1The Psychiatric Institute, Department of Psychiatry, The University of Illinois at Chicago, Chicago, Illinois 60612; and 2Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Mienville, Jean-Marc, Irina Maric, Dragan Maric, and John R. Clay. Loss of IA Expression and Increased Excitability in Postnatal Rat Cajal-Retzius Cells. J. Neurophysiol. 82: 1303-1310, 1999. Although an important secretory function of Cajal-Retzius (CR) cells has been discovered recently, the precise electrical status of these cells among other layer I neurons in particular and in cortical function in general is still unclear. In this paper, early postnatal CR cells from rat neocortex were found to express an inactivating K current whose molecular substrate is likely to be the Kv1.4 channel. Both electrophysiological and immunocytochemical experiments revealed that expression of this A-type current is down-regulated in vivo and virtually disappears by the end of the second postnatal week. At this time, CR cells have become capable of evoked repetitive firing, and their action potentials are larger and faster, yet these electrical properties still appear incompatible with a role in cortical network function, as inferred from comparisons with other cortical neurons. Also at this time, a large proportion of CR cells display spontaneous spiking activity, which suggests the possibility of additional roles for these cells. We conclude that the loss of A channels along with an increase in Na channel density shape the changes in excitability of postnatal CR cells, in terms of both the patterns of evoked firing and the emergence of spontaneous spiking.
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INTRODUCTION |
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Neocortical Cajal-Retzius (CR) cells represent a
near perfect system for the developmental neurobiologist, owing to a
relatively short life span and to reliable anatomic identifiers
(Hestrin and Armstrong 1996;
Marín-Padilla 1984
; Mienville and Pesold 1999
; Ramón y Cajal 1891
; Retzius
1893
), which may allow in extenso follow-ups of various
parameters involved in the function and fate of these cells.
Surprisingly, it is not until the past three years that papers on the
physiology of these cells have begun to appear (Hestrin and
Armstrong 1996
; Mienville 1998
; Mienville and Barker 1997
; Mienville and Pesold 1999
;
Schwartz et al. 1998
; Zhou and Hablitz
1996
). Emerging from these reports is a repertoire of voltage-
and ligand-gated ion channels that would make these cells akin to
"regular" cortical neurons, were it not for the notable immaturity
of some of their membrane electrical properties (Hestrin and
Armstrong 1996
; Mienville 1998
; Mienville
and Pesold 1999
). The possibility that CR cells constitute a
special type of glial cells has been raised in the past (see
König and Schachner 1981
; and
Marín-Padilla 1984
, for historical overviews).
Although such an idea would not be incompatible with the
above-mentioned repertoire of ion channels (Barres
1991
), other features of CR cells including the presence of an
axon, the expression of specific neuronal markers (König
and Schachner 1981
), and the ability to fire action potentials
(APs) (Hestrin and Armstrong 1996
; Zhou and
Hablitz 1996
) have definitively established their neuronal nature.
It is still unclear, however, whether CR cells participate in the same
functions, including rapid transfer and processing of information, and
are part of the same networks as those usually associated with cortical
neurons. In that line of thought, Zhou and Hablitz
(1996) did not detect any obvious difference in properties between postnatal CR cells and other layer I neurons, and
Ekstrand et al. (1996)
even proposed a role for CR cells
in fast feed-forward inhibition. Against such roles, however, is the
observation that 1) postnatal CR cells do not appear to have
synaptic inputs (König and Marty 1981
), consistent
with their lack of spontaneous synaptic activity (A. Kriegstein,
personal communication; J.-M. Mienville, personal observations), and
2) they disappear before full maturation of the cortex
(Del Río et al. 1995
; Mienville and
Pesold 1999
). Early on, Noback and Purpura
(1961)
expressed doubts as to a role of CR cells in electrical
signaling and conjectured that their complex morphology may involve
"some obscure functions." As it turns out, light recently has been
shed on such functions with the discovery that CR cells synthesize and
secrete a large protein, Reelin, crucial for neuronal migration and
proper cortical lamination (D'Arcangelo et al. 1995
).
