Most active single cells in the V Z are not neurons. A , Coronal slice at E15 that was imaged for spontaneous [C a 2 ϩ ] i increases and subsequently for TuJ1 immunoreactivity. Lef t , A single optical section with cells active during the imaging period circled . Right , An average of 50 serial 1 ␮ m sections of the same area after processing for T uJ1 immunoreactivity. In only one case was a T uJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging ( arrow ). Dashed lines approximate the boundary of the VZ. B , An E19 slab imaged for spontaneous [C a 2 ϩ ] i increases and subsequently for TuJ1 immunoreactivity. Lef t , A single optical section ϳ 15 ␮ m from the ventricular surface with cells that were active during the imaging period circled . Right , An average of 50 serial 1 ␮ m sections of the same area after processing for T uJ1 immunoreactivity. There were many more TuJ1-labeled cells at E19 than at E15, and in several instances cells active during C a 2 ϩ imaging were T uJ1-positive ( arrows ).

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Patterns of Intracellular Calcium Fluctuation in Precursor Cells of the Neocortical Ventricular Zone

Article

Full-text available

Aug 1998

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 mic...

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... to drug access. Cells near the ventricular surface of the slice were mon- itored, and activity was still present in the TTX-containing solu- tion (data not shown). We also found that activity persisted in solutions that contained, in addition to TTX, the nonspecific voltage-gated Ca 2 ϩ channel (VGCC) blocker lanthanum (50 ␮ M ), the GABA A receptor blocker BMI (20 ␮ M ), the non-NMDA glutamate receptor blocker CNQX (20 ␮ M ), and the NMDA receptor blocker AP-5 (100 ␮ M ). Figure 3 A depicts the levels of activity for three cells in an E19 slice before and after the addition of the inhibitors. From these data, we conclude that neural activity, VGCC activation, and amino acid neurotransmit- ter receptor activation are not required for the spontaneous [Ca 2 ϩ ] i increases in individual VZ cells. To test whether the [Ca 2 ϩ ] i increases were dependent on extracellular Ca 2 ϩ , we performed experiments in Ca 2 ϩ - containing and Ca 2 ϩ -free (0 Ca 2 ϩ and 2 m M EGTA) ACSF solutions. Embryonic cortical slabs were preincubated in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from an E17 neocortical slab is illustrated in Figure 7 A . This example shows nine sequential pseudocolored images before, during, and after the coordinated event. The activity seems to originate with 1 or 2 cells ( arrow ) and spreads outward to 14 –16 neighboring cells. Figure 7 B shows the time course of [C a 2 ϩ ] i change in eight of these cells and the combined mean value for all cells together ( inset ). There was a close spatiotemporal coupling of ...

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... activ- ity. Slabs were subsequently preincubated in a solution containing TTX (2 ␮ M ), to block sodium-dependent action potentials, for a minimum of 3 min and then reimaged in the continued presence of TTX. Similar levels of activity were present before and after immersion in TTX (data not shown). A second series of experi- ments was performed using brain slices to circumvent the possi- bility that the intact ventricular surface provided a barrier to drug access. Cells near the ventricular surface of the slice were mon- itored, and activity was still present in the TTX-containing solu- tion (data not shown). We also found that activity persisted in solutions that contained, in addition to TTX, the nonspecific voltage-gated Ca 2 ϩ channel (VGCC) blocker lanthanum (50 ␮ M ), the GABA A receptor blocker BMI (20 ␮ M ), the non-NMDA glutamate receptor blocker CNQX (20 ␮ M ), and the NMDA receptor blocker AP-5 (100 ␮ M ). Figure 3 A depicts the levels of activity for three cells in an E19 slice before and after the addition of the inhibitors. From these data, we conclude that neural activity, VGCC activation, and amino acid neurotransmit- ter receptor activation are not required for the spontaneous [Ca 2 ϩ ] i increases in individual VZ cells. To test whether the [Ca 2 ϩ ] i increases were dependent on extracellular Ca 2 ϩ , we performed experiments in Ca 2 ϩ - containing and Ca 2 ϩ -free (0 Ca 2 ϩ and 2 m M EGTA) ACSF solutions. Embryonic cortical slabs were preincubated in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from ...

