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The Journal of Neuroscience, September 15, 2002, 22(18):7903-7912
Nicotinic 
7 Receptor Clusters on Hippocampal GABAergic
Neurons: Regulation by Synaptic Activity and Neurotrophins
Hideki
Kawai*,
Wagner
Zago*, and
Darwin K.
Berg
Neurobiology Section, Biology Division, University of California,
San Diego, La Jolla, California 92093-0357
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ABSTRACT |
Nicotinic acetylcholine receptors containing the 
7 gene product
are expressed at substantial levels in the hippocampus. Because of
their specific locations and their high relative calcium permeability, the receptors not only mediate cholinergic transmission in the hippocampus but also influence signaling at noncholinergic synapses. We
have used fluorescently labeled -bungarotoxin to image
7-containing receptors on hippocampal neurons and to examine their
regulation in culture. The highest levels of staining for such
receptors were most commonly found on GABAergic interneurons identified immunohistochemically. The receptors were distributed in clusters on
the soma and dendrites and were localized in part at GABAergic synapses. A 3 d blockade of electrical activity with tetrodotoxin or NMDA receptors with APV dramatically reduced the proportion of
GABAergic neurons expressing high levels of receptor staining and
reduced the mean number of distinguishable receptor clusters on
individual neurons. Blockade of either GABAA receptors with bicuculline or nicotinic receptors with D-tubocurarine had
no effect, although exposure to nicotine could increase the level of
receptor staining. Anti-BDNF and anti-NGF antibodies produced decrements equivalent to those of tetrodotoxin and APV, whereas addition of BDNF and NGF each increased staining levels and increased the number of distinguishable receptor clusters on GABAergic neurons. The exogenous neurotrophins could not, however, overcome the effects of
either tetrodotoxin or APV. The results indicate that both NMDA
receptor activation and the neurotrophins BDNF and NGF are necessary to
sustain the distribution patterns of 7-containing nicotinic
receptors on GABAergic hippocampal neurons.
Key words:
nicotinic; acetylcholine; receptors; hippocampal; BDNF; NGF; bungarotoxin
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INTRODUCTION |
The hippocampus has been the subject
of intense study examining mechanisms of synaptic plasticity. Central
to this has been the analysis of glutamatergic transmission. Recent
evidence suggests that nicotinic signaling may also contribute
significantly to hippocampal function. Nicotinic acetylcholine
receptors containing the 7 gene product (7-nAChRs) are expressed
at some of their highest CNS levels in the hippocampus. Such receptors
have a high relative permeability to calcium (Bertrand et al., 1993
;
Seguela et al., 1993) and can modulate transmitter release both from
GABAergic and glutamatergic terminals in the hippocampus (Gray et al.,
1996; Alkondon et al., 1997a; Radcliffe and Dani, 1998; Maggi et al., 2001). The receptors also act postsynaptically to mediate excitatory input (Alkondon et al., 1998; Frazier et al., 1998; Hefft et al., 1999;
Jones et al., 1999). Depending on the neurons involved, 7-nAChR
activation can lead to either inhibition or disinhibition of
hippocampal output and can have divergent effects on synaptic plasticity as well (Fujii et al., 2000; Ji and Dani, 2000; Alkondon and
Albuquerque, 2001; Buhler and Dunwiddie, 2001, 2002; Ji et al.,
2001).
The consequences of 7-nAChR activation are likely to depend
critically on receptor location and the calcium-dependent processes resident at those sites. Ultrastructural analysis suggests that 7-nAChRs are concentrated both presynaptically and postsynaptically at almost all synapses in the CA1 stratum radiatum of the hippocampus (Fabian-Fine et al.,. 2001). The anatomical distribution is consistent with reports that 7-nAChR activation can enhance both GABAergic and
glutamatergic transmission, but further suggests that the modulation
may be more complex than previously recognized. How 7-nAChRs become
located at such sites is unknown.
Immunohistochemical staining of rat hippocampal neurons in culture
reveals 7-nAChR clusters on neuronal somata and dendrites. The
clusters often colocalize with synaptotagmin stained as a marker for
presynaptic terminals (Zarei et al., 1999). Recently it has been shown
that 7-nAChR levels on hippocampal neurons in culture can be
regulated by neuregulins and that the neuregulin-induced increases in
receptors also produce enhanced modulation of transmitter release (Liu
et al., 2001). These results suggest that rat hippocampal cultures can
be used to identify conditions that control the distribution of
7-nAChRs and influence their localization at synaptic sites.
We show here that 7-nAChRs can be imaged on hippocampal neurons
using fluorescently labeled -bungarotoxin (-Bgt) and that the
receptors are concentrated in clusters located in part at GABAergic
synapses. The receptor clusters can also be found at the distal tips of
filopodia-like dendritic extensions. Because synaptic activity and
endogenous neurotrophins can both influence synapse formation on
hippocampal neurons (McAllister et al., 1999; Schuman, 1999;
Bolton et al., 2000; Schinder and Poo, 2000; Turrigiano and Nelson,
2000; Murthy et al., 2001; Tyler and Pozzo-Miller, 2001), we examined
their effects on 7-nAChR clusters. We find that transmission via
NMDA receptors but not via GABAA or nicotinic receptors, is required to sustain the pattern of 7-nAChRs.
Endogenous BDNF and NGF are also required, but exogenous neurotrophins
cannot compensate for NMDA receptor blockade, although they can
increase cluster number when acting alone.
