Psychopharmacology

, Volume 195, Issue 3, pp 325–332

Evaluation of serotonin, noradrenaline and dopamine reuptake inhibitors on light-induced phase advances in hamster circadian activity rhythms

Authors

    • Department of BiologyValdosta State University
  • Mark J. Millan
    • Psychopharmacology DepartmentInstitut de Recherches Servier
Original Investigation

DOI: 10.1007/s00213-007-0903-z

Cite this article as:
Gannon, R.L. & Millan, M.J. Psychopharmacology (2007) 195: 325. doi:10.1007/s00213-007-0903-z

Abstract

Rationale

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for the treatment of anxiodepressive states that are often associated with perturbed circadian rhythms including, in certain patients, phase advances. Surprisingly, the influence of SSRIs upon circadian activity rhythms has been little studied in experimental models.

Objectives

Accordingly, this study examined the ability of SSRIs to modulate the phase-setting properties of light on circadian activity rhythms in hamsters. Their actions were compared to those of the mixed serotonin/noradrenaline reuptake inhibitor (SNRI), venlafaxine, the selective noradrenaline reuptake inhibitor, reboxetine, and the dopamine reuptake inhibitor, bupropion.

Materials and methods

Wheel-running activity rhythms were recorded in male Syrian hamsters. Drugs were administered systemically before a light stimulus that was used to advance the timing of the hamster running rhythms.

Results

Four chemically diverse SSRIs, citalopram (1–10 mg/kg, intraperitoneally), fluvoxamine (1–10), paroxetine (1–10), and fluoxetine (10 and 20), all robustly and significantly inhibited the ability of light to phase advance hamster circadian wheel-running activity rhythms. Their actions were mimicked by venlafaxine (1–10) that likewise elicited a marked reduction in phase advances. Conversely, reboxetine (1–20) and bupropion (1–20) did not exert significant effects.

Conclusions

These data suggest that suppression of serotonin (but not noradrenaline or dopamine) reuptake by SSRIs and SNRIs modifies circadian locomotor activity rhythms in hamsters. Further, they support the notion that an inhibitory influence upon the early-morning light-induced advance in circadian activity contributes to the therapeutic effects of serotonin uptake inhibitors in certain depressed patients.

Keywords

DepressionSuprachiasmatic nucleusBiological rhythms

Introduction

Circadian rhythmicity of physiological mechanisms is a core process in all species, from simple single-celled organisms to humans (Johnson et al. 1996; Panda et al. 2002). In mammals, the master circadian pacemaker is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, and the pacemaker is itself entrained to the environmental light/dark cycle by photic information arriving from the eye via the retinohypothalamic tract (Johnson et al. 1988). The SCN pacemaker cells then entrain peripheral oscillators to this lighting schedule through direct and humoral mechanisms (Albrecht and Eichele 2003; Cailotto et al. 2005; Guo et al. 2006; Kriegsfeld and Silver 2006).

The timing of the SCN pacemaker is also modulated by input from the intergeniculate leaflet nucleus of the thalamus and from the midbrain raphe nuclei, although the precise function of these two inputs is poorly understood (Morin and Allen 2006). Serotonergic terminals from the raphe form a dense plexus within the rodent SCN (Morin 1999; Hay-Schmidt et al. 2003). Accordingly, there is likewise a high density of serotonin (5-HT) transporters (SERT) within this structure (Sur et al. 1996; Amir et al. 1998; Legutko and Gannon 2001).

The high concentration of SERT in the SCN is of potential clinical significance inasmuch as selective 5-HT reuptake inhibitors (SSRIs) have long been prescribed for the treatment of depressive and anxiodepressive states, which are characterized by marked alterations in circadian rhythms (Healy and Waterhouse 1995; Duncan 1996; Millan 2003, 2006; Walter 2005). One interpretation of depression and its treatment incorporates the notion that circadian rhythms are phase advanced in relation to the environmental light/dark cycle and that antidepressants may be effective, at least in part, by restoring the proper phase relationship between the SCN pacemaker and the light/dark cycle (Healy and Waterhouse 1995; Duncan 1996; Wirz-Justice 2006). In this regard, several classes of serotonergic agonist acting within the SCN inhibit light-induced phase advances of circadian activity rhythms in rodents (Pickard et al. 1996; Kennaway and Moyer 1998; Weber et al. 1998; Gannon 2001; Sprouse et al. 2005). It would, therefore, be predicted that a SSRI-induced increase in synaptic levels of 5-HT in the SCN should similarly suppress light-induced phase shifts. Such an effect would be consistent with the above-evoked hypothesis that antidepressants abrogate the pathological advancement of circadian rhythms in a subpopulation of depressed patients.

