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Volume 16, Number 9,
Issue of May 1, 1996
pp. 2956-2971
Copyright ©1996 Society for Neuroscience
Regulation of  7 Nicotinic Acetylcholine Receptors
in the Developing Rat Somatosensory Cortex by Thalamocortical
Afferents
Ron S. Broide1,
Richard T. Robertson2, and
Frances M. Leslie1
Departments of 1 Pharmacology and 2 Anatomy
and Neurobiology, College of Medicine, University of California,
Irvine, California 92717
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Distributions of  7 nicotinic acetylcholine
receptor (nAChR) mRNA and
[125I]-bungarotoxin (-BTX) binding sites
in the developing rat somatosensory cortex were characterized in
relation to acetylcholinesterase (AChE) histochemical staining of
thalamocortical terminals to investigate the role of this receptor in
cortical development. Using quantitative in situ
hybridization and receptor autoradiography, elevated levels of mRNA and
binding-site expression were first detected at postnatal day 1 (P1) in
deep and superficial layers, just beneath the AChE-stained
thalamocortical terminals. Onset of expression occurred ~1 d after
ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and
binding-site expression exhibited a disjunctive, barrel-like pattern in
layer IV and, more clearly, in layer VI. The mRNA and binding-site
expressions peaked at ~1 week postnatal and then declined to adult
levels. Unilateral electrolytic or cytochemical lesions placed in the
thalamic ventrobasal complex at P0 (just as thalamocortical afferents
are innervating the cortex) and at P6 (when the somatotopic map is well
established) resulted in a marked reduction of
7 nAChR mRNA and
[125I]-BTX binding-site levels in layers IV
and VI, indicating their regulation by thalamocortical afferents. With
P6 lesions, this reduction was observed as early as 6 hr postlesion.
These results suggest that 7 nAChRs are
localized primarily on cortical cells in rat somatosensory cortex and
provide further evidence for thalamocortical influence on cortical
ontogeny. These data also suggest a role for cholinergic systems during
a critical period of cortical synaptogenesis.
Key words:
nicotinic;
bungarotoxin;
cholinergic;
thalamocortical;
barrels;
cortical development
INTRODUCTION
Considerable attention has been focused on how
regions of the neocortex, particularly primary sensory regions, acquire
their unique characteristics (Rakic, 1988 ; O'Leary, 1989). In the
rodent, one region of the neocortex that has been studied extensively
in relation to this issue is the primary somatosensory cortex (S1).
This region contains discrete aggregates of neurons in layer IV known
as ``barrels'' (Woolsey and Van der Loos, 1970), which reflect the
pattern of vibrissae on the rodent's face (Killackey and Leshin,
1975). These barrels are innervated by clusters of ventrobasal (VB)
thalamic afferents arranged in a somatotopic pattern. Much evidence
suggests that early S1 differentiation is strongly influenced by these
afferents (for review, see O'Leary et al., 1994).
Numerous studies have analyzed both pre- and postsynaptic markers to
determine the earliest appearance of barrel patterns in the S1
(Robertson, 1987; McCandlish et al., 1989; Rhoades et al., 1990;
Jhaveri et al., 1991; Paysan et al., 1994). Such studies have
demonstrated a periphery-related pattern in the S1 as early as
postnatal day 1 (P1). One recent study, examining acetylcholinesterase
(AChE) labeling of the developing thalamocortical projection (Schlaggar
and O'Leary, 1994), has reported a somatotopic pattern of thalamic
terminals in the S1 by birth. This early somatotopic pattern suggests
that thalamocortical afferents convey the patterning information to
developing S1.
Fuchs (1989) has demonstrated that
[125I]-bungarotoxin (-BTX) binding
exhibits a transient pattern of expression within developing rat
sensory cortex. In the S1, this unique columnar pattern is associated
with the whisker barrel field in layer IV, but shows a more intense
barrel-like expression in layer VI. Several studies have suggested
that, in the rat CNS, -BTX binds to receptors composed of
7 nicotinic acetylcholine receptor (nAChR)
subunits (Alkondon and Albuquerque, 1993; Seguela et al., 1993). We
have recently shown a corresponding transient pattern of
7 nAChR mRNA expression in developing S1,
which also exhibits a more intense pattern in layer VI (Broide et al.,
1995). This transient pattern of nAChR expression in deeper S1 laminae,
coinciding with the period of thalamocortical ingrowth, suggests an
intrinsic organization within the cortex that either helps guide
innervating afferents or is induced by them.
To address this issue further, we have used quantitative in
situ hybridization and receptor autoradiography to study the
developmental expression of 7 nAChR mRNA and
[125I]-BTX binding sites in relation to
AChE-labeled thalamic terminals in alternate sections from the same
brain. We find that thalamocortical afferents appear before the
transient pattern of 7 nAChR distribution. In
addition, by interrupting this pathway both at the time that
thalamocortical afferents are beginning to innervate the cortex and
during a period when the somatotopic map has been well established, we
demonstrate a dynamic regulation of 7 nAChR
mRNA and protein levels by thalamocortical afferents. This study
provides further evidence for thalamocortical influence on cortical
development and suggests a possible role for the cholinergic system
during this period of synaptogenesis.
MATERIALS AND METHODS
All chemicals were obtained from Sigma (St. Louis, MO) unless
otherwise mentioned.
Animals. Timed pregnant Sprague-Dawley rats (Simonsen,
Gilroy, CA) were used for this study. Animals were mated during a 2 hr
period, and a sperm-positive vaginal smear started embryonic day 0 (E0). Rat pups were born early on E22, and the first 24 hr period after
birth was termed P0. Rats aged P0, P1, P2, P3, P5, P7, P10
(n = 6-8 per age), P15, and P70 (n = 4 per age)
were used for studying the postnatal time course of
7 receptor pattern formation. Pups aged P0
were used for young animal lesion studies and allowed to survive for 5 d postlesion (n = 5), whereas pups aged P6 were used for
older animal lesion studies and allowed to survive for periods varying
from 6 hr to 4 d postlesion (n = 3-4 per time point).
