Pregnant Sprague-Dawley rats (E17-E18) were anaesthetized with CO₂ and the fetuses were removed and placed in ice-cold Hank's Balanced Salt Solution without Ca²⁺ or Mg²⁺ (HBSS). The brains of fetuses were removed and placed into cold HBSS for further dissection. Using a dissecting microscope and blunt dissection, the meninges were gently separated away. The cerebral cortex was then removed and each cortical hemisphere was cut into four pieces and trypsinized in HBSS at 37°C. Following trypsinization, cells were separated by trituration through the opening of a fire polished Pasteur pipette. The suspensions were then passed through a 70 μm cell strainer. The dissociated cells were added to the bottom of 35mm glass-bottomed petri dishes previously coated with polyethyleneimine (50% solution, Sigma, St. Louis) diluted 1:1000 in borate buffer. The cortical neurons were then allowed to attach to the surface at 37°C, 5% CO₂ in 2 ml of MEM solution (Gibco, BRL) supplemented with 10% (v/v) heat-inactivated fetal bovine serum. After 3-6 hrs, solutions were replaced with fresh supplemented MEM which was later replaced (24 hrs) with Neurobasal medium (Gibco, BRL) supplemented with 2% B-27.

All imaging experiments were performed in HEPES medium containing the following (in mM): 130 NaCl, 5 KCl, 8 MgSO₄, 1 Na₂HPO₄, 25 Glucose, 20 HEPES, 1 Na-Pyruvate; pH adjusted to 7.4. Cortical cells grown in glass-bottomed petri dishes were washed with fresh HEPES medium. The cells were then incubated at 37°C for 30 min with ER-Tracker Red (Molecular Probes, Carlsbad, CA) and Newport Green (Molecular Probes, Carlsbad, CA) or ZinPyr-1 (Neurobiotex, Galveston, TX). Cells were incubated with 10 μM of the specified fluorescent Zn²⁺ indicator either alone or in conjunction with ER-Tracker red for 30 min then were washed 3x with fresh HEPES medium and placed into a custom 35mm stage adapter and continuously perfused with fresh HEPES medium on the stage of a Zeiss LSM 510 (confocal) microscope. Cultures were examined using a Plan-Neofluar 100x/1.3 NA oil immersion objective. For ER-Tracker Red excitation was done with a HeNe Laser line of 543nm and an LP560nm emission filter. Newport Green and ZinPyr-1 were imaged using an Argon Laser line of 488nm for excitation and a BP505-550nm emission filter. Separate fluorescent channels were employed for each indicator, and channels were scanned sequentially to minimize crosstalk. Cells were imaged by serial z-scans progressively from bottom to top, in increments of 500 nm. Colocalization of ER-Tracker Red and Newport Green or ZinPyr-1 was measured using Zeiss software 14. Colocalization was determined by Pearson's correlation coefficient and considered significant when (p ≤ 0.05). Time series measurements of fluorescence intensity were done with image capture at 10 sec intervals, and changes in intensity were measured using Zeiss LSM 510 image analysis software. Fluorescence measurements were background subtracted, normalized to starting values, and expressed as F/Fo.

To directly activate IP₃Rs we used the membrane-permeable UV light-sensitive caged IP₃ analogue, ci-IP3/PM (D-2,3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy) benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl)ester (SiChem. Bremen, Germany) 1516. Cells in brain hippocampal slices were simultaneously loaded by incubation with caged IP₃ and Newport Green for 30 min at 37°C, then washed and incubated for an additional 30 min to allow for complete de-esterification.

IP₃ was photoreleased by flashes of 364 nm light focused uniformly throughout the field of view.

Thapsigargin (Molecular Probes, Carlsbad, CA), the IP₃K inhibitor N²-(m-trifluorobenzyl),N⁶-(p-nitrobenzyl)purine (Calbiochem, Cat. No. 406170), N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) (Molecular Probes, Carlsbad, CA), the Ins(1,4,5)P₃ receptor blocker 2-aminoethoxydiphenyl-borate (2-APB), were applied by bath application in HEPES medium.

