depression, obsessive compulsive disorder, aggression, and anxiety (Blier and de Montigny, 1999; Judd, 1995; Nestler et al., 2002). Fourteen different 5-HT receptor subtypes have been identified and grouped in families from 5-HT1 to 5-HT7 (Nichols and Nichols, 2008). It has been shown, in both human and animal studies, that 5-HT1A (Blier et al., 1993; Nishi et al., 2009), 5-HT1B (Benjamin et al., 1990; Clark et al., 2002;Kaiyala et al., 2003; O'Connor et al., 1994; Saudou et al., 1994; Sari, 2004), 5-HT2A (Bhagwagar et al., 2006), and a 5-HT transporter (5-HTT) (Bhagwagar et al., 2007) play an important role in affective disorders, and in the action of antidepressants. It has been demonstrated that dysfunction in the brain serotonergic system relates to mood and behavior related disorders (Soares and Mann, 1997). We reported that buspirone, a drug with antidepressant properties (Goldberg and Finnerty, 1982; Schweizer et al., 1986; Kennett et al., 1987; Watanabe et al., 2006), needs to be administered at a dose of 20 mg/kg/day for the alleviation of behavioral hyperactivity of OBX rats (Sato et al., 2008), and that there is a difference in the density of 5-HT1A receptors between the OBX and sham operated rats treated with saline. The normalization of behavioral hyperactivity in the open field is taken to indicate antidepressant action of a particular drug. Given the importance of other parameters of brain 5-HT neurotransmission (see above), the fact that buspirone has antidepressant properties, and the known relationship between some of these properties and behavior, it is important to study these parameters in the OBX rat model of depression. Buspirone is a 5-HT1A partial agonist at post-synaptic sites and a full agonist at the pre-synaptic receptor sites (De Vivo and Maayani, 1986; Hoyer and Boddeke, 1993). This drug also displays antagonistic properties at the D2 receptors (Piercey et al., 1994), and its metabolite 1-(2-pyrimidinyl-piperazine) is an α2-adrenergic antagonist (Caccia et al., 1986). Buspirone has both anxiolytic and antidepressant properties (Kennett et al., 1987). In addition, many full and partial agonists of 5-HT1A receptors help alleviate depressive symptoms in animal models of depression (Lucki et al., 1994), suggesting their importance in affective disorders. Animal models of major depressive disorder (MDD) should have three different values/validities (Willner and Mitchell, 2002): (1) face validity (how closely the model resembles the psychiatric condition); (2) construct validity (the consistency of the model with the theoretical rationale); and (3) predictive validity (how closely the action of the drugs in the model resemble the actions of these drugs in the human disease). The OBX rat model of depression possesses all three of these values (Kelly et al., 1997a,b).

The OBX rat model of depression has been widely accepted as a good rat model with many similarities to the agitated form of human depression (Jesberger and Richardson, 1988; Willner, 1990; Kelly et al., 1997a,b). The bilateral removal of the olfactory bulbs in rats produces behavior, endocrine, neurochemical and neuronal connectivity changes, which resemble deficiencies in humans with depression. These changes can be reversed by chronic, but not acute, treatment with antidepressants, including buspirone (Fabre, 1990; Willner, 1990; Watanabe et al., 2006; Hasegawa et al., 2005). It has been shown that the OBX syndrome is not the result of anosmia. Rather, it is the result of widespread, long-lasting changes in synaptic connectivity and neuronal activity in the brain, especially the olfactory-limbic circuitry (Kelly et al., 1997a,b; Zhou et al., 1998). The lesion induced reorganization in the limbic and cortical brain structures appears to be responsible for the secondary behavioral abnormalities present two weeks following OBX (van Rizen and Leonard, 1990). Because these behavioral deficiencies are normalized by chronic, and not acute, treatments with SSRIs, the impairment of the brain serotonergic system may play an important role in the mediation of behavioral and neurochemical changes observed in the OBX rats (Song and Leonard, 1995; Grecksch et al., 1997; Kelly et al., 1997a,b). It has also been found that brain 5-HT synthesis is elevated throughout the brain following OBX surgery (Watanabe et al., 2003; 2006; Hasegawa et al., 2005), an abnormal brain 5-HT concentration (Lumia et al., 1992; Zhou et al., 1998), and limbic region receptor expression (Zhou et al., 1998). The importance of the imbalance in the brain serotonergic system is further confirmed in the OBX rats by the 5,7dihydroxytryptamine lesion of the olfactory bulbs, producing the full behavioral profile of OBX syndrome (Cairncross et al., 1979). Many psychotropic drugs, other than antidepressants, have no effect on the normalization of OBX-induced changes (Song and Leonard, 2005). 5-HT1B receptors act as autoreceptors and/or heteroreceptors in the 5-HT neuron terminal regions (e.g., the basal ganglia, cortices, dorsal subiculum, and amygdala) (Nichols and Nichols, 2008). The mechanism of the 5-HT1B receptors’ involvement in the pathophysiology of depression and/or the action of antidepressants is not yet clear, but some reports suggest the down regulation and/or desensitization of these receptors by antidepressants (Blier et al., 1988; O'Connor et al., 1994; Davidson and Stamford, 1995). Significant differences in the densities of 5-HT1B receptors are reported between another rat model of depression, the Flinders Sensitive Line (FSL), and normal Sprague-Dawley rats (Nishi et al., 2009), suggesting that these receptors may play an important role in the rat model of depression and quite possibly in human depression itself. In addition, the importance of 5-HT1B receptors has already been established in the pathogenesis of migraine headaches and the action of antimigraine medications (Lance,

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1991; Raskin, 1991; Sakai et al., 2008). Because of the involvement of the 5-HT1B receptors in the pathophysiology of brain disorders, similar involvement could be expected in the OBX rat model of depression. 5-HT2A receptors are postsynaptic heteroreceptors in most of the 5-HT projection areas, particularly the neocortex (Pazos et al., 1987). 5-HT2 receptor densities were measured in OBX rats (Earley et al., 1994) in several brain regions using autoradiography, but only a limited number of brain regions were investigated. For example, the densities of 5-HT2A receptors in a very small number of limbic structures were examined. In addition, brain stem regions, the regions from which all 5-HT cell bodies initiate, were not included in that study. Given this information, it was important to perform a detailed study of 5-HT2A receptors in the OBX rat model of depression. The concentration of 5-HT in the synaptic cleft is controlled by an interplay between 5-HT release (Kiss, 2008), reuptake by a 5-HT transporter (5-HTT) (Rudnick and Clark, 1993), synthesis, and degradation. It has been proposed that the balance between these processes is altered in psychiatric disorders (Fuller and Wong, 1990). Post-mortem studies of the human brain yields very valuable information, but the heterogeneity of the samples often created discordant results. Because of these inevitable difficulties, studies in animal models of depression, including the OBX rat model, can help us obtain a better understanding of the mechanisms by which depressive symptoms develop and their subsequent alleviation through treatment. Also, treatment with antidepressants alter brain 5-HT receptor binding in depressed humans (Meyer et al., 1999; 2001; Yatham et al., 1999), normal rats (Hrdina and Vu, 1993; Blier and de Montigny, 1990; Jolas et al., 1994; Esteban et al., 1999) and in the OBX rat model of depression (Earley, et al., 1994). However, we have yet to develop a solid understanding of the relationship between the neurochemical and behavioural changes in both the OBX rat model and human

depression, and how these neurochemical changes relate to the behavioural changes. Based on previous studies (see above), the working hypotheses of the present studies were the following: 1) The densities of 5-HT1B receptors/autoreceptors in raphe nuclei, which control 5-HT synthesis and release (Nichols and Nichols, 2008), will be greater in the OBX rats treated with saline than in the sham rats treated with saline. These changes will be part of the brain 5-HT system homeostatic response to elevated 5-HT synthesis (Watanabe et al., 2003) and elevated extracellular levels of 5-HT (Zhou et al., 1998) in OBX rats; 2) The densities of the 5-HT2A receptors, which are primarily heteroreceptors, will not be different in the limbic structures of the OBX and sham rats treated with saline, and some normalization (equalization of relative differences) of the 5-HT2A receptor densities in the motor brain structures will occur with a higher dose of buspirone, the same dose which produced the open field normalization; 3) OBX rats treated with saline, assumed to resemble the naïve OBX rats, will have elevated densities of 5-HTT throughout the brain, as found in the frontal cortex by Zhou et al. (1998), and the elevation will be particularly pronounced in the brain limbic and motor areas; 4) Assuming a relationship between the normalization of behavior in the OBX rats with a buspirone treatment of 20 mg/kg/day (Sato et al., 2008), and other serotonergic parameters (e.g., 5-HT synthesis; Watanabe et al., 2006), this treatment will produce changes in some of the 5-HT receptors (e.g., 5-HT1B and 5-HT2A sites in the brain motor structures) in the OBX rats; 5) Buspirone treatments will not produce great changes in the sham operated animals (these animals are essentially normal rats and, as such, no antidepressant activity of buspirone should be present); and 6) The changes in these 5-HT parameters will be most pronounced in the brain limbic regions, associated with mood related behavior, and in the motor regions, associated with movement related behavior.

