Patterns of intracellular calcium fluctuation in precursor cells of the neocortical ventricular zone - PubMed (original) (raw)

Patterns of intracellular calcium fluctuation in precursor cells of the neocortical ventricular zone

D F Owens et al. J Neurosci. 1998.

Abstract

Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.

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Figures

Fig. 1.

Fig. 1.

Most imaged cells in the VZ are proliferative neocortical precursor cells. A, Single optical section of a fluo-3 AM-loaded coronal E16 brain slice. Large numbers of cells load with the Ca2+ indicator.B, Single optical section of an E16 coronal brain slice pulsed for 6 hr with BrDU to label cells in S, G2, and M phases of the cell cycle. C, Single optical section of a coronal E17 brain slice stained for the neuronal marker TuJ1. Scale bar: A_–_C, 50 μm. D, Single optical section of an E17 brain slice stained for the radial glia marker vimentin. E, Schematic representation of the experimental preparation. A section of an intact neocortical hemisphere (cortical slab) is removed, loaded with fluo-3 AM, and placed ventricular surface down in an imaging chamber attached to the stage of an inverted confocal microscope. F, Representative view of the_VZ_ from an E17 cortical slab.

Fig. 2.

Fig. 2.

Individual cells display intermittent [Ca2+]i transients._A_1, A microscopic field from an E17 slab imaged in an optical plane ∼20 μm from the ventricular surface. Circles indicate cells that were active over a continuous imaging period of ∼20 min.A_2, A cell before (Rest), during (Peak), and after (Return) a spontaneous [Ca2+]i increase.A_3, Activity graphs of the_numbered cells (1–4) shown in_A_1. Calcium transients ranged from relatively fast events (cell 3) to slow events (cell 2). The inset for cell 3 is an expanded plot of the event with measured values indicated by filled circles. Scale bar in_inset, 10 sec. A minority of cells showed multiple transients (e.g., cell 4) over the imaging period. B, Faster sampling showing that the single-cell events generally occurred over many seconds._B_1, A cell sampled every second._B_2, A cell sampled every 0.215 sec.C, A developmental increase in the number and frequency of single-cell events but no change in duration._C_1, The similar mean durations of [Ca2+]i transients at E15 and E19._C_2, A significant increase in the percentage of active cells/field/trial at E19. _Asterisk_indicates a significant difference._C_3, At E19, a larger percentage of cells with multiple transients than at E15. The _inset_displays the mean frequency per trial of all cells analyzed at the two ages. There was an increase in mean frequency at E19.Asterisk indicates a significant difference.D, Spontaneous [Ca2+]ifluctuations observed in cells throughout the VZ._D_1, A fluo-3-loaded coronal brain slice at E16 with the area imaged indicated by a box._D_2, Higher magnification image with active cells indicated by circles.

Fig. 3.

Fig. 3.

Mechanisms of spontaneous [Ca2+]i fluctuation in VZ cells. A, Three cells (solid,dashed, and dotted lines) near the ventricular surface of a coronal slice at E19. Activity persisted in the presence (solid horizontal bar) of TTX (2 μ

m

), La3+ (50 μ

m

), BMI (20 μ

m

), CNQX (20 μ

m

), and AP-5 (100 μ

m

). B, Three cells at E16 recorded in Ca2+ (2 m

m

) ACSF (control) and after ∼20 min of perfusion with Ca2+-free/2 m

m

EGTA ACSF. There were no obvious differences in the behavior of the [Ca2+]i transients. C, Representative examples of activity in three cells (solid lines) under control conditions and three cells after exposure to thapsigargin (5 μ

m

). Spontaneous activity in VZ cells was abolished after exposure to thapsigargin.

Fig. 4.

Fig. 4.

Most active single cells in the VZ_are not neurons. A, Coronal slice at E15 that was imaged for spontaneous [Ca2+]i increases and subsequently for TuJ1 immunoreactivity. Left, A single optical section with cells active during the imaging period_circled. Right, An average of 50 serial 1 μm sections of the same area after processing for TuJ1 immunoreactivity. In only one case was a TuJ1-positive cell body present where an active cell was seen during Ca2+imaging (arrow). Dashed lines approximate the boundary of the VZ. B, An E19 slab imaged for spontaneous [Ca2+]i increases and subsequently for TuJ1 immunoreactivity. Left, A single optical section ∼15 μm from the ventricular surface with cells that were active during the imaging period circled.Right, An average of 50 serial 1 μm sections of the same area after processing for TuJ1 immunoreactivity. There were many more TuJ1-labeled cells at E19 than at E15, and in several instances cells active during Ca2+ imaging were TuJ1-positive (arrows).