VB thalamic lesions. Rat pups aged P0 were anesthetized by
hypothermia, whereas those aged P6 were anesthetized with Metofane
(Pitman-Moore, Mundelein, IL). Unilateral lesions of the VB thalamic
complex were made using the following coordinates: an
anterior-posterior position half the distance between bregma and
lambda sutures. For P0 rat pups, 1.5 mm lateral from the medial suture
and 3.0 mm ventral from the pial surface. For P6 pups, 2.1-2.3 mm
lateral from the medial suture and 3.5-3.7 mm ventral from the pial
surface. In 5 P0 animals and 20 P6 animals, the tip of a stainless
steel electrode was placed in the VB complex. Electrolytic lesions were
made by passing a 0.9 mA positive current for 20 sec. In four P6
animals, the VB received lesions made by injection of 0.25-0.5 µl of
50 mM NMDA. This cytotoxic agent was used because
its receptor is one of the earliest glutamatergic receptor subtypes
that is expressed in the developing rat thalamus (Laurie and Seeburg,
1994; Monyer et al., 1994). These injections were made using a glass
micropipette with a 100 µm beveled tip attached to a 10 µl
microsyringe. Animals were sutured and allowed to recover from the
anesthetic before being returned to the dam.
Tissue preparation. Rats were decapitated and their brains
were quickly removed. Cerebral cortices were separated from the
brainstem, flattened between two microscope slides, and then frozen by
immersion in isopentane at a temperature below  20°C. These
flattened cortices were sectioned tangentially. A tissue block (which
included the thalamus) from lesioned brains was frozen and sectioned
for lesion verification. In some cases, whole brains were frozen in
isopentane at 20°C for 30 sec for cutting in the transverse plane.
Brain tissue was stored at 70°C until use. Twenty micrometer tissue
sections were cryostat cut and mounted onto either gelatin-coated
slides (for [125I]-BTX binding, AChE
histochemistry, or Nissl staining) or slides with an additional coating
of poly-L-lysine (for in situ
hybridization) kept at 20°C. Slide-mounted sections for
[125I]-BTX binding were stored desiccated at
4°C for 2 hr and then stored at 20°C until use. Sections for
in situ hybridization and AChE staining were post-fixed with
4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 1 hr at 22°C. Sections were then washed in PBS and air dried. Tissue
sections for AChE staining were processed immediately, whereas those
for in situ hybridization were stored desiccated at 20°C
until use. Slide-mounted tissue sections for Nissl staining were
post-fixed with 10% buffered formalin and stained with cresyl
violet.
Probe preparation. A 2.1 kb cDNA encoding the entire rat
7 nAChR subunit (Genbank accession number
M85273[GenBank]) cloned into pBluescript II SK+ was
obtained from Dr. Jim Boulter at the Salk Institute, San Diego, CA
(Broide et al., 1995). 35S-labeled uridine
triphosphate (UTP; Dupont NEN, Boston, MA) was used in synthesizing
cRNA riboprobes for in situ hybridization. These probes were
further subjected to alkaline hydrolysis using the method of Cox et al.
(1984) to yield products with average sizes of 600 bases.
In situ hybridization. Tissue sections were processed
for in situ hybridization according to a modification of the
method described by Simmons et al. (1989). Briefly, sections were
preincubated with 0.1 µg/ml proteinase K (Boehringer Mannheim,
Indianapolis, IN) for 10 min at 22°C, acetylated, dehydrated through
graded ethanols, and then air dried. Slide-mounted sections were
incubated for 18 hr at 60°C with a hybridization solution [50%
formamide (Fluka BioChemika, Ronkonkoma, NY), 10% dextran sulfate
(Pharmacia, Piscataway, NJ), 0.02% Ficoll, 0.02% polyvinyl
pyrolidone, 0.02% bovine serum albumin, 500 µg/ml tRNA, 10 mM dithiothreitol (Boehringer Mannheim), 0.3 M NaCl, 10 mM Tris, pH 8.0, and 1 mM EDTA, pH 8.0] containing cRNA probes
labeled with [35S]UTP (Dupont NEN) (1 × 107
cpm/ml) in the antisense orientation. Sections were then incubated with
RNase A (20 µg/ml) for 30 min at 37°C, followed by 4 × 5 min
high-stringency washes of decreasing salinity (2-0.5× SSC buffer) at
22°C, and a 30 min wash in 0.1× SSC at 70°C. Tissue sections were
dehydrated, dried in a stream of cool air, and apposed to  -max film
(Amersham, Arlington Heights, IL) for 3 d at 4°C.
[125I]-BTX binding. Slide-mounted sections
for [125I]-BTX binding were processed
according to a modification of the method described by Fuchs (1989).
Cortical sections were preincubated in a solution containing 120 mM NaCl and 50 mM Tris-HCl,
pH 7.4, for 15 min at 22°C. Slides were then incubated at 22°C for
2 hr in the same buffer containing 5 nM
[125I]-BTX (specific activity >200 Ci/mmol,
Amersham). Sections were washed at 4°C in two 10 min buffer rinses
followed by 30 sec in distilled water. Tissue sections were dried in a
stream of cool air, stored desiccated for 1 d at 22°C, and then
apposed to radiation-sensitive Hyperfilm (Amersham) for 4 d.
