Unwinding the molecular basis of interval and circadian timing

Abstract

Neural timing mechanisms range from the millisecond to diurnal, and possibly annual, frequencies. Two of the main processes under study are the interval timer (seconds-to-minute range) and the circadian clock. The molecular basis of these two mechanisms is the subject of intense research, as well as their possible relationship. This article summarizes data from studies investigating a possible interaction between interval and circadian timing and reviews the molecular basis of both mechanisms, including the discussion of the contribution from studies of genetically modified animal models. While there is currently no common neurochemical substrate for timing mechanisms in the brain, circadian modulation of interval timing suggests an interaction of different frequencies in cerebral temporal processes.

Keywords: circadian system; cortico-striatal circuits; dopamine; glutamate; interval timing; serotonin; suprachiasmatic nuclei.

Figure 1

(A) Main components of the circadian timing system. Circadian rhythms consists of three main components: (i) an input pathway integrating exogenous signals to synchronize circadian phase and period, (ii) a central oscillator that generates the endogenous circadian signal, and (iii) an output pathway driving circadian periodicity and coupling of biological processes. (B) Molecular mechanisms of circadian timing. The molecular mechanisms of circadian rhythms can be illustrated by the transcription of the Period (Per1and Per2) and Cryptochrome (Cry1, Cry2) genes that are activated by heteromeric complexes containing CLOCK and BMAL1 proteins that act through the E-box regulatory sequences of their target genes. The newly synthesized PER and CRY proteins are translocated into the nucleus, where they inhibit BMAL1–CLOCK activity, and therefore, their own transcription. Clock and Bmal1 both contain basic helix–loop–helix (bHLH) motifs for DNA binding at their N-terminus and Per–Arnt–Sim (PAS) domains. The controlled degradation of PER and CRY proteins by the ubiquitin pathway decreases their protein levels and results in an oscillation of their mRNA and protein levels. During this negative transcriptional feedback loop many of the clock proteins become posttranslationally modified by phosphorylation and ubiquitination (Reppert and Weaver, 2002). This core oscillation is augmented and stabilized by a secondary loop involving two orphan nuclear receptor proteins, REV-ERBα and RORA. Both are activated in phase with the Per and Cry genes by CLOCK and BMAL1, but in turn they affect Bmal1 expression (Preitner et al., 2002). While RORA has a positive role, REV-ERBα is a suppressor of Bmal1, and they coordinate action through RORE regulatory sequences. A positive feedback loop is built by the stimulated transcription of BMAL1 by PER2. Protein phosphorylation events are essential contributors to these feedback loops. Two members of the casein kinase I family (CKIε and CKIδ) phosphorylate PER proteins in order to (i) target them for ubiquitin-mediated proteasomal degradation, and (ii) modulate their nuclear import. A mutation of CKIε shortens rhythm in hamsters (Lowrey et al., 2000) and a mutation of CKIδ shortens rhythm in humans (Xu et al., 2005). The result of these complex regulatory pathways is that the mRNA and protein levels of most circadian genes – except Clock and CKIε – oscillate with a 24-h period. Importantly, the CLOCK–BMAL1 heterodimer regulates the transcription of many clock-controlled genes (CCGs), which in turn influence a wide array of physiological functions external to the oscillatory mechanism. This mediates the output function of the clock, thereby controlling food intake, hormonal synthesis and release, body temperature, metabolism, and many other functions.

Figure 2

Relationships of different neurons in the striatum and neurotransmitter signaling involved in interval timing. (A) Schematic representation of the relationship among oscillatory cortical inputs, medium spiny neurons, cholinergic interneurons, glutamatergic afferents, and dopaminergic axons projecting from the substantia nigra pars compacta (SNpc) to the striatum as specified by the Striatal Beat Frequency model of interval timing. The direct output pathway to the globus pallidus – external (GPe) and internal (GPi) segments, and substantia nigra reticulata (SNr) as well as the indirect pathway to the GPe are indicated. Relevant neurotransmitters = acetylcholine (ACh), dopamine (DA), γ-aminobutyric acid (GABA), glutamate (GLU). (B) Detail of dopaminergic, glutamatergic, and cholinergic input to a striatonigral medium spiny neuron as well as the principal signal transduction pathways modulating the contribution of striatal spiny neurons to interval timing. Abbreviations: AC, adenyl cyclase; ACh, acetylcholine; AMPAR, AMPA receptor; CB1, cannabinoid receptor type 1; CRE, cyclic-AMP-response element; CREB, CRE binding protein; DA, dopamine; DAG, 1,2-diacylglycerol; DARPP-32, camp-regulated phosphoprotein of 32 kDa; D1R, dopamine D1 receptor; D2R, dopamine D2 receptor; EC, endocannabinoids; GABA, γ-aminobutyric acid; Glu, glutamate; GP, globus pallidus; IP3, inositol 1,4,5 trisphosphate; M1R, muscrinic acetylcholine receptor 1; M2R, muscarinic acetylcholine receptor 2; mGluR, metabotropic glutamate receptor; NMDAR, N-methyl-d-aspartic acid receptor; NOS, nitric oxide synthase; PKA, protein kinase A; PKC, protein kinase C; SNpc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; TrKR, tyrosine kinase receptor.