Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder, major depressive disorder and schizophrenia – An autoradiography study

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Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder, major depressive disorder and schizophrenia – An autoradiography study Emma R. Veldmana,n,1, Marie M. Svedberga,1, Per Svenningssonb, Johan Lundberga a

Center for Psychiatry Research, Department of Clinical Neuroscience, Karolinska Institutet & Stockholm Health Care Services, Stockholm County Council, Stockholm, Sweden b Center for Molecular Medicine, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Received 26 December 2016; received in revised form 17 February 2017; accepted 21 February 2017

KEYWORDS

Abstract

Serotonin 1B receptor; Autoradiography; Bipolar disorder; Major depressive disorder; Schizophrenia; Anterior cingulate cortex

The serotonin 1B receptor has recently received more interest as a possible new target for pharmacological treatment of psychiatric disorders. However, the exact mechanisms of action remain unclear. This study aimed to examine the binding distribution and levels of the serotonin 1B receptor in-depth in the anterior cingulate cortex (ACC) and provide more insight in its functional role in bipolar disorder (BD), major depressive disorder (MDD) and schizophrenia (SZ). Serotonin 1B receptor binding distribution was visualized with high resolution autoradiography (ARG), using the radioligand [3H]AZ10419369, in postmortem ACC tissue from patients diagnosed with BD (n =14), MDD (n =12), SZ (n =13) and healthy subjects (n =13). Moreover, a quantification of receptor binding was made with ARG, in relation to patient group, age and gender. In all subject groups a significantly higher specific binding of serotonin 1B receptor was measured in the outer ACC layers compared to the inner ACC layers. Correlation analysis with ARG binding patterns of several radioligands resulted in a significant correlation with glutamatergic N-methyl-D-aspartate receptor binding in the outer layers. No significant difference was found between subject groups in binding levels and distribution. In female subjects a significantly lower receptor binding was found than in male subjects, which was most profound in patients diagnosed with MDD.

n Correspondence to: Centre for Psychiatry Research, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital R5:0, 171 76 Stockholm, Sweden. E-mail address: [email protected] (E.R. Veldman). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.euroneuro.2017.02.011 0924-977X/& 2017 Elsevier B.V. and ECNP. All rights reserved.

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

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E.R. Veldman et al. The binding distribution of the serotonin 1B receptor found in this study supports a role in glutamate transmission in the ACC and was not shown to be significantly altered in BD, MDD or SZ. A gender difference in serotonin 1B receptor binding was found. & 2017 Elsevier B.V. and ECNP. All rights reserved.

1.

Introduction

Psychiatric disorders are among the major causes of disease burden worldwide (Whiteford et al., 2015) of which bipolar disorder (BD), major depressive disorder (MDD) and schizophrenia (SZ) together form the largest group of contributors (World Health Organization, 2008). A major problem for these patients is the lack of new, more efficacious treatment options (Whiteford et al., 2015), which is explained by the insufficient understanding of pathophysiology of these disorders. Several studies support the involvement of the serotonin (5-hydroxytryptamine, 5-HT) neurotransmission in BD, MDD and SZ (Spies et al., 2015). Having a modulatory function on neural activity, the 5-HT system is distributed from cell bodies in the brainstem to the thalamus, basal ganglia, limbic and neocortical structures (Berger et al., 2009). Based on their pharmacological profile, 5-HT receptors can be categorized in 14 currently known types of 5-HT receptors (Alexander et al., 2015). The 5-HT1B receptor has recently been suggested as a putative target for new psychotropic drugs based on a potential role in several psychiatric disorders (Ruf and Bhagwagar, 2009); activity towards the 5-HT1B receptor might explain the antidepressant and antipsychotic effect of vortioxetine (BangAndersen et al., 2011) and asenapine (Shahid et al., 2008), respectively. 5-HT1B receptors are negatively coupled to adenylyl cyclase (Maroteaux et al., 1992) and exist in two forms: as autoreceptors, they exhibit an inhibitory role on 5-HT release, while 5-HT1B heteroreceptors are located on non-serotonergic neurons and regulate release of other neurotransmitters (Ruf and Bhagwagar, 2009; Sari, 2004). Both types of receptors have been found to be widely distributed throughout the human brain, although the heteroreceptors have a higher abundancy (Moret and Briley, 2000; Sari, 2004). 5-HT1B mRNA expression is mostly found in the striatum, cerebral cortex, raphe nuclei and thalamus (Varnäs et al., 2005). However, with in vitro autoradiography (ARG) and in vivo positron emission tomography (PET) highest 5-HT1B receptor binding was found in substantia nigra and globus pallidus, followed by striatum and cerebral cortex, lower binding in other subcortical areas such as the amygdala, and binding was nearly absent in cerebellar cortex (Nord et al., 2014; Pierson et al., 2008; Varnäs et al., 2011, 2005, 2001). This apparent deviation in findings can result from the fact that the 5-HT1B receptor is localized on axon terminals (Sari, 2004); hence, 5-HT1B autoreceptors can be found presynaptically in all projections originating from the raphe nuclei towards the foreand midbrain, while 5-HT1B heteroreceptors can be mostly found on projections from striatal, cortical and thalamic origin (Varnäs et al., 2005). The 5-HT1B receptor has been