AChE staining. AChE histochemistry was performed according
to a modification of the method of Hedreen et al. (1985). Tissue
sections were incubated at 22°C for 10 d in a solution containing
0.132% acetylthiocholine iodide, 50 mM sodium
acetate, 0.057 mM
tetraisopropylpyrophosphoramide, 2.3 mM copper
sulfate, and 2.3 mM copper glycine. Reaction
product was then developed by a 15 sec incubation in 1% ammonium
sulfide (Fisher Scientific, Pittsburgh, PA).
Data analysis. Autoradiograms were quantified with a
computer-based image analysis system (MCID, Imaging Research, St.
Catherine, Ontario, Canada) using calibrated standards of reference. A
calibration curve of optical density against radioligand concentration
(either dpm/mg tissue for in situ hybridization or fmol/mg
tissue for binding films) was constructed using
[14C] brain paste standards of known
radioactivity. The curve was calibrated for reading
[125I] emissions, as described by Miller and
Zahniser (1987). Optical densities in discrete regions of cortical
autoradiographic images were measured, and corresponding values of
radioactivity were determined by interpolation from the standard curve.
The specific activities of cRNA probes used in this study were not
determined; therefore, these concentration measurements do not
represent the absolute levels of mRNA in the tissue.
Density measurements from the primary barrel cortex were obtained by
drawing a border around barrel rows A-E (Woolsey and Van der Loos,
1970). When measuring for partial lesions, only the area affected by
the lesion, as evaluated from adjacent AChE-stained sections, was
measured. At ages of P5 and older, tangential sections through layer IV
were identified as those adjacent to AChE-stained sections showing the
barrel-field pattern. Sections through layer VI were identified as
those showing the highest density of mRNA and binding levels within the
deeper cortical laminae (Broide et al., 1995) and just superficial to
the white matter. Other brain regions were identified using the atlas
of Paxinos and Watson (1986).
Because cerebral cortex is very thin, tangential tissue sections were
conserved for data analysis. Sections were not taken for incubation
with sense-oriented riboprobes or with unlabeled competitive drugs for
nonspecific binding assessment. Instead, measurements were taken from
the perirhinal cortex, adjacent to S1 and auditory cortex, as an
internal control in assessing and subtracting background labeling
within each experiment. All data were examined by two-way and one-way
ANOVA, followed by Newman-Keuls post hoc comparisons.
RESULTS
Emergence of 7 nAChR pattern in the S1
Transverse sections at the level of the caudate putamen revealed
an emerging laminar pattern of 7 nAChR mRNA
and [125I]-BTX binding-site distribution in
the developing S1 (Fig. 1). Two cortical layers
exhibited low-to-moderate mRNA and binding-site expression in all
cortical regions throughout development. For 7
nAChR mRNA, these corresponded to the subplate and cell-dense cortical
plate, whereas [125I]-BTX binding-site
expression was observed in the subplate and marginal zone, or layer I. These layers, which are present throughout cortical maturation,
exhibited a homogeneous expression of mRNA and binding sites when
viewed in the tangential plane (data not shown).
Fig. 1.
Distributions of AChE-labeled thalamocortical
afferents, 7 nAChR mRNA, and
[125I]-BTX binding sites in the developing
S1. Autoradiographic images (7
mRNA and -BTX binding sites) and bright-field
photomicrographs (AChE and Nissl) of adjacent
series of transverse sections through somatosensory cortex of rats at
P1 (A-D), P3 (E-H), P5 (I-L), and
P10 (M-P). Two clusters of AChE-labeled thalamocortical
terminals are indicated at P1 with small arrows.
Arrowheads mark the emerging layers that show a transient
distribution of mRNA and binding sites. Positions of cortical layers
are indicated on Nissl-stained sections. SP, Subplate;
CP, cortical plate. Scale bar, 300 µm.
[View Larger Version of this Image (133K GIF file)]
The focus of the present investigation, however, was on the
emerging cortical layers between the subplate and the cell-dense
cortical plate, which exhibited higher mRNA and binding-site expression
in the S1 than in adjacent cortical regions during development. The
complete laminar pattern was observed at ~1 week postnatal. An
analysis of individual cortical laminae in the tangential plane also
showed the emergence of a somatotopic pattern of mRNA and binding-site
expression at corresponding ages (Figs. 2, 3, 4, 5).
Fig. 2.
7 nAChR mRNA and
[125I]-BTX binding-site distribution in the
primary barrel region of superficial and deep laminae of P1 cortex.
Tangential sections through the cortex at the level of superficial
(A, C, E) and deep (B, D, F) laminae, showing
AChE staining (A, B), 7 mRNA
(C, D), and [125I]-BTX
binding-site (E, F) distributions. The primary barrel region
is indicated within arrowheads. CPu, Caudate
putamen; PRh, perirhinal cortex. Scale bar, 1 mm.
[View Larger Version of this Image (197K GIF file)]
Fig. 3.
Delineation of the primary barrel region in
superficial and deep laminae of P3 cortex by 7
nAChR mRNA and [125I]-BTX binding-site
expression. Tangential sections through the cortex at the level of
superficial (A, C, E) and deep (B, D, F) laminae,
showing AChE staining (A, B), 7
nAChR mRNA (C, D), and [125I]-BTX
binding-site (E, F) distributions. The primary barrel region
(area within arrowheads) and the five barrel rows
(a-e) are indicated. CPu, Caudate putamen;
PRh, perirhinal cortex. Scale bar, 1 mm.
[View Larger Version of this Image (200K GIF file)]
Fig. 4.
Delineation of the primary barrel region in
superficial and deep laminae of P5 cortex by 7
nAChR mRNA and [125I]-BTX binding-site
expression. Tangential sections through the cortex at the level of
superficial (A, C, E) and deep (B, D, F) laminae,
showing AChE staining (A, B), 7
nAChR mRNA (C, D), and [125I]-BTX
binding-site (E, F) distributions. The primary barrel region
(area within arrowheads) and the five barrel rows
(a-e) are indicated. A1, Auditory cortex;
CPu, caudate putamen; PRh, perirhinal cortex.