Fig. 1 – A schematic presentation of the regions read as Amy-BL, Amy-BM, Amy-Me (part A), RD-d, RD-v, and RD-lat (Part B). These were adapted from the cross-sections given in reference (Paxinos and Watson, 1997; with permission).

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2.

Results

Outlines for the quantization of some brain regions are provided in Fig. 1, and a summary of comparisons made is summarized in Table 1. A set of autoradiograms depicts regional distributions and heterogeneities of the 5-HT1B (Fig. 2) and 5-HT2A (Fig. 3) receptors, as well as 5-HTT (Fig. 4), in the sham and OBX rats treated with saline or buspirone (20 mg/kg/day; -B20). A qualitative evaluation of these images clearly shows that all of these parameters of 5-HT neurotransmission are quite heterogeneous throughout the rat brain. The quantitative values of the specific bindings to different receptors are provided in Tables 2–4, and are expressed in pmol/mg-tissue.

2.1.

Densities of 5-HT1B receptors

The quantitative values of the receptor densities in 39 brain regions and the pineal body are provided in Table 2 for each of the sham operated and OBX group of rats. To obtain a better appreciation of the effects, a bar graph showing the effects of saline (differences between the SHX-SAL and OBX-SAL groups) and the treatments of 20 mg/kg/day (differences between the SHX-B20 and OBX-B20 groups) in a priori selected brain regions is presented in Fig. 5. From the same graph, one can notice a large effect of the OBX surgery in Amy, AONu, ThV, LC and the dorsal raphe. Similarly, significant effects of buspirone treatment (20 mg/kg/day) were observed in the Amy, AONu, CA1, CA3, Rmag, R-med, and VTA. One can see (Table 2) that the effect of a 10 mg/kg/day treatment produced a smaller effect on the

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density of the 5-HT1B receptor in the sham operated rats (significant in 16 out of 23 a priori selected regions; SHX-SAL group compared to the SHX-B10 group) than that produced in the OBX rats (significant in all 26 a priori selected regions; the OBX-SAL group compared to the OBX-B10 group), but overall, in both groups, the effects were significant (in the sham group, F (1,10) = 8.6; p < 0.02; in the OBX group, F(1,10) = 783; p < 0.001). In the sham rats, 20 mg/kg/day of buspirone decreased the 5HT1B receptor densities (when comparing the SHX-B10 and SHXB20 groups). A statistical comparison of the effect produced by a 10 mg/kg/day treatment in the sham and OBX rats in a priori selected regions revealed a significantly greater effect in the OBX than sham operated rats (the difference between the OBX-SAL and OBX-B10 groups was greater than the difference between the SHX-SAL and SHX-B10 groups; F(1,30) = 323; p < 0.001). Two-way ANOVA analyses of data from all six groups in a priori selected brain regions revealed significant differences between groups (F(5,30)= 548; p < 0.001) and the group-brain region interaction (group*region interaction F(125,750)=1378; p <0.001). The Newman-Keuls post hoc test on brain region specific ANOVA revealed significant differences in all 23 brain regions when the SHX-SAL and SHX-B20 groups were compared, and in all brain regions, except the CA3 region of the hippocampus, when the OBX-SAL and OBX-B20 groups were compared (Table 2). The statistical evaluation of other brain regions not included in the a priori selected regions revealed significant differences in all of them when the SHX-SAL and SHX-B20, and OBX-SAL and OBX-B20 groups were compared (Table 2). The two-way ANOVA comparison between the SHX-SAL and OBX-SAL groups revealed significant differences in the 5-HT1B

Fig. 2 – Representative autoradiograms exemplifying the heterogeneity of the 5-HT1B receptor distributions in the Sham-SAL, OBX-SAL, Sham-B20, and OBX-B20 rats.

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Fig. 3 – Representative autoradiograms exemplifying the heterogeneity of the 5-HT2A receptor distributions in the Sham-SAL, OBX-SAL, Sham-B20, and OBX-B20 rats.

site densities in many limbic structures (Amy-BL, Amy-BM, Amy-Me, AN, AONu, CA3, Cx-PL, DS, and ThV) and all of the brain stem structures, except the VTA (Table 2). In the brain regions responsible for movement, there was only a significant difference in the SNr between the SHX-SAL and OBX-SAL groups. A statistical comparison between the SHX-B20 and OBX-B20 groups revealed significant differences between the groups (F(1,10) = 9.2; p < 0.02) and group*region interaction (F(25,250)= 97; p < 0.001), with significant differences revealed following a post hoc correction in 21 (not different were LC and SNc) out of 23 a priori selected brain regions (Table 2). A statistical comparison between the SHX-B10 and OBX-B10 groups revealed significant differences between the groups (F(1,10) = 766; p < 0.001) and group*region interaction (F(25,250)=2011; p < 0.001). A post hoc analysis found significant differences in all a priori selected brain regions. Additional exploratory evaluations in other brain regions showed significant effects in all other brain regions (Table 2).

A formal statistical test of the relative effects of the sham and OBX rats that received the 20 mg/kg/day buspirone treatment by comparing the effects relative to the saline group revealed that the effect in the sham and OBX rats (SHX-SAL minus SHX-B20, compared to OBX-SAL minus OBX-B20) is not significantly different (p> 0.5). However, the absolute changes produced in the OBX rats by the 20 mg/kg/day buspirone treatment were greater than those produced in the sham operated rats, and in some regions (CA3, ThV, and MG), the effects were opposite: there was a reduction in the sham group and an elevation in the OBX rats.

2.2.

Densities of 5-HT2A receptors

The quantitative values of the receptor densities in 38 brain regions and the pineal body are provided in Table 3 for each of the sham operated and OBX groups of rats. As above, the main statistical evaluations were performed on a priori selected

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Fig. 4 – Representative autoradiograms exemplifying the heterogeneity of the 5-HTT site distributions in the Sham-SAL, OBX-SAL, Sham-B20, and OBX-B20 rats.

brain regions. Two-way ANOVA analyses on the data from all six groups revealed a significant group-brain region interaction (group*region interaction F(125,750)=154; p < 0.001), as well as a significant group effect (F(5,30)=157.5; p < 0.001). Further statistical comparisons using planned comparisons revealed, overall, significantly greater 5-HT2A receptor density in the OBX-SAL rats relative to the SHX-SAL group (F(1,30)=146; p < 0.001), with significant differences in 20 brain regions out of the 23 a priori selected, when the densities in these two groups were compared. There were no significant differences in the CxCin, LC and Rpo. Buspirone treatment of 10 mg/kg/day produced an overall increase of the 5-HT2A receptor in the sham operated rats (SHX-SAL compared to SHX-B10; F(1,30) = 72.5; p < 0.001). The same treatment also produced an overall increase in the 5-HT2A receptor density in the OBX rats (OBX-SAL compared to OBX-B10; F(1,30)=145; p < 0.001). Comparisons of the densities between the SHX-B10 and OBX-B10 groups in the a priori selected brain regions revealed significant differences in all of the brain regions except the Cx-PL. The treatment with 20 mg/kg/day of buspirone produced an overall increase in the density of the 5-HT2A receptors in both the sham (F(1,30) = 472; p < 0.001) and OBX (F(1,30)=11.4; p < 0.003) rats, but the increase was greater in the sham operated rats (F(1,30)=168; p < 0.001; comparing differences between the SHX-SAL and SHX-B20 groups to those between the OBX-SAL and OBX-B20 groups). Post hoc comparisons between the SHX-B20 and OBX-B20 groups revealed significant differences in all a priori selected brain regions, with the densities being lower in the OBX-B20 rats. The buspirone treatment (20 mg/kg/day) produced large effects in some limbic

structures (e.g., Amy, Hipp, Cx-PL, and Hyp), as well as in the brain dopaminergic regions (SN, VTA) (Fig. 6). Statistical comparisons of the 5-HT2A receptor densities and significant differences obtained in other brain regions between the different groups are summarized in Table 3.

