Platelet [3H]paroxetine and [3H]lysergic acid diethylamide binding in seasonal affective disorder and the effect of bright light therapy

Platelet [3H]Paroxetine and [3H]Lysergic Acid Diethylamide Binding in Seasonal Affective Disorder and the Effect of Bright Light Therapy Khristina Smedh, Olav Spigset, Per Allard, Tom Mjo¨rndal, and Rolf Adolfsson Background: Seasonal affective disorder (SAD) has been regarded as a melatonin disorder, but the pathophysiological mechanisms of SAD are to a large extent unclarified. Serotonergic mechanisms have also been studied, but they have shown inconsistent results. Methods: We have compared [3H]paroxetine and [3H]lysergic acid diethylamide (LSD) binding in platelets from 23 SAD patients and 23 controls. Then SAD patients had 4 weeks of light therapy. On the last treatment day new blood samples were drawn. Symptoms before and after light treatment were measured by SIGH-SAD. Results: Bmax for paroxetine binding before light treatment was higher in SAD patients compared to controls and also higher in responders than in nonresponders. Bmax decreased significantly during light treatment. We also found a negative correlation between the two Bmax values before but not after light treatment. There was a negative correlation between Bmax for paroxetine binding before treatment and clinical status after treatment. Patients with reduced Bmax for LSD binding after treatment had a better clinical treatment response. Conclusions: The present study indicates that serotonin receptor parameters might be suitable in the prediction of clinical response to light treatment. Biol Psychiatry 1999;45:464 – 470 © 1999 Society of Biological Psychiatry Key Words: Seasonal, affective, light, serotonin, [3H]paroxetine, [3H]lysergic acid diethylamide

Introduction

A

causal relationship between depression and light was proposed already in 1946 (Marx 1946). Light treatment for patients with seasonal affective disorder (SAD) (Lewy et al 1982; Rosenthal et al 1984a) is now well

From the Department of Psychiatry (KS, PA, RA); and Department of Clinical Pharmacology (OS, TM), Umeå Universitet, Umeå, Sweden. Address reprint requests to Prof. Rolf Adolfsson, Department of Psychiatry, Umeå University, S-901 87 Umeå, Sweden. Received July 12, 1996; revised May 12, 1997; revised December 14, 1997; accepted January 21, 1998.

© 1999 Society of Biological Psychiatry

established. The pathophysiological mechanisms, however, are still to a large extent unknown. It has been suggested that SAD patients have a disturbed circadian rhythm (Lewy et al 1988; Souetre et al 1985) and that bright light would produce a phase shift, thereby overcoming the depressive state (Lewy et al 1987; Terman et al 1989); however, other studies found no such relationships (e.g., Wirz-Justice et al 1993). Others have postulated that melatonin is involved in the pathophysiology of SAD (Lewy et al 1987). Some studies have shown a relationship between melatonin suppression and level of depression (Lieberman et al 1985; Wetterberg 1991), whereas other studies failed to show such a relationship (Rao et al 1992; Wirz-Justice et al 1990). Research on the pathophysiology of SAD has also focused on the biogenic amines, including serotonin (Anderson et al 1992; Oren et al 1994; Rudorfer et al 1993). The “atypical” symptoms, e.g., carbohydrate craving, which are often present in SAD patients, have been explained by serotonin deficiency (Wallin and Rissanen 1994; Wurtman 1993). It has also been suggested that SAD patients would have a limited availability of serotonin in the winter, and that bright light would increase this level (O’Rourke et al 1987). A recent study showed that light increases available serotonin in hamster brain (Penev et al 1997). Moreover, there is evidence that d-fenfluramine (O’Rourke et al 1989) and fluoxetine (Ruhrman et al 1993), which increase serotonergic activity, exert an antidepressant effect in SAD patients. Results from some studies point at changes at the receptor or at the postreceptor levels. Abnormal behavioral and hormonal responses to m-chlorophenylpiperazine, a postsynaptic serotonin (5-HT)2A as well as 5-HT2C receptor agonist, are compatible with a possible deficiency in serotonergic transmission in SAD patients (Garcia-Borreguero et al 1995; Jacobsen et al 1994). A low number of [3H]imipramine binding sites has been reported in SAD patients (Sza´do´czky et al 1989, 1991). In a study using [3H]paroxetine as ligand (Ozaki et al 1994), no differences were found between SAD patients and healthy controls, and no changes were observed in SAD patients following light 0006-3223/99/$19.00 PII S0006-3223(98)00069-9