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MATERIALS AND METHODS |
Cell cultures. Hippocampal cultures were prepared
from 19-d-old Sprague Dawley rat embryos as previously described for
cortical cultures (Kawai and Berg, 2001). Hippocampi were removed
rapidly under stereomicroscopic observation, cut into small pieces, and digested with 20 U/ml of papain (Worthington, Lakewood, NJ) in HBSS
(Invitrogen, Gaithersburg, MD) containing 0.7 mM
CaCl2, 0.35 mM EDTA, 0.01 mg/ml DNase I, 2 mg/ml L-cysteine, 10 mM HEPES, and 1 mM sodium
pyruvate, at 37°C for 30 min. The tissue segments were then
transferred to Neurobasal medium (Invitrogen) containing 4.4 mM sodium bicarbonate and 21.6 mM NaCl, and triturated with a fire-polished
Pasteur pipette in the presence of 0.01 mg/ml DNase I. After
centrifugation for 5 min at 800 × g, the cells were
resuspended in Neurobasal medium with 2% (v/v) B-27 supplement, 0.5 mM L-glutamine, 50 U/ml
penicillin, and 50 µg/ml streptomycin (Invitrogen). The cells were
plated at 2 × 104 per 12 mm glass
coverslip previously coated with poly-D-lysine (>300 kDa; Sigma, St. Louis, MO). The cultures initially received the
medium described above plus 10% (v/v) heat-inactivated horse serum
(Gemini Bio-Products, Woodland, CA). Subsequent feeding occurred twice
weekly, each time replacing half the volume with medium lacking horse
serum. On day 6, the cultures received 5 µM
cytosine-
-D-arabinofuranoside to inhibit
further proliferation of non-neuronal cells. The cultures were
maintained in a humidified tissue culture incubator with 95% air and
5% CO2 until use. Where indicated, the following
compounds were applied to the cells during the last 3 d in
culture: 1 µM tetrodotoxin (TTX), 50 µM
D-(
)-2amino-5-phosphono-valeric acid (APV), 0.5 µM nicotine, 10 µM
D-tubocurarine (D-TC), 100 ng/ml BDNF (recombinant BDNF; Chemicon, Temecula, CA), 100 ng/ml NGF
(2.5S; Invitrogen, Carlsbad, CA), 5 ng/ml neuregulin (recombinant HRG1-1 EGF-like domain; R & D Systems, Minneapolis, MN), 10 µg/ml chicken anti-BDNF functional-blocking polyclonal antibodies (Promega, Madison, WI), and 1 µg/ml sheep anti-NGF function-blocking polyclonal antibodies (Chemicon). Bicuculline (20 µM) was
applied for the last 7 d as was nicotine where indicated. To
assess possible nonspecific effects of anti-neurotrophin antibodies,
IgGs raised in sheep and chicken were incubated in the cultures and
compared in the same experiments.
Labeling surface 7-nAChRs. For imaging studies,
cells maintained for 19-20 d in culture were washed twice with
Neurobasal medium plus 0.1% BSA and then incubated in the same medium
containing 100 nM of Alexa Fluor 488 -bungarotoxin (Alexa488--Bgt; Molecular Probes, Eugene, OR) at
37°C for 45 min. Nonspecific binding was assessed by incubating the
cells in either nicotine (1 mM), -Bgt (5 µM), or methyllycaconitine (MLA; 5 µM) 10 min before and during the labeling with
Alexa488--Bgt. The cells were then washed four times in 2 ml of
Neurobasal medium and fixed for 20 min at room temperature in 4%
paraformaldehyde in PBS. For binding studies, the cells were incubated
with 5 nM
125I--Bgt (260-390 cpm/fmol; Amersham
Biosciences, Buckinghamshire, UK) for 1 hr at room temperature, rinsed,
scraped in non-ionic detergent, and quantified for retained
radioactivity with a gamma counter as previously described (Kawai and
Berg, 2001). Nonspecific binding was determined by including 0.5 µM -Bgt (Molecular Probes) with the
125I--Bgt; nonspecific binding did not
exceed 12% of total binding and was subtracted in all cases.
Fluorescence immunocytochemistry. For immunolabeling, fixed
cells were permeabilized with 0.1% Triton X-100 in PBS and then incubated with antibodies. These included a mouse anti-glutamic acid
decarboxylase (GAD) monoclonal antibody (mAb; clone GAD-6 for GAD65,
1:1000; Boehringer Mannheim, Indianapolis, IN), a rabbit anti-GAD
polyclonal antibody (for GAD65/67, 1:500, Chemicon), a rabbit
anti-GABAA receptor
(GABAAR) 1-subunit polyclonal antibody (1:500; Upstate Biotechnology, Lake Placid, NY), a mouse anti-MAP-2 mAb
(1:1000; Sigma), a goat anti-7-nAChR polyclonal antibody (SC14447,
1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and both goat and
rabbit anti-vesicular ACh transporter polyclonal antibodies (60596E,
1:1000; PharMingen International, San Diego, CA; Phoenix
Pharmaceuticals, Belmont, CA). Nonspecific antibody binding was
minimized by treatment with 5% donkey serum in PBS for 30 min at room
temperature. Primary antibodies were diluted in 0.1% Triton X-100 PBS
and incubated with the cells for 1 hr at 37°C. Cells were washed
three times (10 min intervals) in PBS and then incubated for 1 hr at
room temperature with secondary antibodies raised in goat or donkey and
conjugated to Cy3 or Cy5 fluorophores (1:500 dilution in 0.1% Triton
X-100 PBS; Jackson ImmunoResearch, West Grove, PA). The cells were then
washed three times (10 min intervals) in PBS and mounted using
anti-fade mounting solution (Vectashield; Vector Laboratories,
Burlingame, CA).
Image acquisition and quantification. Digital images of
fluorescently labeled cells were collected using a CCD camera mounted on a Zeiss Axiovert (63× oil-immersion objective, 1.4 numerical aperture lens) and equipped with SlideBook deconvolving software (Intelligent Imaging Innovations, Santa Monica, CA). Reconstructed images were generated from z-axis stacks of 10 0.5 µm
deconvolved optical sections. Controls in which one or more primary
antibodies were omitted showed no significant cross-contamination among
fluorescence channels. This approach allowed images to be obtained and
analyzed in cases where confocal microscopy proved insufficient.
Quantification of colocalized signal was performed by normalizing the
intensity data among fluorescence channels (maximal intensity, 100%),
followed by subtracting the background fluorescence. Control cultures
in each experiment were used to set the intensity ranges to be
measured; these same ranges were then applied to all images captured in the experiment. Maximum intensities were evaluated by either of two
methods with similar outcomes. One was to scan a number of the
brightest spots on control cells (
10 per neuron, 8 neurons per
experiment), recording the single highest pixel intensity within each
spot, and then averaging such values to define a maximum. The
alternative was to display the entire distribution of pixel intensities
seen for a field of view containing a control neuron (8 neuron fields
analyzed) and to set the maximum to include >99% of the pixel values.
