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The Journal of Neuroscience, February 15, 1998, 18(4):1419-1427
Coordination of Neuronal Activity in Developing Visual Cortex by
Gap Junction-Mediated Biochemical Communication
Karl
Kandler and
Lawrence C.
Katz
Howard Hughes Medical Institute and Department of Neurobiology,
Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
During brain development, endogenously generated coordinated
neuronal activity regulates the precision of developing synaptic circuits (Shatz and Stryker, 1988 ; Weliky and Katz, 1997). In the
neonatal neocortex, a form of endogenous coordinated activity is
present as locally restricted intercellular calcium waves that are
mediated by gap junctions (Yuste et al., 1992). As in other neuronal
and non-neuronal systems, these coordinated calcium fluctuations may
form the basis of functional cell assemblies (for review, see Warner,
1992; Peinado et al., 1993b). In the present study, we investigated the
cellular mechanisms that mediate the activation of neuronal domains and
the propagation of intercellular calcium waves in slices from neonatal
rat neocortex. The occurrence of neuronal domains did not depend on
intercellular propagation of regenerative electrical signals because
domains persisted after blockade of sodium and calcium-dependent action
potentials. Neuronal domains were elicited by intracellular infusion of
inositol trisphosphate (IP3) but not of calcium,
indicating the involvement of IP3-related second-messenger
systems. Pharmacological stimulation of metabotropic glutamate
receptors, which are linked to the production of
IP3, elicited similarly coordinated calcium
increases, whereas pharmacological blockade of metabotropic glutamate
receptors dramatically reduced the number of neuronal domains.
Therefore, the propagating cellular signal that causes the occurrence
of neuronal domains seems to be inositol trisphosphate but not calcium.
Because coordination of neuronal calcium changes by gap junctions is
independent of electrical signals, the function of gap junctions
between neocortical neurons is probably to synchronize biochemical
rather than electrical activity.
Key words:
gap junction; visual cortex; inositol trisphosphate; calcium; metabotropic glutamate receptor; development; thapsigargin
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INTRODUCTION |
In the mammalian visual
system, neuronal activity seems to be required for the emergence of
several basic circuits, including the segregation of retinal ganglion
fibers into eye-specific layers in the lateral geniculate nucleus and
the development of ocular dominance columns and orientation columns in
the primary visual cortex (for review, see Katz and Shatz, 1996).
Because all of these structures can emerge before visual experience
(Shatz, 1983; Chapman et al., 1996; Horton and Hocking, 1996),
formation is likely to be driven by endogenously generated activity
patterns (Shatz and Stryker, 1988; Weliky and Katz, 1997). Coordinated, endogenous activity has been described both in the immature retina in
the form of traveling activity waves mediated by nicotinergic synaptic
transmission (Galli and Maffei, 1988; Meister et al., 1991; Feller et
al., 1996) and in the early postnatal neocortex in the form of neuronal
domains (Yuste et al., 1992).
In brain slices prepared from the early postnatal rat neocortex,
neuronal domains occur as spontaneous, locally restricted intercellular
calcium waves that originate in one or a few centrally located cells
from which the waves propagate over a distance of 50-100 µm (Yuste
et al., 1992, 1995). Propagation of these calcium waves is mediated by
gap junctions and not by synaptic transmission (Yuste et al., 1995).
Because neuronal domains are circular in tangential slices and often
radially elongated in coronal slices, it has been proposed that
neuronal domains partition the immature neocortex into columnar patches
of coordinated activity. The intercellular signals underlying this form
of coordinated activity, however, are obscure.
Gap junctions allow the passage of electrical currents and small
molecules up to ~1 kDa between coupled cells (for review, see, e.g.,
Bennett et al., 1991). In the developing neocortex, electrical coupling
between neurons has been demonstrated in the ventricular zone (Lo Turco
and Kriegstein, 1991) and in the cortical plate (Connors et al., 1983).
Coupling is also most likely responsible for the intercellular
propagation of calcium spikes in the presence of potassium channel
blockers (Yuste et al., 1995) and for mediating the junctional spread
of membrane depolarizations in cultured cortical neurons (Charles et
al., 1996; Peinado et al., 1993b).
Despite these examples, electrical coupling between neurons in the
neocortex seems to be too weak to be responsible for the synchronization of neuronal activity during the occurrence of a
neuronal domain (Connors et al., 1983; Peinado et al., 1993b). An
alternative possibility is that gap junctions synchronize neuronal activity by coordinating biochemical activity rather than electrical activity. In non-neuronal cells, gap junctions mediate intercellular biochemical communication via the direct exchange of second-messenger molecules (Boitano et al., 1992; Allbritton and Meyer, 1993; for review, see Sanderson, 1995), thus raising the possibility that neuronal activity might be coordinated by biochemical communication through gap junctions.
