Introduction

Circadian rhythms in mammals are regulated by a master clock located in the suprachiasmatic nucleus (SCN) of the brain (Klein et al. 1991; Jagota et al. 2000; Jagota 2006). Serotoninergic neurotransmission is important in mammalian circadian clock function (Mistleberger et al. 2000). Several serotonin receptor subtypes have been localized in the SCN (Moyer and Kennaway 1999). The SCN controls and regulates the rhythmic production and discharge of serotonin derivative, melatonin (hormonal message for darkness) from pineal gland via multisynaptic efferent pathways. The nocturnal rise in melatonin production from serotonin is a result of increase in the activity of serotonin N-acetyl transferase (NAT) (Klein et al. 1997). Disruptions of the biological rhythms can impair the health and result in sleep disorders of elderly, insomnia, dementia, affective illness (mood and depression), hypothalamic tumors, heart problems, and problems associated with congenital blindness, jet lag, shift work, and space etc. (Moore 1991; Jagota 2005).

The complex interaction between alcohol and biological clock (the pacemaker) has become a rapidly expanding area in chronopharmacology (Spanagel et al. 2005). Serotonin plays an important role in mediating alcohol’s effects in brain (Lovinger 1999). Alcohol consumption is related with changes in levels of various neurotransmitters such as norepinephrine, GABA, glutamate, dopamine, and noradrenalin etc. (Zarcone 1978; Gewiss et al. 1991; Kawahara et al. 1993; Prospero et al. 1994; Littleton 1998). The specific mechanism underlying the relationships between neurotransmitter function, alcohol and sleep disturbances is obscure. Some drugs such as disulfiram, naltrexone, and acamprostate have been used as anticraving medications and help in overcoming withdrawal symptoms. These drugs have been related with many adverse reactions (Petrakis and Krystal 1997; Oncken et al. 2001; Oscar et al. 2003; Verge et al. 2006). In recent years the development of new medications to treat alcohol dependence has initiated a new era in alcoholism treatment.

The curcumin (1,7-bis (4-hydroxy-3-methoxy phenyl)-1, 6-hetadiene-3, 5-dione), a major yellow phenolic active curcuminoid present in turmeric used in the diet, is non-toxic and protective pharmaceutical, neutraceutical, and phytoceutical agent. It has a plethora of beneficial effects such as antioxidant, anti-inflammatory, anti-carcinogenic, anti-viral, and anti-infectious effects etc. (Arajuo and Leon 2001; Aggarwal et al. 2003; Joe et al. 2004). We report here, the effect of ethanol drinking and its withdrawal on daily rhythms of neurotransmitter serotonin and its metabolite, 5-HIAA in SCN and Pineal and the effect of curcumin on ethanol induced changes in 5-HT and 5-HIAA daily rhythms.

Materials and Methods

Ninety-day-adult male Wistar rats were maintained at 23 ± 1°C with LD, 12:12 (lights on: 06:30 A.M. (Zeitgeber time (ZT)-0) and lights off: 6:30 P.M. (ZT-12)) for 2 weeks prior to experiment. Food and water were provided ad libitum. All experiments were performed as per Institutional Animal Ethics. The rats were separated into four groups—(1) control; (2) ethanol drinking; (3) ethanol withdrawal; (4) curcumin treated.

Group 1 animals were supplied food and water ad libitum. Group 2 were offered for 15 days under the two bottle-free choice regimen with unlimited access of ethanol (10% v/v in tap water) and water. Food pellets were always available. Bottles refilled everyday with a fresh solution and their positions interchanged at random to avoid development of position preference. In Group 3, after ethanol drinking for 15 days as in Group 2, ethanol withdrawal was followed for 15 days, i.e., only food and water were provided ad libitum. In Group 4 also, after ethanol drinking as in Group 2 for 15 days was given 0.002% curcumin (Sigma) in diet for 15 days ad libitum.

Brains were dissected from all the experimental rats (Group 1–4), following anesthesia at various time points such as ZT-0, 6, 12, 18, and 24. Pineal gland was separated and SCN was carefully punched out with the help of scalpel from 500-μ brain slices which were made using tissue chopper (Prosser and Gillete 1989).