The two methods usually agreed within 10%.
Measurements of cluster size, area, and number were performed using
ImagePro 3.0 software (Media Cybernetics, Silver Spring, MD). Clusters
were counted along dendritic segments (
45 µm, starting 5 µm
from the soma) and compared for colocalization with other markers after
merging the appropriate fluorescent images. Threshold values of 0.05 µm2 (five contiguous pixels) and 50% of
maximal intensity were chosen for defining clusters. A minimum overlap
of 0.05 µm2 (five pixels) was taken as
threshold for defining colocalization of two fluorescent signals. Data
were collected from two to four dendrites per neuron, and a total of
five to seven randomly selected GAD-positive neurons per coverslip.
Coverslips were usually analyzed in duplicate per experiment. Values
were normalized to controls (percentage of controls) and are shown as
mean ± SEM of three to nine separate experiments. Statistical
differences between two means were determined using the two-tailed
Student's t test; comparisons among more than two were
determined by ANOVA. To assess the amount of codistribution that might
be expected for two markers based on chance, we measured the fraction
of the dendritic length (along a line segment) occupied by each and
then multiplied these two values together to calculate the fraction
predicted for random overlap. Nonrandom codistribution of two markers
(e.g., staining for 7-nAChR clusters and GAD-positive terminals) was
that amount of codistribution significantly greater than the amount
predicted for random overlap.
In some experiments cells were divided into categories based on overall
Alexa488--Bgt labeling, using the following criteria. "Unlabeled" cells were those with staining intensities not
significantly different from negative controls, i.e., cells
co-incubated with a competing ligand. "Moderate" cells were those
in which individual 7-nAChR clusters could be distinguished on the
soma and dendrites as described above. The range of staining
intensities varied considerably among moderate neurons but the
mean pixel intensity for receptor clusters was ~150 (arbitrary
units), after subtracting a nonspecific background level of 15-20.
"High" cells were those in which perimeter labeling was too
extensive to distinguish individual clusters. The mean pixel intensity
along the perimeter of such cells was ~400, after subtracting a
nonspecific background of 15-20.
Materials. Unless otherwise indicated, all drugs and
compounds were purchased from Sigma.
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RESULTS |
Imaging 7-nAChRs on hippocampal neurons
Staining intact rat hippocampal neurons in culture with
Alexa488--Bgt yielded specific labeling readily visualized with
fluorescence optics and deconvolving software. The staining represented
7-nAChRs because it could be blocked not only by nicotine but also
by either unlabeled -Bgt or MLA (Fig.
1, top row). The cells were
identified as neurons by costaining with MAP-2 (Fig. 1, middle
row). Only surface 7-nAChRs were labeled with the
Alexa488--Bgt because the toxin binding was performed on living
cells before fixation and permeabilization; scanning in the
z-axis confirmed that the staining was confined to the cell
perimeter. When the labeled cells were subsequently fixed,
permeabilized, and costained with a goat anti-7-nAChR antibody
(Paysan et al., 2000), the Alexa488--Bgt staining codistributed
with antibody labeling on the perimeter; additional antibody staining
was found inside the cell, indicating intracellular receptor protein as
well (Fig. 1, bottom row). Binding studies with
125I--Bgt to quantify 7-nAChRs on
the cells showed that the number of surface receptors increased with
culture age for at least the first 3 weeks, as reported previously
(Barrantes et al., 1995; Samuel et al., 1997). All subsequent
experiments were performed on cells during the third week of
culture.
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Figure 1.
Specificity of Alexa488--Bgt labeling of
7-nAChRs on hippocampal neurons in culture. Top row,
Hippocampal neurons stained for 7-nAChRs with Alexa488--Bgt (100 nM, 488--Bgt, green) in the absence
(A) or presence of nicotine
(B), -Bgt (C), or MLA
(D). Middle row, Same neurons as
above, costained with antibodies (red) for MAP-2
(E-H). The staining with Alexa488--Bgt is
specific for 7-nAChRs and reveals receptor clusters distributed over
the cell body and along the proximal dendrites. Nonspecific labeling
represented <10% of average intensity along dendrites. Bottom
row, Cell stained first (green) with
Alexa488--Bgt (I), then fixed,
permeabilized, and costained (red) with anti-7-nAChR
antibodies (J), and shown in overlay
(K), revealing surface clusters of receptor that
colabeled with both markers (arrows). Antibody staining
also showed intracellular receptor protein not seen with the
Alexa488--Bgt staining performed on the intact cell. Substituting
nonimmune IgG for the anti-receptor antibody produced no staining
(L); the cell occupied approximately the same
position and area as the cell in I-K. Scale bars:
A-H, 20 µm; I-L, 5 µm.
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The types of hippocampal neurons expressing detectable levels of
7-nAChRs were assessed by immunostaining. MAP-2 staining was used
again to identify neurons in the cultures (Fig.
2A-C). Costaining for
GAD revealed that 17 ± 2% were GABAergic interneurons (mean ± SEM; n = 14 experiments; one or two coverslips per
experiment; 15 fields of view per coverslip). The remaining 83% were
presumed to be glutamatergic pyramidal neurons, consistent with the
composition in vivo (Olbrich and Braak, 1985; Freund and
Buzsaki, 1996). Staining cultures first with Alexa488--Bgt, and
then rinsing, fixing, permeabilizing, and staining for GAD indicated
that GABAergic interneurons were much more likely to display readily
detectable levels of 7-nAChR labeling than were the GAD-negative
neurons. This is consistent with previous findings (Liu et al., 2001). Subsequent experiments targeted the GAD-positive cells.
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Figure 2.
Cell categories for 7-nAChR labeling on
GABAergic hippocampal neurons. Top row, Hippocampal
neurons, defined by immunostaining for MAP-2 (A),
included examples of both GAD-positive and GAD-negative cells as
putative GABAergic and glutamatergic neurons, respectively
(B), seen in overlay (C).
Second row, Example of GAD-positive neuron expressing
moderate levels of 7-nAChR labeling (D-F).