SAD, Bright Light, and Platelet Serotonin

therapy. In another study using the same ligand (Mellerup et al 1993), the number of serotonin uptake sites, however, decreased significantly following light therapy. [3H]Lysergic acid diethylamide (LSD) binding to 5-HT2A receptors has, to our knowledge, not been studied in SAD. As a part of a larger clinical study comparing treatment response between light room and portable light visor, we have examined [3H]paroxetine and [3H]LSD binding in platelets from SAD patients and healthy controls matched for age and gender. Moreover, the effects of therapeutic bright light on the binding parameters were studied. Our hypothesis is that both platelet paroxetine and LSD binding in SAD differs from controls, and that bright light will decrease these differences.

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Table 1. Description of the Light Room and the Light Visor Equiments Used in the Study Light room Equipment

Light administration Lamps and color Temperature (K) Illuminance (lux)

Luminance (candela)

A 10-m2 room without windows; white walls, ceiling, and floor Reflected light Full spectrum fluorescent tubes 5400 700 measured 1.2 m above the floor Average: 360/m2

Light Visor A portable, headmounted light visor Direct light Krypton incandescent bulbs 2800 1500 measured 5 cm from the light source Average 22300/m2

Methods and Materials Twenty-three outpatients, 19 women and 4 men, with a mean age of 44.0 years (range 26 – 69 years), participated in the study. Twenty-two patients fulfilled the DSM-IV criteria for major depression recurrent type with seasonal variation, and 1 had a depressive type bipolar disorder also with seasonal variation. The seasonal variations in all patients were confirmed by the selfrating questionaire Seasonal Pattern Assessment Questionaire (SPAQ) (Rosenthal et al 1984b). Twenty of the patients had not taken psychotropic drugs for at least 6 months prior to the study. Three patients had ongoing psychopharmacologic treatment (zimelidine, lithium, and buspirone, respectively). Zimelidine, which is available for license prescription in Sweden, and lithium treatment had not been changed the last 6 months and buspirone treatment the last month prior to study start. The severity of depression was measured by the SIGH-SAD-SR scale (Williams and Link 1988), a combination of Hamilton Depression Rating Scale and more specific winter depression items. There was a fixed time interval for ratings and blood samples, at the first treatment day and after 4 weeks of light treatment, to control for cyclic monthly mood changes, i.e., premenstrual syndrome (e.g., Wetzel et al 1975; Halbreicht and Endicott 1985), which otherwise could influence the results. The study was approved by the regional Ethics Committee at the University of Umeå.

Control Subjects The matched control group consisted of 19 women and 4 men with a mean age of 43.6 years (range 21–73 years). They were all healthy as assessed by medical history, physical examination, and routine blood chemistry tests. All volunteers had been entirely drug-free for at least 4 weeks before entering the study. To avoid a systematic effect of possible circannual fluctuations (Spigset et al, in press), blood sampling was performed during the same period of time as for the patients, from October to February.

Light Treatment Light was administered in the morning with two different techniques. Both light treatment methods are used as antidepres-

sive treatment in seasonal affective disorder, although the efficacy of the light visor is not clarified (Joffe et al 1993; Levitt et al 1996; Rosenthal et al 1993; Stewart et al 1990; Teicher 1995), and in all light treatment methods there are discussions of the placebo effect (e.g., Levitt et al 1996). Patients were randomly given treatment either 90 min in a lightroom (n 5 13) (Kjellman et al 1993) or 40 min with a portable head-mounted device (Light Visor™) (n 5 10) (Stewart et al 1990). Treatment was given 5 days per week for 2 consecutive weeks and thereafter 2 days per week. A full description of the equipments is seen in Table 1.

Blood Sampling Blood was sampled from the antecubital vein with a 20-gauge needle and collected into polyethylene tubes containing 1.6 mg ethylenediamidetetraacetate (EDTA) per milliliter blood. Total blood volume obtained was 37.5 mL for [3H]LSD binding and 22.5 mL for [3H]paroxetine binding. All samples were taken between 9 and 11 AM.