Serotonin and 5-HIAA levels were assayed by using HPLC-EC method (Mefford et al. 1980; Grady et al. 1984). The tissue sample was homogenized with 100 μl of 0.1 N perchloric acid containing sodium bisulfate (1 mM). After homogenization the tissue samples were sonicated for approximately 5 s. The centrifugation was done at 12,800g for 10 min to remove tissue debris. The supernatant was filtered through 0.22-μ syringe filters and then clear supernatant was applied to the chromatography system (Waters, USA) by using eluant: 10% methanol; 0.1 M citric acid; 0.1 M sodium acetate, 50 mg/l EDTA (pH 4.1). The protein estimation was done by using Bradford’s method (Bradford 1976).

Statistical Analysis

Data was analyzed using Jandel Scientific Sigma stat software by the analysis of variance (ANOVA) and student’s t-test.

Results

Effect of Curcumin on Ethanol Induced Changes in 5-HT and 5-HIAA Daily Rhythms in SCN

Group 1 (control) showed daily rhythms in 5-HT and 5-HIAA levels. 5-HT levels measured at various time points such as ZT-0, 6, 12, 18, and 24 were 19.37 ± 1.05, 39.29 ± 2.58, 24.26 ± 5.26, 8.65 ± 0.90, and 20.01 ± 1.86 μmol/g protein, respectively (Fig. 1A) and 5-HIAA levels were 2.84 ± 0.67, 8.76 ± 1.66, 12.05 ± 4.32, 2.79 ± 0.46, and 2.93 ± 0.89 μmol/g protein, respectively (Fig. 1B). The 5-HT levels were maximum at subjective mid-day (ZT-6) and minimum at subjective mid-night (ZT-18) whereas 5-HIAA levels were maximum at ZT-12 and minimum at ZT-0. The maximum:minimum ratio (daily pulses) for 5-HT and 5-HIAA were similar (Fig. 1C). The 5-HT/5-HIAA ratio was maximum at ZT-0/24, i.e., at onset of light and minimum at ZT-12, i.e., at onset of darkness (Table 1).

Fig. 1
figure 1

Effect of curcumin treatment in SCN of ethanol treated 90 day rats. Group 1: control, Group 2: ethanol drinking for 15 days, Group 3: ethanol withdrawal for 15 days after ethanol drinking for 15 days, Group 4: curcumin treatment for 15 days after ethanol drinking for 15 days. (A) 5-HT rhythms: rhythmicity persists with a significant increase in 5-HT levels upon ethanol drinking for 15 days, ethanol withdrawal does not result in restoration of 5-HT levels and curcumin treatment results in restoration though with a phase delay of 6 hours for maximum levels. (B) 5-HIAA rhythms: ethanol drinking resulted in increase in 5-HIAA levels with a phase shift by 6 h for maximum levels, ethanol withdrawal did not result in restoration of phase or levels and curcumin treatment resulted in restoration of 5-HIAA levels as well as phase. (C) Daily pulses of 5-HT and 5-HIAA levels: ethanol drinking resulted in significant increase in daily 5-HT pulse, ethanol withdrawal resulted in significant decrease whereas curcumin treatment resulted in restoration though with fluctuation. p a  ≤ 0.05, p b  ≤ 0.05, and p c  ≤ 0.05 (whereas a, b, c refers to comparison between Groups 1 and 2, 1 and 3, and 1 and 4, respectively). Vertical bars indicate mean ± S.E., n = 6

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Table 1 Effect of curcumin on 5-HT/5-HIAA ratio in SCN in alcohol treated rat
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5-HT levels at various time points ZT-0, 6, 12, 18, and 24 in Group 2 (ethanol drinking) were 20.33 ± 3.09, 116.10 ± 7.44, 45.73 ± 6.02, 10.25 ± 0.42, and 18.93 ± 1.412 μmol/g protein, respectively (Fig. 1A) whereas 5-HIAA levels were 34.37 ± 1.48, 37.53 ± 1.27, 37.85 ± 1.64, 100.52 ± 2.30, and 26.68 ± 1.09 μmol/g protein, respectively (Fig. 1B). 5-HT levels showed significant increase at ZT-6 and 12 (p a ≤ 0.05) though there was no significant difference at ZT-0, 18, 24. Interestingly 5-HIAA levels showed significant elevation as compared to Group 1 at all time points with about 50 times increase at subjective mid-night (ZT-18) (p a ≤ 0.05). The daily pulses of 5-HT were significantly high as compared to control (p a ≤ 0.05) (Fig. 1C). The 5-HT/5-HIAA ratio was maximum at subjective mid-day (ZT-6) and minimum at subjective mid-night (ZT-18), i.e., there was a phase delay by about 6 h as compared to control. In addition, the 5-HT/5-HIAA ratio was significantly different at all time points except ZT-12 from controls (p a ≤ 0.05) (Table 1).