Scale bar, 1 mm.
[View Larger Version of this Image (197K GIF file)]
Fig. 5.
Delineation of the primary barrel region in
superficial and deep laminae of P10 cortex by
7 nAChR mRNA and
[125I]-BTX binding-site expression.
Tangential sections through the cortex at the level of superficial
(A, C, E) and deep (B, D, F) laminae, showing
AChE staining (A, B), 7 nAChR mRNA
(C, D), and [125I]-BTX
binding-site (E, F) distributions. The primary barrel region
(area within arrowheads) and the five barrel rows
(a-e) are indicated. A1, Auditory cortex;
CPu, caudate putamen; PRh, perirhinal cortex;
V1, visual cortex. Scale bar, 1 mm.
[View Larger Version of this Image (198K GIF file)]
Postnatal day 0
Transverse sections at P0 revealed AChE-stained thalamocortical
terminals at the base of the cortical plate (data not shown). When
viewed in the tangential plane, the pattern of these AChE-stained
terminals within the S1 appeared disjunctive. In some littermates,
higher levels of 7 nAChR mRNA and
[125I]-BTX binding-site expression were
observed in the primary barrel region of the S1 than in adjacent
cortical regions (data not shown). However, these patches of higher
mRNA and binding expression were not consistently seen in all animals
at this age.
Postnatal day 1
At P1, the developing neocortex is comprised of layers VI and V
(Fig. 1D), with layer IV neurons starting to aggregate at
the bottom of the cell-dense cortical plate (Ignacio et al., 1995).
Transverse sections at this age revealed AChE-positive thalamocortical
afferents localized at the base of the cortical plate (Fig.
1A). When viewed in the tangential plane, this AChE staining
exhibited a disjunctive pattern (Fig. 2A), as previously
shown for this age (Schlaggar and O'Leary, 1994). This disjunctive
pattern of AChE staining was only observed in superficial layers of the
cortex, presumably at the level of the cortical plate, and was not seen
in sections of deeper cortical laminae (Fig. 2B).
P1 was the earliest age at which levels of 7
nAChR mRNA and [125I]-BTX binding-site
expression could consistently be detected as higher in the S1 than in
adjacent cortical regions. This elevated expression of mRNA and binding
was observed clearly within two layers (Fig. 1B,C). The
laminar boundaries of this S1-specific expression were better defined
by mRNA, whereas binding-site expression appeared more evenly
distributed across both layers. When compared with the adjacent
Nissl-stained section (Fig. 1D), the superficial expression
was localized to layer V, just below AChE-stained thalamocortical
afferents (Fig. 1A), whereas the deeper expression was
localized to upper layer VI (Fig. 1B,C).
In the tangential plane, an emerging pattern of
7 nAChR mRNA and
[125I]-BTX binding-site expression,
corresponding to the primary barrel region (Fig. 2C,E), was
detectable in laminae just ventral to the AChE staining pattern (Fig.
2A). However, more extensive somatotopic patterns of mRNA
and binding-site expression were seen in sections through deeper
cortical laminae (Fig. 2D,F), which did not exhibit
somatotopic AChE staining (Fig. 2B). Although the
superficial patterns of mRNA and binding sites at this age were in
layer V (Fig. 1A-D), the deeper patterns were at the level
of layer VI, as indicated by the presence of large areas of the caudate
(Fig. 2D,F) (Broide et al., 1995). At this age, both the
mRNA and binding patterns were observed as patches with no apparent
further differentiation (Fig. 2C-F).
Postnatal day 3
By P3, layer IV has differentiated from the cortical plate (Fig.
1H). At this age, AChE-stained thalamic terminals were found
distributed mainly in layer IV, with some staining extending into the
cortical plate (Fig. 1E). The layer IV staining exhibited a
distinct barrel pattern when viewed in the tangential plane (Fig.
3A). This somatotopic labeling was not
visible in deeper cortical sections. Instead, a pattern of slightly
negative AChE staining was often observed delineating the primary
barrel region within these deeper layers (Fig. 3B).
At this age, disjunctive patterns of 7 nAChR
mRNA and [125I]-BTX binding-site
distribution were visible in the primary barrel region. In the
transverse plane, laminar patterns of mRNA and binding-site
distribution were observed similar to those seen at P1 (Fig.
1F,G), with highest levels in layer V and upper layer VI.
Although AChE-stained thalamocortical terminals had reached layer IV
(Fig. 1E), little or no mRNA or binding-site expression was
observed in this layer. Tangential sections often exhibited patterns of
7 nAChR mRNA and
[125I]-BTX binding-site expression in the
primary barrel region of superficial (Fig. 3C,E) and deep
layers (Fig. 3D,F) that indicated a subtle row-like
organization. As illustrated in Figure 3, this pattern was better
defined by binding (Fig. 3E,F) than by mRNA expression (Fig.
3C,D).
Postnatal day 5
Layer III could be distinguished from the cortical plate by P5
(Fig. 1L). Adjacent transverse sections again showed
AChE-positive thalamocortical terminals localized to layer IV and
extending into layer III (Fig. 1I), whereas a barrel-like
distribution of these terminals was seen in the tangential plane (Fig.
4A). This AChE-stained pattern was not
observed in deeper laminae, but showed a distinct, negative staining
pattern of the primary barrel region within layer VI (Fig.
4B).
At this age, 7 nAChR mRNA and
[125I]-BTX binding-site expression was
observed in deep layer IV, at the base of AChE-stained thalamic
terminals (Fig. 1J,K). As in P1 and P3, mRNA and
binding-site distribution was still observed in layer V and upper layer
VI, with layer VI exhibiting highest levels of expression. In the
tangential plane, somatotopic patterns of mRNA and binding-site
distribution were clearly detectable in the S1 region (Fig. 4).