Naturally, this per se does not exclude other roles for CR cells in
connection with their possible synaptic outputs (see
DISCUSSION). Meanwhile, one may ask the questions: is the
firing capacity of CR cells compatible with the fast signaling required
for higher cortical function? If not, could it be linked to Reelin
secretion? If not, what could be its purpose? These are some of the
questions addressed, from a developmental perspective, in the present
paper. In connection with the marked increase in the excitability of
late CR cells, we first provide anatomic and functional evidence for a
postnatal down-regulation of an inactivating K current likely to be
carried by Kv1.4 channels.
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METHODS |
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All experiments were conducted with Fisher rat pups at three developmental stages corresponding to postnatal day (P) 1, 5, and 12-13. Animals were handled in accordance with guidelines set out by the National Institutes of Health.
Electrophysiology
In situ patch-clamp, single-pipette methods were used in the
somatic whole cell and cell-attached recording configurations (Stuart et al. 1993). Brain slices were prepared, and CR
cells were identified as previously described (Mienville
1998
; Mienville and Barker 1997
;
Mienville and Pesold 1999
). Any given experiment was
performed on two to five slices from at least two litters. For
quantitative voltage-clamp experiments (i.e., Fig.
1), the extracellular solution was
exactly as given by Mienville and Barker (1997)
and was
designed to prevent Na and Ca currents. Pipette solution contained (in
mM) 146.7 KCl, 10 HEPES, and 3.3 KOH (pH 7.2). Evoked APs were studied
in whole cell current clamp, whereas spontaneous firing was studied
with cell-attached methods at a pipette potential of 0 mV; in both
cases, the extracellular solution was regular Ringer (e.g.,
Mienville 1998
). This solution was also used to fill
on-cell pipettes, while a K-gluconate-based solution (Mienville
1998
) was used for current-clamp experiments. During the
latter, voltage responses to depolarizing current increments were
obtained from a membrane potential of
80 mV maintained through constant DC injection. In this paper the term "suprathreshold" refers to a stimulus that evokes maximal AP frequency. APs were abolished by 1 µM tetrodotoxin (TTX; n = 9),
suggesting the primary involvement of Na channels in their generation.
Two cases displaying obvious "Ca shoulders" were eliminated from
the analysis.
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Signal amplification was through a List EPC-7 unit. Voltage- and current-clamp stimulation protocols, including P/4 leak subtraction, as well as data acquisition and analysis, were provided by pCLAMP 6 (Axon Instruments, Foster City, CA). On-cell activity was stored on videotape (Instrutech, Port Washington, NY), and spikes were later counted with the PAT module of SES software (courtesy of Dr. John Dempster) after low-pass filtering at 500 Hz to avoid spurious counts due to membrane noise.
The drug 4-aminopyridine (4-AP; Sigma, St. Louis, MO) was dissolved directly in the extracellular solution and applied through the bath perfusion, while TTX (Sigma) was applied locally. Results are given as means ± SE. Student's t-test and ANOVA were used when comparing two or more than two groups, respectively, unless otherwise noted.
Immunocytochemistry
Neonatal rats were anesthetized with CO2 and perfused intracardially with 5-10 ml of phosphate-buffered saline (PBS), followed by 5-10 ml of ice-cold fixative (4% paraformaldehyde in PBS). The brains were removed, left in fixative for 24 h, and embedded in 30% sucrose in PBS for 2-3 days. They were then frozen in liquid nitrogen-cooled isopentane and cut serially in 16-µm thick coronal sections. The sections were rinsed three times in PBS, preblocked for 2 h at room temperature with PBS containing 10% normal rat serum (NRS), 10% normal donkey serum (NDS), and 0.25% Triton X-100, and incubated overnight at room temperature in primary antibodies diluted in PBS/10%NRS/10%NDS. The primary antibodies were rabbit affinity-purified polyclonal anti-Kv1.4 antibody (Alomone Labs, Jerusalem, Israel) at a dilution of 1:50, rabbit affinity-purified polyclonal anti-Kv4.2C antibody (provided by Dr. James S. Trimmer, SUNY, Stony Brook, NY) at a dilution of 1:250, and mouse anti-reelin (G10, provided by Dr. André Goffinet, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium) at a dilution of 1:10,000. The sections were washed three times in PBS and reacted with appropriate biotin-conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA) for 2 h at room temperature. After three washings with PBS, the sections were incubated with streptavidin-conjugated horseradish peroxidase (Jackson ImmunoResearch Laboratory) for 1 h at room temperature. The immunostaining was visualized by developing the sections in 3-amino-9-ethylcarbazole (AEC; Sigma) substrate (25 mg AEC in 100 ml acetate buffer) containing 0.01% H2O2 for 5-15 min at room temperature. We checked for nonspecific staining by subjecting control sections to the above protocol, except for the omission of primaries from the dilution medium, and did not detect any staining in these sections. The results were viewed with transmission light microscopy (Zeiss Axiophot, Thornwood, NY).