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... ity. Slabs were subsequently preincubated in a solution containing TTX (2 ␮ M ), to block sodium-dependent action potentials, for a minimum of 3 min and then reimaged in the continued presence of TTX. Similar levels of activity were present before and after immersion in TTX (data not shown). A second series of experi- ments was performed using brain slices to circumvent the possi- bility that the intact ventricular surface provided a barrier to drug access. Cells near the ventricular surface of the slice were mon- itored, and activity was still present in the TTX-containing solu- tion (data not shown). We also found that activity persisted in solutions that contained, in addition to TTX, the nonspecific voltage-gated Ca 2 ϩ channel (VGCC) blocker lanthanum (50 ␮ M ), the GABA A receptor blocker BMI (20 ␮ M ), the non-NMDA glutamate receptor blocker CNQX (20 ␮ M ), and the NMDA receptor blocker AP-5 (100 ␮ M ). Figure 3 A depicts the levels of activity for three cells in an E19 slice before and after the addition of the inhibitors. From these data, we conclude that neural activity, VGCC activation, and amino acid neurotransmit- ter receptor activation are not required for the spontaneous [Ca 2 ϩ ] i increases in individual VZ cells. To test whether the [Ca 2 ϩ ] i increases were dependent on extracellular Ca 2 ϩ , we performed experiments in Ca 2 ϩ - containing and Ca 2 ϩ -free (0 Ca 2 ϩ and 2 m M EGTA) ACSF solutions. Embryonic cortical slabs were preincubated in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from an E17 ...

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... in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from an E17 neocortical slab is illustrated in Figure 7 A . This example shows nine sequential pseudocolored images before, during, and after the coordinated event. The activity seems to originate with 1 or 2 cells ( arrow ) and spreads outward to 14 –16 neighboring cells. Figure 7 B shows the time course of [C a 2 ϩ ] i change in eight of these cells and the combined mean value for all cells together ( inset ). There was a close spatiotemporal coupling of [C a 2 ϩ ] i change within the cell group. In this example, the propagation rate of [C a 2 ϩ ] spread from the first cell to neighboring cells was ϳ 8 ␮ m/sec (see below). These events were found to occur infrequently; over the course of our experiments, we observed 21 such events under standard imaging conditions, and in only two instances did the same cell cluster demonstrate a second coordinated [Ca 2 ϩ ] i increase. We observed up to four spontaneous coordinated [Ca 2 ϩ ] i events in a single cortical slab, and in all cases the events were spatially distinct. In clusters in which all or most participating cells were captured in the field of view ( n ϭ 13), the number of cells ranged from 4 to 20, with an average of 9.5 Ϯ 1.33 cells. In cases in which the entire event duration was captured ( n ϭ 10), the average duration of the [Ca 2 ϩ ] i increase was 50.1 Ϯ 4.37 sec. It should be noted that the cell numbers reported here are based on optical sections that cut through the radially oriented clusters at right angles (see Fig. 8 ...

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... levels of activity were present before and after immersion in TTX (data not shown). A second series of experi- ments was performed using brain slices to circumvent the possi- bility that the intact ventricular surface provided a barrier to drug access. Cells near the ventricular surface of the slice were mon- itored, and activity was still present in the TTX-containing solu- tion (data not shown). We also found that activity persisted in solutions that contained, in addition to TTX, the nonspecific voltage-gated Ca 2 ϩ channel (VGCC) blocker lanthanum (50 ␮ M ), the GABA A receptor blocker BMI (20 ␮ M ), the non-NMDA glutamate receptor blocker CNQX (20 ␮ M ), and the NMDA receptor blocker AP-5 (100 ␮ M ). Figure 3 A depicts the levels of activity for three cells in an E19 slice before and after the addition of the inhibitors. From these data, we conclude that neural activity, VGCC activation, and amino acid neurotransmit- ter receptor activation are not required for the spontaneous [Ca 2 ϩ ] i increases in individual VZ cells. To test whether the [Ca 2 ϩ ] i increases were dependent on extracellular Ca 2 ϩ , we performed experiments in Ca 2 ϩ - containing and Ca 2 ϩ -free (0 Ca 2 ϩ and 2 m M EGTA) ACSF solutions. Embryonic cortical slabs were preincubated in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from an E17 neocortical slab is illustrated in Figure 7 A . This example shows nine sequential pseudocolored images before, during, and after the coordinated event. The activity seems to originate with 1 or 2 cells ( arrow ) and ...