linked to several behavior traits in animals, such as feeding behavior (Heslop and Curzon, 1999), aggression (Saudou et al., 1994) and anxiety (Hoplight et al., 2005). In humans, polymorphisms of the 5-HT1B receptor have also been linked to aggression (Zouk et al., 2007). However, in PET no direct correlation with 5-HT1B receptor binding has been found, but rather a correlation with anger and psychopathy in violent offenders (Da Cunha-Bang et al., 2016). A negative correlation of 5-HT1B receptor binding with age has been found in several brain areas using PET in many, but not all studies (Matuskey et al., 2012; Nord et al., 2014; Tiger et al., 2016, 2014; Varrone et al., 2014). 5-HT1B mRNA expression differences between suicide patients and control subjects has been reported to be moderated by gender in the orbitofrontal cortex and hippocampus (Anisman et al., 2008); however, in healthy subjects no gender differences have been found in 5-HT1B receptor binding with PET (Matuskey et al., 2012). The anterior cingulate cortex (ACC) is of interest in BD, MDD and SZ for its central role in emotional and cognitive systems which are suggested to be malfunctioning in these diseases (Phillips et al., 2003; Stevens et al., 2011). Several abnormalities have been found in ACC of psychiatric patients, e.g. altered fMRI activities are seen after exposure to emotional stimuli or during the execution of cognitive tasks in patients with BD (Drevets et al., 1997), MDD (Fitzgerald et al., 2008) and SZ (Heckers et al., 2004) comparing to control subjects. Like other areas in the human cortex, the ACC is divided into separate layers, which all have different features, e.g. different neuron and glial densities (Höistad et al. 2013). The supragranular layers (lamina I-III) mainly consist of intracingulate and other intracortical connections; lamina I has mainly afferent projections and connections in lamina II and III are bidirectional, among which are the efferent projections from the amygdala (Vogt, 1993). Most outward connections towards subcortical areas and intracingulate connections originate in the infragranular layers (lamina V-VI) (Allman et al., 2001). Specific for the ACC is the absence of lamina IV, while lamina V is strongly developed (Allman et al., 2001). The latter has many projections towards the prefrontal cortex and the amygdala, explaining the important role of ACC in cognition and emotion (Paus, 2001; Vogt, 1993). As seen in other cortical areas, the ACC has many glutamatergic and GABA-ergic neurons, next to relatively many dopaminergic, norepinephric and serotonergic neurons, which originate from the midbrain, locus coeruleus and raphe nuclei, respectively (Vogt, 1993). Layer specific abnormalities in receptor count, interneuron density and axon innervation have been found in ACC of patients with BD, MDD and SZ, e.g. laminar specific decrease in interneuron densities were found in ACC of SZ patients comparing with healthy controls (Benes, 2000). However, no laminar difference in total

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder neuron density was found in dorsal ACC (Höistad et al. 2013) or in all ACC layers combined (Stark et al. 2004) of SZ patients compared to control subjects. Previous imaging studies concerning the 5-HT1B receptor have mostly been analyzing distribution throughout the brain, however only sparse detailed information concerning distribution within cortical areas of the human brain has been obtained. By using the highly specific radioligand [3H]AZ10419369 (Pierson et al., 2008) for high resolution ARG, the current study aims to give more insight in the 5-HT1B receptor distribution throughout the anterior cingulate cortex. Moreover, a comparison is made of patients with BD, MDD, SZ and healthy subjects to explore the role of 5-HT1B receptor distribution in pathophysiology, in relation to age and gender.