In the present study, we elucidated the cellular mechanisms by which
gap junctions coordinate neuronal behavior in the developing rat
neocortex by using calcium-imaging techniques in brain slices. We found
that neuronal domains are initiated by stimulation of metabotropic
glutamate receptors and by intracellular increase in the
second-messenger molecule inositol trisphosphate
(IP3) that releases calcium from intracellular
stores. In addition to its role as an intracellular messenger,
IP3 also seems to be the intercellular signal molecule that
diffuses between coupled cells and therefore underlies the propagation
of neuronal calcium waves. This cascade of events strongly supports the
hypothesis that neuronal gap junctions in the developing neocortex
coordinate the biochemical activity among coupled cell assemblies.
Parts of this paper have been published previously in abstract form
(Kandler and Katz, 1995).
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MATERIALS AND METHODS |
Slices (300-400 µm thick) were prepared from the visual
cortex of postnatal day 0 (P0)-P8 rats and were stained with fura-2 AM
(Molecular Probes, Eugene, OR) as described previously (Yuste et al.,
1995) with the only difference that Mg2+-containing
artificial CSF (ACSF) (composition in mM: NaCl 124, MgSO4 1.3, CaCl2 3.1, KCl 5, KH2PO4 1.25, glucose 10, and NaHCO3 26, pH 7.4, when bubbled with 95% O2/5%
CO2) was used during both staining and imaging.
Calcium imaging was performed at room temperature (21-25°C) using an
upright microscope (Axioskope; Zeiss) equipped with an intensified
charge-coupled device camera (Hamamatsu) coupled to an image processor
(Imaging Technologies Series 151). Single excitation images (385 nm)
were acquired every 1-10 sec, and after background subtraction, the
average of 16 frames was stored on an optical disk recorder (Panasonic
TQ 2028F) for off-line analysis. Individual frames were digitized and
further processed using the program National Institutes of Health Image
(ftp://zippy.nimh.gov/pub/nih-image). To minimize phototoxicity caused
by prolonged illumination, we elicited neuronal domains by transiently
decreasing the temperature of the superfusing ACSF by 3-5°C, as
described previously (Yuste et al., 1995). For domain detection, a
reference image before a temperature drop (TD) was subtracted from
subsequent images. Subtracted images were then smoothed by convolution
with a 7 × 7 Gaussian kernel, and intensity changes were
expressed as  F/F (in percent). A neuronal
domain was defined as a decrease in the fura-2 signal in more than five
neighboring neurons occurring in the same frame. The size of domains
was measured by applying a threshold of twice the SD of the pixel
values in the image (Yuste et al., 1995). All values are expressed as
the arithmetic mean ± SEM.
Conventional whole-cell patch-clamp recording techniques (Blanton et
al., 1989) were used to form gigaohm seals on neurons under visual
control (Axioskope equipped with 63× and 40× objectives and Nomarski
optics; Zeiss). Patch pipettes (4-9 M ) were filled with internal
solution (composition in mM: cesium gluconate 110 or KCl
130, MgCl2 1, CaCl2 1, EGTA 11, and HEPES 10, pH 7.2; in some experiments the solution also contained 50-100
µM fura-2 pentapotassium). For intracellular
IP3 infusions, 1 mM inositol 1,4,5-trisphosphate or 50-100 µM inositol
2,4,5-trisphosphate, a nonhydrolyzable analog, was added to the
internal solution. Relatively high IP3 concentrations were
used to ensure the rapid intracellular infusion of a sufficient amount
of IP3. For calcium injections, the internal solution
contained no EGTA and contained 1 mM CaCl2 and
in some cases 200 µM fluo-3 pentapotassium. In some
cases, intracellular calcium was increased by the activation of
voltage-gated calcium channels by a train (10-40 sec) of depolarizing voltage steps (50 msec in duration; 10 Hz). These depolarizations consistently elicited regenerative action potentials that, because of
the presence of 2-5 µM TTX, were interpreted as calcium
action potentials. After the formation of gigaohm seals, image
capturing was started and followed by rupture of the cell membrane.
To deplete intracellular calcium stores with thapsigargin, we dissolved
fura-2 AM in DMSO that also contained 1 mM thapsigargin (Sigma, St. Louis, MO), resulting in a final concentration of 10 µM thapsigargin in the fura-2 AM-staining solution.