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RESULTS |
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IA progressively disappears in postnatal CR cells
Our preceding work (Mienville and Barker 1997) had
shown an increase in IA functional
expression by embryonic CR cells. The present results on postnatal CR
cells demonstrate the inverse trend, i.e., a decrease in
IA functional expression (Fig. 1,
A-C). This decrease is manifested both in terms of the
proportion of cells expressing IA
(Fig. 1B), most P13 cells altogether lacking it (Fig.
1A, bottom traces), and in terms of current density in those
older cells that still express it (Fig. 1C; Table
1). Simultaneous analysis of the delayed
rectifier current indicates no change in maximum current density during
this particular period (Fig. 1D; Table 1). A close look at
the conductance-voltage (G-V) curves (Fig. 1, C
and D) reveals subtle, development-related differences among
the two other parameters, namely the potential of half-maximum activation, V1/2, and the steepness of
the curve, k (Table 1). These differences could be due to a
number of factors affecting channel function, including heteromeric
assembly of main (
) subunits, accessory (
) subunit expression, or
posttranslational modifications. These aspects are beyond the scope of
the present work and are not discussed further.
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To obtain information on the molecular identity of CR cell
IA, we performed immunocytochemical
experiments to investigate the expression of Kv1.4 and Kv4.2 proteins,
two inactivating K channels widely expressed in brain (Serodio
et al. 1994). Kv1.4 was found to be expressed by CR cells (Fig.
2) in all of four series of experiments.
On the contrary, we were never able, during three separate experiments,
to detect Kv4.2 in these cells, whereas this subunit clearly was
expressed in P13 hippocampus (data not shown). These results are
consistent with a previous study showing labeling of neocortex by Kv1.4
but not by Kv4.2, both channels being present in hippocampus
(Sheng et al. 1992
). Figure 2A shows Kv1.4
staining in P1 cortex. Staining is particularly intense on mostly
horizontally oriented cells, consistent with the CR cell phenotype as
also indicated by the presence of Reelin-immunopositive cells in sister
sections (right inset). Despite robust evidence for an
axonal localization of Kv1.4 in telencephalon (Sheng et al.
1992
; Song et al. 1998
), we find that the
strongest labeling occurs on the proximal dendritic swelling of CR
cells. In that respect, CR cells resemble dorsal cochlear nucleus
neurons in which Kv1.4 was also localized to dendrites and somata
(Juiz et al. 1996
). At P5, Kv1.4 staining is still
substantial in layer I (Fig. 2B), but labeling of CR cells
appears somewhat fainter (inset). At P13, Kv1.4 staining is
practically absent from layer I (Fig. 2C). Rare CR cells
still present at this stage (bottom inset) (see also
Mienville and Pesold 1999
) show very faint staining for
Kv1.4 antibody (top inset), whereas layer II/III cells are distinctly labeled. Concerning the latter, the limited resolution of
light microscopy does not permit conclusive localization of Kv1.4, but
the label surrounding cell bodies is not incompatible with axon
terminal staining, as seen in globus pallidus neurons (Song et
al. 1998
).
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Firing properties switch from single to repetitive during postnatal development
Our primary goal here was to investigate possible relationships
between developmental changes in IA
expression and CR cell excitability. A striking result of this inquiry
was the fundamental differences observed in terms of 1) the
maximum number of APs triggered by a 250-ms pulse (Fig.
3), and 2) the
waveform pattern of individual APs (Fig.
