Jin Hee Hong

Two-photon fluorescent probes for intracellular free zinc ions in living tissue.

Zinc is a vital component of enzymes and proteins. In the brain, a few millimoles of intracellular free Zn ions are stored in the presynaptic vesicles, released with synaptic activation, and seem to modulate excitatory neurotransmission. To understand the biological roles of Zn, a variety of fluorescent probes derived from quinoline (TSQ, Zinquin, and TFLZn) and fluorescein (FluZn-3, Znpyr, ZnAF, etc.) have been developed. However, most of them require a rather short excitation wavelength or suffer from pH sensitivity. To visualize the biological activity deep inside living tissue (> 80 mm) without the interference of surface preparation artifacts, it is crucial to use two-photon microscopy (TPM), which utilizes two photons of lower energy for the excitation. Recently, TPM has gained much interest from biologists because it offers a number of advantages in biological imaging, including increased penetration depth, localized excitation, and prolonged observation time. However, efficient two-photon (TP) probes for Zn appear to be rare. Furthermore, although a few pH-resistant sensors for Zn have been reported, they require either microinjection for cellular applications or use a significant amount of ethanol as co-solvent because of the poor water solubility. An efficient TP probe for Zn should have sufficient water solubility to stain the cells, high selectivity for Zn ions, significant TP cross section, pH resistance, and high photostability. In this context, we extend our earlier work and present new TP probes for intracellular free Zn ions (AZn1 and AZn2) derived from 2-acetyl-6-(dimethylamino)naphthalene (acedan) as the fluorophore and N,N-di-(2-picolyl)ethylenediamine (DPEN) as the Zn chelator. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the design of TP fluorescent probes for the membrane and metal ions, and DPEN is a well-known receptor for Zn. Herein, we report that AZn1 and AZn2 are capable of imaging the intracellular free Zn ions in live cells for a long period of time and in living tissue at a depth of > 80 mm without mistargeting and photobleaching problems. The synthesis of AZn1 and AZn2 is shown in Scheme 1. The water solubilities of AZn1 and AZn2 are about 3.0 mm, which is sufficient for staining the cell (see the Supporting

Two-Photon Fluorescent Turn-On Probe for Lipid Rafts in Live Cell and Tissue

We report a new two-photon fluorescence turn-on probe 6-[(E)-3-oxo-1-dodecenyl]-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (CL2) that is designed specifically for visualizing lipid rafts in living cells and tissues. This probe emits much brighter two-photon excited fluorescence in lipid rafts than in non-raft domains and allows direct visualization of the lipid rafts in the live cells and pyramidal neuron layer of the CA1 region at a depth of 100-250 mum in live tissues using two-photon microscopy.

Two-photon fluorescent probes for acidic vesicles in live cells and tissue.

enzymes and secretory proteins exhibiting a variety of functions. To determine their functions, a variety of membrane-permeable fluorescent pH and lysosomal probes have been developed, some of which are commercially available. However, use of these probes with one-photon microscopy (OPM) requires excitation with short-wavelength light (ca. 350–550 nm) that limits their application in deeptissue imaging, owing to the shallow penetration depth (less than 80 mm) as well as to photobleaching, photodamage, and cellular autofluorescence. To overcome these problems, it is crucial to use two-photon microscopy (TPM). TPM employs two near-infrared photons for excitation and offers a number of advantages over OPM, including increased penetration depth (greater than 500 mm), localized excitation, and prolonged observation time. The extra penetration depth that TPM affords is of particular interest in tissue imaging, because surface preparation artifacts such as damaged cells extend over 70 mm into the tissue interior. However, most of the OP fluorescent probes presently used for TPM have small TP action cross sections (Fd) that limit their usage. Although a TP pH probe with appreciable Fd (ca. 42 GM) has been reported, the utility of this probe in TPM imaging has not been verified. Therefore, there is a need to develop an efficient TP probe that can visualize acidic vesicles deep inside tissue for a long period of time. To design an efficient TP probe for acidic vesicles, we chose acedan as the TP fluorophore, because acedan-derived TP probes for Mg (AMg1) and Ca (ACa1) exhibited high photostability as well as significant TP action cross sections for the bright TPM image at low probe concentration, thus allowing the detection of the metal ions deep inside live tissues for over 1100 s. We have introduced an aniline, o-methoxy aniline (pKa(BH ) 4), or tertiary amine (pKa(BH ) 10) substituent as the proton-binding site through an amide linkage to the fluorophore. It is expected that AH1 and AH2 would emit TP-excited fluorescence (TPEF) upon protonation at pH< 4, whereas AL1 would emit TPEF in the acidic vesicles, where it should accumulate as the protonated form. Herein, we report that these probes are capable of imaging the acidic vesicles in live cells and living tissues at greater than 100 mm depth without mistargeting and photobleaching problems. Moreover, AL1 can visualize the transportation of the acidic vesicles in the hippocampal cornu ammonis CA3 region for a long period of time with the use of TPM. AH1, AH2, and AL1 were prepared in 47–77% yields by reactions of 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene and a p-phenylenediamine derivative or N,Ndimethylethylenediamine (see the Supporting Information). The solubilities of AH1, AH2, and AL1 in water are in the range of 5.0–9.0 mm, which are sufficient to stain the cells (Figure S2 in the Supporting Information). The fluorescence spectra of AH1, AH2, and AL1 show gradual bathochromic shifts with solvent polarity (ET) in the order 1,4-dioxane< DMF<EtOH<H2O (Figure S1 and Table S1 in the Supporting Information). The large bathochromic shifts with increasing solvent polarity indicate the utility of these molecules as polarity probes. TP action cross section was determined by investigating the TPEFof the probes using fluorescein as the reference (see the Supporting Information). The TP action spectra of AH1, Scheme 1. The structures of AH1, AH2, and AL1.