Third row, GAD-positive neuron expressing high level of
7-nAChR labeling (G-I). Bottom
row, Unlabeled GAD-positive neuron
(J-L). The relative proportions of GAD-positive
cells in unlabeled, moderate, and high categories for 7-nAChR
labeling were 32 ± 4, 53 ± 4, and 15 ± 2%,
respectively. Values represent mean ± SEM of 12 experiments (360 cells). Scale bar, 20 µm.
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Examining all GABAergic neurons in the cultures indicated that 68 ± 4% (mean ± SEM; n = 12) of them expressed
levels of 7-nAChRs sufficient for imaging. These could be further
divided into cells having moderate labeling (53 ± 4% of the
GAD-positive neurons) and those with high labeling (15 ± 2% of
the GAD-positive neurons). Moderate cells ranged in the level of
staining intensity but shared the feature of displaying discrete
receptor clusters on the soma and dendritic surfaces (Fig.
2D-F). High cells were stained more brightly,
and the staining was so extensive that receptor clusters usually could
not be distinguished (Fig. 2G-I). The remaining GAD-positive neurons (32 ± 4%) were classified as unlabeled
(Fig. 2J-L) because they were equivalent to neurons
in which specific binding had been blocked with a receptor antagonist.
Clusters of 7-nAChRs at GABAergic synapses and on
filopodia-like extensions
Further analysis of GAD-positive neurons with moderate 7-nAChR
staining demonstrated that a significant number of the 7-nAChR clusters were localized at GAD-positive presynaptic terminals abutting
the dendrites (Fig. 3A-C).
The GAD-positive terminals appeared as discrete entities and were
clearly distinct from the soma staining produced by the GABAergic
neuron itself. Quantitative analysis (see below) indicated the
proportion of 7-nAChR clusters located at such terminals to be about
a fifth of the total receptor clusters. This value was five times
greater than the random overlap to be expected from the proportions of
dendritic surface area occupied by 7-nAChR clusters on the one hand
and by GAD-positive terminals on the other. No evidence for cholinergic
terminals was revealed in the cultures, as detected by staining cells
for the vesicular ACh transporter (data not shown).
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Figure 3.
Clusters of 7-nAChRs and their location at
GABAergic synapses. Cells stained with Alexa488--Bgt for surface
7-nAChRs (A, D, G) and costained for GAD (B,
H) and/or 1-GABAAR subunit (E,
I) were viewed in overlay (C, F,
J). Large boxes are expansions of the
small boxes; arrows indicate examples of
codistributed markers. A significant proportion of 7-nAChRs clusters
on GABAergic neurons (18 ± 4%, n = 11) are
present at GABAergic synapses defined by GAD staining and colocalize
with 1-GABAARs. Scale bars: A-F, 20 µm; G-J, 10 µm.
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In some cases the 7-nAChR clusters tightly overlapped with the
presynaptic staining for GAD, suggesting the receptors may have been
presynaptic. In other cases the 7-nAChR clusters were immediately
adjacent to but only partially overlapping with terminal GAD staining,
suggesting that the receptor clusters were mostly postsynaptic.
Examining sequential optical sections in the z-axis supported this interpretation (data not shown). Further evidence for a
postsynaptic location came from staining the cultures for GABAA receptors using antibodies specific for the
GABAA 1 subunit. Costaining with
Alexa488--Bgt for 7-nAChRs and with the anti-1 antibody
indicated numerous occasions of complete overlap (Fig. 3D-F). Triple staining for 7-nAChRs,
1-GABAARs, and GAD provided clear examples of
complete overlap of the two receptor types juxtaposed to GAD-positive
terminals (Fig. 3G-J).
Perhaps most convincing for the hypothesis that 7-nAChR clusters can
often be postsynaptic was the following observation. GAD-positive
neurons that did not themselves express 7-nAChRs (unlabeled category
of neurons) lacked 7-nAChR clusters even at dendritic sites
receiving GAD-positive terminals. Examining a total of 126 dendrites on
unlabeled neurons (28 neurons, 13 experiments) yielded no detectable
7-nAChR clusters, although 2618 GAD-positive terminals were
identified as abutting on the dendrites. In contrast, examining 108 dendrites from neurons with moderate Alexa488--Bgt staining (44 neurons, 14 experiments) yielded 3691 receptor clusters, and 664 of
them were associated with GAD-positive terminals which themselves
numbered 1702 in all. The simplest interpretation is that most, if not
all, of the 7-nAChR clusters detectable at GABAergic synapses on
moderate neurons are provided by the postsynaptic neuron. Presynaptic
7-nAChRs are likely to be present (Alkondon et al., 1997a; Maggi et
al., 2001) but may have been too sparse to score as a cluster with the
current imaging criteria. A much less likely explanation for the result
would be that GAD terminals cannot express presynaptic 7-nAChRs for
some reason if the postsynaptic neuron is unable to do so. Taken
together, the present results suggest a postsynaptic location for many
of the 7-nAChR clusters observed here at GABAergic synapses, but
this conclusion should be considered tentative until confirmed by
ultrastructural analysis.
GAD-positive neurons with high 7-nAChR labeling did not permit
resolution of individual receptor clusters in most cases, but examining
the cells yielded an additional interesting result. In many cases,
7-nAChRs were found sharply concentrated at the distal tips of
filopodia-like extensions emanating from the dendrites (Fig.
4). Such 7-nAChR clusters were not
associated with 1-GABAARs or GAD-stained
presynaptic terminals.
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Figure 4.
Clusters of 7-nAChRs at tips of filopodia-like
structures emanating from dendrites of GABAergic interneurons.
A, Isolated GABAergic neuron with high level of
Alexa488--Bgt labeling (green) costained for
GAD (red) and shown in overlay. B, The
boxed region in A is shown in expansion.
The 7-nAChR clusters present at the tips of the filopodia-like
extensions (arrows) are not associated with GAD
staining. Scale bars: A, 20 µm; B, 5 µm.