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... of experi- ments was performed using brain slices to circumvent the possi- bility that the intact ventricular surface provided a barrier to drug access. Cells near the ventricular surface of the slice were mon- itored, and activity was still present in the TTX-containing solu- tion (data not shown). We also found that activity persisted in solutions that contained, in addition to TTX, the nonspecific voltage-gated Ca 2 ϩ channel (VGCC) blocker lanthanum (50 ␮ M ), the GABA A receptor blocker BMI (20 ␮ M ), the non-NMDA glutamate receptor blocker CNQX (20 ␮ M ), and the NMDA receptor blocker AP-5 (100 ␮ M ). Figure 3 A depicts the levels of activity for three cells in an E19 slice before and after the addition of the inhibitors. From these data, we conclude that neural activity, VGCC activation, and amino acid neurotransmit- ter receptor activation are not required for the spontaneous [Ca 2 ϩ ] i increases in individual VZ cells. To test whether the [Ca 2 ϩ ] i increases were dependent on extracellular Ca 2 ϩ , we performed experiments in Ca 2 ϩ - containing and Ca 2 ϩ -free (0 Ca 2 ϩ and 2 m M EGTA) ACSF solutions. Embryonic cortical slabs were preincubated in normal (2 m M Ca 2 ϩ ) or Ca 2 ϩ -free ACSF for at least 30 min before imaging, and comparisons were made of the activity in both conditions. In some experiments, slabs and slices were imaged first in normal ACSF, followed by imaging of the same area ϳ 20 –30 min after exchange of normal ACSF for Ca 2 ϩ -free ACSF. Results from several experiments showed that in all cases similar levels of activity were present in VZ cells in both Ca 2 ϩ and Ca 2 ϩ -free conditions. Figure 3 B shows graphs of [Ca 2 ϩ ] i changes from three cells in an E16 slice recorded in both normal and Ca 2 ϩ -free ACSF bath solutions. There were no obvious differences in the behavior of the [Ca 2 ϩ ] i transients under the two imaging conditions. In experiments using E19 slabs, we found the mean transient duration was 45.4 Ϯ 3.6 sec ( n ϭ 80) in Ca 2 ϩ -free conditions, and the average frequency was 1.60 Ϯ 0.1 transients per imaging trial ( n ϭ 65). Comparison of these pa- rameters with those obtained at E19 under conditions of standard extracellular Ca 2 ϩ (2 m M ) showed no significant differences (mean duration at E19 was 38.6 Ϯ 3.0 sec; mean frequency was 1.64 Ϯ 0.1 transients per imaging trial). These results suggest that the majority of spontaneous [Ca 2 ϩ ] i fluctuations in VZ cells may be mediated by Ca 2 ϩ released from intracellular stores. To test this possibility directly, we examined cortical slabs before and after incubation in an ACSF solution containing thapsigargin (5 ␮ M ), a Ca 2 ϩ -ATPase inhibitor that depletes intracellular Ca 2 ϩ stores (Thastrup et al., 1990). Fluo- 3-loaded cortical slabs were first imaged to establish baseline levels of activity. The slabs were subsequently incubated in thap- sigargin for at least 15 min and then reimaged in the continued presence of thapsigargin. After treatment with thapsigargin, spontaneous activity in VZ cells was almost completely abol- ished; only a few cells were seen to produce spontaneous [Ca 2 ϩ ] i fluctuations in multiple imaging trials from two separate slabs (Fig. 3 C ). This effect was not caused by cell injury or death because VZ cells still produce [C a 2 ϩ ] i increases in the presence of thapsigargin when exposed to agents that depolarize the cells (data not shown). Although immunohistochemical analysis suggests that the major- ity of imaged cells are precursor cells (see Fig. 1), similar spon- taneous [C a 2 ϩ ] i fluctuations have been observed in postmitotic neurons, including migrating cerebellar granule cells (Komuro and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994; Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et al., 1992; Owens et al., 1996). We therefore combined Ca 2 ϩ imaging and T uJ1 labeling to confirm the identity of spontane- ously active VZ cells. The neuronal marker T uJ1 has been used previously to identif y immature neurons in the neocortical VZ (Menezes and Luskin, 1994; O’Rourke et al., 1997). We first imaged both slabs and slices of embryonic cortex to observe cells that displayed spontaneous [Ca 2 ϩ ] i fluctuations. The tissue was subsequently fixed and stained for TuJ1 immunoreactivity, and the same regions were reimaged (see Materials and Methods). Consistent with results reported in the mouse (Menezes and Luskin, 1994), we found little or no TuJ1 immunoreactivity in the VZ on E15 (approximately E13 in the mouse); however, in these same slices, many cells throughout the depth of the VZ had spontaneous [Ca 2 ϩ ] i fluctuations (Fig. 4 A ). Figure 4 A shows a coronal slice from an E15 embryo that was imaged for spontane- ous [Ca 2 ϩ ] i increases and then for TuJ1 immunoreactivity. The cells outlined with circles were active during Ca 2 ϩ imaging (Fig. 4 A , left ), and the corresponding cell locations are indicated in the