slices derived of 52 individuals between 25 and 68 years old, of which 14 diagnosed with BD, 12 with MDD, 13 with SZ and 13 control subjects (the original collection consisted of material of 15 subjects of each group). This study was approved by the Ethics Committee at Karolinska Institutet (reg. no. 2014/1366-31). Demographic and clinical data of the subjects and physical features of the brain material can be found in Tables 1 and 2, respectively. A mild, negative correlation was found with postmortem interval (PMI) and 5-HT1B receptor binding (punadjusted =0.02, r= 0.33), which did not remain significant after correction for multiple comparisons. Further information on the study subjects and brain material can be found in the supplementary section. Fresh frozen postmortem tissue was sectioned on a Cryostat at 14 mm thickness. Subsequently, tissue slides were mounted on glass slides and stored at 70 1C.

2.3.

2.

3

[3H]AZ10419369 in vitro autoradiography

Experimental procedures

2.1.

Materials

[3H]AZ10419369 (specific activity 21 Ci/mmol), radiochemical purity 499%, was synthesized at Karolinska Institutet, department of Clinical Neuroscience. Kodak NTB emulsion was purchased from VWR, Sweden. All other chemicals were of analytical grade.

2.2.

Human postmortem brain tissue

Human postmortem brain tissue was obtained from the Stanley Foundation, Bethesda, USA, who collected brains with family permission. Tissue originated from ACC from the Neuropathology Consortium brain collection (Torrey et al., 2000), of which the present project had access to the following subset: single brain

Table 1

Slides were thawed at room temperature and incubated in 1.5 nM [3H]AZ10419369 (Pierson et al., 2008), in Tris–HCl 50 mM, pH 7.4 incl. 4 mM MgCl and 4 mM CaCl2 at room temperature for 60 min. The choice of radioligand and concentration used in the current study was based on previously reported high specificity towards the 5-HT1B receptor (495%) and in vitro estimates of Kd (dissociation constant), respectively (Pierson et al., 2008). In a duplicate container 10 mM 5-HT was added to establish nondisplaceable binding. After incubation, slides were washed 3  10 min in Tris–HCl 50 mM, pH 7.4 at 4 1C, followed by a brief wash in distilled water. Slices were dried in room temperature and placed together with autoradiographic micro scale standards (American Radiolabeled Chemicals Inc.), and exposed to phosphor imaging plates (Fujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan). The resulting images were processed in a Fujifilm BAS-5000 phosphor imager (Fujifilm, Tokyo, Japan).

Demographics and clinical characteristics of patients and controls (mean7SD). Bipolar disorder Major disorder

Gender

female male

Age (year) Age at onset of disease (year) Duration of disease (year) Antipsychotic medication (g)a Antidepressant medication (n) Mood stabilizing medication (n, lithium:other) Substance abuse (n) Never Current Past Severity of substance useb Severity of alcohol useb Suicidal death (n) Psychosis (n) Family history of disease None (n) Psychosis Depression Unknown

depressive Schizophrenia Control

Total

5 9 43.6711.1 21.678.6 21.179.2 21.8724.6 7 4:5

4 8 47.1710.4 34.7714.6 12.5711.9 0 10 1:0

4 9 43.2713.5 21.676.4 21.5712.2 42.2750.4 5 2:1

5 8 50.179.7 NA NA 0 0 0

18 34 45.9711.3 25.6711.7 18.6711.6 16.4732.6 22 7:6

6 5 3 1.972.0 2.171.9 8 10 5 6 2 1

7 4 1 1.372.1 2.172.2 6 0 2 1 7 2

8 4 1 1.471.9 1.471.5 4 13 4 8 1 0

10 1 2 0.270.6 1.271.1 0 0 13 0 0 0

31 14 7 1.271.8 1.971.7 18 23 24 15 10 3

NA = not applicable. a Cumulative lifetime use in fluphenazine equivalents (Torrey, 1988). b From a scale of 0–5 (mean7SD).

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Table 2

Physical features of brain material (mean7SD).