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Activity-dependent regulation of 7-nAChR levels on
GABAergic neurons
Electrical activity was necessary to sustain the 7-nAChR
staining levels found on GABAergic hippocampal neurons. This could be
seen by examining the distribution of cells among 7-nAChR labeling
categories as described above for control neurons. Treating cultures
for 3 d with TTX to block voltage-gated sodium channels produced a
dramatic reduction in the cells with high labeling for 7-nAChRs and
a corresponding increase in the proportion of unlabeled GAD-positive
neurons (Fig. 5). Moderate cells appeared unchanged in number but did shift to lower overall levels of labeling, best revealed by quantifying the number of 7-nAChR clusters (see below). The TTX treatment produced no decrement in the total number of
neurons identified by MAP-2 staining (99 ± 5% of control values; mean ± SEM, n = 5 experiments with triplicate
cultures; total of 1579 cells examined) or in the number of GABAergic
neurons identified by GAD staining (98 ± 4% of control
values).
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Figure 5.
Activity-dependent regulation of 7-nAChR
labeling. The distribution of GABAergic neurons among unlabeled,
moderate, and high 7-nAChR labeling categories after treatment
during the third week of culture with TTX (1 µM, 3 d), APV (50 µM, 3 d), bicuculline (20 µM, 7 d), nicotine (0.5 µM, 3 and
7 d), or D-TC (10 µM, 3 d). Values
are expressed as a percentage of controls from the same experiment
(horizontal line) and represent the mean ± SEM of
three to nine experiments. The level of 7-nAChR labeling on
GABAergic neurons depends on electrical activity and NMDA receptor
activation but not on endogenous GABAA or nicotinic
receptor signaling. Addition of nicotine, however, can enhance the
level of labeling. Statistical differences were determined by ANOVA;
*p < 0.05, ** p < 0.01, ***p < 0.001.
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Blockade of NMDA receptor function produced the same effect as TTX. A
3 d exposure to APV almost completely eliminated cells from the
high category; a concomitant increase occurred in unlabeled cells (Fig.
5). APV produced no change in the total number of neurons (92 ± 8% of control values; n = 3 experiments) and no change
in the number of GAD-positive neurons (101 ± 8% of control values). Blockade of GABAergic transmission with bicuculline for as
long as 7 d, on the other hand, had no effect on the proportions of GAD-positive neurons scoring either as moderate or high for 7-nAChR labeling. Blockade of nicotinic transmission with
D-TC also had no effect, although low
concentrations of nicotine either for a 3 or 7 d period increased
the proportion of GAD-positive neurons with high 7-nAChR labeling.
The results indicate that endogenous activation of NMDA receptors is
essential to sustain the 7-nAChR patterns detectable here on
GABAergic hippocampal neurons. Endogenous GABAergic and nicotinic
signaling are much less important, although chronic nicotine exposure
can increase 7-nAChR staining.
A different outcome was obtained in one respect when measuring the
total number of 7-nAChRs on the surface of all hippocampal cells in
culture. Using 125I--Bgt binding to
quantify 7-nAChRs on intact cells showed that the 3 d TTX
treatment produced no change overall (99 ± 10% of control
values; mean ± SEM; n = 3 experiments, three
cultures per experiment). The bicuculline treatment (7 d) produced a
small nominal increase (131 ± 21% of control values;
n = 3), but the difference was not significant
(p > 0.1). The divergence between these results
with TTX and those obtained by fluorescence analysis of 7-nAChRs on
GAD-positive neurons may have arisen from either of two sources.
GAD-positive neurons are a minority of the neuronal population and may
have been overshadowed by receptor changes on the population as a
whole. Alternatively, the treatments may have altered the distribution
of receptors, changing the proportions that were sufficiently
concentrated to be detected by fluorescence imaging. In any case, the
results clearly demonstrate the importance of monitoring receptor
distribution on individual neurons rather than simply measuring overall
levels in heterogeneous cultures.
Effects of activity on 7-nAChR clusters
To obtain a finer grain analysis of activity-dependent 7-nAChR
regulation, we again examined 7-nAChR clusters on GAD-positive neurons with moderate receptor labeling. Moderate cells were chosen because, as stated above, they displayed numerous clusters that could
be readily resolved. GAD staining was used to distinguish GABAergic
presynaptic terminals contacting the dendrites of such neurons, whereas
Alexa488--Bgt staining was used to visualize 7-nAChR clusters on
the cells. Threshold values of five contiguous pixels (0.05 µm2) were set both for the minimum area
defining an 7-nAChR cluster or GAD-positive presynaptic terminal and
for the minimum area defining overlap or codistribution of the two
labels. Threshold values defining the minimum labeling intensity
acceptable for a cluster or terminal were set at half the maximal
intensities over background seen for moderate cells in control cultures
in the same experiment.
By these criteria the 3 d treatment with either TTX or APV
produced no change in the mean size either of GAD-positive terminals abutting the neurons or of 7-nAChR clusters themselves (Fig. 6). Both treatments did, however, produce
large decreases in the number of 7-nAChR clusters (Fig. 6) without
changing the number of GAD-positive terminals (data not shown).
Bicuculline treatment (7 d) increased the mean size of GAD-positive
terminals but had no effect on the size or number of 7-nAChR
clusters. Nicotine treatment for 3 d had no effect on GAD-positive
terminals but increased both the mean size and number of 7-AChR
clusters (Fig. 6). Neither bicuculline nor nicotine significantly
altered the proportion of 7-nAChR clusters found at GABAergic
synapses. Thus, 25 ± 4 and 22 ± 8% (n = 4 experiments) of the 7-nAChR clusters on GABAergic neurons from
bicuculline- and nicotine-treated cultures, respectively, were located
at GAD-positive terminals, whereas 18 ± 4% (n = 11 experiments) were so in control cultures. It was not possible to
determine whether TTX or APV preferentially affected 7-nAChR
clusters at GABAergic synapses because of the small residual numbers of
clusters in these cases. When sufficient clusters were present for
analysis (e.g., controls and cells treated with bicuculline, D-TC, or nicotine), the incidence of
colocalization for 7-nAChR clusters and GAD-positive terminals was
at least five times that expected from random occurrence based on the
proportion of dendritic surface occupied by each population.
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Figure 6.