[3H]LSD Binding Assay Membrane preparation for [3H]LSD binding assay was performed using a modification of the method of Geaney et al (1984). In short, platelet-rich plasma (PRP) was obtained by centrifugation at 180 g for 15 min at 20°C. PRP was then centrifuged at 1200 g for 10 min at 10°C. The platelet pellet was stored frozen at least at 270°C until use. At the day of analysis, the platelet pellet was resuspended in hypotonic TRIS– buffer (5 mmol/L TRIS–HCl, 0.1% EDTA), gently homogenized by 20 strokes of a hand-driven glass/glass homogenizer, and thereafter centrifuged at 30,000 g for 15 min. The resulting membrane pellet was washed once more in hypotonic TRIS– buffer, homogenized and centrifuged as above, and then resuspended in the incubation buffer (50 mmol/L TRIS–HCl, 120 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L MgCl2). [3H]LSD with a specific activity of 76.7 Ci/mmol was purchased from DuPont N.E.N. (Boston, MA). Two hundred microliters membrane suspension, 25 mL [3H]LSD, and 25 mL

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Table 2. Clinical Results, Bmax, and Kd for [3H]Paroxetine and [3H]LSD Binding before and after Light Treatment and in Healthy Controls p values

Clinical status (SIGH-SAD) Room (n 5 13) Visor (n 5 10) p value (room/visor) Bmax paroxetine bindinga (fmol/mg protein) Kd paroxetine bindinga,b (nmol/L) Bmax LSD binding (fmol/ mg protein) Kd LSD binding (nmol/L)

Before treatment (mean 6 SD)

After treatment (mean 6 SD)

Controls (n 5 23) (mean 6 SD)

Before/ after treatment

Before treatment (SAD/controls)

28.1 6 8.1 26.8 6 8.2 29.8 6 7.9

17.6 6 9.7 14.8 6 10.3 21.2 6 7.8

— — —

— — —

909 6 147

834 6 130

765 6 160

,.001 ,.001 .024 .067 .006

.002

0.058 6 0.043

0.046 6 0.013

0.039 6 0.01

.165

.076

28.2 6 8.8

25.3 6 8.0

24.3 6 4.5

.030

.083

1.03 6 0.46

0.88 6 0.38

1.10 6 0.46

.043

.550

a

Three patients missing for administrative reasons. One sample excluded due to technical problems.

b

incubation buffer were incubated in microtiter plates for 4 hours at 37°C. Seven concentrations of [3H]LSD ranging from 0.25 to 2.5 nmol/L were used. Specific binding was defined as total binding minus binding in the presence of 300 nmol/L Spiperone (Sigma, St Louis, MO). All [3H]LSD binding assays were performed in triplicate. The binding was terminated by filtration through Whatman GF/F filters using a cell harvester. The filters were washed for 30 sec with 50 nmol/L TRIS– buffer and dried. The radioactivity trapped by the filters was determined by liquid scintillation spectroscopy. Total bound [3H]LSD did not exceed 2% of the total radioactivity.

[3H]Paroxetine Binding Assay Platelet-rich plasma was obtained by low-speed centrifugation at 180 g for 15 min at 20°C. The volume of each sample was estimated, and the same amount of assay buffer (50 mmol/L TRIS–HCl, 120 mmol/L NaCl, 5 mmol/L KCl, pH 7.4) was added (1:1). After centrifugation at 3000 g for 20 min at 4°C, the pellet was resuspended in 20 mL ice-cold assay buffer, recentrifugated at 3000 g for 10 min at 4°C, and homogenized in 10 1 10 mL of the buffer, using a Kinematica Polytron homogenizer (Luzern, Switzerland), setting 6 for 7 sec. After a final centrifugation at 15,000 g for 10 min at 4°C, the pellet was stored at least at 270°C until assay. At the day of analysis, the platelet pellet was resuspended in assay buffer to a final volume of 35 mL. The homogenates were incubated for 60 min at 25°C in a total volume of 1600 mL. The incubation volume contained 750 mL of tissue homogenate, 750 mL of radioligand, and 100 mL of buffer or drug. Nine concentrations of [3H]paroxetine (33.3 Ci/mmol, Du Pont N.E.N., Boston, MA) ranging from 0.01 to 1.2 nmol/L were used. Specific binding was defined as total binding minus binding in the presence of 10 mmol/L citalopram (H. Lundbeck and Co. A/S, Copenhagen, Denmark) (Andersson and Marcusson 1990). All [3H]paroxetine binding assays were performed in duplicate. After the addition of 6 mL ice-cold buffer, the homogenates were