Cells labeled with intracellular fluorescent Zn²⁺ indicators and examined under basal conditions showed consistent regions of elevated fluorescent intensity in the soma and processes, and particularly in a region that was identified as the endoplasmic reticulum by a fluorescent marker for the organelle. The elevated levels of Zn²⁺ were seen to represent labile or free Zn²⁺ because they were sensitive to both low and high affinity fluorescent Zn²⁺ indicators Newport Green (K_DZn²⁺ ≈ 10^-6M) (Figure 1A) and ZinPyr-1(K_DZn²⁺ ~ 10^-9M) (Figure 1B). Before imaging, cells were washed three times with fresh medium to remove excessive fluorescent residues. Another reason for using ZinPyr-1 is that, unlike AM forms of fluorescence indicators, it is highly lipophilic and remains in the organelle, being sequestrated with zinc. These indicators are essentially insensitive to Ca²⁺ and their fluorescence to Zn²⁺ are not altered in the presence of Ca²⁺ 1718. Fluorescence was sensitive to quenching by the membrane permeable Zn²⁺-chelator TPEN (K_DZn²⁺ ~ 10^-17 M) (Figure 2). The same cells were also loaded with ER-tracker Red, a marker for the ER, to determine the localization of the intracellular fluorescent Zn²⁺ indicators. We performed serial z-scans using confocal microscopy of cortical neurons loaded with ER-tracker Red and either Newport Green or ZinPyr-1 (Figure 1A, B). Both fluorescent Zn²⁺ indicators showed significant colocalization with the ER Tracker Red. Colocalization was abolished upon exposure to 10 μM TPEN (Figure 2), indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ in the basal condition and were located within the lumen of the ER.

To assess the overall dynamics of Zn²⁺ release from thapsigargin-sensitive stores, cells incubated with Newport Green were exposed to thapsigargin, which inhibits SERCA pump. The application of thapsigargin depletes the ER Ca²⁺ stores in cells and raises cytosolic Ca²⁺ concentration. The experiments above suggested that Zn²⁺ may be also transported into and sequestered in ER. If this was true, the accumulation of Zn²⁺ by blocking SERCA with thapsigargin should also produce Zn²⁺ signals. Indeed, exposure to thapsigargin resulted in a gradual increase of cytosolic or intracellular Zn²⁺ (Figure 3), which was sensitive to the Zn²⁺ chelator TPEN (Figure 4).

In the next two tests we determined that the source of this thapsigargin-induced Zn²⁺ increases. One possible source is the influx of extracellular Zn²⁺. To remove extracellular Zn²⁺, we applied a membrane impermeable Zn²⁺ chelator CaEDTA. As shown in figure 4, thapsigargin-induced Zn²⁺ rises were unchanged in the presence of CaEDTA (1 mM). Next, we examined the contribution of extracellular Ca²⁺ on thapsigargin-induced elevation of intracellular Zn²⁺. We found that the removal of extracellular Ca²⁺ had no effect on the thapsigargin-induced Zn²⁺ rises (Figure 4). Taken together, these results indicated that thapsigargin-induced increases in intracellular Zn²⁺ were not dependent on either extracellular Zn²⁺or extracellular Ca²⁺, and were entirely of intracellular origin.

The endoplasmic reticulum is a well established site of intracellular Ca²⁺ storage and release. IP₃ can trigger the release of Ca²⁺ from intracellular stores by binding to and activating its receptor (IP₃R) located on regions of the ER. When IP₃ binds to and activates IP₃Rs, the channel portion of the receptor opens and Ca²⁺ is released from the ER to the cytosol. The experiment was therefore performed utilizing ci-IP3/PM, a cell-permeable form of caged IP₃ to directly induce Zn²⁺ release 1516192021. In neurons loaded with the Zn²⁺ fluorophore Newport Green and caged IP₃, IP₃ uncaging resulted in a rapid increase in intracellular Zn²⁺, which persisted for 30 s and followed by a roughly exponential decay (Figure 5A &5B). This experiment demonstrated that the Zn²⁺ response was due to the activation of IP₃ receptors.

A significant route of IP₃ metabolism is the conversion of IP₃ into inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P₄) by the enzyme Ins(1,4,5)P₃ 3-kinase (IP₃-3Kinase or IP₃-3K) 22. Inhibition of the IP₃-3K has been shown to elevate intracellular levels of IP₃ by halting its conversion into Ins(1,3,4,5)P4 23. Here, to examine the effects of IP₃ signaling on intracellular Zn²⁺ dynamics, N²-(m-trifluorobenzyl),N⁶-(p-nitrobenzyl)purine, a membrane-permeable inhibitor of IP₃-3K was employed to confirm the results observed using the caged IP₃. Upon bath application of the inhibitor, Newport Green fluorescence showed a gradual, significant increase (Figure 5C &5D), supporting an IP₃ mediated process involved in the release of Zn²⁺.