Activity-dependent regulation of 7-nAChR
clusters. Shown are the normalized values for the mean size of either
GAD-positive terminals or 7-nAChR clusters or the mean number of
7-nAChR clusters distributed along the dendrites of GABAergic
neurons having moderate levels of 7-nAChR labeling. Cultures were
treated for 3 d with TTX (1 µM), APV (50 µM), bicuculline (20 µM), or nicotine (0.5 µM). Both TTX and APV decrease while nicotine increases
the number of 7-nAChR clusters; only nicotine increases mean cluster
size. Only bicuculline increases mean GAD terminal size, but it has no
effect on the mean size or number of 7-nAChR clusters. Values are
expressed as a percentage of controls from the same experiment
(horizontal line) and represent the mean ± SEM of
four to nine experiments. Controls had 45 ± 5 7-nAChR clusters
per 100 µm dendritic length and mean sizes of 0.18 ± 0.02 and
0.34 ± 0.02 µm2 for the 7-nAChR clusters
and GAD-positive terminals, respectively (n = 7 experiments). Statistical differences were determined by ANOVA;
*p < 0.05, ** p < 0.01, ***p < 0.001.
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The results indicate that elimination of action potentials or blockade
of NMDA receptors shifts cells to lower categories of labeling and
reduces the number of detectable 7-nAChR clusters. Analyzing the
distribution of clusters after triply staining for 7-nAChRs, GAD,
and 1-GABAARs yielded an additional
conclusion. On neurons that expressed both classes of receptors,
1-GABAAR clusters were found associated with
58 ± 12% of the GAD-positive terminals abutting the dendrites
(eight cells, four experiments). On the same neurons, 7-nAChR
clusters were associated with 38 ± 18% of the GAD-positive
terminals. Both kinds of receptor clusters co-localized at 29 ± 18% of the GAD-positive terminals. This value is consistent with the
two classes of receptors distributing independently at the synapses and
indicates that their presence is not mutually exclusive.
Neurotrophin-dependent regulation of 7-nAChRs
The neurotrophins BDNF and NGF are both produced by hippocampal
neurons and play important roles in synaptic development and plasticity
in the hippocampus (McAllister et al., 1999; Schuman, 1999; Schinder
and Poo, 2000). Both can enhance overall excitation in the cultures
(Bolton et al., 2000; Tyler and Pozzo-Miller, 2001; Kovalchuk et al.,
2001) and are regulated in turn by synaptic activity (Zafra et al.,
1991; Lauterborn et al., 2000; Hartmann et al., 2001). Neuregulin
upregulates functional 7-nAChRs on hippocampal neurons in culture
(Liu et al., 2001), and BDNF can induce neuregulin release in other
systems (Loeb and Fischbach, 1997; Loeb et al., 2002). These
observations motivated us to test the effects of neuregulin in the
imaging assays used here and to examine the roles of BDNF and NGF in
7-nAChR regulation. Exposing hippocampal neurons to neuregulin
(1-1 isoform) for 3 d produced significant increases in the
proportion of GAD-positive neurons expressing high 7-nAChR labeling
(Fig. 7). This mirrored the neuregulin
effects reported previously on 7-nAChR-mediated whole-cell responses
(Liu et al., 2001). Compensatory decreases were seen in the unlabeled
and moderate categories of GAD-positive neurons (data not shown).
Treatment with either BDNF or NGF produced the same increases as
neuregulin (Fig. 7). No further increase was seen when BDNF and
neuregulin were combined (192 ± 22, 175 ± 25, and 163 ± 13% for BDNF, neuregulin, and BDNF + neuregulin treatments, respectively, normalizing the data for control values; mean ± SEM
with n = 4 cultures, 80 cells). Anti-BDNF and anti-NGF
antibodies each significantly decreased the proportion of highly
labeled GAD-positive neurons (Fig. 7). The results suggest that
endogenous BDNF and NGF contribute to 7-nAChR levels on GABAergic
neurons. Neither BDNF nor NGF, when added to the cultures however,
could overcome the effects of either TTX or APV (Fig. 7). Increasing the concentration of BDNF 10-fold did not alter the outcome (data not
shown). Neuregulin was also ineffective against TTX.
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Figure 7.
Neurotrophin regulation of 7-nAChR labeling.
The distribution of GABAergic neurons among 7-nAChR labeling
categories was analyzed as in Figure 5 after treating cells for 3 d with neuregulin (5 nM), BDNF (100 ng/ml), NGF (100 ng/ml), chicken anti-BDNF antibodies, sheep anti-NGF antibodies, both
chicken and sheep nonimmune IgG (negative controls), and where
indicated, either TTX (1 µM) or APV (50 µM)
as well. Shown here are the results obtained for cells in the high
labeling category. Values are expressed as a percentage of controls
from the same experiment (horizontal line) and represent
the mean ± SEM of four to nine experiments. Neuregulin, BDNF, and
NGF each elevate the level of 7-nAChR labeling while anti-BDNF, and
anti-NGF decrease it. The neurotrophins are unable to overcome the
negative effects of either TTX or APV. Neuregulin was unable to
overcome TTX (not significantly different from TTX alone; TTX data from
Fig. 5). Statistical differences were determined by ANOVA; **
p < 0.01, ***p < 0.001.
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Neurotrophin effects were further evaluated by examining the number and
location of 7-nAChR clusters. GABAergic neurons with moderate
7-nAChR labeling were again chosen because individual receptor
clusters could be readily quantified. BDNF, NGF, and neuregulin each
had little effect on the size or number of GAD-positive terminals (data
not shown), but each increased the number of 7-nAChR clusters (Fig.
8). BDNF also caused a small increase in
the mean size of 7-nAChR clusters (data not shown). Treating the
neurons with either anti-BDNF or anti-NGF antiserum had the opposite
effect, reducing the numbers of 7-nAChR clusters (Fig. 8). Nonimmune IgG used as negative controls had no effect. Importantly, neither BDNF
nor NGF were able to overcome the effects of either TTX or APV on the
number of 7-nAChR clusters (Fig. 8).
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Figure 8.
Neurotrophin regulation of 7-nAChR clusters.