Brain hemisphere

Right Left

pH Relative mass of brain (g) Postmortem interval (h)a Storage time (days)b

Bipolar disorder Major depressive disorder Schizophrenia Control

Total

7 7 6.270.2 1434.17175.7 33.1716.6 605.17167.8

23 29 6.270.2 1471.47149.0 29.5714.0 519.27249.3

5 7 6.170.2 1453.37130.4 27.3712.0 426.97289.6

5 8 6.270.3 1478.87114.9 33.9715.6 664.17189.5

6 7 6.370.2 1520.87165.8 23.378.6 367.17236.6

a

Interval between death and freezing of the brain tissue. Storage time does not account for storage at laboratory were experiments were carried out (which has same extra storage time for all samples). b

Regions of interest (ROIs) were determined manually of each section on the scanned images using Multi Gauge 3.2 phosphorimager software (Fujifilm, Tokyo, Japan). For analysis of binding throughout the cortical area, approximately 1/3 of cortical layer outermost and approximately 1/3 of area most adjacent towards white matter was taken. The signal on the scanned images was given as average counts per pixel and the mean pixel values of the ROIs were transformed into radioactivity values and binding density (pmol/mg tissue wet weight), calibrated with micro scale standards. Values of binding density for total grey matter in each slice and layers within the cortical area were derived, after which specific binding was determined by subtracting the level of nonspecific binding from the total binding.

2.4.

High resolution autoradiography

High resolution microscopic emulsion ARG was performed in order to visualize the cellular distribution of the radioligand in the ACC. Slides were subjected to [3H]AZ10419369 in vitro ARG at 1.5 nM and were dipped into NTB emulsion at 42 1C. The slides were air-dried at room temperature and exposed in the dark at 4 1C for 3–4 weeks. Slides were then developed with Dokumol developer, rinsed in water, fixed with Vario Fix in room temperature and washed with water. Counterstaining of Nissl substance, located in the cytoplasma of neurons, was performed to make an approximation of the different layers of the cortical area which were included in the measurements of laminar distribution. In brief, slides were stained with cresyl violet (Histolab, Göteborg, Sweden) and dehydrated consecutively in increasing concentrations of ethanol. Sections were immersed in xylene, dried and mounted. Representative photomicrographs, for visual inspection of the binding distribution, were acquired using a slide-scanning microscope with a 10x objective and brightfield settings (MetaSystems, Alltlussheim, Germany).

2.5.

Statistical analysis

Statistical analysis was performed using R Studio 0.99.489 (R Studio, Inc 2015). ANOVA tests were carried out for all demographic and physical variables (Tables 1 and 2), together with a Tukey Post-Hoc test to examine all pair wise comparisons and to correct for multiple comparisons. η2 was calculated as measurement of effect size. The same tests were performed to compare specific binding between subject groups and to compare patients versus control subjects. A paired Student's t-test was performed to calculate differences between specific binding in cortical layers and Cohen's D was calculated to determine the effect size. Mann-Whitney tests were used to calculate differences for comparisons of genders, where data showed non-normal distribution. For this data Cliff's δ was calculated to determine the effect size. When analyzing

differences between subject groups, p-values were adjusted for multiple comparisons using Bonferroni's method. Pearson's correlation coefficient was calculated to test for correlation of specific binding with age or any other known factor in the studied population.

3. 3.1.

Results [3H]AZ10419369 specific binding

A significantly higher specific binding was measured in the outer layers of the ACC compared to the inner layers for all subjects together (190730 and 151732 pmol/g mean7SD respectively) (Figure 1a) and BD, MDD, SZ and control subject groups separately (Figure 1b). No significant intergroup differences were found for layer differences (Table 3). No significant difference in specific binding in total grey matter of ACC was measured for patients diagnosed with BD, MDD and SZ compared to control subjects, nor was any significant difference in specific binding found between any of the patient groups (Table s1, supplementary section). This finding could not be explained by any of the individual factors shown in Tables 1 and 2. Visual inspection guided by Nissl staining showed nonhomogeneous binding with higher signal in the supragranular layers compared to the infragranular layers, which was seen in all subject groups (Figure 2). A detailed view of the difference in binding pattern in the cortical layers is demonstrated as higher density of silver grains in the outermost cortical area, decreasing towards the innermost cortical area (Figure 3).