2.3.

Densities of 5-HTT

The quantitative values of the 5-HT transporter densities in 38 brain regions and the pineal body are provided in Table 4, both in the sham and OBX rats, for saline and the 20 mg/kg/day buspirone group (note that the two brain regions were divided into three parts; see the Methods section). Because there was no significant effect of the 10 mg/kg/day buspirone treatment in either the sham or OBX rats, and it was reported that behavior is normalized only with a 20 mg/kg/day treatment, the actual transporter densities in rats treated with 10 mg/kg/ day are not provided. In addition, the transporter densities in the 10 mg/kg/day buspirone group were more or less the same as that for the 20 mg/kg/day group. This suggests that neurochemical changes related to this site occur before the behaviour changes. A two-way ANOVA comparison of the effect of the sham operated rats for a priori selected brain regions, with the SHX-SAL group compared to the SHX-B20 group, revealed a significant group*region interaction; F(23,230)= 192; p < 0.001. A post hoc comparison revealed a significant effect of buspirone treatment in all regions except the Amy-BL, AONu, CA1, CxEn, DS, SNr, SNc, and RD-v. A similar comparison between the OBX-SAL and OBX-B20 groups revealed significant group*region interaction: F(23,230)=174; p < 0.001. A post hoc planned comparison revealed a significant effect of the

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Table 1 – Groups compared. Group

Compared to

SHX-SAL SHX-SAL OBX-SAL Difference between SHX-SAL and SHX-B10 Difference between SHX-SAL and SHX-B20 SHX-B10

SHX-B20

OBX-SAL (effect of the OBX surgery) SHX-B10; SHX-B20 (effect of buspirone) OBX-B10; OBX-B20 (effect of buspirone) Difference between OBX-SAL and OBX-B10 (magnitude of the effect compared) Difference between OBX-SAL and OBX-B20 (magnitude of the effect compared) OBX-B10 (compares densities in two treated groups to see if normalization occurred) OBX-B20 (compares densities in two treated groups to see if normalization occurred)

treated with saline, and between the Sham-B20 and OBX-B20 groups, suggesting a small influence of OBX surgery and treatment on the transporter densities; 4) buspirone treatment had a profound effect on the 5-HT2A receptors in both the sham and OBX rats, and the effects were greater in the sham rats. Differences between the Sham-SAL and OBX-SAL groups suggest a profound effect of OBX surgery on the densities of 5-HT2A receptors; and 5) the previously reported normalization of hyperactivity in open field (the behavioral change related to the antidepressant action of buspirone), with a chronic treatment of 20 mg/kg/day of buspirone (Sato et al., 2008), correlates with the changes in the 5-HT1B and 5-HT2A receptors, particularly those in the brain limbic structures (Tables 2 and 3).

3.1. The effect of OBX and buspirone on 5-HT1B receptor density buspirone treatment in all of the regions except the Amy-BL, CA3, DS, RD-later, RD-v, and RD-d. Differences between the Sham-SAL and OBX-SAL groups, and the Sham-B20 and OBX-B20 groups, are presented as a graph in Fig. 7. The most prominent effect of the buspirone treatment was observed in the Rpo (OBX-SAL group compared to OBX-B20 group), while the most prominent effect of the OBX surgery (SHX-SAL group compared to OBX-SAL group) was found in the CA1 and CA3 layers of the hippocampus. In addition to evaluating the buspirone effect, the effect of surgery (the sham of OBX) could be evaluated, assuming there is no differential influence in the two groups of the mini pump itself on the results. Two-way ANOVA comparisons between the SHX-SAL and OBX-SAL groups revealed an overall significant group*region interaction (F(23,230)=123; p < 0.001). The post hoc planned comparison revealed significant differences in the AONu, CA1, CA3, and DS (Table 4). It should be noted that in other brain regions, the densities of 5-HTT are greater in the OBX-SAL group than in the SHX-SAL group, but not significantly (Table 4). A statistical comparison between the SHX-B20 and OBX-B20 groups revealed a significant group*region interaction (F(23,230) = 84.6; p < 0.001). Following post hoc correction, significant differences were found in the ThV, and Rpo among a priori selected regions (Table 4). Data suggest that buspirone treatments result in the elevation of 5-HTT densities, both in the sham operated controls and OBX rats.

3.

Discussion

The most important findings related to our hypotheses are the following: 1) OBX-SAL rats have a different (greater) overall density of 5-HT1B receptors than the SHX-SAL rats, with important differences in densities found in the limbic, motor and brain stem regions (e.g., Amy, AONu, LC and parts of raphe; Table 2). It is interesting to note the large reductions in 5-HT1B densities produced by the buspirone treatment (20 mg/kg/day) in the AONu, CA1, CX-PL, CxCin, CxEn, R-mag, R-med, and VTA (OBX-SAL relative to OBX-B20); 2) OBX-SAL rats have significantly higher densities of 5-HT2A receptors than SHX-SAL rats (Table 3); 3) 5-HTT densities are different between the SHX-SAL and OBX-SAL groups in only a few brain regions (Table 4). There are relatively small differences between the Sham and OBX rats

In the present study, it appears that OBX produced a region specific elevation of the 5-HT1B sites (OBX-SAL group compared to the SHX-SAL group (Table 2)). This finding is similar to the one in another rat model of depression, the Flinders Sensitive Line (FSL) of rats, where it was found that the density of 5-HT1B receptors is elevated, relative to normal rats (Nishi et al., 2009). This finding is opposite to that which reported a reduction of densities of the 5-HT1A receptors in almost all brain regions investigated in OBX rats, relative to the sham operated rats, treated with saline (Sato et al., 2008). Together, the data suggest that 5-HT1B receptors, which are generally presynaptic at the 5-HT terminals, are influenced much less than postsynaptic 5-HT1A receptors on 5-HT projection regions, and that 5-HT1B receptors are similarly affected in both the FSL and OBX rat models of depression. Because the 5-HT1B receptors in the terminal regions of 5-HT neurons are also present as heteroceptors on cholinergic neurons (Cassel et al., 1995), and the fact that OBX results in a partial de-enervation of cholinergic neurons (Yoshimura and Ueki, 1977; Earley et al., 1994; Kelly et al., 1997a,b; Han et al., 2008), the reduction in the densities of 5-HT1B receptors in many brain regions is not surprising. It is not clear which 5-HT receptors mediate the anxiety-like effects. 5-HT1A receptors have been extensively studied in relation to anxiety-like effects (Critchley et al., 1992), but, based on clinical and animal studies, there is the possibility that 5-HT1B receptors are also involved (Benjamin et al., 1990; Pellow et al., 1987). Similar to the present results (e.g., reduction of 5-HT1B receptors by buspirone), Neumaier et al. (1996) reported a reduction in the 5-HT1B mRNA in the normal rat dorsal raphe following fluoxetine treatment (an antidepressant treatment). However, there are several other studies that report no changes following treatment with fluoxetine or citalopram in the 5-HT1B autoreceptors (Gobbi et al., 1997). This apparent discrepancy (a large reduction was reported here) could also be related to the use of different drugs than that used here, and the fact that the present study was performed on a rat model of depression.