The numbers of 7-nAChR clusters per 100 µm dendritic length on
GABAergic neurons are expressed as a percentage of controls from the
same experiment. Values represent the mean ± SEM of four to nine
experiments. Conditions and agents are the same as in Figure 7.
Neuregulin, BDNF, and NGF each increase the total number of 7-nAChR
clusters, whereas anti-BDNF and anti-NGF decrease them. The
neurotrophins are unable to overcome the negative effects of either TTX
or APV. Statistical differences were determined by ANOVA;
*p < 0.05, ***p < 0.001.
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A qualitatively similar pattern was seen when counting only
7-nAChR clusters localized at GAD-positive terminals. Whereas control cells had 8.0 ± 1.0 such "co-clusters" per 100 µm
dendritic length (32 neurons, 10 experiments), cells treated with TTX,
APV, anti-BDNF, or anti-NGF each had much reduced numbers (2.7 ± 1.0, 0.5 ± 0.3, 2.8 ± 0.3, and 1.0 ± 0.4, respectively). In fact for several of the treatments, the number of
7-nAChR clusters at GAD-positive terminals was not significantly
different from that predicted for random occurrence based on the total
number of clusters and GAD-positive terminals along the dendrite. Both
BDNF and NGF increased the number of clusters at GAD-positive terminals
(16 ± 3 and 12 ± 2/100 µm, respectively) but neither
could overcome the effects of APV (1.2 ± 0.2 and 0.6 ± 0.3, respectively). The overall results, both from cluster analysis on
moderate cells and from distribution analysis of cells among labeling
categories, are in agreement. Neurotrophins are necessary, but not
sufficient in the absence of either electrical activity in general or
NMDA receptor activation in particular, to sustain 7-nAChR
distribution patterns on the neurons.
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DISCUSSION |
The principal findings reported here are first that
fluorescently-labeled -Bgt can be used to visualize 7-nAChRs on
the surface of hippocampal neurons in culture and that the receptors are arranged in clusters localized in part at GABAergic synapses. Second, spontaneous electrical activity and NMDA receptor activation are essential for sustaining the receptor clusters, including those at
GABAergic synapses. Third, endogenous BDNF and NGF also are required,
but exogenous BDNF or NGF cannot overcome the downregulation caused by
blockade of NMDA receptors. Several models can account for the results
including the possibilities that BDNF and NGF act upstream of NMDA
receptors, perhaps increasing excitation in the cultures as suggested
in other situations (Bolton et al., 2000; Tyler and Pozzo-Miller, 2001;
Kovalchuk et al., 2001) or that they are required in parallel with
activity. GABAergic neurons have been widely recognized as targets of
7-nAChR signaling. Presynaptic 7-nAChRs on the neurons modulate
GABA release while somatodendritic 7-nAChRs mediate synaptic input
to the neurons and influence their participation in hippocampal
circuits (Alkondon et al., 1997a, 1998; Frazier et al., 1998; Hefft et
al., 1999; Jones et al., 1999; Ji and Dani, 2000; Alkondon and
Albuquerque, 2001; Buhler and Dunwiddie, 2001, 2002; Ji et al., 2001;
Maggi et al., 2001). Patch-clamp recording suggests that GABAergic
interneurons have the highest whole-cell 7-nAChR responses in
hippocampal cultures (Liu et al., 2001). The present studies show that
GABAergic neurons express the highest incidence of surface 7-nAChRs
detectable by fluorescence imaging, a feature that facilitated the
analysis of receptor distribution on the cells. The receptors can also be found on glutamatergic neurons, however, and recent ultrastructural studies in situ show them to be localized at glutamatergic
synapses as well (Fabian-Fine et al., 2001; Levy and Aoki, 2002).
Costaining for GAD and 7-nAChRs made clear the nonrandom
distribution of 7-nAChRs clusters at presumed GABAergic synapses defined by GAD-positive terminals abutting the dendrites. Triply staining for GAD, 7-nAChRs, and 1-GABAARs
indicated that the two types of receptors were not mutually exclusive
in their clustering at individual GABAergic synapses. This is quite
different from the pattern reported for misdirected receptors on
isolated neurons that are constrained to receive autapses as their only
innervation (Rao et al., 2000). In that case inappropriate receptors
could be found at some synapses (e.g., GABA receptors opposite
glutamatergic terminals) but not colocalized with correct receptors
(i.e., glutamate receptors) at the same synapses. In the present
studies the cultures were heterogeneous, the neurons were multiply
innervated, and the 7-nAChR clusters did not exclude the expected
transmitter receptor.
Certainly presynaptic 7-nAChRs would be expected at hippocampal
GABAergic synapses, judging from electrophysiological studies. The
7-nAChR clusters imaged here, however, seemed more likely to be
postsynaptic for the most part. This was suggested by the distribution
of receptors in three-dimensional space, as seen in stacks of optical
sections and by the comparative distributions of 7-nAChRs,
GABAA-1 receptors, and presynaptic GAD
staining in triple-stain experiments. It was also strongly suggested by the finding that detectable 7-nAChR clusters were not associated with GAD-positive terminals if the terminals contacted dendrites from
neurons not expressing detectable 7-nAChRs themselves. Alternative explanations for this latter finding exist but are less plausible. The
imaging studies do not, of course, exclude the presence of presynaptic
7-nAChRs but do suggest that the staining intensity associated with
them is usually less than the threshold set here for defining
fluorescently labeled 7-nAChR clusters. Indeed, the ultrastructural
studies cited above indicate the presence of both presynaptic and
postsynaptic 7-nAChR clusters in the adult hippocampus.
Ultrastructural analysis will probably also be required here to
distinguish unambiguously between presynaptic and postsynaptic
7-nAChRs in culture.
Neuronal activity had pronounced effects on the pattern of 7-nAChRs.
A 3 d exposure to TTX did not alter the total number of surface
7-nAChRs in culture as measured by
125I--Bgt binding, but did markedly
reduce 7-nAChR staining levels and cluster abundance on GABAergic
neurons. The treatment may have dispersed 7-nAChR clusters, causing
receptor density to be subthreshold for detection by fluorescence.