4). We will first address the
first set of differences. Figure 3A depicts
characteristic observations made at P1 and
P13. At P1, 9/12 (75%) cells fired a
single AP on suprathreshold stimulation, whereas at
P12-13 only 1/16 (6%) cell fired a single AP. Intermediate
proportions (7/17 cells; 41%) were noted at P5.
The mean number of APs observed at each stage is reported in
Fig. 3B. The maximum number attained was 7, which
corresponds to a frequency of 28 Hz. This is well below sustained
frequencies observed in both mature and immature pyramidal neurons,
which may range up to 50-100 Hz (McCormick and Prince 1987), and over an order of magnitude lower than those recorded in mature GABAergic interneurons, which may reach 600 Hz
(Connors and Gutnick 1990
; McCormick et al.
1985
).
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In an attempt to correlate AP number with
IA expression, we coupled each
current-clamp recording to a voltage-clamp recording consisting of a
single pulse to +40 mV preceded by a prepulse to 120 mV. Note that
+40 mV is reasonably close to ENa, so
that contamination of IA by
INa was probably minimal. Indeed, the
resulting traces closely resembled, in their development-related
pattern, unsubtracted traces obtained in conditions of K current
isolation (e.g., those displayed on the left of Fig. 1A).
The ratio peak/steady-state current (P/SS) was thus used to provide an
index of IA expression. Although a
significant relationship was found at P5 between this ratio
and AP number (Fig. 3C), no significant correlation was found at any other stage (not shown). This would make sense if the
maturation of P5 cells showed greater variability (the
so-called "range of talent") for both parameters than P1
or P12-13 cells, for which correlations are expected to be
"noisier."
APs mature in postnatal CR cells but fail to acquire properties of adult cortical neurons
The next set of analyses examined changes in various parameters of
individual APs during postnatal development of CR cells. As previously
noted by Zhou and Hablitz (1996), APs increased in
amplitude and decreased in duration (Fig. 4, A and
B) in a manner consistent with an increase in Na channel
density. From P1 to P12-13, amplitude as well as
depolarization and repolarization rates increased (Fig. 4A),
the latter two parameters apparently more so than AP amplitude because
half-amplitude width as well as rise and fall times decreased (Fig.
4B). Zhou and Hablitz (1996)
proposed the
conclusion that layer I neurons, including CR cells, eventually acquire
mature APs. Unless layer I differs widely from other cortical layers
with respect to the mature properties of its constituents, we would
have to disagree on the conclusion concerning CR cells. In our hands,
the various AP parameters of CR cells were substantially different from
those published for mature, nonlayer I cortical neurons. For example,
McCormick et al. (1985)
and McCormick and Prince
(1987)
reported values in the range 80-100 mV for AP
amplitude, 300-400 V/s for rate of rise, and 0.3-0.8 ms for
half-amplitude width. Although their work was performed at 35-37°C,
these values may be contrasted with those of P12-13 CR
cells reported in Fig. 4, A and B. Interestingly, in the study of Hestrin and Armstrong (1996)
, the mean
half-width for late postnatal CR cells (3.9 ms) was substantially
larger than that for other layer I neurons (1.2-1.7 ms).
In view of likely relationships between repetitive firing and Na
channel density (Lockery and Spitzer 1992), we tested
for correlations between AP number and waveform parameters related to
Na channel density. Significant correlations were only found with AP
amplitude (Fig. 4C). This would be consistent with AP amplitude being a more accurate predictor of Na channel density than
parameters involving time, which may also depend on distribution (e.g.,
somatic vs. neuritic).
Effects of 4-AP
To directly test the involvement of
IA in the firing pattern of CR cells,
we attempted to induce repetitive firing in P1 cells by
blocking IA with 4-AP. Addition of 5 mM 4-AP to the bath perfusion virtually abolished
IA, as illustrated in Fig.
5A. Compared with a value of
2.50 ± 0.25 (mean ± SE) noted in control P1
cells, the P/SS ratio was 1.59 ± 0.06 in 4-AP, similar to the
1.55 ± 0.07 value for P12-13 CR cells, suggesting
some degree of delayed rectifier inactivation in CR cells (Fig.