TuJ1-stained section (Fig. 4 A , right ). In only one case was a clear TuJ1-positive cell body present where an active cell was seen during Ca 2 ϩ imaging (Fig. 4 A , arrow on right ). Furthermore, there were no TuJ1-stained cell bodies near the VZ surface where the active cells in tissue slab experiments were seen. These results suggest that the majority of spontaneous single-cell activ- ity is mediated by precursor cells that do not express the TuJ1 antigen. As neurogenesis proceeds, there is an increase in the number of postmitotic neurons that are TuJ1-positive in the VZ (Menezes and Luskin, 1994). Therefore, it is possible that at later develop- mental periods (e.g., E19) the VZ contains both neurons and precursor cells that both display spontaneous [Ca 2 ϩ ] i fluctua- tions. This may be reflected in the greater number of active cells seen in the VZ at E19 compared with E15 (Fig. 2 C ). To address this, we imaged E19 slabs and subsequently stained them for TuJ1 immunoreactivity. Many more fibers and cell bodies were stained with TuJ1 in the VZ at E19 than at E15, and a greater number of spontaneously active cells was found in regions in which there was positive TuJ1 staining (Fig. 4 B , arrows on right ). This result suggests that at later stages of neurogenesis the VZ can contain both precursor cells and neurons that display spontaneous [Ca 2 ϩ ] fluctuations. A second distinctive form of spontaneous [Ca 2 ϩ ] i behavior was observed in VZ cells at the ventricular surface. Intracellular Ca 2 ϩ fluctuations were seen in pairs of adjacent cells whose nuclei often protruded from the surface of the slab. Figure 5 A shows one such doublet in an E19 cortical slab before, during, and after the [Ca 2 ϩ ] i increase. Figure 5 B displays a line graph of three doublet events from the same slab shown in Figure 5 A ( arrows indicate transients shown in Fig. 5 A ). These events were highly synchronized; the peak of the transients occurred at the same time point, and the duration of the events were nearly identical in both cells (Fig. 5). For example, in a sample of 15 of these doublet events, the average duration of one cell of the pair was 36.7 Ϯ 3.1 sec, whereas the other was 37.1 Ϯ 3.7 sec. Doublet events were observed in tissue slabs at or near the ventricular surface at all ages examined (E15–E20). The rounded morphology of these cells and their location at the ventricular surface suggested that they might be M-phase cells in the process of cytokinesis. By using the vital stain syto-11 to label cellular DNA (Chenn and McConnell, 1995), we found that cell doublets were in the same tissue plane as pairs of daughter cells in various stages of mitosis. For example, Figure 6 A shows an optical section of the ventricular surface of an E16 cortical slab stained with fluo-3. Many of the stained cells were in adjacent pairs, had rounded morphology, and displayed synchronized spontaneous [C a 2 ϩ ] i fluctuations. Imaging in the same focal plane after incubation in syto-11 revealed multiple pairs of daughter cells with patterns of condensed chromatin characteris- tic of mitotically active cells (Fig. 6 B ). This pattern of staining was only observed at the ventricular surface of the slab. When focusing deeper into the VZ, only a diffuse nuclear staining was seen, a result similar to that found in slices of ferret cortex (Chenn and McConnell, 1995). Furthermore, when focusing at this deeper level, doublet events were not observed. In addition, negatively stained condensed chromatin could sometimes be seen in the nuclei of fluo-3-labeled cell doublets, as shown in the inset of Figure 6 B . Not surprisingly, synchronous cell pairs were also found to be T uJ1-negative (Fig. 6 C ). Furthermore, during long imaging trials, we were able to observe cell division (Fig. 6 D ). In the example illustrated in Figure 6 D 1 , the dividing cell first appeared rounded, but over the next 20 min, an equatorial con- striction and cleavage plane appeared in the cell, and condensed chromatin separated in a polarized manner. Changes in [Ca 2 ϩ ] i in both daughter cells were synchronized (Fig. 6 D 2 ). These observations confirmed that at least some, if not all, cells exhib- iting synchronized [C a 2 ϩ ] i fluctuations were mitotically active daughter cells. Finally, as with the single-cell events seen in VZ cells, doublets could occur in C a 2 ϩ -free AC SF (Fig. 6 E ), sug- gesting that these events are also mediated by the release of Ca 2 ϩ from intracellular stores. The third pattern of spontaneous [C a 2 ϩ ] i fluctuation consisted of coordinated [C a 2 ϩ ] i increases in groups of neighboring VZ cells. An example of such an event from an E17 neocortical slab is illustrated in Figure 7 A . This example shows nine sequential pseudocolored images before, during, and after the coordinated event. The activity seems to originate with 1 or 2 cells ( arrow ) and spreads outward to 14 –16 neighboring cells. Figure 7 B shows the time course of [C a 2 ϩ ] i ...