Unless otherwise noted, all chemicals were purchased from Sigma.
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RESULTS |
In the following sections, we first describe experiments that
demonstrate that neuronal calcium waves result from the release of
calcium from intracellular stores, suggesting that neuronal domains are
generated by the intercellular propagation of biochemical rather than
electrical signals. We next show that the initiation of neuronal
domains involves stimulation of metabotropic glutamate receptors, which
are linked to the production of intracellular IP3. Finally,
we demonstrate that IP3, not calcium ions, acts as
the propagating signal between coupled cells.
Changes in the intracellular calcium concentration were monitored in
brain slices from the occipital cortex of early postnatal rats stained
with the calcium indicator fura-2 AM. All experiments were conducted in
the presence of the sodium channel antagonist tetrodotoxin (2-5
µM) to block sodium-dependent action potentials and
synaptic transmission. Because spontaneous neuronal domains occur
sporadically at long intervals of ~4 min, the acquisition of a
sufficient number of domains for quantification would have required
continuous imaging over several hours. To minimize photobleaching and
phototoxicity associated with prolonged illumination periods, we used
small transient temperature drops (3-5°C) to elicit neuronal domains. Previous studies have demonstrated that TD-elicited neuronal domains are indistinguishable from spontaneously occurring neuronal domains (Yuste et al., 1995). Consistent with these studies, TD consistently triggered the appearance of neuronal domains (average, 4.5 ± 1.1 domains per TD; n = 17 slices) that
were randomly located throughout the cortical depth and that covered an
average area of 2782 ± 193 µm2
(n = 79 domains) (Figs.
1A,
2).
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Figure 1.
Neuronal domains in fura-2-stained slices in
neonatal rat visual cortex. Slices were illuminated with 385 nm light
at which fura-2 emission decreases with increasing calcium
concentration. Video images were taken before (left) and
during (middle) the occurrence of neuronal domains. On
the right, the changes in the fura-2 fluorescence are
expressed in pseudocolor as F/F (in
percent) and overlaid onto the images shown in the
middle. Each individual image is the average of 16 background-subtracted single frames taken at video rate. The pial
surface is indicated by the arrowheads; the white matter
is identified by the dashed line. A,
Neuronal domains elicited by temperature drop under control conditions (2 µM TTX). Coronal slice of a P3 rat. B,
Neuronal domains elicited by temperature drop in the presence of 2 mM Ni2+ and 2 µM TTX.
These neuronal domains are similar in size and shape to those observed
under control conditions, indicating that neither sodium-dependent
action potentials nor extracellular Ca2+ entry is
required for the occurrence of neuronal domains. Coronal slice of a P2
rat.
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Figure 2.
Effects of nickel, thapsigargin, and t-ACPD on the
number and size of neuronal domains. A, Average number
of neuronal domains elicited by temperature drop under control
conditions and in the presence of 2 mM nickel chloride
(Ni) or 10 µM thapsigargin
(thap). Blockade of voltage-gated calcium channels with
2 mM nickel had no effect on the number of domains. In
contrast, depletion of intracellular calcium stores with 10 µM thapsigargin almost completely abolished neuronal
domains (p < 0.01, Student's
t test). B, Average size of neuronal
domains elicited by temperature drop under control conditions and in
the presence of 2 mM nickel (Ni) and
elicited by bath application of t-ACPD (40-100 µm). Neuronal domains
elicited by t-ACPD were smaller (p < 0.01)
than were domains elicited by temperature drop. In all cases, the bath
solution contained 2 µM TTX. Numbers above
bars indicate the number of slices
(A) or number of neuronal domains
(B). Asterisks indicate a
significant difference (p  0.05; student's
t test).