1A) (Mienville and Barker 1997). Elimination
of IA did not induce repetitive firing
behavior (Fig. 5B), as neither the proportion of cells
firing single spikes (25/31; 81%) nor mean AP number (1.5 ± 0.2)
were different from control (P = 0.60; cf. results
above). On the other hand, 4-AP did have substantial effects on
individual AP parameters (Fig. 5, C and D).
Specifically, amplitude, rise time and half-amplitude width were all
increased. Because the rate of rise did not change, the increase in
rise time and half-width appears as a mere consequence of the increase
in amplitude. Ribera and Spitzer (1990)
also observed an
increase in spike overshoot on application of 4-AP. The increase in
fall time was not significant (P = 0.11) despite the
apparent lack of change in fall rate. This might be due to insufficient A/D resolution for fall rate with respect to sampling interval (software measures maximum dV/dt between 2 sampling points), as suggested by the very small SE of fall rates
(Figs. 4A and 5C). These results nevertheless
point to a lack of effect of IA on AP
repolarization (see Fig. 5B).
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Because there is no reason to suspect that 4-AP potentiates
INa, it seems likely that one effect
of IA is to dampen AP amplitude, owing
to its fast activation partially overlapping with that of INa. (Thus the developmental increase
in AP amplitude may be related in part to the loss of
IA.) This raised the possibility that
IA may interfere with AP threshold. In
cultured neurons, it was shown that IA
increases both the threshold potential and rheobase for AP generation
(Segal et al. 1984). The effect on threshold might be
due to simultaneous and mutually antagonistic activations of IA and
INa (compare threshold potentials for
IA and AP in Figs. 1C and
6), whereas the effect on rheobase might stem from
IA's shunting conductance. Focusing
on the former aspect, we found that the threshold potential for AP
generation was significantly lower in P12-13 CR cells than
in both P1 or P5 cells, yet it failed to change
in P1 cells exposed to 4-AP (Fig.
6). This might be due to the tight
dependence of threshold on INa
density, which may hinder detection of
IA's effects.
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Spontaneous firing occurs late in the development of CR cells
We have extended our initial observation of a lack of spontaneous
firing in embryonic CR cells (Mienville 1998) to the
perinatal period: spontaneous firing never occurred in P1
cells; it occurred in only 12/62 (19%) P5 cells, and in as
many as 19/30 (63%) P12 cells (Fig.
7A). Consistent with these
results, Schwartz et al. (1998)
counted an average of
15% P1-8 CR cells displaying spontaneous Ca transients.
Spontaneous firing frequencies were similar in P5 and
P12 cells (Fig. 7B), and were well below
maximally sustainable rates (see above).
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DISCUSSION |
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Our previous work (Mienville and Barker 1997)
showed an increased contribution of IA
to the total K current evoked in embryonic day
(E) 21 versus E18 CR cells. The
present paper shows that this trend is reversed in postnatal CR cells,
with a perinatal peak of IA expression
subsequently falling down to a virtual loss at a time when few of these
cells are remaining in cortex (Mienville and Pesold
1999
). Such a decline is unlikely to reflect ongoing cell
degeneration (Del Río et al. 1995
) because the
delayed rectifier appears to remain functional, and other channels are
even up-regulated in late CR cells (Mienville 1998
;
Mienville and Pesold 1999
). Similar inverted-U patterns
of K current expression have been described in nonmammalian muscle
cells (Ribera and Spitzer 1991
; Shidara and
Okamura 1991
).
Our immunocytochemical data strongly suggest that Kv1.4 is the
molecular substrate of CR cell IA.
First, we found that Kv1.4 antibody consistently stained CR cells;
second, staining intensity seemed to closely follow the time course of
IA expression. Third, Kv4.2 protein
was never expressed by CR cells. Nevertheless, we cannot exclude that
other IA-producing subunits, such as
Kv4.1 (Serodio et al. 1994; Song et al.
1998
), contribute to CR cell IA; this would imply that different
subunits can be down-regulated simultaneously, which is not
unprecedented (Tsaur et al. 1992
). In rat hippocampus,
Kv1.4 expression does not occur until P5 and subsequently
increases toward adult intensities (Maletic-Savatic et al.