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Plasticity of Dopaminergic Phenotype and Locomotion in Larval Zebrafish Induced by Brain Excitability Changes during the Embryonic Period

Article

Full-text available

Jun 2023

During the embryonic period, neuronal communication starts before the establishment of the synapses with alternative forms of neuronal excitability, called here embryonic neural excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs, but the global consequences for developing organisms are not all understood. Here, we monitored calcium (Ca ²⁺ ) transients in the telencephalon of zebrafish embryos as a proxy for ENE to assess the efficacy of transient pharmacological treatments to either increase or decrease ENE. Increasing or decreasing ENE at the end of the embryonic period promoted an increase or a decrease in the numbers of dopamine (DA) neurons, respectively. This plasticity of dopaminergic specification occurs in the subpallium (SP) of zebrafish larvae at 6 d postfertilization (dpf), within a relatively stable population of vMAT2-positive cells. Nondopaminergic vMAT2-positive cells hence constitute an unanticipated biological marker for a reserve pool of DA neurons that can be recruited by ENE. Modulating ENE also affected larval locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted hyperlocomotion of larvae at 6 dpf, reminiscent of zebrafish endophenotypes reported for attention deficit hyperactivity disorders (ADHDs). These results provide a convenient framework for identifying environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification.

Calcium and activity-dependent signaling in the developing cerebral cortex

Article

Full-text available

Sep 2022
DEVELOPMENT

Calcium influx can be stimulated by various intra- and extracellular signals to set coordinated gene expression programs into motion. As such, the precise regulation of intracellular calcium represents a nexus between environmental cues and intrinsic genetic programs. Mounting genetic evidence points to a role for the deregulation of intracellular calcium signaling in neuropsychiatric disorders of developmental origin. These findings have prompted renewed enthusiasm for understanding the roles of calcium during normal and dysfunctional prenatal development. In this Review, we describe the fundamental mechanisms through which calcium is spatiotemporally regulated and directs early neurodevelopmental events. We also discuss unanswered questions about intracellular calcium regulation during the emergence of neurodevelopmental disease, and provide evidence that disruption of cell-specific calcium homeostasis and/or redeployment of developmental calcium signaling mechanisms may contribute to adult neurological disorders. We propose that understanding the normal developmental events that build the nervous system will rely on gaining insights into cell type-specific calcium signaling mechanisms. Such an understanding will enable therapeutic strategies targeting calcium-dependent mechanisms to mitigate disease.

Embryonic cortical layer 5 pyramidal neurons form an active, transient circuit motif perturbed by autism-associated mutations

Preprint

Full-text available

Aug 2022