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Activation of neuronal domains depends on the release of calcium
from intracellular stores
To address the cellular mechanisms that underlie the activation of
neuronal domains, we first determined the sources of calcium responsible for the increase in free intracellular calcium
concentration ([Ca2+]i). The
occurrence of a neuronal domain begins in one or a few "trigger
cells" from which a calcium wave propagates radially (Yuste et al.,
1995). Because gap junctions are permeable to electrical current as
well as to small second-messenger molecules (Bennett and Goodenough,
1978; Saez et al., 1989; Bennett et al., 1991), intercellular calcium
waves could be generated either by the intercellular propagation of
electrical signals, such as calcium spikes (Yuste et al., 1995), or by
the intercellular diffusion of chemical signals, such as calcium ions
or small second-messenger molecules (Saez et al., 1989; Boitano et al.,
1992; for review, see Sanderson, 1995). To distinguish between these
two possibilities, we applied TDs in the presence of 2-5
mM nickel chloride, which, at these concentrations, blocks
both low voltage- and high voltage-activated calcium channels (Gu et
al., 1994). Although nickel completely blocked depolarization-induced
calcium spikes (0.5 mM NiCl and 2 µM TTX;
n = 5 neurons; data not shown), TDs in the presence of
nickel still consistently elicited neuronal domains with an average
frequency and area indistinguishable from those of controls (frequency,
5.2 ± 1.0 domains/TD; n = 14 slices;
p > 0.1, Student's t test; area, 3190 ± 279 µm2; n = 72 domains;
p > 0.1, Student's t test; Figs.
1B, 2).
These findings indicate that the junctional propagation of fast
electrical signals such as calcium action potentials and that calcium
entry via voltage-gated calcium channels are not required for the
activation of neuronal domains. We next tested whether neuronal domains
are elicited by the release of calcium from internal stores by
depleting intracellular calcium stores with thapsigargin (10 µM), an endoplasmic reticular
Ca2+-ATPase inhibitor. In thapsigargin-treated
slices, the number of neuronal domains decreased dramatically from
4.5 ± 1.1 domains/TD (n = 17 slices) to 0.8 ± 1.1 domains/TD (n = 24 slices; p < 0.01; Fig. 2A). Thapsigargin had no effect on calcium
increases in single cells resulting from KCl-induced depolarizations
(60 mM) (n = 8 slices; data not shown).
Taken together, these results demonstrate that the activation of
cortical neuronal domains requires calcium release from intracellular
stores.
Activation of metabotropic glutamate receptors elicits
neuronal domains
Because the activation of neuronal domains results from calcium
release from intracellular stores, we next investigated whether neuronal domains could be elicited by neurotransmitters that activate internal calcium stores. Because the metabotropic glutamate receptors mGluR1 and mGluR5 are abundant in the immature neocortex and are linked
to the production of IP3 (Dudek et al., 1989; Fotuhi et al., 1993; Bevilacqua et al., 1995) and to the release of calcium from
intracellular stores (for review, see Berridge, 1993), we tested
whether stimulation of mGluR elicits neuronal domains. Bath application
of the agonist
(1S,3R) 1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD; 40-100 µM), which increases
IP3 in cortical neurons (Challiss et al., 1994), triggered
numerous neuronal domains throughout the cortical depth (5.2 ± 2.7 domains/treatment; n = 9 slices) (in Fig.
3A, several domains in
upper layers are shown). Although the overall shape of
t-ACPD-elicited domains resembled spontaneous (Yuste et al., 1995) and
TD-elicited domains (Fig. 1), they were somewhat smaller than were
those elicited by TD (average area, 1963 µm2 ± 198; n = 47 domains) (Fig. 2B), a
finding for which we currently cannot offer a plausible
explanation.
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Figure 3.
A, Activation of the metabotropic
glutamate agonist t-ACPD elicits neuronal domains. The
left and middle images were taken before
and during the application of 40 µM t-ACPD. In the
right image, changes in fura-2 emission are pseudocolor
coded as F/F (in percent) and overlaid
onto the middle image. Each individual image is the
average of 16 background-subtracted frames. Numerous neuronal domains
are visible, as are individual cells, the
[Ca2+]i of which increased by
activation of metabotropic glutamate receptors. B,
t-ACPD-elicited neuronal domains depend on functional gap junctions. In
the presence of the gap junction blocker octanol (1 mM),
application of 200 µM t-ACPD elicits only single-cell responses but no neuronal domains. The pial surface is to the upper right in A and up in
B. A, Coronal slice of a P3 rat.
B, Coronal slice of a P4 rat.
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In addition to neuronal domains, t-ACPD also elicited isolated calcium
responses in individual neurons (Fig. 3A), perhaps because
of subthreshold IP3 concentrations in these neurons or because of the existence of a subpopulation of (highly coupled) neurons
capable of eliciting neuronal domains.
Because of temporal limitations of our imaging system (maximum frame
rate, 1 Hz), we could not exclude the possibility that t-ACPD-elicited
domains resulted from direct, simultaneous activation of a group of
neighboring neurons rather than from "triggering" of a few cells.