Although the superficial mRNA pattern was often patchy with limited
definition (Fig. 4C), the
[125I]-BTX binding pattern was distinctly
barrel-like at this level (Fig. 4E). These sections were
directly adjacent to those exhibiting an AChE-stained barrel pattern
(Fig. 4A), indicating them to be at the level of layer IV.
At the level of layer VI, a more intense barrel-like distribution was
evident for both mRNA and binding-site expression (Fig.
4D,F). Within layer V, only
[125I]-BTX binding exhibited a barrel-like
pattern, but it was faint and lacked the definition observed in layers
IV and VI (data not shown).
Postnatal day 10
By P10, all cortical layers can be distinguished in the
Nissl-stained section (Fig. 1P). Transverse sections through
S1 at this age showed clusters of AChE-reactive thalamocortical
afferents throughout layer IV and extending slightly into layer III
(Fig. 1M). In addition, there was some light AChE staining
in superficial layer VI and deep layer V. When viewed in the tangential
plane of section, an AChE-stained barrel pattern was visible in layer
IV (Fig. 5A). A similar, but faint AChE
staining pattern was also observed in layer VI (Fig.
5B).
At this age, mRNA expression was highest in deep layer IV and upper
layer VI (Fig. 1N). Within layer V, mRNA expression was now
diminished and exhibited only scattered cellular labeling. In contrast,
[125I]-BTX binding was distributed in layers
IV-VI in a columnar pattern (see Fig. 1O) that corresponded to the
pattern of AChE-stained thalamic terminals (Fig. 1M). In the
tangential plane, barrel patterns of both 7
mRNA and [125I]-BTX binding were observed at
the level of layer IV (Fig. 5C,D). Although most intense in
deep layer IV, these patterns spanned the entire layer, corresponding
directly to the AChE staining pattern (Fig. 1M-P). A
barrel-like pattern of [125I]-BTX binding
was also evident within layer VI (Fig. 5F); however, the
pattern of mRNA expression in this layer was no longer disjunctive.
Instead, a patch of mRNA expression slightly higher than in the
adjacent cortex was observed (Fig. 5E).
Quantitative analysis of 7 nAChR ontogeny
At P1, the levels of 7 mRNA and
[125I]-BTX binding in both superficial and
deep laminae of S1 were not substantially elevated above surrounding
cortex (Fig. 6). By P5, mRNA and binding-site levels in
the barrel region exhibited a 200-400% increase in the superficial
and deep laminae, with levels in layer VI higher than those in layer
IV. Both mRNA and binding in layer VI peaked at P5-P7 and then
declined significantly to adult levels
(F7,25 = 10.698;
F7,25 = 8.191; p < 0.0001;
one-way ANOVA). Within layer IV, there was a more delayed peak of mRNA
and binding-site expression (Fig. 6), declining after P10 to adult
levels (F7,26 = 10.435;
F7,26 = 24.611; p < 0.0001;
one-way ANOVA). Levels of 7 nAChR mRNA and
[125I]-BTX binding in the adjacent
perirhinal cortex did not show significant changes throughout cortical
development (data not shown).
Fig. 6.
Density of 7 nAChR mRNA
and [125I]-BTX binding-site expression in
the primary barrel region of the somatosensory cortex during postnatal
development. The mRNA (A) and binding-site (B)
densities were determined in superficial (squares) and deep
(circles) cortical laminae. Data represent the mean ± SEM
for three to six animals.
[View Larger Version of this Image (15K GIF file)]
Effects of electrolytic thalamic lesions at P6
To examine the dynamics of regulation by thalamocortical
afferents, electrolytic lesions were placed in the VB thalamic complex
of P6 animals. After a 6 hr to 4 d survival period, cortical
hemispheres were qualitatively and quantitatively analyzed for mRNA and
protein expression within the S1. Transverse sections of thalamus were
first assessed by light microscopy to verify location and extent of VB
lesions. Figure 7A shows a unilateral
electrolytic lesion of the left VB complex from an animal lesioned on
P6 and killed 1 d later. This is an example of a partial lesion, which
destroyed the dorsal half of the ventral posterior medial (VPM) and the
ventral posterior lateral (VPL) thalamic nuclei. As illustrated in
Figure 7E, this partial lesion resulted in a loss in
AChE-positive staining of rows A-D of the primary barrel field on the
ipsilateral cortical hemisphere, while sparing row E and some of row D. The control hemisphere exhibited a normal pattern of AChE staining
(Fig. 7C).
Fig. 7.
Effects of thalamic lesions on AChE-stained
thalamocortical terminals in somatosensory cortex. A,
Bright-field photomicrograph of a transverse section through the
thalamic VB complex, 1 d after placement of a unilateral, electrolytic
lesion. Note that only the ventral halves of the ventral posterior
medial (VPM) and ventral posterior lateral (VPL)
nuclei are visible in the left hemisphere. B, Bright-field
photomicrograph of a transverse section through the VB complex, 1 d
after placement of a unilateral, cytochemical lesion. Note that most of
the VB complex, as well as the posterior thalamic nucleus
(Po), has been destroyed. C, E, Photomicrographs
of AChE-stained thalamocortical terminals at the level of layer IV of
the control (C) and lesioned (E) cortical
hemispheres from the animal shown in A. D, F,
Photomicrographs of AChE-stained thalamocortical terminals at the level
of layer IV of the control (D) and lesioned (F)
hemispheres from the animal shown in B. A1,
Auditory cortex. Scale bar, 1 mm.
[View Larger Version of this Image (176K GIF file)]
A similar decline in 7 nAChR mRNA and
[125I]-BTX binding-site expression in the S1
region of the lesioned hemisphere was observed (Fig. 8).