In view of the above, it is surprising that the effects of SSRIs on circadian rhythms have received little attention (Klemfuss and Kripke 1994; Healy and Waterhouse 1995; Duncan 1996). Accordingly, in the present study, we the influence of citalopram, fluoxetine, fluvoxamine, and paroxetine (upon light-induced phase shifts) in circadian activity rhythms in hamsters. This model was selected in view of its broad utilization in previous studies (Pickard et al. 1996; Van Reeth et al. 1997; Kennaway and Moyer 1998; Weber et al. 1998; Gannon 2003; Sprouse et al. 2005; Gannon and Millan 2005, 2006b; Lall and Harrington 2006) and because altered diurnal rhythms in hamsters provoke changes in affective responses, including depressive-like behaviors (Prendergast and Nelson 2005). SSRIs were compared to venlafaxine, a mixed inhibitor of 5-HT and noradrenaline (NA) reuptake (SNRI), reboxetine, a preferential NA reuptake inhibitor, and bupropion, a preferential inhibitor of dopamine (DA) reuptake (Ascher et al. 1995; Millan et al. 2000; Wong et al. 2000; Bymaster et al. 2001; Morilak and Frazer 2004; Walter 2005; Millan 2006).

Materials and methods

One-month-old male Syrian hamsters were obtained from Charles River Laboratories (Kingston, NY) and were housed in a 14-h:10-h light/dark cycle for at least 2 weeks before the start of experiments. Food and water was provided ad libitum at all times. The principles of laboratory animal care were followed according to guidelines specified by the institutional animal care committee of Valdosta State University and the Animal Welfare Act of the United States. At the beginning of each experiment, hamsters were transferred to individual cages equipped with running wheels and housed under conditions of constant darkness (DD). Hamster wheel-running activity was recorded by a computer using magnets and magnetic switches attached to the wheel and the lid of the cage, respectively. The activity data were sorted and analyzed using the ClockLab system from Actimetrics (Wilmette, IL).

Hamsters were allowed to free-run in DD for approximately 10 days before being removed from their cage under dim red light (<1 lux) and exposed to a 10-min pulse of dim white light (20 lux) and then returned to their cage. The light pulse was administered at circadian time (CT) 19, which is 7 h after the onset of wheel-running activity at CT 12. Each hamster was given an intraperitoneal injection of either drug or vehicle (between subjects experimental design) 45 min before the light pulse (CT 18.25). For non-photic controls, no light pulse was administered, and hamsters were returned to their cage after injections. Hamsters were allowed to free-run for an additional 10 days after light exposure to conclude the experiment. This method typically produces phase advances in wheel-running activity rhythms that average between 1 and 1.5 h. Lines are fit through the activity onsets using the software from Actimetrics for the 5 days preceding the light pulse and days 6–10 after the light pulse. The time difference in the extrapolated intersection of these two lines on the day of the light pulse determines the magnitude of the phase advance. The results were analyzed using SigmaStat software to conduct analysis of variance (ANOVA) tests and post-hoc analysis.

All of the compounds used in this study were synthesized (not purchased) by chemists at the Institute de Recherches Servier (Paris, France). Bupropion, citalopram, fluvoxamine, paroxetine, reboxetine, and venlafaxine were dissolved in water (vehicle). Fluoxetine was dissolved in 10% dimethylsulfoxide (also used as vehicle in that experiment).