We attempted to address this possibility by injecting t-ACPD locally,
but because of the dimensions of neuronal domains (diameters,
~50-100 µm), it was impossible to distinguish between seemingly
coordinated calcium increases resulting from extracellular diffusion of
injected t-ACPD and genuine neuronal domains resulting from
intercellular calcium waves. Therefore, we applied t-ACPD (100-200
µM) in the presence of the gap junction blocker octanol. In seven slices, bath application of t-ACPD in the presence of 1 mM octanol elicited calcium changes only in individual
cells and never triggered neuronal domains (Fig. 3B). This
indicates that t-ACPD-elicited domains were caused by stimulation of
individual cells that acted as trigger cells to elicit gap
junction-mediated neuronal domains. The application of the inactive
enantiomer cis-ACPD (100-500 µM) did not
trigger neuronal domains, nor did it increase the cytosolic calcium
concentration in individual neurons (n = 5 slices; data
not shown). All the effects of t-ACPD were blocked by the metabotropic
glutamate receptor antagonist (+)- -methyl-4-carboxyphenylglycine [(+)-MCPG; 1 mM; n = 3 slices; data not
shown].
To test whether the occurrence of neuronal domains depends on the
activation of mGluRs, we applied temperature drops while blocking
mGluRs. Bath application of the mGluR antagonist (+)-MCPG (1 mM) substantially reduced the number of neuronal domains
(MCPG, 1.3 ± 0.5 domains/TD; n = 21 slices;
control, 5.4 ± 1.1 domains/TD; n = 7 slices;
p < 0.01; Fig.
4A), indicating that
mGluR activation is critically involved in the initiation of neuronal
domains. If mGluR activation is also involved in the propagation of
domains, one would expect that (+)-MCPG also decreases the size of
domains. However, neuronal domains that persisted in the presence of
(+)-MCPG were similar in size to controls (MCPG, 3756 ± 390 µm2; n = 29 domains; age matched
control, 3851 ± 246 µm2; n = 53 domains; p > 0.1; Fig. 4B),
suggesting that mGluR activation is responsible for the initiation of
domains but not for the continued propagation of interneuronal calcium
waves.
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Figure 4.
Metabotropic glutamate receptors are required for
initiation of neuronal domains. A, Bath application of 1 mM (+)-MCPG dramatically reduced the number of neuronal
domains (p < 0.01, Student's
t test). B, Bath application of 1 mM (+)-MCPG does not affect the size of the neuronal
domains that persist, indicating that metabotropic glutamate receptors
are only involved in the initiation of neuronal domains.
Numbers above bars indicate the number of
slices (A) or number of neuronal domains
(B). An asterisk indicates a
significant difference (p 0.01; student's
t-test).
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Increases in intracellular inositol trisphosphate, but not calcium,
trigger neuronal domains
To test the participation of IP3 in neuronal domains
directly, we infused IP3 through patch pipettes into
individual cells. These IP3
infusions produced calcium waves 2-30 sec after the rupture of the
cell membrane and influx of IP3 (Fig. 5). These calcium
waves always originated from the injected cell and covered an average
area of 2164 ± 353 µm2 (n = 18 domains) that closely resembled the size of neuronal domains
elicited by TD (2782 ± 193 µm2;
n = 79 domains; p > 0.1, Student's
t test). Patching neurons without IP3 in the
pipette solution never triggered neuronal domains. These experiments
demonstrate that the stimulation of an individual cell can trigger a
neuronal domain and that IP3 alone is sufficient to trigger
neuronal domains.
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Figure 5.
Neuronal domains are elicited by increasing
[IP3]i but not
[Ca2+]i. A, Neuronal
domains elicited by intracellular infusion of inositol
1,4,5-trisphosphate are shown. Changes in the fura-2 fluorescence
signal are pseudocolor coded as F/F
(in percent) and superimposed on background-subtracted video
frames. The arrow points to the filled
cell in the first frame that was taken 2 sec (2)
before rupture of the cell membrane (frame 0).
Increasing [IP3]i triggered a concentric
calcium wave with a diameter of ~80 µm. Tangential slice of a P1
rat. B, Increasing
[Ca2+]i in a single cell
(arrow) by depolarizing voltage steps failed to elicit
intercellular calcium waves. The cell was depolarized from a holding
potential of 70 mV to +10 mV by a 40 sec train consisting of
50-msec-long depolarizations delivered at 10 Hz. The neuron was also
filled with 100 µM fluo-3 to visualize its basic
structure (right). Coronal slice of a P3 rat.