3.2.

Gender

In the whole study group, men had significantly higher [3H] AZ10419369 binding than women (p= 0.01 and Cliff's δ = 0.43) (Figure 4A; Table s1, supplementary section). Within each subject group, gender difference in [3H] AZ10419369 binding was shown to be highest in patients with MDD (Figure 4B), which remained significant after correction for multiple comparisons (padjusted = 0.02 and Cliff's δ= 1). Differences in specific binding between genders in BD, SZ and controls were not significant. Furthermore, no significant differences in specific binding between subject groups or between patients and controls were found when

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder

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Figure 1 Specific binding of [3H]AZ10419369 in outer and inner layers. A. All subject groups together; B. Subject groups separately, BD: Bipolar Disorder, MDD: Major Depressive Disorder, SZ: Schizophrenia, C: Control subjects. Box represents interquartile range with horizontal line representing the median, the vertical lines represent the minimum and maximum values within 1.5 times the interquartile range distance from the 1st and 3rd quartile, respectively. Outliers are represented as single dots.

Table 3 Differences in [3H]AZ10419369 binding between outer and inner layers of ACC grey matter; compared in the BD, MDD, SZ and control group. Mean difference is expressed as percentage of specific binding in whole grey matter of each slice.

Mean difference 95% CI padjusted Effect size (Cohen's D)

Bipolar disorder

Major depressive disorder

Schizophrenia

Control

Total

27.7% 7 8.3% 1.86  10 3.06

22.7% 7 5.4% 1.23  10 2.56

22.1% 7 5.1% 1.64  10 2.80

22.3% 7 5.3% 2.65  10 2.14

23.8% 7 6.2% o1.1  10–15 2.66

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data for men and women were analyzed separately (Figures 2 and 4b).

3.3.

Age

Overall, a tendency towards a positive correlation of specific binding with age was seen. The strongest positive correlation was seen in the BD group (r= 0.54), which was not significant after correction for multiple comparisons. On the contrary, the control group showed a tendency towards negative correlation of specific binding with age. Specific binding in inner and outer cortical area followed similar patterns to those find in total slice with regards to age and gender (data not shown).

4. 4.1.

Discussion Laminar distribution of [3H]AZ10419369

The aim of the present study was to examine the 5-HT1B receptor binding distribution in the ACC. The results show a significant difference in [3H]AZ10419369 specific binding between outer and inner layers of the ACC in patients diagnosed with BD, MDD, SZ and control subjects. Our findings in control subjects are in line with what was previously noticed with visual inspection in a postmortem study with 3 healthy subjects, where 5-HT1B receptor

5

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binding was visualized with [3H]GR 125743 ARG (Varnäs et al., 2004, 2001). To our knowledge no findings on binding distribution of the 5-HT1B receptor in ACC of BD, MDD and SZ have previously been reported. Between different subject groups, no significant differences were found in layer distribution, nor was this affected by gender or age. The current study uses microscopic emulsion ARG to study 5-HT1B receptor distribution in detail. While in vivo PET currently can reach a resolution of a few millimeters (Van Velden et al., 2009), in vitro ARG has a resolution of 750– 100 mm and microscopic emulsion ARG even reaches a resolution of 10–50 mm (Schmidt and Smith, 2005; Solon, 2012), making this a highly suitable technique to study laminar receptor distributions. The outer layer of ACC sections measured in this study consists of lamina I, II and a part of lamina III; the inner layer consists of lamina VI and a part of lamina Vb, based on previously published data on laminar height of the same material as used in the current study (Cotter et al., 2001). In general, lamina I, and to a lesser extent lamina II and III, consists mostly of axon terminals from pyramidal neurons from other cortical connections and deeper lying laminas within the cingulate cortex, known to be mostly glutamatergic (Vogt et al., 1995). Moreover, it has been shown that glutamate-immunoreactive processes are more present in supragranular layers of ACC than in infragranular layers (Benes et al., 1992a, 1992b). Therefore, the high 5-HT1B receptor binding implies a large contribution to measured

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

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Figure 2 Autoradiogram of [3H]AZ10419369 in vitro binding in human, postmortem anterior cingulate cortex. Higher magnification of the gray matter shows laminar binding distribution in: A. Bipolar disorder; B. Major depressive disorder; C. Schizophrenia; D. Control subjects.