3.2. The effect of OBX and buspirone on 5-HT2A receptor density One can interpret the measured densities of 5-HT2A receptors in the SHX-SAL and OBX-SAL rats in the present study as being in limited agreement with the results reported in a small

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Table 2 – 5-HT1B receptor densities (unit) in sham and OBX rats treated with saline or buspirone (10 or 20 mg/kg/day). Values are shown as mean ± SD. Region Amy-BL Amy-BM Amy-Me AN AONu CA1 CA3 CxCin CxEn Cx-PL DS Hyp SepN ThV Brain Stem LC R-mag R-med RD-d RD-lat RD-v Rpo VTA Motor Structure CPl GP SNc SNr Other Brain Regions Clustrum CPGP CPm CxA CxF CxP CxSM CxV DTh IC LG MFB MG SC Sch Scx PB

SHX SAL (N = 6)*

SHX B10 (N = 6)‡

SHX B20 (N = 6)

OBX SAL (N = 6)$

OBX B10 (N = 6)

OBX B20 (N = 6)&

21.9 ± 2.0¶ 23.1 ± 2.0¶ 22.6 ± 2.0¶,# 69.5 ± 4.3¶,# 9.0 ± 1.0¶ 8.4 ± 1.1# 4.2 ± 0.6¶,# 17.1 ± 1.6# 44.6 ± 3.1# 25.7 ± 2.1¶,# 334.3 ± 10.8¶,# 42.2 ± 3.0# 60.5 ± 3.8# 3.9 ± 0.6¶,#

23.8 ± 1.8 22.2 ± 1.6 18.2 ± 1.5 64.1 ± 3.6 8.9 ± 0.9 4.5 ± 0.6 0.4 ± 0.2 13.7 ± 1.2 35.2 ± 2.4 16.0 ± 1.3 279.5 ± 8.9 32.6 ± 2.2 50.8 ± 3.0 0.2 ± 0.1

6.9 ± 0.6† 6.6 ± 0.5† 5.8 ± 0.5† 20.2 ± 1.1† 2.4 ± 0.2† 1.3 ± 0.2† 0.96 ± 0.04† 3.1 ± 0.3† 9.8 ± 0.7† 5.2 ± 0.4† 77.1 ± 2.4† 11.7 ± 0.7† 14.0 ± 0.8† 0.17 ± 0.03†

31.1 ± 2.5 36.4 ± 2.7 31.4 ± 2.4 76.6 ± 4.5 12.9 ± 1.3 9.2 ± 1.1 3.2 ± 0.5 17.7 ± 1.7 44.6 ± 3.2 29.5 ± 2.3 297.3 ± 9.1 42.7 ± 3.0 56.8 ± 3.6 0.7 ± 0.1

15.6 ± 1.0 13.7 ± 0.9 10.1 ± 0.8 28.1 ± 1.5 4.6 ± 0.4 3.1 ± 0.4 0.8 ± 0.1 5.5 ± 0.5 15.7 ± 1.0 8.9 ± 0.7 93.1 ± 2.7 17.6 ± 1.1 19.1 ± 1.1 0.1 ± 0.0

12.1 ± 0.9 11.3 ± 0.9 10.0 ± 0.8 18.3 ± 1.1 7.7 ± 0.8 5.8 ± 0.7 3.2 ± 0.5 8.5 ± 0.8 14.5 ± 1.0 11.2 ± 0.9 66.9 ± 2.2 13.1 ± 0.9 15.7 ± 1.0 1.8 ± 0.3

39.2 ± 2.9¶ 4.5 ± 0.6¶,# 17.7 ± 1.7¶,# 59.9 ± 3.8¶,# 55.1 ± 3.5¶ 52.1 ± 3.7¶,# 12.9 ± 1.3¶,# 9.2 ± 1.1

38.9 ± 2.5 3.6 ± 0.5 14.2 ± 1.3 53.2 ± 3.1 51.7 ± 3.0 47.5 ± 3.1 9.5 ± 0.9 9.2 ± 1.0

13.3 ± 0.8 0.9 ± 0.1† 2.6 ± 0.3† 16.2 ± 0.9† 14.6 ± 0.9† 13.1 ± 0.9† 2.1 ± 0.2† 1.3 ± 0.2†

57.3 ± 3.6 5.9 ± 0.8 14.5 ± 1.5 76.5 ± 4.2 73.6 ± 4.2 69.6 ± 4.3 11.0 ± 1.2 8.4 ± 1.1

20.5 ± 1.2 2.3 ± 0.3 4.9 ± 0.5 23.7 ± 1.3 22.4 ± 1.3 20.9 ± 1.3 3.4 ± 0.4 2.4 ± 0.3

13.3 ± 0.9 3.5 ± 0.5 9.9 ± 1.0 18.3 ± 1.1 18.2 ± 1.1 17.2 ± 1.1 3.3 ± 0.4 5.7 ± 0.7

36.9 ± 2.6 204.1 ± 11.3 65.6 ± 4.8 386.0 ± 13.8

10.2 ± 0.7† 57.7 ± 3.2† 18.4 ± 1.4 99.3 ± 3.8†

39.9 ± 3.1 192.4 ± 10.0 66.3 ± 5.2 339.8 ± 13.1

15.7 ± 1.1 67.8 ± 3.3 26.9 ± 2.0 114.5 ± 4.1

13.2 ± 1.0 45.3 ± 2.5 17.5 ± 1.5 92.9 ± 3.7

24.3 ± 1.7 41.3 ± 3.0 44.3 ± 3.0 6.6 ± 0.7 13.3 ± 1.2 9.1 ± 0.9 13.8 ± 1.2 6.7 ± 0.7 13.0 ± 1.2 1.9 ± 0.3 10.1 ± 1.0 13.6 ± 1.2 1.5 ± 0.3 65.2 ± 3.6 34.2 ± 2.2 8.4 ± 0.9 0.8 ± 0.2

6.8 ± 0.5† 10.8 ± 0.8† 13.8 ± 0.9 1.4 ± 0.2† 3.4 ± 0.3† 2.2 ± 0.2† 3.1 ± 0.3† 1.6 ± 0.2† 4.6 ± 0.4† 1.1 ± 0.2† 2.7 ± 0.3† 3.6 ± 0.3† 0.3 ± 0.1† 20.2 ± 1.1† 7.5 ± 0.5† 2.0 ± 0.2† 0.3 ± 0.1†

31.3 ± 2.4 43.7 ± 3.4 49.4 ± 3.7 10.6 ± 1.2 17.2 ± 1.7 13.1 ± 1.4 17.7 ± 1.7 9.4 ± 1.1 16.3 ± 1.6 5.5 ± 0.8 14.7 ± 1.5 14.0 ± 1.5 2.7 ± 0.4 83.0 ± 4.7 44.0 ± 3.1 11.1 ± 1.2 0.9 ± 0.2

12.0 ± 0.8 17.1 ± 1.2 20.0 ± 1.3 3.2 ± 0.3 5.4 ± 0.5 4.1 ± 0.4 6.5 ± 0.6 2.8 ± 0.3 7.0 ± 0.6 1.3 ± 0.2 5.2 ± 0.5 5.4 ± 0.5 0.2 ± 0.1 28.2 ± 1.4 10.2 ± 0.7 3.4 ± 0.4 0.2 ± 0.0

11.8 ± 0.9 13.0 ± 1.0 14.5 ± 1.0 6.6 ± 0.7 8.3 ± 0.8 7.9 ± 0.8 8.7 ± 0.8 6.2 ± 0.7 8.1 ± 0.8 0.8 ± 0.2 7.8 ± 0.8 7.9 ± 0.8 3.2 ± 0.5 17.8 ± 1.1 11.6 ± 0.9 6.6 ± 0.7 2.9 ± 0.5

38.7 ± 3.1 205.0 ± 12.3 65.6 ± 5.4 387.1 ± 14.7¶ 28.1 ± 2.2¶,# 41.9 ± 3.4 49.3 ± 3.7# 9.4 ± 1.1# 16.7 ± 1.6# 12.8 ± 1.3# 17.2 ± 1.7# 9.4 ± 1.0# 17.4 ± 1.7# 8.7 ± 1.1¶,# 15.1 ± 1.6# 16.5 ± 1.6¶,# 4.7 ± 0.7¶,# 83.1 ± 4.7# 45.8 ± 3.1# 11.7 ± 1.2# 1.7 ± 0.3

¶ Significantly different from the value in the OBX_SAL group. ‡ All values are significantly different from the values in the OBX-B10 group. † Significantly different from the value in the OBX-B20 group. # Significantly different from the value in the SHX-B10 group. * All values are significantly different from the values in the SHX-B20 group. $ All values are significantly different from the values in the OBX-B10 group. & All values are significantly different from the values in the OBX-SAL group except the value in the CA3.