In Group 3 (ethanol withdrawal), the 5-HT levels at various time points such as ZT-0, 6, 12, 18, and 24 were 28.36 ± 1.049, 76.51 ± 3.18, 68.25 ± 5.55, 70.30 ± 2.89, and 31.00 ± 2.37 μmol/g protein, respectively which were significantly high as compared to controls at all time points (p b ≤ 0.05) (Fig. 1A). The levels remained significantly high not only at subjective mid-day (ZT-6) but also after onset of darkness (ZT-12) as well as subjective mid-night (ZT-18). The 5-HIAA levels on withdrawal were also not restored to normal and were significantly high (p b ≤ 0.05) as compared to control. These were 53.85 ± 3.11, 41.58 ± 1.56, 69.86 ± 3.36, 102.76 ± 3.31, and 50.65 ± 1.192 μmol/g protein at ZT-0, 6, 12, 18, and 24, respectively (Fig. 1B). The daily pulses for 5-HT were significantly different as compared to control (p b ≤ 0.05) (Fig. 1C). The 5-HT/5-HIAA ratio was maximum at ZT-6 and minimum at ZT-0 and the ratio was reduced in amplitude significantly at ZT-0, 6, 18, 24 (p b ≤ 0.05) and phase delayed by 12 h (Table 1).

Group 4 (curcumin treated) animals showed decrease in 5-HT and 5-HIAA as compared to Group 2 and Group 3. The 5-HT levels at ZT-0, 6, 12, 18, and 24 were 1.45 ± 0.71, 2.52 ± 0.54, 17.11 ± 14.69, 4.64 ± 2.47, and 1.85 ± 0.43 μmol/g protein (Fig. 1A) and 5-HIAA were 2.73 ± 0.96, 3.58 ± 0.88, 3.88 ± 1.64, 4.45 ± 1.13, and 1.044 ± 0.230 μmol/g protein (Fig. 1B) respectively. The 5-HT levels though appeared reduced but the maximum levels were at ZT-12. The levels were significantly different at ZT-0, 6, and 24 (p c ≤ 0.05). Interestingly, there was no significant difference in the daily pulses of 5-HT and 5-HIAA as compared to control therefore daily pulses were restored though Standard Error was high for serotonin pulses. The 5-HT/5-HIAA ratio was maximum at ZT-12 i.e. onset of darkness and minimum at ZT-0 i.e. onset of light (Fig. 1C). The maximum 5-HT/5-HIAA ratio was not significantly different from that of control. Rhythmicity appeared to be restored, though with a phase reversal (Table 1).

Effect of curcumin on ethanol induced changes in 5-HT and 5-HIAA daily rhythms in Pineal

5-HT and 5-HIAA levels were measured in Pineal similarly as in SCN. In Group 1 (control), at various time points ZT-0, 6, 12, 18, and 24, 5-HT levels were 206.03 ± 39.79, 291.61 ± 58.38, 179.44 ± 28.13, 28.87 ± 12.50, and 203.28 ± 13.25 μmol/g protein, respectively (Fig. 2A) whereas 5HIAA levels were 11.98 ± 1.54, 18.00 ± 1.81, 10.76 ± 1.12, 2.79 ± 0.72, and 12.08 ± 1.35 μmol/g protein, respectively (Fig. 2B). The 5-HT and 5-HIAA levels showed daily rhythms and were maximum at ZT-6 and minimum at ZT-18 respectively as in SCN. The 5-HT and 5-HIAA daily pulses were significantly different (Fig. 2C). The 5-HT/5-HIAA ratio was maximum at ZT-0/24, i.e., onset of light and minimum at ZT-18, i.e., mid subjective night (Table 2).