Results

SSRIs and the SNRI, venlafaxine

All the SSRIs used in this study, as well as the SNRI, venlafaxine, inhibited light-induced phase advances in hamster wheel-running rhythms, albeit with different efficacies and potencies. Citalopram produced the largest inhibition (Fig. 1) while paroxetine and venlafaxine were the most potent, producing significant effects at doses of 5 mg/kg (Fig. 2). Fluoxetine was the least effective compound of the test group (Fig. 2). Citalopram inhibited light-induced phase advances by approximately 75% at the highest dose tested (Fig. 1). The mean phase advance produced by light and vehicle was 1.0 ± 0.1 h, and the light-induced advance seen after injection of 10 mg/kg citalopram was 0.24 ± 0.1 h (mean ± SEM; one-way ANOVA, F[3, 32] = 14.7, P < 0.001). Fluoxetine was ineffective at inhibiting light-induced phase shifts at a dose of 10 mg/kg but did inhibit phase advances by 32% at 20 mg/kg (Fig. 2). The mean phase advance with vehicle was 1.2 ± 0.1 h and with 20 mg/kg fluoxetine was 0.8 ± 0.1 h (mean ± SEM; one-way ANOVA, F[2, 36] = 3.6, P < 0.05). Fluvoxamine inhibited light-induced phase advances by only 32% at a dose of 10 mg/kg (Fig. 2). The mean phase advance with vehicle was 1.5 ± 0.1 h and with 10 mg/kg fluvoxamine was 1.02 ± 0.1 h (mean ± SEM; one-way ANOVA, F[3, 19] = 3.9, P < 0.05). Paroxetine displayed a dose-dependent inhibition of light-induced phase advances with a maximal inhibition of approximately 60% at a dose of 10 mg/kg (Fig. 2). The mean phase advance with vehicle was 1.4 ± 0.1 h and with 10 mg/kg paroxetine was 0.5 ± 0.2 (mean ± SEM; one-way ANOVA, F[3, 25] = 8.9, P < 0.001. Venlafaxine inhibited light-induced phase advances of hamster activity rhythms by approximately 40% at both 5-mg/kg and 10-mg/kg doses (Fig. 2). The mean phase advance with vehicle was 1.1 ± 0.1 h and with 10 mg/kg venlafaxine was 0.65 ± 0.1 h (mean ± SEM; one-way ANOVA, F[3, 21] = 5.3, P < 0.01).
https://static-content.springer.com/image/art%3A10.1007%2Fs00213-007-0903-z/MediaObjects/213_2007_903_Fig1_HTML.gif
Fig. 1

Citalopram inhibition of light-induced phase advances in hamster circadian activity rhythms. The actograms below the bar chart show 15 consecutive days of wheel-running activity over a 24-h period. The height of the bars on each day represents relative numbers of wheel revolutions in the 6-min period. The star is the time of the light pulse. Numbers in parentheses below the bars in the chart is the number of hamsters for each dose. Asterisk, P < 0.001 from vehicle, post-hoc Student–Newman–Keuls

https://static-content.springer.com/image/art%3A10.1007%2Fs00213-007-0903-z/MediaObjects/213_2007_903_Fig2_HTML.gif
Fig. 2

SSRI and SNRI inhibition of light-induced phase advances in hamster circadian activity rhythms. a, b, dAsterisk, P < 0.05 from vehicle, casterisk, P < 0.001, double asterisk, P < 0.05 from vehicle, post-hoc Student–Newman–Keuls

Therefore, at equivalent doses of 10 mg/kg, the effectiveness of the SSRIs and SNRI at inhibiting light-induced phase advances in hamster wheel-running activity rhythms is ranked as follows:
$$ citalopram > paroxetine > venlafaxine > fluvoxamine > fluoxetine. $$

Selective NA and DA reuptake inhibitors

In contrast to the SSRIs, neither reboxetine nor bupropion had any effect on light-induced phase advances of hamster circadian activity rhythms (Table 1). In a separate study, higher doses (20 mg/kg) of reboxetine and bupropion also failed to inhibit light-induced phase advances: vehicle = 0.8 ± 0.1 h, reboxetine = 1.2 ± 0.2 h, bupropion = 1.1 ± 0.2 h (mean ± SEM; one-way ANOVA, F[2, 11] = 1.3, P = 0.3).
Table 1

Bupropion and reboxetine do not inhibit light-induced phase advances of hamster circadian activity rhythms

Vehicle

1 mg/kg

5 mg/kg

10 mg/kg

Bupropion

 1.3 ± 0.3 h, n = 6

0.9 ± 0.3 h, n = 5

1.2 ± 0.2 h, n = 5

1.1 ± 0.1 h, n = 6

Reboxetine

 1.6 ± 0.1 h, n = 6

1.5 ± 0.1 h, n = 6

1.9 ± 0.1 h, n = 6

1.5 ± 0.1 h, n = 6

Nonphotic effects

None of the compounds used in this report elicited any phase changes, advances, or delays, in wheel-running rhythms when injected without light at CT 19 (Table 2).
Table 2

There were no nonphotic effects of the compounds used in this report when injected without light at CT 19

