Elsevier

Brain Research

Volume 1129, 19 January 2007, Pages 34-42
Brain Research

Research Report
Diurnal differences in dopamine transporter and tyrosine hydroxylase levels in rat brain: Dependence on the suprachiasmatic nucleus

https://doi.org/10.1016/j.brainres.2006.10.063Get rights and content

Abstract

Time of day can influence cocaine-seeking behavior in rats, and this influence may depend on the suprachiasmatic nucleus (SCN). We used western blots to measure expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and caudate in sham and SCN-lesioned (SCNx) rats at ZT4 (Zeitgeber time 4, 4 h after lights-on) or ZT20 (8 h after lights-off). In the mPFC, DAT levels were lower at ZT20 than at ZT4 in sham but not SCNx rats. In the NAc, DAT expression was higher at ZT20 than at ZT4, and this effect was blunted in SCNx rats. Caudate DAT levels were unaffected by time of day or SCNx. Levels of TH did not change in mPFC with time of day or SCN lesion; TH levels were higher at ZT20 in the NAc, with a trend towards higher levels at this time in SCNx rats. Caudate TH expression was slightly elevated at ZT20 in sham but not in SCNx rats. We conclude that DAT and TH within these brain regions exhibit diurnal variation and dependence on the SCN. The results may have implications for new strategies to maximize pharmacological treatments for drug addiction.

Introduction

Recently, we demonstrated diurnal differences in long-term sensitization to cocaine (Sleipness et al., 2005a, Sleipness et al., 2005b) and diurnal differences in cocaine-conditioned place preference (Sleipness et al., 2005a, Sleipness et al., 2005b). A growing body of evidence shows that there are diurnal differences in cocaine-seeking behavior (Abarca et al., 2002, Akhisaroglu et al., 2004, Hummel and Unterwald, 2002, Roberts et al., 2002, Yuferov et al., 2005). Additionally, time of day can serve as a contextual cue in sensitization to methamphetamine (Arvanitogiannis et al., 2000) and conditioned place preference (Ralph et al., 2002).

One possible physiological mechanism leading to diurnal variation in cocaine-related behaviors is a change in dopaminergic neurotransmission occurring within brain regions such as the caudate, nucleus accumbens (NAc), and medial prefrontal cortex (mPFC), three areas known to mediate behavioral responses to cocaine exposure (Baker et al., 2003, Di Ciano et al., 2001, Fuchs et al., 2006). Indeed, several investigators have reported daily fluctuations in dopamine levels in the caudate and NAc (Castaneda et al., 2004, Paulson and Robinson, 1994, Paulson and Robinson, 1996, Schade et al., 1995).

A growing literature points to clock genes, transcription factors expressed rhythmically over a 24 h period (Harms et al., 2004, Hastings and Herzog, 2004, Reppert and Weaver, 2002), as mediators of diurnal changes in dopaminergic transmission and daily patterns of cocaine-seeking behavior. For example, several brain regions implicated in drug-seeking behavior express clock genes, including the ventral tegmental area (VTA), NAc, striatum, and mPFC (Abe et al., 2002, Amir et al., 2006, McCkung et al., 2005). The expression of clock genes in these brain regions may be synchronized by the body's own master circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus (SCN) (Moga et al., 1995). The SCN itself utilizes these clock genes to generate daily rhythms in behavior (Okamura et al., 2002, Reppert and Weaver, 2002). Thus, the SCN could produce time-of-day variation in dopaminergic brain regions via neural projections to these sites by altering proteins that regulate dopaminergic signaling. Alternatively, clock gene activity within circuitry involved in drug reward may produce these time-of-day effects independently of the SCN.

Two proteins critical for dopaminergic signaling are tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and the dopamine transporter (DAT), which removes extracellular dopamine from the synapse (Calligaro and Eldefrawi, 1987, Mateo et al., 2004). In mice (gb (GeneBank): AAHY01107653; gb: CAAA01132564.1), rats (gb: AABR03000077.1; gb: AAHX01009104.1), and humans (gb: AADC01048382.1; gb: AADB02013527.1), the promoter regions of the genes encoding both of these proteins contain E-box sequences to which the CLOCK/BMAL1 complex, both members of the clock family of transcription factors mentioned above, is capable of binding (Kawarai et al., 1997, Weber et al., 2004). Thus, TH and DAT levels may be regulated according to the phase of the light:dark cycle, in turn generating the diurnal differences in extracellular dopamine levels. Evidence supporting a role for CLOCK in the regulation of TH protein and activity levels was provided by McClung et al. (2005) who reported that TH and CLOCK are co-expressed in VTA dopamine neurons, and that the expression of TH mRNA and protein as well as phosphorylated TH is greatly enhanced in mice lacking the circadian gene clock. The same group also found VTA neuron activity to be influenced by CLOCK, since animals lacking this gene had higher basal and burst-firing rates and were hyperactive compared with wild type mice.

While the above studies suggest the presence of a diurnal variation in dopaminergic activity, E-boxes bind not only clock proteins, but also non-circadian transcription factors, such as the Mad1/Max heterodimer (Montage et al., 2005). As such, it is possible that expression of these key dopamine-regulating proteins may in fact not change with the time of day. Therefore, the present experiments were performed to determine whether there were diurnal differences in TH and DAT protein levels in the mPFC, NAc, and caudate, and furthermore, to determine the dependence of any diurnal variation in TH and DAT expression on the SCN.

Section snippets

Confirmation of SCN lesion

A representative thionin-stained section, taken at the level of the SCN from a sham and SCNx rat, is shown in Fig. 1A. Electrolytic lesion destroyed the SCN in the animal shown in the bottom panel. Fig. 1B shows representative Fast Fourier transformed locomotor activity data from the same two animals shown in Fig. 1A, and from an incompletely SCN-lesioned (SCNi) rat for comparison. Sham and SCNi rats showed robust 24 h rhythms while the SCNx rat exhibited no 24 h periodicity. The success rate

Discussion

To our knowledge, this is the first study to investigate the influence of time of day and the role of the SCN on the expression of DAT and TH protein levels in the rat forebrain. Collectively, the data reveal both a time- and brain region-dependent reliance on the SCN of DAT and TH expression within dopaminergic terminal brain regions. Overall, the most robust changes observed were those in the NAc for both DAT and TH; both of these molecules were more abundant at ZT20. While DAT expression was

Animals and drugs

Male Sprague–Dawley rats (n = 32; Harlan, Indianapolis, IN) weighing 280–300 g were used. Animals were kept on a 12 h light:12 h dark (12L:12D) photoperiod with ad libitum access to food and water. The experiments conformed to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996 and were approved by the Washington State University Institutional Animal Care and Use Committee. Ketamine and xylazine were obtained from Sigma (St.

Acknowledgments

The authors are grateful to Dr. Abdur Rehman and Ms. Melissa Forquer for technical assistance and to Ms. Jenny Baylon for editorial suggestions. This work was supported by Washington State Initiative 171 (WSU Alcohol and Drug Abuse Research Program).

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