Fig. 2
figure 2

Effect of curcumin treatment in Pineal of ethanol treated 90 day rats. Group 1–4 same as in Fig. 1. (A) 5-HT rhythms: ethanol drinking resulted in increase in 5-HT levels, ethanol withdrawal did not result in restoration and curcumin treatment resulted in restoration of maximum 5-HT levels. (B) 5-HIAA rhythms: ethanol drinking resulted in increase in 5-HIAA levels with phase delay of 6 h, ethanol withdrawal restored the phase but 5-HIAA levels were as high as 15 times compared to control and curcumin treatment resulted in restoration of phase as well as 5-HIAA levels. (C) Daily pulses of 5-HT and 5-HIAA levels: ethanol drinking resulted in significant decrease in daily 5-HT and 5-HIAA pulses though levels were high in ethanol withdrawal, daily 5-HT, and 5-HIAA pulse was restored and curcumin treatment resulted in restoration of 5-HIAA daily pulse though 5-HT pulse decreased significantly. p a  ≤ 0.05, p b  ≤ 0.05, and p c  ≤ 0.05 (whereas a, b, c same as in Fig. 1). Vertical bars indicate mean ± S.E., n = 6

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Table 2 Effect of curcumin on 5-HT/5-HIAA ratio in Pineal in alcohol treated rat
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The 5-HT levels at various time points ZT-0, 6, 12, 18, and 24 in Group 2 (ethanol drinking) were 149.13 ± 5.43, 398.43 ± 30.35, 442.19 ± 26.14, 453.06 ± 22.71, and 141.08 ± 10.15 μmol/g protein respectively (Fig. 2A) and 5-HIAA were 60.12 ± 1.85, 63.40 ± 2.64, 85.94 ± 2.70, 35.35 ± 1.32, and 61.59 ± 0.81 μmol/g protein, respectively (Fig. 2B). The basal levels of 5-HIAA were significantly high as compared to control at all time points though rhythmicity persisted (p a  ≤ 0.05). 5-HT and 5-HIAA daily pulses were significantly different as compared to controls (p a ≤ 0.05) (Fig. 2C). The 5-HT/5-HIAA ratio was maximum at mid night (ZT-18) phase advanced by 12 h or phases were reversed and minimum at ZT-0/24. In addition the amplitude in 5-HT/5-HIAA ratio was significantly reduced at ZT-0, 6, 12, and 24 (p a ≤ 0.05) (Table 2).

In Group 3 (ethanol withdrawal), 5-HT levels were 83.28 ± 5.45, 436.44 ± 10.01, 331.23 ± 14.49, 412.72 ± 19.40, and 88.27 ± 4.448 μmol/g protein, respectively (Fig. 2A) and 5-HIAA levels were 28.56 ± 1.07, 132.95 ± 2.13, 49.26 ± 0.69, 52.39 ± 1.85, and 24.93 ± 0.93 μmol/g protein, respectively (Fig. 2B). Neither rhythmicity nor amplitude of 5-HT and 5-HIAA was restored. The 5-HT as well as 5-HIAA values were significantly high as compared to control at all time points (p a ≤ 0.05) though daily pulses were not significantly different from control. The maximum levels were 15 times higher as compared to control (p b ≤ 0.05) (Fig. 2C). The 5-HT/5-HIAA ratio was maximum at ZT-18 and minimum at ZT-0 and significantly different at all time points from control group (p b ≤ 0.05) (Table 2).

Group 4 (curcumin treated) animals showed 5-HT levels at ZT-0, 6, 12, 18, and 24 were 59.35 ± 14.31, 191.40 ± 44.21, 92.08 ± 10.49, 91.25 ± 14.36, and 64.15 ± 6.218 μmol/g protein, respectively (Fig. 2A) and 5-HIAA as 10.08 ± 4.63, 11.394 ± 4.02, 4.04 ± 1.10, 2.18 ± 0.63, and 8.77 ± 0.61 μmol/g protein, respectively (Fig. 2B). The 5-HIAA levels appeared similar to control and were different only at ZT-12 (p c ≤ 0.05) with restoration of rhythmicity. The 5-HT levels also showed decreased levels at ZT-0, 12, and 24 (p c ≤ 0.05), with restoration of rhythmicity. Daily pulse of 5-HT were significantly different (p c ≤ 0.05) but 5-HIAA daily pulses were not significantly different from control (Fig. 2C). The 5-HT/5-HIAA ratio was maximum at ZT-18 and minimum at ZT-0/24 with restoration of rhythmicity, though advanced by 6 h. The maximum ratio was 2.6 times more as compared to control (p c ≤ 0.05). Also at ZT-0, 6 and 24, 5-HT/5-HIAA ratio was significantly different as compared to control (p c ≤ 0.05) (Table 2).