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that embryonic layer 5 pyramidal neurons, identified through single cell transcriptomics, display two phases of circuit assembly in vivo. At E14.5, a multi-layered circuit motif, composed of a single layer 5 cell type, forms. This motif is transient, switching to a second circuit motif, involving all three types, by E17.5. In vivo targeted single cell recordings and two-photon calcium imaging of embryonic layer 5 neurons reveal that, in both phases, neurons have active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses. Embryonic layer 5 neurons strongly express autism-associated genes, and perturbing these genes disrupts the switch between the two motifs. Hence, layer 5 pyramidal neurons form transient active pyramidal-to-pyramidal circuits, at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

Diazepam binding inhibitor governs neurogenesis of excitatory and inhibitory neurons during embryonic development via GABA signaling

Article

Aug 2022
NEURON

Of the neurotransmitters that influence neurogenesis, gamma-aminobutyric acid (GABA) plays an outstanding role, and GABA receptors support non-synaptic signaling in progenitors and migrating neurons. Here, we report that expression levels of diazepam binding inhibitor (DBI), an endozepine that modulates GABA signaling, regulate embryonic neurogenesis, affecting the long-term outcome regarding the number of neurons in the postnatal mouse brain. We demonstrate that DBI is highly expressed in radial glia and intermediate progenitor cells in the germinal zones of the embryonic mouse brain that give rise to excitatory and inhibitory cells. The mechanism by which DBI controls neurogenesis involves its action as a negative allosteric modulator of GABA-induced currents on progenitor cells that express GABAA receptors containing γ2 subunits. DBI’s modulatory effect parallels that of GABAA-receptor-mediating signaling in these cells in the proliferative areas, reflecting the tight control that DBI exerts on embryonic neurogenesis.

[Ca2+] fluctuation mediated by T-type Ca2+ channel is required for the differentiation of cortical neural progenitor cells

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Full-text available

Jun 2022
DEV BIOL

The fluctuation of intracellular calcium concentration ([Ca²⁺]i) is known to be involved in various processes in the development of central nervous system, such as the proliferation of neural progenitor cells (NPCs), migration of intermediate progenitor cells (IPCs) from the ventricular zone (VZ) to the subventricular zone (SVZ), and migration of immature neurons from the SVZ to cortical plate. However, the roles of [Ca²⁺]i fluctuation in NPC development, especially in the differentiation of the self-renewing NPCs into neuron-generating NPCs and immature neurons have not been elucidated. Using calcium imaging of acute cortical slices and cells isolated from mouse embryonic cortex, we examined temporal changes in the pattern of [Ca²⁺]i fluctuations in VZ cells from E12 to E16. We observed intracellular Ca²⁺ levels in Pax6-positive self-renewing NPCs decreased with their neural differentiation. In E11, Pax6-positive NPCs and Tuj1-positive immature neurons exhibited characteristic [Ca²⁺]i fluctuations; few Pax6-positive NPCs exhibited [Ca²⁺]i transient, but many Tuj1-positive immature neurons did, suggesting that the change in pattern of [Ca²⁺]i fluctuation correlate to their differentiation. The [Ca²⁺]i fluctuation during NPCs development was mostly mediated by the T-type calcium channel and blockage of T-type calcium channel in neurosphere cultures increased the number of spheres and inhibited neuronal differentiation. Consistent with this finding, knockdown of Cav3.1 by RNAi in vivo maintained Pax6-positive cells as self-renewing NPCs, and simultaneously suppressing their neuronal differentiation of NPCs into Tbr1-positive immature neurons. These results reveal that [Ca²⁺]i fluctuation mediated by Cav3.1 is required for the neural differentiation of Pax6-positive self-renewing NPCs.