Similar to the pattern of AChE-stained thalamocortical terminals, there
was a partial loss in the pattern of mRNA and binding, consistent with
the extent of lesion. In both layers IV (Fig. 8B,F) and VI
(Fig. 8D,H), only rows A-D were affected, whereas row E and
part of row D were spared. Patterns of expression in the control
hemisphere were unaffected (Fig. 8A,C,E,G).
Fig. 8.
Autoradiographic images of
7 nAChR mRNA (A-D) and
[125I]-BTX binding-site (E-H)
distributions in the control and lesioned S1 of an animal 1 d after a
unilateral, electrolytic lesion at postnatal day 6. Adjacent,
tangential sections through the cortex at the level of layers IV
(A, B, E, F) and VI (C, D, G, H). The primary
barrel region (area within arrowheads) and the five barrel
rows (a-e) are indicated on the control hemisphere. Note
that only rows d and e of the primary barrel
region are visible on the lesioned side. A1, Auditory
cortex; CPu, caudate putamen; PRh, perirhinal
cortex. Scale bar, 1 mm.
[View Larger Version of this Image (136K GIF file)]
Time course of lesion
The loss in AChE staining of thalamocortical terminals within
layer IV of the lesioned cortex was not complete until 24 hr
postlesion. Animals killed at earlier time points still exhibited an
AChE-stained barrel pattern, indicating that thalamic afferents had not
completely degenerated (data not shown).
In contrast, quantitative analysis indicated that VB lesions resulted
in a 33% decrease in levels of 7 nAChR mRNA
expression in layer IV of the S1 as early as 6 hr postlesion (Fig.
9A). By 12 hr postlesion, a maximal 50%
reduction was observed that was significantly different from control
(p < 0.05; Newman-Keuls). There was a slightly
delayed reduction in [125I]-BTX binding
(Fig. 9B), with a 15% decrease at 12 hr postlesion and a
significant 52% reduction at 1 d (p < 0.05). By 3 d
postlesion, mRNA levels in layer IV of the control and lesioned
hemispheres were no longer significantly different and showed an
increasing trend, whereas binding-site levels continued to show a
significant difference. By the fourth day postlesion, distributions of
7 nAChR mRNA and
[125I]-BTX binding sites exhibited a
somatotopic pattern in layer IV of the lesioned S1, similar to that of
control (Fig. 10C,D,G,H). However,
AChE-labeled thalamic terminals on the lesioned side remained absent at
this time period (Fig. 10A,B).
Fig. 9.
Density of 7 nAChR mRNA
and [125I]-BTX binding-site expression
presented over time for the primary barrel region in the somatosensory
cortex of electrolytically lesioned animals. The mRNA (A, C)
and binding-site (B, D) densities were determined at the
level of layers IV (A, B) and VI (C, D) of
control (triangles) and lesioned (circles)
hemispheres. Animals received unilateral lesions as described in
Materials and Methods. Data represent the mean ± SEM for three to four
animals. *, p < 0.05,  , p < 0.01,  , p < 0.001 significantly different from
the control hemisphere.
[View Larger Version of this Image (27K GIF file)]
Fig. 10.
Photomicrographs and autoradiographic images of
AChE staining (A, B), 7 mRNA
(C-F), and [125I]-BTX
binding-site (G-J) distributions in the control and
lesioned S1 of an animal 4 d after a unilateral, electrolytic lesion at
postnatal day 6. Adjacent, tangential sections through the cortex at
the level of layers IV (A, C, E, G, I) and VI (B, D,
F, H, J). The primary barrel region (area within
arrowheads) and the five barrel rows (a-e) are
indicated. A1, Auditory cortex; CPu, caudate
putamen; PRh, perirhinal cortex. Scale bar, 1 mm.
[View Larger Version of this Image (123K GIF file)]
In layer VI, a 75% decrease in 7 nAChR mRNA
expression was observed in S1 of the lesioned hemisphere as early as 6 hr postlesion (p < 0.01) (Fig. 9C),
whereas [125I]-BTX binding-site levels were
significantly decreased by 48% at 12 hr postlesion
(p < 0.05) (Fig. 9D). After the first
day, levels of mRNA and binding sites in layer VI of the lesioned side
showed a significant increase (F5,28 = 2.58; F5,29 = 3.467; p < 0.05;
two-way ANOVA), but remained lower than the control side until the
fourth day postlesion. By this time, somatotopic patterns of
7 nAChR mRNA and
[125I]-BTX binding-site expression were
observed to delineate the primary barrel region on the lesioned side,
similar to that of control (Fig. 10E,F,I,J).
Effects of VB cytochemical lesions
To determine whether the 7 nAChR mRNA and
[125I]-BTX binding-site loss represented
retrograde degeneration of corticothalamic neurons, the VB thalamic
complex of P6 animals was injected with NMDA, so that thalamocortical
neurons would be killed but corticothalamic projections could be
spared. Transverse sections of thalamus were again assessed by light
microscopy to verify location and extent of VB lesions. Figure
7B shows a unilateral cytochemical lesion of the left VB
complex of the thalamus from an animal that survived 1 d postlesion.
This was a nearly complete lesion that destroyed both the VPM and VPL
as well as the posterior (Po) thalamic nuclei. The lesion
resulted in a total loss of barrel-like AChE staining in the
ipsilateral cortical hemisphere (Fig. 7F), while not
affecting terminals on the control side (Fig. 7D). A similar
decline in 7 mRNA and
[125I]-BTX binding-site levels was observed
in the lesioned cortex (Table 1). Levels of mRNA were
decreased by 69% (p < 0.05) and 84%
(p < 0.001), whereas binding levels were decreased
by 33% (p < 0.01) and 55% (p < 0.001) in layers IV and VI, respectively.