rapidly filtered through Whatman GF/C filters using a 24channel cell harvester (Brandel, Geithersburg, MD). Finally, the filters were washed with three 6-mL rinses of the buffer. The radioactivity trapped by the filters was determined by liquid scintillation spectroscopy. Total bound [3H]paroxetine did not exceed 5% of the total radioactivity. As in the [3H]LSD binding experiments, protein content was assayed by the method of Lowry et al (1951) with modifications suggested by Markwell et al (1978). We have used two different methods for LSD binding and paroxetine binding because we have followed the methods originally described for the separation of platelet for paroxetine binding (Andersson and Marcusson 1990) and LSD binding (Geaney et al 1984) as closely as possible.

Statistical Methods The binding characteristics (Bmax and Kd) for [3H]LSD and [3H]paroxetine binding were calculated from Scatchard analysis of the specific binding data according to the method of least squares linear regression. For comparative statistics, paired and unpaired two-tailed Student’s t tests and Pearson’s regression analysis were applied. p values less than .05 were regarded as statistically significant.

Results SIGH-SAD-SR depression scores were significantly reduced after light treatment in both light therapy conditions (Table 2). There were no differences in treatment outcome or binding parameters between patients with or without psychopharmacologic treatment. Bmax and Kd for paroxetine binding and LSD binding before and after light treatment, as well as in the control group, are also shown in Table 2. Bmax for paroxetine binding was significantly higher in the SAD group before

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Table 3. Bmax and Kd for [3H]Paroxetine and [3H]LSD Binding in Light Treatment Responders and Nonresponders Before treatment

Responders (n 5 8) Nonresponders (n 5 13) p value

After treatment

Bmax paroxetine bindinga

Kd paroxetine bindinga

Bmax LSD binding

Kd LSD binding

Bmax paroxetine binding

Kd paroxetine binding

Bmax LSD binding

Kd binding

1006 6 146

0.049 6 0.007

25.5 6 8.7

1.18 6 0.61

914 6 162

0.043 6 0.010

20.3 6 6.9

0.86 6 0.18

857 6 124

0.063 6 0.053

29.5 6 8.9

0.96 6 0.38

789 6 86

0.048 6 0.015

27.4 6 7.6

0.88 6 0.44

.330

.309

.031

.391

.046

.901

.027

b

.481

For response at least 50% reduction in SIGH-SAD-SR score. a Three patients missing for administrative reasons. b Sample excluded due to technical problems.

light treatment compared to the control group (p 5 .002), while this difference did not reach statistical significance for LSD binding (p 5 .083). Following light treatment, Bmax for paroxetine binding and LSD binding were significantly reduced (p 5 .006 and .030, respectively), and not different from the control group. Also Kd for LSD binding was significantly reduced (p 5 .043) following light therapy. With a response definition of at least 50% reduction in SIGH-SAD-SR score, Bmax for paroxetine binding was significantly higher in responders than in nonresponders before treatment (p 5 .027) (Table 3). For Bmax LSD binding there were no such differences. When comparing responders and nonresponders Bmax for paroxetine binding was similarly reduced after light treatment, whereas the level of Bmax for LSD binding was significantly lower after treatment in responders than in nonresponders (p 5 .046). There was a significant negative correlation between Bmax for paroxetine binding and Bmax for LSD binding before treatment (r 5 2.46; p 5 .039) (Figure 1) but not after treatment (r 5 .087; p 5 .72) and not in the control

group (r 5 .132; p 5 .53). Correlations between Bmax for paroxetine binding and depression scores are presented in Table 4. Bmax for paroxetine binding before as well as after treatment was significantly negatively correlated with SIGH-SAD-SR score after treatment. Patients with decreased Bmax values for LSD binding after treatment (n 5 18) had a mean reduction in SIGHSAD-SR score of 45.5%, while the group without decreased Bmax (n 5 5) had a mean reduction of 10.2% (p 5 .012). For Bmax for paroxetine binding there were no clinical differences between these subgroups (42.2% and 35.8% reduction in SIGH-SAD-SR score, respectively, p 5 .663). There were no significant correlations between Kd for paroxetine or LSD binding and SIGH-SAD-SR score.