Early Developmental PMCA2b Expression Protects From Ketamine-Induced Apoptosis and GABA Impairments in Differentiating Hippocampal Progenitor Cells

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May 2022

PMCA2 is not expressed until the late embryonic state when the control of subtle Ca ²⁺ fluxes becomes important for neuronal specialization. During this period, immature neurons are especially vulnerable to degenerative insults induced by the N-methyl-D-aspartate (NMDA) receptor blocker, ketamine. As H19-7 hippocampal progenitor cells isolated from E17 do not express the PMCA2 isoform, they constitute a valuable model for studying its role in neuronal development. In this study, we demonstrated that heterologous expression of PMCA2b enhanced the differentiation of H19-7 cells and protected from ketamine-induced death. PMCA2b did not affect resting [Ca ²⁺ ] c in the presence or absence of ketamine and had no effect on the rate of Ca ²⁺ clearance following membrane depolarization in the presence of the drug. The upregulation of endogenous PMCA1 demonstrated in response to PMCA2b expression as well as ketamine-induced PMCA4 depletion were indifferent to the rate of Ca ²⁺ clearance in the presence of ketamine. Yet, co-expression of PMCA4b and PMCA2b was able to partially restore Ca ²⁺ extrusion diminished by ketamine. The profiling of NMDA receptor expression showed upregulation of the NMDAR1 subunit in PMCA2b-expressing cells and increased co-immunoprecipitation of both proteins following ketamine treatment. Further microarray screening demonstrated a significant influence of PMCA2b on GABA signaling in differentiating progenitor cells, manifested by the unique regulation of several genes key to the GABAergic transmission. The overall activity of glutamate decarboxylase remained unchanged, but Ca ²⁺ -induced GABA release was inhibited in the presence of ketamine. Interestingly, PMCA2b expression was able to reverse this effect. The mechanism of GABA secretion normalization in the presence of ketamine may involve PMCA2b-mediated inhibition of GABA transaminase, thus shifting GABA utilization from energetic purposes to neurosecretion. In this study, we show for the first time that developmentally controlled PMCA expression may dictate the pattern of differentiation of hippocampal progenitor cells. Moreover, the appearance of PMCA2 early in development has long-standing consequences for GABA metabolism with yet an unpredictable influence on GABAergic neurotransmission during later stages of brain maturation. In contrast, the presence of PMCA2b seems to be protective for differentiating progenitor cells from ketamine-induced apoptotic death.

Timing as a Mechanism of Development and Evolution in the Cerebral Cortex

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Full-text available

Dec 2021
BRAIN BEHAV EVOLUT

Laura R. Fenlon

One of the biggest mysteries in neurobiology concerns the mechanisms responsible for the diversification of the brain over different time scales i.e. during development and evolution. Subtle differences in the timing of biological processes during development, e.g. onset, offset, duration, speed and sequence, can trigger large changes in phenotypic outcomes. At the level of a single organism, altered timing of developmental events can lead to individual variability, as well as malformation and disease. At the level of phylogeny, there are known interspecies differences in the timing of developmental events, and this is thought to be an important factor that drives phenotypic variation across evolution, known as heterochrony. A particularly striking example of phenotypic variation is the evolution of human cognitive abilities, which has largely been attributed to the development of the mammalian-specific neocortex and its subsequent expansion in higher primates. Here, I review how the timing of different aspects of cortical development specifies developmental outcomes within species, including processes of cell proliferation and differentiation, neuronal migration and lamination, and axonal targeting and circuit maturation. Some examples of the ways that different processes might “keep time” in the cortex are explored, reviewing potential cell-intrinsic and -extrinsic mechanisms. Further, by combining this knowledge with known differences in timing across species, timing changes that may have occurred during evolution are identified, which perhaps drove the phylogenetic diversification of neocortical structure and function.