Figure 3 Images of Nissl staining (pointed out with arrow) and binding pattern of [3H]AZ10419369 through emulsion coating (black dots represent silver grains) in brightfield illumination with 10  magnification and 3.5  magnification by post-processing. A. Autoradiographic image from radioactivity on Phosphor Imager plate, with squares marking location of images B-D (from left to right); B. Outer layers; C. Middle layers; D. Inner layers. Bar shows equivalent of 10 μm for Figure 3B-D.

binding signal in these laminae originating from 5-HT1B heteroreceptors on glutamatergic neurons. Interestingly, previous studies measuring 5-HT1B receptor mRNA found highest densities in lamina II and III, following lamina VI, lowest binding in lamina V and no binding in lamina I (Varnäs et al., 2005). As 5-HT1B receptors are found to be situated on axon terminals, co-localization of 5-HT1B receptor mRNA implies the localization of this receptor on small GABA-ergic interneurons, which are abundant in lamina II and III (Vogt, 2009). Findings in the current study are not likely to represent the binding of autoreceptors. Although projections from the raphe nuclei terminate in ACC with small unmyelinated axons throughout all laminas and large

myelinated axons in lamina I (Vogt, 1993), 5-HT1B receptors have hitherto only been found on terminal areas of fine, unmyelinated axons (Sari, 2004). 5-HT1B autoreceptors, situated on neurons originating from the raphe nuclei, are therefore likely to be uniformly distributed throughout ACC laminas. To further explore our findings, our data was compared with previous ARG findings in ACC tissue of the same subjects (available in the Stanley Neuropathology Consortium Integrative Database, http://sncid.stanleyresearch. org; (Kim and Webster, 2010)) on all 8 different ligands for which complete data was available in supragranular and infragranular layers (for binding sites on the

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Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder

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Figure 4 Specific binding of [3H]AZ10419369 in: A. Male and female subjects; B. Each subject group, separated by gender, BD: Bipolar Disorder, MDD: Major Depressive Disorder, SZ: Schizophrenia, C: Control subjects. Box represents interquartile range with horizontal line representing the median, the vertical lines represent the minimum and maximum values within 1.5 times the interquartile range distance from the 1st and 3rd quartile, respectively. Outliers are represented as single dots.

N-methyl-D-aspartate [NMDA], Kainate, Substance P, Mus carinic 1/4 and nicotine Acetylcholinergic receptor). A moderate, but significant correlation was only found between 5-HT1B receptor binding and NMDA receptor intra channel site binding (ligand: [3H]MK801) (outer layers: r= 0. 41, punadjusted = 0.003 inner layers: r= 0.37, punadjusted = 0. 008) in all subjects, see Figure s1 in the supplementary section. After correction for multiple comparisons for all ligands and both layers, only the correlation in outer layers remained significant (padjusted = 0.04, with a statistical power of 87%). [3H]MK801 has previously been shown to be an antagonist for the NMDA receptor with high specificity (Reynolds, 2001). This ionotropic glutamate receptor is mostly situated on pyramidal dendrites and has an impor tant function in postsynaptic excitatory synaptic transmis sion, activated by glutamate (Blanke et al., 2009; Zito and Scheuss, 2010). It should be noted that methods for defining outer and inner layers differed slightly and the availability of ARG data on different receptors was limited. Hence, further studies relating 5-HT1B receptors to the NMDA-receptor and glutamate transmission in human sub jects are needed. The 5-HT1B receptor binding pattern found in the current study mostly implies a localization of these receptors on pyramidal glutamatergic neurons, originating from intracortical connections or from the amygdala (Vogt, 1993). The involvement of the 5-HT1B receptor in glutamatergic transmission in ACC has previously been shown in rat, where 5HT1B activation on pyramidal neurons originating in lamina V resulted in presynaptic inhibition of glutamate release, with no effect on GABA release (Tanaka and North, 1993); and in non-human primates, where an increase of 5-HT1B receptor binding was seen in the nucleus accumbens and ventral pallidum after administration of the NMDA-antagonist ketamine (Yamanaka et al., 2014). Glutamate neurotransmission has shown to play an important role in BD, MDD and SZ: altered glutamate concentrations were found in ACC of MDD patients (Auer et al., 2000) and in supragranular layers in ACC of SZ patients (Benes et al., 1992a, 1992b) compared to

healthy controls. Moreover, alterations in NMDA-associated proteins were found in prefrontal cortex of BD and SZ patients (Beneyto and Meador-Woodruff, 2007). Serotonergic modulation of glutamergic transmission has recently been suggested as a promising alternative approach for pharmacological treatment of depression (Pehrson and Sanchez, 2014). Our results suggest the 5-HT1B receptor to be a stable target in BD, MDD and SZ.

