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Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

ABSTRACT: Mammalian circadian rhythm is maintained by the suprachiasmatic nucleus (SCN) via an intricate set of neuropeptides and other signaling molecules. In this work, peptidomic analyses from two times of day were examined to characterize variation in SCN peptides using three different label-free quantitation approaches: spectral count, spectra index and SIEVE. Of the 448 identified peptides, 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptides with significant (adjusted p-value < 0.01) differential peptide abundances between daytime and nighttime, including multiple peptides derived from secretogranin II, cocaine and amphetamine regulated transcript, and proprotein convertase subtilisin/kexin type 1 inhibitor. Interestingly, more peptides were analyzable and had significantly different abundances between the two time points using the spectral count and spectral index methods than with a prior analysis using the SIEVE method with the same data. The results of this study reveal the importance of using the appropriate data analysis approaches for label-free relative quantitation of peptides. The detection of significant changes in so rich a set of neuropeptides reflects the dynamic nature of the SCN and the number of influences such as feeding behavior on circadian rhythm. Using spectral count and spectral index, peptide level changes are correlated to time of day, suggesting their key role in circadian function.

SUBMITTER: Southey BR

PROVIDER: S-EPMC3886391 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

Southey Bruce R BR Lee Ji Eun JE Zamdborg Leonid L Atkins Norman N Mitchell Jennifer W JW Li Mingxi M Gillette Martha U MU Kelleher Neil L NL Sweedler Jonathan V JV

Analytical chemistry 20131216 1

Mammalian circadian rhythm is maintained by the suprachiasmatic nucleus (SCN) via an intricate set of neuropeptides and other signaling molecules. In this work, peptidomic analyses from two times of day were examined to characterize variation in SCN peptides using three different label-free quantitation approaches: spectral count, spectra index and SIEVE. Of the 448 identified peptides, 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptide ...[more]

PMID: 24313826

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Project description:Key pointsLight-responsive neurones in the rat suprachiasmatic nucleus discharge with a harmonic distribution of interspike intervals, whereas unresponsive neurones seldom do. This harmonic patterning has a fundamental frequency of close to 30 Hz, and is the same in light-on cells as in light-off cells, and is unaffected by exposure to light. Light-on cells are more active than light-off cells in both subjective day and subjective night, and both light-on cells and light-off cells respond more strongly to changes in light intensity during the subjective night than during the subjective day. Paired recordings indicate that the discharge of adjacent light-responsive cells is very tightly synchronized. The gap junction inhibitor carbenoxolone increases the spontaneous activity of suprachiasmatic nucleus neurones but does not block the harmonic discharge patterning.AbstractThe suprachiasmatic nucleus (SCN) of the hypothalamus has an essential role in orchestrating circadian rhythms of behaviour and physiology. In the present study, we recorded from single SCN neurons in urethane-anaesthetized rats, categorized them by the statistical features of their electrical activity and by their responses to light, and examined how activity in the light phase differs from activity in the dark phase. We classified cells as light-on cells or light-off cells according to how their firing rate changed in acute response to light, or as non-responsive cells. In both sets of light-responsive neurons, responses to light were stronger at subjective night than in subjective day. Neuronal firing patterns were analysed by constructing hazard functions from interspike interval data. For most light-responsive cells, the hazard functions showed a multimodal distribution, with a harmonic sequence of modes, indicating that spike activity was driven by an oscillatory input with a fundamental frequency of close to 30 Hz; this harmonic pattern was rarely seen in non-responsive SCN cells. The frequency of the rhythm was the same in light-on cells as in light-off cells, was the same in subjective day as at subjective night, and was unaffected by exposure to light. Paired recordings indicated that the discharge of adjacent light-responsive neurons was very tightly synchronized, consistent with electrical coupling.

Project description:The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.

Dataset Information

Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

Publications

Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

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