Spontaneous Synchronous Network Activity in the Neonatal Development of mPFC in Mice

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Full-text available

Jan 2021
DEV NEUROBIOL

Spontaneous Synchronous Network Activity (SSA) is a hallmark of neurodevelopment found in numerous central nervous system structures, including neocortex. SSA occurs during restricted developmental time windows, commonly referred to as critical periods in sensory neocortex. Although part of the neocortex, the critical period for SSA in the medial prefrontal cortex (mPFC) and the underlying mechanisms for generation and propagation are unknown. Using Ca²⁺ imaging and whole cell patch‐clamp in an acute mPFC slice mouse model, the development of spontaneous activity and SSA was investigated at cellular and network levels during the two first postnatal weeks. The data revealed that developing mPFC neuronal networks are spontaneously active and exhibit SSA in the first two postnatal weeks, with peak synchronous activity at postnatal days (P)8‐9. Networks remain active but are desynchronised by the end of this two‐week period. SSA was driven by excitatory ionotropic glutamatergic transmission with a small contribution of excitatory GABAergic transmission at early timepoints. The neurohormone oxytocin desynchronised SSA in the first postnatal week only without affecting concurrent spontaneous activity. By the end of the second postnatal week, inhibiting GABAA receptors restored SSA. These findings point to the emergence of GABAA receptor‐mediated inhibition as a major factor in the termination of SSA in mouse mPFC.

Glycine Receptor Inhibition Differentially Affect Selected Neuronal Populations of the Developing Embryonic Cortex, as Evidenced by the Analysis of Spontaneous Calcium Oscillations

Article

Full-text available

Oct 2020
INT J MOL SCI

The embryonic developing cerebral cortex is characterized by the presence of distinctive cell types such as progenitor pools, immature projection neurons and interneurons. Each of these cell types is diverse on itself, but they all take part of the developmental process responding to intrinsic and extrinsic cues that can affect their calcium oscillations. Importantly, calcium activity is crucial for controlling cellular events linked to cell cycle progression, cell fate determination, specification, cell positioning, morphological development and maturation. Therefore, in this work we measured calcium activity in control conditions and in response to neurotransmitter inhibition. Different data analysis methods were applied over the experimental measurements including statistical methods entropy and fractal calculations, and spectral and principal component analyses. We found that developing projection neurons are differentially affected by classic inhibitory neurotransmission as a cell type and at different places compared to migrating interneurons, which are also heterogeneous in their response to neurotransmitter inhibition. This reveals important insights into the developmental role of neurotransmitters and calcium oscillations in the forming brain cortex. Moreover, we present an improved analysis proposing a Gini coefficient-based inequality distribution and principal component analysis as mathematical tools for understanding the earliest patterns of brain activity.

NAD+-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior

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Full-text available

Oct 2020

Abdallah Elkhal

Intrinsic defects within blood vessels from the earliest developmental time points can directly contribute to psychiatric disease origin. Here, we show that nicotinamide adenine dinucleotide (NAD+), administered during a critical window of prenatal development, in a mouse model with dysfunctional endothelial γ-aminobutyric acid type A (GABAA) receptors (Gabrb3 endothelial cell knockout mice), results in a synergistic repair of impaired angiogenesis and normalization of brain development, thus preventing the acquisition of abnormal behavioral symptoms. The prenatal NAD+ treatment stimulated extensive cellular and molecular changes in endothelial cells and restored blood vessel formation, GABAergic neuronal development, and forebrain morphology by recruiting an alternate pathway for cellular repair, via previously unknown transcriptional mechanisms and purinergic receptor signaling. Our findings illustrate a novel and powerful role for NAD+ in sculpting prenatal brain development that has profound implications for rescuing brain blood flow in a permanent and irreversible manner, with long-lasting consequences for mental health outcome.

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