number of the brain regions by Earley et al. (1994). In general, the 5-HT2A receptor densities reported here are greater, but there is strong accordance with the relative values for the few structures measured in both studies. It is important to note that in the present study, a significant difference was found between the SHX-SAL and OBX-SAL groups of rats in all of the limbic brain regions, except the cingulate cortex, but including

the amygdala, AONu, Hyp, AN, and CPl, while Earley et al. (1994) did not find any significant differences in these limbic structures. In addition, we found significant differences in other limbic structures (Table 3) that were not evaluated by Earley et al. (1994). From the presented data, one can conclude that the densities of the 5-HT 2A receptors are greatly influenced by OBX surgery as well as buspirone treatment. It

36

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Table 3 – 5-HT2A receptor densities (unit) in sham and OBX rats treated with saline or buspirone (10 or 20 mg/kg/day). Values are shown as mean ± SD. Limbic Structures Amy-BL Amy-BM Amy-Me AN AONu CA1 CA3 Cx-PL CxCin CxEn DS Hyp SepN ThV Brain Stem LC R-mag R-med RD RD-d RD-lat RD-v Rpo VTA Motor Structures CPGP CPl GP SNc SNr Other Brian Regions Clustrum CPm CxA CxF CxP CxS CxSM CxV DTh IC LG MFB MG PB SC Sch

SHX SAL (N = 6)*

SHX B10 (N = 6)

SHX B20 (N = 6)

OBX SAL (N = 6)$

OBX_B10 (N = 6)

OBX B20 (N = 6)

5.5 ± 0.3¶,# 9.5 ± 0.3¶,# 7.6 ± 0.2¶,# 29.1 ± 1.4¶ 20.3 ± 0.6¶,# 7.0 ± 0.2¶,# 9.7 ± 0.2¶,# 41.1 ± 2.3¶,# 25.3 ± 1.4 9.9 ± 0.5¶,# 9.4 ± 0.2¶,# 10.8 ± 0.3¶,# 6.6 ± 0.2¶,# 7.2 ± 0.2¶,#

13.9 ± 0.3‡ 13.5 ± 0.3‡ 12.1 ± 0.3‡ 28.7 ± 1.3‡ 21.4 ± 0.5‡ 12.5 ± 0.3‡ 13.7 ± 0.3‡ 35.9 ± 1.8 25.4 ± 1.1‡ 17.6 ± 0.4‡ 10.3 ± 0.2‡ 13.5 ± 0.3‡ 13.1 ± 0.3‡ 6.7 ± 0.1‡

21.5 ± 0.7† 19.4 ± 0.5† 18.8 ± 0.6† 32.7 ± 1.7† 24.9 ± 0.7† 18.4 ± 0.5† 20.3 ± 0.6† 32.0 ± 1.9† 26.8 ± 1.6† 23.6 ± 0.8 17.4 ± 0.4† 20.0 ± 0.6 17.6 ± 0.4† 15.8 ± 0.5†

18.1 ± 0.5 16.8 ± 0.4 14.9 ± 0.4 27.0 ± 1.3 22.9 ± 0.5 14.9 ± 0.3 16.4 ± 0.3 31.1 ± 1.7 26.5 ± 1.5 23.2 ± 0.7 14.5 ± 0.3 16.4 ± 0.4 13.6 ± 0.3 13.5 ± 0.4

22.9 ± 0.6 21.7 ± 0.5 19.0 ± 0.5 32.3 ± 1.6 24.0 ± 0.6 20.8 ± 0.4 21.4 ± 0.5 35.1 ± 1.9 28.4 ± 1.7 25.7 ± 0.8 19.7 ± 0.5 23.5 ± 0.5 17.7 ± 0.4 15.4 ± 0.5

19.7 ± 0.6& 18.1 ± 0.4& 15.1 ± 0.4 27.5 ± 1.2 21.0 ± 0.5& 14.9 ± 0.2 17.0 ± 0.3& 28.5 ± 1.4& 23.2 ± 1.2& 22.4 ± 0.7 15.7 ± 0.4& 20.1 ± 0.4& 14.8 ± 0.3& 14.1 ± 0.3&

23.5 ± 1.1# 8.6 ± 0.2¶,# 9.2 ± 0.3¶,# 12.8 ± 0.5¶,# 11.4 ± 0.3¶,# 10.0 ± 0.3¶,# 16.4 ± 0.6¶,# 10.9 ± 0.3# 6.6 ± 0.2¶,#

26.2 ± 1.7‡ 4.8 ± 0.1‡ 13.7 ± 0.4‡ 19.8 ± 0.7‡ 18.9 ± 0.6‡ 18.8 ± 0.4‡ 22.2 ± 0.8‡ 11.2 ± 0.3‡ 10.4 ± 0.3‡

32.5 ± 2.4† 11.0 ± 0.3† 16.2 ± 0.6† 25.2 ± 1.2† 25.0 ± 1.0† 24.8 ± 0.9† 26.8 ± 1.5 15.2 ± 0.6† 12.4 ± 0.5

23.1 ± 1.1 8.1 ± 0.2 12.7 ± 0.4 19.6 ± 0.7 18.8 ± 0.5 18.8 ± 0.5 21.2 ± 0.8 11.2 ± 0.3 10.2 ± 0.3

37.7 ± 1.8 13.4 ± 0.3 17.3 ± 0.6 26.3 ± 1.0 25.4 ± 0.9 25.3 ± 0.7 28.4 ± 1.2 14.4 ± 0.4 11.0 ± 0.4

29.2 ± 1.5& 10.1 ± 0.2& 15.3 ± 0.4& 22.9 ± 0.8& 22.3 ± 0.7& 21.5 ± 0.6& 25.3 ± 1.1& 14.0 ± 0.3& 12.4 ± 0.4&

19.5 ± 1.2¶,# 14.3 ± 0.6¶,# 5.6 ± 0.2¶,# 6.7 ± 0.3¶,# 7.3 ± 0.2¶,#

21.9 ± 1.3‡ 19.7 ± 0.9‡ 7.4 ± 0.2‡ 12.1 ± 0.4‡ 10.7 ± 0.2‡

27.8 ± 2.3† 26.4 ± 1.6† 12.9 ± 0.4† 17.0 ± 0.8† 16.1 ± 0.6†

21.9 ± 1.5 20.4 ± 1.0 10.1 ± 0.3 13.5 ± 0.5 12.0 ± 0.3

28.3 ± 2.3 25.6 ± 1.6 12.4 ± 0.5 15.7 ± 0.6 15.2 ± 0.5

24.7 ± 1.5& 22.8 ± 1.0& 10.4 ± 0.3 14.4 ± 0.5& 13.8 ± 0.4&

39.0 ± 1.9¶,# 17.2 ± 0.8¶,# 18.8 ± 0.9¶,# 20.0 ± 1.3¶,# 20.6 ± 1.1¶ 26.5 ± 1.1# 26.3 ± 1.4¶,# 19.7 ± 0.9¶ 7.4 ± 0.2¶,# 11.2 ± 0.3 6.9 ± 0.2¶,# 6.4 ± 0.2¶,# 7.4 ± 0.2¶,# 39.6 ± 1.1¶ 14.9 ± 0.3¶ 8.5 ± 0.2¶

31.8 ± 1.5‡ 23.4 ± 1.2‡ 17.6 ± 0.8‡ 22.3 ± 1.0‡ 20.5 ± 0.9‡ 24.8 ± 1.0‡ 28.5 ± 1.2‡ 18.8 ± 0.7‡ 8.1 ± 0.2‡ 11.0 ± 0.3‡ 12.4 ± 0.3‡ 8.5 ± 0.2‡ 12.4 ± 0.3‡ 30.5 ± 1.0‡ 15.9 ± 0.4‡ 11.9 ± 0.2‡

34.8 ± 1.7† 28.5 ± 2.0† 23.7 ± 1.3† 27.2 ± 1.7† 25.1 ± 1.3† 28.2 ± 1.5† 30.9 ± 1.7† 24.6 ± 1.2† 20.7 ± 0.6† 14.4 ± 0.6 17.9 ± 0.6† 14.1 ± 0.5† 17.9 ± 0.6† 40.3 ± 1.5† 22.1 ± 0.6† 20.3 ± 0.6†