Effects of electrolytic thalamic lesions at P0
To examine the influence of thalamocortical innervation on the
developmental pattern of 7 nAChR expression in
the S1 cortex, electrolytic lesions were placed in the VB thalamic
complex of P0 rat pups. After a 5 d survival, cortical hemispheres were
qualitatively and quantitatively analyzed for mRNA and protein
expression within the S1. At this age, a barrel-like expression of
7 mRNA and
[125I]-BTX binding sites within layers IV
and VI is clearly visible in the control, unlesioned cortical
hemisphere (Fig. 11C,E,G,I). Both partial
and complete VB lesions resulted in a loss of barrel-like AChE staining
in the ipsilateral cortex (Fig. 11B), corresponding to the
extent of lesion. A similar decline in 7
nAChR mRNA and [125I]-BTX
binding-site expression in the S1 region of the lesioned hemisphere was
observed in layer IV (Fig. 11D,H) and layer VI (Fig.
11F,J). Levels of mRNA were significantly decreased
by 65% (p < 0.001) and 79% (p < 0.001), whereas binding levels were decreased by 82%
(p < 0.001) and 81% (p < 0.001) in layers IV and VI, respectively (Table 2).
Fig. 11.
Photomicrographs and autoradiographic images of
AChE staining (A, B), 7 mRNA
(C-F), and [125I]-BTX
binding-site (G-J) distributions in the control and
lesioned S1 of an animal 5 d after a unilateral, electrolytic lesion at
postnatal day 0. Adjacent, tangential sections through the cortex at
the level of layers IV (A, C, E, G, I) and VI (B, D,
F, H, J). The primary barrel region (area within
arrowheads) and the five barrel rows (a-e) are
indicated. A1, Auditory cortex; CPu, caudate
putamen; PRh, perirhinal cortex. Scale bar, 1 mm.
[View Larger Version of this Image (127K GIF file)]
Lesion controls
Measurements of 7 mRNA and
[125I]-BTX binding-site levels in auditory
or perirhinal cortex of either electrolytic or cytochemically damaged
animals showed no significant difference between the lesioned and
control hemispheres (data not shown). In addition, animals that
received electrolytic or cytochemical lesions in regions of the
thalamus adjacent to VB did not exhibit any decrease in
7 nAChR mRNA or
[125I]-BTX binding-site levels in the S1.
Finally, lesions that affected only a portion of VB affected only
corresponding parts of S1.
DISCUSSION
The present study demonstrates transient patterns of
7 nAChR mRNA and protein expression during the
early postnatal period of rat S1 development in deep and superficial
laminae. Initial expression occurs ~1 d later than the barrel pattern
exhibited by AChE-stained thalamocortical terminals (Schlaggar and
O'Leary, 1994). The deeper pattern of receptor expression was
localized consistently to upper layer VI, whereas the superficial
patterns gradually shifted from deep to more superficial laminae, after
the growing thalamocortical axons, and eventually localized in layer
IV. Expression peaked at P7-P10 and declined thereafter to
adult levels. Placement of lesions in the VB thalamic complex at P0
(just as thalamocortical afferents are innervating the cortex)
(Catalano et al., 1991) and at P6 (after the somatotopic map in the S1
has been well established) (Killackey et al., 1995) resulted in a
significant decrease in 7 nAChR mRNA and
protein expression in both layers IV and VI of the ipsilateral
S1. With P6 lesions, this decline in receptor expression was observed
as early as 6 hr postlesion. A complete loss of AChE-stained thalamic
terminals, however, was not evident until 1 d postlesion. These data
suggest a regulation of 7 nAChRs on cortical
neurons in the S1 by thalamocortical afferents.
Methodological issues
Alternate sections from the same brain were processed for AChE
histochemistry, 7 nAChR mRNA expression, and
[125I]-BTX binding. This experimental design
was chosen to eliminate inter-animal variability and to permit detailed
comparison of the ontogeny of AChE-labeled thalamocortical afferents
and 7 nAChR. Experimental conditions were
optimized for receptor analysis and involved the use of cryostat-cut,
post-fixed tissue sections. Standard AChE processing conditions were
appropriately modified to allow visualization of signal in nonperfused
tissue. Although not optimized for enzymatic analysis, the
developmental pattern of AChE that was observed is similar to that
reported by Schlaggar and O'Leary (1994).
To conserve tissue, sections for receptor analysis were not processed
for nonspecific binding or sense hybridization. Previous studies from
our laboratory have shown that nonspecific binding or hybridization
represents <35% and 6% of total signal in this region, respectively
(Broide et al., 1995). Rather than use separate sections as nonspecific
controls, labeling within the perirhinal cortex region of the
experimental section was measured and subtracted as background, since
labeling within this region did not change significantly with age or
lesion.
Ontogeny of 7 nAChRs in relation to
thalamic afferents
Whereas layer VI of S1 is labeled by AChE at P0, consistent
expression of 7 nAChRs was not observed in
this region until P1. Our observation that 7
receptor expression follows the pattern delineated first by AChE
suggests that expression of 7 nAChRs in the S1
may be induced by ingrowing thalamocortical afferents.
One feature of 7 nAChR expression in
developing S1 is the somatotopic representation observed in both deep
and superficial laminae. Whereas the deep pattern is consistently
observed in layer VIa, a superficial pattern develops behind ingrowing
thalamocortical terminals. In the lower tier, the barrel patterns of
7 nAChR mRNA and protein expression become
clearly evident at P3-P5. Earlier studies have demonstrated that
ingrowing thalamocortical axons arborize in this region in a
somatotopic pattern (Agmon et al., 1993; Schlaggar and O'Leary, 1994).