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Previous studies in non-neuronal cells have shown that intercellular
calcium waves can be mediated by the diffusion of the second messengers
IP3 or calcium ions (Saez et al., 1989; Christ et al.,
1992; for review, see Sanderson, 1995). To investigate the nature of
the propagating molecule that causes the occurrence of cortical
neuronal domains, we tested whether neuronal domains could be elicited
by increasing intracellular [Ca2+]i
without directly changing [IP3]i.
[Ca2+]i was increased by intracellular
infusion of calcium through a patch pipette that contained 1 mM Ca2+ (n = 14 cells)
or by activating voltage-gated calcium channels (Giffin et al., 1991)
with depolarizing electrical current injections (+60 mV; 100 msec; 5 Hz; 5-10 sec; n = 12 neurons). Both approaches consistently increased calcium levels in single neurons, but changes in
[Ca2+]i were always restricted to the
patched cell and never triggered propagating intercellular waves (Fig.
5B). Thus an increase in [IP3]i is
required both for eliciting and propagating neuronal calcium waves.
As calcium-induced calcium release (CICR) is a prominent calcium
release mechanism in cerebellar Purkinje neurons (Kano et al., 1995)
and a component of calcium wave propagation between retinal Mueller
cells (Keirstead and Miller, 1995), we assessed its contribution to
intracellular calcium waves in cortical neurons. Blockage of CICR with
ryanodine (100 µM) had no significant effect on the size
(ryanodine, 2330 ± 203 µm2;
n = 38; control, 2782 ± 193 µm2; n = 79; p > 0.1) or the frequency (ryanodine, 6.33 ± 1.0 domains/TD; control,
4.5 ± 1.1 domains/TD; n = 17; p > 0.1) of neuronal domains. This indicates that CICR is not required
for the generation of neuronal domains.
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DISCUSSION |
In the present study, we investigated the cellular mechanisms that
generate coordinated neuronal activity in the form of gap junction-mediated intercellular calcium waves (neuronal domains) in the
early developing neocortex. Our data suggest that initiation of
neuronal domains consists of stimulation of metabotropic glutamate receptors, an increase in [IP3]I, and
release of calcium from intracellular stores. Propagation of neuronal
activity involves the intercellular diffusion and probably the partial
regeneration of IP3. In contrast to other forms of early
coordinated activity, such as retinal waves (Meister et al., 1991;
Feller et al., 1996), neuronal domains are coordinated by biochemical
rather than electrical activity.
Neuronal domains are generated by the intercellular diffusion of
inositol trisphosphate and the release of calcium from internal
stores
The results of this study indicate that neuronal domains in the
developing neocortex are caused by propagation of the intercellular diffusion of the second messenger IP3 rather than by the
intercellular spread of electrical signals or of calcium ions. This is
supported by the following observations. First, neuronal domains
persist when sodium- and calcium-dependent action potentials are
blocked with TTX (Fig. 1A; Yuste et al., 1995) and
nickel (Fig. 1B), arguing against propagation of
regenerative electrical signals as the responsible mechanism. Second,
neuronal domains are not elicited by single-cell depolarizations (Fig.
5B) and are not affected by blocking voltage-gated calcium
channels with high concentrations of nickel, indicating that neuronal
domains are not generated by passive electrotonic spread of
depolarizations and subsequent activation of voltage-gated calcium
channels. Third, neuronal domains are abolished after depletion of
intracellular calcium stores with thapsigargin, indicating that
neuronal domains depend on the release of calcium from internal stores.
Fourth, neuronal domains are elicited by increasing the intracellular
IP3 concentration, either by stimulation of metabotropic
glutamate receptors (Fig. 3A) or by intracellular
IP3 infusions (Fig. 5A), indicating that IP3 is sufficient for triggering neuronal domains. Finally,
neuronal domains are not elicited by increases in the intracellular
calcium concentration produced by either direct infusion of calcium or by activation of voltage-gated calcium channels (Fig. 5B),
indicating that calcium is neither the intracellular trigger nor the
signal that diffuses through gap junctions.
Intercellular second-messenger waves have been described previously in
a variety of non-neuronal systems (for review, see, e.g., Katz, 1995)
and between leech neurons (Wolszon et al., 1994). In these systems, the
propagating second-messenger molecule has been characterized as either
IP3 (for review, see Sanderson, 1995) or calcium (Wolszon
et al., 1994). The results of the present study suggest that
IP3, or perhaps one of its metabolites (for review,
see Berridge, 1993), acts as the intercellular propagating molecule
that causes a neuronal domain. According to our results, calcium acts
as the responding molecule downstream from IP3 because increasing intracellular IP3, but not calcium alone,
was sufficient to trigger neuronal domains (Fig. 5). Although one could
argue that the failure of calcium infusions to elicit neuronal domains might be the result of calcium-induced uncoupling (Baux et al., 1978;
Peracchia, 1978; Rao et al., 1987), such uncoupling does not occur with
calcium levels as they are achieved by calcium spikes (Yuste et al.,
1995) or by prolonged depolarizations, such as those routinely used for
biocytin injections to visualize dye coupling in these same cells
(Peinado et al., 1993a). Because single-cell depolarization also failed
to trigger neuronal domains (Fig. 5B), calcium ions alone
can neither trigger nor propagate cortical neuronal calcium waves.