36.5 ± 1.6 21.4 ± 1.2 21.4 ± 1.1 24.2 ± 1.3 23.6 ± 1.1 25.4 ± 1.1 28.1 ± 1.3 21.3 ± 0.9 15.4 ± 0.4 11.3 ± 0.4 13.2 ± 0.4 11.5 ± 0.3 13.7 ± 0.4 27.9 ± 0.8 18.7 ± 0.4 14.5 ± 0.3

40.0 ± 1.6 28.7 ± 1.9 24.5 ± 1.3 27.3 ± 1.5 30.0 ± 1.4 28.7 ± 1.4 33.4 ± 1.6 24.8 ± 1.1 21.2 ± 0.6 12.3 ± 0.4 19.9 ± 0.6 14.0 ± 0.4 16.4 ± 0.5 37.8 ± 1.2 24.4 ± 0.6 20.8 ± 0.5

31.6 ± 1.2& 23.8 ± 1.2& 21.2 ± 1.1 21.5 ± 1.1& 22.5 ± 1.0 24.3 ± 1.0 26.3 ± 1.1& 22.6 ± 1.0& 17.8 ± 0.4& 14.1 ± 0.4& 14.8 ± 0.4& 11.4 ± 0.3 14.9 ± 0.4& 38.0 ± 1.3& 19.1 ± 0.5 15.9 ± 0.3&

¶ Significantly different from the value in the OBX_SAL group. ‡ Significantly different from the values in the OBX-B10 group. † Significantly different from the value in the OBX-B20 group. # Significantly different from the value in the SHX-B10 group. * All values are significantly different from the values in the SHX-B20 group except the value in CxCin. $ All values are significantly different from the values in the OBX-B10 group except the value in CxCin. & Significantly different from the values in the OBX-SAL group.

is also important to note that the densities of the 5-HT2A receptors in the OBX-B20 group are not significantly different in the a priori selected brain regions from those found in the SHX-B20 group. This suggests a possible correlation between open field normalization (Sato et al., 2008) and the changes in 5-HT2A receptors. It was similarly reported that desipramine

treatment normalizes open field behavior (Earley et al., 1994) and produces a reduction in the densities of the 5-HT2 receptors ( Earley et al., 1994), which is opposite to the effect of buspirone in the present study. This, combined with the present findings, suggest that the normalization of open field behavior can be related to either the reduction or elevation of

37

BR A IN RE S E A RCH 1 3 45 ( 20 1 0 ) 2 8 –4 4

Table 4 – 5-HTT densities (unit) in sham and OBX rats treated with saline or buspirone (10 or 20 mg/kg/day). Values are shown as mean ± SD.

Limbic Structures Amy Amy-BL Amy-BM Amy-Me AN AONu CA1 CA3 Cx-PL CxCin CxEn DS Hyp SepN ThV Brain Stem LC R-mag R-med RD RD-d RD-lat RD-v Rpo VTA Motor Structures CPGP CPl GP SNc SNr Other Brain Regions Clustrum CPm CxA CxF CxP CxS CxSM CxV DTh IC LG MFB MG PB SC Sch

SHX-SAL (N = 6)

SHX-B20 (N = 6)

OBX_SAL (N = 6)

OBX_B20 (N = 6)

110.1 ± 7.6 105.2 ± 7.1 111.7 ± 8.0 113.2 ± 7.7 102.9 ± 7.3 108.9 ± 6.8¶ 93.4 ± 7.7¶ 89.3 ± 7.0¶ 126.1 ± 7.6 113.2 ± 8.1 114.9 ± 8.9 88.5 ± 6.4¶ 117.8 ± 8.4 103.0 ± 6.2 96.6 ± 7.1

99.6 ± 5.7* 106.6 ± 6.1 96.1 ± 5.4* 96.0 ± 5.5* 93.7 ± 5.3* 111.6 ± 5.6 94.1 ± 5.1 97.9 ± 5.1* 113.8 ± 5.6* 102.4 ± 5.9* 108.6 ± 5.6 89.1 ± 4.6 98.5 ± 4.9* 88.8 ± 4.7* 78.6 ± 4.6‡,*

109.5 ± 7.9 108.0 ± 7.8 110.7 ± 8.0 109.9 ± 7.9 105.8 ± 8.7 119.7 ± 8.0 109.5 ± 8.5 105.9 ± 8.3 132.7 ± 8.0 115.7 ± 7.6 113.3 ± 8.9 98.1 ± 6.7 110.6 ± 8.0 105.1 ± 6.6 93.1 ± 7.0

96.8 ± 5.3& 102.7 ± 5.5 92.6 ± 5.0& 95.1 ± 5.4& 96.0 ± 5.6& 109.2 ± 6.2& 93.9 ± 5.1& 97.2 ± 5.0 110.2 ± 5.6& 99.3 ± 5.9& 103.6 ± 5.6& 92.7 ± 5.2 94.7 ± 4.8& 86.4 ± 4.7& 72.0 ± 4.7&

134.0 ± 9.0 84.9 ± 5.9 115.5 ± 8.0 131.9 ± 9.4 138.7 ± 9.6 126.9 ± 8.9 132.1 ± 9.1 106.2 ± 7.9 77.6 ± 5.9

117.2 ± 6.6* 63.6 ± 4.4* 87.2 ± 6.0* 121.5 ± 7.9* 126.1 ± 8.1* 109.0 ± 6.0* 126.6 ± 8.1 88.7 ± 6.3‡,* 64.6 ± 5.9*

130.2 ± 8.3 90.0 ± 6.6 105.8 ± 7.5 129.2 ± 9.4 130.2 ± 10.1 126.2 ± 9.1 131.8 ± 9.0 99.1 ± 7.3 83.4 ± 7.0

117.6 ± 6.3& 64.1 ± 4.3& 81.5 ± 6.2& 122.2 ± 7.7& 125.6 ± 7.8 116.8 ± 6.3 126.5 ± 7.9 71.0 ± 5.2& 71.1 ± 5.7&

95.7 ± 6.5 92.4 ± 6.0 87.0 ± 5.9 86.0 ± 5.8 89.8 ± 5.8

81.0 ± 6.3* 80.1 ± 5.2* 69.1 ± 5.1* 80.4 ± 5.3 89.1 ± 5.3

101.2 ± 7.2 97.5 ± 6.7 93.0 ± 6.8 91.2 ± 6.9 94.8 ± 6.7

84.6 ± 6.9& 79.0 ± 5.0& 66.0 ± 5.1& 77.4 ± 5.5& 86.3 ± 5.4&

110.4 ± 7.4 97.4 ± 6.6 107.9 ± 9.8 111.7 ± 10.0¶ 111.2 ± 8.7 113.8 ± 9.6 113.8 ± 8.8 131.1 ± 11.2 122.2 ± 8.3 104.1 ± 8.3 96.1 ± 7.0¶ 93.2 ± 6.4 97.4 ± 7.2 97.2 ± 6.8 118.6 ± 8.1 123.0 ± 6.4

99.6 ± 5.3* 85.6 ± 5.8* 104.8 ± 8.4 104.5 ± 6.7 101.0 ± 5.8‡,* 103.6 ± 7.9 102.1 ± 7.2* 116.8 ± 7.5* 100.9 ± 5.6‡,* 76.3 ± 6.4* 85.1 ± 5.2* 80.8 ± 4.9* 83.3 ± 5.2* 173.9 ± 8.6* 104.5 ± 5.6* 97.7 ± 4.8‡,*

114.6 ± 7.9 100.8 ± 6.8 116.5 ± 10.4 125.6 ± 9.7 133.8 ± 10.6 118.2 ± 9.5 123.3 ± 10.0 130.6 ± 10.6 119.6 ± 8.0 96.1 ± 7.8 108.2 ± 8.0 90.0 ± 6.9 101.3 ± 7.4 93.7 ± 6.3 116.5 ± 8.1 120.0 ± 7.5

104.4 ± 5.8& 83.5 ± 5.4& 109.0 ± 9.4 104.8 ± 7.2& 111.3 ± 6.8& 103.7 ± 8.3& 101.2 ± 7.0& 118.1 ± 8.1& 91.8 ± 6.0& 72.4 ± 6.0& 82.0 ± 5.4& 78.3 ± 4.7& 83.6 ± 5.6& 181.9 ± 9.6 108.8 ± 6.0 106.9 ± 5.4&

¶ Significantly different from the value in the OBX_SAL group. † Significantly different from the value in the OBX-B20 group. * Significantly different from the values in the SHX-SAL group. & Significantly different from the values in the OBX-SAL group.