Discussion It is important to notice the relatively small sample size in our study, especially when responders are compared with nonresponders, and also to consider the use of multiple t tests. The use of platelets as a model for brain should always be interpreted with caution; it has not consistently lived up to the promise of reflecting central nervous system activity. Table 4. Correlations between SIGH-SAD-SR and Bmax for Paroxetine and LSD Binding before and after Light Treatment SIGH-SAD-SR before treatment

3

3

Figure 1. Bmax for [ H]paroxetine binding and [ H]LSD binding in patients with seasonal affective disorder before light treatment (r 5 2.46; p 5 .039).

Bmax paroxetine binding before treatment Bmax paroxetine binding after treatment Bmax LSD binding before treatment Bmax LSD binding after treatment

SIGH-SAD-SR after treatment

r

p

r

p

2.271

.248

2.449

.047

2.449

.047

2.548

.012

.041

.863

.094

.693

.080

.737

.341

.141

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The effects of light therapy on serotonin receptor parameters in patients with SAD have earlier been investigated using [3H]imipramine (Sza´do´czky 1989,1994) as radioligands and have yielded somewhat inconsistent results. These inconsistencies may be due to diagnostic, geographic, or methodologic diversity, the use of different ligands, a small number of subjects, medications, different treatment times, normal variability in age and gender, and diurnal or seasonal variations. In the present study, there were no age or gender differences between patients and controls, and platelets were obtained at the same time of day and during the same period of the year. Since some results indicate that the menstrual cycle may affect at least 5-HT2A receptor status (Spigset and Mjo¨rndal 1997), a 4-week sampling interval might therefore be appropriate. In an earlier study using paroxetine as ligand (Mellerup et al 1993), patients who responded to light therapy had a significant reduction in Bmax following treatment. In contrast, patients who did not respond to light therapy were found to have a lower Bmax prior to treatment, and Bmax did not change significantly following treatment. Although that study did not use stringent criteria for the diagnosis of SAD, had a short treatment period of 1–2 weeks, and included a number of drug-treated patients (26% medicated by benzodiazepines; 10% by antidepressants), the results are principally the same as in our study. In another study using paroxetine as radioligand (Ozaki et al 1994), no differences in Bmax for paroxetine binding were found between SAD patients before treatment and healthy controls, and no effects were observed after light treatment. There were many differences between this study and the study by Ozaki, for example treatment time, geographic localization, and basal measures. We found a significant negative correlation between Bmax for paroxetine and for LSD binding in SAD patients before treatment, a difference, however, that disappeared during treatment and was not present in the control group. One may speculate that a reduced serotonergic activity, as postulated in SAD patients, may control the regulation of both the uptake site and the 5-HT2A receptor. In contrast, such conditions might not be present when the serotonin activity is above a threshold, as in controls and in SAD patients after light therapy. The most interesting clinical aspect could be the difference in Bmax for paroxetine binding in responders and nonresponders before treatment. We also found a lower Bmax for LSD binding in responders than in nonresponders after treatment. Perhaps a high number of uptake sites before treatment is necessary for a postsynaptic upregulation after treatment. Studies in a larger number of subjects are warranted to study if Bmax for [3H]paroxetine is valuable as a predictor for the effect of light treatment.

The project was supported in part by a grant from the Swedish Society for Medical Research, So¨derstro¨m–Ko¨ningska Foundation, and The Joint Committee for Northern Sweden (Samverkansna¨mnden). Dr Spigset was the recipient of a fellowship in clinical pharmacology, funded by Merck Sharp & Dohme, Sweden. [3H]paroxetine and citalopram were generous gifts from Novo Nordisk Pharma AB, Malmo¨, Sweden, and Lundbeck and Co A/S, Copenhagen, Denmark, respectively. We acknowledge the technical assistance by Ingrid Persson and Margareta Danielsson and the clinical assistance by RN Britta Lo¨fgren and RN Kerstin Granberg.

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