Vehicle

20 mg/kg fluoxetine

10 mg/kg fluvoxamine

10 mg/kg citalopram

10 mg/kg venlafaxine

Vehicle

10 mg/kg paroxetine

20 mg/kg reboxetine

20 mg/kg bupropion

0.07 ± 0.09 h, n = 5

0.02 ± 0.06 h, n = 4

(−)0.05 ± 0.1 h, n = 5

(−)0.04 ± 0.08 h, n = 5

0.07 ± 0.06 h, n = 5

(−)0.14 ± 0.13, n = 4

(−)0.1 ± 0.04, n = 4

0.0 ± 0.05, n = 5

(−)0.02 ± 0.09, n = 4

Discussion

In this study, four chemically distinct SSRIs, citalopram, fluoxetine, fluvoxamine, and paroxetine, inhibited light-induced phase advances of hamster circadian wheel-running rhythms by 32–75% when injected late in the subjective night (Figs. 1 and 2). Moreover, their actions were mimicked by the SNRI, venlafaxine, whereas bupropion and reboxetine, preferential inhibitors of NA and DA reuptake, respectively, were ineffective (Table 1). These results, consistent with the predominance of serotonergic vs dopaminergic and adrenergic input to the SCN, indicate that increasing synaptic levels of 5-HT but not NA or DA inhibits the ability of light to phase advance the circadian pacemaker.

Unfortunately, there is no obvious “ranking” of the efficacy of potencies of SSRIs in depression (Masand and Gupta 1999; Millan 2006) that can be correlated with their relative activities in modulating circadian rhythms in hamsters. However, among these SSRIs, citalopram is the most selective vs transporters for NA and DA (Walter 2005; Millan 2006), perhaps accounting for its particularly marked efficacy (Fig. 1). In any case, the coherent pattern of data acquired in this study is important because little information is available concerning the influence of antidepressants on circadian rhythms. Nonetheless, while the tricyclic imipramine did not affect light-induced phase shifts in hamsters (Refinetti and Menaker 1993), the inhibitory influence of the monoamine oxidase inhibitor clorgyline was attributed to elevated levels of 5-HT, in line with the present findings (Duncan 1996). As regard to SSRIs and SNRIs, the effect of citalopram, fluvoxamine, paroxetine, and venlafaxine upon circadian activity rhythms have never been documented. A single paper in mice failed to demonstrate any effect of paroxetine on light-induced expression of the circadian clock genes mPer1 or mPer2 in the SCN (Takahashi et al. 2002). As concerns to fluoxetine, available data are sparse and rather contradictory. Chronic oral administration did not affect the light-entrained phase or the free-running period in hamsters (Klemfuss and Kripke 1994), but fluoxetine was provided in drinking water, an imprecise method of drug administration. Conversely, chronic injection of fluoxetine decreased and delayed the timing of hypothalamic temperature rhythms in hamsters, an observation consistent with the present work (Gao et al. 1992; Duncan et al. 1995). In mice, chronic fluoxetine administration shortened the circadian period of wheel running activity (Possidente et al. 1992) and reversed the period-lengthening effect of olfactory bulbectomy (Possidente et al. 1996). In addition, acute administration of fluoxetine inhibited light-induced phase delays in circadian wheel-running rhythms in mice (Challet et al. 2001). Collectively, the present and previous data indicate that fluoxetine generally inhibits the influence of light on circadian activity rhythms in hamsters and mice. In contrast, in rats—a species in which serotonergic input to the circadian system mimics the effects of light (Kohler et al. 2000)—chronic fluoxetine administration did not affect circadian wheel-running activity rhythms in subjects housed under constant darkness (Wollnik 1992). Further, fluoxetine induced nonphotic phase shifts in a rat hypothalamic slice preparation during the day after preloading of tryptophan to offset the loss of serotonergic input from the raphe (Sprouse et al. 2006). However, the relationship of day-time phase shifts in slice preparations to entrainment processes of the intact circadian system in vivo is uncertain.

In theory, SSRIs might act in raphe nuclei projecting to the SCN to prevent phase advances. However, blockade of raphe-localized SERT would increase 5-HT1A autoreceptor inhibition of raphe neuronal firing and attenuate serotonergic output from the raphe (Millan et al. 2000). Consequently, light-induced phase advances of hamster circadian activity rhythms should be increased, as 5-HT input to the SCN is inhibitory (Weber et al. 1998; Gannon 2003; Gannon and Millan 2006a). In addition, there is extensive SERT immunoreactivity within the SCN of hamsters and other rodents (Sur et al. 1996; Amir et al. 1998; Legutko and Gannon 2001). It seems more likely, therefore, that SSRIs exert their actions within the SCN to enhance extracellular 5-HT and thereby inhibit light-induced phase shifts of hamster activity rhythms (Weber et al. 1998).