5-HT2, but produced by two rather different drugs. The changes in the hypothalamus are probably influenced by rather dense projections from the olfactory bulbs to the hypothalamus (Haberly and Price, 1978), with reciprocal connections between the hypothalamus and amygdala (Cowan et al., 1965). The results of the present study indicate

additional limbic structures being significantly affected by the OBX surgery, the Cx-PL, CPm, as well as the entire dorsal raphe and different parts thereof. The lesion study with 5,7-dihydroxytryptamine (5,7-DHT) clearly showed that the brain 5-HT system is pivotal for the development of behavior deficiencies found in the OBX rats (Cairncross et al., 1979).

38

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Fig. 5 – A graphical representation of differences in the 5-HT1B receptors between the Sham-SAL and OBX-SAL groups, and the Sham-B20 and OBX-B20 groups in the a priori selected brain regions. The abbreviations of the brain structures are explained in the list of abbreviations. The differences are given relative to the sham group values.

Because many 5-HT2A receptors are found in high densities on the cholinergic neurons in the hippocampus and likely other brain regions, and the fact that OBX produces a significant degeneration of the cholinergic system (Yoshimura and Ueki, 1977; Earley et al., 1994; Kelly et al., 1997a,b; Han et al., 2008), the elevation of 5-HT2A receptors could be related to an upregulation of 5-HT2A sites on cells surviving denervation, particularly in the brain regions connected to the olfactory bulbs (e.g., AMY, Cx-PL, AONu, Hyp). With respect to 5-HT2A binding in humans, discrepant findings have been reported, including a decrease (Biver et al., 1997), no difference (Meltzer et al., 1999),or an increase in 5-HT2A binding (Meyer et al., 2003)in depressed patients compared to healthy controls. The proposed reasons for these discrepancies have been the heterogeneity of the clinical samples regarding demographic variables, subtypes of depression, previous treatments, current mood states, as well as differences in brain imaging techniques, such as the choice of the radioactive ligand, image acquisition and data analysis (see Stockmeier 2003 for review). It could be stated that the results found in the OBX rats (present data) support an increase in the 5-HT2A receptors in a depressed state. Binding to 5-HT2A receptors (note that binding potential was measured in positron emission tomographic studies) is decreased following paroxetine and desipramine treatments in subjects with major depression (Meyer et al., 2001; Yatham et al., 1999), which is opposite to the findings reported here, but with different drug. The main reason for this could be the fact that the present study was performed in an animal model of depression and the fact that buspirone is a different antidepressant than paroxetine and desipramine. A reduction in 5-HT2A receptors has also been reported in the OBX rats following treatments with mianserine or desipramine (Earley et al., 1994), again suggesting the drug specific changes in the density of these sites. It was previously shown that citalopram (SSRI) and buspirone have different effects on brain 5-HT synthesis, another important parameter of brain neurotransmission, in the OBX and Sham operated rats (Watanabe et al., 2006; Hasegawa et al., 2005).

3.3.

The effect of OBX and buspirone on 5-HTT density

Highly significant differences (elevation in OBX-SAL group) in the densities of 5-HTT between the SHX-SAL and OBX-SAL groups were found, which is in limited agreement with the reported elevation of paroxetine labeled sites in a few brain regions in OBX rats (e.g., frontal cortex; Zhou et al. (1998). The main difference between the measurements reported here and those of Zhou et al. (1998) is the fact that the rats in the present study were treated with saline for 14 days, making them older than those used in Zhou et al.'s study. This may be important because studies have shown that the density of 5-HTT is reduced with age (van Dyck et al., 2000). However, an increase in young humans (Makkonen et al., 2008) has also been reported. In addition, the measurements of Zhou et al. (1998) were performed on dissected tissue samples, while the present data were obtained from autoradiographic measurements which should have much better anatomical resolution and tissue type identification - the resolution is in an order of 20 micron. Several antidepressants have been shown to reduce the density of SERT in the rat brain (Benmansour et al., 1999; Gould et al., 2007). The elevation of 5-HTT density could also be related to sprouting of the degenerated terminals (Zhou et al., 1998) and the elevation of 5-HT synthesis (Watanabe et al., 2003; 2006). Further, the elevation of 5-HT synthesis found in the OBX rats (Watanabe et al., 2003; 2006) could be associated with the increase in 5-HTT to maintain the homeostasis of 5-HT. The need for the elevation of 5-HTT could be somewhat reduced by the fact that the dopamine transporter can, under certain conditions, take up 5-HT into the dopaminergic neurons (Norrholm et al., 2007) where monoamine oxidase-A, which has 5-HT as a preferred substrate, is present. Similarly, depressed patients show a decreased density of platelet 5-HT uptake, which is also found in OBX rats (Butler et al., 1988). The measurements of the [3H] imipramine binding reported both a decrease (Jesberger and Richardson, 1986) and increase (Al-Khatib et al., 1988) in the densities of the transporter.

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39

Fig. 6 – A graphical representation of differences in the 5-HT2A receptors between the Sham-SAL and OBX-SAL groups, and the Sham-B20 and OBX-B20 groups in the a priori selected brain regions. The abbreviations of the brain structures are explained in the list of abbreviations. The differences are given relative to the sham group values.

In line with the present results on the differences in 5-HTT between the OBX-SAL and SHX-SAL rats are two studies that examined 5HTT BP using selective carbon-11 labeled 3-amino4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile ([11C]DASB). They found no changes in 5HTT BP (Meyer et al., 2004; Bhagwagar et al., 2007). However, in one study, increased 5HTT BP was found in the thalamus, insula, striatum and periaqueductal area in individuals with MDD (Cannon et al., 2007), and in the other study, decreased 5HTT BP was found in the thalamus of individuals with MDD (Reimold et al., 2008).

4.

Experimental procedures

4.1.

Animals

Male Sprague-Dawley rats (Charles River Canada, St. Constant, Quebec, Canada) weighing between 180-240 g were used. The

rats used for the measurements described here were the same ones used for the behavioral and 5-HT1A receptor density measurements reported previously (Sato et al., 2008), where the details of different treatments and behavioral testing (open field) were provided. The rats were housed in the animal facility (room temperature 22 °C and a 12 h day-night cycle) for a minimum of 3 days before being used in the experiments. On the first day of the experiment, the OBX and sham operations were performed under light isoflurane anesthesia (1-3%), as detailed previously (Sato et al., 2008). After the surgery, the rats were housed in pairs in a cage for two weeks, as that period is sufficient for recovery from surgery and the development of the OBX syndrome (Kelly et al., 1997a,b). The rats were then randomly assigned into the buspirone (-B10 treated with 10 mg/kg/day and -B20 treated with 20 mg/kg/ day) or saline (-SAL) groups and the treatments were commenced. After two weeks of either treatment, the rats were decapitated and the brains were removed and frozen in

Fig. 7 – A graphical representation of differences in the 5-HTT sites between the Sham-SAL and OBX-SAL groups, and the Sham-B20 and OBX-B20 groups in the a priori selected brain regions. The abbreviations of the brain structures are explained in the list of abbreviations. The differences are given relative to the sham group values.

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4.3.

Autoradiographic procedures

in which the bindings were done contained prazosin hydrochloride (1 μM; to block binding to adrenergic receptors) and tetrabenazine (100 nM; to block binding to the vesicular transporter). Non-specific binding was done in a bath which, in addition to these two drugs, contained methysergide (1 μM). Preincubation was performed for 5 min in the Tris buffer, while incubations (bindings) were done in the buffer with the above noted drugs and [3H]-labelled kentanserine (specific activity=28.8 Ci/mmol) for 60 min. Following incubation, the slides were washed two times for 30 min in an ice cold buffer, and rinsed for 10 sec in cold deionized water. After washing, the slides were dried in a stream of cool air overnight and stored for another day in a refrigerator at 4 °C in a box containing desiccant. Subsequently, the labelled brain cross sections were contacted with [3H]-sensitive fluorescence plates (Fuji; YBIP2025TR) for up to eight weeks along with tritium standards. The densities in the different brain regions in the images were quantified in 38 brain regions and the pineal body (5-HT2A and 5-HTT) bilaterally in at least three consecutive brain coronal sections using the Rat Brain Atlas (Paxinos and Watson, 1997).