Fig. 5.

Fig. 5.

Pairs of VZ cells at or near the ventricular surface show synchronized increases in [Ca2+]i. A, An example of a doublet event from an E19 cortical slab before (_A_1), during (_A_2), and after (A_3) a [Ca2+]i increase. B, A three-dimensional graphic representation of three highly synchronized doublet events. The arrows indicate transients shown in_A. All cells are from the same slab.

Fig. 6.

Fig. 6.

Synchronously active cell pairs are M-phase cells in the process of cell division. A, A fluo-3-loaded E16 slab imaged at the ventricular surface. Notice the presence of a great many cells apparently in the state of mitosis. B, Syto-11 staining and imaging of the VZ surface displaying patterns of condensed chromatin. The inset shows condensed chromatin visible with fluo-3 loading in the doublet indicated by_arrows_ in A. C, Doublet event (arrows) in an E19 slab during Ca2+imaging (_C_1) and after subsequent TuJ1 staining (_C_2), demonstrating that doublets are not neurons. _D_1, A dividing E17 VZ cell observed over a 20 min period. Each image (1–4) is separated by ∼5 min._D_2, Spontaneous fluctuations in [Ca2+]i that were synchronized in both daughter cells. E, Doublets occurring in Ca2+-free ACSF, suggesting that these events are mediated by the release of Ca2+ from intracellular stores. Inset shows images before (1), during (2), and after (3) the doublet event.

Fig. 7.

Fig. 7.

Top. Coordinated increases in [Ca2+]i occur in clusters of neighboring VZ cells. A, A spontaneously active VZ cluster at E17. This example shows nine sequential pseudocolored images taken every 4 sec before, during, and after a cluster event.B, The time course of the [Ca2+]i change plotted for eight of the cells from the cluster shown in A. _Arrow_indicates putative trigger cell. The time course of the mean value for all of the cells is shown in the inset.C, Coordinated cell activity occurring in Ca2+-free ACSF and propagating at ∼10 μm/sec._C_1, A cluster event in Ca2+-free/2 m

m

EGTA ACSF with a putative trigger cell (cell 1) and two follower cells (cells 2 and 3) labeled.C_2, Activity graph for all of the cells in the cluster with the onset of the Ca2+transients for the three labeled cells displayed in the_inset. Measuring the distance of each follower cell from the trigger cell and the time of onset of the [Ca2+]i increase in each cell allowed estimation of the rate of signal propagation.

Fig. 9.

Fig. 9.

Spontaneous [Ca2+]i fluctuations in developing neurons. _A_1, MZ of an E15 cortical slice during Ca2+ imaging (left) and after TuJ1 staining (right). The MZ contained many active cells, some of which had the morphological features of Cajal-Retzius neurons (arrows), and contained a high density of TuJ1-stained cells. A_2, Activity graph of an_MZ cell shown in _A_1.B, Activity graph of an E16 CP cell._C_1, Presumptive migrating neuron in the IZ (arrows) of an E16 coronal brain slice during Ca2+ imaging (left) and after TuJ1 staining (right). Pial surface is to the top right-hand corner. _C_2, Activity graph of the cell shown in _C_1.D, An E17 MZ cell recorded in Ca2+ (2 m

m

) ACSF and after ∼30 min of perfusion with Ca2+-free ACSF. Transients failed to appear in the Ca2+-free condition but returned once normal ACSF was reperfused (Wash).

Fig. 10.

Fig. 10.

Schematic of cell types demonstrating different patterns of spontaneous [Ca2+]ifluctuations. Numbers refer to cells demonstrating single-cell events (1), double-cell events (2), and coordinated multicell cluster events (3). Single active cells are primarily TuJ1-negative proliferative cells but could also include members of clusters that display independent [Ca2+]i fluctuations (?) and postmitotic neurons in the process of migration, particularly during the later stages of neurogenesis. Single-cell activity might also include radial glia cells. Cell pairs demonstrating synchronized activity are mitotically active precursor cells. Active cell clusters correspond to groups of gap junction-coupled cells that include precursor cells in G1, G2, and S and at least one radial glia cell. Spontaneous [Ca2+]i fluctuations are also seen in neurons of the IZ, CP, and_MZ_ (shaded cells).

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