Dataset Information

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

ABSTRACT:

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms.

Methodology/principal findings

We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%.

Conclusions/significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.

SUBMITTER: Hong JH

PROVIDER: S-EPMC2835761 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Publications

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

Hong Jin Hee JH Min Cheol Hong CH Jeong Byeongha B Kojiya Tomoyoshi T Morioka Eri E Nagai Takeharu T Ikeda Masayuki M Lee Kyoung J KJ

PloS one 20100310 3

<h4>Background</h4>Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms.<h4 ...[more]

PMID: 20224788

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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

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

Background

Methodology/principal findings

Conclusions/significance

Publications

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

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