4.3.1.

5-HT1B receptor

4.3.3.

a bowl of cold 2-Methylbutane (-20 °C). The brains were kept at –80 °C until they were cut and used for the receptor binding study. All of the surgical procedures and experiments were performed with the approval of the Animal Care Committee of the Montreal Neurological Institute of McGill University, and were done according to the procedures of the Canadian Council on Animal Care.

4.2.

Chemicals

Buspirone-hydrochloride (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) was dissolved in saline (0.9% NaCl). Two weeks following the OBX or sham operation, either 10 (-B10) or 20 (-B20) mg/(kg day) of buspirone was administered subcutaneously for 14 days by minipump. The treatments, either with saline or the proper concentration of buspirone in saline, were delivered by an osmotic minipump (Alzet osmotic minipump model 2ML2; ALZA, Palo Alto, CA, USA) at a rate of 0.12 mL/day.

The frozen brains were cut into 20 µm thick coronal sections at a temperature of-22 °C in a cryostat Leica CM3000 (Leica Microsystems, Bensheim, Germany). Sections were then thawmounted on adhesion intensified glass slides (Superfrost Plus GOLD, Erie Scientific Company, Portsmouth, NH, USA), and, following that, kept at 4 °C with desiccant until the day of the binding assay. The protocol used for the quantification of the 5-HT1B receptors was, more or less, the protocol described earlier by Pazos and Palacios (1985). 5-HT1B receptors were visualized using [3H]5-HT with a specific activity of 30 Ci/mmol. The binding of the ligand to the 5-HT1A sites was blocked by 0.1 μM of 8-OH-DPAT (a selective 5-HT1A agonist) and the binding to the 5-HT2C and 5-HT1C sites was blocked by 0.5 nM of mesulergine. Preincubation was performed in the buffer (Tris HCl buffer 0.17 M; pH= 7.6) for 30 min at room temperature. The preincubation was followed by incubation in the same buffer with the above mentioned components and [3H]5-HT (2 nM) for 60 min at room temperature. The incubation was followed by washing in a cold buffer and rinsing in cold deionized water (4 °C) for 10 sec. Nonspecific binding was performed in the presence of cold 5-HT (1 μM). The washed specimens were dried under a stream of cool air followed by overnight drying in a refrigerator at 4 °C. Next, the specimens were contacted along with tritium standards for 6-8 weeks to tritium sensitive fluorescence plates (Fuji; YBIP2025TR) at 4 °C. The densities in the different brain regions in the images were quantified in 38 brain regions and the pineal body bilaterally in at least three consecutive brain coronal sections by the aid of the Rat Brain Atlas (Paxinos and Watson, 1997).

4.3.2.

5-HT2A receptor

The protocol used was adopted from the one described previously by O'Dell et al. (1990), and thus will only be briefly described here. Preincubation and binding were performed in a 50 mM Tris buffer (pH = 7.4) at room temperature. The baths

5-HTT sites

The protocol used for the quantification of the 5-HTT was as described previously (Habert et al., 1985). Preincubation was performed for 30 min in 50 mM TRISx HCl pH = 7.4 at room temperature to remove the endogenous 5-HT. This was followed by incubation in 50 mM TRISx HCl (pH = 7.4) containing 120 mM NaCl and 5 mM KCl for 60 min at room temperature using approximately 2 nM 3H-paroxetine (specific activitiy = 24.4 Ci/mmol). The nonspecific binding was determined in the presence of 10 μM of fluoxetin. The washing was done for 5 min in an ice cold buffer followed by three rinses (for 10 sec each) in a cold buffer. The slices were dried under a stream of cool air and left overnight in a refrigerator at 4 °C. The slices were then stored in the boxes in the refrigerator until contacted with a tritium sensitive fluorescence plate (Fuji, YBIP2025TR) along with tritium standards. Contacting was done for 6-8 weeks after which time the plates were scanned in a BAS-5000 scanner (Fuji; YBBAS5000). The densities in the different brain regions in the images were quantified in 38 brain regions and the pineal body bilaterally in at least three consecutive brain coronal sections by the aid of the Rat Brain Atlas (Paxinos and Watson, 1997). In all of the experiments, the quantitative measurements of the bound radioactivity were determined with an aid of a standard calibrated imaging system (MCID/M4, Image Research Co., St. Catherines, Ontario, Canada). The readings were performed bilaterally and on at least three consecutive slices, and the average values were used for further calculations. The brain areas were identified using the Rat Brain Atlas (Paxinos and Watson, 1997). The difference between the total and non-specific binding, which varied between 15% and 30% of the total binding, was used as a specifically bound ligand. These specific bindings were corrected for radioactivity in each incubation bath.

4.3.4.

A priori selected regions

Neurochemical (e.g., glucose utilization, 5-HT synthesis, receptor densities, dopaminergic and noradrenergic systems

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deficiencies) differences between patients with depression and normal controls in the limbic structures (e.g., amygdala, cingulate and entorhinal cortices, dorsal and ventral hippocampus, hypothalamus, and ventral thalamus), brain stem (e.g., raphe and ventral tegmental area), and motor brain structures (e.g., caudate-putamen lateral part, and substantia nigra compacta and reticulata) have been reported by many investigators. Because of these reports, the brain regions consisting of these three groups were a priori selected: limbic structures (amygdala, accumbens, anterior olfactory nucleus, cingulate and entorhinal cortices, dorsal subiculum, dorsal and ventral hippocampus, hypothalamus, septal nucleus, and ventral thalamus), brain stem (dorsal, median, magnus and pontine raphe, locus coeruleus, and ventral tegmental area), and the motor brain regions (caudate-putamen lateral part, globus pallidus, and substantia nigra compacta and reticulata). The measurements were also performed in additional brain structures to determine if there were effects of buspirone on these neurochemical parameters. Abnormalities in the frontal cortex of depressed subjects (Mayberg, 2003; Phillips et al., 2003), as well as in the OBX rat model of depression (Kelly et al., 1997a; Zhou et al., 1998), have been reported. As a result of these findings, and the possibility of obtaining a better understanding of the etiology of affective disorders and/ or the actions of antidepressants, other brain regions containing reasonable densities for these sites were investigated (e.g., cortical regions, IC, LG, Clustrum, CPm, MG, Sch). Three different parts of the amygdala (Amy); Amy-Me, Amy-BL and Amy-BM (Fig. 1B), and three parts of the dorsal raphe; dorsal (DR-d), ventral (DR-v), and lateral (DR-lat) (Fig. 1A), were separately quantified as exemplified in Fig. 1.

41

dorsal raphe (DR) into ventral (DR-v), lateral (DR-lat) raphe into, dorsal (DR-d), and amygdala (Amy) into medial (Amy-Me), basomedial (Amy-BM), and basolateral (Amy-BL). In general, a 10 mg/kg/day buspirone treatment produces very little effect, or the effect was almost the same as that produced with a 20 mg/kg/day treatment, and therefore the images obtained with 10 mg/kg/day are not presented.

5.

Conclusion

The data presented suggest that the alteration of the 5-HT1B and 5-HT2A receptors is very important in the development of depression and the subsequent alleviation of symptoms through treatment. Because buspirone treatment produced significant effects in the densities of both of these sites, the study of these sites will not likely yield unique conclusions of treatment effects. Although the 5-HTT sites do not play an important role, the small changes observed may still be relevant or potentially significant.

Acknowledgments This research was supported, in part, by grants from the Croatian Ministry of Science, Education and Sports (219-1081970-2032), and the Canadian Institute for Health Research (MOP-42438). The authors wish to thank Ms.Valerie Ann Cherneski for her editorial help.

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4.3.5.

Statistical comparisons

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