Acta Pharmacologica Sinica

Full-length article

Acta Pharmacologica Sinica 2006 July; 27 (7): 848-852

Ca²⁺ sparks and Ca²⁺ glows in superior cervical ganglion neurons¹

Li-jun YAO^2,3,4, Gang WANG^3,4,, Kun-fu OU-YANG³, Chao-liang WEI³, Xian-hua WANG³, Shi-rong WANG³, Wei YAO³, Hong-ping HUANG³, Jian-hong LUO², Cai-hong WU³, Jie LIU³, Zhuan ZHOU^3,5, He-ping CHENG^3,5

²Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou 310031, China; ³Institute of Molecular Medicine and State Key Laboratory of Biomembrane Engineering, Peking University, Beijing 100871, China

¹This work was supported by grants from the National Basic Research Program of China (ZZ and HC) and the National Natural Science Foundation of China (ZZ, HC, JL, XHW).
⁴The authors contribute equally to this work.
⁵Correspondence to Drs He-ping CHENG or Zhuan ZHOU.
Phn/Fax 86-10-6276-5957.
E-mail chengp@pku.edu.cn
zzhou@pku.edu.cn
Received 2006-05-18
Accepted 2006-05-25
doi: 10.1111/j.1745-7254.2006.00402.x

Abstract

Aim: Ca²⁺ release from the endoplasmic reticulum (ER) is an integral component of neuronal Ca²⁺ signaling. The present study is to investigate properties of local Ca²⁺ release events in superior cervical ganglion (SCG) neurons. Methods: Primary cultured SCG neurons were prepared from neonatal rats (P3_P7). Low concentration of caffeine was used to induce Ca²⁺ release from the ER Ca²⁺ store, and intracellular Ca²⁺was recorded by high-resolution line scan confocal imaging and the Ca²⁺ indicator Fluo-4. Results: Two populations of local Ca²⁺ release events with distinct temporal characteristics were evoked by 1.5 mmol/L caffeine near the surface membrane in the soma and the neurites of SCG neurons. Brief events similar to classic Ca²⁺ sparks lasted a few hundreds of milliseconds, whereas long-lasting events displayed duration up to tens of seconds. Typical somatic and neurite sparks were of 0.3- and 0.52-fold increase in local Fluo-4 fluorescence, respectively. Typical Ca²⁺ glows were brighter (DF/F₀ approximately 0.6), but were highly confined in space. The half maximum of full duration of neurite sparks was much longer than those in the soma (685 vs 381 ms). Conclusion: Co-existence of Ca²⁺ sparks and Ca²⁺ glows in SCG neurons indicates distinctive local regulation of Ca²⁺ release kinetics. The local Ca²⁺ signals of variable, site-specific temporal length may bear important implications in encoding a "memory" of the trigger signal.

Key words

superior cervical ganglion neurons; Ca²⁺ sparks; ryanodine receptors; caffeine

Extract

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Introduction
Ca²⁺ is an ubiquitous intracellular messenger and regulates a great variety of physiological processes including excitability, secretion, development, learning and memory^[1]. In neurons, cytoplasmic Ca²⁺ elevation results primarily from two kinds of Ca²⁺ sources. One is the extracellular Ca²⁺entry via voltage- and receptor-operated Ca²⁺ channels, the other is the Ca²⁺release from intracellular Ca²⁺stores via ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP₃R) in the endoplasmic reticulum (ER).
Ca²⁺ sparks as elementary Ca²⁺ release signals were first described in quiescent rat heart cells^[2]. Since then, Ca²⁺ sparks have been found in many types of excitable and nonexcitable cells, such as skeletal and smooth muscle cells^[3_5], hippocampal neurons^[6], dorsal root ganglion (DRG) neurons^[7], and hypothalamic neurons^[8]. The high Ca²⁺ microdomains associated with Ca²⁺ sparks may stimulate high-threshold Ca²⁺-dependent signaling processes in the vicinity of the release channels. As well, spatially and temporally coordinated activation of Ca²⁺ sparks gives rise to propagating Ca²⁺ waves or near synchronous Ca²⁺ transients throughout the cell.
While Ca²⁺ sparks have been extensively investigated in all three types of muscles, only limited information from a few types of neurons^[6_8] is available as to the spatial and temporal architecture of intracellular Ca²⁺ signaling. Since space-time organization of Ca²⁺ signals is critical to the efficacy, specificity and diversity of Ca²⁺ signaling, much remains to be learnt about organization of intracellular Ca²⁺ signals in different types of neurons. In this regard, superior cervical ganglion (SCG) neuron has been extensively used in the study of various aspects of synaptic transmission including synaptic plasticity (a cellular mechanism of "memory") in which cytosolic Ca²⁺ plays an important role^[9]. In the present study, we investigated local Ca²⁺ release events in rat SCG neurons and analyzed their spatial and temporal properties with the aid of high resolution confocal micro-scopy.

