ABSTRACT
The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.
Subject(s)
Adenosine Triphosphate/pharmacology , Neural Inhibition/drug effects , Neurons/drug effects , Suprachiasmatic Nucleus/cytology , Synaptic Transmission/drug effects , Animals , Animals, Newborn , Biophysical Phenomena/drug effects , Calcium/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Gene Expression Regulation/drug effects , In Vitro Techniques , Male , Patch-Clamp Techniques , Platelet Aggregation Inhibitors/pharmacology , Purinergic Agents/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Purinergic P2X/genetics , Receptors, Purinergic P2X/metabolism , Sodium Channel Blockers/pharmacology , Synaptic Potentials/drug effects , Tetrodotoxin/pharmacology , gamma-Aminobutyric Acid/pharmacologyABSTRACT
The molecular mechanism underlying circadian rhythmicity within the suprachiasmatic nuclei (SCN) of the hypothalamus has two light-sensitive components, namely the clock genes Per1 and Per2. Besides, light induces the immediate-early gene c-fos. In adult rats, expression of all three genes is induced by light administered during the subjective night but not subjective day. The aim of the present study was to ascertain when and where within the SCN the photic sensitivity of Per1, Per2 and c-fos develops during early postnatal ontogenesis. The specific aim was to find out when the circadian clock starts to gate photic sensitivity. The effect of a light pulse administered during either the subjective day or the first or second part of the subjective night on gene expression within the rat SCN was determined at postnatal days (P) 1, 3, 5 and 10. Per1, Per2 and c-fos mRNA levels were assessed 30 min, 1 and 2 h after the start of each light pulse by in situ hybridization histochemistry. Expression of Per1 and c-fos was light responsive from P1, and the responses began to be gated by the circadian clock at P3 and P10, respectively. Expression of Per2 was only slightly light responsive at P3, and the response was not fully gated until P5. These data demonstrate that the light sensitivity of the circadian clock develops gradually during postnatal ontogenesis before the circadian clock starts to control the response. The photoinduction of the clock gene Per2 develops later than that of Per1.
Subject(s)
Biological Clocks/genetics , Cell Cycle Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Suprachiasmatic Nucleus/metabolism , Transcription Factors/metabolism , Aging/genetics , Aging/radiation effects , Animals , Animals, Newborn , Biological Clocks/radiation effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/radiation effects , Female , Gene Expression Regulation/physiology , Gene Expression Regulation/radiation effects , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/radiation effects , Light , Light Signal Transduction/genetics , Light Signal Transduction/radiation effects , Male , Neurons/radiation effects , Nuclear Proteins/genetics , Nuclear Proteins/radiation effects , Period Circadian Proteins , Photic Stimulation , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/radiation effects , RNA, Messenger/metabolism , RNA, Messenger/radiation effects , Rats , Rats, Wistar , Suprachiasmatic Nucleus/radiation effects , Transcription Factors/genetics , Transcription Factors/radiation effectsABSTRACT
The molecular clockwork underlying the generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) develops gradually during ontogenesis. The authors' previous work has shown that rhythms in clock gene expression in the rat SCN are not detectable at embryonic day (E) 19, start to form at E20 and develop further via increasing amplitude until postnatal day (P) 10. The aim of the present work was to elucidate whether and how swiftly the immature fetal and neonatal molecular SCN clocks can be reset by maternal cues. Pregnant rats maintained under a light-dark (LD) regimen with 12 h of light and 12 h of darkness were exposed to a 6-h delay of the dark period and released into constant darkness at different stages of the fetal SCN development. Adult rats maintained under the same LD regimen were exposed to an identical shifting procedure. Daily rhythms in spontaneous c-fos, Avp, Per1, and Per2 expression were examined within the adult and newborn SCN by in situ hybridization. Exposure of adult rats to the shifting procedure induced a significant phase delay of locomotor activity within 3 days after the phase shift as well as a delay in the rhythms of c-fos and Avp expression within 3 days and Per1 and Per2 expression within 5 days. Exposure of pregnant rats to the shifting procedure at E18, but not at E20, delayed the rhythm in c-fos and Avp expression in the SCN of newborn pups at P0-1. The shifting procedure at E20 did, however, induce a phase delay of Per1 and Per2 expression rhythms at P3 and P6. Hence, 5 days were necessary for phase-shifting the pups' SCN clock by maternal cues, be it the interval between E18 and P0-1 or the interval between E20 and P3, while only 3 days were necessary for phase-shifting the maternal SCN by photic cues. These results demonstrate that the SCN clock is capable of significant phase shifts at fetal developmental stages when no or very faint molecular oscillations can be detected.