The presence of DA transporters in the SCN in rodents has not been reported, and dopaminergic input to the adult SCN is minimal (Duffield et al. 1999; Ishida et al. 2002; Yan et al. 2006). In line with these observations, the DA reuptake inhibitor bupropion did not affect circadian activity rhythms. Nonetheless, cerebral levels of DA vary diurnally in hamsters (Ozaki et al. 1993), while lesions of the SCN eliminate diurnal changes in the density of DA transporters in the cortex and nucleus accumbens of rats (Sleipness et al. 2007). Furthermore, DA reuptake inhibitors might indirectly influence circadian rhythms by actions in the retina or pineal gland, which secretes melatonin (Witkovsky 2004; Doi et al. 2006; Phansuwan-Pujito et al. 2006; Yan et al. 2006). In view of these comments and the efficacy of bupropion in seasonal depression (Dilsaver et al. 1992; Modell et al. 2005), further examination of the interrelationship between DA transporters and circadian rhythms would appear justified.

Although there are reports of [3H]NA reuptake in the SCN (Meyer 1983), no definitive evidence for NA transporters in this structure is available, and its noradrenergic innervation is sparse (Jacomy and Bosler 1995). Indeed, reboxetine was ineffective in the present study. However, a further NA reuptake inhibitor, desipramine, shortened the circadian period in hamsters and rats (Wollnik 1992; Klemfuss and Kripke 1994), and chronic administration of the α2-adrenoceptor agonist clonidine inhibited light-induced phase shifts of hamster activity rhythms (Dwyer and Rosenwasser 2000). Moreover, a possible influence of NA upon circadian rhythms could be mediated via modulation of pineal generation of melatonin (Drijfhout et al. 1996; Sun et al. 2002). Therefore, it may be premature to discount effects of NA reuptake inhibitors upon circadian rhythms.

Circadian rhythm dysfunction has frequently been linked to depressive states that are variously accompanied by phase advances, phase delays, or a dampening of the amplitude of temporally correct rhythms (Duncan 1996; Gorka et al. 1996; Healy and Waterhouse 1995; Millan 2006). The phase advance model of depression is based upon the observation that night-time cortisol and prolactin peaks in depressed patients as well as awakening times are advanced (Keller et al. 2006; Lewy et al. 2006; Linkowski 2003; Millan 2006; Wirz-Justice 2006). It is unknown if phase advances reflect an abnormally short circadian period and/or aberrant response of circadian rhythms to entrainment by light. However, the latter is unlikely because there is no evidence that rhythms in depressed patients “free-run” in response to the internal circadian pacemaker. Therefore, modulation of the influence of light upon circadian rhythms appears a reasonable strategy, and SSRIs and SNRIs would benefit depressed patients demonstrating phase advances.

In seasonal depression, circadian rhythms are generally delayed (Linkowski 2003; Magnusson and Boivin 2003; Murray et al. 2005; Putilov and Danilenko 2005), and the use of SSRIs and SNRIs would be contraindicated. The same holds for a subset of depressed patients responding poorly to fluoxetine that display “reversed” diurnal mood swings (worse in the evening), possibly because of a phase delay (Joyce et al. 2005). A more suitable compound to treat phase delays of seasonal and major depressive disorders would be, for example, the melatonin agonist/5-HT2C antagonist, agomelatine (Van Reeth et al. 1997; Krauchi et al. 1997; Leproult et al. 2005; Millan 2005; Fuchs et al. 2006), which is effective in seasonal-affective disorder (Pjrek et al. 2007). Alternatively, low-efficacy 5-HT1A receptor ligands such as S15535 may be helpful (Gannon and Millan 2006a; Lall and Harrington 2006).

In conclusion, diverse SSRIs abrogate light-induced circadian phase advances in hamsters. Their effects are mimicked by the SNRI, venlafaxine, which likewise suppresses 5-HT reuptake, but not by reboxetine and bupropion, preferential inhibitors of NA and DA reuptake, respectively. These findings are consistent with the enrichment of serotonergic fibers and 5-HT vs NA and DA transporters in the SCN. It would be interesting to extend the present work to chronic drug administration and to experimental models of depressed states. In addition, SSRIs may have activity at different times of the circadian cycle than those reported here for late subjective night. Moreover, the specific subtypes of 5-HT receptor mediating the actions of SSRIs remain to be identified. Collectively, the present data support the notion that reversing phase advances may be a useful component of antidepressant activity for certain (though not all) depressive states.

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