Materials and methods
Cell culture SCG was removed from neonatal rats (P3-P7) and dissociated using previously described methods^[10]. Briefly, ganglions were dissected and incubated in Ca²⁺ free solution containing collagenase (1.5 mg/mL) and trypsin (0.5 mg/mL) at 37 ºC for 45 min. Then the dispersed cells were plated on poly-L-lysine coated glass culture dishes and primarily cultured with DMEM (Gibco) containing 10% fetal bovine serum (FBS), nerve growth factor (20 ng/mL; 2.5 s) and maintained at 37 ºC in a 5% CO₂ incubator. The neurons of 3_10 cultured days were prepared for Ca²⁺ imaging experiments.
Ca²⁺ free solution contained (mmol/L): 109 NaCl, 5.4 KCl, 23.8 NaHCO₃, 10 NaH₂PO₄, 7.28 Na-Hepes, 17.72 H-Hepes, 10 glucose (pH 7.4). DMEM for cell culture contained 13.4 mg/mL DMEM, 44 mol/L NaHCO₃, 100 IU/mL penicillin G, 100 µg/mL streptomycin, 0.6% vitamin C and 10% FBS. The standard bath solution for SCG contained (mmol/L): 141 NaCl, 2.8 KCl, 1 MgCl₂, 2 CaCl₂, 10 H-Hepes (pH 7.4).
DMEM and FBS were purchased from Gibco. All other chemicals were from Sigma, unless otherwise specified. All experiments were conducted at room temperature (22_24 ºC).
Line scan imaging SCG neurons were loaded with Fluo-4 AM (5 µmol/L, 15 min) (Molecular Probes). Fluo-4 was excited at 488 nm and the emitted fluorescence was collected at wavelengths >505 nm, with a Zeiss 510 inverted confocal microscope (40 oil immersion lens of numerical aperture 1.3). The horizontal and axial resolutions were set at 0.4 and 1.5 mm, respectively. Rectilineal scan, curve scan and 2D (xy) imaging modes were used to measure Ca²⁺ dynamics, while the transmission channel image of the cell were recorded simultaneously. Image processing and data analysis were performed using IDL 6.0 software (Research Systems, Boulder, CO) and Igor software 4.03 (WaveMetrix).
Local perfusion system Solutions were puffed locally onto the cell via an RCP-2B multichannel microperfusion system (INBIO, Wuhan, China), which allowed fast (<100 ms) electronic change of local solutions between seven solution channels. The tip (100 µm diameter) of the puffer pipette was located about 120 µm from the cell. As determined by the conductance tests, the solution around a cell under study was fully controlled by the application solution, provided the application flow speed was 100 µL/min or greater. All pharmacological experiments met this criterion^[11].

Results
Subsurface local Ca²⁺ release events in the soma of superior cervical ganglion neurons Primary cultured SCG neurons were examined with confocal microscopy in conjunction with the Ca²⁺ indicator, Fluo-4. To monitor subsurface Ca²⁺ release events that participate in bidirectional Ca²⁺ signaling between the plasma membrane and the ER, high-resolution curve scan images were obtained by setting the scan trajectory along the periphery of the soma. Application of low concentration of caffeine (1.5 mmol/L), which sensitizes RyR opening, evoked a flurry of local Ca²⁺ transients at punctuated sites in quiescent cells (Figure 1A). Local release events apparently consisted of heterogeneous populations, two examples of which are shown in Figure 1A. At site 1, a train of repetitive release events similar to classic Ca²⁺ sparks were observed, and individual sparks were characterized by a rapid rise and a quasi-exponential decay (Figure 1B[a]). By contrast, release at site 2 displayed a prolonged rise time, a sustained plateau followed by a slow decay, with an overall release duration greater than 20 s (Figure 1B[b]). We named these long-lasting local release events (duration longer than 5 s) "Ca²⁺ glows". The majority subsurface release sites were of the spark rather than the glow type (66% spark sites from nine neurons).
On average, somatic Ca²⁺ sparks displayed 0.30±0.01 fold-increase of local Fluo-4 fluorescence (DF/F₀, n=36 events from nine cells), whereas Ca²⁺ glows tended to have greater peak amplitude (0.61±0.13, n=5 events). The spatial width, indexed by the full width of half maximum (FWHM) at the peak Ca²⁺ level, was 1.79±0.19 and 0.66±0.21 mm for Ca²⁺ sparks and Ca²⁺ glows, respectively. The co-existence of Ca²⁺ sparks and Ca²⁺ glows in the soma of SCG neuron indicates distinctly different release kinetics at different release sites.
Neurite Ca²⁺ sparks and Ca²⁺ glows in SCG neurons Next, we used rectilineal scan imaging method to visualize local Ca²⁺ events in neurites, with the scan line placed in parallel with the neurite of interest (Figure 2A). Both Ca²⁺ sparks and Ca²⁺ glows were evoked by 1.5 mmol/L caffeine (Figure 2B, 2C), as was the case in the soma. However, the percent of Ca²⁺ glow sites was significantly greater in neurites (56/83 or 67%) than in the soma (c² test, P<0.05). Furthermore, 42 out of 56 release sites displayed repetitive Ca²⁺ sparks on top of the Ca²⁺ glows (Figure 2B, site 1).
Thapsigargin blocked caffeine-evoked Ca²⁺ sparks To validate that caffeine-evoked Ca²⁺ release events arise from local intracellular Ca²⁺ release from the ER, 10 µmol/L thapsigargin (TG), an inhibitor of the ER Ca²⁺-ATPase was applied 30 min prior to caffeine application. Under these experimental conditions, neither Ca²⁺ sparks nor Ca²⁺ glows were observed upon caffeine application (Figure 3). This result confirms the notion that both Ca²⁺ sparks and Ca²⁺ glows reflect local Ca²⁺ release from the ER.
Comparison between somatic and neurite Ca²⁺ sparks We analyzed the amplitude, FWHM, and the half maximum of full duration (FDHM) from 55 neurite Ca²⁺ sparks and 36 somatic Ca²⁺ sparks. Histogram distributions of these spark parameters are shown in Figure 4. The SCG spark amplitude in the neurites was greater than in the soma (DF/F₀ 0.52 vs 0.30, P<0.01), and was much smaller than that in cardiac or skeletal muscles where Ca²⁺ sparks were first described. The lateral extensions of neurite and somatic Ca²⁺ sparks were similar (~1.8 mm). However, the FDHM of Ca²⁺ sparks was significantly smaller in the soma than in the neurites (381 vs 685 ms, P<0.05).