Possible reasons for this could include permeation selectivity of
cortical neuronal gap junctions (for review, see Veenstra, 1996) or
differences in the diffusion properties of IP3 and
Ca2+ (for review, see Kasai and Peterson, 1994).
Neuronal domains are initiated by activation of metabotropic
glutamate receptors
Since the discovery of spontaneous neuronal domains, the
physiological signals responsible for their initiation have remained obscure. The results from the present study strongly suggest that the
major physiological trigger for neuronal domains is glutamate acting
via metabotropic glutamate receptors. Stimulation of mGluRs with the
agonist t-ACPD elicited neuronal domains (Fig. 3A), whereas blockade of mGluRs by the antagonist (+)-MCPG dramatically reduced the
number of neuronal domains (Fig. 4). The fact that (+)-MCPG did not
completely prevent the occurrence of all neuronal domains (Fig.
4A) may be attributed to an incomplete blockade of
all mGluRs or to the existence of additional trigger mechanisms such as
other neurotransmitter receptors capable of increasing intracellular IP3 concentrations (Kendall and Nahorski, 1987; Simpson et
al., 1995). A likely candidate for mediating glutamate-elicited
neuronal domains is the metabotropic glutamate receptor mGluR5, which
is highly expressed in early postnatal cortex and is coupled to the production of IP3 (Abe et al., 1992; Catania et al., 1994).
These receptors could then be activated either by ambient glutamate or
by glutamate spontaneously released from immature synapses (Lo Turco et
al., 1994; Kim et al., 1995).
Although neuronal domains can be readily observed in slices, whether
they also occur in vivo has been questioned, because a
physiologically plausible triggering mechanism was absent. Our finding
that glutamate, the major excitatory cortical neurotransmitter, can
initiate neuronal domains in slices, however, makes the presence of
neuronal domains in vivo more plausible.
Proposed model for the initiation and propagation of cortical
calcium waves
Based on our results, we propose the following model for the
initiation and propagation of cortical calcium waves (Fig.
6). Neuronal domains are initiated by
glutamate, probably released from developing synapses, which acts on
metabotropic glutamate receptors. These in turn are linked to
G-proteins that activate phospholipase C. Stimulation of this cascade
results in production of IP3 and the subsequent release of
calcium from intracellular IP3-sensitive stores. In
addition to its role of mediating this direct calcium response,
IP3, or one of its metabolites (Jia et al., 1995;
for review, see Berridge, 1993), most likely also acts as the
intercellular messenger that is responsible for intercellular wave
propagation. The propagation of calcium waves over many cells, without
significant decrement (Fig. 5A; Yuste et al., 1995), implies the involvement of regenerative mechanisms. Although the specific nature of these mechanisms remains to be determined for neuronal domains, calcium-induced increases in IP3 receptor
sensitivity (Bezprozvanny et al., 1991) and in phospholipase C activity
(Meyer, 1991) could provide the necessary positive feedback loops.
View larger version (38K):
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|
Figure 6.
Model for the initiation and propagation of
interneuronal calcium waves underlying neuronal domains. Waves are
initiated either by ambient or synaptically released glutamate
(Glu; open triangles) that acts on
metabotropic glutamate receptors (mGluR). This
stimulates the G-protein (G)-phospholipase C
(PLC) cascade that results in the production of inositol
trisphosphate (IP3; filled
circles). IP3 activates
IP3 receptors
(IP3R; small filled
ovals) and thereby causes calcium release (filled
squares) from intracellular stores. In addition,
IP3 also diffuses through gap junctions
(GJ) into neighboring neurons where it causes
calcium release. In coupled cells, IP3 could
be regenerated by calcium-mediated positive feedback loops
(dashed arrows), including the sensitization of
IP3R and stimulation of
PLC.