Discussion

Serotonin has been shown to play a major role in the regulation of circadian pacemaker (Morin 1999; Mistleberger et al. 2000; Rea and Pickard 2000). This is in agreement with our work as there is a significant increase in serotonin and its metabolite 5-HIAA levels upon ethanol drinking in SCN and in Pineal. The ethanol induced shifts in 5-HT and 5-HIAA rhythms in SCN and in Pineal observed in present study is in agreement with earlier workers who have reported ethanol induced shifts in per 1 and per 2 (important molecular components of the clock) in various brain regions including SCN (Chen et al. 2004; Spanagel et al. 2005). It is also in agreement with reports that alcohol ingestion alters the phase, amplitude or abolish the expression of circadian rhythms in a variety of physiological and behavioral functions, including locomotor activity, body temperature (Baird et al. 1998), sleep (Ehlers and Slawecki 2000), food intake (Barr 1988), secretion of the stress related hormone, corticosterone and other functions (Rajakrishnan et al. 1999; El-Mas and Abdel-Rahman 2000). Alcohol consumption has also been earlier related with reduction in synthesis of several hypothalamic neuropeptides within the SCN (Madeira and Paul-Barbosa 1999) and alteration in the free running circadian period (Mistleberger and Nadeau 1992).

The long-lasting alcohol tolerance has been related to multifunctional neurotransmitters like serotonin, norepinephrine and dopamine (Tabakoff and Hoffman 1996; Valenuzuela and Harris 1997). We report here alcohol induced phase shifts and increase in 5-HT and 5-HIAA levels. In addition, acute alcohol consumption is also related with the release of serotonin, GABA, and taurine, and result in increased chloride flux and decreased neuronal excitability in rat brain (Yoshimoto et al. 1992; Dahchour et al. 1994; LeMarquand et al. 1994). However, some workers have reported decreased serotonin level upon chronic ethanol drinking (Carmichael and Israel 1975; Michaelis et al. 1978).

The ethanol withdrawal resulted in the alteration in 5-HT and 5-HIAA levels as well as rhythms in Pineal and SCN. This is in agreement with earlier reports where ethanol withdrawal has been associated with phase advances of circadian rhythms in body temperature (Kodama et al. 1988), rapid eye movement (REM) sleep (Imatoh et al. 1986) and the levels of 5-HIAA (Sano et al. 1993, 1994) and phase delays in blood cortisol, a key stress hormone (Iranmanesh et al. 1989). It has been reported that both cortisol and melatonin rhythms might severely get abolished upon ethanol withdrawal (Fonzi et al. 1994; Mukai et al. 1998; Danel and Touiton 2006). The cerebral hyperactivity during ethanol withdrawal in some studies (Glue and Nutt 1990; Grant et al. 1990) can be related to altered 5-HT, 5-HIAA levels and rhythms upon ethanol withdrawal in the present study.

As curcumin is known to have antioxidant, anti-inflammatory, anti-carcinogenic properties (Arajuo and Leon 2001; Aggarwal et al. 2003; Joe et al. 2004), we looked into its protective effects on alcohol induced alterations in 5-HT and 5-HIAA levels and rhythms in SCN and Pineal. We report, here, that curcumin influences the clock and helps in restoring the levels of neurotransmitter 5-HT and its metabolite 5-HIAA. Alcohol induced changes in 5-HT and 5-HIAA rhythms in SCN and Pineal are sensitive to curcumin treatment. More experiments are in progress in our laboratory to study the influence of this compound, but certainly the results in this study indicate curcumin provides a viable food based as well as chrono-pharmacologic approach to ethanol induced alteration in 5-HT, 5-HIAA levels and daily rhythms.