Two-Photon Fluorescent Probes for Long-Term Imaging of Calcium Waves in Live Tissue

2-Acetyl-6-(dimethylamino)naphthalene-derived two-photon fluorescent Ca2+ probes (ACa1-ACa3) are reported. They can be excited by a 780 nm laser beam, show 23-50-fold enhancement in one- and two-photon excited fluorescence in response to Ca2+, emit fourfold stronger two-photon excited fluorescence than Oregon Green 488 BAPTA-1 upon complexation with Ca2+, and can selectively detect intracellular free Ca2+ ions in live cells and living tissues with minimum interference from other metal ions and membrane-bound probes. Moreover, these probes are capable of monitoring calcium waves at a depth of 120-170 microm in live tissues for 1100-4000 s using two-photon microscopy with no artifacts of photobleaching.

Circadian waves of cytosolic calcium concentration and long-range network connections in rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the master clock in mammals governing the daily physiological and behavioral rhythms. It is composed of thousands of clock cells with their own intrinsic periods varying over a wide range (20–28 h). Despite this heterogeneity, an intact SCN maintains a coherent 24 h periodic rhythm through some cell‐to‐cell coupling mechanisms. This study examined how the clock cells are connected to each other and how their phases are organized in space by monitoring the cytosolic free calcium ion concentration ([Ca2+]c) of clock cells using the calcium‐binding fluorescent protein, cameleon. Extensive analysis of 18 different organotypic slice cultures of the SCN showed that the SCN calcium dynamics is coordinated by phase‐synchronizing networks of long‐range neurites as well as by diffusively propagating phase waves. The networks appear quite extensive and far‐reaching, and the clock cells connected by them exhibit heterogeneous responses in their amplitudes and periods of oscillation to tetrodotoxin treatments. Taken together, our study suggests that the network of long‐range cellular connectivity has an important role for the SCN in achieving its phase and period coherence.

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

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 “Ca2+ spikes” (i.e., [Ca2+]c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ 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 Ca2+ 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 Ca2+ spikes was increased to 13∼14%. Conclusions/Significance Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]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 Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN.

Cardiac beat-to-beat alternations driven by unusual spiral waves

Alternans, a beat-to-beat temporal alternation in the sequence of heartbeats, is a known precursor of the development of cardiac fibrillation, leading to sudden cardiac death. The equally important precursor of cardiac arrhythmias is the rotating spiral wave of electro-mechanical activity, or reentry, on the heart tissue. Here, we show that these two seemingly different phenomena can have a remarkable relationship. In well controlled in vitro tissue cultures, isotropic populations of rat ventricular myocytes sustaining a temporal rhythm of alternans can support period-2 oscillatory reentries and vice versa. These reentries bear “line defects” across which the phase of local excitation slips rather abruptly by 2π, when a full period-2 cycle of alternans completes in 4π. In other words, the cells belonging to the line defects are period-1 oscillatory, whereas all of the others in the bulk medium are period-2 oscillatory. We also find that a slowly rotating line defect results in a quasi-periodic like oscillation in the bulk medium. Some key features of these phenomena can be well reproduced in computer simulations of a nonlinear reaction-diffusion model.

Spiral wave drift and complex-oscillatory spiral waves caused by heterogeneities in two-dimensional in?vitro cardiac tissues

Understanding spiral reentry wave dynamics in cardiac systems is important since it underlies various cardiac arrhythmia including cardiac fibrillation. Primary cultures of dissociated cardiac cells have been a convenient and useful system for studying cardiac wave dynamics, since one can carry out systematic and quantitative studies with them under well-controlled environments. One key drawback of the dissociated cell culture is that, inevitably, some spatial inhomogeneities in terms of cell types and density, and/or the degree of gap junction connectivity, are introduced to the system during the preparation. These unintentional spatial inhomogeneities can cause some non-trivial wave dynamics, for example, the entrainment dynamics among different spiral waves and the generation of complex-oscillatory spiral waves. The aim of this paper is to quantify these general phenomena in an in vitro cardiac system and provide explanations for them with a simple physiological model having some realistic spatial inhomogeneities incorporated.

Spiral reentry waves in confluent layer of HL-1 cardiomyocyte cell lines

Cardiac excitation waves that arise in heart tissues have long been an important research topic because they are related to various cardiac arrhythmia. Investigating their properties based on intact animal whole hearts is important but quite demanding and expensive. Subsequently, dissociated cardiac cell cultures have been used as an alternative. Here, we access the usefulness of cardiomyocyte cell line HL-1 in studying generic properties of cardiac waves. Spontaneous wave activities in confluent populations of HL-1 cells are monitored using a phase-contrast optical mapping system and a microelectrode array recording device. We find that high-density cultures of HL-1 cells can support well-defined reentries. Their conduction velocity and rotation period both increase over few days. The increasing trend of rotation period is opposite to the case of control experiments using primary cultures of mouse atrial cells. The progressive myolysis of HL-1 seems responsible for this difference.

Serotonin-2C receptor involved serotonin-induced Ca 2+ mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca2+ sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K+ or 5-HT increased cytosolic Ca2+ in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca2+ mobilisations. Consistently, Ca2+ imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca2+ mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.