4.2.

Comparison between subject groups

No significant difference in 5-HT1B receptor binding throughout the grey matter was found between subject groups. This result could not be explained by differences in physical features of the brain tissue, such as PMI. As several PET studies have previously been performed using [11C] AZ10419369 (Nord et al., 2014; Tiger et al., 2016), this study provides an opportunity to compare in vitro with in vivo findings. The current findings are in contrast with our previous findings using PET and [11C]AZ10419369 (Tiger et al., 2016), where significantly lower 5-HT1B receptor binding was found in ACC of patients with MDD compared to healthy controls, in vivo. In this PET study, patients were unmedicated, in contrast with the current study where 10 out of 12 MDD patients received pharmacotherapeutic treatment for their illness. Due to too few untreated MDD patients in this study, a medication effect could not be studied. No significant effect of type or use of mood stabilizers and antidepressant on specific binding was found in the patient groups; however, information on compliance and dosage is lacking. The type of prescribed antipsychotics was unknown; however, since commonly used atypical antipsychotics have been shown to have strong serotonergic effects (Meltzer, 1999), a possible effect on 5-HT1B receptor binding was tested. A correlation with lifetime dose of antipsychotics could not be made, as not all antipsychotics show a linear dose-response curve (Davis and Chen, 2004). Instead the outcomes of patients with and without

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

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antipsychotic treatment where compared, which resulted in no significant difference of 5-HT1B receptor binding between those two groups. We have previously reported no significant change in 5-HT1B binding in ACC of patients with MDD who were successfully treated with cognitive behavioral therapy (Tiger et al., 2014). Whether antidepressant drug treatment has the same effect on 5-HT1B binding is not known in humans. Previously, alterations in 5HT1B receptor binding after single administration of antidepressant compounds have been reported (Nord et al., 2013; Yamanaka et al., 2014). An alternative explanation of the divergence between ARG and PET findings could be the fact that [11C]AZ10419369 binding has been shown to be sensitive to pharmacologically induced changes in endogenous serotonin levels (Finnema et al., 2012). Based on the serotonin focused theory on the mechanism of action of several antidepressants (Lapin and Oxenkrug, 1969), related to increasing of extracellular serotonin concentrations, this would however not explain our findings: theoretically a lower measured 5-HT1B receptor binding would then be expected. Lastly, since in the previous PET study patients with severe MDD and suicidality were excluded, our current findings could be explained by the possibility of 5-HT1B receptor distribution being a state marker of depression, altering with severity of the disease, rather than a trait. This however, requires further studies. In the current study, no significant differences in 5-HT1B receptor binding were found between suicide victims and patients with other causes of death. It should be noted that for each studied subject one brain slice was available and that tissue of all subjects cover a smaller area (mostly pregenual ACC, see supplementary material) than the complete ACC which was studied in vivo in the previously mentioned PET study. Several studies show altered findings in subgenual ACC in relation to BD and MDD, which are absent in pregenual ACC (Drevets et al. 2008). It should be further studied whether 5-HT1B receptor alterations in MDD occur throughout the ACC or are specifically located in a subregion. Another previous study on the 5-HT1B receptor involve the protein p11 (S100a10), an adaptor protein which facilitates 5-HT1B receptor function (Svenningsson et al., 2013, 2006). P11 mRNA and protein levels are decreased in ACC of MDD patients comparing to healthy controls (Svenningsson et al., 2006). Likewise, p11 mRNA, along with 5-HT1B receptor mRNA, is reduced in frontal cortices from suicidal victims who suffered from MDD compared to controls (Anisman et al., 2008). As mentioned previously, 5-HT1B receptors have found to be situated on axon terminals (Sari, 2004). Therefore, the deviation from findings in the current study is likely to be caused by alterations in 5-HT1B receptor density in projection areas of the ACC. This is in line with Varnäs et al. (2005) who reported mismatches of 5-HT1B receptor binding and mRNA expression in several human cerebral regions.