Discussion
The ER Ca²⁺ signaling plays pluripotent roles in vital neuronal physiological and pathophysiological processes, such as neuronal excitability, neurotransmitter release, somatic secretion, synaptic plasticity, gene expression, neuronal growth and survival as well as circadian rhythms^[12]. The specificity and versatility of Ca²⁺ signaling are in part determined by the spatiotemporal mode of local Ca²⁺ release. In the present study, we found that, in addition to Ca²⁺ sparks, there is a new class of local Ca²⁺ release events, namely Ca²⁺ glows, in SCG neurons. Once activated, local Ca²⁺ release in a glow lasts for many seconds and up to tens of seconds, as if there is no mechanism of release termination. Ca²⁺ sparks and Ca²⁺ glows can be observed both in the subsurface layer of the soma and the neurites of different thickness. However, the glow or spark type of response appeared to be a site-specific property, for preliminary data show that the response of a given site is rather stereotypic during repeated caffeine applications (interval ~20_30 s).
The similarities and differences among SCG, DRG, hippocampal and hypothalamic neurons reinforce the notion that the space-time architecture of intracellular Ca²⁺ signaling is highly neuronal cell type-specific. The presence of Ca²⁺ glows in neurons bears important ramifications. First, it indicates that inactivation of the release mechanism is non-existent or very weak. This is reminiscent of the situation in DRG neurons where type 3 RyR display little Ca²⁺-dependent inactivation and rapidly repetitive Ca²⁺sparks can be readily activated at given sites^[7,13]. Second, there must be a rapid Ca²⁺refilling and recycling mechanism to prevent exhaustion of local ER Ca²⁺ store, sustaining release in a Ca²⁺glow. Functionally, the long-lasting Ca²⁺ release will retain a "memory" of the trigger signal well beyond the trigger duration. Indeed, we noticed that Ca²⁺ glows persisted for at least 10 s after washout of caffeine (data not shown). Hence, our finding may shed some new light on the encoding of cellular "memory" via Ca²⁺-dependent mechanisms.

Conclusions
At present, the exact mechanism that confers a site the Ca²⁺ glow property remains elusive. Whether a release site can switch dynamically from a spark site to a glow site or vice versa is also an intriguing possibility that needs to be addressed over an extended timescale. The exact physiological role of the subsurface local Ca²⁺ glows, as well as Ca²⁺ sparks in the soma and in the neurites of SCG neurons, also warrants future investigation. Among others, subsurface Ca²⁺ is able to modulate the processes of membrane excitability^[14,15], exocytosis^[7] and synaptic transmission. In addition, sustained elevation of local Ca²⁺ will lead to the activation of Ca²⁺-dependnet kinases, which are implicated in the long-term neuronal plasticity^[16].

Acknowledgments
We thank Lin-ling HE and Lie-cheng WANG for their assistance in cell preparation.

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