The consistent feature in our data is that growing thalamocortical
axons appear organized in a somatotopic pattern in deeper layers and
eventually in layer IV, and that a somatotopic pattern of
7 nAChR mRNA and
[125I]-BTX binding appears to follow the
thalamocortical pattern by ~1 d.
Effects of thalamic lesions
Our lesion data have confirmed the critical influence of ingrowing
thalamocortical afferents on 7 nAChR
expression in layers IV and VI. Electrolytic ablation of neurons in the
thalamic VB nucleus of P6 rat pups, after the somatotopic map in the S1
has been well established, results in a rapid loss of
7 nAChR expression in both cortical layers.
The loss in 7 mRNA expression is maximally
decreased within 12 hr postlesion before the disappearance of AChE
labeling of thalamocortical terminals. A corresponding but slightly
delayed loss of protein is maximal within 24 hr. Cytochemical lesions
of the VB at P6 also result in a significant and rapid decline in mRNA
and protein expression in both layers IV and VI, confirming the
specificity of this effect and eliminating the possible involvement of
the reciprocal corticofugal pathway, which develops in parallel with
the thalamocortical axons (De Carlos and O'Leary, 1992; Miller et al.,
1993).
After the initial downregulation after P6 lesions, however, some
recovery of 7 nAChR expression is observed
such that with longer postlesion periods, the differences between
control and lesion sides are no longer statistically significant.
Furthermore, there is some re-emergence of the somatotopic pattern in
both layers even though the loss of AChE-labeled terminals is complete.
Thus, after the critical period of cortical development,
7 nAChR expression is dynamically modulated
and not completely dependent on thalamocortical innervation.
Electrolytic lesions placed in the VB of P0 pups at the time when
thalamocortical afferents are just beginning to innervate the cortex
results in a lack of the normal 7 nAChR
developmental pattern in layers IV and VI of the ipsilateral S1. These
results suggest that during the critical period of cortical
development, ontogeny of 7 nAChR expression is
dependent on thalamocortical innervation. Taken together, these lesion
data indicate that there is a very tight coupling between the
thalamocortical pathway and cortical 7 nAChR
expression, and further suggest that these ingrowing afferents imprint
a somatotopic pattern in both layers IV and VI.
The rapid loss of radioligand binding observed after placement of
lesions may indicate a very rapid turnover of receptor protein. A high
rate of turnover of peripheral nAChRs has previously been observed at
the developing neuromuscular junction (Michler and Sakmann, 1980;
Reiness and Weinberg, 1981). Alternatively, the observed binding
changes may reflect alterations in receptor affinity or rapid
internalization to an intracellular receptor pool (Stollberg and Berg,
1987).
Evidence for 7 nAChRs on
thalamocortical afferents
Our present data suggest that 7 nAChRs in
developing S1 are localized primarily to cortical cells. However, in
rat, 7 mRNA is also expressed in the VB
thalamic complex (Broide et al., 1995), which could suggest that a
proportion of 7 nAChR expression in layer IV
is on presynaptic terminals. Consistent with this hypothesis is our
observation that the pattern of protein expression in layer IV is more
discrete than that of mRNA. However, the ontogeny of protein expression
in layer IV is later than that of AChE-labeled thalamic afferents and
is more consistent temporally with the expression of mRNA in this
region. Electron microscopic studies will be required to further
clarify this issue.
Role of 7 nAChRs in S1 ontogeny
Much recent data implicate the 7 nAChR in
mechanisms underlying cellular plasticity. Previous studies have shown
this receptor to be developmentally regulated (Couturier et al., 1990;
Broide et al., 1995) and that activation of this receptor induces
Ca2+ influx (Vijayaraghavan et al., 1992; Seguela
et al., 1993; Zhang et al., 1994) and subsequent neurite retraction
(Chan and Quik, 1993; Pugh and Berg, 1994). Thus, the transient
expression during the critical period of rat S1 development is
consistent with a developmental role in establishing cortical
circuitry.
For this hypothesis to be valid, there must be a source of endogenous
ACh within cortex during this period of early postnatal development. A
transient expression of cholinergic neurons within rat sensory cortex
has been reported to peak during the perinatal period (Dori and
Parnavelas, 1989). Also, cortical ingrowth of basal forebrain afferents
in this species has been observed as early as P0 (Calarco and
Robertson, 1995). DiI-labeling studies have indicated that early
postnatal development of basal forebrain fibers coincides temporally
with the expression of 7 nAChRs, which we
document in the present study. Whereas ChAT expression in these basal
forebrain afferents has not been detected before P5 (Kiss and Patel,
1992), the cells of origin are ChAT-positive at the time of birth
(Gould et al., 1991). It is therefore possible that ACh may be released
from basal forebrain axons during early postnatal development.
Electrophysiological studies will be required to address this issue
further.
Our present data, indicating expression of a cholinergic receptor in
association with AChE-labeled thalamocortical afferents, suggest the
possibility that functional cholinergic synapses may exist during this
critical period of cortical development. Thus, a functional triad may
be formed between basal forebrain afferents, thalamocortical afferents,
and cortical target cells. If, as extensive literature suggests
(O'Leary et al., 1994), thalamocortical afferents stimulate
differentiation of cortical structure and function, the
7 nAChR may serve as an early transducer of
this process.
FOOTNOTES
Received Oct. 10, 1995; revised Jan. 31, 1996; accepted Feb. 5, 1996.
This work was supported by Public Health Service Grant NS30109. We
thank Dr. Jim Boulter for providing the 7 cDNA
and Drs. Aric Agmon and Ursula Winzer-Serhan for their comments on this
manuscript.
Correspondence should be addressed to Dr. F. M. Leslie, Department of
Pharmacology, College of Medicine, University of California, Room 360, MSII, Irvine, CA 92717.
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, p < 0.01,
, p < 0.001 significantly different from the control hemisphere.