1995
), which, taking our results into account, suggests varying
spatiotemporal regulations of Kv1.4 in brain. At present, the
mechanisms underlying such regulations can only be speculative, although promising leads already have been explored, as in the case of
the down-regulation of Kv4.2 in hippocampus (Tsaur et al.
1992
). CR cells thus may provide a unique mammalian model for
future studies of the mechanisms involved in the differential regulation of ion channel expression.
The observed loss of IA expression has
prompted us to investigate possible consequences on the excitability of
CR cells. In view of the complexity deriving from the simultaneous (and
opposite) changes in IA and
INa densities during CR cell
development, we have initiated computer simulations based in part on a
Goldman-Hodgkin-Katz formalism for K channel I-V curves
(Clay 1998). Preliminary results (Mienville et
al. 1999
) indicate that up-regulation of
INa and downregulation of
IA during the P1-P13
period are both required for repetitive firing to occur. Ribera
and Spitzer (1990)
observed the reverse phenomenon in
developing amphibian neurons, in which IA's late emergence coincides with a
switch from repetitive to single AP firing. Simulation studies
performed in the same laboratory (Lockery and Spitzer
1992
) also established that such a switch requires simultaneous
manipulation of both IA and
INa. These results can explain most of
our present data, including the correlations between AP number and P/SS
ratio (Fig. 3C) or AP amplitude (Fig. 4C), as
well as our failure to induce repetitive firing with 4-AP due to
inadequate INa density at
P1. Even more interestingly, our simulations predict the
late emergence of spontaneous firing at the relatively low resting
potential of CR cells (Mienville and Pesold 1999
).
An important observation is that the broad, low-amplitude APs and the
slow firing rate of CR cells do not seem adequate for fast information
processing in the cortex (Connors and Gutnick 1990;
McCormick et al. 1985
). What then could be the purpose
of this firing? One naturally may be tempted, given the secretory role
of CR cells, to infer that APs constitute stimuli for Reelin exocytosis. However, this is highly unlikely because during the perinatal migratory period, when Reelin would be expected to be most
actively involved, CR cells not only are essentially silent, but also
seem unable of sustained firing. Given the fact that Reelin is related
to extracellular matrix proteins (D'Arcangelo et al.
1995
), it rather is likely that its secretion is constitutive, i.e., regulated at the synthetic level (Alberts et al.
1994
). This view seems confirmed by antisense experiments
currently in progress in our laboratory (Lacor et al.
1999
). These considerations do not diminish our interest in the
spontaneous activity of CR cells. The latter also appear capable of
synthesizing neurotransmitters (Del Río et al.
1995
; Imamoto et al. 1994
) and of making
axodendritic contacts with cortical cells (Del Río et
al. 1995
; Marín-Padilla 1984
). Thus in
addition to their initial role in chemically driven migration, CR cells
subsequently might become instrumental in activity-dependent circuit
formation (Katz and Shatz 1996
). Particularly appealing
in this scenario is the fact that it would fit the tight dependence
between ion channel expression, spontaneous activity, and network
maturation (see Moody 1998
, for review). In Ascidian embryos, for example, the simultaneous disappearance of an inward rectifier K current and appearance of a high-threshold Ca current initiate spontaneous activity, which in turn allows expression of
another ion channel species necessary for mature signaling (Moody 1998
). In the case of CR cells, it is the
simultaneous increase in INa and
disappearance of IA that would be
responsible for the emergence of spontaneous activity. Among the issues
that remain to be resolved are the regulation of
IA expression, the mechanisms that set
firing frequency, and the possible consequences of CR cell spontaneous
activity on the maturation of cortical circuits.
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ACKNOWLEDGMENTS |
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We are grateful to Drs. James Trimmer and André Goffinet for the generous gift of antibodies, W. S. Liu and S. Thomas for help with tissue fixation and staining, Dr. Christine Pesold for helpful advice, and Drs. John Larson and Erminio Costa for critical reading of the manuscript.
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FOOTNOTES |
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Address for reprint requests: J.-M. Mienville, Dept. of Psychiatry, m/c 912, The Psychiatric Institute, The University of Illinois at Chicago, 1601 W. Taylor St., Chicago, IL 60612.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 March 1999; accepted in final form 11 May 1999.
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REFERENCES |
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