4.3.

Gender

The present study shows significantly higher 5-HT1B receptor binding density in the ACC in male subjects than female subjects, which was found to be driven by MDD patients as further analysis showed a significant gender difference only

in this group. Although no significant differences were found in PMI-values between female and male subjects, slight differences could explain part of this effect; median (interquartile range) of PMI was 28.5 h (23.5–40.0 h) and 26.5 (19.0–31.8 h), respectively. The results could not be explained by any of the other individual factors (see Tables 1 and 2). However, the relatively small sample size should be taken into account. Interestingly, specifically MDD is known to have a strong gender difference in prevalence with female preponderance, which is not present in BD and SZ (Whiteford et al., 2015). Clinical manifestations of MDD are known to show strong gender differences (Piccinelli and Wilkinson, 2000) and differences in physical markers are reported, e.g. a PET study showed significantly lower serotonin transporter binding in midbrain of male MDD patients comparing to healthy controls, which was not seen in female patients (Ruhé et al., 2009). Previously, no regional gender difference in 5-HT1B receptor binding was found in 48 control subjects, using [11C]P943 PET (Matuskey et al., 2012), and in 11 MDD patients, using [11C]AZ10419369 PET (Tiger et al., 2016). Future studies with larger sample sizes are required to clarify our findings.

4.4.

Age

A positive correlation with age was seen in BD patients, which did not survive correction for multiple comparisons. Considering the relatively small sample size of BD patients, this effect should be further studied in larger sample sizes to draw any relevant conclusions. No significant correlation between 5-HT1B receptor binding and age was detected in MDD patients, in contrast with our previous findings with PET, where a negative correlation was found in ACC of MDD patients (Tiger et al., 2016). Previously, a negative correlation was also seen in 51 control subjects (Nord et al., 2014). Control subjects in the current study showed a tendency towards negative correlation with age; this effect could possibly be stronger with a larger sample size. The results were not affected by the demographic factors of the subjects and physical factors of the brain tissue mentioned in Tables 1 and 2.

4.5.

Conclusion

In conclusion, in this study a significantly higher 5-HT1B receptor binding was found with ARG in supragranular layers of ACC compared to infragranular layers. This binding pattern showed a correlation with previous findings on the binding pattern of NMDA receptor, a possible modulatory role of 5-HT1B receptors on glutamate transmission in ACC should be further investigated. No differences were found in 5-HT1B receptor binding distribution or total binding in grey matter between patients with BD, MDD, SZ and healthy subjects. However, a significant difference in 5-HT1B receptor binding was found between genders, mostly profound in MDD patients. Future PET-studies could give complementary information to clarify a possible gender difference of 5-HT1B binding in BD, MDD, SZ and healthy subjects.

Please cite this article as: Veldman, E.R., et al., Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of.... European Neuropsychopharmacology (2017), http://dx.doi.org/10.1016/j.euroneuro.2017.02.011

Distribution and levels of 5-HT1B receptors in anterior cingulate cortex of patients with bipolar disorder

Role of funding source The study was supported by the Swedish Research Council (5232013-2982); a donation by Birgitta and Sten Westerberg; Stiftelsen Söderström-Königska sjukhemmet; The Swedish Society of Medicine; and Karolinska Institutet, the Stockholm Centre for Psychiatric Research and Education. The funders had no role in study design, data collection and analysis, in the writing of the report, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, in the writing of the report, decision to publish, or preparation of the manuscript.

Contributors MM Svedberg managed study and protocol design and ER Veldman performed the literature searches and analyses. ER Veldman, MM Svedberg and J Lundberg wrote the first draft of the manuscript. P Svenningsson provided the material and was involved in the planning of this study. All authors contributed to and have approved the final manuscript.

Conflict of interest None.

Acknowledgment The excellent technical assistance of Siv Eriksson, Åsa Södergren, Dr Nicholas Mitsios and Dr Jan Mulder is greatly acknowledged. Postmortem brain tissue was kindly donated by the Stanley Foundation Brain Bank, courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert Yolken.

Appendix A.

Supplementary material

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/ j.euroneuro.2017.02.011.

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