Alternatively, reductions in the number of 7-nAChRs on GABAergic
neurons may have been overshadowed by increases in receptors on other
cell populations in the cultures. Blockade of NMDA receptors with APV produced reductions at least as severe as did TTX, but it would be
premature to conclude that all of the regulatory effects of neuronal
activity were mediated through NMDA receptors.
Surprisingly, blockade of GABAA receptors with
bicuculline had no effect on 7-nAChR staining patterns even though a
significant fraction of the 7-nAChR clusters were located at
GABAergic synapses. The normal GABA-mediated maturation of the chloride
gradient should already have occurred before the bicuculline treatment
(Ganguly et al., 2001). Blockade of nicotinic receptors with
D-TC also had no obvious effect, although the culture
medium contained sufficient choline to partially desensitize the
receptors (Alkondon et al., 1997b). Absent from the cultures was the
septal input which provides the major cholinergic innervation of the
hippocampus in vivo, and staining for vesicular ACh
transporter failed to detect cholinergic terminals in the cultures.
Chronic nicotine exposure, however, did upregulate the level of
staining, consistent with other studies on CNS 7-nAChRs (Barrantes
et al., 1995; Peng et al., 1997; Kawai and Berg, 2001), and suggests
that cholinergic input could play a role in vivo.
Perhaps most interesting are the effects of BDNF and NGF. Both were
attractive candidates for mediating activity-dependent regulation of
7-nAChR clusters. BDNF and NGF are expressed in the hippocampus and
both can influence synapse formation (McAllister et al., 1999; Schuman,
1999; Schinder and Poo, 2000). BDNF can enhance neuregulin release at
the neuromuscular junction, and neuregulin upregulates functional
7-nAChRs on hippocampal neurons (Loeb and Fischbach, 1997; Liu et
al., 2001; Loeb et al., 2002). Moreover, chronic excitation increases
the expression of BDNF and NGF by hippocampal cells. The present
studies show that BDNF and NGF can each increase the number of
7-nAChR clusters on GABAergic hippocampal neurons and that anti-BDNF
and anti-NGF antisera each reduce the number. The latter results argue
strongly for a neurotrophin role in regulating 7-nAChRs.
Neither BDNF nor NGF, when supplied exogenously, could overcome the
effects of either TTX or APV. One possibility, as cited above, is that
the neurotrophins act upstream of NMDA receptors. But neurotrophins are
known to have multiple effects and sites of action in the hippocampus
(McAllister et al., 1999; Schuman, 1999; Schinder and Poo, 2000).
Accordingly, other possibilities that must be considered are that the
neurotrophins are necessary but not sufficient or that they must act
locally or that their signaling is compromised somehow by NMDA receptor
blockade (e.g., trk receptor downregulation). Still it is attractive to
consider that a neurotrophin-mediated enhancement of activity might
drive neuregulin release which, in turn, regulates 7-nAChRs. BDNF
and neuregulin are not additive in their effects, and preliminary results suggest that exogenous neuregulin can overcome the effects of
anti-BDNF antiserum on 7-nAChR cluster abundance (W. M. Zago and D. K. Berg, unpublished results). These observations
support a downstream site of action for neuregulin. Where NMDA
receptors participate in the pathway and exactly how the neurotrophins
and neuregulin exert their effects have yet to be determined.
Chronic blockade of excitatory activity in hippocampal
cultures increases the capacity for excitation by altering both
presynaptic and postsynaptic elements at glutamatergic synapses
(Schinder and Poo, 2000; Turrigiano and Nelson, 2000; Murthy et al.,
2001). This homeostatic adaptation depends on AMPA receptor signaling rather than on NMDA receptors. NMDA receptors commonly mediate other
types of activity-dependent synaptic plasticity in the hippocampus. Nonetheless, the TTX- and APV-mediated reductions seen here in 7-nAChR clusters on GABAergic neurons could be viewed as homeostatic compensation if the receptor clusters normally enhance GABAergic inhibitory signaling. Although this is certainly true for presynaptic 7-nAChRs (Alkondon et al., 1997a; Maggi et al., 2001), it is less
clear for postsynaptic 7-nAChRs at GABAergic synapses. Because GABAergic neurons, however, were the focus of the present study, it
might be argued that any reduction in excitatory input to the cells,
including that via 7-nAChRs, could represent a compensatory mechanism because it would ultimately reduce output from these inhibitory neurons.
Key questions remain about the physiological roles of 7-nAChR
clusters at noncholinergic synapses. The fact that such clusters persist in the adult suggests they provide a continuing function. Particularly interesting are postsynaptic 7-nAChR clusters at GABAergic synapses because the postsynaptic clusters should oppose the
effect of presynaptic 7-nAChRs on inhibitory signaling. This opposition would be acute if both classes of receptors are activated in
the same time frame, as should occur if ACh participates in volume
transmission made possible by the diffuse cholinergic projection of the
medial septum to the hippocampus (McKinney et al., 1983; Frotscher and
Leranth, 1985; Descarries et al., 1997; Descarries, 1998; Zoli et al.,
1999) (but see Turrini et al., 2001). Equally intriguing are the
7-nAChR clusters found at the tips of filopodia-like extensions
emanating from GABAergic neuron dendrites. Dendritic filopodia are
candidates for synaptic precursor structures (Fiala et al., 1998;
Jontes and Smith, 2000; Cline, 2001). What 7-nAChRs do for such
structures will be interesting to determine.
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FOOTNOTES |
Received May 9, 2002; revised June 27, 2002; accepted July 3, 2002.
*
H.K. and W.Z. contributed equally to this work.
This work was supported by National Institutes of Health Grants NS12601
and NS35469 and by Tobacco-Related Disease Research Program Grant
9RT-0221. We thank Xiao-Yun Wang for preparation of the hippocampal
cultures and Lynn Ogden for performing the binding studies. H.K. is an
American Heart Association Postdoctoral Fellow.
Correspondence should be addressed to Darwin K. Berg, Neurobiology
Section, Biology Division, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0357. E-mail: dberg{at}ucsd.edu.
H. Kawai's present address: Department of Neuroscience, Brown
University, 190 Thayer Street, Providence, RI 02912.
| |
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TOP
ABSTRACT
INTRODUCTION