|
|
The cellular mechanisms of intercellular neuronal calcium waves in the
developing neocortex closely resemble mechanisms that have been
proposed for calcium waves in non-neuronal systems (for review, see
Sanderson, 1995). In this respect, young neurons, still at a stage when
they are sparsely connected by synapses (Miller, 1988), use mechanisms
typical of nonelectrically excitable cells to establish cellular
communication and coordinate behavior. Currently, there are only a few
studies that address the cellular mechanisms of gap junction-mediated
intercellular calcium waves between mammalian neurons, despite the
presence of extensive neuronal coupling early in development. Recently
Charles et al. (1996) proposed a model for intercellular calcium waves
between cultured cortical neurons and GT1-1-immortalized neurons. In
contrast to our model, their model proposes that neuronal calcium waves
propagate by the intercellular spread of membrane depolarizations and
the subsequent activation of voltage-gated Ca2+
channels and influx of extracellular calcium. However, despite some
similarities between calcium waves in slices and calcium waves in
cultured cells, such as propagation speed [~100 µm/sec in slices
(Yuste et al., 1995) and 100-200 µm/sec in cultures (Charles et al.,
1996)] or area of propagation (~3000 µm2 in
slices and 50-100 cells in culture), there also exist several basic
differences between these waves. Waves in cortical slices are initiated
by mGluRs, whereas those in cultures require mechanical stimulation.
Moreover, waves in slices are TTX-insensitive, whereas those in
cultures are TTX-sensitive. These fundamental differences indicate that
gap junctions between cortical neurons can support different types of
calcium waves depending on the environmental conditions. Because
coupling strength and type of expressed connexins are regulated by
factors such as neuromodulators (for review, see Bennett et al., 1991)
or injury (Gutnick et al., 1985; Rohlmann et al., 1994; Balice-Gordon
et al., 1996), the coupling situation in cultured cortical cells might
be quite different than that in acute brain slices.
Direct intercellular diffusion of second messengers: an alternative
route of neuronal communication
Since the discovery of electrical synapses by Furshpan and Potter
(1959) almost 40 years ago, neuronal gap junctions have primarily been
viewed as the basis of electrical synapses dedicated to electronically
coupling cells. However, electrical coupling between developing neurons
is generally very weak, raising doubts about their contribution to
neuronal synchronization (for review, see Katz, 1995; Kandler, 1997).
In contrast to the small changes in the electrical potential that such
coupling can elicit, we found that regenerative biochemical waves,
spreading over considerable distances, can be supported by such
apparently weak coupling. The direct exchange of IP3
between neurons provides another avenue, besides the conventional role
of electrical activity, by which neuronal behavior can be synchronized
among members of neuronal assemblies. Although our data do not exclude
the possibility that weak electrical communication between cortical
neurons (Connors et al., 1983; Lo Turco and Kriegstein, 1991; Peinado
et al., 1993b) coexists with biochemical communication, biochemical
communication seems to be the main functional route by which coupled
cortical neurons synchronize their behavior. Because neuronal coupling is most prominent before and during the major period of synapse formation (Connors et al., 1983; Lo Turco and Kriegstein, 1991; Peinado
et al., 1993a; Kim et al., 1995) and is inversely correlated with
synaptic activity in ferrets (Kandler and Katz, 1998), coordination of
biochemical activity across large cell assemblies is likely to
influence cortical development before or during early stages of synapse
formation but not after the emergence of bona fide circuits. Because of
the widespread effects of second messengers on essential cellular
processes such as neuronal differentiation and gene expression
(Spitzer, 1995; Finkbeiner and Greenberg, 1996), the direct control of
biochemical activity can coordinate a much wider range of cellular
functions than would be possible by the coordination of electrical
activity alone.
| |
FOOTNOTES |
Received Aug. 1, 1997; revised Oct. 15, 1997; accepted Dec. 2, 1997.
This research was supported by National Institutes of Health Grants
NS32396 to L.C.K. and EY06730 to K.K. and by postdoctoral grants to
K.K. from the Deutscher Akademischer Austauschdienst and the Alexander
von Humboldt Stiftung. We thank Don C. Lo, Tobias Meyer, and Tom Tucker
for critical comments on an earlier version of this manuscript and
Darin Nelson for developing data acquisition and analysis software.
Correspondence should be addressed to Dr. Karl Kandler, Department of
Neurobiology, Box 3209, Duke University Medical Center, Durham, NC
27710.
Dr. Kandler's present address: Department of Neurobiology, University
of Pittsburgh School of Medicine, E 1440 Biomedical Science Tower,
Pittsburgh, PA 15261.
| |
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Top
Abstract
Introduction
